U.S. patent application number 13/476809 was filed with the patent office on 2012-09-13 for microdevices containing photorecognizable coding patterns and methods of using and producing the same.
This patent application is currently assigned to Aviva Biosciences Corporation. Invention is credited to Depu Chen, Jing Cheng, Mingxian Huang, Litian Liu, David M. Rothwarf, Wei Shao, Baoquan Sun, Guoliang Tao, Xiaobo Wang, Lei WU, Junquan Xu, Weiping Yang.
Application Number | 20120228386 13/476809 |
Document ID | / |
Family ID | 28452381 |
Filed Date | 2012-09-13 |
United States Patent
Application |
20120228386 |
Kind Code |
A1 |
WU; Lei ; et al. |
September 13, 2012 |
MICRODEVICES CONTAINING PHOTORECOGNIZABLE CODING PATTERNS AND
METHODS OF USING AND PRODUCING THE SAME
Abstract
This invention relates generally to the field of moiety or
molecule analysis, isolation, detection and manipulation and
library synthesis. In particular, the invention provides a
microdevice, which microdevice comprises: a) a substrate; and b) a
photorecognizable coding pattern on said substrate. Preferably, the
microdevice does not comprise an anodized metal surface layer.
Methods and kits for isolating, detecting and manipulating
moieties, and synthesizing libraries using the microdevices are
also provided. The invention further provides two-dimensional
optical encoders and uses thereof. In certain embodiments, the
invention provides a microdevice, which microdevice comprises: a) a
magnetizable substance; and b) a photorecognizable coding pattern,
wherein said microdevice has a preferential axis of magnetization.
Systems and methods for isolating, detecting and manipulating
moieties and synthesizing libraries using the microdevices are also
provided.
Inventors: |
WU; Lei; (San Diego, CA)
; Wang; Xiaobo; (San Diego, CA) ; Tao;
Guoliang; (San Diego, CA) ; Xu; Junquan; (San
Diego, CA) ; Cheng; Jing; (Beijing, CN) ;
Huang; Mingxian; (San Diego, CA) ; Sun; Baoquan;
(Shangdong Province, CN) ; Shao; Wei; (Beijing,
CN) ; Liu; Litian; (Beijing, CN) ; Chen;
Depu; (Beijing, CN) ; Rothwarf; David M.; (La
Jolla, CA) ; Yang; Weiping; (San Diego, CA) |
Assignee: |
Aviva Biosciences
Corporation
San Diego
CA
|
Family ID: |
28452381 |
Appl. No.: |
13/476809 |
Filed: |
May 21, 2012 |
Related U.S. Patent Documents
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12760379 |
Apr 14, 2010 |
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13476809 |
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11841935 |
Aug 20, 2007 |
7718419 |
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12760379 |
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11230411 |
Sep 20, 2005 |
7262016 |
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11841935 |
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10104571 |
Mar 21, 2002 |
7015047 |
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11230411 |
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Current U.S.
Class: |
235/488 |
Current CPC
Class: |
B01J 2219/0059 20130101;
C40B 40/10 20130101; G01N 33/54386 20130101; B01J 2219/00659
20130101; B01J 2219/00729 20130101; B01J 2219/00549 20130101; B01J
2219/0074 20130101; B01L 2400/0415 20130101; B82Y 30/00 20130101;
B01J 2219/00468 20130101; B01J 2219/00596 20130101; B01L 3/5027
20130101; B01J 2219/00457 20130101; B01J 2219/00592 20130101; B01J
2219/00722 20130101; B01J 2219/00556 20130101; B01J 2219/00743
20130101; B01J 2219/00702 20130101; B01L 2200/0647 20130101; B82Y
10/00 20130101; B01J 2219/00707 20130101; C40B 40/12 20130101; B01J
2219/00731 20130101; B01J 2219/0072 20130101; B01J 2219/00576
20130101; Y10T 428/24917 20150115; B01L 3/502761 20130101; B03C
5/028 20130101; C07K 1/047 20130101; B01J 2219/00542 20130101; B01L
2400/043 20130101; B01J 2219/00547 20130101; G06K 19/06046
20130101; G01N 33/54366 20130101; G06K 19/06196 20130101; B01J
2219/00497 20130101; B82Y 5/00 20130101; B01L 3/50273 20130101;
B01J 2219/00725 20130101; B01J 2219/00585 20130101; C07B 2200/11
20130101; B01J 2219/00655 20130101; B01J 2219/00502 20130101; B01L
2300/0819 20130101; B01J 2219/005 20130101; B01J 2219/0054
20130101; B01J 2219/00734 20130101; C40B 40/06 20130101; B01J
19/0046 20130101; G06K 19/06009 20130101 |
Class at
Publication: |
235/488 |
International
Class: |
G06K 19/00 20060101
G06K019/00; G06K 19/06 20060101 G06K019/06; G06K 19/02 20060101
G06K019/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2001 |
CN |
01104318.0 |
Claims
1-11. (canceled)
12. An encoded microdevice comprising a longest linear dimension of
not more than 500 microns; a photorecognizable coding pattern; and
top and bottom layers enclosing the photorecognizable coding
pattern.
13. An encoded microdevice comprising a longest linear dimension of
not more than 500 microns; a photorecognizable coding pattern; and
top and bottom layers encapsulating the photorecognizable coding
pattern.
14. The microdevice of claim 12, wherein the photorecognizable
coding pattern comprises a 2-D barcode.
15. The microdevice of claim 12, wherein the microdevice further
comprises a magnetic material.
16. The microdevice of claim 12, wherein the photorecognizable
coding pattern is detectable though the enclosing layers.
17. The microdevice of claim 12, wherein the photorecognizable
coding pattern is lithographically patterned.
Description
[0001] The present application is a continuation of U.S.
application Ser. No. 11/841,935, filed on Aug. 20, 2007, which has
been allowed, and which is a continuation of U.S. application Ser.
No. 11/230,411, filed on Sep. 20, 2005, now U.S. Pat. No.
7,262,016, which is a divisional of U.S. application Ser. No.
10/104,571, filed on Mar. 21, 2002, now U.S. Pat. No. 7,015,047,
which is a continuation-in-part of U.S. patent application Ser. No.
09/924,428, filed Aug. 7, 2001, now abandoned. The content of each
of these U.S. patents and applications is incorporated by reference
herein in its entirety.
TECHNICAL FIELD
[0002] This invention relates generally to the field of moiety or
molecule analysis, isolation, detection, analysis, manipulation and
chemical synthesis. In particular, the invention provides a
microdevice, which microdevice comprises: a) a substrate; and b) a
photorecognizable coding pattern on said substrate. Preferably, the
microdevice does not comprise an anodized metal surface layer.
Methods and kits for isolating, detecting, analyzing and
manipulating moieties, and synthesizing compounds or libraries
using the microdevices are also provided. The invention further
provides two-dimensional optical encoders and uses thereof.
BACKGROUND ART
[0003] Micro array technology has revolutionized the biotechnology
industry. Its ability to process large number of biological samples
in parallel is unprecedented. The current micro array technologies
can be generally categorized into two groups. One group is based on
a two-dimensional solid support system, on which all the biological
reactions and signal detections are completed (see e.g.,
"Large-scale identification, mapping and genotyping of
single-nucleotide polymorphisms in the human genome" by Wang, D.
G., et al., Science (1998) 280:1077-1082). The other group utilizes
microparticles as reaction platform. One example of such technology
is the fluorescent particle technology or three-dimensional micro
array (see e.g., "Multiplexed particle-based flow cytometric
assays" by Vignali, D. A., J. Immunol. Methods (2000) 243:243-255;
"Multiplexed analysis of human cytokines by use of the FlowMetrix
system" by Oliver, K. G., et al., Clinical Chemistry (1998)
44:2057-2060; and U.S. Pat. Nos. 6,057,107, 5,981,180 and
5,736,330). Limitations have been observed on both types of
technologies. Biological reaction conducted on the two-dimensional
based technology platform is limited by molecule diffusion. In
general, a longer reaction time is required for the two-dimensional
reaction platforms. The three-dimensional fluorescence particle
technology has problems in the complexity of the technology and
limitation on numbers of particle encoding, e.g., only hundreds or
thousands of encoding are available. In addition, the detection of
two-color fluorescence levels on the microparticles requires
sophisticated instrumentation.
[0004] WO 00/16893 discloses a system for carrying out parallel
bioassays. Microfabricated labels are made to each carry a
biochemical test, many different labels are mixed together with an
analyte sample. A device that reads the individual labels isolates
the results of the individual tests. The microfabricated labels
have a surface layer of anodized metal and are produced by
anodizing, lithographic patterning and etching steps. Aluminum is
the preferred metal.
[0005] In modern pharmaceutical industry, a very important approach
for developing new drugs is through the screening of
combinatorially synthesized compound libraries. Combinatorial
chemistry is high-throughput, rapid and "synchronized" method that
can synthesize the structurally-similar compounds and derivatives
of a lead compound. While previous synthesis methods are primarily
based on individual compound synthesis, combinatorial chemistry is
capable of synthesizing thousands to tens of thousands compounds in
serial and parallel fashions (Dolle, Journal of Combinatorial
Chemistry (2000) 2:383-433).
DISCLOSURE OF THE INVENTION
[0006] In one aspect, the present invention is directed to a
microdevice, which microdevice comprises: a) a substrate; and b) a
photorecognizable coding pattern on said substrate. Preferably, the
microdevice does not comprise an anodized metal surface layer,
e.g., an anodized aluminium surface layer.
[0007] In another aspect, the present invention is directed to a
method for isolating a moiety, which method comprises: a) providing
a microdevice comprising a substrate, a photorecognizable coding
pattern on said substrate and a binding partner that is capable of
binding to a moiety to be isolated; b) contacting a sample
containing or suspected of containing said moiety with said
microdevice provided in step a) under conditions allowing binding
between said moiety and said binding partner; and c) recovering
said microdevice from said sample, whereby the identity of said
isolated moiety is assessed by photoanalysis (or optical analysis)
of said photorecognizable coding pattern. Preferably, the
microdevice used in the method does not comprise an anodized metal
surface layer, e.g., an anodized aluminium surface layer.
[0008] In still another aspect, the present invention is directed
to a method for isolating a plurality of moieties, which method
comprises: a) providing a plurality of microdevices each comprising
a substrate, a photorecognizable coding pattern on said substrate
and a binding partner that is capable of binding to one type of
moieties to be isolated; b) contacting a sample containing or
suspected of containing said moieties with said microdevice
provided in step a) under conditions allowing binding between said
moieties and their corresponding binding partners; and c)
recovering a plurality of microdevices from said sample, whereby
the identity of said isolated moiety is assessed by photoanalysis
(or optical analysis) of said photorecognizable coding pattern.
Preferably, at least one of the microdevices used in the method
does not comprise an anodized metal surface layer, e.g., an
anodized aluminium surface layer. More preferably, at least 50% or
all of the microdevices used in the method do not comprise an
anodized metal surface layer, e.g., an anodized aluminium surface
layer.
[0009] In yet another aspect, the present invention is directed to
a method for manipulating a moiety, e.g., in a microfluidic
application, which method comprises: a) providing a microdevice
comprising a substrate, a photorecognizable coding pattern on said
substrate and a binding partner that is capable of binding to a
moiety to be manipulated; b) coupling said moiety to said
microdevice provided in step a) via binding between said moiety and
said binding partner to form a moiety-microdevice complex; and c)
manipulating said moiety-microdevice complex with a physical force,
preferably in a chip format, thereby said moiety is manipulated.
The above method for manipulating a moiety can be readily extended
to manipulating multiple moieties by using multiple microdevices,
each of which is targeted to one type of moieties to be
manipulated. Preferably, the microdevice used in the method does
not comprise an anodized metal surface layer, e.g., an anodized
aluminium surface layer.
[0010] In yet another aspect, the present invention is directed to
a kit for manipulating a moiety, e.g. in a microfluidic
application, which kit comprises: a) a microdevice comprising a
substrate, a photorecognizable coding pattern on said substrate and
a binding partner that is capable of binding to a moiety to be
manipulated; and b) a chip on which a moiety-microdevice complex
can be manipulated. Preferably, the microdevice used in the kit
does not comprise an anodized metal surface layer, e.g., an
anodized aluminium surface layer.
[0011] In yet another aspect, the present invention is directed to
a method for detecting a moiety, which method comprises: a)
providing a microdevice comprising a substrate, a photorecognizable
coding pattern on said substrate and a binding partner that is
capable of binding to a moiety to be detected; b) contacting a
sample containing or suspected of containing said moiety with said
microdevice provided in step a) under conditions allowing binding
between said moiety and said binding partner; and c) detecting
binding between said moiety and said binding partner, whereby the
presence or amount of said moiety is assessed by analysis of
binding between said moiety and said binding partner and the
identity of said moiety is assessed by photoanalysis (or optical
analysis) of said photorecognizable coding pattern. Preferably, the
microdevice used in the method does not comprise an anodized metal
surface layer, e.g., an anodized aluminium surface layer. The above
method for detecting a moiety can be readily extended to detecting
multiple moieties by using multiple microdevices, each of which is
targeted to one type of moieties to be manipulated.
[0012] In yet another aspect, the present invention is directed to
an array of microdevices for detecting moieties, which array
comprises a plurality of microdevices located, positioned or
immobilized on a surface, e.g., a chip, each of said microdevices
comprises a photorecognizable coding pattern on a substrate and a
binding partner that is capable of binding to a moiety to be
detected. Preferably, at least one of the microdevices used in the
array does not comprise an anodized metal surface layer, e.g., an
anodized aluminium surface layer. More preferably, at least 50% or
all of the microdevices used in the array do not comprise an
anodized metal surface layer, e.g., an anodized aluminium surface
layer.
[0013] In yet another aspect, the present invention is directed to
a method for synthesizing a library, which method comprises: a)
providing a plurality of microdevices, each of said microdevices
comprises a substrate and a photorecognizable coding pattern on
said substrate, wherein said photorecognizable coding pattern
corresponds to an entity to be synthesized on said microdevice; and
b) synthesizing said entities on said microdevices, wherein said
microdevices are sorted after each synthesis cycle according to
said photorecognizable coding patterns, whereby a library is
synthesized, wherein each of said microdevices contains an entity
that corresponds to a photorecognizable coding pattern on said
microdevice and the sum of said microdevices collectively contains
a plurality of entities that is predetermined before the library
synthesis. Preferably, at least one of the microdevices used in the
method does not comprise an anodized metal surface layer, e.g., an
anodized aluminium surface layer. More preferably, at least 50% or
all of the microdevices used in the method do not comprise an
anodized metal surface layer, e.g., an anodized aluminium surface
layer.
[0014] In yet another aspect, the present invention is directed to
a method for generating an antibody library, which method
comprises: a) contacting a library synthesized by the
above-described method with a plurality of antibodies; and b)
selecting and/or recovering antibodies that specifically bind to
the entities of the library synthesized by the above-described
method. Preferably, at least one of the microdevices used in the
method does not comprise an anodized metal surface layer, e.g., an
anodized aluminium surface layer. More preferably, at least 50% or
all of the microdevices used in the method do not comprise an
anodized metal surface layer, e.g., an anodized aluminium surface
layer.
[0015] In yet another aspect, the present invention is directed to
a two-dimensional optical encoder, which encoder comprises: a) a
substrate; and b) a microfabricated or micromachined
two-dimensional optical code on said substrate. Preferably, the
two-dimensional optical encoder does not comprise an anodized metal
surface layer, e.g., an anodized aluminium surface layer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates encoding examples of microdevices
(microstructures) wherein the microdevices are in rectangular shape
and the holes are introduced along the middle lines of the
structures.
[0017] FIG. 2 illustrates another example of the microdevices
(microstructures) wherein the microdevices are in circular disc
shape on which holes are produced.
[0018] FIG. 3 illustrates another example of the microdevices
(microstructures) wherein the microdevices are in circular disc
shape on which holes are produced and on which
orientation/alignment markers are also fabricated.
[0019] FIGS. 4A and 4B shows a MicroDisk, an exemplary microdevice
of the present invention, containing a 2D Barcode with the
numerical representation below.
[0020] FIG. 5 shows MicroDisks distributed on the surface of a
slide.
[0021] FIGS. 6A and 6B shows formation of chains caused by presence
of a weak magnetic field in the plane (generated by Alnico C-shaped
magnet).
[0022] FIG. 7 shows large number of MicroDisks standing on edge in
the presence of a strong magnetic field perpendicular to the plane
(generated by Neodymium disk-shaped magnet).
[0023] FIGS. 8A and 8B shows 2 MicroDisks.
[0024] FIG. 9 shows orientation of MicroDisks following magnetic
manipulation.
[0025] FIG. 10A-D shows results of a covalent attachment
experiment.
[0026] FIG. 11A-D shows results of a bioassay experiment.
[0027] FIGS. 12A and 12B shows further results of a bioassay
experiment determining the amount of fluorescence signal from both
types of MicroDisks in the same measurement.
[0028] FIG. 13 shows an exemplary fabrication process for making
one type of microdevices (or encoding particles) of the present
invention. FIG. 13A shows preparation of the substrate; FIG. 13B
shows deposition of the sacrificial layer; FIG. 13C shows
deposition of the first layer; FIG. 13D shows deposition of the
second layer; FIG. 13E shows patterning of the second layer; FIG.
13F shows deposition of the third layer; FIG. 13G shows patterning
of the first and the third layers; and FIG. 13H shows etching of
the sacrificial layer.
[0029] FIG. 14 is a schematic diagram showing chemical synthesis
process using 2-D optical encoders, an exemplary microdevice of the
present invention.
[0030] FIG. 15A-C illustrates three different coding methods for
2-D optical encoders.
[0031] FIG. 16 is a schematic diagram showing sorting and analyzing
of the 2-D optical encoder.
[0032] FIG. 17 is a schematic diagram showing a process using 2-D
optical encoders to detect "unknown" substances.
MODES OF CARRYING OUT THE INVENTION
[0033] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections that follow.
A. Definitions
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications referred to herein are incorporated by reference in
their entirety. If a definition set forth in this section is
contrary to or otherwise inconsistent with a definition set forth
in applications, published applications and other publications that
are herein incorporated by reference, the definition set forth in
this section prevails over the definition that is incorporated
herein by reference.
[0035] As used herein, "a" or "an" means "at least one" or "one or
more."
[0036] As used herein, "a photorecognizable coding pattern" refers
to any coding pattern that can be detected and/or assessed by
photoanalysis (optical analysis). Any photorecognizable property
can be used as the characteristics of the coding pattern. For
example, the photorecognizable coding pattern can be the material
composition of the substrate itself, a hole in the substrate or a
substance immobilized on the substrate, said substance having an
optical refractive property that is different from the optical
refractive property of the substrate. The versatility of the
photorecognizable coding pattern can be based on the shape, number,
position distribution, optical refractive property, material
composition, or a combination thereof, of the substrate, the
hole(s), or the substance(s) located, deposited or immobilized on
the substrate. To facilitate optical analysis (or photoanalysis) of
encoding patterns, certain microdevices may incorporate
"orientation" marks or alignment markers. For example, for the
microdevices having thin circular disk shapes, the microdevices
lying flat on either of its major surfaces will look identical,
causing difficulties in identification. Therefore, the orientation
markers can be used for indicating which major surface is up and
for helping decode the patterns.
[0037] As used herein, "a photorecognizable coding pattern on said
substrate" means that the photorecognizable coding pattern is
located on, in, or within (or inside) the substrate so that the
photorecognizable coding pattern is optically detectable. For
example, the photorecognizable coding pattern can be located on the
surface or on top of the substrate. The photorecognizable coding
pattern can also be located within or inside the substrate. In
other embodiments, the substrate may have multiple layers and the
photorecognizable coding pattern can be located on the surface
layer, on top of the surface layer, or can be located within or
inside one or more layers.
[0038] As used herein, "the photorecognizable coding pattern is
fabricated or microfabricated on the substrate" means the use of
any microfabrication or micromachining methods to produce or
generate encoding patterns on the substrate. Various semiconductor
fabrication protocols such as, pattern masking, photolithography,
wet etching, reactive-ion-etching and deep-reactive-ion-etching,
etc., can be used.
[0039] As used herein, "chip" refers to a solid substrate with a
plurality of one-, two- or three-dimensional micro structures or
micro-scale structures on which certain processes, such as
physical, chemical, biological, biophysical or biochemical
processes, etc., can be carried out. The micro structures or
micro-scale structures such as, channels and wells, electrode
elements, electromagnetic elements, are incorporated into,
fabricated on or otherwise attached to the substrate for
facilitating physical, biophysical, biological, biochemical,
chemical reactions or processes on the chip. The chip may be thin
in one dimension and may have various shapes in other dimensions,
for example, a rectangle, a circle, an ellipse, or other irregular
shapes. The size of the major surface of chips of the present
invention can vary considerably, e.g., from about 1 mm.sup.2 to
about 0.25 m.sup.2. Preferably, the size of the chips is from about
4 mm.sup.2 to about 25 cm.sup.2 with a characteristic dimension
from about 1 mm to about 5 cm. The chip surfaces may be flat, or
not flat. The chips with non-flat surfaces may include channels or
wells fabricated on the surfaces.
[0040] As used herein, "physical field," e.g., used itself or used
as "physical field in a region of space" or "physical field is
generated in a region of space" means that the region of space has
the following characteristics. When a moiety, alone or bound to a
microdevice via a binding partner, of appropriate properties is
introduced into the region of space (i.e. into the physical field),
forces are produced on the moiety, the microdevice or both, as a
result of the interaction between the moiety and/or microdevice and
the field. A moiety can be manipulated within a field via the
physical forces exerted on the moiety by the field. Exemplary
fields include electric, magnetic, acoustic, optical and velocity
fields. In the present invention, physical field always exists in a
medium in a region of space, and the moiety to be manipulated is
suspended in, or is dissolved in, or more generally, is placed in
the medium. Typically, the medium is a fluid such as aqueous or
non-aqueous liquids, or a gas. Depending on the field
configuration, an electric field may produce electrophoretic forces
on charged moieties, or may produce conventional dielectrophoretic
forces and/or traveling wave dielectrophoretic forces on charged
and/or neutral moieties. Magnetic field may produce magnetic forces
on magnetic moieties. Acoustic field may produce acoustic radiation
forces on moieties. Optical field may produce optical radiation
forces on moieties. Velocity field in the medium in a region of
space refers to a velocity distribution of the medium that moves in
the region of the space. Various mechanisms may be responsible for
causing the medium to move and the medium at different positions
may exhibit different velocities, thus generating a velocity field.
Velocity field may exert mechanical forces on moieties in the
medium.
[0041] As used herein, "medium (or media)" refers to a fluidic
carrier, e.g., liquid or gas, wherein a moiety, alone or bound to a
microdevice via a binding partner, is dissolved, suspended or
contained.
[0042] As used herein, "microfluidic application" refers to the use
of microscale devices, e.g., the characteristic dimension of basic
structural elements is in the range between less than 1 micron to 1
cm scale, for fluidic manipulation and process, typically for
performing specific biological, biochemical or chemical reactions
and procedures. The specific areas include, but are not limited to,
biochips, i.e., chips for biologically related reactions and
processes, chemchips, i.e., chips for chemical reactions, or a
combination thereof. The characteristic dimensions of the basic
elements refer to the single dimension sizes. For example, for the
microscale devices having circular shape structures (e.g. round
electrode pads), the characteristic dimension refers to the
diameter of the round electrodes. For the devices having thin,
rectangular lines as basic structures, the characteristic
dimensions may refer to the width or length of these lines. As used
herein, "microfluidic application" also encompass a process wherein
the moiety is manipulated directly by a desirable force. It is not
necessary that the force acts on the fluid to move the fluid and
the movement of the fluid effects the manipulation of the moiety.
For example, a moiety having a magnetic property can be manipulated
by a magnetic force directly while the fluid may not be moved by
the magnetic force. In other examples, the force can act on the
fluid first and the movement of the fluid will effect the
manipulation of the moiety. For example, a micropump can be used to
move fluid, which in turn moves or manipulates the moiety contained
in the fluid.
[0043] As used herein, "built-in structures on said substrate of a
chip" means that the structures are built into the substrate or the
structures are located on the substrate or the structures are
structurally linked to the substrate of the chip. In one
embodiment, the built-in structures may be fabricated on the
substrate. For example, as described in "Dielectrophoretic
manipulation of cells using spiral electrodes by Wang, et al.,
Biophys. J. (1997) 72:1887-1899", spiral electrodes are fabricated
on a glass substrate. Here the spiral electrodes are "built-in"
structures on the glass substrate. In another embodiment, the
"built-in" structures may be first fabricated on one substrate and
the structure-containing first substrate may then be attached or
bound to a second substrate. Such structures are "built-in"
structures not only on the first substrate but also on the second
substrate. In still another embodiment, the built-in structures may
be attached or bound to the substrate. For example, thin,
electrically-conductive wires may be used as electrodes for
producing electric field. These electric wires may be bound or
attached to a glass substrate. In this case, the
electrically-conductive wires are "built-in" structures on the
glass substrate. Throughout this application, when it is described
that "built-in" structures on the chip or on the substrate are
capable of generating physical forces and/or physical fields or
these structures generate physical forces and/or physical fields,
these structures are used in combination with external signal
sources or external energy sources.
[0044] As used herein, "structures internal to said apparatus"
means that the structures are integral parts of and structurally
linked to other parts of the apparatus, or the structures are not
separated or separable from other structural elements of the
apparatus. For example, such internal structures can be
microfabricated or otherwise attached to the substrate or other
structural element(s) of the apparatus. Any "built-in structures on
said substrates" described above are "structures internal to said
apparatus" as long as the said apparatus comprise the substrates.
Any built-in structures on a chip are "structures internal to said
apparatus" as long as the said apparatus comprise the chip.
Throughout this application, when it is described that "internal"
structures of apparatus are capable of generating physical forces
and/or physical fields or these structures generate physical forces
and/or physical fields, these structures are used in combination
with external signal sources or external energy sources.
[0045] As used herein, "micro-scale structures" means that the
scale of the internal structures of the apparatus for exerting
desired physical forces must be compatible with and useable in
microfluidic applications and have characteristic dimensions of
basic structural elements in the range from about 1 micron to about
20 mm scale.
[0046] As used herein, "moiety" refers to any substance whose
analysis, isolation, manipulation, measurement, quantification or
detection using the present microdevice is desirable. Normally, the
dimension (or the characteristic dimensions) of the moiety should
not exceed 1 cm. For example, if the moiety is spherical or
approximately spherical, the dimension of the moiety refers to the
diameter of the sphere or an approximated sphere for the moiety. If
the moiety is cubical or approximately cubical, then the dimension
of the moiety refers to the side width of the cube or an
approximated cube for the moiety. If the moiety has an irregular
shape, the dimension of the moiety may refer to the average between
its largest axis and smallest axis. Non-limiting examples of
moieties include cells, cellular organelles, viruses, particles,
molecules, e.g., proteins, DNAs and RNAs, or an aggregate or
complex thereof.
[0047] Moieties to be analyzed, isolated, manipulated, measured,
quantified or detected include many types of particles--solid
(e.g., glass beads, latex particles, magnetic beads), liquid (e.g.,
liquid droplets) or gaseous particles (e.g., gas bubble), dissolved
particles (e.g., molecules, proteins, antibodies, antigens, lipids,
DNAs, RNAs, molecule-complexes), suspended particles (e.g., glass
beads, latex particles, polystyrene beads). Particles can be
organic (e.g., mammalian cells or other cells, bacteria, virus, or
other microorganisms) or inorganic (e.g., metal particles).
Particles can be of different shapes (e.g., sphere, elliptical
sphere, cubic, discoid, needle-type) and can be of different sizes
(e.g., from nano-meter-size gold sphere, to micrometer-size cells,
to millimeter-size particle-aggregate). Examples of particles
include, but are not limited to, biomolecules such as DNA, RNA,
chromosomes, protein molecules (e.g., antibodies), cells, colloid
particles (e.g., polystyrene beads, magnetic beads), any
biomolecules (e.g., enzyme, antigen, hormone etc). One specific
type of particles refers to complexes formed between moieties and
their binding partners, as described in a co-pending US patent
application entitled "METHODS FOR MANIPULATING MOIETIES IN
MICROFLUIDIC SYSTEMS" (U.S. patent application Ser. No. 09/636,104,
by Wang, et al., filed on Aug. 10, 2000). The examples of such
complexes include particle-particle complexes, particle-molecule
complexes (e.g., cell-magnetic bead complexes formed by binding of
the cells onto antibody-coated beads through the interaction
between the antigens or protein molecules on cell surfaces and the
antibody molecules immobilized on the magnetic bead surfaces; DNA
molecule-magnetic bead complexes formed by immobilizing DNA
molecules on magnetic bead surfaces, or protein
molecule-polystyrene bead complexes formed by covering polystyrene
bead surfaces with protein molecules). The methods disclosed in a
co-pending US patent application "METHODS FOR MANIPULATING MOIETIES
IN MICROFLUIDIC SYSTEMS" (U.S. patent application Ser. No.
09/636,104, by Wang, et al., filed on Aug. 10, 2000) may be used
for manipulating moieties and/or binding partner-moiety complexes
in the devices and apparatus in the present invention. The
co-pending US patent application "METHODS FOR MANIPULATING MOIETIES
IN MICROFLUIDIC SYSTEMS" (U.S. patent application Ser. No.
09/636,104) by Wang, et al., filed on Aug. 10, 2000 is incorporated
by reference in their entirety. These moieties can be isolated,
manipulated, measured, quantified or detected using a microdevice
of the present application.
[0048] As used herein, "plant" refers to any of various
photosynthetic, eucaryotic multi-cellular organisms of the kingdom
Plantae, characteristically producing embryos, containing
chloroplasts, having cellulose cell walls and lacking
locomotion.
[0049] As used herein, "animal" refers to a multi-cellular organism
of the kingdom of Animalia, characterized by a capacity for
locomotion, nonphotosynthetic metabolism, pronounced response to
stimuli, restricted growth and fixed bodily structure. Non-limiting
examples of animals include birds such as chickens, vertebrates
such fish and mammals such as mice, rats, rabbits, cats, dogs,
pigs, cows, ox, sheep, goats, horses, monkeys and other non-human
primates.
[0050] As used herein, "bacteria" refers to small prokaryotic
organisms (linear dimensions of around 1 micron) with
non-compartmentalized circular DNA and ribosomes of about 70S.
Bacteria protein synthesis differs from that of eukaryotes. Many
anti-bacterial antibiotics interfere with bacteria proteins
synthesis but do not affect the infected host.
[0051] As used herein, "eubacteria" refers to a major subdivision
of the bacteria except the archaebacteria. Most Gram-positive
bacteria, cyanobacteria, mycoplasmas, enterobacteria, pseudomonas
and chloroplasts are eubacteria. The cytoplasmic membrane of
eubacteria contains ester-linked lipids; there is peptidoglycan in
the cell wall (if present); and no introns have been discovered in
eubacteria.
[0052] As used herein, "archaebacteria" refers to a major
subdivision of the bacteria except the eubacteria. There are three
main orders of archaebacteria: extreme halophiles, methanogens and
sulphur-dependent extreme thermophiles. Archaebacteria differs from
eubacteria in ribosomal structure, the possession (in some case) of
introns, and other features including membrane composition.
[0053] As used herein, "virus" refers to an obligate intracellular
parasite of living but non-cellular nature, consisting of DNA or
RNA and a protein coat. Viruses range in diameter from about 20 to
about 300 nm Class I viruses (Baltimore classification) have a
double-stranded DNA as their genome; Class II viruses have a
single-stranded DNA as their genome; Class III viruses have a
double-stranded RNA as their genome; Class IV viruses have a
positive single-stranded RNA as their genome, the genome itself
acting as mRNA; Class V viruses have a negative single-stranded RNA
as their genome used as a template for mRNA synthesis; and Class VI
viruses have a positive single-stranded RNA genome but with a DNA
intermediate not only in replication but also in mRNA synthesis.
The majority of viruses are recognized by the diseases they cause
in plants, animals and prokaryotes. Viruses of prokaryotes are
known as bacteriophages.
[0054] As used herein, "fungus" refers to a division of eucaryotic
organisms that grow in irregular masses, without roots, stems, or
leaves, and are devoid of chlorophyll or other pigments capable of
photosynthesis. Each organism (thallus) is unicellular to
filamentous, and possesses branched somatic structures (hyphae)
surrounded by cell walls containing glucan or chitin or both, and
containing true nuclei.
[0055] As used herein, "binding partners" refers to any substances
that bind to the moieties with desired affinity or specificity.
Non-limiting examples of the binding partners include cells,
cellular organelles, viruses, particles, microparticles or an
aggregate or complex thereof, or an aggregate or complex of
molecules, or specific molecules such as antibodies, single
stranded DNAs. The binding partner can be a substance that is
coated on the surface of a microdevice of the present invention.
Alternatively, the binding partner can be a substance that is
incorporated, e.g., microfabricated, into the material composition
of the surface layer or bulk structure of the microdevice. The
material composition of the surface layer or bulk structure of a
microdevice may possess binding affinity to certain moiety, and
thus functioning a binding partner itself.
[0056] As used herein, "an element that facilitates and/or enables
manipulation of the microdevice and/or a moiety/microdevice
complex" refers to any substance that is itself manipulatable or
makes the moiety/microdevice complex manipulatable with the desired
physical force(s). Non-limiting examples of the elements include
cells, cellular organelles, viruses, particles, microparticles or
an aggregate or complex thereof, or an aggregate or complex of
molecules.
[0057] As used herein, "microparticles" refers to particles of any
shape, any composition, any complex structures that are
manipulatable by desired physical force(s) in microfluidic settings
or applications. One example of microparticles is magnetic beads
that are manipulatable by magnetic forces. Another example of
microparticles is a cell that is manipulatable by an electric force
such as a traveling-wave dielectrophoretic force. The
microparticles used in the methods can have a dimension from about
0.01 micron to about ten centimeters. Preferably, the
microparticles used in the methods have a dimension from about 0.01
micron to about several thousand microns. Examples of the
microparticles include, but are not limited to, plastic particles,
polystyrene microbeads, glass beads, magnetic beads, hollow glass
spheres, particles of complex compositions, microfabricated
free-standing microstructures, etc. The microdevice of the present
invention is an example of a microparticle. Other particles include
cells, cell organelles, large biomolecules such as DNA, RNA and
proteins etc.
[0058] As used herein, "manipulation" refers to moving or
processing of the moieties, and the microdevices disclosed in the
present invention, which results in one-, two- or three-dimensional
movement of the moiety and/or the microdevices. manipulation can be
conducted in chip or non-chip format. When conducted in a chip
format, it can be conducted within a single chip or between or
among multiple chips, or on a substrate or among substrates of an
apparatus. "Manipulation" of moieties and/or the microdevices can
also be performed in liquid containers. Non-limiting examples of
the manipulations include transportation, focusing, enrichment,
concentration, aggregation, trapping, repulsion, levitation,
separation, sorting, fractionation, isolation, or linear or other
directed motion of the moieties. For effective manipulation, the
characteristics of the moiety and/or the microdevices to be
manipulated and the physical force used for manipulation must be
compatible. For example, microdevices with certain magnetic
properties can be used with magnetic force. In a specific example,
the microdevice can comprise one or more types of magnetic
materials, such ferro- or ferri-magnetic materials in the middle of
the substrate. Exemplary ferro- or ferri-magnetic materials can be
nickel metal or CoTaZr (Cobalt-Tantalum-Zirconium) alloy.
Similarly, microdevices with electric charge(s) can be used with
electrostatic (i.e. electrophoretic) force. In the case of
manipulating microdevice-binding partner-moiety complexes, the
characteristics of the moiety, or its binding partner or the
microdevices, and the physical force used for manipulation must be
compatible. For example, moiety or its binding partner or the
microdevices with certain dielectric properties to induce
dielectric polarization in the moiety or its binding partner or the
microdevices can be used with dielectrophoresis force.
[0059] As used herein, "the moiety is not directly manipulatable"
by a particular physical force means that no observable movement of
the moiety can be detected when the moiety itself not coupled to a
binding partner or a microdevice is acted upon by the particular
physical force.
[0060] As used herein, "physical force" refers to any force that
moves the moieties or their binding partners or the corresponding
microdevices without chemically or biologically reacting with the
moieties and the microdevice and/or binding partners, or with
minimal chemical or biological reactions with the microdevices,
binding partners and the moieties so that the biological/chemical
functions/properties of the microdevices, binding partners and the
moieties are not substantially altered as a result of such
reactions. Throughout the application, the term "forces" or
"physical forces" always means the "forces" or "physical forces"
exerted on a moiety or moieties, the binding partner(s) and/or the
microdevice(s). The "forces" or "physical forces" are always
generated through "fields" or "physical fields". The forces exerted
on moieties, the binding partner(s) and/or the microdevice(s) by
the fields depend on the properties of the moieties, the binding
partner(s) and/or the microdevice(s). Thus, for a given field or
physical field to exert physical forces on a moiety, it is
necessary for the moiety to have certain properties. While certain
types of fields may be able to exert forces on different types of
moieties having different properties, other types of fields may be
able to exert forces on only limited type of moieties. For example,
magnetic field can exert magnetic forces only on magnetic
particles, e.g., microdevices or moieties having certain magnetic
properties, but not on other microdevices or particles, e.g.,
polystyrene beads. The magnetic microdevices can be made by, e.g.,
incorporating magnetic materials such as ferro- or ferri-magnetic
materials, into the microdevices. On the other hand, a non-uniform
electric field can exert physical forces on many types of moieties
such as polystyrene beads, cells, and also magnetic particles. It
is not necessary for the physical field to be able to exert forces
on different types of moieties or different moieties. But it is
necessary for the physical field to be able to exert force on at
least one type of moiety or at least one moiety, the binding
partner(s) and/or the microdevice(s).
[0061] As used herein, "electric forces (or electrical forces)" are
the forces exerted on moieties, the binding partner(s) and/or the
microdevice(s) by an electric (or electrical) field.
[0062] As used herein, "magnetic forces" are the forces exerted on
moieties, the binding partner(s) and/or the microdevice(s) by a
magnetic field.
[0063] As used herein, "acoustic forces (or acoustic radiation
forces)" are the forces exerted on moieties, the binding partner(s)
and/or the microdevice(s) by an acoustic field.
[0064] As used herein, "optical (or optical radiation) forces" are
the forces exerted on moieties, the binding partner(s) and/or the
microdevice(s) by an optical field.
[0065] As used herein, "mechanical forces" are the forces exerted
on moieties, the binding partner(s) and/or the microdevice(s) by a
velocity field.
[0066] As used herein, "the moiety to be manipulated is
substantially coupled onto the surface of the binding partner"
means that a certain percentage, and preferably a majority, of the
moiety to be manipulated is coupled onto the surface of the binding
partner and can be manipulated by a suitable physical force via
manipulation of the binding partner in the microdevice. Ordinarily,
at least 0.5% of the moiety to be manipulated is coupled onto the
surface of the binding partner. Preferably, at least 1%, 2%, 3%,
4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the moiety
to be manipulated is coupled onto the surface of the binding
partner. The percentage of the coupled moiety includes the
percentage of the moiety coupled onto the surface of a particular
type of binding partner or a plurality of binding partners. When a
plurality of binding partners is used, the moiety can be coupled
onto the surface of the plurality of binding partners
simultaneously or sequentially.
