U.S. patent application number 11/503401 was filed with the patent office on 2010-10-14 for apparatus for assay, synthesis and storage, and methods of manufacture, use, and manipulation thereof.
Invention is credited to Colin Brenan, Robert Hess, Tanya S. Kanigan, John Linton, Can Ozbal.
Application Number | 20100261159 11/503401 |
Document ID | / |
Family ID | 46332237 |
Filed Date | 2010-10-14 |
United States Patent
Application |
20100261159 |
Kind Code |
A1 |
Hess; Robert ; et
al. |
October 14, 2010 |
Apparatus for assay, synthesis and storage, and methods of
manufacture, use, and manipulation thereof
Abstract
The invention features methods of making devices, or "platens",
having a high-density array of through-holes, as well as methods of
cleaning and refurbishing the surfaces of the platens. The
invention further features methods of making high-density arrays of
chemical, biochemical, and biological compounds, having many
advantages over conventional, lower-density arrays. The invention
includes methods by which many physical, chemical or biological
transformations can be implemented in serial or in parallel within
each addressable through-hole of the devices. Additionally, the
invention includes methods of analyzing the contents of the array,
including assaying of physical properties of the samples.
Inventors: |
Hess; Robert; (Arlington,
MA) ; Linton; John; (Lincoln, MA) ; Kanigan;
Tanya S.; (Cambridge, MA) ; Brenan; Colin;
(Marblehead, MA) ; Ozbal; Can; (Cambridge,
MA) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Family ID: |
46332237 |
Appl. No.: |
11/503401 |
Filed: |
August 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10315832 |
Dec 10, 2002 |
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11503401 |
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09975496 |
Oct 10, 2001 |
6716629 |
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10315832 |
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60707501 |
Aug 11, 2005 |
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60239538 |
Oct 10, 2000 |
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60268894 |
Feb 14, 2001 |
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60284710 |
Apr 18, 2001 |
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Current U.S.
Class: |
435/6.14 ;
427/240; 427/402; 435/29; 435/305.2; 435/325; 435/7.1; 506/9 |
Current CPC
Class: |
B01J 2219/00511
20130101; B01J 2219/00423 20130101; G01N 30/82 20130101; B01J
19/0046 20130101; B01J 2219/00387 20130101; B01L 3/5025 20130101;
B01J 2219/00351 20130101; B01J 2219/0036 20130101; B01J 2219/00673
20130101; B01J 2219/00389 20130101; B01J 2219/00648 20130101; G01N
30/466 20130101; B01J 2219/00369 20130101; B01L 3/0268 20130101;
B01L 13/02 20190801; B01J 2219/00479 20130101; B01J 2219/00587
20130101; B01J 2219/00585 20130101; B01J 2219/00317 20130101; B01L
3/5085 20130101; B01L 3/0262 20130101; B01J 2219/0043 20130101;
B01J 2219/00495 20130101; B01L 2300/0845 20130101; C12M 23/12
20130101; B01J 2219/005 20130101; B01J 2219/00653 20130101; G01N
30/6095 20130101; B01J 2219/00319 20130101; B01J 2219/00596
20130101; B01L 3/50255 20130101; B01J 2219/00659 20130101; G01N
30/6091 20130101; C40B 60/14 20130101; G01N 2030/8417 20130101;
B01L 2200/0657 20130101; B01L 3/50857 20130101; B01L 3/0244
20130101 |
Class at
Publication: |
435/6 ;
435/305.2; 435/325; 427/402; 427/240; 435/29; 506/9; 435/7.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/14 20060101 C12M001/14; C12N 5/00 20060101
C12N005/00; B05D 1/36 20060101 B05D001/36; C12Q 1/02 20060101
C12Q001/02; C40B 30/04 20060101 C40B030/04; G01N 33/53 20060101
G01N033/53 |
Claims
1-72. (canceled)
73. A cell chip comprising first and second platens, each having a
plurality of through-holes, and a porous membrane, wherein the
platens are aligned such that the through-holes of the first platen
are substantially aligned with the through-holes of the second
platen and the membrane is sandwiched in between the two
platens.
74. The cell chip of claim 73, wherein the membrane comprises pores
that are no more than half the through-hole diameter.
75. The cell chip of claim 73, wherein the membrane comprises pores
of between about 0.2-250 .mu.m.
76-77. (canceled)
78. The cell chip of claim 77, wherein the membrane comprises
aluminum oxide or polycarbonate.
79-80. (canceled)
81. The cell chip of claim 79, wherein the polycarbonate is coated
with fibronectin, laminin, collagen, or another substrate that
supports cell adhesion.
82. The cell chip of claim 73, wherein the platens comprise
polystyrene or a metal selected from the group consisting of gold,
Tungsten or stainless steel.
83-84. (canceled)
85. The cell chip of claim 73, wherein the chip further comprises a
gasket that seals off individual wells.
86. The cell chip of claim 85, wherein the gasket is removable.
87. The cell chip of claim 73, wherein the chip further comprise a
hydrophobic compound that prevents lateral diffusion.
88. (canceled)
89. The cell chip of claim 73, wherein one platen comprises a
flexible biocompatible material selected from the group consisting
of silicone, polypropylene, or rubber and the other platen is a
rigid platen that supports the flexible platen.
90. (canceled)
91. The cell chip of claim 73, wherein the two platens are attached
by raised surfaces on one platen that fit into a recessed surface
on the other platen.
92-94. (canceled)
95. The cell chip of claim 73, further comprising a solid support
in contact with the first platen.
96. The cell chip of claim 95, wherein the solid support is a
microscope slide.
97. The cell chip of claim 96, further comprising a coverslip in
contact with the second platen.
98. The cell chip of claim 97, wherein the coverslip, microscope
slide, and cell chip are secured together.
99. A cell chip comprising in order from top to bottom: (a) a
coverslip in contact with a spacer; (b) a spacer that separates the
covership from a first platen; (c) a first platen having a
plurality of through-holes; (d) a gasket comprising a plurality of
through-holes that provides a seal between the first platent and
the membrane; (e) a porous membrane comprising aluminum oxide and
having pores between 0.1 and 1 .mu.m sandwiched between the gasket
and the second platen; (f) a second platent having a plurality of
through holes; (g) a solid support in contact with the second
platen.
100. The cell chip of claim 99, wherein the chip further comprises
a fastener that holds the various components together.
101. The cell chip of claim 99, wherein the membrane comprises a
uniform structure of pores that are 0.2 .mu.m in diameter.
102. The cell chip of claim 99, wherein the platens comprise
tungsten, gold, or stainless steel.
103. A method of culturing a cell on a cell chip, the method
comprising: (a) providing a cell chip of claim 73 or 99 comprising
cell culture medium; (b) contacting the porous membrane with a
cell; and (c) incubating the cell under conditions suitable for
cell survival.
104. The method of claim 103, wherein the conditions comprise
contacting the cell chip a gas permeable liquid.
105. The method of claim 104, wherein the gas permeable liquid is
perfluorodecalin.