[0067] As used herein, "the moiety to be manipulated is completely
coupled onto the surface of the binding partner" means that at
least 90% of the moiety to be manipulated is coupled onto the
surface of the binding partner in the microdevice. Preferably, at
least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the
moiety to be manipulated is coupled onto the surface of the binding
partner. The percentage of the coupled moiety includes the
percentage of the moiety coupled onto the surface of a particular
type of binding partner or a plurality of binding partners. When a
plurality of binding partners is used, the moiety can be coupled
onto the surface of the plurality of binding partners
simultaneously or sequentially.
[0068] As used herein, "intracellular moiety" refers to any moiety
that resides or is otherwise located within or attached to a cell,
i.e., located in the cytoplasm or matrix of cellular organelles,
attached to any intracellular membrane, resides or is otherwise
located within periplasma, if there is one, or resides in or is
otherwise located on the cell surface, i.e., attached on the outer
surface of the cytoplasm membrane or cell wall, if there is
one.
[0069] As used herein, "said photorecognizable coding pattern
corresponds to an entity to be synthesized on said microdevice"
means that the entity to be synthesized on a particular microdevice
is predetermined according to the photorecognizable coding pattern
on that microdevice. The coding pattern can determine the entity to
be synthesized on a microdevice in different ways. For example, a
coding pattern can have multiple digits and each digit determines a
particular synthesis reaction and the collection of all digits
collectively determines all synthesis reactions, and hence the
identity of the entity to be synthesized. Alternatively, a coding
pattern can be an "intact" pattern, i.e., the entire pattern, not a
portion or a digit of the pattern, determines the entire synthesis
reactions on the microdevice, and hence the identity of the entity
to be synthesized.
[0070] As used herein, "said microdevices are sorted after each
synthesis cycle according to said photorecognizable coding
patterns" means that the synthetic steps or orders for making an
entity on a particular microdevice are predetermined according to
the photorecognizable coding pattern on that microdevice and after
each synthesis cycle, the photorecognizable coding patterns on the
microdevice is assessed for directing the next synthetic step or
order.
[0071] As used herein, "sample" refers to anything which may
contain a moiety to be analyzed, isolated, manipulated, measured,
quantified or detected by the present microdevices and/or methods.
The sample may be a biological sample, such as a biological fluid
or a biological tissue. Examples of biological fluids include
urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral
spinal fluid, tears, mucus, amniotic fluid or the like. Biological
tissues are aggregates of cells, usually of a particular kind
together with their intercellular substance that form one of the
structural materials of a human, animal, plant, bacterial, fungal
or viral structure, including connective, epithelium, muscle and
nerve tissues. Examples of biological tissues also include organs,
tumors, lymph nodes, arteries and individual cell(s). The sample
may also be a mixture of target analyte or enzyme containing
molecules prepared in vitro. The sample may also be an
environmental or agricultural sample derived from air, water such
as river, lake, or ocean, soil, mountains or forests, etc.
[0072] As used herein, a "liquid (fluid) sample" refers to a sample
that naturally exists as a liquid or fluid, e.g., a biological
fluid. A "liquid sample" also refers to a sample that naturally
exists in a non-liquid status, e.g., solid or gas, but is prepared
as a liquid, fluid, solution or suspension containing the solid or
gas sample material. For example, a liquid sample can encompass a
liquid, fluid, solution or suspension containing a biological
tissue, biological cells or other types of biological
molecules.
[0073] As used herein the term "assessing (or assessed)" is
intended to include quantitative and qualitative determination of
the identity of a moiety, e.g., a protein or nucleic acid, present
in the sample, and also of obtaining an index, ratio, percentage,
visual or other value indicative of the identity of a moiety in the
sample. Assessment may be direct or indirect.
B. Microdevices
[0074] In one aspect, the present invention is directed to a
microdevice, which microdevice comprises: a) a substrate; and b) a
photorecognizable coding pattern on said substrate. Preferably, the
microdevice does not comprise an anodized metal surface layer,
e.g., an anodized aluminium surface layer.
[0075] Any suitable substrate can be used in the microdevice. For
example, the substrate can comprise silicon, e.g., silicon dioxide
or silicon nitride, plastic, glass, ceramic, rubber, polymer, in
its internal structure or on its surface, and a combination
thereof. The substrate can comprise multiple layers such as 3, 4 or
more layers. For example, a substrate can have 3 layers. The top
and the bottom layers can be made of same material, e.g., SiO.sub.2
(or glass) and the middle layer can contain magnetic material(s).
Alternatively, the top and the bottom layers can have different
materials.
[0076] The substrate can comprises a surface that is hydrophobic or
hydrophilic. The substrate can be in any suitable shape such as
sphere, square, rectangle, triangle, circular disc, cube-like
shape, cube, rectangular parallelepiped (cuboid), cone, cylinder,
prism, pyramid, right circular cylinder and other regular or
irregular shape. The substrate can be in any suitable dimension(s).
For example, the thickness of the substrate can be from about 0.1
micron to about 500 microns. Preferably, the thickness of the
substrate can be from about 1 micron to about 200 microns. More
preferably, the thickness of the substrate can be from about 1
micron to about 50 microns. In a specific embodiment, the substrate
is a rectangle having a surface area from about 10 squared-microns
to about 1,000,000 squared-microns (e.g., 1000 micron by 1000
micron). In another specific embodiment, the substrate is a
circular disc having a diameter from about 10 microns to about 500
microns. In still another specific embodiment, the substrate is in
a cube-like shape having a side width from about 10 microns to
about 100 microns. In yet another specific embodiment, the
substrate is in an irregular shape having a single-dimension from
about 1 micron to about 500 microns. In a preferred embodiment, the
substrate is a composite comprising silicon, metal film and polymer
film. In another preferred embodiment, the substrate can comprise a
silicon layer and a metal layer, e.g., an aluminum layer. More
preferably, the metal layer can comprise a magnetic material, such
as nickel metal or CoTaZr (Cobalt-Tantalum-Zirconium) alloy.
[0077] The photorecognizable coding pattern can be based on any
suitable photorecognizable (optical) property constructed on the
substrate. For example, the photorecognizable coding pattern can be
a photorecognizable (optical) property constructed on the material
composition of the substrate itself, a hole in the substrate or a
substance located, deposited or immobilized on the substrate, said
substance having an optical refractive property that is different
from the optical refractive property of the substrate. The
substrate can be patterned. In addition, the surface layer of the
substrate or microdevice can be modified. The versatility of the
photorecognizable coding pattern can be caused by the shape,
number, position distribution, optical refractive property,
material composition, or a combination thereof, of the substrate,
the hole(s), or the substance(s) located, deposited or immobilized
on the substrate. In one exemplary microdevice, the substrate can
have 4 layers. The top and the bottom layers can be made of same
material, e.g., SiO.sub.2 (or glass). One of the middle layers can
contain magnetic material(s), e.g., magnetic alloys. The other
middle lay can contain a photorecognizable coding pattern as a
encoding layer. Preferably, the magnetic layer and the encoding
layer does not substantially overlap, or not overlap at all, to
ensure optical detection of the photorecognizable coding pattern in
the encoding layer. Alternatively, the top and the bottom layer can
have different materials. Exemplary patterns include numbers,
letters, structures, 1-D and 2-D barcodes.
[0078] Although the microdevice can comprise a single
photorecognizable coding pattern, it can also comprise a plurality
of photorecognizable coding patterns, e.g., a plurality of the
holes and/or a plurality of the substances.
[0079] To facilitate optical analysis (or photo-analysis) of
encoding patterns, certain microdevices may incorporate
"orientation" marks or alignment markers. For example, for the
microdevices having thin circular disk shapes, the microdevices
lying flat on either of its major surfaces will look identical,
causing difficulties in identification. Therefore, the orientation
markers can be used for indicating which major surface is being
looked at when the microdevices are lying up and for helping decode
the patterns.
[0080] The photorecognizable coding pattern can be constructed on
the substrate according to any methods known in the art. For
example, the photorecognizable coding pattern can be fabricated or
microfabricated on the substrate. Any suitable fabrication or
microfabrication method can be used including lithography such as
photolithography, electron beam lithography and X-ray lithography
(WO 96/39937 and U.S. Pat. Nos. 5,651,900, 5,893,974 and
5,660,680). For example, the fabrication or microfabrication
methods can be used directly on the substrate to produce desirable
patterns such as numbers, letters, structures, 1-D and 2-D
barcodes.
[0081] If a substance having an optical refractive property that is
different from the optical refractive property of the substrate is
used as the photorecognizable coding pattern, the substance can be
deposited or immobilized on the substrate by any suitable methods
known in the art. For example, the substance can be deposited or
immobilized on the substrate by evaporation or sputtering methods.
The substance can be deposited or immobilized on the substrate
directly or via a linker, e.g., a cleavable linker. The fabrication
or microfabrication methods can be used on the substances deposited
on the substrate to produce desirable patterns such as numbers,
letters, structures, 1-D and 2-D barcodes. The substance can be
immobilized deposited or on the substrate via a covalent or a
non-covalent linkage. The substance can be deposited or immobilized
on the substrate via specific or non-specific binding. Preferably,
the linkage between the substance and the substrate can be a
cleavable linkage such as a linkage cleavable by a chemical,
physical or an enzymatic treatment.
[0082] In choosing the type, materials, compositions, structures
and sizes of the microdevices, these properties or parameters of
the microdevices should be compatible with the isolation,
manipulation or detection format in the specific applications. For
example, the microdevices may be used to isolate target
analyte-molecules (e.g. proteins) from a molecule mixture. If the
isolation uses dielectrophoretic forces, then the microdevices
should have the desired dielectric properties. If the
isolation/manipulation utilizes magnetic forces, then the
microdevices should have incorporated magnetic materials such as
ferro- or ferri-magnetic materials.
[0083] The microdevice can also comprise a binding partner that is
capable of binding to a moiety to be isolated, manipulated or
detected. Preferably, the binding partner specifically binds to the
moiety. Throughout this application, whenever the binding partners
are described or used, they are always coupled onto the
microdevices of the present inventions. For example, when the
complexes between the binding partners and the moieties to be
manipulated are discussed, the complexes between the moieties and
the binding partners that are coupled on the microdevices are
referred to.
[0084] Any suitable binding partner including the binding partners
disclosed in the co-pending U.S. patent application Ser. No.
09/636,104, filed Aug. 10, 2000 and Ser. No. 09/679,024, filed Oct.
4, 2000, the disclosures of which are incorporated by reference in
its entirety, can be used. For example, the binding partners can be
cells such as animal, plant, fungus or bacterium cells; cellular
organelles such as nucleus, mitochondria, chloroplasts, ribosomes,
ERs, Golgi apparatuses, lysosomes, proteasomes, secretory vesicles,
vacuoles or microsomes; viruses, microparticles or an aggregate or
complex thereof. Other binding partners may be molecules that have
been immobilized on the microdevices' surfaces. For example,
antibodies can be immobilized or bound on to the microdevices'
surfaces. The antibody-bound microdevices can then be used to
capture and bind to target proteins in a molecule mixture or to
capture and bind to target cells in a cell mixture. Oligo-dT (e.g.
25 mer of T) can be immobilized onto the microdevices' surfaces.
The oligo-dT bound microdevices can then be used to capture mRNA
from a molecule mixture. Other molecules may be used as binding
partners for capturing or binding DNA molecules. Nucleic acid
fragments, e.g., DNA, RNA, PNA segments of specific sequences, may
be used to hybridize to target nucleic acid, DNA, RNA or PNA,
molecule.
[0085] Preferably, the microparticles used in the present
microdevices have a dimension from about 0.01 micron to about
several thousand microns. Non-limiting examples of the
microparticles used in the microdevices include plastic particles,
polystyrene microbeads, glass beads, magnetic beads, hollow glass
spheres, metal particles, particles of complex compositions,
microfabricated free-standing microstructures (e.g., Design of
asynchronous dielectric micrometers by Hagedorn et al., in Journal
of Electrostatics, 1994, Volume: 33, Pages 159-185). Particles of
complex composition refer to particles that comprise or consist of
multiple compositional elements, for example, a metallic sphere
covered with a thin layer of non-conducting polymer film. In anther
example, the particles may comprise a plastic sphere covered with a
conductive polymer layer, which is in turn covered by an insulating
polymer layer.
[0086] In choosing binding partners, the type, material,
composition, structure and size of the binding partners may need to
be compatible with the isolation, manipulation or detection format
in the specific applications. This is especially important when the
properties of the microdevices can not be controlled to fit
specific applications. For example, magnetic beads may be used as
binding partners if the means for manipulating
moiety-binding-partner-microdevices are magnetic field-based. Beads
having appropriate dielectric properties may be used if
dielectrophoretic field is used for manipulating
moiety-binding-partner-microdevices. However, if the microdevices
comprise an element that facilities manipulation by a desirable
force, the binding partner does not need to contain such an
element. For example, if a microdevice contains a magnetic
material, it is not necessary for a binding partner to have any
magnetic materials for manipulation via a magnetic force.
Similarly, if a microdevice contains a conductive material, it is
not necessary for a binding partner to have any conductive
materials for manipulation via a dielectrophoretic force.
[0087] The choice of the beads is further related with specific
isolation, manipulation or detection details. For example, for
separating target moiety from a mixture of molecules and particles
by dielectrophoresis manipulation, binding partner's or
microdevice's dielectric properties should be significantly
different from those of molecules and particles so that when
binding partners are coupled with the target moiety, the
moiety-binding-partner-microdevices complexes may be selectively
manipulated by dielectrophoresis. In an example of separating
target cancer cells from a mixture of normal cells, the cancer
cells may have similar dielectric properties to those of normal
cells and all the cells behave similarly in their dielectrophoretic
responses, e.g., negative dielectrophoresis. In this case, the
binding partners or the microdevice preferably should be more
dielectrically-polarizable than their suspending medium and will
exhibit positive dielectrophoresis. Thus, such microdevices-binding
partners-cancer-cell complexes can be selectively manipulated
through positive dielectrophoresis forces while other cells
experience negative dielectrophoresis forces.
[0088] The microdevice can comprise a single binding partner.
Alternatively, it can be used in a high throughput analysis and can
comprise a plurality of binding partners capable of binding or
specifically binding to different moieties to be isolated,
manipulated or detected.
[0089] The microdevice can further comprise an element that
facilitates and/or enables manipulation of the microdevice and/or a
moiety/microdevice complex. Any suitable element can be used. For
example, the element can be magnetic materials to facilitate and/or
enable manipulation by magnetic force, conductive or insulating
materials to facilitate and/or enable manipulation by
dielectrophoresis force, materials having high or low acoustic
impedance to facilitate and/or enable manipulation by acoustic
force, or charged materials to facilitate and/or enable
manipulation by electrostatic force, etc. The element can be a
cell, a cellular organelle, a virus, a microparticle, an aggregate
or complex of molecules and an aggregate or complex thereof. In
addition, the binding partners disclosed above and disclosed in the
co-pending U.S. patent application Ser. No. 09/636,104, filed Aug.
10, 2000 can also be used as the element(s) that facilitates and/or
enables manipulation of the microdevice and/or a moiety/microdevice
complex.
[0090] The element can facilitate and/or enable manipulation of the
microdevice and/or a moiety/microdevice complex by any suitable
physical force including the physical forces disclosed in the
co-pending U.S. patent application Ser. No. 09/636,104, filed Aug.
10, 2000. For instances, a dielectrophoresis force, a
traveling-wave dielectrophoresis force, a magnetic force such as
one effected via a magnetic field generated by a ferromagnetic
material or one effected via a microelectromagnetic unit, an
acoustic force such as one effected via a standing-wave acoustic
field or a traveling-wave acoustic field, an electrostatic force
such as one effected via a DC electric field, a mechanical force
such as fluidic flow force, or an optical radiation force such as
one effected via an optical intensity field generated by laser
tweezers, can be used.
[0091] Dielectrophoresis refers to the movement of polarized
particles, e.g., microdevices, microdevice-moiety complex, or
microdevice-binding partner-moiety complex, in a non-uniform AC
electrical field. When a particle is placed in an electrical field,
if the dielectric properties of the particle and its surrounding
medium are different, dielectric polarization will occur to the
particle. Thus, the electrical charges are induced at the
particle/medium interface. If the applied field is non-uniform,
then the interaction between the non-uniform field and the induced
polarization charges will produce a net force acting on the
particle to cause particle motion towards the region of strong or
weak field intensity. The net force acting on the particle is
called dielectrophoretic force and the particle motion is
dielectrophoresis. Dielectrophoretic force depends on the
dielectric properties of the particles, particle surrounding
medium, the frequency of the applied electrical field and the field
distribution.
[0092] Traveling-wave dielectrophoresis is similar to
dielectrophoresis in which the traveling-electric field interacts
with the field-induced polarization and generates electrical forces
acting on the particles. Particles, e.g., microdevices,
microdevice-moiety complex, or microdevice-binding partner-moiety
complex, are caused to move either with or against the direction of
the traveling field. Traveling-wave dielectrophoretic forces depend
on the dielectric properties of the particles and their suspending
medium, the frequency and the magnitude of the traveling-field. The
theory for dielectrophoresis and traveling-wave dielectrophoresis
and the use of dielectrophoresis for manipulation and processing of
microparticles may be found in various literatures (e.g.,
"Non-uniform Spatial Distributions of Both the Magnitude and Phase
of AC Electric Fields determine Dielectrophoretic Forces by Wang et
al., in Biochim Biophys Acta Vol. 1243, 1995, pages 185-194",
"Dielectrophoretic Manipulation of Particles by Wang et al, in IEEE
Transaction on Industry Applications, Vol. 33, No. 3, May/June,
1997, pages 660-669", "Electrokinetic behavior of colloidal
particles in traveling electric fields: studies using yeast cells
by Huang et al, in J. Phys. D: Appl. Phys., Vol. 26, pages
1528-1535", "Positioning and manipulation of cells and
microparticles using miniaturized electric field traps and
traveling waves. By Fuhr et al., in Sensors and Materials. Vol. 7:
pages 131-146", "Dielectrophoretic manipulation of cells using
spiral electrodes by Wang, X-B. et al., in Biophys. J. Volume 72,
pages 1887-1899, 1997", "Separation of human breast cancer cells
from blood by differential dielectric affinity by Becker et al, in
Proc. Natl. Acad. Sci., Vol., 92, January 1995, pages 860-864").
The manipulation of microparticles with dielectrophoresis and
traveling wave dielectrophoresis includes
concentration/aggregation, trapping, repulsion, linear or other
directed motion, levitation, and separation of particles. Particles
may be focused, enriched and trapped in specific regions of the
electrode reaction chamber. Particles may be separated into
different subpopulations over a microscopic scale. Particles may be
transported over certain distances. The electrical field
distribution necessary for specific particle manipulation depends
on the dimension and geometry of microelectrode structures and may
be designed using dielectrophoresis theory and electrical field
simulation methods.
[0093] The dielectrophoretic force F.sub.DEPz acting on a particle
of radius r subjected to a non-uniform electrical field may be
given, under dipole approximation, by
F.sub.DEPz=2.pi..di-elect cons..sub.mr.sup.3.chi..sub.DEP
VE.sub.rms.sup.2{right arrow over (a)}.sub.z
[0094] where E.sub.rms is the RMS value of the field strength,
.di-elect cons..sub.m is the dielectric permitivity of the medium.
.chi..sub.DEP is the particle dielectric polarization factor or
dielectrophoresis polarization factor, given, under dipole
approximation, by
.chi. DEP = Re ( p * - m * p * + 2 m * ) , ##EQU00001##
[0095] "Re" refers to the real part of the "complex number". The
symbol
x * = x - j .sigma. x 2 .pi. f ##EQU00002##
is the complex permitivity (of the particle x=p, and the medium
x=m). The parameters .di-elect cons..sub.p and .sigma..sub.p are
the effective permitivity and conductivity of the particle,
respectively. These parameters may be frequency dependent. For
example, a typical biological cell will have frequency dependent,
effective conductivity and permitivity, at least, because of
cytoplasm membrane polarization.
[0096] The above equation for the dielectrophoretic force can also
be written as
F.sub.DEPz=2.pi..di-elect
cons..sub.mr.sup.3.chi..sub.DEPV.sup.2p(z){right arrow over
(a)}.sub.z
[0097] where p(z) is the square-field distribution for a
unit-voltage excitation (V=1 V) on the electrodes, V is the applied
voltage.
[0098] There are generally two types of dielectrophoresis, positive
dielectrophoresis and negative dielectrophoresis. In positive
dielectrophoresis, particles are moved by dielectrophoresis forces
towards the strong field regions. In negative dielectrophoresis,
particles are moved by dielectrophoresis forces towards weak field
regions. Whether particles exhibit positive and negative
dielectrophoresis depends on whether the particles are more or less
polarizable than the surrounding medium.
[0099] Traveling-wave DEP force refers to the force that is
generated on particles or molecules due to a traveling-wave
electric field. A traveling-wave electric field is characterized by
the non-uniform distribution of the phase values of AC electric
field components.
[0100] Here we analyze the traveling-wave DEP force for an ideal
traveling-wave field. The dielectrophoretic force F.sub.DEP acting
on a particle of radius r subjected to a traveling-wave electrical
field E.sub.TWD=E cos(2.pi.(ft-z/.lamda..sub.0){right arrow over
(a)}.sub.x (i.e., a x-direction field is traveling along the
z-direction) is given, under dipole approximation, by
F.sub.TWD=-2.pi..di-elect
cons..sub.mr.sup.3.zeta..sub.TWDE.sup.2{right arrow over
(a)}.sub.z
[0101] where E is the magnitude of the field strength, .di-elect
cons..sub.m is the dielectric permitivity of the medium.
.zeta..sub.TWD is the particle polarization factor, given, under
dipole approximation, by
.zeta. TWD = Im ( p * - m * p * + 2 m * ) , ##EQU00003##
[0102] "Im" refers to the imaginary part of the "complex number".
The symbol
x * = x - j .sigma. x 2 .pi. f ##EQU00004##
[0103] is the complex permitivity (of the particle x=p, and the
medium x=m). The parameters .di-elect cons..sub.p and .sigma..sub.p
are the effective permitivity and conductivity of the particle,
respectively. These parameters may be frequency dependent.
[0104] Particles such as biological cells having different
dielectric property (as defined by permitivity and conductivity)
will experience different dielectrophoretic forces. For
traveling-wave DEP manipulation of particles (including biological
cells), traveling-wave DEP forces acting on a particle of 10 micron
in diameter can vary somewhere between 0.01 and 10,000 pN.
[0105] A traveling wave electric field can be established by
applying appropriate AC signals to the microelectrodes
appropriately arranged on a chip. For generating a
traveling-wave-electric field, it is necessary to apply at least
three types of electrical signals each having a different phase
value. One method to produce a traveling wave electric field is to
use four phase-quardrature signals (0, 90, 180 and 270 degrees) to
energize four linear, parallel electrodes patterned on the chip
surface. This set of four electrodes forms a basic, repeating unit.
Depending on the applications, there may be more than two such
units that are located next to each other. This will produce a
traveling-electric field in the space above or near the electrodes.
As long as electrode elements are arranged following certain
spatially sequential orders, applying phase-sequenced signals will
result in establishment of traveling electrical fields in the
region close to the electrodes.
[0106] Both dielectrophoresis and traveling-wave dielectrophoresis
forces acting on particles, e.g., microdevices, microdevice-moiety
complex, or microdevice-binding partner-moiety complex, depend on
not only the field distributions (e.g., the magnitude, frequency
and phase distribution of electrical field components; the
modulation of the field for magnitude and/or frequency) but also
the dielectric properties of the particles and the medium in which
particles are suspended or placed. For dielectrophoresis, if
particles are more polarizable than the medium (e.g., having larger
conductivities and/or permitivities depending on the applied
frequency), particles will experience positive dielectrophoresis
forces and be directed towards the strong field regions. The
particles that are less polarizable than the surrounding medium
will experience negative dielectrophoresis forces and be directed
towards the weak field regions. For traveling wave
dielectrophoresis, particles may experience dielectrophoresis
forces that drive them in the same direction as the field is
traveling direction or against it, dependent on the polarization
factor .zeta..sub.TWD. The following papers provide basic theories
and practices for dielectrophoresis and
traveling-wave-dielectrophoresis: Huang, et al., J. Phys. D: Appl.
Phys. 26:1528-1535 (1993); Wang, et al., Biochim. Biophys. Acta.
1243:185-194 (1995); Wang, et al., IEEE Trans. Ind. Appl.
33:660-669 (1997).
[0107] Microparticles, e.g., microdevices, microdevice-moiety
complex, or microdevice-binding partner-moiety complex, may be
manipulated with magnetic forces. Magnetic forces refer to the
forces acting on a particle due to the application of a magnetic
field. In general, particles have to be magnetic or paramagnetic
when sufficient magnetic forces are needed to manipulate particles.
We consider a typical magnetic particle made of super-paramagnetic
material. When the particle is subjected to a magnetic field B, a
magnetic dipole .mu. is induced in the particle
.mu. _ = V p ( .chi. p - .chi. m ) B _ .mu. m , = V p ( .chi. p -
.chi. m ) H _ m ##EQU00005##
[0108] where V.sub.p is the particle volume, and .chi..sub.p and
.chi..sub.m are the volume susceptibility of the particle and its
surrounding medium, .mu..sub.m is the magnetic permeability of
medium, H.sub.m is the magnetic field strength. The magnetic force
F.sub.magnetic acting on the particle is determined by the magnetic
dipole moment and the magnetic field gradient:
F.sub.magnetic=-0.5V.sub.p(.chi..sub.p-.chi..sub.m){right arrow
over (H)}.sub.m V{right arrow over (B)}.sub.m),
[0109] where the symbols "" and " V" refer to dot-product and
gradient operations, respectively. Clearly, whether there is
magnetic force acting on a particle depends on the difference in
the volume susceptibility between the particle and its surrounding
medium. Typically, particles are suspended in a liquid,
non-magnetic medium (the volume susceptibility is close to zero)
thus it is necessary to utilize magnetic particles (its volume
susceptibility is much larger than zero). The particle velocity
.nu..sub.particle under the balance between magnetic force and
viscous drag is given by:
v particle = F _ magnetic 6 .pi. r .eta. m ##EQU00006##
[0110] where r is the particle radius and .eta..sub.m is the
viscosity of the surrounding medium. Thus to achieve sufficiently
large magnetic manipulation force, the following factors should be
considered: (1) the volume susceptibility of the magnetic particles
should be maximized; (2) magnetic field strength should be
maximized; and (3) magnetic field strength gradient should be
maximized.
[0111] Paramagnetic particles are preferred whose magnetic dipoles
are induced by externally applied magnetic fields and return to
zero when external field is turned off. For such applications,
commercially available paramagnetic or other magnetic particles may
be used. Many of these particles range from submicron (e.g., 50
nm-0.5 micron) up to tens of microns. They may have different
structures and compositions. One type of magnetic particle has
ferromagnetic materials encapsulated in thin polymer layer, e.g.,
polystyrene. Another type of magnetic particle has ferromagnetic
nanoparticles filled into the poles of porous beads e.g.,
polystyrene beads. The surface of both types of these particles can
be polystyrene in nature and may be modified to link to various
types of molecules. In still another type of magnetic particle,
ferro-magnetic materials can be incorporated uniformly into the
particles during the polymerization process.
[0112] The manipulation of magnetic particles, microdevices,
microdevice-moiety complex, or microdevice-binding partner-moiety
complex, requires the generation of magnetic field distribution
over microscopic scales. One desirable feature of a particle to be
manipulated by magnetic force is that the particle has large
magnetic susceptibility. Another desirable feature is that the
particle has small residue magnetic polarization after the applied
magnetic field/force is turned off. One approach for generating
such magnetic fields is the use of microelectromagnetic units. Such
units can induce or produce magnetic fields when an electrical
current is applied. The on/off status and the magnitude of the
electrical current applied to each unit will determine the magnetic
field distribution. The structure and dimension of the
microelectromagnetic units may be designed according to the
requirement of the magnetic field distribution. Manipulation of
magnetic particles includes the directed movement, focusing and
trapping of magnetic particles. The motion of magnetic particles in
a magnetic field is termed "magnetophoresis". Theories and practice
of magnetophoresis for cell separation and other applications may
be found in various literatures (e.g., Magnetic Microspheres in
Cell Separation, by Kronick, P. L. in Methods of Cell Separation,
Volume 3, edited by N. Catsimpoolas, 1980, pages 115-139; Use of
magnetic techniques for the isolation of cells, by Safarik I. And
Safarikova M., in J. of Chromatography (1999) 722(B):33-53; A fully
integrated micromachined magnetic particle separator, by Ahn, C.
H., et al., in J. of Microelectromechanical systems (1996)
5:151-157).
[0113] Microparticles, e.g., microdevices, microdevice-moiety
complex, or microdevice-binding partner-moiety complex, may be
manipulated using acoustic forces, i.e., using acoustic fields. In
one case, a standing-wave acoustic field is generated by the
superimposition of an acoustic wave generated from an acoustic wave
source and its reflective wave. Particles in standing-wave acoustic
fields experience the so-called acoustic radiation force that
depends on the acoustic impedance of the particles and their
surrounding medium. Acoustic impedance is the product of the
density of the material and the velocity of acoustic-wave in the
material. Particles with higher acoustic impedance than the
surrounding medium are directed towards the pressure nodes of the
standing wave acoustic field. Particles experience different
acoustic forces in different acoustic field distributions.
[0114] One method to generate an acoustic wave source is to use
piezoelectric material. These materials, upon applying electrical
fields at appropriate frequencies, can generate mechanical
vibrations that are transmitted into the medium surrounding the
materials. One type of piezoelectric material is piezoelectric
ceramics. Microelectrodes may be deposited on such ceramics to
activate the piezoelectric ceramic and thus to produce appropriate
acoustic wave fields. Various geometry and dimensions of
microelectrodes may be used according to the requirements of
different applications. Reflective walls are needed to generate a
standing-wave acoustic field. Acoustic wave fields of various
frequencies may be applied, i.e., fields at frequencies between kHz
and hundred megahertz. In another case, one could use a
non-standing wave acoustic field, e.g., a traveling-wave acoustic
field. A traveling-wave acoustic field may exert forces on
particles (see e.g., see, "Acoustic radiation pressure on a
compressible sphere, by K. Yoshioka and Y. Kawashima in Acustica
(1955) 5:167-173"). Particles not only experience forces from
acoustic fields directly but also experience forces due to
surrounding fluid because the fluid may be induced to move by the
traveling-wave acoustic field. Using acoustic fields, particles may
be focused, concentrated, trapped, levitated and transported in a
microfluidic environment. Another mechanism for producing forces on
particles in an acoustic field is through acoustic-induced fluid
convection. An acoustic field produced in a liquid may induce
liquid convection. Such convection is dependent on the acoustic
field distribution, properties of the liquid, and the volume and
structure of the chamber in which the liquid is placed. Such liquid
convection will impose forces on particles placed in the liquid and
those forces may be used for manipulating particles. One example
where such manipulating forces may be exploited is for enhancing
the mixing of liquids or the mixing of particles in a liquid. For
the present invention, such convection may be used to enhance the
mixing of the binding partners coupled onto the microdevices with
moiety in a suspension and to promote the interaction between the
moiety and the binding partners.
[0115] A standing plane wave of ultrasound can be established by
applying AC signals to the piezoelectric transducers. For example,
the standing wave spatially varying along the z axis in a fluid can
be expressed as:
.DELTA.p(z)=p.sub.0 sin(kz)cos(.omega.t)
[0116] where .DELTA.p is acoustic pressure at z, p.sub.0 is the
acoustic pressure amplitude, k is the wave number (2.pi./.lamda.,
where .lamda., is the wavelength), z is the distance from the
pressure node, .omega. is the angular frequency, and t is the time.
According to the theory developed by Yoshioka and Kawashima (see,
"Acoustic radiation pressure on a compressible sphere, by K.
Yoshioka and Y. Kawashima in Acustica (1955) 5:167-173"), the
radiation force F.sub.acoustic acting on a spherical particle in
the stationary standing wave field is given by (see "Studies on
particle separation by acoustic radiation force and electrostatic
force by Yasuda, K., et al., in Jpn. J. Appl. Physics (1996)
35:3295-3299")
F acoustic = - 4 .pi. 3 r 3 kE acoustic A sin ( 2 kz )
##EQU00007##
[0117] where r is the particle radius, E.sub.acoustic is the
average acoustic energy density, A is a constant given by
A = 5 .rho. p - 2 .rho. m 2 .rho. p + .rho. m - .gamma. p .gamma. m
##EQU00008##
[0118] where .rho..sub.m and .rho..sub.p are the density of the
particle and the medium, .gamma..sub.m and .gamma..sub.p are the
compressibility of the particle and medium, respectively. A is
termed herein as the acoustic-polarization-factor.
[0119] When A>0, the particle moves towards the pressure node
(z=0) of the standing wave.
[0120] When A<0, the particle moves away from the pressure
node.
[0121] Clearly, particles of different density and compressibility
will experience different acoustic-radiation-forces when placed
into the same standing acoustic wave field. For example, the
acoustic radiation force acting on a particle of 10 micron diameter
can vary somewhere between 0.01 and 1,000 pN, depending on the
established acoustic energy density distribution.
[0122] Piezoelectric transducers are made from "piezoelectric
materials" that produce an electric field when exposed to a change
in dimension caused by an imposed mechanical force (piezoelectric
or generator effect). Conversely, an applied electric field will
produce a mechanical stress (electrostrictive or motor effect) in
the materials. They transform energy from mechanical to electrical
and vice-versa. The piezoelectric effect was discovered by Pierre
Curie and his brother Jacques in 1880. It is explained by the
displacement of ions, causing the electric polarization of the
materials' structural units. When an electric field is applied, the
ions are displaced by electrostatic forces, resulting in the
mechanical deformation of the whole material.
[0123] Microparticles, e.g., microdevices, microdevice-moiety
complex, or microdevice-binding partner-moiety complex, may be
manipulated using DC electric fields. A DC electric field can exert
an electrostatic force on charged particles. The force depends on
the charge magnitude and polarity of the particles as well as on
the magnitude and direction of the field. The particles with
positive and negative charges may be directed to electrodes with
negative and positive potentials, respectively. By designing a
microelectrode array in a microfluidic device, electric field
distributions may be appropriately structured and realized. With DC
electric fields, microparticles may be concentrated (enriched),
focussed and moved (transported) in a microfluidic device. Proper
dielectric coating may be applied on to DC electrodes to prevent
and reduce undesired surface electrochemistry and to protect
electrode surfaces.
[0124] The electrostatic force F.sub.E on a particle in an applied
electrical field E.sub.z{right arrow over (a)}.sub.z can be given
by
F.sub.E=Q.sub.pE.sub.z{right arrow over (a)}.sub.z
[0125] where Q.sub.p is the effective electric charge on the
particle. The direction of the electrostatic force on a charged
particle depends on the polarity of the particle charge as well as
the direction of the applied field.
[0126] Thermal convection forces refer to the forces acting on
particles, e.g., microdevices, microdevice-moiety complex, or
microdevice-binding partner-moiety complex, due to the
fluid-convection (liquid-convection) that is induced by a thermal
gradient in the fluid. Thermal diffusion in the fluid drives the
fluid towards thermal equilibrium. This causes a fluid convection.
In addition, the density of aqueous solutions tends to decrease
with increasing temperature. Such density differences are also not
stable within a fluid resulting in convection. Thermal convection
may be used to facilitate liquid mixing. Directed thermal
convection may act as an active force.
[0127] Thermal gradient distributions may be established within a
chip-based chamber where heating and/or cooling elements may be
incorporated into the chip structure. A heating element may be a
simple joule-heating resistor coil. Such a coil could be
microfabricated onto the chip. As an example, consider a coil
having a resistance of 10 ohm Applying 0.2 A through the coil would
result in 0.4 W joule heating-power. When the coil is located in an
area <100 micron.sup.2, this is an effective way of heat
generation. Similarly, a cooling element may be a Peltier element
that could draw heat upon applying electric potentials.
[0128] As an exemplary embodiment, the microdevices of the present
invention may be used on a chip which incorporates an array of
individually addressable heating elements. These heating elements
may be positioned or structurally arranged in certain order so that
when each, some or all of the elements are activated, thermal
gradient distributions will be established to produce thermal
convection. For example, if one heating element is activated,
temperature increases in the liquid in the neighborhood of that
element will induce fluid convection. In another exemplary
embodiment, the chip may comprise multiple, interconnected heating
units so that these units can be turned on or off in a synchronized
order. Yet, in another example, the chip may comprise only one
heating element that can be energized to produce heat and induce
thermal convection in the liquid fluid.
[0129] Other physical forces may be applied. For example,
mechanical forces, e.g., fluidic flow forces, may be used to
transport microparticles, e.g., microdevices, microdevice-moiety
complex, or microdevice-binding partner-moiety complex. Optical
radiation forces as exploited in "laser tweezers" may be used to
focus, trap, levitate and manipulate microparticles. The optical
radiation forces are the so-called gradient-forces when a material
(e.g., a microparticle) with a refractive index different from that
of the surrounding medium is placed in a light gradient. As light
passes through a polarizable material, it induces fluctuating
dipoles. These dipoles interact with the electromagnetic field
gradient, resulting in a force directed towards the brighter region
of the light if the material has a refractive index larger than
that of the surrounding medium. Conversely, an object with a
refractive index lower than the surrounding medium experiences a
force drawing it towards the darker region. The theory and practice
of "laser tweezers" for various biological application are
described in various literatures (e.g., "Making light work with
optical tweezers, by Block S. M., in Nature, 1992, Volume 360,
pages 493-496"; "Forces of a single-beam gradient laser trap on a
dielectric sphere in the ray optics regime, by Ashkin, A., in
Biophys. J., 1992, Volume 61, pages 569-582"; "Laser trapping in
cell biology, by Wright et al., in IEEE J. of Quantum Electronics,
1990, Volume 26, pages 2148-2157"; "Laser manipulation of atoms and
particles, by Chu S. in Science, 1991, Volume 253, pages 861-866").
The light field distribution and/or light intensity distribution
may be produced with built-in optical elements and arrays on a chip
and external optical signal sources, or may be produced with
built-in electro-optical elements and arrays on a chip and the
external structures are electrical signal sources. In the former
case, when the light produced by the optical signal sources passes
through the built-in optical elements and arrays, light is
processed by these elements/arrays through, e.g., reflection,
focusing, interference, etc. Optical field distributions are
generated in the regions around the chip. In the latter case, when
the electrical signals from the external electrical signal sources
are applied to the built-in electro-optical elements and arrays,
light is produced from these elements and arrays and optical fields
are generated in the regions around the chip.