106. The method of claim 104, wherein the cell chip further
comprises a hydrophobic fluid in contact with the cell culture
medium, wherein the hydrophobic liquid is selected from the group
consisting of perfluorodecalin, silicone oil or and mineral oil
107. (canceled)
108. A method of constructing a cell chip of claim 73 or 99 the
method comprising: (a) filling a first platen having a plurality of
through-holes with cell culture medium; (b) contacting the first
platen with a porous membrane; (c) contacting the membrane with a
second platen having a plurality of through-holes, such that the
through-holes are substantially aligned, thereby constructing a
cell chip.
109. The method of claim 108, the cell chip further comprising a
solid support in contact with the first platen.
110. The method of claim 109, wherein the cell chip further
comprises a spacer in contact with the second platen, wherein the
spacer is in contact with a cover slip.
111. The method of claim 108, wherein the cell chip further
comprises a gasket sandwiched between the first platen.
112. The method of claim 108, wherein the gasket comprises a
flexible material or a biocompatible elastomer selected from the
group consisting of teflon, silicone, or rubber.
113-114. (canceled)
115. The method of claim 108, wherein the filling is accomplished
by placing the first platen on a solid support and centrifuging the
platen and solid support.
116. The method of claim 108, wherein the first platen is contacted
with a gasket and cell medium is then overlayed on the platen.
117. A method for identifying an agent having a desired biological
activity, the method comprising: (a) contacting a cell chip of
claim 73 or 99 comprising a cell with a platen comprising an agent;
(b) contacting the cell with the agent; and (c) detecting an
alteration in the cell, thereby identifying an agent having a
desired biological activity.
118. The method of claim 117, wherein the agent is present in cell
growth medium, or is contacted with the cell using a slotted pin or
syringe, or by adding the cell to a well comprising the agent.
119. The method of claim 117, wherein the agent is a polypeptide,
nucleic acid molecule, or small compound.
120. The method of claim 117, wherein the nucleic acid molecule is
an siRNA, microRNA, or an aptamer.
121. The method of claim 117, wherein the alteration is an
alteration in gene expression, polypeptide expression, cell growth,
proliferation or survival, in the intracellular localization of a
cellular component, morphological change, or change in
motility.
122. The method of claim 117, wherein the alteration is detected in
an immunoassay, an enzymatic assay, highthroughput gene expression
profiling, reverse transcriptase polymerase chain reaction
(RT-PCR), quantitative PCR, real time PCR, methylation, or high
content screening (HCS) using quantitative fluorescence microscopy
and automated image acquisition.
123. The method of claim 117, wherein the high content screening
detects alterations in protein translocation.
124. The method of claim 117, wherein the cell is lysed and the
proteins or nucleic acid molecules are bound on a binding
surface.
125. The method of claim 117, wherein the binding surface is a weak
cationic exchange medium.
126. The method of claim 122, wherein the bound proteins or nucleic
acid molecules are analyzed for a characteristic selected from the
group consisting of sequence, molecular weight, binding
characteristic, and expression level.
127. The method of claim 122, wherein the binding characteristic is
detected in an immunoassay or by polypeptide binding.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/707,501, which was filed Aug. 11, 2005; and is a
continuation-in-part of U.S. patent application Ser. No.
10/315,832, which was filed on Dec. 10, 2002, which is a divisional
application of U.S. patent application Ser. No. 09/975,496, which
was filed on Oct. 10, 2001, and is now issued as U.S. Pat. No.
6,716,629, and which claims the benefit of U.S. Provisional
Application No. 60/239,538, filed Oct. 10, 2000, U.S. Provisional
Application No. 60/268,894, filed Feb. 14, 2001, and U.S.
Provisional Application No. 60/284,710, filed Apr. 18, 2001; each
of the foregoing applications are hereby incorporated by reference
in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to devices for molecular synthesis,
storage and screening, and other chemical, biochemical, biological,
and physical experiments, and to methods of making, using, and
manipulating the same.
BACKGROUND OF THE INVENTION
[0003] High throughput methods for creating and analyzing chemical
and biochemical diversity play a vital role in technologies
including drug discovery and development. Specific applications of
high throughput methods include drug discovery, optimization of
reaction conditions (e.g., conditions suitable for protein
crystallization), genomics, proteomics, genotyping, polymorphism
analysis, examination of RNA expression profiles in cells or
tissues, sequencing by hybridization, and recombinant enzyme
discovery.
[0004] Rapid, high throughput methods for synthesizing (e.g., using
combinatorial chemistry methods) and screening large numbers of
these compounds for biological and physicochemical properties are
desired, for example, to increase the speed of discovery and
optimization of drug leads.
[0005] Similarly, due in part to the large amount of sequence data
from the human genome project, efforts are underway to rapidly
obtain x-ray crystallography data for the protein products of many
newly discovered genes. One of the rate limiting steps in this
process is the search for appropriate solution conditions (e.g.,
pH, salt concentration) to cause protein crystallization. There is
also a need to determine the function of each of the newly
discovered genes (i.e., "functional genomics") and to map
protein-protein interactions (i.e., "proteomics"). Given the large
number of human genes, protein modifications, and protein binding
partners, higher throughput methods are desired.
[0006] Another advance in biotechnology is the creation of surfaces
with high-density arrays of biopolymers such as oligonucleotides or
peptides. High-density oligonucleotide arrays are used, for
example, in genotyping, polymorphism analysis, examination of RNA
expression profiles in cells or tissues, and hybridization-based
sequencing methods as described, for example, in U.S. Pat. Nos.
5,492,806, 5,525,464, and 5,667,972 to Hyseq, Inc. Arrays
containing a greater number of probes than currently provided are
desirable.
[0007] The process of discovering and improving recombinant enzymes
for industrial or consumer use has emerged as an important economic
activity in recent years. A desire to discover very rare,
activity-improving mutations has further stimulated the search for
higher throughput screening methods. Such methods often require
screening 100,000 to 1,000,000 members of a genetic library in
parallel, and then rapidly detecting and isolating promising
members for further analysis and optimization.
[0008] One of the challenges in the development of high throughput
methods is that conventional liquid handling techniques such as
pipetting, piezoelectric droplet dispensing, split pin dispensing,
and microspritzing are generally unsuitable for rapidly loading or
transferring liquids to or from plates of high density (e.g.,
plates having more than about 384 wells). For example, these
techniques can cause substantial splashing, resulting, for example,
in contamination of neighboring wells and loss of sample volume.
Also, as the number of wells increases, the time necessary to
reformat compounds from the previous generation of plates to the
higher density plates generally increases, thus limiting the
utility of higher density plates. Evaporation can also be
problematic with times greater than a few seconds. Moreover,
entrapped air bubbles can result in inconsistencies in the loading
of small fluid volumes (e.g., less than about one microliter).
[0009] Significant bottlenecks in high throughput screening efforts
include library storage, handling, and shipping. As the number of
compounds in a library increases, the number of 96- or 384-well
plates, and the total volume needed to store the libraries, also
increases. For compounds that are stored in frozen solvent such as
DMSO or water, thawing, dispensing, and refreezing pose the hazard
of crystallization, precipitation, or degradation of some
compounds, making it difficult to dispense accurate quantities in
the future. Having samples stored in low-density plates requires a
time consuming step of reformatting the samples into high-density
plates before the high-density technology can be utilized.
SUMMARY OF THE INVENTION
[0010] The invention features methods of making devices, or
"platens", having a high-density array of through-holes, as well as
methods of cleaning and refurbishing the surfaces of the platens.
The invention further features methods of making high-density
arrays of chemical, biochemical, and biological compounds, having
many advantages over conventional, lower-density arrays. The
invention includes methods by which many physical, chemical or
biological transformations can be implemented in serial or in
parallel within each addressable through-hole of the devices.
Additionally, the invention includes methods of analyzing the
contents of the array, including assaying of physical properties of
the samples.
[0011] In various embodiments, the reagents can be contained within
the through-holes by capillary action, attached to the walls of the
through-holes, or attached to or contained within a porous material
inside the through-hole. The porous material can be, for example, a
gel, a bead, sintered glass, or particulate matter, or can be the
inner wall of a through-hole that has been chemically etched. In
particular embodiments, the arrays can include individual
molecules, complexes of molecules, viruses, cells, groups of cells,
pieces of tissue, or small particles or beads. The members of the
arrays can also, for example, function as transducers that report
the presence of an analyte (e.g., by providing an easily detected
signal), or they can function as selective binding agents for the
retention of analytes of interest. Using these methods, arrays
corresponding to a large plurality of human genes (e.g., using
nucleic acid probes) can also be prepared.
[0012] On embodiment of the invention features a method of making a
platen of a desired thickness having a plurality of through-holes.
The method includes the steps of (a) providing a plurality (e.g.,
2, 3, 5, 8, 10, 100, 1000 or more) of plates having upper and lower
surfaces, wherein one or both of the upper and lower surfaces of at
least some of said plurality of plates has continuous,
substantially parallel grooves running the length of said surfaces;
(b) bonding the upper surfaces of all but one of said plurality of
plates to the lower surfaces of the other plates (i.e., the upper
surface of the first plate is bonding to the lower surface of the
second plate; the upper surface of the second plate is bonded to
the lower surface of the third plate; and so on; the upper surface
of the last plate is not bonded to anything else); and (c) if
necessary to achieve the desired thickness, slicing the platen
substantially perpendicularly to the through-holes, thereby
creating a platen of a desired thickness having a plurality of
through-holes. Step c) can optionally be repeated make a plurality
of platens. . By "a plurality of through-holes" is meant at least 2
(e.g., 2, 5, 10, 20, 25, 50, 100, 200, 250, 500, 25,000, 50,000 or
more). For example, a platen the size of a conventional microscope
slide may have about 3,072 holes, while a platen the size of a
microtiter plate may have about 24,576. The number of through-holes
on a microtiter plate can be 50,000, 100,000, 200,000 or more. Of
course, the number of through-holes will vary depending on the
diameter of the hole and the size of the platen. For example, where
the through-hole has a diameter of less than about 400 micrometers,
the through-hole density is at least 1.6 through-holes per square
millimeter.
[0013] The plates can be made from any material that can be bonded
(e.g., plastic, metal, glass, or ceramic), and each can have a
thickness from, e.g., about 0.01 mm to 2.0 mm, preferably 0.1 mm to
1 mm; the grooves have a depth from, e.g., 0.005 mm to 2.0 mm
(i.e., less than the thickness of the plates); and the grooves can
have a width from, e.g., 0.1 mm to 1.0 mm.
[0014] The plates can be bonded in a configuration in which the
grooves of one plate are substantially parallel to the grooves of
each of the other plates, or can be bonded so that the grooves of
certain plates are perpendicular to, or at acute angles to, the
grooves of certain other plates.
[0015] In another embodiment, the invention features a device for
the immobilization of probes, cells, or solvent. The device
includes a platen (optionally having hydrophobic upper and lower
surfaces) having a plurality of through-holes (e.g., from the upper
surface to the lower surface), where at least some of the
through-holes contain a porous material such as a gel (e.g.,
polyacrylamide), silica, sintered glass, or polymers for the
immobilization of probes, cells, or solvent.
[0016] In still another embodiment, the invention features a method
of making a platen having opposing hydrophobic surfaces and a
plurality of hydrophilic through-holes. The method includes the
steps of: (a) coating a plate with a material (e.g., gold, silver,
copper, gallium arsenide. metal oxides, or alumina) that reacts
with amphiphilic molecules (e.g., alkane thiols, alkanephosphates,
alkane carboxylates); (b) forming through-holes in the plate (e.g.,
by micromachining methods such as drilling, electrospark discharge
machining (EDM), punching, stamping, or etching; and (c) treating
(e.g., dipping or spraying) the plate with a solution or vapor of
an amphiphilic molecule to provide a platen having hydrophobic
coating on surfaces of the platen but not on the walls of the
through-holes. The invention also includes the platens made by this
method, as well as a method of regenerating the hydrophobic coating
on the platen after use. This method includes the steps of (a)
removing residual hydrophobic coating, if any (e.g., by washing the
platen with oxidant, reductant, acid, base, or detergent, or by
heating, electropolishing, irradiating, or burning); and (b)
treating the platen with a solution or vapor of an amphiphilic
molecule to regenerate the hydrophobic coating.
[0017] In yet another embodiment, the invention features a method
of selectively making a coating on the surfaces of a platen having
a plurality of through-holes. The method includes the steps of: (a)
selectively coating the surfaces of the platen with a material that
reacts with amphiphilic molecules; and (b) treating the platen with
a solution or vapor of an amphiphilic molecule to regenerate the
hydrophobic coating.
[0018] Still another embodiment of the invention features a platen
having two opposing surfaces and a plurality of through-holes
extending between the surfaces. The surfaces have different
chemical properties relative to the walls of the through-holes,
such that the walls and surfaces can be independently
functionalized. For example, the walls can be coated with gold
(e.g., by coating the entire platen, including both the walls and
the opposing surfaces with gold, and then electropolishing the
surfaces to remove the gold therefrom), allowing the walls to be
rendered hydrophobic upon treatment with alkane thiols. Conversely,
the surfaces (but not the walls) could be coated with metal oxides
so that alkanephosphates can be bound thereto.
[0019] In another embodiment, the invention features a method of
making a plastic platen of a desired thickness, having
through-holes. The method features the steps of: a) potting a
plurality of capillaries (e.g., glass or plastic capillaries) in
the through-holes of a stack of platens comprising at least two
platens having through holes; b) separating adjacent platens by a
distance equal to the desired thickness; c) injecting a
plastic-forming material into the space between the separated
platens; d) forming (e.g., heat-setting or curing) the plastic; and
e) slicing at the interface between the platens and the plastic to
form the chips. The plastic-forming material can be, for example, a
photo-, thermo-, or chemical-curable material such as a UV-curable
material, e.g., polymethylmethacrylate (PMMA), polystyrene, or
epoxy, and the forming step can entail exposing the material to
ultraviolet light; or the plastic-forming material can be a molten
thermoplastic material and the forming step can involve cooling the
material.
[0020] In still another embodiment, the invention features a method
of making a plastic chip of a desired thickness, having
through-holes. The method features the steps of: a) potting a
plurality of fibers or wires in the through-holes of a stack of
platens comprising at least two platens having through holes; b)
separating adjacent platens by a distance equal to the desired
thickness; c) injecting a plastic-forming material into the space
between the separated platens; d) forming the plastic; e)
withdrawing the fibers or wires from the plastic to form
through-holes; and f) slicing at the interface between the platens
and the plastic to form the chips.