[0130] Although the microdevices can comprise a single element,
they may also be used in high throughput analysis and preferably
comprise a plurality of elements, each of the elements facilitates
and/or enables manipulation of the microdevice and/or the
moiety/microdevice complex by a different physical force. For
example, the element can be a magnetic material for manipulation by
a magnetic force, a conductive or insulating material for
manipulation by a dielectrophoresis force, a material having high
or low acoustic impedance for manipulation by acoustic force,
and/or a charged material for manipulation by a electrostatic
force, etc.
[0131] In a preferred embodiment, the microdevice comprises a
binding partner that is capable of binding or specifically binding
to a moiety to be isolated, manipulated or detected and an element
that facilitates and/or enables manipulation of the microdevice
and/or the moiety/microdevice complex. More preferably, the
microdevice(s) comprises a plurality of binding partners, each of
the binding partners is capable of binding or specifically binding
to a different moiety to be isolated, manipulated or detected and a
plurality of the elements, each of the elements facilitates and/or
enables manipulation of the microdevice and/or the
moiety/microdevice complex by a different physical force.
[0132] The microdevice can further comprise a detectable marker or
a molecular tag. Exemplary detectable markers include dyes,
radioactive substances and fluorescent substances. Exemplary
detectable molecular tags include nucleic acid, oligonucleotide,
protein and peptide sequences.
[0133] In a specific embodiment, the present invention is directed
to a microdevice that does not comprise a porous surface. In
another specific embodiment, the present invention is directed to a
microdevice that comprises a metal or metal alloy layer and a
non-metal surface layer. In still another specific embodiment, the
present invention is directed to a microdevice that comprises a
hole as the photorecognizable coding pattern and said hole does not
penetrate through the entire depth of the substrate.
C. Methods, Kits and Arrays for Analyzing, Isolating, Manipulating
and Detecting Moieties
[0134] In one aspect, the present invention is directed to a method
for isolating a moiety, which method comprises: a) providing a
microdevice comprising a substrate, a photorecognizable coding
pattern on said substrate and a binding partner that is capable of
binding, and preferably specifically binding, to a moiety to be
isolated; b) contacting a sample containing or suspected of
containing said moiety with said microdevice provided in step a)
under conditions allowing binding between said moiety and said
binding partner; and c) recovering said microdevice from said
sample, whereby the identity of said isolated moiety is assessed by
photoanalysis of said photorecognizable coding pattern. Preferably,
the microdevice used in the method does not comprise an anodized
metal surface layer, e.g., an anodized aluminium surface layer.
[0135] Any moiety including the moieties disclosed in the above
Section B can be isolated by the present method. For example, the
moiety to be isolated can be a cell, a cellular organelle, a virus,
a molecule and an aggregate or complex thereof.
[0136] Although the present method can be used to isolate a single
moiety, it is preferably to be used in high throughput analysis and
preferably a plurality of moieties are isolated by using a
plurality of microdevices, each of the microdevices contains a
binding partner that is capable of binding to a member of the
plurality of the moieties.
[0137] A moiety in any suitable sample can be isolated. Preferably,
the moiety to be isolated is contained in a fluid sample.
[0138] The isolation can be conducted in any suitable apparatus or
device. For example, the isolation can be conducted in a liquid
container such as a beaker, a flask, a cylinder, a test tube, a
microcentrifuge tube, a centrifugation tube, a culture dish, a
multiwell plate and a filter device. Alternatively, the isolation
can be conducted in a chip format.
[0139] The method can further comprise a step of recovering said
isolated moiety from said microdevice.
[0140] In another aspect, the present invention is directed to a
method for manipulating a moiety, e.g., in a microfluidic
application, which method comprises: a) providing a microdevice
comprising a substrate, a photorecognizable coding pattern on said
substrate and a binding partner that is capable of binding, and
preferably specifically binding, to a moiety to be manipulated; b)
coupling said moiety to said microdevice provided in step a) via
binding between said moiety and said binding partner to form a
moiety-microdevice complex; and c) manipulating said
moiety-microdevice complex with a physical force in a chip format,
thereby said moiety is manipulated. Preferably, the microdevice
used in the method does not comprise an anodized metal surface
layer, e.g., an anodized aluminium surface layer. Alternatively,
the above manipulation method can be conducted in an off-chip
format, e.g., in a liquid container.
[0141] Preferably, the manipulation is effected through a
combination of a structure that is external to the chip and a
structure that is built-in in the chip. For example, chips and
structures internal and external to the chips that are disclosed in
the co-pending U.S. patent application Ser. No. 09/636,104, filed
Aug. 10, 2000 and Ser. No. 09/679,024, filed Oct. 4, 2000, the
disclosures of which are incorporated by reference in its entirety,
can be used in the present method. For example, the methods can be
used on silicon, silicon dioxide, silicon nitride, plastic, glass,
ceramic, photoresist or rubber chips. In addition, the methods can
be used on a chemchip, i.e., on which chemical reactions are
carried out, a biochip, i.e., on which biological reactions are
carried out, or a combination of a biochemchip.
[0142] The physical forces used in the present methods are effected
through a combination of the structure that is external to the chip
and the structure that is built-in in the chip. The external
structures are energy sources that can be connected to the built-in
structures for energizing the built-in structures to generate a
physical force such as dielectrophoresis force, magnetic force,
acoustic force, electrostatic force, mechanical force or optical
radiation force. The built-in structures comprise a single unit or
a plurality of units. Each unit is, when energized and in
combination with the external structure, capable of effecting the
physical force on the binding partner. In the case of a plurality
of units, the built-in structure may further comprise the means for
selectively energizing any one of the plurality of units.
[0143] In one example, when magnetic force is used to manipulate a
complex of a moiety (e.g., DNA molecules) and a microdevice
comprising its binding partner, the electromagnetic chip disclosed
in the co-pending U.S. patent application Ser. No. 09/399,299,
filed Sep. 16, 1999, which is incorporated by reference in its
entirety, can be used in the methods. Typically, such
electromagnetic chips with individually addressable
micro-electromagnetic units comprise: a substrate; a plurality of
micro-electromagnetic units on the substrate, each unit capable of
inducing a magnetic field upon application electric current; a
means for selectively energizing any one of a plurality of units to
induce a magnetic field therein. Preferably, the electromagnetic
chips further comprise a functional layer coated on the surface of
the chips for immobilizing certain types of molecules. In this
example of magnetic manipulation of moiety-binding
partner-microdevice complexes, microelectromagnetic units are the
built-in structures internal to the chip and the electrical current
source that is connected to the microelectromagnetic units is the
structures external to the chip. When the electric current from the
external current source is applied to the microelectromagnetic
units, magnetic fields will be generated in the regions around the
microelectromagnetic units and magnetic forces will be produced on
magnetic particles that are present in the region around the
microelectromagnetic units. Typically, for the case of the
manipulation force being magnetic force, the built-in structures
are electromagnetic units that are incorporated on the chip and the
external structures are the electrical signal sources (e.g.,
current sources). When the appropriately designed and fabricated
electromagnetic units are energized by the electrical signal
sources, magnetic fields are generated in the regions around the
chip. When the microdevice-binding partner-moiety complexes are
subjected to such magnetic fields, magnetic forces are produced on
them, and such forces are dependent on the magnetic field
distribution, the magnetic properties of the microdevices or the
binding partner or microdevice-binding partner-moiety complexes and
the magnetic properties of the medium that surrounds the
microdevices or microdevice-binding partner-moiety complexes.
[0144] In another example, when dielectrophoresis force and
traveling-wave dielectrophoresis force are used to manipulate a
complex of a moiety (e.g., protein molecules) and its binding
partner coupled onto a microdevice (e.g., antibodies can be coupled
onto microdevices' surfaces, allowing for binding of protein
molecules), a spiral electrode array on a glass chip, together with
a phase-quardrature AC electrical signal source, can be used in the
method (see "Dielectrophoretic manipulation of cells using spiral
electrodes by Wang, X-B., et al., in Biophys. J. (1997)
72:1887-1899"). In this example of dielectrophoretic manipulation
of moiety-binding partner-microdevice complexes, a spiral electrode
array is a built-in structure internal to the chip and the AC
electrical signal source that is connected to the spiral electrodes
is the structure external to the chip. When AC electrical signals
of appropriate phases from the external signal source are applied
to the spiral electrode array, electrical fields will be generated
in the regions around the spiral electrode array. Dielectrophoretic
and traveling-wave dielectrophoretic forces will be produced on
moiety-binding partner-microdevice complexes that are present in
the region around the spiral electrode array. Typically, for the
case of the manipulation force being dielectrophoresis and/or
dielectrophoresis force, the built-in structures are the electrode
elements and electrode arrays that are incorporated on a chip and
the external structures are electrical signal sources. When the
appropriately designed electrode elements and electrode arrays are
energized by the electrical signal sources, non-uniform electrical
fields are generated in the regions around the chip. When the
microdevice or microdevice-binding partner-moiety complexes are
subjected to such non-uniform electrical fields, dielectrophoresis
and/or traveling-wave dielectrophoresis forces acting on the
microdevices or microdevice-binding partner-moiety complexes are
produced. Such forces are dependent on the interaction between the
electrical field distributions and field induced dielectric
polarization in microdevices, microdevice-moiety complex, or
microdevice-binding partner-moiety complex, etc.
[0145] In still another example, when acoustic force is used to
manipulate a complex of a moiety (e.g., cells) and its binding
partner coupled onto microdevices (e.g., antibodies immobilized on
microdevices' surfaces, allowing for binding of cells), the phased
array of piezoelectric transducers described in U.S. Pat. No.
6,029,518 by Oeftering, R. can be used in the methods. In this
example of acoustic manipulation of moiety-binding
partner-microdevice complexes, the phased array of piezoelectric
transducers is a built-in structure internal to the chip and the AC
electrical signal source that is connected to the phased array is
the structure external to the chip. When AC electrical signals from
the external signal source are applied to the phased array of
piezoelectric transducers, acoustic wave will be generated from the
piezoelectric transducers and transmitted into the regions around
the piezoelectric transducer. Depending on the chamber structure
comprising such a piezoelectric transducer, when moieties and
moiety-binding partner-microdevice complexes in a liquid suspension
are introduced into the chamber, acoustic radiation forces will be
produced on moieties and moiety-binding partner-microdevice
complexes. Typically, for the case of the manipulation force being
acoustic forces, the built-in structures are the piezoelectric
elements or structures that are incorporated on a chip and the
external structures are electrical signal sources. When the
appropriately designed piezoelectric elements or structures are
energized by the electrical signal sources, acoustic waves are
generated from piezoelectric elements or structures and
acoustic-wave fields are produced in the regions around the chip.
When the microdevices or microdevice-binding partner-moiety
complexes are subjected to such acoustic fields, acoustic forces
are produced on the microdevices or microdevice-binding
partner-moiety complexes and such forces are dependent on
acoustic-wave field distribution, acoustic properties of the
microdevices or microdevice-binding partner-moiety complexes and
acoustic properties of the medium that surrounds the microdevices
or microdevice-binding partner-moiety complexes.
[0146] For the case of the manipulation force being electrostatic
force, the built-in structures are the electrode elements and
electrode arrays that are incorporated on a chip and the external
structures are electrical signal sources (e.g., a DC current
source). When the appropriately designed electrode elements and
electrode arrays are energized by the electrical signal sources,
electrical fields are generated in the regions around the chip.
When the microdevice or microdevice-binding partner-moiety
complexes are subjected to electrical fields, electrostatic forces
acting on the microdevices or microdevice-binding partner-moiety
complexes are produced. Such forces depend on the electrical field
distributions and charge properties of the microdevices or
microdevice-binding partner-moiety complexes.
[0147] For the case of the manipulation force being optical
radiation force, in one example, the built-in structures are the
optical elements and arrays that are incorporated on a chip and the
external structures are optical signal sources (e.g., a laser
source). When the light produced by the optical signal sources
passes through the built-in optical elements and arrays, optical
fields are generated in the regions around the chip and the optical
field distribution is dependent on the geometrical structures,
sizes and compositions of the built-in optical elements and arrays.
When the microdevices or microdevice-binding partner-moiety
complexes are subjected to optical fields, optical radiation forces
acting on the binding partners or binding partner-moiety complexes
are produced. Such forces depend on the optical field distributions
and optical properties of the binding partners or binding
partner-moiety complexes. In other examples, the built-in
structures are the electro-optical elements and arrays that are
incorporated on a chip and the external structures are electrical
signal sources (e.g., a DC current source). When electrical signals
from the external electrical signal sources are applied to the
built-in electro-optical elements and arrays, light is produced
from these elements and arrays and optical fields are generated in
the regions around the chip. When the microdevices or
microdevice-binding partner-moiety complexes are subjected to
optical fields, optical radiation forces acting on the microdevices
or microdevice-binding partner-moiety complexes are produced. Such
forces depend on the optical field distributions and optical
properties of the microdevices or microdevice-binding
partner-moiety complexes.
[0148] For the case of the manipulation force being mechanical
force, the built-in structures may be the electro-mechanical
elements/devices that are incorporated on a chip and the external
structures are electrical signal sources (e.g., a DC current
source). The electromechanical devices may be a microfabricated
pump that can generate pressure to pump fluids. When the
appropriately designed electro-mechanical elements/devices are
energized by the electrical signal sources, mechanical forces are
exerted on the fluid that is introduced to the spaces around the
chip (e.g., on the chip). Thus, the microdevices or
microdevice-binding partner-moiety complexes in the fluid will
experience mechanical forces. The forces on microdevices or
microdevice-binding partner-moiety complexes depend on the
mechanical forces on the fluid and depend on the size, composition
and geometry of the microdevices or microdevice-binding
partner-moiety complexes.
[0149] Any moiety including the moieties disclosed in the above
Section B can be manipulated by the present method. For example,
the moiety to be isolated can be a cell, a cellular organelle, a
virus, a molecule and an aggregate or complex thereof.
[0150] The manipulation can be effected via any suitable physical
force such as a dielectrophoresis, a traveling-wave
dielectrophoresis, a magnetic, an acoustic, an electrostatic, a
mechanical, an optical radiation and/or a thermal convection
force.
[0151] The present method can be used for any type of suitable
manipulation. Exemplary manipulations include transportation,
focusing, enrichment, concentration, aggregation, trapping,
repulsion, levitation, separation, fractionation, isolation and
linear or other directed motion of the moiety.
[0152] In a preferred embodiment, the moiety is not directly
manipulatable by a physical force. In another preferred embodiment,
neither the moiety nor the binding partner is directly
manipulatable by a physical force, and the microdevice contains an
element that makes the microdevice or the moiety-microdevice
complex manipulatable. Any such element including the elements
disclosed in the above Section B can be used in the present
method.
[0153] Although the present method can be used to manipulate a
single moiety, it is preferably to be used in a high throughput
analysis and preferably a plurality of moieties is manipulated.
Preferably, the plurality of moieties is manipulated via a
plurality of corresponding microdevices. The plurality of moieties
can be manipulated sequentially or simultaneously.
[0154] The present method can also comprise a step of recovering
said manipulated moiety from said microdevice and/or said chip. The
present method can further comprise a step of assessing the
identity of the manipulated moiety by photoanalysis of the
photorecognizable coding pattern of the microdevice. The present
method can still further comprise a step of assessing the identity
of the recovered moiety by photoanalysis of the photorecognizable
coding pattern of the microdevice.
[0155] In still another aspect, the present invention is directed
to a kit for manipulating a moiety, e.g., in a microfluidic
application, which kit comprises: a) a microdevice comprising a
substrate, a photorecognizable coding pattern on said substrate and
a binding partner that is capable of binding, and preferably
specifically binding, to a moiety to be manipulated; and b) a chip
on which a moiety-microdevice complex can be manipulated.
Preferably, the kit can further comprise instruction(s) for
coupling the moiety to the microdevice and/or for manipulating the
moiety-microdevice complex on the chip. Also preferably, the
microdevice used in the kit does not comprise an anodized metal
surface layer, e.g., an anodized aluminium surface layer.
[0156] In yet another aspect, the present invention is directed to
a method for detecting a moiety, which method comprises: a)
providing a microdevice comprising a substrate, a photorecognizable
coding pattern on said substrate and a binding partner that is
capable of binding, and preferably specifically binding, to a
moiety to be detected; b) contacting a sample containing or
suspected of containing said moiety with said microdevice provided
in step a) under conditions allowing binding between said moiety
and said binding partner; and c) detecting binding between said
moiety and said binding partner, whereby the presence or amount of
said moiety is assessed by analysis of binding between said moiety
and said binding partner and the identity of said moiety is
assessed by photoanalysis of said photorecognizable coding pattern.
Preferably, the microdevice used in the method does not comprise an
anodized metal surface layer, e.g., an anodized aluminium surface
layer.
[0157] The binding between the moiety and the binding partner can
be detected by any suitable methods, devices or instruments. For
example, the moiety can be labeled, e.g., with fluorescent,
radioactive, enzymatic or other chemical labels. The moiety can be
labeled before its binding with the binding partner or after its
binding with the binding partner. In another example, the
absorbance or other optical properties of the moiety can be used in
detecting its binding with the binding partner. In still another
example, the molecular weight of the moiety can be used in
detecting its binding with the binding partner, e.g., by mass
spectrometry such as MALDI-TOF. The detecting methods based on the
labeling of the moiety can be conducted in a direct labeling
method, i.e., the moiety to be detected is labeled, or in a
competitive assay format, i.e., a labeled moiety or moiety analog
is added to the sample containing a moiety to be detected. In yet
another example, the moiety is cleaved off or recovered from, or
isolated or purified from the moiety-binding partner complex before
the detection. Any suitable methods, e.g., HPLC, can be used to
isolate or purify the moiety.
[0158] Any moiety including the moieties disclosed in the above
Section B can be detected by the present method. For example, the
moiety to be detected can be a cell, a cellular organelle, a virus,
a molecule and/or an aggregate or complex thereof.
[0159] Although the present method can be used to detect a single
moiety, it is preferably to be used in a high throughput analysis
and preferably a plurality of moieties is detected by using a
plurality of microdevices, each of the microdevices contains a
binding partner that is capable of binding to a member of the
plurality of the moieties. The plurality of moieties can be
detected sequentially or simultaneously.
[0160] The detection can be conducted in any suitable apparatus or
device. For example, the detection can be conducted in a liquid
container such as a beaker, a flask, a cylinder, a test tube, a
microcentrifuge tube, a centrifugation tube, a culture dish, a
multiwell plate and/or a filter device. Alternatively, the
microdevice is placed or immobilized on a surface and the detection
can be conducted in a chip format. Preferably, a plurality of
microdevice is placed or immobilized on a surface and the detection
can be conducted in a chip format.
[0161] A moiety in any suitable sample can be detected. Preferably,
the moiety to be detected is contained in a fluid sample.
[0162] In yet another aspect, the present invention is directed to
an array for detecting moieties, which array comprises a plurality
of microdevices positioned, deposited or immobilized on a surface,
e.g., a chip, each of said microdevices comprises a
photorecognizable coding pattern on a substrate and a binding
partner that is capable of binding, and preferably specifically
binding, to a moiety to be detected. Preferably, at least one of
the microdevices used in the array does not comprise an anodized
metal surface layer, e.g., an anodized aluminium surface layer.
More preferably, at least 50% or all of the microdevices used in
the array do not comprise an anodized metal surface layer, e.g., an
anodized aluminium surface layer. The microdevices can be
positioned, deposited or immobilized on the surface or chip using
any suitable methods such as being positioned on a surface by a
magnetic force.
[0163] The present methods can be used for analyzing, isolating,
manipulating or detecting any types of moieties when the moieties
are involved in certain processes, such as physical, chemical,
biological, biophysical or biochemical processes, etc., in a chip
format or non-chip format. Moieties can be cells, cellular
organelles, viruses, molecules or an aggregate or complex thereof.
Moieties can be pure substances or can exist in a mixture of
substances wherein the target moiety is only one of the substances
in the mixture. For example, cancer cells in the blood from
leukemia patients, cancer cells in the solid tissues from patients
with solid tumors and fetal cells in maternal blood from pregnant
women can be the moieties to be isolated, manipulated or detected.
Similarly, various blood cells such as red and white blood cells in
the blood can be the moieties to be isolated, manipulated or
detected. DNA molecules, mRNA molecules, certain types of protein
molecules, or all protein molecules from a cell lysate can be
moieties to be isolated, manipulated or detected.
[0164] Non-limiting examples of cells include animal cells, plant
cells, fungi, bacteria, recombinant cells or cultured cells.
Animal, plant cells, fungus, bacterium cells to be isolated,
manipulated or detected can be derived from any genus or subgenus
of the Animalia, Plantae, fungus or bacterium kingdom. Cells
derived from any genus or subgenus of ciliates, cellular slime
molds, flagellates and microsporidia can also be isolated,
manipulated or detected. Cells derived from birds such as chickens,
vertebrates such fish and mammals such as mice, rats, rabbits,
cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeys and other
non-human primates, and humans can be isolated, manipulated or
detected by the present methods.
[0165] For animal cells, cells derived from a particular tissue or
organ can be isolated, manipulated or detected. For example,
connective, epithelium, muscle or nerve tissue cells can be
isolated, manipulated or detected. Similarly, cells derived from an
accessory organ of the eye, annulospiral organ, auditory organ,
Chievitz organ, circumventricular organ, Corti organ, critical
organ, enamel organ, end organ, external female genital organ,
external male genital organ, floating organ, flower-spray organ of
Ruffini, genital organ, Golgi tendon organ, gustatory organ, organ
of hearing, internal female genital organ, internal male genital
organ, intromittent organ, Jacobson organ, neurohemal organ,
neurotendinous organ, olfactory organ, otolithic organ, ptotic
organ, organ of Rosenmuller, sense organ, organ of smell, spiral
organ, subcommissural organ, subformical organ, supernumerary
organ, tactile organ, target organ, organ of taste, organ of touch,
urinary organ, vascular organ of lamina terminalis, vestibular
organ, vestibulocochlear organ, vestigial organ, organ of vision,
visual organ, vomeronasal organ, wandering organ, Weber organ and
organ of Zuckerkandl can be isolated, manipulated or detected.
Preferably, cells derived from an internal animal organ such as
brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,
blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,
intestine, testis, ovary, uterus, rectum, nervous system, gland,
internal blood vessels, etc can be isolated, manipulated or
detected. Further, cells derived from any plants, fungi such as
yeasts, bacteria such as eubacteria or archaebacteria can be
isolated, manipulated or detected. Recombinant cells derived from
any eucaryotic or prokaryotic sources such as animal, plant, fungus
or bacterium cells can also be isolated, manipulated or detected.
Cells from various types of body fluid such as blood, urine,
saliva, bone marrow, sperm or other ascitic fluids, and
subfractions thereof, e.g., serum or plasma, can also be isolated,
manipulated or detected.
[0166] Isolatable, manipulatable or detectable cellular organelles
include nucleus, mitochondria, chloroplasts, ribosomes, ERs, Golgi
apparatuses, lysosomes, proteasomes, secretory vesicles, vacuoles
or microsomes. Isolatable, manipulatable or detectable viruses
include intact viruses or any viral structures, e.g., viral
particles, in the virus life cycle that can be derived from viruses
such as Class I viruses, Class II viruses, Class III viruses, Class
IV viruses, Class V viruses or Class VI viruses.
[0167] Isolatable, manipulatable or detectable molecules can be
inorganic molecules such as ions, organic molecules or a complex
thereof. Non-limiting examples of ions include sodium, potassium,
magnesium, calcium, chlorine, iron, copper, zinc, manganese,
cobalt, iodine, molybdenum, vanadium, nickel, chromium, fluorine,
silicon, tin, boron or arsenic ions. Non-limiting examples of
organic molecules include amino acids, peptides, proteins,
nucleosides, nucleotides, oligonucleotides, nucleic acids,
vitamins, monosaccharides, oligosaccharides, carbohydrates, lipids
or a complex thereof.
[0168] Any amino acids can be isolated, manipulated or detected by
the present methods. For example, a D- and a L-amino-acid can be
isolated, manipulated or detected. In addition, any building blocks
of naturally occurring peptides and proteins including Ala (A), Arg
(R), Asn (N), Asp (D), Cys (C), Gln (O), Glu (E), Gly (G), H is
(H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P) Ser (S),
Thr (T), Trp (W), Tyr (Y) and Val (V) can be isolated, manipulated
or detected.
[0169] Any proteins or peptides can be isolated, manipulated or
detected by the present methods. For example, membrane proteins
such as receptor proteins on cell membranes, enzymes, transport
proteins such as ion channels and pumps, nutrient or storage
proteins, contractile or motile proteins such as actins and
myosins, structural proteins, defense protein or regulatory
proteins such as antibodies, hormones and growth factors can be
isolated, manipulated or detected. Proteineous or peptidic antigens
can also be isolated, manipulated or detected.
[0170] Any nucleic acids, including single-, double and
triple-stranded nucleic acids, can be isolated, manipulated or
detected by the present methods. Examples of such nucleic acids
include DNA, such as A-, B- or Z-form DNA, and RNA such as mRNA,
tRNA and rRNA.
[0171] Any nucleosides can be isolated, manipulated or detected by
the present methods. Examples of such nucleosides include
adenosine, guanosine, cytidine, thymidine and uridine. Any
nucleotides can be isolated, manipulated or detected by the present
methods. Examples of such nucleotides include AMP, GMP, CMP, UMP,
ADP, GDP, CDP, UDP, ATP, GTP, CTP, UTP, dAMP, dGMP, dCMP, dTMP,
dADP, dGDP, dCDP, dTDP, dATP, dGTP, dCTP and dTTP.
[0172] Any vitamins can be isolated, manipulated or detected by the
present methods. For example, water-soluble vitamins such as
thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxine,
biotin, folate, vitamin B.sub.12 and ascorbic acid can be isolated,
manipulated or detected. Similarly, fat-soluble vitamins such as
vitamin A, vitamin D, vitamin E, and vitamin K can be isolated,
manipulated or detected.
[0173] Any monosaccharides, whether D- or L-monosaccharides and
whether aldoses or ketoses, can be isolated, manipulated or
detected by the present methods. Examples of monosaccharides
include triose such as glyceraldehyde, tetroses such as erythrose
and threose, pentoses such as ribose, arabinose, xylose, lyxose and
ribulose, hexoses such as allose, altrose, glucose, mannose,
gulose, idose, galactose, talose and fructose and heptose such as
sedoheptulose.
[0174] Any lipids can be isolated, manipulated or detected by the
present methods. Examples of lipids include triacylglycerols such
as tristearin, tripalmitin and triolein, waxes, phosphoglycerides
such as phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, phosphatidylinositol and cardiolipin,
sphingolipids such as sphingomyelin, cerebrosides and gangliosides,
sterols such as cholesterol and stigmasterol and sterol fatty acid
esters. The fatty acids can be saturated fatty acids such as lauric
acid, myristic acid, palmitic acid, stearic acid, arachidic acid
and lignoceric acid, or can be unsaturated fatty acids such as
palmitoleic acid, oleic acid, linoleic acid, linolenic acid and
arachidonic acid.
D. Methods for Synthesizing a Library and Uses Thereof
[0175] In yet another aspect, the present invention is directed to
a method for synthesizing a library, which method comprises: a)
providing a plurality of microdevices, each of said microdevices
comprises a substrate and a photorecognizable coding pattern on
said substrate, wherein said photorecognizable coding pattern
corresponds to an entity to be synthesized on said microdevice; and
b) synthesizing said entities on said microdevices, wherein said
microdevices are sorted after each synthesis cycle according to
said photorecognizable coding patterns, whereby a library is
synthesized, wherein each of said microdevices contains an entity
that corresponds to a photorecognizable coding pattern on said
microdevice and the sum of said microdevices collectively contains
a plurality of entities that is predetermined before the library
synthesis. Preferably, at least one of the microdevices used in the
method does not comprise an anodized metal surface layer, e.g., an
anodized aluminium surface layer. More preferably, at least 50% or
all of the microdevices used in the method do not comprise an
anodized metal surface layer, e.g., an anodized aluminium surface
layer.
[0176] The microdevice used in the present method can comprise any
suitable substrate. For example, the substrate can comprise
silicon, e.g., silicon dioxide or silicon nitride, plastic, glass,
ceramic, rubber, polymer and a combination thereof. The substrate
can comprises a surface that is hydrophobic or hydrophilic. The
substrate can be in any suitable shape such as sphere, square,
rectangle, triangle, circular disc, cube-like shape, cube,
rectangular parallelepiped (cuboid), cone, cylinder, prism,
pyramid, right circular cylinder and other regular or irregular
shape. The substrate can be in any suitable dimension(s). For
example, the thickness of the substrate can be from about 0.1
micron to about 500 microns. Preferably, the thickness of the
substrate can be from about 1 micron to about 200 microns. More
preferably, the thickness of the substrate can be from about 1
micron to about 50 microns. In a specific embodiment, the substrate
is a rectangle having a surface area from about 10 squared-microns
to about 1,000,000 squared-microns (e.g., 1,000 micron by 1,000
micron). In another specific embodiment, the substrate is a
circular disc having a diameter from about 10 microns to about 500
microns. In still another specific embodiment, the substrate is in
a cube-like shape having a side width from about 10 microns to
about 100 microns. In yet another specific embodiment, the
substrate is in an irregular shape having a single-dimension from
about 1 micron to about 500 microns. In a preferred embodiment, the
substrate is a composite comprising silicon, metal film and polymer
film.
[0177] The microdevice used in the present method can comprise a
photorecognizable coding pattern based on any suitable
photorecognizable (optical) property constructed on the substrate.
For example, the photorecognizable coding pattern can be
photorecognizable (optical) property constructed on the material
composition of the substrate itself, a hole in the substrate or a
substance immobilized on the substrate, said substance having an
optical refractive property that is different from the optical
refractive property of the substrate. The versatility of the
photorecognizable coding pattern can be caused by the shape,
number, position distribution, optical refractive property,
material composition, or a combination thereof, of the substrate,
the hole(s), or the substance(s) immobilized on the substrate.
[0178] Although the microdevice used in the present method can
comprise a single photorecognizable coding pattern, it can also be
used in a high throughput synthesis and can comprise a plurality of
photorecognizable coding pattern, e.g., a plurality of the holes
and/or a plurality of the substances.
[0179] The photorecognizable coding pattern can be constructed on
the substrate according to any methods known in the art. For
example, the photorecognizable coding pattern can be fabricated or
microfabricated on the substrate. Any suitable fabrication or
microfabrication methods can be used including lithography such as
photolithography, electron beam lithography and X-ray lithography
(WO 96/39937 and U.S. Pat. Nos. 5,651,900, 5,893,974 and
5,660,680). If a substance having an optical refractive property
that is different from the optical refractive property of the
substrate is used as the photorecognizable coding pattern, the
substance can be positioned, deposited or immobilized on the
substrate by any suitable methods known in the art. For example,
the substance can be positioned, deposited or immobilized on the
substrate by any suitable methods such as evaporation or
sputtering. The substance can be positioned, deposited or
immobilized on the substrate directly or via a linker, e.g., a
cleavable linker. The substance can be positioned, deposited or
immobilized on the substrate via a covalent or a non-covalent
linkage. The substance can be positioned, deposited or immobilized
on the substrate via a specific or a non-specific binding.
Preferably, the linkage between the substance and the substrate can
be a cleavable linkage such as a linkage cleavable by a chemical,
physical or an enzymatic treatment.
[0180] The microdevice used in the present method can further
comprise an element that facilitates and/or enables manipulation of
the microdevice and/or a moiety/microdevice complex. Any suitable
element can be used. For example, the element can be a cell, a
cellular organelle, a virus, a microparticle, an aggregate or
complex of molecules and an aggregate or complex thereof. The
element can facilitate and/or enable manipulation of the
microdevice and/or a moiety/microdevice complex by any suitable
physical force such as a dielectrophoresis, a traveling-wave
dielectrophoresis, a magnetic, an acoustic, an electrostatic, a
mechanical, an optical radiation and a thermal convection force.
For example, the element can be a magnetic material for
manipulation by a magnetic force, a conductive or insulating
material for manipulation by a dielectrophoresis force, a material
with high or low acoustic impedance for manipulation by a acoustic
force or a charged material for manipulation by an electrostatic
force.
[0181] Although the microdevice used in the present method can
comprise a single element, it can also be used in a high throughput
analysis and can comprise a plurality of the elements, each of the
elements facilitates and/or enables manipulation of the microdevice
and/or the moiety/microdevice complex by a different physical
force.
[0182] The microdevice used in the present method can further
comprise a detectable marker or a molecular tag. Exemplary
detectable markers include dye, radioactive substance and
fluorescent substance. Exemplary detectable molecular tags include
nucleic acid, oligonucleotide, protein and peptide sequences.
[0183] Any number of suitable entity(ies) can be synthesized on a
single microdevice. For example, a single entity or a plurality of
entities can be synthesized on a single microdevice. Preferably, a
single entity is synthesized on a single microdevice.
[0184] The present method can be used to synthesize any kind of
library. For example, the synthesized entities can be peptides,
proteins, oligonucleotides, nucleic acids, vitamins,
oligosaccharides, carbohydrates, lipids, small molecules, or a
complex or combination thereof. Preferably, the synthesized library
comprises a defined set of entities that are involved in a
biological pathway, belongs to a group of entities with identical
or similar biological function, expressed in a stage of cell cycle,
expressed in a cell type, expressed in a tissue type, expressed in
an organ type, expressed in a developmental stage, entities whose
expression and/or activity are altered in a disease or disorder
type or stage, or entities whose expression and/or activity are
altered by drug or other treatments.
[0185] In a specific embodiment, the synthesized library comprises
a defined set of nucleic acid, e.g., DNA or RNA, fragments such as
a defined set of nucleic acid fragments that cover an entire
genome, e.g., the entire human genome sequence. Preferably, each of
the nucleic acid fragments in the synthesized library comprises at
least 10, 15, 20, 25, 50, 75, 100, 200, or 500 nucleotides.
[0186] In another specific embodiment, the synthesized library
comprises a defined set of protein or peptide fragments such as a
defined set of protein or peptide fragments that cover protein or
peptide sequences encoded by an entire genome, e.g., the entire
human genome sequence. Preferably, each of the protein or peptide
fragments in the synthesized library comprises at least 5, 10, 15,
20, 25, 50, 75, 100, 150, 200, 300, 400 or 500 amino acid
residues.
[0187] In still another specific embodiment, a library that is
synthesized according to the above-described method is
provided.
[0188] In yet another specific embodiment, a method for generating
an antibody library is provided, which method comprises: a)
contacting a library synthesized by the above-described method with
a plurality of antibodies; and b) selecting and/or recovering the
antibodies that specifically bind to the entities of the library
synthesized according to the above-described method. Any suitable
antibodies can be used in the present method. For example,
plurality of antibodies used in the present method is a phage
display library (See U.S. Pat. Nos. 6,127,132 and 6,174,708).
E. The Microfabricated Two-Dimensional Optical Encoders and their
Uses
[0189] In yet another aspect, the present invention is directed to
an example of a microdevice of the present invention, a
two-dimensional optical encoder and uses thereof.
[0190] In a specific embodiment, the present invention is directed
to a two-dimensional optical encoder, which encoder comprises: a) a
substrate; and b) a microfabricated or micromachined
two-dimensional optical code on said substrate. Preferably, the
two-dimensional optical encoder does not comprise an anodized metal
surface layer, e.g., an anodized aluminium surface layer.
[0191] Any suitable material can be used in the substrate.
Preferably, the substrate comprises silicon, silicon dioxide,
glass, plastic, polymer, magnetic material, carbon, metal, oxidized
metal or a composite thereof.
[0192] Any suitable pattern or substance or composites can be used
as the two-dimensional code. Preferably, the two-dimensional code
is a grating, an aperture-based code or a black-white line-segment
code.
[0193] In another specific embodiment, the present invention is
directed to a carrier for chemical synthesis, which carrier
comprises a surface suitable for chemical synthesis, said surface
comprises a microfabricated or micromachined two-dimensional
optical code, and said optical code identifies a chemical reaction
to be conducted on said surface and/or product of said chemical
reaction. Preferably, the carrier does not comprise an anodized
metal surface layer, e.g., an anodized aluminium surface layer.
[0194] The carrier can have any suitable shape. For example, the
carrier can be a cube, a rectangular parallelepiped (cuboid), a
cone, a cylinder, a prism, a pyramid and a right-angled circular
cylinder. Preferably, the carrier does not comprise an anodized
metal surface layer, e.g., an anodized aluminium surface layer. The
carrier can comprise a spherical portion and a flat portion,
wherein said flat portion comprises a microfabricated or
micromachined two-dimensional optical code and said spherical
portion is used for chemical synthesis. Also preferably, the
non-coding region of the carrier can further comprises a chemical
layer linked to the carrier surface via a cleavable linker, e.g.,
an optically cleavable, an enzymatically cleavable and/or a
thermally cleavable linker, and said cleavable linker allows for
subsequent chemical synthesis reactions.
[0195] In still another specific embodiment, the present invention
is directed to a carrier for labeling a substance, which carrier
comprises a surface for binding or linking a substance, and a
microfabricated or micromachined two-dimensional optical code on
said surface, said optical code is used for identifying said
substance linked or coupled to said carrier. The carrier can have
any suitable shape. For example, the carrier can be a cube, a
rectangular parallelepiped (cuboid), a cone, a cylinder, a prism, a
pyramid and a right-angled circular cylinder. Preferably, the
carrier comprises a spherical portion and a flat portion, wherein
said flat portion comprises a microfabricated or micromachined
two-dimensional optical code and said spherical portion is used for
linking or coupling the substance.
[0196] In still another specific embodiment, the present invention
is directed to a method for conducting chemical synthesis on the
above-described two-dimensional optical encoder, which method
comprises, based on optical code on said encoder, introducing said
encoder into a corresponding reaction chamber and allowing a
predetermined chemical synthesis reaction to be conducted on said
encoder. Preferably, the method further comprises the following
steps: a) mixing a plurality of the two-dimensional optical
encoders, each encoder having a unique optical code representing
the corresponding synthesis reaction(s) to be conducted and/or
product(s) to be synthesized on said encoder; b) chemically
modifying the non-encoding regions of the surface of the encoders;
c) continuously passing the optical encoders through a sorting
device capable of identifying the optical code on said optical
encoders, and transporting or sorting the optical encoders into
corresponding reaction chambers based on their optical codes; d)
performing the chemical synthesis procedures on said optical
encoders in their corresponding reaction chambers; and e) after
each step of the chemical synthesis, mixing the optical encoders
and sorting the encoders in a sorting device into new,
corresponding reaction chambers again based on the optical codes on
said encoders and the subsequent requisite synthesis steps for said
encoders, performing a new step of the chemical synthesis until all
requisite synthesis steps are performed.
[0197] The sorting device used in the method can comprise a
microchannel that allows the passage of one and only one optical
encoder at a time. The encoder suspended in a liquid solution is
manipulated or controlled to pass through the microchannel via an
applied force, and the encoder is monitored or detected by a
code-reader that is located in the vicinity of the
microchannel.