[0021] Still another embodiment of the invention is a method of
creating a chemical array.
[0022] The method includes the steps of: a) providing a platen
having a plurality of through-holes and two opposing surfaces; b)
applying a mask to one or both surfaces of the platen to block at
least some of the through-holes, while leaving other through-holes
open; c) exposing a surface of the platen to a reagent (e.g., e.g.,
a liquid, a gas, a solid, a powder, a gel, a solution, a suspension
such as a slurry, a cell culture, a virus preparation, or
electromagnetic radiation; e.g., by spraying the platen with a
solution or suspension of the reagent, or by condensing, pouring,
depositing, or dipping the reagent onto the platen) so that the
reagent enters at least one of the open through-holes; and d)
repeating steps b) and c) (e.g., at least once, generally at least
three times; for creation of nucleic acid arrays, the steps can be
repeated four times the length of the desired nucleic acid chains;
for creation of protein arrays, the steps can be repeated twenty
times the length of the desired peptide chains) with at least one
different mask and at least one different reagent to create a
chemical array. The masks can be reusable or disposable, and can be
applied mechanically (e.g., robotically) or manually. The mask can,
in some cases, initially include the reagent (e.g., absorbed onto
or contained within it). The mask can be flexible or rigid, for
example, and can be made of a polymer, an elastomer, paper, glass,
or a semiconductor material. The mask can, for example, include
mechanical valves, pin arrays (e.g., posts, pistons, tubes, plugs,
or pins), or gas jets. In some cases, the "applying" step forms a
hermetic seal between the mask and the platen. The mask can also be
translated (e.g., moved between the repetitions of the method) to
expose different through-holes. In some cases, the mask has
co-registration pins and holes such that alignment of pins and
holes in the mask register with the through-holes in the platen. In
these cases, multiple masks can be made part of a flexible tape,
and the multiple masks are registered with the through-holes of the
platen by advancing the tape (e.g., the masks can be on a spool,
ribbon, or roll, and can be advanced in a manner analogous to the
advancing of film in a camera). Arrays created by any of these
methods are also considered to be an aspect of the invention.
[0023] In yet another embodiment, the invention features a method
of creating a chemical array. The method includes the steps of: a)
providing a platen having a plurality of through-holes and two
opposing surfaces; b) applying a mask that has one or more reagents
on its surface to one or both surfaces of the platen to transfer
the reagent from the mask to at least some of the through-holes;
and c) repeating step b) with at least one different mask and at
least one different reagent to create a chemical array.
[0024] The invention also features a method for separating samples
within a chemical array in a platen. The method includes the steps
of a) providing a platen having a plurality of through-holes and
two opposing surfaces; b) electrophoretically transporting a
charged reagent into at least some of the through-holes by placing
the platen into an electrophoresis apparatus containing the reagent
and applying an electric field parallel to the through-holes; and
c) repeating step b) with at least one different reagent to create
a chemical array.
[0025] In still another embodiment, the invention features a method
of creating a spatially addressable array. The method includes the
following steps: a) providing a platen having a spatially
addressable plurality of discrete through-holes each having an
inner wall, wherein said platen has opposing hydrophobic surfaces;
and b) covalently or non-covalently immobilizing at least one
reagent or probe on the inner walls of at least some of the
through-holes or on a bead contained within at least one of the
through-holes to form a spatially addressable array. In this
method, the through-holes can be either non-communicating (i.e.,
the contents of adjacent through-holes do not mix with each other)
or selectively communicating (i.e., the walls of at least some of
the through-holes act as semi-permeable membranes) through-holes.
In some cases, the method can also include the step of: c) flowing
reagents (e.g., monomers, wash solutions, catalysts, terminators,
denaturants, activators, polymers, cells, buffer solutions,
luminescent and chromatogenic substrate solutions, beads, heated or
cooled liquids or gases, labelled compounds, or reactive organic
molecules) into or through a predetermined subset of the through
holes.
[0026] Yet another embodiment of the invention is a method of
creating a stochastic array. The method includes a) providing a
platen having a plurality of through-holes; and b) applying each of
a plurality of reagents to the through-holes in a random or
semi-random manner (e.g., spatially random or random with respect
to distribution of reagents) to create a stochastic array. The
"applying" step can include, for example, providing a plurality of
dispensing devices addressing at least some of the through-holes,
dispensing different combinations of reagent solutions (e.g., as
solutions, neat, or in suspension) into each through-hole, and
repositioning the dispensing devices at least once to address a
different set of through-holes. In this case, the method can also
involve dispensing a fluid that is immiscible with the reagent
solutions into at least one through-hole.
[0027] In another embodiment, the invention features a method of
identifying combinations of reagents having a biological, chemical
or physical property of interest. The method involves, for example,
the use of radiolabelled probes, or the measurement of
chemiluminescence. The method features the steps of: a) creating a
stochastic array using the above method; b) assaying the stochastic
array for combinations having a property of interest; and c)
identifying the reagents that have the property of interest.
Non-limiting examples of properties of interest include catalysis
(see, e.g., Weinberg et al., Current Opinion in Solid State &
Materials Science, 3:104-110 (1998)); binding affinity for a
particular molecule (see, e.g., Brandts et al., American Laboratory
22:3041 (1990); or Weber et al., J. Am. Chem. Soc. 16:2717-2724
(1994)); ability to inhibit particular chemical and biochemical
reactions; thermal stability (see, e.g., Pantaliano et al., U.S.
Pat. Nos. 6,036,920 and 6,020,141); luminescence (see, e.g.,
Danielson et al., Nature 389:944-948 (1997)); crystal structure
(see, e.g., Hindeleh et al., Journal of Materials Science
26:5127-5133 (1991)); crystal growth rate; diastereoselectivity
(see, e.g., Burgess et al., Angew. Chem. 180:192-194 (1996));
crystal quality or polymorphism; surface tension; (see, e.g.,
Erbil, J. Phys. Chem. B., 102:9234-9238 (1998)); surface energy
(see, e.g., Leslot et al., Phys. Rev. Lett. 65:599-602 (1990));
electromagnetic properties (see, e.g., Briceno et al., Science
270:273-275 (1995); or Xiang et al., Science 268:1738-1740 (1995));
electrochemical properties (see, e.g., Mallouk et al., Extended
Abstracts; Fuel Cell seminar: Orlando, Fla., 686-689 (1996)); and
optical properties (see, e.g., Levy et al., Advanced Materials
7:120-129 (1995)); toxicity, antibiotic activity, binding, and
other biological properties; fluorescence and other optical
properties; and pH, mass, binding affinity, and other chemical and
physical properties.
[0028] In another embodiment yet, the invention features a method
of loading a platen having a plurality of through-holes, where the
platen has opposing surfaces (e.g., the surfaces are hydrophobic
and the through-holes have hydrophilic walls). The method includes
the steps of: a) dipping the platen into a liquid sample (e.g., a
neat liquid, a solution, a suspensions, or a cell culture) that
includes a sample to be loaded into the through-holes, thereby
loading at least some of the through-holes with the sample; and b)
passing the platen through a liquid that has an affinity for the
surfaces of the platen but that is immiscible with the liquid
sample, thereby cleaning the surface of the platen of excess sample
mixture (e.g., by adding, on top of the sample mixture, the
immiscible liquid, where the liquid has a lower density than the
sample mixture (e.g., mineral oil); and removing the platen from
the sample mixture through the liquid; device comprising a barrier
between the sample and the liquid).