[0198] Any suitable physical force can be used in the present
method. For example, the applied force on the optical encoder, or
substances linked thereto, can be a traveling-wave
dielectrophoresis force, a traveling-wave magnetic field-force or a
traveling-wave acoustic wave-induced force, whereby said applied
force causes the encoders to pass through the microchannel and be
sorted. In another example, the applied force on the optical
encoder, or substances linked thereto, can be an electroosmotic
pumping force, a mechanical pumping force and/or an
electrohydrodynamic pumping force, said applied forces are applied
to the solution liquid of the reaction system, and said solution
liquid carries the optical encoder and the linked substances
through the microchannel.
[0199] After the identification of the optical codes on the optical
encoders via the sorting device, the encoders can be transported,
based on the optical code signals that are read-out from the
encoder, to different reaction chambers that are linked to the
microchannels.
[0200] In yet another specific embodiment, the present invention is
directed to a chip, which chip comprises a plurality of the above
microfabricated two-dimensional optical encoders, each encoder
having biological and chemical substance(s) linked thereto, and
said biological and chemical substance(s) are capable of being
identified by the optical code on each optical encoder. Preferably,
at least one of the optical encoders used in the chip does not
comprise an anodized metal surface layer, e.g., an anodized
aluminium surface layer. More preferably, at least 50% or all of
the optical encoders used in the chip do not comprise an anodized
metal surface layer, e.g., an anodized aluminium surface layer.
[0201] Any biological and chemical substance(s) can be linked to
the present chips. For example, DNA, RNA, peptide, protein,
antibody, antigen, sugar, lipid, cytokine, hormone, cell, bacteria,
virus and a composite thereof can be linked to the present
chips.
[0202] In yet another specific embodiment, the present invention is
directed to a method for measuring and/or detecting a substance,
which method comprises: a) labeling a substance to be measured
and/or detected; b) providing a plurality of the above chips, each
of said chips having immobilized thereto a different biological or
chemical entity and the identity of said entity corresponding to
the optical code of said chip; c) binding and/or reacting the
labeled substance with said plurality of chips provided in step b);
d) conducting a wash to remove substances that do not bind and/or
react with said entities on said chips; e) passing said washed
chips sequentially through a device to detect and measuring labels
of said substances attached to said chips and to decode the code on
the chip, thereby measuring and/or detecting the type or quantities
of said substances.
[0203] In exemplary embodiments, the present invention discloses a
large-scale chemical synthesis control method. In this method, the
2-D optical encoders serve as carriers. Based on the code on the
2-D optical encoders, these encoders are manipulated and
transported to different reaction chambers for different synthesis
reactions.
[0204] Preferably, the large-scale chemical synthesis control
method includes:
[0205] 1) using different optical codes to denote the different
synthesis reactions and related production. Mixing the different
2-D optical encoders and modifying the surface;
[0206] 2) using a sorting device to readout the codes on the 2-D
optical encoders, then based on the first code on the encoders,
transporting these encoders to related reaction chambers;
[0207] 3) after the reaction cycle is complete, mixing all 2-D
optical encoders and then using sorting device to sort again. Based
on the second code on the encoders, transporting these encoders to
related reaction chambers; and
[0208] 4) repeating step 3, each time reading the next code on the
encoders, until finishing all the synthesis reaction.
[0209] Alternatively, each optical encoder can contain an intact
code which identifies the entire synthesis steps/procedures and the
product to be synthesized on that particular optical encoder. This
way, it is not necessary to decode each digit of a code after each
synthesis cycle.
[0210] Here, the sorting device has a small channel. Each time only
one encoder in the solution can be manipulated to go through the
channel. At the same time, the readout system will read the code on
the 2-D optical encoder and decode it.
[0211] The applied forces for manipulating the 2-D optical encoders
or the linked substances include, but are not limit to, electric
force, magnetic force, acoustic force and mechanical force. These
applied forces can control and manipulate the 2-D optical encoders
and the linked substances in the solution and make sure each time
only one encoder in the solution can go through the channel.
[0212] After the readout system in the sorting device decodes the
code on the 2-D optical encoder, the 2-D optical encoders are
transported to different reaction chambers. Each time based on the
different decoding signal, the sorting device can connect the
related reaction chamber with the microchannel and let the 2-D
optical encoder go into the chamber for next synthesis
reaction.
[0213] The present invention also discloses a chip. This chip
includes many microfabricated 2-D optical encoders. Each 2-D
optical encoder can bind with one kind of biological substance or
chemical substance, also the code on each 2-D optical encoder can
specifically denote the biological material or chemical material
which binds to this 2-D optical encoder. The non-coded surface
region of the 2-D optical encoder can be modified with functional
layer for biological material or chemical material binding.
[0214] In this chip application, the biological substances include,
but are not limited to, DNA, RNA, peptide, protein, antigen,
antibody, monosaccharide, oligosaccharide, carbohydrate, lipid,
hormone and the complex thereof, cell, virus and so on. A common
format for the biological material or chemical material is a probe
used in the chip.
[0215] The present invention also discloses a method using the
present chip to detect different substances in a sample. This
method includes:
[0216] 1) labeling the "unknown" substances in a sample;
[0217] 2) providing 2-D optical encoders, wherein each 2-D optical
encoder binds with one kind of biological substance or chemical
substance, and the code on each 2-D optical encoder can
specifically denote the bound biological material or chemical
material;
[0218] 3) mixing the 2-D optical encoders and reacting them with
"unknown" substances in the sample; and
[0219] 4) after the reaction, manipulating and transporting these
2-D optical encoders to a readout system one by one. When a 2-D
optical encoder with labeled substance goes through the readout
system, the labeled substance will trigger the readout system and
the readout system will read and decode the code on this 2-D
optical encoder. Then the class and quantity of the "unknown"
material binding on the 2-D optical encoder surface will be
ascertained.
[0220] The label for "unknown" substances includes, but are not
limited to, fluorescence label, isotope label, etc.
[0221] In this present detection method, since 2-D optical encoders
serve as the carriers for biological substances or chemical
substances, it is easy to determine the identity and quantity of
unknown substances and also it is easy to conduct the high
throughput screening for reaction products.
[0222] The present 2-D optical encoders can be used in a wide
variety of fields such as chemistry, pharmaceutical industry and
biotechnology.
[0223] Since each kind of 2-D optical encoder has a specific code
that can distinguish it from other 2-D optical encoders, 2-D
optical encoders can be used to label and control the compound
synthesis process. FIG. 14 is the schematic diagram showing the
synthesis process for different compounds using 2-D optical
encoders. For example, using three different 2-D optical encoders
(M1, M2 and M3) to synthesis three different compounds: compound W1
(a-b-c), compound W2 (a-c-c), and compound W3 (b-a-c). Here a, b, c
are different products coming from different synthesis reactions.
The code for 2-D optical encoder M1 is 123, for 2-D optical encoder
M2 is 133, for 2-D optical encoder M3 is 213. Code 1 defines
synthesis reaction a, code 2 defines synthesis reaction b, and code
3 defines synthesis reaction c. These three different kinds of 2-D
optical encoders are mixed in one chamber. After modifying the
surfaces of these 2-D optical encoders, the cleavable linkers are
bound to the encoder surface. These cleavable linkers include, but
are not limited to, optically cleavable linker, enzymatically
cleavable linker, and thermally cleavable linker, etc. The
following synthesis reactions are conducted on these linkers and
the reaction products are connected to these linkers. Then the
sorting device is used to sort these 2-D optical encoders. Based on
the codes on these 2-D optical encoders, the 2-D optical encoders
are transported to related reaction chambers. For example, at the
first sorting process, the sorting device decodes the first code on
the 2-D optical encoder. 2-D optical encoders M1 and M2 are
transported to chamber a to carry out synthesis reaction a. 2-D
optical encoder M3 is transported to chamber b to carry out
synthesis reaction b. After finishing the first-round synthesis
reaction, all the 2-D optical encoders are mixed and then sorted
again. Based on the second code on the 2-D optical encoders, 2-D
optical encoder M3 is transported to chamber a to carry out
synthesis reaction a. 2-D optical encoder M1 is transported to
chamber b to carry out synthesis reaction b and 2-D optical encoder
M2 is transported to chamber c to carry out synthesis reaction c.
Following this rule, after complete of each synthesis reaction, all
the 2-D optical encoders are mixed again. Then based on the related
code on these 2-D optical encoders, the sorting device can
transport them to related chambers to carry out related synthesis
reactions. When all the synthesis reaction are completed, the
desired compounds are linked to 2-D optical encoders. Through
reading the codes on these 2-D optical encoders, it is easy to know
the identity of the compounds. Also these compounds are linked to
2-D optical encoders by cleavable linkers so that it is easy to
recover the compounds.
[0224] In FIG. 14, each rectangle with three letters (a, b and c)
is a 2-D optical encoder. Here the letters are the codes for 2-D
optical encoder. The capital letter in the black circle denotes the
production of related synthesis reaction.
[0225] In FIG. 15, there are three different optical coding
methods. FIG. 15(A) illustrates bar codes. FIG. 15(B) illustrates
grating codes. FIG. 15(C) illustrates hole codes.
[0226] FIG. 16 illustrates sorting of a sector sphere encoder 1,
i.e., an encoder having a spherical portion and a flat portion. The
codes are located on the flat surface of this sector sphere. And
the other surface of this sector sphere contains the substrate for
compound synthesis. The density of this kind of 2-D optical encoder
is nearly the same as the solution. So the 2-D optical encoder can
float in solution and the flat surface of this 2-D optical encoder
will always face up. The sorting device has a microchannel 2 and
each time only one 2-D optical encoder can go through this
microchannel 2. A readout system 3 is located around the
microchannel 2. When the sorting device sorts the mixed 2-D optical
encoders, the 2-D optical encoders in the solution will go through
the microchannel 2 quickly. The readout system 3 will read and
decode the code on each 2-D optical encoder. And then the 2-D
optical encoders are transported to different reaction chambers
behind the sorting device. Each time based on the different
decoding signal; the sorting device can connect the related
reaction chamber with the microchannel 2 and let the 2-D optical
encoder go into the chamber for next synthesis reaction.
[0227] The carrier or 2-D optical encoder showed in FIG. 16 can be
of any suitable shape such as a cube, a rectangular parallelepiped
(cuboid), a cone, a cylinder, a prism, a pyramid and a right-angled
circular cylinder. The applied forces for manipulating the 2-D
optical encoders or the linked substances include, but are not
limit to, electric forces, magnetic forces, acoustic forces and
mechanic forces. These applied forces can control and manipulate
the 2-D optical encoders or the substances on these encoders in the
solution and make sure each time only one encoder in the solution
can go through the microchannel 2 in the sorting device. Also
applied forces can be selected from electroosmotic pumping forces,
mechanical pumping forces, and electrohydrodynamic pumping forces.
These applied forces are applied to the solution liquid of the
reaction system, and the solution liquid will carry the 2-D optical
encoders and the linked substances through the microchannel on
sorting device.
[0228] The present 2-D optical encoders also can be used to make
different kinds of chips, such as DNA chip, protein chip and
polysaccharide chips.
[0229] The 2-D optical encoders can be used to fabricate a chip,
e.g., a biochip. First, many kinds of different 2-D optical
encoders 1 can be prepared. These 2-D optical encoders have a
modified functional layer linked to the non-coding surface region.
And the functional layer is used for immobilizing the biological or
chemical substances. Examples of the functional layer include, but
are not limited to, a molecular monolayer, a membrane, a gel, a
porous or non-porous material layer. The functional layer may be an
additional layer adhered to the surface of 2-D optical encoder
(through microfabrication method). Alternatively, the functional
layer may be formed by direct chemical-modification of the surface
molecules of the 2-D optical encoder. Preferably, the functional
layer should have minimal or no non-specific bindings to molecules
other than ligand molecules, and should allow efficient binding or
attachment of the necessary biological substances or chemical
substances. The functional layer may be a hydrophilic or
hydrophobic molecular monolayer, a hydrophilic or hydrophobic
membrane, a hydrophilic or hydrophobic gel, a polymer layer, porous
or non-porous materials and/or the composite of these materials.
Molecular monolayer refers to single molecular layer (for example,
Langmuir-Blodgett film). For immobilizing nucleic acid probes,
binding materials such as nitrocellulose or nylon may be used as in
Southern or northern blots. Proteins and peptides can be bound by
various physical (e.g., hydrophobic) or chemical approaches. For
example, specific receptors such as antibodies or lectins can be
incorporated into the functional layer for binding target molecules
of protein or peptide-types. Depending on the intended targets and
the assays or reactions to be carried out by the biochip, different
molecules can be incorporated into the functional layer for binding
target molecules. These molecules incorporated in the functional
layer for binding target molecules are referred to as the
functional groups. Examples of the functional groups include, but
are not limited to, aldehydes, carbodiimides, succinimydyl esters,
antibodies, receptors, and lectins. The functional groups also
include chemical groups or molecular sites that are formed through
chemical modification on the 2-D optical encoder surface
molecules.
[0230] For example the 2-D optical encoder can be used in the
fabrication of a protein chip. Each kind of proteins as probes will
be immobilized on the functional layer of different kinds of 2-D
optical encoders. The codes of the 2-D optical encoders will
specifically denote the proteins immobilized on the 2-D optical
encoders. So the classes of the protein immobilized on the 2-D
optical encoders can be easily identified through decoding the code
on the 2-D optical encoder. These 2-D optical encoders can be used
to detect the "unknown" protein. First, the "unknown" proteins in
the sample solution are labeled with fluorescence. Then a plurality
of 2-D encoders with different substances are loaded and reacted
with "unknown" proteins. After the stringency control wash, these
2-D optical encoders are manipulated to go through the detection
system one by one.
[0231] FIG. 17 is a schematic diagram showing an exemplary
detection system of the present invention. This detection system is
similar to sorting device shown in FIG. 16. The detection system
has a microchannel 2. The dimension of this microchannel 2 is fit
for 2-D optical encoders. That means each time only one 2-D optical
encoder can go through this microchannel. The forces induced by
various effects such as traveling-wave dielectrophoresis,
traveling-wave magnetic field, traveling-wave acoustic wave,
mechanic force induced by fluid motion, etc., can control and
manipulate the 2-D optical encoders or the substances on these
encoders in the solution and make sure each time only one encoder
in the solution can go through the microchannel 2 in the detection
system. Also applied forces can be selected from various pumping
forces such as electroosmotic pumping forces, mechanical pumping
forces, and electrohydrodynamic pumping forces. These forces are
applied to the solution liquid of the reaction system, and the
solution liquid will carry the 2-D optical encoders and the linked
substances through the microchannel on sorting device. There are
two windows on the same location of the microchannel. Above the up
window there is a readout system 3 and below the bottom window
there is a fluorescence detection system 4. When the 2-D optical
encoder goes through the microchannel 2, if there is "unknown"
protein from a sample binding with the protein probe immobilized on
the 2-D optical encoder, the fluorescence detection system 4 will
detect the fluorescence signal and trig the readout system 3 to
read and decode the code on this 2-D optical encoder. Then based on
the decoding result, the class of "unknown" protein may be
ascertained. The fluorescent signal detected by the detection
system 4 can also be used to determine the quantity of the protein
in the sample.
[0232] The advantage of this kind of biochip is that users can
immobilize different kinds of probe, e.g., proteins probes, to
different microfabricated 2-D optical encoders by themselves. So it
is easy for users to construct different probe libraries.
[0233] To measure and detect the "unknown" substance in a sample,
the "unknown" substance molecules may be labeled with fluorescence
or isotope. After the reaction, the 2-D optical encoders will go
through the detection system one by one. The detection system will
ascertain if there is reaction between the probe on the 2-D optical
encoder with the "unknown" substance molecules and determine the
class of this 2-D optical encoder. The signals detected in the
detection systems 3 and 4 can be used, alone or in combination, to
determine the presence, absence or amount of an analyte, e.g., a
target protein, in the sample.
F. Examples
1. Information Encoded Fluid Suspendable Microdevice
[0234] In one specific embodiment, the invention is intended to
solve the problems encountered in 2-dimensional micro array systems
as well as 3-D micro particle systems. The invention described
herein is a system compromising a microdevice, individual
microdevices that are information encoded, a detection system and a
data analysis system. It may also include an array system for
application of biological samples.
[0235] The microdevice can be encoded individually using a bar
coding system. Each individual encoded microdevice serves as a
biological reaction and detection platform. The microdevice can be
square or circle or other shape. Its dimension can be 50 micrometer
by 50 micrometer (10 micron to 100 micron). The microdevice can be
thin, e.g., about 100 anstrong to about 1 micron. It can be
biologically compatible and liquid suspensable. The microdevice can
be used in studies of nucleic acid, protein, biochemical reaction,
cell biology, diagnostics and drug screen.
[0236] The microbiochip system can comprise or consist of (a)
individually encoded microdevices, (b) devices that separate the
microdevices, (c) a detection system that reads both coding
information and reaction information and (d) a data processing
system.
[0237] The microdevice can be encoded by a pattern that is located
on the chip. The pattern can be created by making an array of photo
transmissible micro sized holes or by dotted reflection materials.
The processes producing those patterns can be conducted through
fabrication called chemical etching. A series of masks that have
different patterns can be created by computers and produced by
conventional technology. Thin films of inert materials, e.g.,
silicon, glass, metal, ceramic, plastic, etc., can be laid on top
of a flat polymer surface, such as agarose for chemical etching.
Photolithography process can be carried out. After the desired
pattern has been created to produce the patterns of the film, the
polymer layer at the bottom can be removed by the appropriate
method. In the case of agarose, heat is needed to melt the agarose
and release individual microdevices. The size of the microdevices
can be about less than 50, 50-100, or more than 100 .mu.m in
diameters and can be circle, square, rectangle or any other shape.
The information holes or spots on the microdevice may be numbered.
The microdevice can be modified by chemical process to obtain
desired surface chemistry suitable for biological reactions.
[0238] After the microdevices have reacted with biochemical
analytes the microdevices can be separated by a microdevice
separation chamber. In this chamber, microdevices in solution can
be isolated and separated in a narrow thin micro channel and lined
up one by one in the channel. Then the individual microdevice can
be transported to a detection zone for analysis of the coding
information and reaction information.
[0239] The detection system can comprise or consist of an optical
detector, an analyte detector, e.g., a fluorescence detector and
data analysis software. The optical detector will detect the light
transmission pattern of the individual microdevice to decode the
encoded information. The fluorescence detector will detect the
fluorescence signal generated from an analyte specific reaction.
Data analysis software will analyze the data and provide two types
of information. One is the identity of the specific analyte on a
microdevice, such as a specific nucleic acid probe, antibody,
specific protein or other moiety of interest, such as a cell, a
bacterium, and a virus. The system will also provide qualitative
and quantitative information regarding the specific analyte on a
microdevice.
[0240] The system can be used for many purposes such as analyzing
nucleic acid hybridization, antibody-antigen interactions,
receptor-ligand interactions, cell sorting, screening of phage
particles that display antibody or binding partners of interest.
The system can also be used for screening hybridoma cells that
carry specific antibody, chemical compound synthesis and screening
and studying other molecular interaction events.
2. Microfabricated Encoding Microparticles for Microfluidic and
Biochip Applications
[0241] In another specific embodiment, the invention concerns
information encoded microparticles and uses thereof. The
microparticles or microdevices are microfabricated structures or
the microdevices disclosed in the present invention. The structures
or microdevices may be a thin, rectangular shaped substrate (e.g.
thickness between <1 micron to >10 micron with major surface
areas between <10 squared-micron and >10,000
squared-microns). Or, the structures may be thin, circular disks
(e.g. thickness between <1 micron to >10 micron with
circle-type surface having diameter between <10 microns and
>500 microns). Or, the structures may have cube-like shapes
(side width between <10 and >100 micron). Or, the
microstructures may have other irregular shapes. The
single-dimensions of the structures may vary between as small as
<1 micron and as large as >500 micron. The micro-fabricated
structures or the microdevices may be from simple material types
such as silicon, plastic, ceramics, metals, or the structures may
be made from composite materials comprising silicon, metal film and
polymer films.
[0242] Preferably, the microfabricated structures or the
microdevices have encoding patterns on the surface or on the body.
The encoding patterns would allow, first of all, many types of
fabricated structures to be made, and secondly, the discrimination
and distinguishing between different microstructures. There may be
a number of methods for incorporating encoding patterns on the
structures. One approach is to incorporate "holes" on the chip
surfaces. For example, on rectangular chips having dimensions of 1
by 5 by 50 microns. Along 50 micron dimensions, there may be 4
holes, spaced 10 microns apart for the center-to-center distance.
The holes may have a diameter of 2 microns. Depending on whether
holes are produced at the particular positions and depending on how
many holes there are on the microstructures or microdevices, there
are total 16 combinations (=2.sup.4). FIG. 1 provides encoding
examples of microstructures or microdevices where the structures
are rectangular shape and the holes are introduced along the middle
lines of the structures.
[0243] Another example of the microstructures or the microdevices
is the circular discs on which holes are produced. Possible
examples are shown in FIG. 2 where the holes are positioned not
symmetrical on the circular disk. The holes are located at four
different diameter positions (r=0 at the center, r=1/4*radius;
r=1/2*radius; r=3/4*radius). Again there will be 16 encoding
combinations--leading to total 16 kinds of microfabricated
structures or microdevices. FIG. 2 shows three examples of such
encoding discs. FIG. 3 shows an example of the microfabricated
microdevices. The circular holes on the disk are used as the
encoding pattern. The rectangular holes on the edges of the disks
are used as orientation markers. During the decoding step, these
orientation markers can be detected, and the relative positions
(and the numbers) of the circular holes with regard to these
orientation markers can be analyzed to decode the encoding patterns
on the microdevices.
[0244] Holes are just one example for making encoding structures or
encoding microdevices. Materials of different optical refractive
properties may also be used. For example, encoding structures may
be fabricated on silicon wafers whilst encoding, small circle-type
disks are made of a metallic material such as aluminum, silver or
gold. As long as the encoding pattern on the structures can be read
through some mechanism, the structures or microdevices can be
utilized as encoding devices. As long as the structures may be
fabricated through certain fabrication procedures and encoding
patterns and features are incorporated on the structures, such
structures may be used for the purpose of encoding for different
types of the structures or microdevices.
[0245] Preferably, in use, these microdevices would have certain
surface chemical properties that would allow them to bind to some
bioanalytes (e.g., cells, DNA, RNA, proteins). For example, the
microdevice surface may have antibodies immobilized so that
proteins can bind to the microdevice surface. In another example,
the microdevice surface may have single stranded DNA attached so
that the single-stranded DNA may then bind to its complementary
strand. In such cases, the microdevices will be used as binding
partners for a number of moieties to be manipulated (see the
co-pending U.S. patent application Ser. No. 09/636,104, filed Aug.
10, 2000). These microdevices can be used for capturing target
cells, binding to target protein, binding to target DNA segments,
binding to target RNA segments, or reacting with any type of
bioanalyte from a mixture solution.
[0246] For example, we could have two types of encoding
microdevices--one is labeled with antibodies (Abs) for T-lymphocyte
(microdevice one) and another type labeled with Abs for
B-lymphocytes (microdevice two). These microdevices will be
incubated with a blood sample or diluted blood sample. The
microdevices may then bind to T-cells and B-cells separately. We
can then use certain methods to isolate these target
cell-microdevice complexes (there are two types, the first type is
T-cell with microdevice one, the second type is B-cell with
microdevice two) from the total cell mixture. We can then use
certain methods to identify the microdevices and sort the
cell-microdevice mixture.
[0247] In another example, we have 100 types of encoding
microdevices--each is labeled with one type of Ab against certain
target proteins. Incubating such microdevices with a protein
mixture solution may result in the target protein molecule coupling
to the surface of the encoding microdevices. We can then use
fluorescently labeled secondary antibodies to label the bound
proteins. We then can measure the fluorescent levels on each
microdevice and simultaneously, and determine which he identity of
each microdevice. We then establish the type of microdevices tested
and fluorescent levels on the microdevices. This would provide
information as to the identity and amount or concentration target
proteins in the test solution.
[0248] In the above example, the fabricated microdevices or the
fabricated microdevices are used in the same way as multiple
microbeads that have been developed for assaying and analyzing
bioanalytes in mixture solutions.
[0249] Preferably, the fabricated microdevices or the microdevices
have desired physical properties such that these physical
properties allow these microdevices to be manipulatable by on-chip
generated physical forces. For example, if dielectrophoresis is
used to control and manipulate microdevices, the microdevices
should have certain dielectric properties so that their properties
are different from those of the solution in which the microdevices
are introduced or suspended. In such cases, dielectrophoresis
theories may be applied to design the materials, sizes, geometries
and compositions of such microfabricated microdevices. In another
example, if magnetophoresis with magnetic fields is used to move
and manipulate the fabricated microdevices, then the microdevices
are expected to have desired magnetic properties, e.g., magnetic
film materials have been introduced into the microdevices.
[0250] Microfabricated encoding microdevices or microdevices can be
fabricated or micromachined with a number of standard procedures.
Photolithographic processes may be used with masks that have
defined patterns. These patterns will correspond to the final
encoding patterns. The steps involved may include steps like
deposition of thin film layers, etching off the thin film at
designated places etc. A number of articles that described certain
fabrication methods that may be used for producing such
microfabricated microdevices are incorporated by reference (e.g.,
"Design of asynchronous dielectric micromortors", by Hagedorn, et
al., J. Elecetrostatics (1994) 33:159-185; "Design considerations
for micromachined electric actuators", by Bart, et al., Sensors and
Acuators (1988) 14:269-292). Appropriate materials with desired
physical properties should be used so that microfabricated
microdevices may be moved or manipulated or controlled by certain
physical forces generated by physical fields. The free standing
microdevices after fabrication will then be modified on their
surfaces so these microdevices could bind to the surfaces of the
microdevices.
[0251] The microfabricated encoding microdevices or the devices are
then chemically or biochemically modified. Common procedures that
are used for modifying solid substrates may be utilized for such
purposes. The modification steps will lead to specific molecules
attached on the microdevices' surfaces. These specific molecules
may include antibodies, DNAs, RNAs, ligands, enzymes etc. In such
cases, these molecules are used as binding partners.
[0252] These microfabricated encoding microdevices having specific
molecules attached to their surfaces may then be used to bind the
target bioanalytes from a solution mixture. After binding to the
target analytes, the target analytes may then be further detected
on these microdevices. For example, individual microdevices can be
detected by determining their types and sorting them out according
to their individual types using, e.g., biochip-based devices--so
that each type of microfabricated microdevices is concentrated or
accumulated into one specific region on a biochip. Then the target
molecules on these microdevices are further labeled and detected.
The labeling may use or involve fluorescent molecules. Detection
may then be based on fluorescent labeling. Another approach may
utilize or involve magnetic beads. Then detection may be magnetic
chip based detection. In another example, individual microdevice
can be detected for determining their types and measuring
fluorescent levels on each microdevice, recording the correlation
information between types of individual microdevice and fluorescent
levels on each microdevice.
[0253] Detection of individual microdevices may involve the use of
instruments such as a microscope, an optical-imager, or a
image-capture system. This can be accomplished by methods or
devices known in the art such as an image-processing and/or pattern
recognition programs.
[0254] Sorting such microfabricated microdevices may also be
possible. For example, we could use our on-chip based microparticle
switch (pumwitch=pumps & switches for microparticle
transportation and sorting). For example, the microparticle
switches disclosed in the co-pending U.S. patent application Ser.
No. 09/678,263 can be used. The on-chip sorting or separation of
microdevices (with the binding partners and the moieties) may use
the methods disclosed in the co-pending U.S. patent application
Ser. No. 09/678,263, which is incorporated by reference in its
entirety. In this case, microfabricated microdevices will be
separated according to their encoding patterns. Microfabricated
encoding microdevices (i.e. microparticles) having the same
patterns will be sorted or moved to the same locations on the chip
by applying appropriate electrical signals, based on the detected
patterns on the microdevices.
[0255] The microfabricated, encoding particles (or the microdevices
or the microdevices) can be used both on biochip or off biochip. In
off-biochip cases, these microfabricated particles (i.e.
microdevices) can be used in a manner similar to that of to
microbeads in current biological/biomedical applications. The
microfbaricated particles (i.e., the microdevices) can be used to
separate cells, isolate target molecules, separate molecules,
transport cells/molecules, etc. Primarily, the particles (i.e., the
microdevices) are used as binding partners for binding to specific
moieties or bioanalytes. In on-chip cases, these microfabricated
encoding particles (i.e. the microdevices) can be used as binding
partners to bind bioanalytes or moieties or other biomolecules. The
on-chip use of these microfabricated encoding particle (i.e., the
microdevices) or microdevices can be the same as the procedure of
manipulating moieties through binding partners as described in the
co-pending U.S. patent application Ser. No. 09/636,104, filed Aug.
10, 2000.
3. Library Synthesis Using Information Encoded Sortable
Particles
[0256] Another specific embodiment is to use microdevices of the
present invention for library synthesis. Information Encoded
Sortable Particles (IESPs) can be used in library synthesis. This
allows vast addressable arrays to be generated for any molecule
that can be synthesized using conventional solid phase methods. For
simplicity DNA library synthesis has been chosen as an example.
[0257] Background
[0258] The one-bead/one-compound procedure is well-established and
permits the use of existing solid phase chemistry, e.g., peptide
and nucleic acid synthesis. The following example will demonstrate
the typical method of preparing DNA on beads using the
one-bead/one-compound approach. In the case of DNA there are 4
bases, A, T, C, and G. Beads are divided randomly into 4 tubes
labeled A, T, C, and G and the corresponding base is chemically
coupled to the beads in each tube. The beads from each tube are
then mixed together and randomly divided into another set of 4
tubes labeled A, T, C and G and the corresponding base is once
again added. This process of dividing, coupling, and mixing is
repeated N times, where N is the length of the individual
oligonucleotide chains. This process as the name implies produces
beads that contain only a single type of compound. Consider a
specific example where N is 10 and 10,000,000 beads, at the end of
the synthesis, the library will consist of all possible 10-mers
(1,048,576 or 4.sup.10) that are represented (on average) 9-10
times in the library. The amount of each compound on the beads is
determined by the chemistry on the bead (number of coupling sites)
and it is possible to have 10.sup.6 or more molecules/bead. Using
the one-bead/one-compound strategy it is therefore straightforward
to generate vast arrays of compounds, but there are major
restrictions. The identity of the compound on any particular bead
is not known or determinable other than through some type of
analysis of the compound on the bead (e.g. sequencing or mass
spectrometry). The ability to represent an entire sequence space is
limited by the physical constraints imposed by the volume of the
beads, i.e., in practice it is difficult to generate or screen bead
libraries larger than .about.10.sup.10. For example, consider a
library of DNA 25-mers, there are over 10.sup.15 25mers and at
least 4 times as many beads would be required to insure that each
25-mer is represented at least once in the final bead library. Even
if 1 micron beads were used, the library would still occupy several
liters. When the number of beads is less than the number of
possible compounds the library no longer represents the entire
sequence space. Since all the steps are random, knowledge of the
specific sequences contained in the library is lost when the number
of compounds exceeds the number of beads.
[0259] Information Encoded Sortable Particles (IESPs)
[0260] Passive Sorting
[0261] If the particles (beads) are encoded then at each step in
the synthesis described in the previous section the identity of
each bead can be determined and that information stored. At the end
of the process, the DNA sequence on each particle will be known to
correspond to a specific particle. This means, that in any assay,
identifying the code on the particle reveals the identity of the
compound on that particle. Such knowledge is essential when
carrying out assays leading to thousands or millions of positive
responses such as occurs in mRNA profiling (it is impractical to
sequence thousands or millions of beads). In addition, because each
synthetic step is recorded, the precise representation within the
library is known. Consequently, even in libraries where the number
of compounds greatly exceeds the number of beads, the identity of
every compound within the library is known.
[0262] Active Sorting
[0263] Sorting can also be active, and in this case instead of
particles being mixed and randomly distributed following each
synthetic step, specific predetermined particles are instead sorted
into specific tubes. In an active sorting procedure the specific
sequences are preassigned to individual particles. For example, the
IESP assigned the sequence ATCGGGTTAA (SEQ ID NO:1) would go to the
A tube in the first step of synthesis then to the T tube in the
second, the C tube in the third, etc. Consequently, active sorting
could be used to generate a library corresponding to any particular
predetermined subset of sequence space. For example, this procedure
could be used to generate a library of 10.sup.6 50-mers all of
which correspond to a sequence in the human genome. This is a very
small and specific subset in a sequence space of over 10.sup.30
(4.sup.50). Active sorting permits the precise determination of the
number of times each compound is represented in the collection of
particles, making it possible to generate arrays containing only
unique representations. Both passive and active sorting procedures,
by identifying the particles containing specific compounds, make it
possible to create specific sub-arrays of the full compound library
without resynthesis, for example, selecting from an IESP library
containing all possible DNA 10-mers only those with a G at position
4 but lacking C at positions 2 and 6. The major source of error in
the generation of these libraries is likely to be due to mistakes
in sorting, i.e., misidentification of a particle during the
synthesis. However, since active sorting directs specific particles
to specific tubes after each synthesis step, an error would have to
occur on the same particle in two consecutive cycles in order to
propagate, greatly reducing the frequency of misidentification
errors.
[0264] Applications
[0265] The examples below represent a few of the many possible
applications of IESPs. IESPs enable the inexpensive manufacture of
vast arrays of known sequence. Apart from ease of synthesis using
IESPs there are a number of general advantages to libraries
produced on particles as opposed to membranes or glass slides used
in competing technologies. Because of the greater freedom in
synthesis it is possible to display compounds on the end of soluble
spacer molecules allowing for more effective presentation of the
library (this can be essential in the case of hydrophobic compounds
which may be otherwise insoluble). Additionally, in assays
involving target binding, isolation of a particular IESP directly
corresponds to purification of its target, raising the possibility
of carrying out secondary analyses following the initial capture
process. Purification procedures can benefit from the ability of
IESPs to be rapidly sorted using a FACS machine to sequester
positives followed by a final sorting and identification using a
slower IESP sorting device.
[0266] DNA Arrays
[0267] DNA arrays for determining mRNA levels could be generated
using IESPs. Such arrays would be expected to be superior to those
of the currently used arrays. Membrane based arrays of synthetic
oligonucleotides are severely limited in the length of the
oligonucleotides that can be displayed. By contrast, particle based
syntheses have no such limitations and oligonucleotide sequences
significantly longer than those used by the membrane based arrays
can be employed to minimize background, e.g., permit much more
stringent hybridization conditions. In addition, the use of longer
oligonucleotides results in other advantages. Minor synthesis
errors do not affect the result (an error in one or two errors in a
50-mer is inconsequential, in a 25-mer it is fatal). Similarly SNPs
in the target DNA will not affect hybridization.
[0268] Peptide Arrays
[0269] One of the more intriguing applications of IESPs, not
achievable using any current technique, is to generate a peptide
library that represents an entire genome. Such peptide arrays would
permit screening of various enzymes in an attempt to identify
physiological substrates such as receptor ligands or kinase
substrates. Existing random peptide libraries are more restricted
because they do not correspond directly to the genome but instead
sample all of sequence space. For example, a peptide array
representing each protein in the human genome by a series of
20-residue peptides which overlap by 10 residues would contain
.about.10.sup.7 peptides, the complete sequence space for all
20-residue peptides is .about.10.sup.26 combinations. Moreover, if
it were possible to make such huge random arrays an overwhelming
amount of the information would be irrelevant to physiological
function (in this example there would be 10.sup.19 as many
nonphysiological peptides as physiological ones). Even in a random
library consisting of all 8-residue peptides, less than 1% of the
peptides are encoded in the human genome.
[0270] Since IESP-generated peptide arrays are synthetic they can
include, in addition to the common amino acids, unnatural, D-amino
acids, and peptide mimetics. Such peptide arrays can be used in
screens for drug leads as discussed in the next subsection.
[0271] Drug Discovery
[0272] One major application for IESPs is in drug discovery. Using
well-established solid-phase techniques it should be possible to
generate arrays of 10.sup.6 or more particles. Such arrays could be
screened against a single drug target using a fluorescence based
detection system. Arraying the IESPs in a monolayer would permit
fluorescence detection and identification to be carried out
simultaneously. Following the initial analysis a new library of
10.sup.6 or more compounds based upon the first screening could be
generated and the assay repeated. With IESP technology 10-100 fold
more information could be determined in a few hours. In addition,
more restrictive libraries can be rapidly generated based upon the
positive results from earlier screenings. The application of such
an iterative process would further enhance the huge competitive
advantage of IESP technology for drug design.
[0273] Information Encoding
[0274] IESP can be used to encode information and to rapidly
retrieve information from complex systems. For example, any type of
synthetic library generated using IESPs could include a specific
DNA tag. Following synthesis, the library could be released from
the particles and the assay carried out in solution. The library
could then be reattached to IESPs by hybridization for
identification. This approach permits interaction in solution
followed by chip capture, thus making it possible to carry out
assays that are difficult or impossible to perform on molecules
bound to a surface. While DNA has been used as an example other
specific interactions could be used, e.g., aptamer array--peptide
interactions. It is important to note that this approach has
significant advantages over competing types of capture approaches
using membranes. In particular, using IESPs any tagging sequence
can be generated. For example, using DNA 20-mers for tagging it is
possible to generate 10.sup.8 tags where each tag differs from
every other by 5 or more bases thus eliminating mismatches and
cross hybridization between tags and targets. In addition, since
the identity of the compound bound to each specific DNA tag is
known, the procedure is easily validated (e.g. can determine if any
of the DNA tags fail to hybridize in good yield prior to the
analysis).
[0275] Soluble tagged IESP generated libraries also makes it
possible to utilize multiple libraries in a single assay, e.g., a
library of antibodies screened against a library of peptides
instead of against a single peptide as is done currently. Another
advantage of DNA tagging is that an amplification/labeling step can
be included prior to decoding to enhance signal strength. This
method is particularly useful when using a library to distinguish
differences among a large number of different targets, e.g.,
identifying synthetic antibodies or ligands that uniquely bind to a
particular cell type.
4. High Throughput Antibody Screen
[0276] Antigen Immobilization of Encoded Microdevice
[0277] Antigen targets can be immobilized on the microdevice
covalently or non-covalently. Antigen target can be peptides,
proteins, nucleic acids, polysaccharides, chemical molecules or
other molecules that can be recognized by an antibody Immobilized
on each microdevice is a unique antigen target, the identity of
which is known. In some instances, it may be beneficial to have a
mixture of more than one target antigen immobilized on a
microdevice. The identities of the components of such antigen
mixtures are also known. Protein targets (1 ng-10 .mu.g) can be
immobilized onto a chemically modified surface of the
microdevices.
[0278] Recombinant Antibody Clones Selection
[0279] A. Antibody Library Construction
[0280] An antibody library is established by recombinant phage
display technologies. Briefly, antibody encoding DNA fragments are
amplified from mRNA preparation from human peripheral blood
lymphocytes, bone marrow cells or spleen cells through reverse
transcription PCR. Mouse antibody fragments can be made from mRNA
preparation from spleen cells. Alternatively, antibody fragments
can be generated by designed total DNA synthesis or semi DNA
synthesis. The DNA fragments encoding IgG heavy chain and light
chain are inserted into a phagemid vector respectively by
recombinant DNA technologies. The phagemid vector contains a gene
encoding filamentous phage (fd) gene III product, which is the C
terminal of the inserted antibody gene. The resulting vectors
containing antibody encoding genes are transformed into an E. coli.
strain (for example TG1) and helper phages are infected into the E.
coli. cells. Phages that display antibody are collected from the
supernatant of the cell culture. Those phage populations serve as
starting materials for the antibody screen.
[0281] B. High Throughout Screening of Antibodies
[0282] The encoded microdevices (up to 1000 different codes) that
have been immobilized with antigens are used to screen antibodies.
In a test tube, 100 .mu.l of microdevices suspended in PBS buffer
was aliquoted. The 100 .mu.l of microdevice solution contains 100
copies of 1000 (from 1 to 1000) different coded microdevices. One
or more than one different peptide antigens, e.g., 10, 100 or 1,000
antigens, is immobilized on each kind of microdevice. The
microdevice mixture is incubated with 100 .mu.l of phage library
(10.sup.10 p.f.u) produced by above mentioned method. Incubation
condition is 2 hr-18 hr at 4.degree. C. This procedure ensures that
the antibody displayed on phage binds to its selected targets.
After incubation, the microdevice suspension is washed with wash
buffer for 5 times to remove non-bound phages. Then, fluorescence
labeled anti-M13 coat protein antibody is incubated with the
microdevices suspended in 100 .mu.l of PBS buffer at 37.degree. C.
for 2 hrs. And then, the microdevices are washed 3 times with wash
buffer.
[0283] C. Detecting and Sorting Individual Microdevice
[0284] The microdevice mixture is suspended in 0.1-1 ml of PBS
buffer. The mixture is loaded into a detection biochip for
fluorescence detection and barcode sorting. The whole process is
performed on the instrument specialized for the detection.
According to the fluorescent signals and encoding information on
each microdevice, individual microdevice with fluorescence signal
is sorted and collected into a micro well on a microtiter plate.
Two information are collected by this process: 1) fluorescence
signal on the microdevice indicating that specific phage carrying
an antibody is bound on the target on chip; and 2) the target on
chip is analyzed by the decoding the pattering information of the
microdevice. Accordingly, specific antibody for given target is
obtained after the sorting.
[0285] D. Antibody Characterization
[0286] Microdevices positive for antibodies are collected into
microtiter plate wells by the sorting process. Phages bound on the
microdevices are released by treatment with proteolytic enzymes or
low pH. Released phages are reinfected into E. coli. cells.
Individual colonies are obtained by plating the infected E. coli.
cells onto nutrient agar plate. Individual colony is selected and
cultured for antibody production. Antibody producing cells are
selected by an ELISA method using specific antigens. Once specific
antibody for a given antigen is obtained, it can be used for
large-scale production of a specific antibody.
5. Exemplary Fabrication Processes
[0287] FIG. 13 shows one example of the fabrication processes for
making one type of the encoding particles. The encoding particles
described here have three layers, i.e., top layer, bottom layer
and/or middle layer with respect to the orientation shown in FIG.
13, with the encoding features located in the middle layer. The top
and bottom layers enclosing the middle layer are of materials that
can be modified to attach suitable molecules. The steps in FIG. 13
and described below are just examples of the fabrication procedures
that could be used for making the encoding particles. Those who are
skilled in the art of microfabrication or micromachining can
readily adopt different procedures/protocols based on the materials
and geometries of the encoding particles to be fabricated.
[0288] As shown in FIG. 13, the exemplary process starts with the
preparation of a solid substrate. The substrate should be
pre-cleaned to make sure that it is suited for the fabrication. An
example of the substrate may be silicon wafer used for
semiconductor fabrications. The clean substrate will then be
deposited or coated with a sacrificial layer. As described later,
the sacrificial layer will be removed at the last step of the
fabrication by methods such as dissolving, etching, etc. Examples
of the sacrificial layer can be metal, e.g., copper,
Si.sub.3N.sub.4, or other materials. When choosing appropriate
materials for the sacrificial layer, it is necessary that the
sacrificial layer can be selectively removed without affecting the
materials used for making the encoding particles themselves. The
sacrificial layer can be of variable thickness, e.g., .about.1
micron. The method for depositing such a sacrificial layer can be
sputtering, evaporation or other methods of deposition. The methods
chosen for deposition depend on factors such as the
sacrificial-layer materials, the thickness of the layer,
availability of the methods in the fabrication labs, etc.
[0289] After forming the sacrificial layer, the bottom layer, i.e.,
the first layer as shown in step 3 of the FIG. 13, of the encoding
particles is then formed or deposited on the sacrificial layer.
This layer can be made of different materials such as silicon
dioxide, aluminum oxide, plastics, polymers, etc. Preferably, the
bottom layer can be readily modified so that molecules of interest
can be attached on the bottom layer surfaces. Various methods can
be used for forming such a layer. For example, sputtering or
evaporation may be used for depositing a silicon dioxide layer.
This bottom layer can be of variable thickness, depending on the
specific design of the encoding particles. This layer can be as
thin as several nanometers, or as thick as many microns or
millimeters. For example, we have fabricated the encoding particles
having a bottom layer thickness of 50 nm, 0.1 micron, 0.3 micron,
0.5 micron or 1 micron made of silicon dioxide.
[0290] After forming the bottom layer, i.e., the first layer as
shown in step 3 of the FIG. 13, of the encoding particles, the
middle layer, i.e., the second layer as shown in step 4 of the FIG.
13, is then formed or deposited on the bottom layer. This layer may
serve various purposes. It may be used for including the encoding
features, as in the case shown in FIG. 13. For example, a metal
layer (e.g., aluminum) may be used and the metal layer will be
patterned using photolithography to make the encoding features such
as lines, dots, squares, numbers, etc. This middle layer may
comprise suitable materials so that the encoding particles have
certain physical properties. For example, this layer may be of
magnetic, ferromagnetic, or ferrimagnetic materials so that the
encoding particles have magnetic properties. For example, nickel
metal or CoTaZr (Cobalt-Tantalum-Zirconium) alloy or other magnetic
materials may be used. Various methods may be used for depositing
or forming such a layer, depending on factors such as materials to
be deposited, thickness of the layer, availability of the methods.
Non-limiting examples are evaporation, sputtering, etc. This layer
can be of variable thickness, depending on the designs and
requirements for the encoding particles. The layer can be as thin
as several nanometers or as thick as many microns or many
millimeters. For example, we have fabricated the encoding particles
with the middle layer thickness of 0.02 micron, 0.05 micron, 0.1
micron, 0.3 micron, 0.5 micron, 1 micron and 3 microns using
various materials including aluminum, nickel, CoTaZr, etc.
[0291] After forming the middle layer via various deposition
methods, the middle layer may then be patterned for producing
required encoding features or producing certain geometrical
patterns in this middle layer (i.e., the second layer as shown in
step 5 of FIG. 13). The encoding features are used for coding each
individual particle. The features may include, but not limited to,
numbers, letters, symbols, lines, squares, 1-D bar codes, 2-D bar
codes. Many commercially available coding patterns can be used
(e.g. Two-dimensional codes in "Automatic I.D. News", October
1995). Patterning of the middle layer can be achieved using
techniques such as photolithography with photomasks. Such
photolithography-based patterning can be done with a number of
methods. Those who are skilled in the art of micro-fabrication can
readily determine and choose or develop appropriate protocols for
patterning this middle layer to produce required encoding
geometries/features, based on the required geometrical sizes of the
patterns and the materials of the middle layer. For example, we can
use chemical etch for patterning a metal layer by first patterning
a coated photoresist layer.
[0292] After patterning the middle layer (i.e., the second layer as
shown in step 5 of FIG. 13), the top layer (i.e., the third layer
as shown in step 6 FIG. 13) may then be formed or deposited on the
middle layer. This layer can be made of a number of materials such
as silicon dioxide, aluminum oxide, plastics, polymers, etc. In
some cases, the top layer can be of the same material as those for
the bottom layer. But this does not have to be the case. The top
layer may be of different materials/compositions from the bottom
layer. Preferably, the top layer materials may be readily modified
so that molecules of interest can be attached or added onto the top
layer surfaces. Various methods can be used for forming such a
layer. For example, sputtering or evaporation may be used for
depositing a silicon dioxide layer. This top layer can be of
variable thickness, depending on the specific designs of the
encoding particles. This layer can be as thin as several
nanometers, or as thick as many micrometers or millimeters. For
example, we have fabricated the encoding particles having a top
layer thickness of 50 nm, 0.1 micron, 0.3 micron, 0.5 micron, 1
micron, 1.5 micron and 1.9 micron made of silicon dioxide. In
designing and choosing the optically encoding particles with the
layered structures similar to those shown in FIG. 13, care should
be taken so that the deposited top layer should cover all the
surfaces of the middle layer, especially when the middle layer may
be metal or other not-inert materials. But this may not always be a
strict requirement. For certain applications, exposure of some
middle layer materials due to non-covered top layer to some
reaction solutions may not be a problem. In such cases, it may not
be necessary to ensure that the coverage of the top layer over the
middle is complete.
[0293] After depositing the top layer by using the appropriate
deposition method, the top layer and the bottom layer may then be
patterned to produce individually non-connecting encoding
particles. If the bottom layer (i.e. the first layer as shown in
step 3 of the figure) and the top layer (i.e., the third layer as
shown in step 6 of FIG. 13) are of the same materials, the
patterning of the top and bottom layer may be performed
simultaneously. But this does not have to be the case. The bottom
layer and the top layer may be patterned in two separate steps,
especially if the top and bottom layers are made of different
materials. For example, we have produced certain encoding particles
with silicon dioxide on both the top and bottom layer. For those
particles, we have used both chemical etch and dry etch methods to
pattern both the top and the bottom layers.
[0294] After patterning the top and bottom layer, the encoding
particles are made but are still attached to the sacrificial layer.
Thus, the last step of the fabrication involves the release of the
fabricated encoding particles by removing or etching away the
sacrificial layer. For example, certain etching solutions (e.g.
acid) can be used to etch a metal sacrificial layer to release the
encoding particles.
[0295] In the above description, we described an exemplary process
for making one type of encoding particles. It is important to know
that the encoding particles can be of different configurations to
the one shown in FIG. 13. For example, the encoding particles in
FIG. 13 are discussed as three-layers, but they can be single
layer, two-layer, four-layer, or even more layers. Also, there
exists quite different fabrication approaches or methods for making
such encoding particles. For example, the fabrication methods
described for making dielectric micromotors in Journal of
Electrostatics (volume 33, pages 159-195, 1994) by Hagedorn et al
can be used or modified for making the optically encoding particles
of the invention.
6. Exemplary Uses of Microdevices
[0296] FIGS. 4-12 illustrates use of exemplary microdevices
MicroDisks.
[0297] FIG. 4 shows a MicroDisk containing a 2D Bar code with the
numerical representation below. MicroDisk is composed of 80.mu.
diameter, 0.5.mu. thick outer layers of SiO.sub.2 with a 70.mu.
diameter 0.5.mu. thick Nickel central layer (see schematic on left
hand side--not drawn to scale). Bright region of encoding pattern
is Nickel; dark region consists of SiO.sub.2. MicroDisk is
illuminated from above. Magnification is 220.times..
[0298] FIG. 5 shows disks randomly distributed on the surface of a
slide. MicroDisks are composed of 80.mu. diameter, 1.0.mu. thick
outer layers of SiO.sub.2 with a 70.mu. diameter 0.3.mu. thick
CoTaZr central layer. Magnification is 44.times.
[0299] FIG. 6 shows formation of chains caused by presence of weak
magnetic field in the plane (generated by Alnico C-shaped magnet).
MicroDisks are composed of 80.mu. diameter, 1.0.mu. thick outer
layers of SiO.sub.2 with a 70.mu. diameter 0.3.mu. thick CoTaZr
central layer. Left panel: Magnification is 44.times.; Right panel:
Magnification is 88.times..
[0300] FIG. 7 shows large number MicroDisks standing on edge in the
presence of strong magnetic field perpendicular to the plane
(generated by Neodymium disk-shaped magnet). MicroDisks are
composed of 80.mu. diameter, 1.9.mu. thick outer layers of
SiO.sub.2 with a 70.mu. diameter 0.1.mu. thick CoTaZr central
layer. Magnification is 44.times..
[0301] FIG. 8 shows 2 MicroDisks. In left panel they are standing
on edge in the presence of a strong magnetic field perpendicular to
the plane (generated by Neodymium disk-shaped magnet). Right hand
panel shows MicroDisks after magnetic field has been removed.
MicroDisks are composed of 80.mu. diameter, 1.0.mu. thick outer
layers of SiO.sub.2 with a 70.mu. diameter 0.3.mu. thick CoTaZr
central layer. Magnification is 88.times..
[0302] FIG. 9 shows orientation of MicroDisks following magnetic
manipulation. MicroDisks are composed of 80.mu. diameter, 1.0.mu.
thick outer layers of SiO.sub.2 with a 70.mu. diameter 0.3.mu.
thick CoTaZr central layer. Magnification is 88.times..
[0303] FIG. 10 shows results of covalent attachment experiment.
MicroDisks were treated with 3-glycidoxypropyltrimethoxy silane and
the resulting epoxide was hydrolyzed with acid to generate a diol
surface. Diol-coated MicroDisks were activated with
2,2,2-Trifluoroethanesulfonyl chloride (tresyl chloride). The upper
panels show the covalent attachment of a fluorophore
(Biocytin-Alexafluor594; Molecular Probes) to the activated
MicroDisk. Lower panels show the results of a parallel reaction
using non-activated diol-coated MicroDisks. The left-hand panels
show bright-field illumination; the right-hand panels show
fluorescent signal. After correction for background, the
fluorescence signal of the activated MicroDisks is over 100.times.
greater than that of the non-activated diol-coated MicroDisks.
MicroDisks are composed of 80.mu. diameter, 1.0.mu. thick outer
layers of SiO.sub.2 with a 70.mu. diameter 0.3.mu. thick CoTaZr
central layer. Magnification is 88.times..
[0304] FIG. 11 shows results of bioassay experiment. Mouse IgG was
covalently linked to tresyl-activated MicroDisks (upper panel).
MicroDisks were then incubated with a fluorescently-labeled
anti-mouse antibody (Alexafluor488 goat anti-mouse IgG; Molecular
Probes). Lower panels show the results of a parallel reaction using
non-activated diol-coated MicroDisks. The left-hand panels show
bright-field illumination; the right-hand panels show fluorescent
signal. After correction for background, the fluorescence signal of
the MicroDisks displaying covalent mouse IgG is over 100.times.
greater than that of the non-activated diol-coated MicroDisks.
MicroDisks are composed of 80.mu. diameter, 1.0.mu. thick outer
layers of SiO.sub.2 with a 70.mu. diameter 0.3.mu. thick CoTaZr
central layer. Magnification is 88.times..
[0305] FIG. 12 shows further results of bioassay experiment
determining the amount of fluorescence signal from both types of
MicroDisks in the same measurement. The left-hand panels show
bright-field illumination; the right-hand panel shows fluorescent
signal. Within each panel MicroDisks containing covalently linked
mouse IgG are on the left side and diol-coated MicroDisks are on
the right side. Magnification is 88.times..
[0306] The above examples are included for illustrative purposes
only and are not intended to limit the scope of the invention. Many
variations to those described above are possible. Examples of these
variations include, but not limited to, the substrate materials for
making the chips, the electrode structures for generating electric
fields, the structure of electromagnetic units for producing
magnetic fields, the structures of piezoelectric elements for
producing acoustic fields, the structures of optical elements for
generating optical fields, the structures of heating/cooling
elements for generating temperature gradient, etc. Since
modifications and variations to the examples described above will
be apparent to those of skill in this art, it is intended that this
invention be limited only by the scope of the appended claims.
TECHNICAL FIELD
[0307] This invention relates generally to the field of moiety or
molecule isolation, detection, manipulation and synthesis. In
particular, the invention provides a microdevice, which microdevice
comprises: a) a magnetizable substance; and b) a photorecognizable
coding pattern, wherein said microdevice has a preferential axis of
magnetization. Systems and methods for isolating, detecting,
manipulating and synthesizing moieties using the microdevices are
also provided.
BACKGROUND ART
[0308] High-density, high throughput biological and biochemical
assays have become essential tools for diagnostic and research
applications, particularly in areas involving the acquisition and
analysis of genetic information. These assays typically involve the
use of solid substrates. Examples of typical quantitative assays
performed on solid substrates include measurement of an antigen by
ELISA or the determination of mRNA levels by hybridization. Solid
substrates can take any form though typically they fall into two
categories--those using spherical beads or those using planar
arrays.
[0309] Planar objects such as slide- or chip-based arrays offer the
advantage of allowing capture molecules, e.g., antibody or cDNA, of
known identity to be bound at spatially distinct positions.
Surfaces are easily washed to remove unbound material. A single
mixture of analytes can be captured on a surface and detected using
a common marker, e.g., fluorescent dye. The identification of
captured analytes is governed by the spatial position of the bound
capture molecule. Archival storage of the array is generally
possible. Because the array corresponds to a stationary flat
surface, detection devices are generally simpler in design and have
lower cost of manufacture than bead reading devices. One of the
difficulties of the planar array approach is the initial
positioning of the capture molecule onto the surface. Techniques
such as robotic deposition (e.g., "Quantitative monitoring of gene
expression patterns with a complementary DNA microarray" by Schena,
et al., Science (1995) 270:467-470), photolithography (e.g.,
"Light-directed, spatially addressable parallel chemical synthesis"
by Fodor, et al., Science (1991) 251:767-773), or ink-jet
technologies (e.g., "High-density oligonucleotide arrays" by
Blanchard, et al., Biosensors Bioelectronics (1996) 6/7:687-690)
are generally used. These methods have a number of limitations.
They require expensive instrumentation to generate high density
arrays (greater than 1000 features/cm.sup.2), and there is no
ability to alter the pattern after manufacture, e.g., replace one
capture cDNA with another, consequently any alterations require a
new manufacturing process and greatly increase expenses. Moreover,
molecules bound to large flat surfaces exhibit less favorable
reaction kinetics than do molecules that are free in solution.
[0310] One way around many of these problems is to use surfaces of
small particles. Spherical beads have been the small particles of
choice because of their uniform symmetry and their minimal
self-interacting surface. Small particles, however, suffer from the
problem of being difficult to distinguish, e.g., a mixture of beads
is not spatially distinct. A number of technologies have been
developed to overcome this problem by encoding beads to make them
distinguishable. Companies such as the Luminex Corporation have
developed methods of doing this by incorporating different mixtures
of fluorescent dyes into beads to make them optically
distinguishable. In a similar manner, other researchers have
developed ways of incorporating other optically distinguishable
materials into beads (e.g., "Quantum-dot-tagged microbeads for
multiplexed optical coding of biomolecules" by Han, et al., Nature
Biotechnology (2001) 19:631-635). Furthermore, quantum dots,
nanometer scale particles that are neither small molecules nor bulk
solids, have also been used for bead identification. Their
composition and small size (a few hundred to a few thousand atoms)
give these dots extraordinary optical properties that can be
readily customized by changing the size or composition of the dots.
Quantum dots absorb light, then quickly re-emit the light but in a
different color. The most important property is that the color of
quantum dots--both in absorption and emission--can be "tuned" to
any chosen wavelength by simply changing their size. Genicon
Sciences Corporation (Their "RLS" particles are of nano-sizes and
have certain "resonance light scattering (RLS) properties) also
developed micro-beads or nano-beads with optically distinguishable
properties. However, in using any of these approaches, it is
difficult to manufacture more than 1,000 or so different encoded
beads.
[0311] Beads are also the format of choice in combinatorial
chemistry. Using the one-bead/one-compound procedure (also known as
the split and mix procedure) (see "The "one-bead-one-compound"
combinatorial library method" by Lam, et al., Chem. Rev. (1997)
97:411-448), it is possible to generate huge libraries containing
in excess of 10.sup.8 different molecules. However, the beads are
not distinguishable in any way other than by identifying the
compound on a particular bead. Labeled "tea bags" which contain
groups of beads displaying the same compound have been used to
distinguish beads. Recently, IROR1 has extended the tea bag
technology to small canisters containing either a radiofrequency
transponder or an optically encoded surface. This technology is
generally limited to constructing libraries on the order of 10,000
compounds, a single canister occupies .about.0.25 mL. Moreover, the
technology is not well suited to high-throughput-screening.
PharmaSeq, Inc. uses individual substrates containing transponders.
These devices are 250.mu..times.250.mu..times.100.mu.. Larger
libraries can be synthesized directly onto a surface to form planar
arrays using photolithographic methods (such as those used by
Affymetrix). However, such techniques have largely been restricted
to short oligonucleotides due to cost considerations and the lower
repetitive yields associated with photochemical synthesis
procedures (see e.g., "The efficiency of light-directed synthesis
of DNA arrays on glass substrates" by Mc Gall, et al., J. Am. Chem.
Soc. (1997) 119:5081-5090). In addition, the available number of
photo-labile protecting groups is severely limited compared to the
tremendous breadth and diversity of chemically labile protecting
groups that have been developed over the past 30+ years for use on
beads. Recently, SmartBeads Technologies has introduced
microfabricated particles (e.g., strip particles having dimensions
of 100.mu..times.10.mu..times.1.mu.) containing bar codes that can
be decoded using a flow-based reader. Microfabricated particles
have the advantage that a nearly infinite number of encoding
patterns can be easily incorporated into them. The difficulty lies
in being able to easily analyze mixtures of encoded particles.
Since such particles tend to be flat objects as opposed to
spherical beads, they tend to be more prone to aggregation or
overlapping as well as being more difficult to disperse.
[0312] Nicewarner-Pena, et al., Science (2001) 294(5540):137-141
recently reported synthesis of multimetal microrods intrinsically
encoded with submicrometer stripes. According to Nicewarner-Pena,
et al., complex striping patterns are readily prepared by
sequential electrochemical deposition of metal ions into templates
with uniformly sized pores. The differential reflectivity of
adjacent stripes enables identification of the striping patterns by
conventional light microscopy. This readout mechanism does not
interfere with the use of fluorescence for detection of analytes
bound to particles by affinity capture, as demonstrated by DNA and
protein bioassays.
[0313] A system incorporating the advantages of planar arrays and
of encoded micro-particles would address many of the problems
inherent in the existing approaches. Illumina, Inc. has attempted
to do this by providing a method of generating arrays of microbeads
using etched glass fibers (e.g., "High-density fiber-optic DNA
random microsphere array" by Ferguson, et al., Anal. Chem. (2000)
72:5618-5624). However, Illumina's oligonucleotide based
fluorescent-encoding microbeads are also limited in the number of
unique representations. BioArray Solutions has used
Light-controlled Electrokinetic Assembly of Particles near Surfaces
(LEAPS) to form arrays of beads on surfaces (WO97/40385). However,
the LEAPS approach is still subject to the same restrictions as
bead-based techniques with respect to the types of available
encoding.
[0314] There exists needs in the art for microdevices and methods
that can take the advantages of both microfabricated particles and
spatially distinct arrays. This invention address these and other
related needs in the art.
DISCLOSURE OF THE INVENTION
[0315] In one aspect, the present invention is directed to a
microdevice, which microdevice comprises: a) a magnetizable
substance; and b) a photorecognizable coding pattern, wherein said
microdevice has a preferential axis of magnetization. In a specific
embodiment, the present microdevice does not comprise Pt, Pd, Ni,
Co, Ag, Cu or Au for encoding purposes. In another specific
embodiment, the present microdevice does not comprise Pt, Pd, Ni,
Co, Ag, Cu or Au.
[0316] In another aspect, the present invention is directed to a
system for forming a microdevice array, which system comprises: a)
a plurality of the microdevices, each of the microdevices
comprising a magnetizable substance and a photorecognizable coding
pattern, wherein said microdevices have a preferential axis of
magnetization; and b) a microchannel array comprising a plurality
of microchannels, said microchannels are sufficiently wide to
permit rotation of said microdevices within said microchannels but
sufficiently narrow to prevent said microdevices from forming a
chain when the major axis of said microdevices is substantially
perpendicular to the major axis of said microchannels when the said
microdevices are subjected to an applied magnetic field. In a
specific embodiment, the microdevice used in the present system
does not comprise Pt, Pd, Ni, Co, Ag, Cu or Au for encoding
purposes. In another specific embodiment, the microdevice used in
the present system does not comprise Pt, Pd, Ni, Co, Ag, Cu or
Au.
[0317] In still another aspect, the present invention is directed
to a method for forming a microdevice array, which method
comprises: a) providing a plurality of the microdevices, each of
the microdevices comprising a magnetizable substance and a
photorecognizable coding pattern, wherein said microdevices have a
preferential axis of magnetization; b) providing a microchannel
array comprising a plurality of microchannels, said microchannels
are sufficiently wide to permit rotation of said microdevices
within said microchannels but sufficiently narrow to prevent said
microdevices from forming a chain when the major axis of said
microdevices is substantially perpendicular to the major axis of
said microchannels when the said microdevices are subjected to an
applied magnetic field; c) introducing said plurality of
microdevices into said plurality of microchannels; and d) rotating
said microdevices within said microchannels by a magnetic force,
whereby the combined effect of said magnetic force and said
preferential axis of magnetization of said microdevices
substantially separates said microdevices from each other. In a
specific embodiment, the microdevice used in the present method
does not comprise Pt, Pd, Ni, Co, Ag, Cu or Au for encoding
purposes. In another specific embodiment, the microdevice used in
the present system does not comprise Pt, Pd, Ni, Co, Ag, Cu or
Au.
[0318] In yet another aspect, the present invention is directed to
a method for forming a microdevice array, which method comprises:
a) providing a plurality of the microdevices, each of the
microdevices comprising a magnetizable substance and a
photorecognizable coding pattern, wherein said microdevices have a
preferential axis of magnetization, on a surface suitable for
rotation of said microdevices; and b) rotating said microdevices on
said surface by a magnetic force, whereby the combined effect of
said magnetic force and said preferential axis of magnetization of
said microdevices substantially separates said microdevices from
each other. In a specific embodiment, the microdevice used in the
present method does not comprise Pt, Pd, Ni, Co, Ag, Cu or Au for
encoding purposes. In another specific embodiment, the microdevice
used in the present system does not comprise Pt, Pd, Ni, Co, Ag, Cu
or Au.
[0319] In yet another aspect, the present invention is directed to
a method for synthesizing a library, which method comprises: a)
providing a plurality of microdevices, each of said microdevices
comprises a magnetizable substance and a photorecognizable coding
pattern, wherein said microdevices have a preferential axis of
magnetization and wherein said photorecognizable coding pattern
corresponds to an entity to be synthesized on said microdevice; and
b) synthesizing said entities on said microdevices, wherein said
microdevices are sorted after each synthesis cycle according to
said photorecognizable coding patterns, whereby a library is
synthesized, wherein each of said microdevices contains an entity
that corresponds to a photorecognizable coding pattern on said
microdevice and the sum of said microdevices collectively contains
a plurality of entities that is predetermined before the library
synthesis. In a specific embodiment, the microdevice used in the
present method does not comprise Pt, Pd, Ni, Co, Ag, Cu or Au for
encoding purposes. In another specific embodiment, the microdevice
used in the present system does not comprise Pt, Pd, Ni, Co, Ag, Cu
or Au. A library that is synthesized according to the above method
is also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0320] FIG. 18 illustrates an example of a microdevice (MicroDisk)
that is rectangular and consists of four regions. Magnetic bars are
shown in light gray. Dark gray region (e.g., made of the material
Aluminum, Al) is an encoding region. The surrounding white edge
(e.g. made of SiO.sub.2) indicates the regions that encapsulate the
magnetic bars and encoding region. Arrow indicates direction of the
external magnetic field. These different regions are also located
separately along the thickness direction. The magnetic bars and the
encoding region are located in the middle, and are encapsulated by
the top and bottom layers that correspond to the surrounding white
edge. In an exemplary microdevice, the MicroDisk contains magnetic
bars comprising soft magnetic material, e.g., CoTaZr or NiFe and is
90.mu. long by 70.mu. wide by 3.2 thick.
[0321] FIG. 19 illustrates examples of possible arrangements of
multiple MicroDisks constrained to a surface in the presence of a
magnetic field whose direction is indicated by the arrow.
[0322] FIG. 20 illustrates a short chain of MicroDisks constrained
to a surface and further constrained in a channel while in the
presence of a magnetic field whose direction is indicated by the
arrow.
[0323] FIG. 21 illustrates the same short chain of MicroDisks shown
in FIG. 20 after the external magnetic field has been rotated by 90
degrees as indicated by the arrow.
[0324] FIG. 22 shows examples of MicroDisks containing different
types of magnetic bars.
[0325] FIG. 23 shows examples of two types of encoding patterns: 2D
datamatrix on the left and four character optical character
recognition (OCR) on the right.
[0326] FIG. 24 shows an exemplary microchannel device containing a
loading region, guiding posts, microchannels, collection areas and
fluidic connections.
[0327] FIG. 25 shows 4 exemplary types of MicroDisks. Images show
MicroDisks after fabrication but before release from the wafer.
Magnification is .about.400.times.. A--Pair of rectangular magnetic
bars, 2D bar code; B--Pair of rectangular magnetic bars with
tapered ends, 3-character OCR code; C--Pair of rectangular magnetic
bars with "three-fingered" ends; 1D bar code; D--Five rectangular
magnetic bars, 4-character OCR code.
[0328] FIG. 26 shows MicroDisks forming linear chains on a glass
surface in the presence of a magnetic field whose direction is
indicated by the arrow. The 2D bar codes are fully exposed in this
chain. Illumination is from below. Magnification is
.about.400.times..
[0329] FIG. 27 shows MicroDisks forming chains with some branching
on a glass surface in the presence of a magnetic field whose
direction is indicated by the arrow. Illumination is from below.
Magnification is .about.400.times..
[0330] FIG. 28 shows MicroDisks constrained to a 130.mu. wide
channel responding to a magnetic field whose direction is indicated
by the arrow. In the upper panel (A), the MicroDisks form a compact
chain. Ninety (90)-degree rotation of the magnetic field as shown
in the lower panel (B) results in the disks fully separating from
each other. Illumination is from above. Magnification is
.about.160.times..
MODES OF CARRYING OUT THE INVENTION
[0331] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections that follow.
A. Definitions
[0332] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications referred to herein are incorporated by reference in
their entirety. If a definition set forth in this section is
contrary to or otherwise inconsistent with a definition set forth
in the patents, applications, published applications and other
publications that are herein incorporated by reference, the
definition set forth in this section prevails over the definition
that is incorporated herein by reference.
[0333] As used herein, "a" or "an" means "at least one" or "one or
more."
[0334] As used herein, "magnetic substance" refers to any substance
that has the properties of a magnet, pertaining to a magnet or to
magnetism, producing, caused by, or operating by means of,
magnetism.
[0335] As used herein, "magnetizable substance" refers to any
substance that has the property of being interacted with the field
of a magnet, and hence, when suspended or placed freely in a
magnetic field, of inducing magnetization and producing a magnetic
moment. Examples of magnetizable substance include, but are not
limited to, paramagnetic, ferromagnetic and ferrimagnetic
substances.
[0336] As used herein, "paramagnetic substance" refers to the
substances where the individual atoms, ions or molecules possess a
permanent magnetic dipole moment. In the absence of an external
magnetic field, the atomic dipoles point in random directions and
there is no resultant magnetization of the substances as a whole in
any direction. This random orientation is the result of thermal
agitation within the substance. When an external magnetic field is
applied, the atomic dipoles tend to orient themselves parallel to
the field, since this is the state of lower energy than
antiparallel position. This gives a net magnetization parallel to
the field and a positive contribution to the susceptibility.
Further details on "paramagnetic substance" or "paramagnetism" can
be found in various literatures, e.g., at Page 169-page 171,
Chapter 6, in "Electricity and Magnetism" by B. I Bleaney and B.
Bleaney, Oxford, 1975.
[0337] As used herein, "ferromagnetic substance" refers to the
substances that are distinguished by very large (positive) values
of susceptibility, and are dependent on the applied magnetic field
strength. In addition, ferromagnetic substances may possess a
magnetic moment even in the absence of the applied magnetic field,
and the retention of magnetization in zero field is known as
"remanence". Further details on "ferromagnetic substance" or
"ferromagnetism" can be found in various literatures, e.g., at Page
171-page 174, Chapter 6, in "Electricity and Magnetism" by B. I
Bleaney and B. Bleaney, Oxford, 1975.
[0338] As used herein, "ferrimagnetic substance" refers to the
substances that show spontaneous magnetization, remanence, and
other properties similar to ordinary ferromagnetic materials, but
the spontaneous moment does not correspond to the value expected
for full parallel alignment of the (magnetic) dipoles in the
substance. Further details on "ferrimagnetic substance" or
"ferrimagnetism" can be found in various literatures, e.g., at Page
519-524, Chapter 16, in "Electricity and Magnetism" by B. I Bleaney
and B. Bleaney, Oxford, 1975.
[0339] As used herein, "a photorecognizable coding pattern" refers
to any coding pattern that can be detected and/or assessed by
photoanalysis (optical analysis). Any photorecognizable property
can be used as the characteristics of the coding pattern. For
example, the photorecognizable coding pattern can be the material
composition of the microdevice or substrate itself, structural
configuration of the microdevice (e.g., a hole in the microdevice
or the substrate or a substance immobilized on the microdevice or
the substrate), said substance having an optical refractive
property that is different from the optical refractive property of
the microdevice or the substrate. The versatility of the
photorecognizable coding pattern can be based on the shape, number,
position distribution, optical refractive property, material
composition, or a combination thereof, of the microdevice or the
substrate, the hole(s), or other structural configurations, or
certain substance(s) located, deposited or immobilized on the
microdevice or the substrate. To facilitate optical analysis (or
photoanalysis) of encoding patterns, certain microdevices may
incorporate "orientation" marks or alignment markers. The
orientation markers can be used for indicating which major surface
is up and for helping decode the patterns. 1-D and/or 2-D bar
coding patterns can also be used as photorecognizable coding
pattern in the present microdevices.
[0340] As used herein, "a photorecognizable coding pattern on said
substrate" means that the photorecognizable coding pattern is
located on, in, or within (or inside) the substrate so that the
photorecognizable coding pattern is optically detectable. For
example, the photorecognizable coding pattern can be located on the
surface or on top of the substrate. The photorecognizable coding
pattern can also be located within or inside the substrate. In
other embodiments, the substrate may have multiple layers and the
photorecognizable coding pattern can be located on the surface
layer, on top of the surface layer, or can be located within or
inside one or more layers.
[0341] As used herein, "the photorecognizable coding pattern is
fabricated or microfabricated on the substrate" means the use of
any microfabrication or micromachining methods to produce or
generate encoding patterns on the substrate. Various
microfabrication or micromachining protocols such as, pattern
masking, photolithography, wet etching, reactive-ion-etching and
deep-reactive-ion-etching, etc., can be used.
[0342] As used herein, "major axis of the microdevice" refers to
the longest dimension of the microdevice. For the microdevices
having a thin round-disk shape, the height of the microdevice
refers to the thickness of the disk. In this case of thin
round-disk shaped microdevices, the major axis refers to any axis
in the plane parallel to the major surfaces of the disk. In one
preferred embodiment of such round-disk shaped microdevices, the
photorecognizable coding patterns are on the plane parallel to the
major surfaces of the disk surface, located on the disk surface, or
within the disk between the two major surfaces. For the
microdevices having a thin rectangular shape, three dimensions are
defined, the major axis (i.e., length), the minor axis (i.e., the
width) and the height (i.e. the thickness of the rectangular
microdevice). In such cases, the major axis of the microdevice is
longer than the minor axis and height of the microdevice. The minor
axis of the microdevice is longer than or equals to the height of
the microdevice. The microdevices may have any other shapes.
[0343] As used herein, "said microdevice has a preferential axis of
magnetization" means that the induced magnetization of the
microdevice under the influence of an applied magnetic field
depends on the relative angles of the direction of the applied
magnetic field and various axes of the microdevices so that when
the microdevices are introduced into a minimum-friction (or little-
or no-friction) medium and/or placed on a minimum-friction (or
little- or no-friction) surface, the microdevice may rotate or
orient itself under the interaction of the applied magnetic field
and the induced magnetization to achieve a minimum energy state or
stable state. When the microdevices introduced into a minimum
friction (or little or no-friction) medium and/or placed on a
minimum-friction (or little- or no-friction) surface are in such a
minimum energy state, the microdevice's axis that is aligned with
the applied magnetic field is the preferential axis of
magnetization. The preferential axis of magnetization is determined
by the geometry of the microdevice, e.g., the ratio between the
dimensions of the major axis and the minor axis, as well as the
composition and structural configuration of the microdevices.
Depending on the geometry of the microdevice, the preferential axis
of magnetization can be a single axis in a particular direction or
multiple axes in multiple directions, or even any axis direction
lying within a plane. Once the dynamic process of inducing
magnetization is over and the microdevice has achieved the minimum
energy state in a magnetic field, the induced magnetization along
the preferential axis of magnetization (in its absolute magnitude)
is larger than or at least equal to induced magnetization along any
other axis of the microdevice. In general, for the microdevices of
the present invention to rotate or orient itself under the
interaction of the applied magnetic field and the induced
magnetization, the induced magnetization (in its absolute
magnitude) along the preferential axis of magnetization of the
microdevice should be at least 20% more than the induced
magnetization of the microdevice along at least one other axis.
Preferably, the induced magnetization (in its absolute magnitude)
along the preferential axis of magnetization of the microdevices of
the present invention should be at least 50%, 70%, or 90% more than
the induced magnetization of the microdevice along at least one
other axis. Even more preferably, the induced magnetization (in its
absolute magnitude) along the preferential axis of the
magnetization of the microdevices of the present invention should
be at least one time, twice, five times, ten times, twenty times,
fifty times or even hundred times more than the induced
magnetization of the microdevice along at least one other axis. The
rotation and orientation of the microdevice under the influence of
the applied magnetic field is a dynamic process and may take some
time to achieve the minimum energy state or stable state. In an
environment where friction or other force, e.g., gravity, exists,
the preferential axis of the magnetization of microdevice may not
align with the applied magnetic field perfectly even when a
steady-state is achieved. Preferably, numerous factors such as the
geometry of the microdevice, the direction and strength of the
applied magnetic field and other factors (e.g., for a microdevice
lying on a support surface, the frictional force that may relate to
the property of the support surface may be a factor) can be
adjusted to ensure that the preferential axis of magnetization of
microdevice is substantially aligned with the applied magnetic
field when a steady-state is achieved. For example, for a
microdevice having a thin round-disk shape with magnetizable
substance inside having a thin disc shape, the preferential axis of
magnetization of such microdevice may lie in the plane parallel to
the major surfaces of the microdevice (and also parallel to the
major surface of the thin disk magnetic substance). When such a
microdevice is subject to an applied magnetic field, even if
initially the thin disk microdevice lies in the plane normal to the
applied magnetic field, the microdevice will re-align itself so
that the thin disk plane will be parallel or close-to-parallel to
the direction of the magnetic field. In another example, the
microdevice has a thin rectangular shape inside which the
magnetizable substance forms a magnetic structure such as a
magnetic rectangular bar whose length, width and thickness are in
the same directions as those of the microdevice itself. The
preferential axis of magnetization of such microdevice may be in
the same direction as the length-direction of the microdevice and
the length direction of the magnetic bar inside the
microdevice.