[0029] The invention also features another method of loading a
platen having a plurality of through-holes, where the platen has
opposing surfaces. The method includes: a) dipping the platen into
a liquid sample comprising a sample to be loaded into the
through-holes, thereby loading at least some of the through-holes
with the sample; and b) contacting the platen with a liquid that
has an affinity for the surfaces of the platen but is immiscible
with the liquid sample, thereby cleaning the surface of the platen
of excess sample mixture.
[0030] The invention also features a method of maintaining the
viability of an aerobic organism in a platen having a plurality of
through-holes. The method includes the steps of: a) loading the
aerobic organism (e.g., a cell or an embryo) into at least some of
the through-holes of the platen, and b) submerging the platen into
a gas permeable liquid. The organism can be, for example, in a
fluid such as a growth medium, in which case the gas permeable
liquid should be immiscible with the fluid. The method can also
include assaying one or more physical properties of the aerobic
organism.
[0031] The gas permeable liquid can be, for example, a fluorocarbon
such as perfluorodecalin, a silicone polymer, or a monolayer (e.g.,
a monolayer of a lipid or high molecular weight alcohol.
[0032] In another embodiment still, the invention features a method
of mixing volatile samples with other samples (whether volatile or
non-volatile). The method include the steps of: a) providing a
platen having a plurality of through-holes; b) optionally loading
some or all of the through-holes with one or more non-volatile
samples (if any); c) loading at least some of the through-holes of
the platen with one or more volatile samples to allow the samples
in each through-hole to mix with other samples in the same
through-hole; and d) submerging the platen in a liquid immiscible
with the volatile samples, where steps b), c) and d) can be
performed in any order. In preferred embodiments, step d) is
performed prior to introduction of volatile samples. The samples to
be mixed can be initially provided in two separate platens that are
contacted while submerged in said immiscible liquid to allow
mixing. The immiscible liquid can be, for example, a fluorocarbon,
a silicone polymer, mineral oil, or an alkane.
[0033] The invention also features a method of mixing an array of
samples. The method entails: a) providing a platen having a
plurality of through-holes, wherein at least some of the through
holes are loaded with a first sample or set of samples; b)
providing a substantially flat surface comprising an array of a
second sample or set of samples, wherein the second sample or set
of samples on the flat surface can be registered (e.g., the second
sample or set of samples can be arranged in a spatial pattern that
allows it to line up with at least some of the through-holes of the
platen) with the sample in the platen; c) registering the platen
with the array of the second sample or set of samples on the flat
surface; and d) contacting the platen with the flat surface,
wherein the sample in the platen is aligned with the sample on the
flat surface. This method can be used, for example, to avoid
cross-contamination; also, registering and contacting can be done
simultaneously. In some cases, either the first or second sample or
set of samples can include one or more probes. The method can also
include the further step of analyzing a physical property (such as
fluorescence or other optical properties, pH, mass, binding
affinity; e.g., using radiolabelled probes and film,
chemiluminescence) of a sample contained in the platen. In some
cases, the flat surface can also include a hydrophobic pattern
matching the pattern of the platen array (e.g., to prevent
cross-contamination).
[0034] In another embodiment, the invention features a method for
transferring a reagent or probe to a receptacle (e.g., into a
bottle, a tube, another platen, a microtiter plate, or a can) from
a specific through-hole of a platen comprising a plurality of
through-holes. The method includes the steps of: a) placing the
platen over the receptacle; and b) applying a burst of gas, liquid,
solid, or a pin (e.g., a piston, a tube, a post, a plug) to the
specific through-hole to transfer the reagent or probe into the
receptacle. The burst of gas, liquid, or solid can be generated,
for example, with a syringe, or by depositing a photodynamic or
photothermal material (carbon black, plastic explosives, water
droplets) in or above the through-hole, and then exposing the
photodynamic or photothermal material to a laser beam of frequency
and intensity suitable to activate the photodynamic or photothermal
material.
[0035] In another embodiment, the invention features a device for
filling or draining through-holes in a platen having a plurality of
through-holes. The device includes: a) a holder adapted to accept
the platen; b) a nozzle having an aperture of a suitable size to
inject a sample into a single through-hole in said platen; and c) a
valve that controls a flow of a sample through said nozzle, wherein
the holder and nozzle can move with respect to each other. The
nozzle can be, for example, positioned so as to contact the platen
(or not). The device can optionally include a microplate (e.g., a
microtiter plate) positioned to receive samples from the platen, as
well as a computer that can control the valve and control the
positions of the holder and nozzle (and, optionally, the
microplate) relative to one other. The optional microplate, the
holder, and the nozzle can, in some cases, be moved independently
of each other in at least two dimensions. Alternatively, the nozzle
can be held in a single position while the holder and nozzle can be
moved independently of each other in at least two dimensions.
[0036] In another embodiment, the invention features a method of
analyzing the kinetics of one or more reactions occurring in at
least one of the through holes of a platen. The method includes: a)
providing a first platen having a plurality of through-holes,
wherein the through-holes are loaded with a first sample or set of
samples; b) introducing the platen into a detection device; c)
introducing a second platen having a plurality of through-holes
into the detection device, wherein the through holes are loaded
with sample or reagent; d) registering and contacting the platens
such that contents of the through-holes of said first platen can
mix with contents of corresponding through-holes of said second
platen; and e) detecting a change in a physical property of the
contents of at least some of the through-holes over time.
[0037] In another embodiment, the invention features a method of
analyzing a physical property of a sample in an array. The method
includes the steps of: a) providing a platen having a plurality of
through-holes, where the through-holes are loaded with a sample; b)
placing the platen between two partially transmitting mirrors; c)
illuminating the samples through one of the mirrors (e.g., with a
laser, atomic lamp, or other light source, including white light
sources); and d) detecting optical output from the sample.
Optionally, mirrors that reflect at only one wavelength and
transmit at all others can be used, and non-linear optical effects
can also be observed. The "imaging" step can involve, for example,
measuring light emanating from the array or measuring light emitted
from the mirror opposite from the illumination source. The platen
can also be placed within a laser cavity, and an optical gain
medium can be positioned between the two mirrors.
[0038] The invention also features a method of measuring sample
output from an array. The method includes the steps of: a)
providing a platen having a plurality of through-holes, wherein the
through-holes are loaded with sample; b) introducing the sample
into an array of capillaries; c) eluting the samples through the
capillaries using pulse pressure, creating a non-continuous flow;
d) spotting the eluting samples onto a surface that is moving
relative to the capillaries (e.g., a web, a tape, a belt, or a
film), wherein the spots are discrete and no mixing of the samples
occurs; and e) analyzing a physical property of the spots.