[0344] As used herein, "the preferential axis of magnetization of
the microdevice is substantially aligned with an applied magnetic
field" means that the angle between the preferential axis of the
magnetization and the applied magnetic field should be 45 degrees
or less. Preferably, the angle between the preferential axis of
magnetization and the applied magnetic field should be 15 degrees
or less. More preferably, the preferential axis of magnetization is
completely aligned with the applied magnetic field. For
microdevices whose preferential axis of magnetization is the major
axis, then "the preferential axis of magnetization of the
microdevice is substantially aligned with an applied magnetic
field" means that the angle between the major axis of the
microdevice and the applied magnetic field should be 45 degrees or
less. For example, for microdevices having thin rectangular shape
and having the major axis as the preferential axis of
magnetization, an applied magnetic field may result in the
formation of a chain of the microdevices along their major axises.
When the applied magnetic field rotates for more than 45 degree
(e.g. 90 degree), the microdevices would also rotate for the same
or similar degrees so that each microdevice in the chain is
substantially separated from each other.
[0345] As used herein, "the preferential axis of magnetization of
the microdevice is substantially aligned with microdevice's major
axis" means that the angle between the preferential axis of the
magnetization and the major axis should be 45 degrees or less.
Preferably, the angle between the preferential axis of
magnetization and the major axis should be 15 degrees or less. More
preferably, the preferential axis of magnetization is completely
aligned with the major axis.
[0346] As used herein, "each microdevice in the chain is
substantially separated from each other" means that the
microdevices are sufficiently separated so that each of the
microdevices can be identified and/or analyzed by its respective
photorecognizable coding pattern. The degree of the separation
among individual microdevices is determined by a number of factors
such as the type, number and/or distribution of the
photorecognizable coding pattern(s), the geometry of the
microdevices, the methods for assessing the photorecognizable
coding pattern(s) and the purpose of the identification and/or
analysis of the microdevices. Certain touch or overlap among
individual microdevices are permissible so long as each of the
microdevices can be identified and/or analyzed by its respective
photorecognizable coding pattern for the intended purpose. In
certain situations, it is preferably that the microdevices are
completely separated from each other without any touching or
overlap.
[0347] As used herein, "said microchannels are sufficiently wide to
permit rotation of said microdevices within said microchannels but
sufficiently narrow to prevent said microdevices from forming a
chain when the major axis of said microdevices is substantially
perpendicular to the major axis of said microchannels" means that
the width of a microchannel equals to or is larger than the longest
dimension of microdevices, e.g., diagonal dimension of a rectangle,
within the microchannel to permit rotation of the microdevices
within the microchannel. At the same time, the width of a
microchannel equals to or is less than 150% of the longest
dimension of microdevices, e.g., diagonal dimension of a rectangle,
within the microchannel to prevent microdevices from forming a
chain (of at least two microdevices) when the major axis of said
microdevices is substantially perpendicular to the major axis of
said microchannels. Preferably, the width of a microchannel equals
to or is less than 150%, 140%, 130%, 120%, 110%, or 105% 102% of
the longest dimension of microdevices. "Sufficiently narrow to
prevent said microdevices from forming a chain" also means that
after the rotation, each microdevice in the chain is substantially
separated from each other as defined above. It is not necessary,
although permissible, that each of the microchannels within a
microchannel array has same width. It is sufficient that each of
the microchannels has a width that is compatible to the
microdevices to be rotated within the microchannel. Here, the major
axis of the microchannel refers to the length direction of the
microchannel.
[0348] As used herein, "the major axis of said microdevices is
substantially perpendicular to the major axis of said
microchannels" means that the angle between the major axis of
microdevices and the major axis of the microchannel that contains
the microdevices equals to or is larger than 45 degrees.
Preferably, the angle between the major axis of microdevices and
the major axis of the microchannels that contains the microdevices
equals to or is larger than 50, 55, 60, 65, 70, 75, 80, 85 and 90
degrees. Here, the major axis of the microchannels refers to the
length direction of the microchannels.
[0349] As used herein, "the height of the microchannels and/or the
constraint on the microdevices by a magnetic field does not allow
the microdevices to stand up within the microchannels" means that
the height of the microchannels alone, the constraint on the
microdevices by a magnetic field alone, or both, may be sufficient
to prevent microdevices from taking a position so that the major
axis of the microdevices is substantially aligned with the height
of the microchannel. In these cases, the dimension of microchannel
is defined by its length, width and height. Its length corresponds
to the major axis of the microchannel. The microchannel height
corresponds to the microchannel axis that is normal to the surface
on which the microchannel is positioned. The microchannel width
refers to the third dimension. The "major axis of the microdevices
is substantially aligned with the height of the microchannel" means
that the angle between the major axis of the microdevices and the
height of the microchannel equals to is less than 45 degrees. When
the constraint on the microdevices by a magnetic field alone is
sufficient to prevent microdevices from taking such a prohibitive
position, the height of the microchannels becomes irrelevant in
this consideration.
[0350] As used herein, "said photorecognizable coding pattern
corresponds to an entity to be synthesized on said microdevice"
means that the entity to be synthesized on a particular microdevice
is predetermined according to the photorecognizable coding pattern
on that microdevice. The coding pattern can determine the entity to
be synthesized on a microdevice in different ways. For example, a
coding pattern can have multiple digits and each digit determines a
particular synthesis reaction and the collection of all digits
collectively determines all synthesis reactions, and hence the
identity of the entity to be synthesized. Alternatively, a coding
pattern can be an "intact" pattern, i.e., the entire pattern, not a
portion or a digit of the pattern, determines the entire synthesis
reactions on the microdevice, and hence the identity of the entity
to be synthesized.
[0351] As used herein, "said microdevices are sorted after each
synthesis cycle according to said photorecognizable coding
patterns" means that the synthetic steps or orders for making an
entity on a particular microdevice are predetermined according to
the photorecognizable coding pattern on that microdevice and after
each synthesis cycle, the photorecognizable coding pattern on the
microdevice is assessed for directing the next synthetic step or
order.
[0352] As used herein, "electrically conductive or dielectrically
polarizable substance" refers to any substance that can be
subjected to dielectrophoresis force under appropriate conditions.
Depending on the dielectric and electric properties of the
substance, the substance may be subject to positive or negative
dielctrophpresis forces under certain conditions. Such conditions
include, but are not limited to, the frequency of the applied
electric field, and the electrical and dielectric property of the
medium in which the substance is placed or introduced.
[0353] As used herein, "optical labeling substance" refers to any
optically detectable substance that can be used to label the
microdevices of the present invention to facilitate and/or enable
detection and/or identification of the microdevices. Quantum-dot is
an example of an optical labeling substance.
[0354] As used herein, "scattered-light detectable particle" refers
to any particle that can emit unique and identifiable
scattered-light upon illumination with light under appropriate
conditions. The nano-sized particles with certain "resonance light
scattering (RLS)" properties are examples of one type of
"scattered-light detectable particle".
[0355] As used herein, "quantum dot" refers to a fluorescent label
comprising water-soluble semiconductor nanocrystal(s). One unique
feature of a quantum dot is that its fluorescent spectrum is
related or determined by the diameter of its nanocrystals(s).
"Water-soluble" is used herein to mean sufficiently soluble or
suspendable in a aqueous-based solution, such as in water or
water-based solutions or physiological solutions, including those
used in the various fluorescence detection systems as known by
those skilled in the art. Generally, quantum dots can be prepared
which result in relative monodispersity; e.g., the diameter of the
core varying approximately less than 10% between quantum dots in
the preparation.
[0356] As used herein, "chip" refers to a solid substrate with a
plurality of one-, two- or three-dimensional micro structures or
micro-scale structures on which certain processes, such as
physical, chemical, biological, biophysical or biochemical
processes, etc., can be carried out. The micro structures or
micro-scale structures such as, channels and wells, electrode
elements, electromagnetic elements, are incorporated into,
fabricated on or otherwise attached to the substrate for
facilitating physical, biophysical, biological, biochemical,
chemical reactions or processes on the chip. The chip may be thin
in one dimension and may have various shapes in other dimensions,
for example, a rectangle, a circle, an ellipse, or other irregular
shapes. The size of the major surface of chips used in the present
invention can vary considerably, e.g., from about 1 mm.sup.2 to
about 0.25 m.sup.2. Preferably, the size of the chips is from about
4 mm.sup.2 to about 25 cm.sup.2 with a characteristic dimension
from about 1 mm to about 7.5 cm. The chip surfaces may be flat, or
not flat. The chips with non-flat surfaces may include channels or
wells fabricated on the surfaces.
[0357] As used herein, "a means for generating a physical force on
said chip" refers to any substance, structure or a combination
thereof that is capable of generating, in conjunction with an
built-in structure on a chip, to generate a desirable physical
force on the chip.
[0358] As used herein, "physical field," e.g., used itself or used
as "physical field in a region of space" or "physical field is
generated in a region of space" means that the region of space has
following characteristics. When a moiety, alone or bound to a
microdevice, of appropriate properties is introduced into the
region of space (i.e. into the physical field), forces are produced
on the moiety, the microdevice or both, as a result of the
interaction between the moiety and/or microdevice and the field. A
moiety can be manipulated within a field via the physical forces
exerted on the moiety by the field. Exemplary fields include
electric, magnetic, acoustic, optical and velocity fields. In the
present invention, physical field always exists in a medium in a
region of space, and the moiety to be manipulated is suspended in,
or is dissolved in, or more generally, is placed in the medium.
Typically, the medium is a fluid such as aqueous or non-aqueous
liquids, or a gas. Depending on the field configuration, an
electric field may produce electrophoretic forces on charged
moieties, or may produce conventional dielectrophoretic forces
and/or traveling wave dielectrophoretic forces on charged and/or
neutral moieties. Magnetic field may produce magnetic forces on
magnetic moieties. Acoustic field may produce acoustic radiation
forces on moieties. Optical field may produce optical radiation
forces on moieties. Velocity field in the medium in a region of
space refers to a velocity distribution of the medium that moves in
the region of the space. Various mechanisms may be responsible for
causing the medium to move and the medium at different positions
may exhibit different velocities, thus generating a velocity field.
Velocity field may exert mechanical forces on moieties in the
medium.
[0359] As used herein, "medium (or media)" refers to a fluidic
carrier, e.g., liquid or gas, wherein a moiety, alone or bound to a
microdevice, is dissolved, suspended or contained.
[0360] As used herein, "microfluidic application" refers to the use
of microscale devices, e.g., the characteristic dimension of basic
structural elements is in the range between less than 1 micron to 1
cm scale, for manipulation and process in a fluid-based setting,
typically for performing specific biological, biochemical or
chemical reactions and procedures. The specific areas include, but
are not limited to, biochips, i.e., chips for biologically related
reactions and processes, chemchips, i.e., chips for chemical
reactions, or a combination thereof. The characteristic dimensions
of the basic elements refer to the single dimension sizes. For
example, for the microscale devices having circular shape
structures (e.g. round electrode pads), the characteristic
dimension refers to the diameter of the round electrodes. For the
devices having thin, rectangular lines as basic structures, the
characteristic dimensions may refer to the width or length of these
lines.
[0361] As used herein, "built-in structures on said substrate of a
chip" means that the structures are built into the substrate or the
structures are located on the substrate or the structures are
structurally linked to the substrate of the chip. In one
embodiment, the built-in structures may be fabricated on the
substrate. For example, as described in "Dielectrophoretic
manipulation of cells using spiral electrodes by Wang, et al.,
Biophys. J. (1997) 72:1887-1899", spiral electrodes are fabricated
on a glass substrate. Here the spiral electrodes are "built-in"
structures on the glass substrate. In another embodiment, the
"built-in" structures may be first fabricated on one substrate and
the structure-containing first substrate may then be attached or
bound to a second substrate. Such structures are "built-in"
structures not only on the first substrate but also on the second
substrate. In still another embodiment, the built-in structures may
be attached or bound to the substrate. For example, thin,
electrically-conductive wires may be used as electrodes for
producing electric field. These electric wires may be bound or
attached to a glass substrate. In this case, the
electrically-conductive wires are "built-in" structures on the
glass substrate. Throughout this application, when it is described
that "built-in" structures on the chip or on the substrate are
capable of generating physical forces and/or physical fields or
these structures generate physical forces and/or physical fields,
these structures are used in combination with external signal
sources or external energy sources.
[0362] As used herein, "micro-scale structures" means that the
scale of the internal structures of the apparatus for exerting
desired physical forces must be compatible with and useable in
microfluidic applications and have characteristic dimension of
basic structural elements in the range from about 1 micron to about
20 mm scale.
[0363] As used herein, "moiety" refers to any substance whose
isolation, manipulation, measurement, quantification, detection or
synthesis using the present microdevice is desirable. Normally, the
dimension (or the characteristic dimensions) of the moiety should
not exceed 1 cm. For example, if the moiety is spherical or
approximately spherical, the dimension of the moiety refers to the
diameter of the sphere or an approximated sphere for the moiety. If
the moiety is cubical or approximately cubical, then the dimension
of the moiety refers to the side width of the cube or an
approximated cube for the moiety. If the moiety has an irregular
shape, the dimension of the moiety may refer to the average between
its largest axis and smallest axis. Non-limiting examples of
moieties include cells, cellular organelles, viruses, particles,
molecules, e.g., proteins, DNAs and RNAs, or an aggregate or
complex thereof.
[0364] Moiety to be isolated, manipulated, measured, quantified,
detected or synthesized includes many types of particles--solid
(e.g., glass beads, latex particles, magnetic beads), liquid (e.g.,
liquid droplets) or gaseous particles (e.g., gas bubble), dissolved
particles (e.g., molecules, proteins, antibodies, antigens, lipids,
DNAs, RNAs, molecule-complexes), suspended particles (e.g., glass
beads, latex particles, polystyrene beads). Particles can be
organic (e.g., mammalian cells or other cells, bacteria, virus, or
other microorganisms) or inorganic (e.g., metal particles).
Particles can be of different shapes (e.g., sphere, elliptical
sphere, cubic, discoid, needle-type) and can be of different sizes
(e.g., nano-meter-size gold sphere, to micrometer-size cells, to
millimeter-size particle-aggregate). Examples of particles include,
but are not limited to, biomolecules such as DNA, RNA, chromosomes,
protein molecules (e.g., antibodies), cells, colloid particles
(e.g., polystyrene beads, magnetic beads), any biomolecules (e.g.,
enzyme, antigen, hormone etc).
[0365] As used herein, "plant" refers to any of various
photosynthetic, eucaryotic multi-cellular organisms of the kingdom
Plantae, characteristically producing embryos, containing
chloroplasts, having cellulose cell walls and lacking
locomotion.
[0366] As used herein, "animal" refers to a multi-cellular organism
of the kingdom of Animalia, characterized by a capacity for
locomotion, nonphotosynthetic metabolism, pronounced response to
stimuli, restricted growth and fixed bodily structure. Non-limiting
examples of animals include birds such as chickens, vertebrates
such fish and mammals such as mice, rats, rabbits, cats, dogs,
pigs, cows, ox, sheep, goats, horses, monkeys and other non-human
primates.
[0367] As used herein, "bacteria" refers to small prokaryotic
organisms (linear dimensions of around 1 micron) with
non-compartmentalized circular DNA and ribosomes of about 70S.
Bacteria protein synthesis differs from that of eukaryotes. Many
anti-bacterial antibiotics interfere with bacteria proteins
synthesis but do not affect the infected host.
[0368] As used herein, "eubacteria" refers to a major subdivision
of the bacteria except the archaebacteria. Most Gram-positive
bacteria, cyanobacteria, mycoplasmas, enterobacteria, pseudomonas
and chloroplasts are eubacteria. The cytoplasmic membrane of
eubacteria contains ester-linked lipids; there is peptidoglycan in
the cell wall (if present); and no introns have been discovered in
eubacteria.
[0369] As used herein, "archaebacteria" refers to a major
subdivision of the bacteria except the eubacteria. There are three
main orders of archaebacteria: extreme halophiles, methanogens and
sulphur-dependent extreme thermophiles. Archaebacteria differs from
eubacteria in ribosomal structure, the possession (in some case) of
introns, and other features including membrane composition.
[0370] As used herein, "virus" refers to an obligate intracellular
parasite of living but non-cellular nature, consisting of DNA or
RNA and a protein coat. Viruses range in diameter from about 20 to
about 300 nm Class I viruses (Baltimore classification) have a
double-stranded DNA as their genome; Class II viruses have a
single-stranded DNA as their genome; Class III viruses have a
double-stranded RNA as their genome; Class IV viruses have a
positive single-stranded RNA as their genome, the genome itself
acting as mRNA; Class V viruses have a negative single-stranded RNA
as their genome used as a template for mRNA synthesis; and Class VI
viruses have a positive single-stranded RNA genome but with a DNA
intermediate not only in replication but also in mRNA synthesis.
The majority of viruses are recognized by the diseases they cause
in plants, animals and prokaryotes. Viruses of prokaryotes are
known as bacteriophages.
[0371] As used herein, "fungus" refers to a division of eucaryotic
organisms that grow in irregular masses, without roots, stems, or
leaves, and are devoid of chlorophyll or other pigments capable of
photosynthesis. Each organism (thallus) is unicellular to
filamentous, and possesses branched somatic structures (hyphae)
surrounded by cell walls containing glucan or chitin or both, and
containing true nuclei.
[0372] As used herein, "binding partners" refers to any substances
that bind to the moieties with desired affinity or specificity.
Non-limiting examples of the binding partners include cells,
cellular organelles, viruses, particles, microparticles or an
aggregate or complex thereof, or an aggregate or complex of
molecules, or specific molecules such as antibodies, single
stranded DNAs. The binding partner can be a substance that is
coated on the surface of the present microdevice. Alternatively,
the binding partner can be a substance that is incorporated, e.g.,
microfabricated, into the material composition of the present
microdevice. The material composition of the present microdevice,
in addition being a substrate, may possess binding affinity to
certain moiety, and thus functioning as a binding partner
itself.
[0373] As used herein, "an element that facilitates and/or enables
manipulation of the microdevice and/or a moiety/microdevice
complex" refers to any substance that is itself manipulatable or
makes the moiety/microdevice complex manipulatable with the desired
physical force(s). Non-limiting examples of the elements include
cells, cellular organelles, viruses, particles, microparticles or
an aggregate or complex thereof, or an aggregate or complex of
molecules. Non-limiting examples of the elements may further
include deposited or other-procedure-produced materials with
specific physical or chemical properties. Metal films made of Au,
Cr, Ti, Pt etc are examples of the elements that can be
incorporated into the microdevices and increase electrical
conductivity of the microdevices. Insulating materials such as
polystyrene, paralene, or other plastic polymers are also examples
of the elements that may be incorporated into the microdevices and
reduce electrical conductivity of the microdevices.
[0374] As used herein, "microparticles" refers to particles of any
shape, any composition, any complex structures that are
manipulatable by desired physical force(s) in microfluidic settings
or applications. One example of microparticles is magnetic beads
that are manipulatable by magnetic forces. Another example of a
microparticle is a cell that is manipulatable by an electric force
such as a traveling-wave dielectrophoretic force. The
microparticles used in the methods can have a dimension from about
0.01 micron to about ten centimeters. Preferably, the
microparticles used in the methods have a dimension from about 0.01
micron to about several thousand microns. Examples of the
microparticles include, but are not limited to, plastic particles,
polystyrene microbeads, glass beads, magnetic beads, hollow glass
spheres, particles of complex compositions, microfabricated
free-standing microstructures, etc. Other particles include cells,
cell organelles, large biomolecules such as DNA, RNA and proteins
etc.
[0375] As used herein, "manipulation" refers to moving or
processing of the moieties, and the microdevices disclosed in the
present invention, which results in one-, two- or three-dimensional
movement of the moiety (and the microdevices). The manipulation can
be conducted off a chip or in a chip format, whether within a
single chip or between or among multiple chips, or on a substrate
or among substrates of an apparatus. "Manipulation" of moieties and
the microdevices can also be performed in liquid containers.
Non-limiting examples of the manipulations include transportation,
focusing, enrichment, concentration, aggregation, trapping,
repulsion, levitation, separation, sorting, fractionation,
isolation or linear or other directed motion of the moieties. For
effective manipulation, the characteristics of the moiety (and the
microdevices) to be manipulated and the physical force used for
manipulation must be compatible. For example, the microdevices with
certain magnetic properties can be used with magnetic force.
Similarly, the microdevices with electric charge(s) can be used
with electrostatic (i.e. electrophoretic) force. In the case of
manipulating microdevices-binding partner-moiety complexes, the
characteristics of the moiety, or its binding partner or the
microdevices, and the physical force used for manipulation must be
compatible. For example, moiety or its binding partner or the
microdevices with certain dielectric properties to induce
dielectric polarization in the moiety or its binding partner or the
microdevices can be used with dielectrophoresis force.
[0376] As used herein, "the moiety is not directly manipulatable"
by a particular physical force means that no observable movement of
the moiety can be detected when the moiety itself not coupled to a
binding partner is acted upon by the particular physical force.
[0377] As used herein, "physical force" refers to any force that
moves the moieties or their binding partners or the corresponding
microdevices without chemically or biologically reacting with the
moieties and the binding partners, or with minimal chemical or
biological reactions with the binding partners and the moieties so
that the biological/chemical functions/properties of the binding
partners and the moieties are not substantially altered as a result
of such reactions. Throughout the application, the term of "forces"
or "physical forces" always means the "forces" or "physical forces"
exerted on a moiety or moieties, the binding partner(s) and/or the
microdevice(s). The "forces" or "physical forces" are always
generated through "fields" or "physical fields". The forces exerted
on moieties, the binding partner(s) and/or the microdevice(s) by
the fields depend on the properties of the moieties, the binding
partner(s) and/or the microdevice(s). Thus, for a given field or
physical field to exert physical forces on a moiety, it is
necessary for the moiety to have certain properties. While certain
types of fields may be able to exert forces on different types of
moieties having different properties, other types of fields may be
able to exert forces on only limited type of moieties. For example,
magnetic field can exert forces or magnetic forces only on magnetic
particles or moieties having certain magnetic properties, but not
on other particles, e.g., polystyrene microdevices. On the other
hand, a non-uniform electric field can exert physical forces on
many types of moieties such as polystyrene microdevices, cells, and
also magnetic particles. It is not necessary for the physical field
to be able to exert forces on different types of moieties or
different moieties. But it is necessary for the physical field to
be able to exert forces on at least one type of moiety or at least
one moiety, the binding partner(s) and/or the microdevice(s).
[0378] As used here in, "electric forces (or electrical forces)"
are the forces exerted on moieties, the binding partner(s) and/or
the microdevice(s) by an electric (or electrical) field.
[0379] As used herein, "magnetic forces" are the forces exerted on
moieties, the binding partner(s) and/or the microdevice(s) by a
magnetic field.
[0380] As used herein, "acoustic forces (or acoustic radiation
forces)" are the forces exerted on moieties, the binding partner(s)
and/or the microdevice(s) by an acoustic field.
[0381] As used herein, "optical (or optical radiation) forces" are
the forces exerted on moieties, the binding partner(s) and/or the
microdevice(s) by an optical field.
[0382] As used herein, "mechanical forces" are the forces exerted
on moieties, the binding partner(s) and/or the microdevice(s) by a
velocity field.
[0383] As used herein, "sample" refers to anything which may
contain a moiety to be isolated, manipulated, measured, quantified
or detected by the present microdevices and/or methods. The sample
may be a biological sample, such as a biological fluid or a
biological tissue. Examples of biological fluids include urine,
blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal
fluid, tears, mucus, amniotic fluid or the like. Biological tissues
are aggregates of cells, usually of a particular kind together with
their intercellular substance that form one of the structural
materials of a human, animal, plant, bacterial, fungal or viral
structure, including connective, epithelium, muscle and nerve
tissues. Examples of biological tissues also include organs,
tumors, lymph nodes, arteries and individual cell(s). The sample
may also be a mixture of target analyte or enzyme containing
molecules prepared in vitro.
[0384] As used herein, a "liquid (fluid) sample" refers to a sample
that naturally exists as a liquid or fluid, e.g., a biological
fluid. A "liquid sample" also refers to a sample that naturally
exists in a non-liquid status, e.g., solid or gas, but is prepared
as a liquid, fluid, solution or suspension containing the solid or
gas sample material. For example, a liquid sample can encompass a
liquid, fluid, solution or suspension containing a biological
tissue.
[0385] As used herein the term "assessing (or assessed)" is
intended to include quantitative and qualitative determination of
the identity and/or quantity of a moiety, e.g., a protein or
nucleic acid, present in the sample or on the microdevices or in
whatever form or state. Assessment would involve obtaining an
index, ratio, percentage, visual or other value indicative of the
identity of a moiety in the sample and may further involve
obtaining a number, an index, or other value indicative of the
amount or quantity or the concentration of a moiety present in the
sample or on the microdevice or in whatever form or state.
Assessment may be direct or indirect.
B. Microdevices and Systems for Forming a Microdevice Array
[0386] In one aspect, the present invention is directed to a
microdevice, which microdevice comprises: a) a magnetizable
substance; and b) a photorecognizable coding pattern, wherein said
microdevice has a preferential axis of magnetization.
[0387] Any suitable magnetizable substance can be used in the
present microdevices. In one example, the magnetizable substance
used in the microdevice is a paramagnetic substance, a
ferromagnetic substance, a ferrimagnetic substance, or a
superparamagnetic substance. In another example, the magnetizable
substance used in the microdevice comprises a metal composition.
Preferably, the metal composition is a transition metal composition
or an alloy thereof such as iron, nickel, copper, cobalt,
manganese, tantalum, zirconium and cobalt-tantalum-zirconium
(CoTaZr) alloy. In a preferred example, the magnetic substance is a
metal oxide Fe.sub.3O.sub.4.
[0388] The present microdevice can further comprise a
non-magnetizable substrate. Any suitable material including
silicon, plastic, glass, ceramic, rubber, polymer, silicon dioxide,
silicon nitride, aluminum oxide, titanium, aluminum, gold and a
combination thereof can be used in the substrate. The magnetizable
substance can be linked to the substrate in any form. For example,
the magnetizable substance can be made part of the substrate or can
be attached or deposited or located on the substrate. In another
example, the magnetizable substance can be located within the
substrate.
[0389] The substrate can be a single layer or can comprise multiple
layers such as 3, 4 or more layers. For example, a substrate can
have 3 layers. The top and the bottom layers can be made of the
same material, e.g., SiO.sub.2 (or glass) and the middle layer can
contain magnetizable material(s). Alternatively, the top and the
bottom layers can have different materials.
[0390] The substrate can comprises a surface that is hydrophobic or
hydrophilic. The substrate can be in any suitable shape such as
rectangle and other regular or irregular shape provided that the
microdevice be made to have a preferential axis of magnetization.
The substrate can be in any suitable dimension(s). For example, the
thickness of the substrate can be from about 0.1 micron to about
500 microns. Preferably, the thickness of the substrate can be from
about 1 micron to about 200 microns. More preferably, the thickness
of the substrate can be from about 1 micron to about 50 microns. In
a specific embodiment, the substrate is a rectangle having a
surface area from about 10 squared-microns to about 1,000,000
squared-microns (e.g., 1000 micron by 1000 micron). In another
specific embodiment, the substrate is in an irregular shape having
a single-dimension from about 1 micron to about 500 microns. In a
preferred embodiment, the substrate is a composite comprising
silicon, metal film and polymer film. In another preferred
embodiment, the substrate can comprise a silicon layer and a metal
layer, e.g., an aluminum layer. More preferably, the metal layer
can comprise a magnetic material, such as nickel metal or CoTaZr
(Cobalt-Tantalum-Zirconium) alloy.
[0391] The photorecognizable coding pattern can be based on any
suitable photorecognizable (optical) property constructed in or on
the microdevice or substrate. For example, the photorecognizable
coding pattern can be the material composition of the microdevice
itself, a hole in the microdevice, or other structural
configurations, or certain substance(s) located, deposited or
immobilized on the microdevice or the substrate, or an optical
labeling substance or an 1-D and/or a 2-D bar coding pattern. The
microdevice or substrate can be patterned. In addition, the surface
layer of the substrate or microdevice can be modified. The
versatility of the photorecognizable coding pattern can be caused
by the shape, number, letters, words, position distribution,
optical refractive property, material composition, or a combination
thereof, of the substrate, the hole(s) or other structure
configurations, or certain substance(s) located, deposited or
immobilized on the microdevice or the substrate. In one exemplary
microdevice, the microdevice or substrate can have 4 layers. The
top and the bottom layers can be made of the same material, e.g.,
SiO.sub.2 (or glass). One of the middle layers can contain
paramagnetic material(s), e.g., magnetic alloys. The other middle
layer can contain a photorecognizable coding pattern as a encoding
layer. Preferably, the paramagnetic layer and the encoding layer do
not substantially overlap, or do not overlap at all, to ensure
optical detection of the photorecognizable coding pattern in the
encoding layer. Alternatively, the top and the bottom layers can
have different materials. Exemplary patterns include numbers,
letters, structures, 1-D and 2-D barcodes.
[0392] Although the microdevice can comprise a single
photorecognizable coding pattern, it can also comprise a plurality
of photorecognizable coding patterns, e.g., a plurality of holes or
other structure configurations, a plurality of numbers, a plurality
of letter, and/or a plurality of the substances.
[0393] To facilitate optical analysis (or photo-analysis) of
encoding patterns, certain microdevices may incorporate
"orientation" marks or alignment markers. For example, for the
microdevices having thin symmetrical shapes, the microdevices lying
flat on either of its major surfaces will look identical, causing
difficulties in identification. Therefore, the orientation marks
can be used for indicating which major surface is being looked at
when the microdevices are lying up and for helping decode the
patterns.
[0394] The photorecognizable coding pattern can be constructed
according to any methods known in the art. For example, the
photorecognizable coding pattern can be fabricated or
microfabricated on a substrate. Any suitable fabrication or
microfabrication method can be used including lithography such as
photolithography, electron beam lithography and X-ray lithography
(WO 96/39937 and U.S. Pat. Nos. 5,651,900, 5,893,974 and
5,660,680). For example, the fabrication or microfabrication
methods can be used directly on a substrate to produce desirable
patterns such as numbers, letters, structures, 1-D and 2-D
barcodes.
[0395] If a substance having an optical refractive property that is
different from the optical refractive property of the substrate is
used as the photorecognizable coding pattern, the substance can be
deposited or immobilized on the substrate by any suitable methods
known in the art. For example, the substance used for
photorecognizable encoding can be deposited or immobilized on the
substrate by evaporation or sputtering methods. The substance can
be deposited or immobilized on the substrate directly or via a
linker. The linker can be any material or molecules that linking
the substance to the substrate. The fabrication or microfabrication
methods can be used on the substances deposited on the substrate to
produce desirable patterns such as numbers, letters, structures,
1-D and 2-D barcodes. The substance can be immobilized or deposited
on the substrate via a covalent or a non-covalent linkage. The
substance can be deposited or immobilized on the substrate via
specific or non-specific binding.
[0396] Any suitable optical labeling substance can be used in the
present microdevices. In a specific embodiment, the optical
labeling substance used in the present microdevices is a metal film
such as Cu, Al, Au, Pt that can be patterned to form
photorecognizable encoding patters such as letters, numbers,
structures or structural configurations, 1-D or 2-D barcodes. In
another specific embodiment, the optical labeling substance used in
the present microdevices is a fluorescent substance, a
scattered-light detectable particle (See e.g., U.S. Pat. No.
6,214,560) and a quantum dot (See e.g., U.S. Pat. No.
6,252,664).
[0397] Any suitable quantum dot can be used in the present
microdevices. In a specific embodiment, the quantum dot used in the
present microdevices comprises a Cd--X core, X being Se, S or Te.
Preferably, the quantum dot can be passivated with an inorganic
coating shell, e.g., a coating shell comprising Y--Z, Y being Cd or
Zn, and Z being S or Se. Also preferably, the quantum dot can
comprise a Cd--X core, X being Se, S or Te, a Y--Z shell, Y being
Cd or Zn, and Z being S or Se, and the quantum dot can further be
overcoated with a trialkylphosphine oxide.
[0398] Any suitable methods can be used to make the CdX core/YZ
shell quantum dots water-soluble (See e.g., U.S. Pat. No.
6,252,664). One method to make the CdX core/YZ shell quantum dots
water-soluble is to exchange the overcoating layer with a coating
which will make the quantum dots water-soluble. For example, a
mercaptocarboxylic acid may be used to exchange with the
trialkylphosphine oxide coat. Exchange of the coating group is
accomplished by treating the water-insoluble quantum dots with a
large excess of neat mercaptocarboxylic acid. Alternatively,
exchange of the coating group is accomplished by treating the
water-insoluble quantum dots with a large excess of
mercaptocarboxylic acid in CHCl.sub.3 solution (Chan and Nie, 1998,
Science 281:2016-2018). The thiol group of the new coating molecule
forms Cd (or Zn)--S bonds, creating a coating which is not easily
displaced in solution. Another method to make the CdX core/YZ shell
quantum dots water-soluble is by the formation of a coating of
silica around the dots (Bruchez, Jr., et al., Science (1998)
281:2013-2015). An extensively polymerized polysilane shell imparts
water solubility to nanocrystalline materials, as well as allowing
further chemical modifications of the silica surface. Generally,
these "water-soluble" quantum dots require further
functionalization to make them sufficiently stable in an aqueous
solution for practical use in a fluorescence detection system (See
e.g., U.S. Pat. No. 6,114,038), particularly when exposed to air
(oxygen) and/or light. Water-soluble functionalized nanocrystals
are extremely sensitive in terms of detection, because of their
fluorescent properties (e.g., including, but not limited to, high
quantum efficiency, resistance to photobleaching, and stability in
complex aqueous environments); and comprise a class of
semiconductor nanocrystals that may be excited with a single peak
wavelength of light resulting in detectable fluorescence emissions
of high quantum yield and with discrete fluorescence peaks (e.g.,
having a narrow spectral band ranging between about 10 nm to about
60 nm).
[0399] The quantum dot used in the present microdevice can have any
suitable size. For example, the quantum dot can have a size ranging
from about 1 nm to about 100 nm.
[0400] The microdevice of the present invention can comprise a
single quantum dot. Alternatively, the microdevice of the present
invention can comprise a plurality of quantum dots. Preferably, the
microdevice of the present invention comprises at least two quantum
dots that have different sizes.
[0401] The microdevice of the present invention can comprise a
single optical labeling substance. Alternatively, the microdevice
of the present invention can comprise a plurality of optical
labeling substances. For example, the microdevice of the present
invention can comprise at least two different types of optical
labeling substances.
[0402] In a specific embodiment, the microdevice of the present
invention comprises an electrically conductive or dielectrically
polarizable substance. Such electrically conductive or
dielectrically polarizable substance incorporated into the
microdevice may alter the overall electrical and/or dielectric
properties of the microdevice, resulting in a change in the
interaction between the microdevice and an applied electrical field
and a change in the electrical field-induced force (e.g.,
dielectrophoretic force, traveling wave dielectrophoretic forces)
acting on the microdevice.
[0403] In choosing the type, materials, compositions, structures
and sizes of the microdevices, these properties or parameters of
the microdevices should be compatible with the isolation,
manipulation, detection or synthesis format in the specific
applications. For example, the microdevices may be used to isolate
target analyte-molecules (e.g. proteins) from a molecule mixture.
If the isolation uses dielectrophoretic forces, then the
microdevices should have the desired dielectric properties. If the
isolation/manipulation utilizes magnetic forces, then the
microdevices should have incorporated magnetic materials such as
ferro- or ferri-magnetic materials.
[0404] The microdevice can also comprise a binding partner that is
capable of binding to a moiety, e.g., a moiety to be isolated,
manipulated, detected or synthesized. Preferably, the binding
partner specifically binds to the moiety. Throughout this
application, whenever the binding partners are described or used,
they are always coupled onto the microdevices of the present
inventions. For example, when the complexes between the binding
partners and the moieties are discussed, the complexes between the
moieties and the binding partners that are coupled on the
microdevices are referred to.
[0405] Any suitable binding partner including the binding partners
disclosed in the co-pending U.S. patent application Ser. No.
09/636,104, filed Aug. 10, 2000 and Ser. No. 09/679,024, filed Oct.
4, 2000, the disclosures of which are incorporated by reference in
its entirety, can be used. For example, the binding partners can be
cells such as animal, plant, fungus or bacterium cells; cellular
organelles such as nucleus, mitochondria, chloroplasts, ribosomes,
ERs, Golgi apparatuses, lysosomes, proteasomes, secretory vesicles,
vacuoles or microsomes; viruses, microparticles or an aggregate or
complex thereof. Other binding partners may be molecules that have
been immobilized on the microdevices' surfaces. For example,
antibodies can be immobilized or bound on to the microdevices'
surfaces. The antibody-bound microdevices can then be used to
capture and bind to target proteins in a molecule mixture or to
capture and bind to target cells in a cell mixture. Oligo-dT (e.g.
25 mer of T) can be immobilized onto the microdevices' surfaces.
The oligo-dT bound microdevices can then be used to capture mRNA
from a molecule mixture. Other molecules may be used as binding
partners for capturing or binding DNA molecules. Nucleic acid
fragments, e.g., DNA, RNA, PNA segments of specific sequences, may
be used to hybridize to target nucleic acid, DNA, RNA or PNA,
molecule. Other binding partners may be molecules or functional
groups that are attached or otherwise bound to the microdevices'
surfaces, resulting in functionalized surfaces to which various
chemical/biochemical/biological reactions can occur. In some
embodiments, these various reactions may allow the moieties to bind
to the microdevices so that the moieties can be manipulated,
isolated, or detected via the use of the microdevices of the
present invention. In some other exemplary embodiments., the
functionalized surfaces allow synthesis reaction to take place on
the microdevices' surfaces. Examples of such synthesis include the
synthesis of nucleic acids, (e.g. DNA, RNA), or the synthesis of
peptides or proteins, etc. Examples of such functionalized surfaces
include, but are not limited to, surfaces derivatized with
carboxyl, amino, hydroxyl, sulfhydryl, epoxy, ester, alkene,
alkyne, alkyl, aromatic, aldehyde, ketone, sulfate, amide, urethane
group(s), or their derivatives thereof.
[0406] The choice of the microdevices is further related to the
specific isolation, manipulation detection or synthesis uses. For
example, for separating target moiety from a mixture of molecules
and particles by dielectrophoresis manipulation, binding partner's
or microdevice's dielectric properties should be significantly
different from those of molecules and particles so that when
binding partners are coupled with the target moiety, the
moiety-binding-partner-microdevices complexes may be selectively
manipulated by dielectrophoresis. In an example of separating
target cancer cells from a mixture of normal cells, the cancer
cells may have similar dielectric properties to those of normal
cells and all the cells behave similarly in their dielectrophoretic
responses, e.g., negative dielectrophoresis. In this case, the
binding partners or the microdevice preferably should be more
dielectrically-polarizable than their suspending medium and will
exhibit positive dielectrophoresis. Thus, such microdevices-binding
partners-cancer-cell complexes can be selectively manipulated
through positive dielectrophoresis forces while other cells
experience negative dielectrophoresis forces.