[0039] The invention also features a method of storing a plurality
of samples in an assay-ready, high-density format. The method
includes the steps of a) providing a platen having a plurality of
through-holes; b) loading the through-holes with the samples (e.g.,
small molecules) dissolved in a mixture comprising two solvents, a
first solvent having a low vapor pressure (e.g., dimethyl sulfoxide
(DMSO)) and a second solvent having a higher vapor pressure
relative to the first solvent (e.g., ethanol; preferably, both
solvents are inert and are able to dissolve the sample); and c)
evaporating the second solvent to result in a plurality of samples
in first solvent (preferably as films on the walls of the
through-holes). The volume of the first solvent in each solution
can be, for example, less than about 25 nl (e.g., less than 10 nl,
1 nl, 250 pl, 100 pl, or even less than about 25 pl; e.g., a
"microdroplet"). In some embodiments, the sample dissolved in the
first solvent forms a film on the wall of a through-hole.
[0040] The invention features a method of forming a high throughput
assay. The method includes: a) providing a platen having a
plurality of through-holes, wherein at least some of the
through-holes contain a sample dissolved in a solvent having a low
vapor pressure (such as a array of samples prepared for storage
according to the above method); b) cooling the platen to a
temperature sufficient to freeze the dissolved sample, c) dipping
the platen into a solution comprising a reagent, wherein the
temperature of the solution is less than the freezing point of the
sample, but greater than the freezing point of the reagent
solution, d) removing the platen from the reagent solution, and e)
warming the platen to a temperature greater than the freezing point
of the sample. The reagent solution can be, for example, an aqueous
solution.
[0041] Yet another embodiment of the invention features a
filtration device, having first and second platens, each having a
plurality of through-holes, and a semi-permeable membrane.
[0042] The platens are aligned such that the through-holes of the
first platen are substantially aligned with the through-holes of
the second platen and the membrane is sandwiched in between the two
platens. Optionally, the platens can have hydrophobic surfaces. The
semi-permeable membrane can be, for example, a nitrocellulose
membrane, or can include a layer of cells. In yet another aspect,
the invention provides a cell chip containing first and second
platens, each having a plurality of through-holes, and a porous
membrane, wherein the platens are aligned such that the
through-holes of the first platen are substantially aligned with
the through-holes of the second platen and the membrane is
sandwiched in between the two platens. By "cell chip" is meant at
least a platen containing a plurality of through-holes and
containing in at least one through-hole a cell and culture
media.
[0043] In another aspect, the invention provides a cell chip
containing in order from top to bottom: (a) a coverslip in contact
with a spacer; (b) a spacer that separates the covership from a
first platen; (c) a first platen having a plurality of
through-holes; (d) a gasket containing a plurality of through-holes
that provides a seal between the first platent and the membrane;
(e) a porous membrane containing aluminum oxide and having pores
between 0.1 and 1 .mu.m sandwiched between the gasket and the
second platen; (f) a second platent having a plurality of through
holes; and (g) a solid support in contact with the second
platen.
[0044] In yet another aspect, the invention provides a method of
culturing a cell on a cell chip, the method involving providing a
cell chip of any previous aspect containing cell culture medium;
contacting the porous membrane with a cell; and incubating the cell
under conditions suitable for cell survival. In one embodiment, the
conditions include contacting the cell chip a gas permeable liquid
(e.g., perfluorodecalin). In yet another aspect, the cell chip
further comprises a hydrophobic fluid (e.g., perfluorodecalin,
silicone oil or mineral oil) in contact with the cell culture
medium.
[0045] In yet another aspect, the invention provides a method of
constructing a cell chip of any previous aspect, the method
involving filling a first platen having a plurality of
through-holes with cell culture medium; contacting the first platen
with a porous membrane; and contacting the membrane with a second
platen having a plurality of through-holes, such that the
through-holes are substantially aligned, thereby constructing a
cell chip. In one embodiment, the cell chip further contains a
solid support in contact with the first platen. In another
embodiment, the cell chip further comprises a spacer in contact
with the second platen, wherein the spacer is in contact with a
cover slip. In yet another embodiment, the cell chip further
comprises a gasket sandwiched between the first platen. In still
another embodiment, the gasket comprises a flexible material (e.g.,
a biocompatible elastomer, such as teflon, silicone, or rubber.
[0046] In yet another aspect, the invention provides a method for
identifying an agent having a desired biological activity, the
method including contacting a cell chip of any previous aspect
containing a cell with a platen containing an agent; contacting the
cell with the agent; and detecting an alteration in the cell,
thereby identifying an agent having a desired biological activity.
In one embodiment, the agent is present in cell growth medium, or
is contacted with the cell using a slotted pin or syringe, or by
adding the cell to a well containing the agent. In another
embodiment, the agent is a polypeptide, nucleic acid molecule
(e.g., siRNA, microRNA, or an aptamer), or small compound. In
another embodiment, the alteration is an alteration in gene
expression, polypeptide expression, cell growth, proliferation or
survival, in the intracellular localization of a cellular
component, morphological change, or change in motility. In yet
another embodiment, the alteration is detected in an immunoassay,
an enzymatic assay, highthroughput gene expression profiling,
reverse transcriptase polymerase chain reaction (RT-PCR),
quantitative PCR, real time PCR, methylation, or high content
screening (HCS) using quantitative fluorescence microscopy and
automated image acquisition. In yet another embodiment, the high
content screening detects alterations in protein translocation. In
yet another embodiment, the cell is lysed and the proteins or
nucleic acid molecules are bound on a binding surface. In yet
another embodiment, the binding surface is a weak cationic exchange
medium. In another embodiment, the bound proteins or nucleic acid
molecules are analysed for a characteristic selected from the group
consisting of sequence, molecular weight, binding characteristic,
and expression level. In another embodiment, the binding
characteristic is detected in an immunoassay or by polypeptide
binding.
[0047] In various embodiments of any of the above aspects, the
membrane comprises pores that are no more than half the
through-hole diameter (e.g., pores of between about 0.2-250 .mu.m)
In various embodiments pores are about 0.2 .mu.m, 0.5 or 1.0 .mu.m
in diameter. In other embodiments of any previous aspect, the
membrane comprises aluminum oxide or polycarbonate. In still other
embodiments, the polycarbonate comprises 1 .mu.m pores. In still
other embodiments of any previous aspect, the membrane is coated
with fibronectin, laminin, collagen, or another substrate that
supports cell adhesion. In still other embodiments of any previous
aspect, the platens comprise metal (e.g., gold, Tungsten or
stainless steel), polystyrene, or a flexible material (e.g.,
silicone, rubber, teflon). In still other embodiments of any
previous aspect, the chip further comprises a gasket that seals off
individual wells, such as a removable gasket. In still other
embodiments, the gasket contains a plurality of through-holes
aligned with those of the platens. In still other embodiments of
any previous aspect, the chip further comprise a hydrophobic
compound that prevents lateral diffusion, such as a hydrophobic
compound that provides a watertight seal between the membrane and
the platen. In still other embodiments of any previous aspect, the
platen comprises a flexible biocompatible material and the other
platen is a rigid platen that supports the flexible platen. In
various embodiments, the flexible platen comprises silicone,
polypropylene, or rubber. In still other embodiments of any
previous aspect, the two platens are attached by raised surfaces on
one platen that fit into a recessed surface on the other platen. In
still other embodiments of any previous aspect, the total thickness
of the cell chip is less than about 10 mm, 5 mm, or 1 mm. In still
other embodiments of any previous aspect, a solid support (e.g., a
microscope slide) in contact with the first platen. In another
embodiment, the chip further containing a coverslip in contact with
the second platen. In still other embodiments, the coverslip,
microscope slide, and cell chip are secured together. In still
other embodiments of any previous aspect, the filling of the chip
with media is accomplished by placing the first platen on a solid
support and centrifuging the platen and solid support. In one
example, the first platen is contacted with a gasket and cell
medium is then overlayed on the platen.
[0048] A "spatially addressable through-hole" has a position and
dimensions that are known to a high degree of certainty (e.g.,
relative to a reference position on the device). The degree of
certainty is sufficient that the through-holes of two platens
placed one on top of the other can align, allowing reagents to
transfer in a parallel fashion. The degree of certainty is also
sufficient such that a sample in any given through-hole can be
retrieved by a robotic device that knows only the position in which
that hole should be found relative to a reference point on the
device. The term "planar array of through-holes" refers to an array
of through-holes on a platen such as that described in PCT
application WO99/34920.
[0049] A "reagent" is a chemical compound, a gas, a liquid, a
solid, a powder, a solution, a gel, a bead, or electromagnetic
radiation.
[0050] The term "probe" or "chemical probe" refers to a chemical,
biological, mechanical, or electronic structure that detects a
specific analyte by a specific binding or catalytic event. The
binding or catalytic event can be transduced into a signal readable
by an operator. One type of chemical probe is an affinity probe
(e.g., a specific nucleic acid that binds to another nucleic acid).
Examples of mechanical probes include a cantilever that has a
ligand immobilized on its surface and a material whose properties
(e.g., strain, inertia, surface tension) change in response to a
chemical or biological event.
[0051] The term "chemical detection event" refers to a chemical
reaction between molecule(s) of interest and probe molecule(s) that
in turn produces a signal that can be observed by an operator. For
example, the hydrolysis of fluorescein di-.beta.-galactoside by the
enzyme .beta.-galactosidase, to produce the fluorescent molecule
fluorescein, is a chemical detection event. In some cases, the
chemical detection event can involve a series of chemical reactions
triggered by an initial interaction of analyte and probe (e.g.,
activation of a signal transduction pathway in a probe cell by the
binding of a ligand to a surface receptor).
[0052] The term "linker molecule" means a molecule that has a high
affinity for or covalently links to the surface of a platen or
bead. The linker molecule can have a spacer segment such as a
carbon chain, and can also have a functional group at its end to
enable attachment of probe molecules covalently or with high
affinity.
[0053] The term "immobilized" means substantially attached at the
molecular level (i.e., through a covalent or non-covalent bond or
interaction).
[0054] The term "photocleavable compound" refers to a compound that
contains a moiety that, when exposed to light, dissociates into
multiple independent molecules.
[0055] The term "small molecule" refers to a molecule having a mass
less than about 3000 daltons.
[0056] The term "hybridization" refers to complementary, specific
binding of two or more molecules (e.g., nucleic acids) to one
another.
[0057] "Solid phase synthesis" refers to a chemical synthesis
process in which at least one of the starting materials in the
synthesis reaction is attached to a solid material such as a
polymer bead, a gelatinous resin, a porous solid, or a planar
surface.
[0058] The term "blotter" refers to a material capable of capturing
excess liquids by absorption.
[0059] The term "bead" means a small particle, generally less than
about 1 mm (e.g., less than about 100 .mu.m) in any dimension, with
the ability to have reagents attached to its surface or stored in
its interior. A bead can be made from one or more of a variety of
materials, including organic polymers, glass, and metals. The
reagent is typically attached to the bead by chemical reaction with
a reactive functional group such as a carboxyl, silanol, or amino
group on its surface. Reagents can, for example, be confined to the
bead by covalent chemical attachment or by physical adsorption to
the bead surface. The bead shape can be nearly spherical,
irregularly shaped, or of an intermediate shape.
[0060] The term "stringency" refers to the degree to which
non-specific molecular interactions are disrupted during a washing
step.
[0061] The term "electrophoretic washing" refers to the removal of
non-specifically bound, ionic molecules from a probe by applying an
electric field.
[0062] "Specific interactions" are interactions between two
molecules resulting from a unique three-dimensional structure of at
least one of the molecules involved. For example, enzymes have
specific interactions with transition state analogues due to their
evolution toward stabilizing reaction intermediates.
[0063] The term "micro-plate" refers to a collection plate used to
transfer the contents of the through-holes of an array, where no
cross contamination of the through-holes occurs in the
transfer.
[0064] The term "micro-droplet" means a drop of liquid having a
volume of 50 nl or less (e.g., less than about 50 nl, 25 nl, 10 nl,
5 nl, 1 nl, 500 pl, 250 pl, 100 pl, 50 pl, or less).
[0065] The term "physical properties" means any measurable property
of an object or system, including electrical, magnetic, optical,
thermal, mechanical, biological, nuclear, and chemical
properties.
[0066] The new methods have numerous advantages. For example, the
new methods allow optimization of processes in a parallel manner.
For instance, synthesis of a particular molecular species often
requires tedious quantitative investigation of different synthetic
methods with a view towards optimizing product yield. Using the new
methods, process parameters can be varied on a
through-hole-by-through-hole basis in the array, and the product
analyzed to determine the protocol best suited for high yield
synthesis.
[0067] Another advantage of the invention over conventional arrays
of chemical probes on a planar substrate is that each chemical
detection event takes place in a physically isolated container
(i.e., the through-hole), allowing amplification of the signal by
catalysis (e.g., releasing detectable molecules into the solution
contained in each through-hole). Such detectable molecules include,
for example, fluorescent products of a fluorogenic enzyme
substrate, and chromogenic products of a chromogenic substrate.
Physical isolation of samples retained in the array also prevents
cross-contamination by eliminating lateral communication between
the through-holes.
[0068] Another advantage of the invention is that each through-hole
can have a precise and known spatial location in the array. Each
through-hole is then spatially addressable, thereby facilitating
the insertion and removal of liquids from each through-hole, the
analysis of the contents of each through-hole, and the alignment of
multiple arrays for highly parallel transfer of reagents.
[0069] Another advantage of the invention is that the relative
volumes of the members of two arrays can be easily adjusted by
changing the depth of one array with respect to the other.
[0070] Still another advantage of the invention is that substances
that bind to chemical probes contained in the through-hole array
can easily be recovered as distinct samples for further analysis.
For example, the bound contents of the well can be eluted onto a
planar substrate for analysis by matrix-assisted laser desorption
and ionization (MALDI) or surface-enhanced laser desorption and
ionization (SELDI) mass spectrometry, or nuclear magnetic resonance
(NMR) spectroscopy. Alternatively, the contents of the through-hole
can be electrosprayed directly from the through-hole into a mass
spectrometer. The contents of the through-hole can also be
crystallized and analyzed with x-ray or electron diffraction
techniques (e.g., to determine crystal structure). This aspect of
the invention allows for sensitive detection of unlabelled
analytes.
[0071] Yet another advantage of the invention is that the samples
can be introduced or removed from the platen by electrophoresis, as
the through-holes can allow for conduction of an electric
field.