[0407] The microdevice can comprise a single binding partner.
Alternatively, it can be used in a high throughput analysis and can
comprise a plurality of binding partners capable of binding or
specifically binding to different moieties to be isolated,
manipulated or detected, or synthesized.
[0408] Since the present microdevice contains magnetizable
substance, the microdevice, microdevice-moiety complex, or
microdevice-binding partner-moiety complex can always be rotated or
otherwise moved or manipulated with magnetic forces. Magnetic
forces refer to the forces acting on a particle due to the
application of a magnetic field. In general, particles have to be
magnetic (e.g. paramagnetic, ferromagnetic) or magnetizable when
sufficient magnetic forces are needed to manipulate particles. We
consider a typical magnetic particle made of super-paramagnetic
material. When the magnetic particle is subjected to a magnetic
field B, a magnetic dipole .mu. is induced in the magnetic
particle
.mu. _ = V p ( .chi. p - .chi. m ) B _ .mu. m , = V p ( .chi. p -
.chi. m ) H _ m ##EQU00009##
[0409] where V.sub.p is the magnetic-particle volume, .chi..sub.p
and .chi..sub.m are the volume susceptibility of the magentic
particle and its surrounding medium, .mu..sub.m is the magnetic
permeability of medium, H.sub.m is the magnetic field strength. The
magnetic force F.sub.magnetic acting on the magentic particle is
determined by the magnetic dipole moment and the magnetic field
gradient:
F.sub.magnetic=-0.5V.sub.p(.chi..sub.p-x.sub.m){right arrow over
(H)} V{right arrow over (B)}.sub.m,
[0410] where the symbols "" and " V" refer to dot-product and
gradient operations, respectively. Clearly, whether there is
magnetic force acting on a particle depends on the difference in
the volume susceptibility between the magnetic particle and its
surrounding medium. Typically, magnetic particles are suspended in
a liquid, non-magnetic medium (the volume susceptibility is close
to zero) thus it is necessary to utilize magnetic particles (its
volume susceptibility is much larger than zero). The velocity
.nu..sub.particle of the magnetic particle under the balance
between magnetic force and viscous drag is given by:
v particle = F _ magnetic 6 .pi. r .eta. m ##EQU00010##
[0411] where r is the particle radius and .eta..sub.m is the
viscosity of the surrounding medium. Thus to achieve sufficiently
large magnetic manipulation force, the following factors should be
considered: (1) the volume susceptibility of the magnetic particles
should be maximized; (2) magnetic field strength should be
maximized; and (3) magnetic field strength gradient should be
maximized
[0412] Paramagnetic substances are preferred whose magnetic dipoles
are induced by externally applied magnetic fields and return to
zero when external field is turned off. Examples of the
paramagnetic substances include the commercially available
paramagnetic or other magnetic particles. Many of these particles
range from submicron (e.g., 50 nm-0.5 micron) up to tens of
microns. They may have different structures and compositions. One
type of magnetic particle has ferromagnetic materials encapsulated
in thin polymer layer, e.g., polystyrene. Another type of magnetic
particle has ferromagnetic nanoparticles filled into the poles of
porous beads e.g., polystyrene beads. The surface of both types of
these particles can be polystyrene in nature and may be modified to
link to various types of molecules. In still another type of
magnetic particle, ferro-magnetic materials can be incorporated
uniformly into the particles during the polymerization process.
Thus, in certain embodiments of the microdevices of the present
invention, these paramagnetic or magnetic particles may be
incorporated into the microdevices so that the microdevices
comprise the magnetizable substances.
[0413] Exemplary embodiments of the magnetizable substance
comprised in the microdevices may include paramagnetic substance,
ferromagnetic substance, ferrimagnetic substance, or
superparamagnetic substance that are directly deposited or
fabricated or incorporated into the microdevices. In one example,
the metal composition such as transition metal composition (e.g.,
iron, nickel, copper, cobalt, manganese, tantalum, zirconium) or an
alloy (e.g., cobalt-tantalum-zirconium (CoTaZr) alloy, iron-nickel
alloy) composition may be deposited into the microdevices. Various
methods such as electroplating (e.g., for making iron-nickel
alloy), sputtering (e.g. for making CoTaZr alloy), can be used for
depositing magnetizable substances. A number of methods for
depositing and/or producing magnetizable substances (e.g. magnetic,
paramagnetic, ferro magnetic substances) are described in the U.S.
patent application Ser. No. 09/685,410, filed on Oct. 10, 2000,
titled "Individually Addressable Micro-Electromagnetic Unit Array
Chips in Horizontal Configurations" and naming Wu et al as
inventors. This patent application Ser. No. 09/685,410 is
incorporated by reference in its entirety.
[0414] The rotation or manipulation of the microdevices,
microdevice-moiety complex, or microdevice-binding partner-moiety
complex, requires the generation of magnetic field distribution
over microscopic scales. One desirable feature of a microdevice is
that it has large magnetic susceptibility. Another desirable
feature is that it has small residue magnetic polarization after
the applied magnetic field/force is turned off. One approach for
generating such magnetic fields is the use of microelectromagnetic
units. Such units can induce or produce magnetic fields when an
electrical current is applied. The on/off status and the magnitude
of the electrical current applied to each unit will determine the
magnetic field distribution. The structure and dimension of the
microelectromagnetic units may be designed according to the
requirement of the magnetic field distribution. Manipulation of the
microdevices, microdevice-moiety complex, or microdevice-binding
partner-moiety complex includes the directed movement, focusing and
trapping of them. The motion of magnetic particles in a magnetic
field is termed "magnetophoresis". Theories and practice of
magnetophoresis for cell separation and other applications may be
found in various literatures (e.g., Magnetic Microspheres in Cell
Separation, by Kronick, P. L. in Methods of Cell Separation, Volume
3, edited by N. Catsimpoolas, 1980, pages 115-139; Use of magnetic
techniques for the isolation of cells, by Safarik I. And Safarikova
M., in J. of Chromatography, 1999, Volume 722(B), pages 33-53; A
fully integrated micromachined magnetic particle separator, by Ahn,
C. H., et al., J. Microelectromechanical Systems (1996)
5:151-157).
[0415] The microdevice can further comprise an element that
facilitates and/or enables manipulation of the microdevice and/or a
moiety/microdevice complex or synthesis on the microdevice. Any
suitable element that can be incorporated to the microdevice and
that can alter certain properties of the microdevice can be used.
For example, the element can be electrically-conductive or
dielectrically-polarizable or electrically-insulating materials to
facilitate and/or enable manipulation by dielectrophoresis force,
materials having high or low acoustic impedance to facilitate
and/or enable manipulation by acoustic force, or charged materials
to facilitate and/or enable manipulation by electrostatic force,
etc. The element can be a material of certain composition, a cell,
a cellular organelle, a virus, a microparticle, an aggregate or
complex of molecules and an aggregate or complex thereof. In
addition, the binding partners disclosed above and disclosed in the
co-pending U.S. patent application Ser. No. 09/636,104, filed Aug.
10, 2000 can also be used as the element(s) that facilitates and/or
enables manipulation of the microdevice and/or a moiety/microdevice
complex or synthesis on the microdevice. Non-limiting examples of
the elements may further include deposited or
other-procedure-produced materials with specific physical or
chemical properties. Metal films made of Au, Cr, Ti, Pt etc are
examples of the elements that can be incorporated into the
microdevices and increase electrical conductivity of the
microdevices. Insulating materials such as polystyrene, paralene,
or other plastic polymers are also examples of the elements that
may be incorporated into the microdevices and reduce electrical
conductivity of the microdevices.
[0416] The element can facilitate and/or enable manipulation of the
microdevice and/or a moiety/microdevice complex by any suitable
physical force including the physical forces disclosed in the
co-pending U.S. patent application Ser. No. 09/636,104, filed Aug.
10, 2000. For instance, a dielectrophoresis force, a traveling-wave
dielectrophoresis force, an acoustic force such as one effected via
a standing-wave acoustic field or a traveling-wave acoustic field,
an electrostatic force such as one effected via a DC electric
field, a mechanical force such as fluidic flow force, or an optical
radiation force such as one effected via an optical intensity field
generated by laser tweezers, can be used.
[0417] Dielectrophoresis refers to the movement of polarized
particles, e.g., microdevices, microdevice-moiety complex, or
microdevice-binding partner-moiety complex, in a non-uniform AC
electrical field. When a particle is placed in an electrical field,
if the dielectric properties of the particle and its surrounding
medium are different, dielectric polarization will occur to the
particle. Thus, the electrical charges are induced at the
particle/medium interface. If the applied field is non-uniform,
then the interaction between the non-uniform field and the induced
polarization charges will produce a net force acting on the
particle to cause particle motion towards the region of strong or
weak field intensity. The net force acting on the particle is
called dielectrophoretic force and the particle motion is
dielectrophoresis. Dielectrophoretic force depends on the
dielectric properties of the particles, particle surrounding
medium, the frequency of the applied electrical field and the field
distribution.
[0418] Traveling-wave dielectrophoresis is similar to
dielectrophoresis in which the traveling-electric field interacts
with the field-induced polarization and generates electrical forces
acting on the particles. Particles, e.g., microdevices,
microdevice-moiety complex, or microdevice-binding partner-moiety
complex, are caused to move either with or against the direction of
the traveling field. Traveling-wave dielectrophoretic forces depend
on the dielectric properties of the particles and their suspending
medium, the frequency and the magnitude of the traveling-field. The
theory for dielectrophoresis and traveling-wave dielectrophoresis
and the use of dielectrophoresis for manipulation and processing of
microparticles may be found in various literatures (e.g.,
"Non-uniform Spatial Distributions of Both the Magnitude and Phase
of AC Electric Fields determine Dielectrophoretic Forces by Wang,
et al., in Biochim Biophys Acta (1995) 1243:185-194",
"Dielectrophoretic Manipulation of Particles by Wang, et al., IEEE
Transaction on Industry Applications (1997) 33:660-669",
"Electrokinetic behavior of colloidal particles in traveling
electric fields: studies using yeast cells by Huang, et al., in J.
Phys. D: Appl. Phys., 26:1528-1535", "Positioning and manipulation
of cells and microparticles using miniaturized electric field traps
and traveling waves. By Fuhr, et al., in Sensors and Materials,
7:131-146", "Dielectrophoretic manipulation of cells using spiral
electrodes by Wang, X-B., et al., in Biophys. J. (1997)
72:1887-1899", "Separation of human breast cancer cells from blood
by differential dielectric affinity by Becker, et al., in Proc.
Natl. Acad. Sci. (1995) 92:860-864"). The manipulation of
microparticles with dielectrophoresis and traveling wave
dielectrophoresis includes concentration/aggregation, trapping,
repulsion, linear or other directed motion, levitation, and
separation of particles. Particles may be focused, enriched and
trapped in specific regions of the electrode reaction chamber.
Particles may be separated into different subpopulations over a
microscopic scale. Particles may be transported over certain
distances. The electrical field distribution necessary for specific
particle manipulation depends on the dimension and geometry of
microelectrode structures and may be designed using
dielectrophoresis theory and electrical field simulation
methods.
[0419] The dielectrophoretic force F.sub.DEPz acting on a particle
of radius r subjected to a non-uniform electrical field may be
given, under dipole approximation, by
F.sub.DEPz=2.pi..di-elect cons..sub.mr.sup.3.chi..sub.DEP
VE.sub.rms.sup.2{right arrow over (a)}.sub.z
[0420] where E.sub.rms is the RMS value of the field strength,
.di-elect cons..sub.m is the dielectric permitivity of the medium.
.chi..sub.DEP is the particle dielectric polarization factor or
dielectrophoresis polarization factor, given, under dipole
approximation, by
.chi. DEP = Re ( p * - m * p * + 2 m * ) , ##EQU00011##
[0421] "Re" refers to the real part of the "complex number". The
symbol
x * = x - j .sigma. x 2 .pi. f ##EQU00012##
is the complex permitivity (of the particle x=p, and the medium
x=m). The parameters .di-elect cons..sub.p and .sigma..sub.p are
the effective permitivity and conductivity of the particle,
respectively. These parameters may be frequency dependent. For
example, a typical biological cell will have frequency dependent,
effective conductivity and permitivity, at least, because of
cytoplasm membrane polarization.
[0422] The above equation for the dielectrophoretic force can also
be written as
F.sub.DEPz=2.pi..di-elect
cons..sub.mr.sup.3.chi..sub.DEPV.sup.2p(z){right arrow over
(a)}.sub.z
[0423] where p(z) is the square-field distribution for a
unit-voltage excitation (V=1 V) on the electrodes, V is the applied
voltage.
[0424] There are generally two types of dielectrophoresis, positive
dielectrophoresis and negative dielectrophoresis. In positive
dielectrophoresis, particles are moved by dielectrophoresis forces
towards the strong field regions. In negative dielectrophoresis,
particles are moved by dielectrophoresis forces towards weak field
regions. Whether particles exhibit positive and negative
dielectrophoresis depends on whether the particles are more or less
polarizable than the surrounding medium.
[0425] Traveling-wave DEP force refers to the force that is
generated on particles or molecules due to a traveling-wave
electric field. A traveling-wave electric field is characterized by
the non-uniform distribution of the phase values of AC electric
field components.
[0426] Here we analyze the traveling-wave DEP force for an ideal
traveling-wave field. The dielectrophoretic force F.sub.DEP acting
on a particle of radius r subjected to a traveling-wave electrical
field E.sub.TWD=E cos(2.pi.(ft-z/.lamda..sub.0)){right arrow over
(a)}.sub.x (i.e., a x-direction field is traveling along the
z-direction) is given, under dipole approximation, by
F.sub.TWD=-2.pi..di-elect
cons..sub.mr.sup.3.zeta..sub.TWDE.sup.2{right arrow over
(a)}.sub.z
[0427] where E is the magnitude of the field strength, .di-elect
cons..sub.m is the dielectric permitivity of the medium.
.zeta..sub.TWD is the particle polarization factor, given, under
dipole approximation, by
.zeta. TWD = Im ( p * - m * p * + 2 m * ) , ##EQU00013##
[0428] "Im" refers to the imaginary part of the "complex number".
The symbol
x * = x - j .sigma. x 2 .pi. f ##EQU00014##
is the complex permitivity (of the particle x=p, and the medium
x=m). The parameters .di-elect cons..sub.p and .sigma..sub.p are
the effective permitivity and conductivity of the particle,
respectively. These parameters may be frequency dependent.
[0429] Particles such as biological cells having different
dielectric properties (as defined by permitivity and conductivity)
will experience different dielectrophoretic forces. For
traveling-wave DEP manipulation of particles (including biological
cells), traveling-wave DEP forces acting on a particle of 10 micron
in diameter can vary between 0.01 and 10000 pN.
[0430] A traveling wave electric field can be established by
applying appropriate AC signals to the microelectrodes
appropriately arranged on a chip. For generating a
traveling-wave-electric field, it is necessary to apply at least
three types of electrical signals each having a different phase
value. One method to produce a traveling wave electric field is to
use four phase-quardrature signals (0, 90, 180 and 270 degrees) to
energize four linear, parallel electrodes patterned on the chip
surface. This set of four electrodes forms a basic, repeating unit.
Depending on the applications, there may be more than two such
units that are located next to each other. This will produce a
traveling-electric field in the space above or near the electrodes.
As long as electrode elements are arranged following certain
spatially sequential orders, applying phase-sequenced signals will
result in establishment of traveling electrical fields in the
region close to the electrodes.
[0431] Both dielectrophoresis and traveling-wave dielectrophoresis
forces acting on particles, e.g., microdevices, microdevice-moiety
complex, or microdevice-binding partner-moiety complex, depend on
not only the field distributions (e.g., the magnitude, frequency
and phase distribution of electrical field components; the
modulation of the field for magnitude and/or frequency) but also
the dielectric properties of the particles and the medium in which
particles are suspended or placed. For dielectrophoresis, if
particles are more polarizable than the medium (e.g., having larger
conductivities and/or permitivities depending on the applied
frequency), particles will experience positive dielectrophoresis
forces and be directed towards the strong field regions. The
particles that are less polarizable than the surrounding medium
will experience negative dielectrophoresis forces and be directed
towards the weak field regions. For traveling wave
dielectrophoresis, particles may experience dielectrophoresis
forces that drive them in the same direction as the field is
traveling direction or against it, dependent on the polarization
factor .zeta..sub.TWD. The following papers provide basic theories
and practices for dielectrophoresis and
traveling-wave-dielectrophoresis: Huang, et al., J. Phys. D: Appl.
Phys. (1993) 26:1528-1535; Wang, et al., Biochim. Biophys. Acta.
(1995) 1243:185-194; Wang, et al., IEEE Trans. Ind. Appl. (1997)
33:660-669.
[0432] Microdevices, microdevice-moiety complex, or
microdevice-binding partner-moiety complex, may be manipulated
using acoustic forces, i.e., using acoustic fields. In one case, a
standing-wave acoustic field is generated by the superimposition of
an acoustic wave generated from an acoustic wave source and its
reflective wave. Particles in standing-wave acoustic fields
experience the so-called acoustic radiation force that depends on
the acoustic impedance of the particles and their surrounding
medium. Acoustic impedance is the product of the density of the
material and the velocity of acoustic-wave in the material.
Particles with higher acoustic impedance than the surrounding
medium are directed towards the pressure nodes of the standing wave
acoustic field. Particles experience different acoustic forces in
different acoustic field distributions.
[0433] One method to generate an acoustic wave source is to use
piezoelectric material. These materials, upon applying electrical
fields at appropriate frequencies, can generate mechanical
vibrations that are transmitted into the medium surrounding the
materials. One type of piezoelectric material is piezoelectric
ceramics. Microelectrodes may be deposited on such ceramics to
activate the piezoelectric ceramic and thus to produce appropriate
acoustic wave fields. Various geometry and dimensions of
microelectrodes may be used according to the requirements of
different applications. Reflective walls are needed to generate a
standing-wave acoustic field. Acoustic wave fields of various
frequencies may be applied, i.e., fields at frequencies between kHz
and hundred megahertz. In another case, one could use a
non-standing wave acoustic field, e.g., a traveling-wave acoustic
field. A traveling-wave acoustic field may exert forces on
particles (see e.g., see, "Acoustic radiation pressure on a
compressible sphere, by K. Yoshioka and Y. Kawashima in Acustica,
1955, Vol. 5, pages 167-173"). Particles not only experience forces
from acoustic fields directly but also experience forces due to
surrounding fluid because the fluid may be induced to move by the
traveling-wave acoustic field. Using acoustic fields, particles may
be focussed, concentrated, trapped, levitated and transported in a
microfluidic environment. Another mechanism for producing forces on
particles in an acoustic field is through acoustic-induced fluid
convection. An acoustic field produced in a liquid may induce
liquid convection. Such convection is dependent on the acoustic
field distribution, properties of the liquid, and the volume and
structure of the chamber in which the liquid is placed. Such liquid
convection will impose forces on particles placed in the liquid and
those forces may be used for manipulating particles. One example
where such manipulating forces may be exploited is for enhancing
the mixing of liquids or the mixing of particles in a liquid. For
the present invention, such convection may be used to enhance the
mixing of the binding partners coupled onto the microdevices with
moiety in a suspension and to promote the interaction between the
moiety and the binding partners.
[0434] A standing plane wave of ultrasound can be established by
applying AC signals to the piezoelectric transducers. For example,
the standing wave spatially varying along the z axis in a fluid can
be expressed as:
.DELTA.p(z)=p.sub.0 sin(kz)cos(.omega.t)
[0435] where .DELTA.p is acoustic pressure at z, p.sub.0 is the
acoustic pressure amplitude, k is the wave number (2.pi./.lamda.,
where .lamda. is the wavelength), z is the distance from the
pressure node, .omega. is the angular frequency, and t is the time.
According to the theory developed by Yoshioka and Kawashima (see,
"Acoustic radiation pressure on a compressible sphere, by K.
Yoshioka and Y. Kawashima in Acustica, 1955, Vol. 5, pages
167-173"), the radiation force F.sub.acoustic acting on a spherical
particle in the stationary standing wave field is given by (see
"Studies on particle separation by acoustic radiation force and
electrostatic force by Yasuda K., et al., in Jpn. J. Appl. Physics
(1996) 35:3295-3299")
F acoustic = - 4 .pi. 3 r 3 kE acoustic A sin ( 2 kz )
##EQU00015##
[0436] where r is the particle radius, E.sub.acoustic is the
average acoustic energy density, A is a constant given by
A = 5 .rho. p - 2 .rho. m 2 .rho. p + .rho. m - .gamma. p .gamma. m
##EQU00016##
[0437] where .rho..sub.m and .rho..sub.p are the density of the
particle and the medium, .gamma..sub.m and .gamma..sub.p are the
compressibility of the particle and medium, respectively. A is
termed herein as the acoustic-polarization-factor.
[0438] When A>0, the particle moves towards the pressure node
(z=0) of the standing wave.
[0439] When A<0, the particle moves away from the pressure
node.
[0440] Clearly, particles of different density and compressibility
will experience different acoustic-radiation-forces when placed
into the same standing acoustic wave field. For example, the
acoustic radiation force acting on a particle of 10 micron diameter
can vary between 0.01 and 1000 pN, depending on the established
acoustic energy density distribution.
[0441] Piezoelectric transducers are made from "piezoelectric
materials" that produce an electric field when exposed to a change
in dimension caused by an imposed mechanical force (piezoelectric
or generator effect). Conversely, an applied electric field will
produce a mechanical stress (electrostrictive or motor effect) in
the materials. They transform energy from mechanical to electrical
and vice-versa. The piezoelectric effect was discovered by Pierre
Curie and his brother Jacques in 1880. It is explained by the
displacement of ions, causing the electric polarization of the
materials' structural units. When an electric field is applied, the
ions are displaced by electrostatic forces, resulting in the
mechanical deformation of the whole material.
[0442] Microdevices, microdevice-moiety complex, or
microdevice-binding partner-moiety complex, may be manipulated
using DC electric fields. A DC electric field can exert an
electrostatic force on charged particles. The force depends on the
charge magnitude and polarity of the particles as well as on the
magnitude and direction of the field. The particles with positive
and negative charges may be directed to electrodes with negative
and positive potentials, respectively. By designing a
microelectrode array in a microfluidic device, electric field
distributions may be appropriately structured and realized. With DC
electric fields, microparticles may be concentrated (enriched),
focussed and moved (transported) in a microfluidic device. Proper
dielectric coating may be applied on to DC electrodes to prevent
and reduce undesired surface electrochemistry and to protect
electrode surfaces.
[0443] The electrostatic force F.sub.E on a particle in an applied
electrical field E.sub.z{right arrow over (a)}.sub.z can be given
by
F.sub.E=Q.sub.pE.sub.z{right arrow over (a)}.sub.z
[0444] where Q.sub.p is the effective electric charge on the
particle. The direction of the electrostatic force on a charged
particle depends on the polarity of the particle charge as well as
the direction of the applied field.
[0445] Thermal convection forces refer to the forces acting on
particles, e.g., microdevices, microdevice-moiety complex, or
microdevice-binding partner-moiety complex, due to the
fluid-convection (liquid-convection) that is induced by a thermal
gradient in the fluid. Thermal diffusion in the fluid drives the
fluid towards thermal equilibrium. This causes a fluid convection.
In addition, the density of aqueous solutions tends to decrease
with increasing temperature. Such density differences are also not
stable within a fluid resulting in convection. Thermal convection
may be used to facilitate liquid mixing. Directed thermal
convection may act as an active force.
[0446] Thermal gradient distributions may be established within a
chip-based chamber where heating and/or cooling elements may be
incorporated into the chip structure. A heating element may be a
simple joule-heating resistor coil. Such a coil could be
microfabricated onto the chip. As an example, consider a coil
having a resistance of 10 ohm. Applying 0.2 A through the coil
would result in 0.4 W joule heating-power. When the coil is located
in an area <100 micron.sup.2, this is an effective way of heat
generation. Similarly, a cooling element may be a Peltier element
that could draw heat upon applying electric potentials.
[0447] As an exemplary embodiment, the microdevices of the present
invention may be used on a chip that incorporates an array of
individually addressable heating elements. These heating elements
may be positioned or structurally arranged in certain order so that
when each, some, or all of the elements are activated, thermal
gradient distributions will be established to produce thermal
convection. For example, if one heating element is activated,
temperature increases in the liquid in the neighborhood of that
element will induce fluid convection. In another exemplary
embodiment, the chip may comprise multiple, interconnected heating
units so that these units can be turned on or off in a synchronized
order. Yet, in another example, the chip may comprise only one
heating element that can be energized to produce heat and induce
thermal convection in the liquid fluid.
[0448] Other physical forces may be applied. For example,
mechanical forces, e.g., fluidic flow forces, may be used to
transport microparticles, e.g., microdevices, microdevice-moiety
complex, or microdevice-binding partner-moiety complex. Optical
radiation forces as exploited in "laser tweezers" may be used to
focus, trap, levitate and manipulate microparticles. The optical
radiation forces are the so-called gradient-forces when a material
(e.g., a microparticle) with a refractive index different from that
of the surrounding medium is placed in a light gradient. As light
passes through a polarizable material, it induces fluctuating
dipoles. These dipoles interact with the electromagnetic field
gradient, resulting in a force directed towards the brighter region
of the light if the material has a refractive index larger than
that of the surrounding medium. Conversely, an object with a
refractive index lower than the surrounding medium experiences a
force drawing it towards the darker region. The theory and practice
of "laser tweezers" for various biological application are
described in various literatures (e.g., "Making light work with
optical tweezers, by Block S. M., in Nature, 1992, Volume 360,
pages 493-496"; "Forces of a single-beam gradient laser trap on a
dielectric sphere in the ray optics regime, by Ashkin, A., in
Biophys. J., 1992, Volume 61, pages 569-582"; "Laser trapping in
cell biology, by Wright, et al., in IEEE J. of Quantum Electronics
(1990) 26:2148-2157"; "Laser manipulation of atoms and particles,
by Chu, S. in Science (1991) 253:861-866"). The light field
distribution and/or light intensity distribution may be produced
with built-in optical elements and arrays on a chip and external
optical signal sources, or may be produced with built-in
electro-optical elements and arrays on a chip and the external
structures are electrical signal sources. In the former case, when
the light produced by the optical signal sources passes through the
built-in optical elements and arrays, light is processed by these
elements/arrays through, e.g., reflection, focusing, interference,
etc. Optical field distributions are generated in the regions
around the chip. In the latter case, when the electrical signals
from the external electrical signal sources are applied to the
built-in electro-optical elements and arrays, light is produced
from these elements and arrays and optical fields are generated in
the regions around the chip.
[0449] Although the microdevices can comprise a single element that
can facilitate and/or enable manipulation of the microdevice by one
type of physical forces or synthesis on the microdevice, they may
also be used in high throughput analysis and preferably comprise a
plurality of elements, each of the elements facilitates and/or
enables manipulation of the microdevice and/or the
moiety/microdevice complex by a different physical force. For
example, the element can be a conductive or insulating material for
manipulation by a dielectrophoresis force, a material having high
or low acoustic impedance for manipulation by acoustic force,
and/or a charged material for manipulation by a electrostatic
force, etc.
[0450] In a preferred embodiment, the microdevice comprises a
binding partner that is capable of binding or specifically binding
to a moiety to be isolated, manipulated, detected or synthesized
and an element that facilitates and/or enables manipulation of the
microdevice and/or the moiety/microdevice complex. More preferably,
the microdevice(s) comprises a plurality of binding partners, each
of the binding partners is capable of binding or specifically
binding to a different moiety to be isolated, manipulated, detected
or synthesized and a plurality of the elements, each of the
elements facilitates and/or enables manipulation of the microdevice
and/or the moiety/microdevice complex by a different physical
force.
[0451] The microdevice can further comprise a detectable marker or
a molecular tag. Exemplary detectable markers include dyes,
radioactive substances and fluorescent substances. Exemplary
detectable molecular tags include nucleic acid, oligonucleotide,
protein and peptide sequences.
[0452] In a specific embodiment, the present microdevice has a thin
rectangular shape and has a major axis (length) to minor axis
(width) ratio of at least about 1.2, and preferably at least about
1.5, and has a thickness (height) smaller than both major axis and
minor axis. In another specific embodiment, the present microdevice
comprises at least two rectangular-shaped strips (or bars) or
near-rectangular-shaped strips (or bars) of the paramagnetic
substance. Preferably, at least two strips (or bars) of the
paramagnetic substance are separated and located on each side of
the microdevice along the major axis of the microdevice. More
preferably, a metal film is processed to have a photorecognizable
pattern that is located between the at least two strips (or bars)
of the paramagnetic substances. More preferably, the metal film
comprises aluminum. Also more preferably, the present microdevice
has unequal number of the paramagnetic substance strip(s) (or bars)
on each side along the major axis of the microdevice. In still
another specific embodiment, the present microdevice comprises two
strips (or bars) of the paramagnetic substance along the major axis
of the microdevice. Preferably, the two strips (or bars) of the
paramagnetic substance have fingers on both ends. In yet another
specific embodiment, the paramagnetic substance in the present
microdevice forms a strip (or bar) along the major axis of the
microdevice and said strip (or bar) has fingers on both ends.
[0453] In another aspect, the present invention is directed to a
system for forming a microdevice array, which system comprises: a)
a plurality of microdevices, each of the microdevices comprising a
magnetizable substance and a photorecognizable coding pattern,
wherein said microdevices have a preferential axis of
magnetization; and b) a microchannel array comprising a plurality
of microchannels, said microchannels are sufficiently wide to
permit rotation of said microdevices within said microchannels but
sufficiently narrow to prevent said microdevices from forming a
chain when the major axis of said microdevices is substantially
perpendicular to the major axis of said microchannels wherein the
said microdevices are subjected to an applied magnetic field.
[0454] In preferred embodiments, the microdevices are manipulated
to be "flat" or "substantially flat" in the microchannels so that
the photorecognizable patterns on the microdevices can be optically
detected or analyzed via the optical means in the direction
substantially-normal to the plane defined by the microchannel
length and width. In preferred embodiments, the height of the
microchannels and/or the constraint on the microdevices by a
magnetic field should be adjusted to prevent the microdevices from
standing up within the microchannels. In a specific embodiment, the
height of the microchannels is less than about 70% of the major
axis of the microdevices.
[0455] The microchannel array can further comprise a staging area
or loading area where the microdevices can be introduced into
and/or an output area or an outlet channel where the microdevices
may be removed from the microchannel array. The microchannel array
can also further comprise a magnetic field generating means capable
of generating a magnetic field suitable for manipulating the
microdevices into, within and/or out of the microchannel array, or
rotating the microdevices within the microchannel array. Any
suitable magnetic field generating means can be used. For example,
the magnetic field generating means can comprise a permanent
magnet, a mobile permanent magnet, an electromagnetic unit, a
ferromagnetic material or a microelectromagenetic unit. The
magnetic field generating means can be located at any suitable
location, e.g., below, within, above and/or near the microchannel
array.
C. Methods for Forming a Microdevice Array
[0456] In still another aspect, the present invention is directed
to a method for forming a microdevice array, which method
comprises: a) providing a plurality of microdevices, each of the
microdevices comprising a magnetizable substance and a
photorecognizable coding pattern, wherein said microdevices have a
preferential axis of magnetization; b) providing a microchannel
array comprising a plurality of microchannels, said microchannels
are sufficiently wide to permit rotation of said microdevices
within said microchannels but sufficiently narrow to prevent said
microdevices from forming a chain when the major axis of said
microdevices is substantially perpendicular to the major axis of
said microchannels wherein said microdevices are subjected to an
applied magnetic field; c) introducing said plurality of
microdevices into said plurality of microchannels; and d) rotating
said microdevices within said microchannels by a magnetic force,
whereby the combined effect of said magnetic force and said
preferential axis of magnetization of said microdevices
substantially separates said microdevices from each other.
[0457] In preferred embodiments, the microdevices are manipulated
to be "flat" or "substantially flat" in the microchannels so that
the photorecognizable patterns on the microdevices can be optically
detected or analyzed via the optical means in the direction
substantially-normal to the plane defined by the microchannel
length and width. In preferred embodiments, the height of the
microchannels and/or the constraint on the microdevices by a
magnetic field should be adjusted to prevent the microdevices from
standing up within the microchannels. In a specific embodiment, the
height of the microchannels is less than about 70% of the major
axis of the microdevices.
[0458] The microdevices can be introduced into the microchannels by
any suitable force. For example, the microdevices can be introduced
into the microchannels by a magnetic force, a fluidic force or a
combination thereof. There are multiple methods for introducing or
loading the microdevices into the channels. In one example, the
microdevices are in the form of the MicroDisks, which have two
major surfaces and a small dimension (small thickness) between the
two major surfaces. MicroDisks are placed in the loading area near
the inlet to the microchannels or channels. A small Neodymium
magnet at the outlet end of the channel is used to draw the
MicroDisks into the channel. The magnet is rotated to facilitate
movement of the MicroDisks into the channels. In one experiment,
the MicroDisk's major surfaces are of dimensions of 90 .mu.m by 70
.mu.m and the MicroDisks are several .mu.m thick. Using the
above-described procedure, it was possible to completely fill five
2 cm long channels (channel widths ranging from 120-160.mu.) with
90.times.70.mu. MicroDisks (containing magnetic strips (or bars)
with the "3-finger" pattern) in less than 3 minutes. The length,
width and height directions of the magnetic strips or bars
correspond, respectively to, the length, width and height
directions of the MicroDisks. Since the rate-limiting step in the
loading or filling process is the MicroDisks moving along the
length of the channel, the number of channels can be increased
without significantly affecting loading or filling time, e.g., two
hundred 2 cm long channels can be filled with about 50,000
MicroDisks within a 3-minute time-period using this procedure.
Channels loaded in this manner may be overloaded such that when the
direction of the applied external magnetic field is perpendicular
to the channel the "perpendicularly arrayed" MicroDisks will be
overlapping. Overlaps can be relieved by alternating the direction
of the applied external magnetic field between perpendicular and
parallel several times. This causes the "chains" to lengthen. In
this example, preferably, MicroDisks are introduced into the
channels or microchannels so that the height direction of the
MicroDisks is substantially aligned with the height direction of
the microchannels or channels. Preferably, MicroDisks that have
been loaded and/or arrayed into the channels or microchannels are
lying flat on the surface of the microchannels.
[0459] In another example, the microdevices are in the form of the
MicroDisks, which have two major surfaces and a small dimension
(small thickness) between the two major surfaces. MicroDisks are
loaded into the microchannels or channels exactly as described in
above example with the addition of a steady flow-rate of liquid
through the channels to increase the efficiency of channel loading.
In this example, preferably, MicroDisks are introduced into the
channels or microchannels so that the height direction of the
MicroDisks is substantially aligned with the height direction of
the microchannels or channels. Preferably, MicroDisks that have
been loaded and/or arrayed into the channels or microchannels are
lying flat on the surface of the microchannels.
[0460] In still another example, the microdevices are in the form
of the MicroDisks, which have two major surfaces and a small
dimension (small thickness) between the two major surfaces. The
MicroDisks comprises magnetic strips or bars, whose length, width
and height directions correspond, respectively, to the length,
width and height directions of the MicroDisks. MicroDisks are
placed in the loading area near the inlet to the channels. A large
Neodymium magnet at the outlet end of the channel is used to draw
the MicroDisks into the channel. The magnet field from this magnet
is perpendicular to the channels. A small Neodymium magnet is
placed above or below the inlet to the channels and rotated to
facilitate movement of the MicroDisks into the channels. A steady
flow-rate of liquid through the channels increases the efficiency
of channel loading. MicroDisks are loaded in their final
"perpendicularly arrayed" form (preferential axis of magnitization
perpendicular to the long (or major) axis of the channel),
minimizing channel overloading and providing a more uniform
arraying pattern. The method of loading or arraying the MicroDisks
in this example will result in the magnetic bars within or on the
MicroDisks being perpendicular to the channel after the MicroDisks
are loaded into the channels or microchannels, i.e. the length
direction of the magnetic strips or bars (i.e. the length direction
of the MicroDisks) will be normal or substantially normal to the
length direction of the microchannels. In this example, preferably,
MicroDisks are introduced into the channels or microchannels so
that the height direction of the MicroDisks is substantially
aligned with the height direction of the microchannels or channels.
Preferably, MicroDisks that have been loaded and/or arrayed into
the channels or microchannels are lying flat on the surface of the
microchannels.
[0461] The microdevices or MicroDisks can be introduced into the
microchannels at any suitable angle. For example, the microdevices
can be introduced into the microchannels by a magnetic field at a
direction such that the angle between the major axis of the
microdevice and the microchannel is about less than 45 degrees. The
direction of the magnetic field will affect the orientation of the
microdevices or MicroDisks and affect the direction of the major
axis of the microdevice. Normally, when the microdevices can freely
rotate or re-orientate, the preferential axis of magnetization is
substantially aligned with the applied magnetic field. For
microdevice or MicroDisk whose preferential axis of magnetization
is the same as, or substantially aligned with, the major axis of
the microdevice, then the major axis of the microdevice is
substantially aligned with the applied magnetic field. It is thus
possible to introduce the microdevices into the microchannels by a
magnetic field at appropriate directions so that the major axis of
the microdevice is angled with respect to the length direction of
the microchannels at degrees less than 45 degree. Preferably, the
microdevices are introduced into the microchannels by a magnetic
field at a direction such that the angle between the major axis of
the microdevice and the microchannel is about less than 40, 35, 30,
25, 20, 15, 10, 5 or 0 degrees.
[0462] The present method can further comprise a step of breaking a
chain formed among the microdevices prior to or concurrent with
introducing the microdevices into the microchannels. This can be
accomplished by any suitable methods, e.g., rotating the direction
of magnetic field between the major and minor axis of the
microdevices.
[0463] Preferably, after microdevices or MicroDisks are loaded
and/or filled into the channels or microchannels, microdevices or
MicroDisks are lying flat on the surface of the microchannels. The
microdevices or MicroDisks can be rotated within the microchannels
for any suitable degrees provided that the rotation is sufficient
to substantially separate the microdevices or MicroDisks from each
other. The separation can be achieved by a single rotation of a
larger degree or by multiple rotations for smaller degrees.
Preferably, the microdevices or MicroDisks are rotated at least 45
degrees. More preferably, the microdevices or MicroDisks are
rotated 90 degrees.
[0464] In a specific embodiment, at least one of the microdevices
binds to a moiety and the method is used to manipulate said moiety.
In another specific embodiment, a plurality of the microdevices
bind to a plurality of moieties and the method is used to
manipulate said plurality of moieties. The present method can be
used for any suitable manipulation of a moiety, e.g.,
transportation, focusing, enrichment, concentration, aggregation,
trapping, repulsion, levitation, separation, fractionation,
isolation and linear or other directed motion of the moiety. In
still another specific embodiment, the present method can further
comprise a step of assessing the identity of the manipulated moiety
by photoanalysis of the photorecognizable coding pattern on the
microdevice to which the moiety binds. Assessment of the identity
of the manipulated moiety may involve obtaining an index, ratio,
percentage, visual or other value indicative of the identity of the
manipulated moiety. In still another specific embodiment, the
present method can further comprise a step of assessing the
quantity of the manipulated moiety by further quantitative means
for analyzing the amount of the manipulated moiety on the
microdevice. The assessment of the quantity of the manipulated
moiety may involve obtaining a number, an index, or other value
indicative of the amount or quantity or the concentration of the
manipulated moiety. In yet another specific embodiment, the present
method can further comprise a step of collecting the microdevice to
which the moiety binds through an outlet channel. The present
method can further comprise a step of recovering the moiety from
the collected microdevice.
[0465] In yet another aspect, the present invention is directed to
a method for forming a microdevice array, which method comprises:
a) providing a plurality of microdevices, each of the microdevices
comprising a magnetizable substance and a photorecognizable coding
pattern, wherein said microdevices have a preferential axis of
magnetization, on a surface suitable for rotation of said
microdevices; and b) rotating said microdevices on said surface by
a magnetic force, whereby the combined effect of said magnetic
force and said preferential axis of magnetization of said
microdevices substantially separates said microdevices from each
other.
[0466] In yet another aspect, the present invention is directed to
a method for forming a microdevice array, which method comprises:
a) providing a plurality of the microdevices, each of the
microdevices comprising a magnetizable substance and a
photorecognizable coding pattern, and having a preferential axis of
magnetization; b) introducing said plurality of microdevices onto a
surface; and rotating said microdevices by a magnetic force to form
chains and clusters, whereby the combined effect of said magnetic
force and said preferential axis of magnetization of said
microdevices substantially separates the microdevices from each
other. In an embodiment of the arraying method, the microdevices
are introduced onto the surface in a liquid suspension. The
microdevice suspension can be added to the surface by a variety of
methods, such as via micropieppetting, or pumping into the
microchanels that are formed on the surface. In another embodiment
of the methods, the surface may comprise grooves with width
dimensions substantially narrower than that of the microdevice.
After the microdevice are arrayed on the surface, the liquid in
which the microdevices are suspended may be removed via the grooves
on the surfaces by various methods such as suction or pumping
out.
[0467] The present methods can be used for analyzing, isolating,
manipulating or detecting any types of moieties when the moieties
are involved in certain processes, such as physical, chemical,
biological, biophysical or biochemical processes, etc., in a chip
format or non-chip format. Moieties can be cells, cellular
organelles, viruses, molecules or an aggregate or complex thereof.
Moieties can be pure substances or can exist in a mixture of
substances wherein the target moiety is only one of the substances
in the mixture. For example, cancer cells in the blood from
leukemia patients, cancer cells in the solid tissues from patients
with solid tumors and fetal cells in maternal blood from pregnant
women can be the moieties to be isolated, manipulated or detected.
Similarly, various blood cells such as red and white blood cells in
the blood can be the moieties to be isolated, manipulated or
detected. DNA molecules, mRNA molecules, certain types of protein
molecules, or all protein molecules from a cell lysate can be
moieties to be isolated, manipulated or detected.
[0468] Non-limiting examples of cells include animal cells, plant
cells, fungi, bacteria, recombinant cells or cultured cells.
Animal, plant cells, fungus, bacterium cells to be isolated,
manipulated or detected can be derived from any genus or subgenus
of the Animalia, Plantae, fungus or bacterium kingdom. Cells
derived from any genus or subgenus of ciliates, cellular slime
molds, flagellates and microsporidia can also be isolated,
manipulated or detected. Cells derived from birds such as chickens,
vertebrates such fish and mammals such as mice, rats, rabbits,
cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeys and other
non-human primates, and humans can be isolated, manipulated or
detected by the present methods.
[0469] For animal cells, cells derived from a particular tissue or
organ can be isolated, manipulated or detected. For example,
connective, epithelium, muscle or nerve tissue cells can be
isolated, manipulated or detected. Similarly, cells derived from an
accessory organ of the eye, annulospiral organ, auditory organ,
Chievitz organ, circumventricular organ, Corti organ, critical
organ, enamel organ, end organ, external female genital organ,
external male genital organ, floating organ, flower-spray organ of
Ruffini, genital organ, Golgi tendon organ, gustatory organ, organ
of hearing, internal female genital organ, internal male genital
organ, intromittent organ, Jacobson organ, neurohemal organ,
neurotendinous organ, olfactory organ, otolithic organ, ptotic
organ, organ of Rosenmuller, sense organ, organ of smell, spiral
organ, subcommissural organ, subformical organ, supernumerary
organ, tactile organ, target organ, organ of taste, organ of touch,
urinary organ, vascular organ of lamina terminalis, vestibular
organ, vestibulocochlear organ, vestigial organ, organ of vision,
visual organ, vomeronasal organ, wandering organ, Weber organ and
organ of Zuckerkandl can be isolated, manipulated or detected.
Preferably, cells derived from an internal animal organ such as
brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,
blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,
intestine, testis, ovary, uterus, rectum, nervous system, gland,
internal blood vessels, etc can be isolated, manipulated or
detected. Further, cells derived from any plants, fungi such as
yeasts, bacteria such as eubacteria or archaebacteria can be
isolated, manipulated or detected. Recombinant cells derived from
any eucaryotic or prokaryotic sources such as animal, plant, fungus
or bacterium cells can also be isolated, manipulated or detected.
Cells from various types of body fluid such as blood, urine,
saliva, bone marrow, sperm or other ascitic fluids, and
subfractions thereof, e.g., serum or plasma, can also be isolated,
manipulated or detected.
[0470] Isolatable, manipulatable or detectable cellular organelles
include nucleus, mitochondria, chloroplasts, ribosomes, ERs, Golgi
apparatuses, lysosomes, proteasomes, secretory vesicles, vacuoles
or microsomes. Isolatable, manipulatable or detectable viruses
include intact viruses or any viral structures, e.g., viral
particles, in the virus life cycle that can be derived from viruses
such as Class I viruses, Class II viruses, Class III viruses, Class
IV viruses, Class V viruses or Class VI viruses.
[0471] Isolatable, manipulatable or detectable molecules can be
inorganic molecules such as ions, organic molecules or a complex
thereof. Non-limiting examples of ions include sodium, potassium,
magnesium, calcium, chlorine, iron, copper, zinc, manganese,
cobalt, iodine, molybdenum, vanadium, nickel, chromium, fluorine,
silicon, tin, boron or arsenic ions. Non-limiting examples of
organic molecules include amino acids, peptides, proteins,
nucleosides, nucleotides, oligonucleotides, nucleic acids,
vitamins, monosaccharides, oligosaccharides, carbohydrates, lipids
or a complex thereof.
[0472] Any amino acids can be isolated, manipulated or detected by
the present methods. For example, a D- and a L-amino-acid can be
isolated, manipulated or detected. In addition, any building blocks
of naturally occurring peptides and proteins including Ala (A), Arg
(R), Asn (N), Asp (D), Cys (C), Gln (O), Glu (E), Gly (G), H is
(H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P) Ser (S),
Thr (T), Trp (W), Tyr (Y) and Val (V) can be isolated, manipulated
or detected.
[0473] Any proteins or peptides can be isolated, manipulated or
detected by the present methods. For example, membrane proteins
such as receptor proteins on cell membranes, enzymes, transport
proteins such as ion channels and pumps, nutrient or storage
proteins, contractile or motile proteins such as actins and
myosins, structural proteins, defense protein or regulatory
proteins such as antibodies, hormones and growth factors can be
isolated, manipulated or detected. Proteineous or peptidic antigens
can also be isolated, manipulated or detected.
[0474] Any nucleic acids, including single-, double and
triple-stranded nucleic acids, can be isolated, manipulated or
detected by the present methods. Examples of such nucleic acids
include DNA, such as A-, B- or Z-form DNA, and RNA such as mRNA,
tRNA and rRNA.
[0475] Any nucleosides can be isolated, manipulated or detected by
the present methods. Examples of such nucleosides include
adenosine, guanosine, cytidine, thymidine and uridine. Any
nucleotides can be isolated, manipulated or detected by the present
methods. Examples of such nucleotides include AMP, GMP, CMP, UMP,
ADP, GDP, CDP, UDP, ATP, GTP, CTP, UTP, dAMP, dGMP, dCMP, dTMP,
dADP, dGDP, dCDP, dTDP, dATP, dGTP, dCTP and dTTP.
[0476] Any vitamins can be isolated, manipulated or detected by the
present methods. For example, water-soluble vitamins such as
thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxine,
biotin, folate, vitamin B.sub.12 and ascorbic acid can be isolated,
manipulated or detected. Similarly, fat-soluble vitamins such as
vitamin A, vitamin D, vitamin E, and vitamin K can be isolated,
manipulated or detected.
[0477] Any monosaccharides, whether D- or L-monosaccharides and
whether aldoses or ketoses, can be isolated, manipulated or
detected by the present methods. Examples of monosaccharides
include triose such as glyceraldehyde, tetroses such as erythrose
and threose, pentoses such as ribose, arabinose, xylose, lyxose and
ribulose, hexoses such as allose, altrose, glucose, mannose,
gulose, idose, galactose, talose and fructose and heptose such as
sedoheptulose.
[0478] Any lipids can be isolated, manipulated or detected by the
present methods. Examples of lipids include triacylglycerols such
as tristearin, tripalmitin and triolein, waxes, phosphoglycerides
such as phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, phosphatidylinositol and cardiolipin,
sphingolipids such as sphingomyelin, cerebrosides and gangliosides,
sterols such as cholesterol and stigmasterol and sterol fatty acid
esters. The fatty acids can be saturated fatty acids such as lauric
acid, myristic acid, palmitic acid, stearic acid, arachidic acid
and lignoceric acid, or can be unsaturated fatty acids such as
palmitoleic acid, oleic acid, linoleic acid, linolenic acid and
arachidonic acid.
D. Methods for Synthesizing a Library and Uses Thereof
[0479] In another aspect, the present invention is directed to a
method for synthesizing a random library, which method comprises:
a) providing a plurality of microdevices, each of said microdevices
comprises a magnetizable substance and a unique photorecognizable
coding pattern, wherein each of said microdevices has a
preferential axis of magnetization and wherein said unique
photorecognizable coding pattern on each of said microdevices
corresponds to an entity to be synthesized on each of the said
microdevices; and b) synthesizing said entities on said
microdevices, wherein said microdevices are identified after each
synthesis cycle according to said unique photorecognizable coding
patterns, whereby a library is synthesized, wherein each of said
microdevices contains an entity that corresponds to said unique
photorecognizable coding pattern on each of the said microdevices
and the sum of said microdevices collectively contains a plurality
of entities. A library that is synthesized according to the above
method is also provided.
[0480] In yet another aspect, the present invention is directed to
a method for synthesizing a library of predetermined sequence,
which method comprises: a) providing a plurality of microdevices,
each of said microdevices comprises a magnetizable substance and a
photorecognizable coding pattern, wherein said microdevices have a
preferential axis of magnetization and wherein said
photorecognizable coding pattern corresponds to an entity to be
synthesized on said microdevice; and b) synthesizing said entities
on said microdevices, wherein said microdevices are sorted after
each synthesis cycle according to said photorecognizable coding
patterns, whereby a library is synthesized, wherein each of said
microdevices contains an entity that corresponds to a
photorecognizable coding pattern on said microdevice and the sum of
said microdevices collectively contains a plurality of entities
that is predetermined before the library synthesis. A library that
is synthesized according to the above method is also provided.
[0481] The microdevices can be sorted by any suitable methods. For
example, the microdevices can be sorted through a microchannel
array comprising a plurality of microchannels, said microchannels
are sufficiently wide to permit rotation of said microdevices
within said microchannels but sufficiently narrow to prevent said
microdevices from forming a chain when the major axis of said
microdevices is substantially perpendicular to the major axis of
said microchannels, via a combined effect of a magnetic force and
the preferential axis of magnetization of the microdevices that
substantially separates the microdevices from each other. The
height of the microchannels and/or the constraint on the
microdevices by a magnetic field should be adjusted to prevent the
microdevices from standing up within the microchannels. In a
specific embodiment, the height of the microchannels is about less
than 70% of the major axis of the microdevices. After the
microdevices are arrayed into the microchannels, photoanalysis of
microdevices is performed to determine photorecognizable coding (or
encoding) pattern of individual microdevice. A method that can
handle individual microdevice is used to manipulate individual
microdevice and to sort them to different
regions/locations/reaction chambers according to their
photorecognizable pattern. For example, a microelectromagnetic
needle that can generate magnetic field at a fine tip-end of the
needle can be used to pick up individual microdevice from their
arrayed channels (in this case, the channels have to be open on the
top side) and move and send/dispense individual microdevices to
different locations/regions/reaction chambers. In another example,
microdevices are moved out from the microchannels by, e.g., a
combination of magnetic forces and fluidic forces, and at the
outlet region of the channel, microdevices can be sent to different
locations by the control of magnetic forces and/or fluidic
forces.
[0482] Sorting can also be accomplished through the use of magnetic
force to specifically capture desired grouping of microdevices
after each step of the synthesis and deposit them into the
appropriate reaction vessel. Microdevices can be arrayed using a
photoresist to form either the top or bottom surface of the
arraying chamber. When exposed to light of the appropriate
wavelength the photoresist in the illuminated regions can be
dissolved exposing the Microdevices in those locations and allowing
them to be removed by magnetic force. A programmable digital
micromirror array (e.g. "Maskless fabrication of light-directed
oligonucleotides microarrays using a digital micromirror array" by
Singh-Gasson, et al., Nature Biotechnology (1999) 17:974-978) or
similar maskless array synthesizer device could be used to direct
the light.
[0483] An alternative and preferred method of sorting utilizing
magnetic force is to use sorting channels. As discussed above,
microdevices having a preferential axis of magnetization when
arrayed in a channel in the presence of a magnetic field will align
and separate due to repulsive magnetic force and can be drawn
through liquid filled channels in a "perpendicularly arrayed" form
(preferential axis of magnetization perpendicular to the long axis
of the channel). A sufficient increase in surface tension will
prevent movement of the microdevice. Such an effect can be
generated by creating an appropriate liquid-liquid (immiscible
liquids such as hexane and water) or gas-liquid interface. For
example consider an arraying channel separated from a series of
sorting channels by a microvalve (the design, manufacture, and use
of such valves are well known to those practiced in the art).
Through an appropriately positioned orifice near the end of the
arraying channel a bubble can be introduced between the final and
the penultimate microdevice. Opening of the valve and application
of a magnetic force will result in only the final microdevice being
drawn through the channel into the sorting channels, others
microdevices will remain trapped behind the bubble. The valve is
then closed and the single disk in the sorting channel can be
directed using magnetic and/or fluidic force to and/or other
physical force (e.g. dielectrophoresis force) the appropriate
reaction vessel. Application of fluidic force (pumping liquid)
drives the bubble out through an appropriately placed outlet at the
end of the channel allowing the microdevices to advance and the
sorting process is then repeated. The size of orifices for gas
delivery and removal must be significantly smaller than the
microdevices. An alternative and potentially more rapid system
would be to introduce bubbles between all of the microdevices
within a channel and to adjust the magnetic field and fluidic force
such that the microdevices move in a segmented fashion through the
channel. This is analogous to the segmented fluid flow approach
widely used by Technicon International, Ltd. to prevent peak
broadening (e.g., U.S. Pat. Nos. 2,797,149 and 3,109,713). A third
parameter in addition to magnetic and fluidic force which can be
adjusted to insure smooth segmented flow of microdevices is the
surface tension of the liquid(s) which can be regulated by the use
the appropriate solvents or additives (e.g. surfactants). The
ability to alter surface tension by choice of solvents is known to
anyone trained in the art.
[0484] In another example, microdevices can be sorted using the
apparatuses (i.e.: particle switches) that can switch and
manipulate particles. U.S. patent application Ser. No. 09/678,263,
filed on Oct. 3, 2000, titled "Apparatus for switching and
manipulating particles and methods of use thereof" describe several
types of devices and apparatuses for switching, sorting and
manipulating particle. The patent application Ser. No. 09/678,263
is incorporated by reference in its entirety. The devices and
apparatuses and the methods of their use can be applied for sorting
microdevices of present invention. For example, traveling-wave
dielectrophoresis can be used as a mechanism for sorting particles
via a particle-switching device. The particle switching device
comprises at least three sets of electrodes which are electrically
independent from each other. The three or more sets of electrodes
are capable of generating respective traveling-wave
dielectrophoresis (twDEP) forces on particles to move the particles
along respective branches when the electrodes in each set of
electrodes are connected to out-of-phase signals, and said branches
are interconnected at a common junction to permit the twDEP forces
to route particles from one of the branches to another of the
branches. The end (other than the common junction of the branches)
of each branch may be used for the inlet (input) and/or outlet
(output) ports. Thus, in this example, the particle sorting device
has at least three inlet (input)/outlets (outputs). Consider an
example where the particle sorting device has one inlet and two
outlet ports. Microdevices of the present invention can be fed into
the inlet port and then transported along the branches within the
particle sorting device to be outputted in one of the two outlets,
depending on the electrical voltage signals applied to the
electrodes. More importantly, for a given microdevice of the
present invention, it is possible to first perform photo-analysis
to determine the photorecognizable coding pattern on the
microdevice and then according to its coding pattern, appropriate
electrical signals can be applied to the electrodes within the
particle sorting device so that the microdevice can be transported
and sorted to one of the two outlet ports. An array of such
particle sorting devices can be used for sorting microdevices into
more than two outlet ports (or more than two output
points/positions). Examples of such multiple
particle-sorting-device used in an array format are also disclosed
in the U.S. patent application Ser. No. 09/678,263, which is
incorporated by reference in its entirety.
[0485] Microdevices can also be sorted using a flow system, which
has one inlet port and multiple outlet ports. The flow system can
transport microdevices from the inlet port to any one of multiple
outlet ports. Each microdevice can be flown through an optical
decoder (in the flow system), which can identify the
photorecognizable coding pattern of the microdevice, and is then
directed to different outlet ports according to the identified
coding pattern on the microdevice by changing the fluid flow
patterns in the flow system.
[0486] Any other sorting method that can sort microdevices
according to their photorecognizable coding patterns can be
used.
[0487] Any number of suitable entity(ies) can be synthesized on a
single microdevice. For example, a single entity or a plurality of
entities can be synthesized on a single microdevice. Preferably, a
single entity is synthesized on a single microdevice.
[0488] The present method can be used to synthesize any kind of
library. For example, the synthesized entities can be peptides,
proteins, oligonucleotides, nucleic acids, vitamins,
oligosaccharides, carbohydrates, lipids, small molecules, or a
complex or combination thereof. Preferably, the synthesized library
comprises a defined set of entities that are involved in a
biological pathway, belongs to a group of entities with identical
or similar biological function, expressed in a stage of cell cycle,
expressed in a cell type, expressed in a tissue type, expressed in
an organ type, expressed in a developmental stage, entities whose
expression and/or activity are altered in a disease or disorder
type or stage, or entities whose expression and/or activity are
altered by drug or other treatments.
[0489] In a specific embodiment, the synthesized library comprises
a defined set of nucleic acid, e.g., DNA or RNA, fragments such as
a defined set of nucleic acid fragments that cover an entire
genome, e.g., the entire human genome sequence. Preferably, each of
the nucleic acid fragments in the synthesized library comprises at
least 2, 3, 5, 10, 15, 20, 25, 50, 75, 100, 200, or 500
nucleotides.
[0490] In another specific embodiment, the synthesized library
comprises a defined set of protein or peptide fragments such as a
defined set of protein or peptide fragments that cover protein or
peptide sequences encoded by an entire genome, e.g., the entire
human genome sequence. Preferably, each of the protein or peptide
fragments in the synthesized library comprises at least 2, 3, 5,
10, 15, 20, 25, 50, 75, 100, 150, 200, 300, 400 or 500 amino acid
residues.
[0491] In still another specific embodiment, a library that is
synthesized according to the above-described method is
provided.
E. Preferred Embodiments
[0492] In one specific embodiment, the present invention is
directed toward a method for arraying microdevices (or MicroDisks)
in predetermined geometries using magnetic forces. A MicroDisk is a
microfabricated particle ranging in size from 1-1000.mu. on a side
and containing one or more strips or bars of magnetic material.
These bars must have the property of having a preferential axis of
magnetization. Such a property is a consequence of the physical
geometry of the magnetic material and, typically, will consist of a
thin film (generally less than 1.mu.) bar having a length to width
ratio of greater than 3. Typically, the preferential axis of
magnetization of a bar is its major axis. An example of a MicroDisk
containing two magnetic strips or bars is shown in FIG. 18. For
example in the presence of a magnetic field as indicated by the
arrow, the MicroDisks will orient or rotate so that the
preferential axis of magnetization will be parallel or
substantially parallel to the field. For such MicroDisks, the
preferential axis of magnetization is aligned with its major axis,
or is its major axis. If not spatially constrained, MicroDisks will
form chains and clusters as shown in FIG. 19 (the arrow indicating
the direction of applied magnetic field). Chains may be constrained
to a channel as shown in FIG. 20. A 90-degree rotation of the
magnetic field once the MicroDisk chains are constrained in a
channel will cause the MicroDisks to rotate and separate as shown
in FIG. 21. The process of steps illustrated in FIGS. 19-21
comprises "magnetic arraying".
[0493] The first step in the process, formation of chains and
clusters, occurs spontaneously in the presence of a magnetic field.
In order to be moved into channels clusters must be disrupted. This
process is accomplished by rotating the magnetic field. Guiding
posts (discussed below in the description of microchannels) may be
used to provide pivot points for the rotating clusters and chains,
thereby facilitating their rearrangement. A series of properly
constructed posts leads to the creation of chains of narrow width.
The chain may be wider than the width of a single MicroDisk.
[0494] Chains can then be moved into channels using magnet force or
fluidic force or a combination of the two. Chains will move along
lines of increasing magnetic field strength. If the
length-direction of the chain (which is substantially aligned with
the preferential axis of magnetization of MicroDisk) aligns with or
substantially aligns with the movement direction, then a smaller
hydrodynamic dragging resistance is exerted on the chains, leading
to a faster movement. On the other hand, it appears that, at least
for individual MicroDisks, larger magnetic force is exerted on the
MicroDisks if the preferential axis of magnetization is
perpendicular or substantially perpendicular to the movement
direction along which the magnetic field is increased in magnitude.
For these reasons, the chains move most efficiently when the
length-direction of the chain is at angles less than 90 degree to
the direction along which the magnetic field is increased in
magnitude, typically around 45 degrees, although the chains can
also move at other degrees. Such magnetic field gradients can be
generated by large permanent magnets or electromagnets as well as
by a series of small electromagnets either within or adjacent to
the surface of the channels. Once the MicroDisks are in the channel
rotation of the magnetic field so that it is perpendicular to the
MicroDisks chain (as well as the channel) results in the individual
MicroDisks rotating to align with the field.
[0495] Selection of optimal dimensions for the MicroDisks and
channels is important. The amount of overlap of MicroDisks in the
chains is dependent on the shape of the magnetic strips or bars
within or on the MicroDisk and the thickness of the MicroDisk. In
the example shown in FIG. 18, disks would be expected to overlap by
20-30% when in the chain configuration. By having a length to width
ratio of 1.22 (90.mu./70.mu.) when the MicroDisks are rotated in
the channel, there is no requirement for a significant change in
the relative positions of the individual MicroDisk's center of mass
within the channel due to the rotation of the magnetic field. By
contrast, circular MicroDisks either would remain overlapped or
would, as a consequence of magnetic repulsion, spread laterally
through channel.
[0496] The optimal width of the channel is controlled by two
factors. The channel must be wide enough to allow the MicroDisks to
rotate, for the example shown in FIG. 18 the diagonal of the
MicroDisk is .about.114.mu.(= {square root over
(90.sup.2+70.sup.2)}) hence this is the minimum width. The channel
should be narrow enough to prevent two disks from forming a chain
when their magnetic bars or their major axis are perpendicular to
the axis of the channel when a magnetic field in the direction
along the channel with is applied. In the example shown in FIG. 18
where the MicroDisk has a dimension of 90 .mu.m by 70 .mu.m for its
major surfaces, assuming an overlap of .about.30%, the length of
two overlapping MicroDisks would be .about.153 .mu.m
(=90+90-90.times.0.3), hence this is the maximum width. For
overlaps of 10% or 20%, the corresponding maximum width would be
171 .mu.m or 162 .mu.m. Channel height is also important since in a
strong magnetic field the MicroDisks will tend to stand upright.
When the constraint on the microdevices by a magnetic field alone
is sufficient to prevent microdevices from taking such a
prohibitive position, the height of the microchannels may become
irrelevant in this consideration. The arraying principles discussed
above and illustrated in FIGS. 19-21 are dependent on the
MicroDisks being constrained to lie flat in a plane. Consequently,
the height of the channels should be less than the narrow dimension
of the MicroDisks. A MicroDisk having angles of elevation slightly
less than 90 degrees with respect to the bottom surface of the
microchannel may be stable if the microchannel is covered with a
lid or otherwise sealed on the top. The minimum angle of elevation
which still permits stable standing of the MicroDisks is dependent
on the strength of the magnetic field, the amount of magnetic
material and its saturation magnetization, as well as the weight
and density of the MicroDisks and the density of the surrounding
fluid. While these values can be determined either empirically or
through modeling, elevation angles less than 45 degrees would
generally result in the Microdisks lying flat in the microchannels.
For these reasons, for the MicroDisk shown in FIG. 18 a maximum
channel height that prevents the MicroDisk standing up in the
channel is .about.50.mu.=(70.mu..times.sin(45)).
[0497] The shape of the magnetic bars (or magnetic strips) within
or on the MicroDisks can be tailored to direct certain types of
chains and clusters to form and to alter the amount of overlap
between MicroDisks. FIG. 22 shows some examples of other types of
bars.
[0498] MicroDisks can be encoded in a variety of ways to make them
individually identifiable. The preferred encoding method is one
generated during the fabrication of the MicroDisks such as 2-D bar
coding or inclusion of optical character recognition (OCR)
characters as shown in FIG. 23.
[0499] Encoded MicroDisks can be fabricated using any methods known
in the art. A typical MicroDisk as shown in FIG. 18 would consist
of four regions. Magnetic bars or strips are shown in light gray.
Dark gray region (e.g., made of the material Aluminum, Al) is an
encoding region. The surrounding white edge indicates the regions
that encapsulate the magnetic bars and encoding region and provide
the surface for modification. This edge could be any simple
material e.g., silicon, ceramic, metal, etc., though a preferred
material is SiO.sub.2. These different regions are also located
separately along the thickness direction. The magnetic bars and the
encoding region are located in the middle, and are encapsulated by
the top and bottom layers that correspond to the surrounding white
edge.
[0500] The magnetic bars within or on the MicroDisks can be
constructed out of any magnetic material. Preferentially, they will
be constructed out of a material of low magneto-restriction, low
remanence, but containing a high saturation magnetization. For
example, CoTaZr alloys meet these criteria. Materials of higher
remanence, e.g., nickel, are compatible with the magnetic arraying
process and may be used. The encoding layer may be constructed out
of any non-magnetic material. For example, aluminum, gold, or
copper could be used. Unlike encoded beads using fluorescent
labels, microfabricated bar codes such as those shown in FIG. 23
have no inherent technological limit to the number of different
codes. FIG. 23 shows a 4-digit OCR representation, considering only
capital and lower case letters and digits (62 characters) results
in over 10.sup.7 possible unique representations for that type of
encoding.
[0501] The ability to array allows rapid reading of encoding
information on the MicroDisk without the need for complex optics
with multiple orientations and flow systems. Arrays are compatible
with long-term storage and archiving. However, unlike conventional
arrays where all captured molecules are fixed to the same surface,
in a MicroDisk array each type of captured molecule is bound to a
different surface. Consequently, individual MicroDisks can also be
used for sequential methods of analysis. For example, following the
initial screening a desired subset of MicroDisks could be reprobed
with a different detection molecule or could be subjected to
another form of analysis, e.g., sequencing or mass spectroscopy.
Capture on a MicroDisk in practical terms corresponds to
purification of the captured molecule. Therefore, MicroDisks, when
coupled with a sorting technology, can be used to purify moieties,
including proteins, DNA, cells, etc.
[0502] Another aspect of this invention is directed towards the
sorting of MicroDisks. Once inside the channel MicroDisks can be
moved either individually or in chains through the channel. These
channels can be branched to direct output towards different
collection chambers using magnetic force. For example, in the case
of DNA synthesis each channel can lead to one of four tubes (A, T,
C, or G). Such directional channels could also be used to isolate
specific subsets of disks for further analysis (See e.g., U.S.
patent application Ser. No. 09/924,428, filed Aug. 7, 2001). Other
sorting methods described in Section D could also be used for
sorting of MicroDisks.
[0503] The term "magnetic bars", in addition to rectangular shapes,
includes rod-like shapes as well as slightly irregular shapes that
still exhibit a preferential axis of magnetization, e.g., elongated
pyramidal shapes. While the examples have been confined to flat
particles (MicroDisks), the microdevices of the present invention
can have any shape including spherical beads. The simplest
microdevice consists of a single magnetic bar that is encoded. This
encoding can be created during fabrication e.g., by
photolithography or it can be added after fabrication of the bar,
e.g., by coupling a fluorophore.
[0504] As defined above arraying consists of displaying
microdevices in an ordered format such that the encoding pattern is
readable. While the preferred form of arraying is for said
microdevices to be in channels where their preferential axis of
magnetization is perpendicular to the major axis or length axis of
the microchannels, the chains of disks shown in FIG. 26 on a glass
surface can already be photoanalyzed or detected for the encoding
patterns of each individual MicroDisk. Furthermore, while the
preferred form of arraying is within channels (as shown in FIGS. 21
and 28), arraying can be carried out on any flat surface e.g., a
glass slide (as shown in FIG. 26). In addition, arrays can be
effectively formed in chains even if adjacent MicroDisks would
overlap. This can be accomplished by employing certain "accessory"
MicroDisks that do not contain an encoding pattern and are
transparent. By adding an appropriate excess of such transparent,
"accessory" MicroDisks to the mxiture of encoded MicroDisks before
chain formation, the probability of two encoded MicroDisks being
adjacent in the chain will be very small. Thus, by simply forming
chains of MicroDisks using a magnetic field, we can effectively
achieving the arraying of the encoded MicroDisks.
[0505] While arraying is generally considered a static process,
this need not be the case. For example, particles can be moved
through channels and the encoding pattern and other information be
read. The encoding pattern and other information can be read by any
suitable sorting instruments e.g., FACS machines, while sorting is
carried out.
[0506] In addition to enabling arraying, microdevices with a
preferential axis of magnetization are able to rotate in a
controlled manner within a channel in response to changes in the
direction of the external applied magnetic field. This rotation
facilitates mixing, thereby enhancing reaction kinetics and
solution uniformity.
F. Examples
Protein Profiling
[0507] Encoded MicroDisks bearing a SiO.sub.2 surface are coated
using a silane to provide activatable functional groups, e.g.,
coating with 3-aminoproplytrimethoxysilane to provide an amine
surface. The functional groups are activated for coupling e.g. an
amine surface is activated using N-hydroxysulfosuccinimide and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and capture
antibodies are covalently linked to the surface through primary
amino groups. Many such encoded MicroDisks, each containing a
different capture antibody, can be made in this manner.
Antibody-containing MicroDisks are then incubated with a sample
containing antigens (or proteins) recognized by the capture
antibodies and biotinylated detection antibodies that recognize
those same antigens (or proteins). After a suitable incubation
time, fluorescently labeled streptavidin is added and after further
incubation, the MicroDisks are arrayed and subjected to analysis on
an optical reader to detect the encoding pattern and on a
fluorescence reader to determine the level of bound antigen (or
protein).
[0508] mRNA/cDNA Profiling
[0509] Encoded MicroDisks encapsulated in SiO.sub.2 are modified
using a silane to generate an aldehyde surface--the preferred
chemistry for linking synthetic oligonucleotides to a surface (see
"Comparison between different strategies of covalent attachment of
DNA to glass surfaces to build DNA microarrays" by Zammatteo et.
al. Anal. Biochem., 280:143-150 (2000)). This can be accomplished
by coating the MicroDisks bearing an --SiO.sub.2 surface with
3-glycidoxyproplytrimethoxysilane and hydrolyzing the resulting
epoxide to a diol. The diol surface is converted to an aldehyde by
periodate oxidation and an amino-tagged synthetic capture
oligonucleotide is covalently linked to the surface. Many such
encoded MicroDisks, each containing a different capture
oligonucleotides, can be made in this manner.
Oligonucleotide-containing MicroDisks are then incubated with a
sample containing fluorescently-labeled cDNA complementary to the
capture oligonucleotide. After a suitable incubation time and
washing steps, the MicroDisks are arrayed and subjected to analysis
on an optical reader to detect the encoding pattern and on a
fluorescence reader to determine the level of bound antigen.
[0510] Library Synthesis
[0511] In the absence of a device or instrument that can sort
individual MicroDisks, library synthesis is random. Using the split
and pool method libraries can be synthesized directly onto the
MicroDisks. After each step in the synthesis, the MicroDisks are
arrayed and optically decoded before proceeding onto the next
synthesis cycle. For example in the case of DNA, after the first
cycle the disks are mixed and divided into four groups, one group
each for A, C, T, and G bases. The four groups are arrayed and
optically decoded and the information is stored. The process is
then repeated for each cycle. At the end of the synthesis the
identity of the oligonucleotide on each MicroDisk is known. In this
method of random library synthesis, no two microdevices should have
same photorecognizable coding/encoding pattern, because two
microdevices with same photorecognizable coding pattern may go
through different synthesis cycles and result in different
synthesized entities with no method to distinguish between them. In
other words, for this example, each microdevice must have a unique
photorecognizable coding pattern. On the other hand, in this method
of random library synthesis, it is possible for two microdevices
having different photorecognizable coding patterns to go through
same synthesis cycles, resulting in their having the same
synthesized entities. The synthesized libraries can be used for
screening. Such a library-synthesis technique could also be used to
generate peptide libraries. Any library typically generated on
beads could be synthesized on MicroDisks. A very large number of
such libraries are known to those practiced in the art of
combinatorial chemistry (e.g. "Comprehensive survey of
combinatorial library synthesis; 1999" by Dolle Journal of
Combinatorial Chemistry, 2:383-433 (2000)). This technique requires
that each MicroDisk contain a unique code.
[0512] A second and more valuable method of library synthesis
involves the use of a sorting step after each synthesis cycle. In
this method, individually encoded MicroDisks are assigned a target
sequence prior to the initiation of library synthesis. After each
step in the synthesis, each MicroDisk is directed to the
appropriate reaction chamber. Procedure and the specific sequences
are preassigned to individual particles. For example, in the case
of synthesizing oligonucleotides an encoded MicroDisk assigned the
sequence ATCAGTCATGCG (SEQ ID NO:1) would go to the A tube in the
first step of synthesis then to the T tube in the second, the C
tube in the third, etc. The complete space of the library is
determined prior to synthesis and may correspond to a subset of the
entire sequence space available e.g., 10.sup.7 specific 50-residue
oligonucleotides out of a sequence space of 10.sup.30, or in the
case of peptides, 10.sup.7 specific 20-residue peptides out of a
sequence space of 10.sup.26. In both of these examples, it
therefore is possible to generate libraries not available by random
synthetic methods (or any methods). Moreover, such techniques can
be used to generate genome-specific libraries, e.g., all 50-residue
oligonucleotides or all 20-residue peptides present in the human
genome. In addition, since the encoded MicroDisks are sorted at
each step it is possible to generate multiple copies of the same
library in a single synthesis because all MicroDisks containing the
same code will be sorted together at each step in the synthesis.
For screening purposes, this means that the number of copies of
individual MicroDisks can be controlled and more importantly,
libraries can be subdivided or mixed with subsets of other
libraries to generate new libraries of known sequence.
[0513] A major implementation in the synthesis of libraries
involves generating a template or scaffold that contains variable
regions. Many researchers and companies (e.g. Affibody, Phylos,
Ribozyme Pharmaceuticals, Somalogic) have utilized such an approach
to generate synthetic antibodies, enzymes, or molecules capable of
specific molecular recognition (e.g. aptamers), enzymatic activity
(e.g. ribozymes), or signaling (e.g. by fluorescence intensity or
fluorescence energy transfer). A common feature of these approaches
is that they rely on the use of enzymes (In vitro or Ex vivo) to
generate secondary libraries and/or to interpret the results. For
example, in the case of aptamer selection, aptamers typically are
generated through the SELEX process (Systematic Evolution of
Ligands by Exponential enrichments--e.g. U.S. Pat. No. 6,048,698).
This involves random synthesis (though flanking regions of specific
"template" sequence are required) and then screening to obtain a
subpopulation with desired binding properties. This subpopulation
is then expanded and randomized by PCR-based methods and screened.
This iterative process of expansion and screening is continued
until an aptamer of desired specificity and affinity has been
generated.
[0514] An alternative approach in which screening and expansion are
carried out using MicroDisks offers two major advantages. The first
is that all requirements that the polymer be amplifiable by an
enzymatic process are removed. Consequently, since the polymers in
each library iteration can be generated exclusively by chemical
synthesis. The polymer can comprise virtually any type or
combination of subunits, e.g. nucleotide, amino acid, small organic
molecule, sugar, protein-nucleic acid, etc. The tremendously
increased diversity of MicroDisk generated libraries enhances the
likelihood of being able to produce molecules that carry out
particular functions under extremes of condition, e.g., using the
molecules in the libraries for capturing proteins under
protein-denaturing conditions. Such libraries can be produced using
conventional bead based synthesis, but screening and production of
further generation libraries becomes rate limiting. In conventional
bead-based synthesis, a small subset is identified by analytical
methods, e.g. mass spectroscopy, and it is impractical to evaluate
the properties of all the members of the library. However, since
the identity of each MicroDisk is known though optical decoding,
all members of a MicroDisk library can be evaluated. For example,
in a library of 10.sup.10 MicroDisks, the binding efficiency of all
members of the library can be determined and a subset of sequences
can be used as starting points to generate the next generation
library. Furthermore, in each library generation information about
the measured properties of all library components is retained,
facilitating the use of computational approaches to select future
generations. Such computational methods benefit greatly from the
ability to incorporate conformational constraints into the library,
e.g., through the use of specific crosslinks or conformationally
constrained subunits. As a result of the huge amount of information
obtained during each library screening cycle, using MicroDisk
technology the conventional random approach is replaced by a guided
systematic one.
[0515] The above examples are included for illustrative purposes
only and are not intended to limit the scope of the invention. Many
variations to those described above are possible. Since
modifications and variations to the examples described above will
be apparent to those of skill in this art, it is intended that this
invention be limited only by the scope of the appended claims.
* * * * *