[0072] Another advantage of the invention is that samples are
accessible from both sides of the platens. This means, for example,
that samples can be removed from the platens by applying pressure,
an air or gas stream, or an explosive charge to a through-hole of
interest and then collecting the material from the opposing face of
the platen. Alternately, samples can be sucked out of the platen
without creating a vacuum. Thus, the volume of the samples in not
limited by the current state-of-the-art microfluidics techniques,
and a minimum quantity of fluid is lost upon the collection of the
sample. A pressure can be applied, for example, in the form of a
solid pin (acting, e.g., as a piston), or in the form of a burst of
inert gas. Another implication of this advantage is that it is
relatively easy to perform electrospray ionization mass
spectrometry directly from the platen. Simultaneous measurement of
luminescence from two spectrally distinct luminescent probes
located in the microchannel array can be performed in either a
trans- or epi-illumination optical configuration, including, for
example, a light source, an optical filter, and a CCD camera.
Optical signals can be collected from both sides of the platen
simultaneously.
[0073] The numerous samples contained in the platen can be rapidly
transferred to a flat surface or membrane, facilitating processes
such as SELDI mass spectrometric analysis and growth of bacterial
cells (e.g., cells contained in the through-holes), to form
individual colonies for storage and further analysis. Transfer from
a planar material to the array can also be accomplished, as in
electroblotting from a polyacrylamide 2-D protein gel into the
array.
[0074] Advantageously, the surface area of the liquid exposed to
the environment is minimized by the high aspect ratio geometry,
thus limiting evaporation.
[0075] Still another advantage of the new methods is that the
sample contained in a given through-hole constitutes a small
thermal mass and can, therefore, reach thermal equilibrium quickly
and uniformly. The fact is relevant, for example, to synthetic
methods that involve heating and/or cooling steps (e.g.,
replication of nucleic acids using the polymerase chain reaction,
PCR).
[0076] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0077] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0078] FIG. 1 is an exploded top corner view of a platen with top
and bottom masks.
[0079] FIG. 2 is a cross-sectional view of an interlocking array
system.
[0080] FIG. 3 is an illustration of a method for transferring small
volume drops into specific through-holes in an array with a pin
array.
[0081] FIG. 4 is an illustration of a method for producing a mask
by use of UV curable epoxy.
[0082] FIG. 5 is an illustration of a method for producing a mask
by use of an array of pins having a precision fit into a matching
array of through-holes.
[0083] FIG. 6 is an illustration of a method for storing liquid
from individual wells from a microtiter plate in a bundle of
capillary tubing for transference into high-density through-hole
arrays.
[0084] FIG. 7 is an illustration of a method for transferring fluid
from wells in a microtiter plate to through-holes in an array using
a flexible member.
[0085] FIG. 8 is a drawing of an array in which the through-hole
cross-sections are shaped to hold only one microsphere per
through-hole, and the longitudinal cross-section is tapered such
that the microsphere sits in the hole either at or below the array
surface.
[0086] FIG. 9 is an illustration of a method for transfer with a
single sampling device with a fast sequential positioning of a
mechanical plunger over the through-holes to be sampled and pushing
the plunger through the hole to transfer the hole's contents to the
well of a microtiter plate located at a small distance below the
through-hole array.
[0087] FIG. 10 is an illustration of a method for transferring
materials from a through-hole in a through-hole array into a well
of a microtiter plate using a gas jet generated by spatially
localized heating with a focused laser beam.
[0088] FIG. 11 is an illustration of the transfer of material from
a through-hole in the array to a well in a microtiter plate with a
gas jet caused by localized ignition of an explosive charge
randomly distributed in a thin sheet overlaid on one surface of the
through-hole array.
[0089] FIG. 12 is an illustration of a sheet with an explosive
charge pattern matching the through-hole positions in the
array.
[0090] FIG. 13 is an illustration of a method to interface
massively parallel HPLC separation with inherently serial
analytical methods such as mass spectrometry.
[0091] FIG. 14 is an illustration of a chromatographic device.
[0092] FIG. 15 is an illustration of a linear MALDI-TOF mass
spectrometer.
[0093] FIG. 16 is an illustration of an array of chamfered
through-holes machined in a block of material, a syringe bank with
the same center-to-center spacing as the through-hole array,
wherein the syringe needles pass through a metal block that is
attached to the syringe bank holder by pneumatically-actuated,
spring-loaded pins.
[0094] FIG. 17 is an illustration of the array of FIG. 16, wherein
the capillary channels are pressurized by the syringes.
[0095] FIG. 18 is an illustration of an array similar to that of
FIG. 17, with the exception that the syringe bank is bolted to the
capillary tube array.
[0096] FIG. 19 is an illustration of an array similar to that of
FIG. 18, with the exception that the syringe bank is bolted to the
capillary tube array.
[0097] FIG. 20 is an illustration of a method of manufacturing a
platen having a plurality of through-holes and two opposing
surfaces, by bonding together multiple grooved surfaces.
[0098] FIG. 21 depicts an array positioned inside an optical
resonator featuring a source of illumination and two partially
reflective surfaces.
[0099] FIG. 22 depicts a device for removal of the contents of a
through-hole array and transfer of those contents. The device has a
nozzle, a stage for holding the through-hole array and a stage for
holding a capture chamber. Movement in two dimensions of the nozzle
or the through-hole array can be achieved.
[0100] FIG. 23 depicts a method for wiping excess fluids from the
surface of the platen. The device has enables a through-hole array
to be loaded with sample and removed through a wiping fluid in an
efficient manner.
[0101] FIG. 24 depicts a device for aligning platens having a
plurality of through-holes inside a detection device, wherein the
platens are held in place through device comprising two pins
attached to a flat base.
[0102] FIG. 25 shows a flow chart of a cell-chip microarray. A
platen of rigid material such as metal or polystyrene with pores of
50-200 .mu.m in diameter is attached to a porous membrane forming
an array of microwells with a porous bottom. Cells are added and
allowed to adhere to the membrane before test compound is added
with a microspotting pin. After incubation, the membrane may be
processed for analysis of protein or mRNA expression, or any assay
or assay component of interest.
[0103] FIG. 26 shows polycarbonate membranes, having 1.0 .mu.m pore
size, soaked in 100 ug/mLl fibronectin and air dried for 2 hours.
The membrane was attached to the bottom of a Petri dish by the
addition of 10% agarose at the edges. PKC.beta.-GFP transfected 293
cells were added at a concentration of 5.times.10.sup.5/mL and
incubated 37.degree. C., 5% CO.sub.2. Images were acquired by
confocal microscopy.
[0104] FIG. 27 shows polycarbonate membranes, 1.0 .mu.m pore size,
soaked in 100 ug/mL fibronectin and air dried for 2 hours. The
membrane was attached to the bottom of a Petri dish by the addition
of 10% agarose at the edges. PKC.beta.-GFP transfected 293 cells
were added at a concentration of 5.times.10.sup.5/mL and incubated
37.degree. C., 5% CO.sub.2. Then, 1 uM
phorbol-12-myristate-13-acetate (PMA) was added. After 1 hour at
37.degree. C., 5% CO.sub.2, images were acquired by confocal
microscopy.
[0105] FIG. 28 shows attachment of cells to the membrane in the
cell-chip device comprised of gold platen and porous membrane
assembled as demonstrated in FIG. 1.
[0106] FIG. 29 shows microspotting on gold platen. A 300 mesh gold
platen (50 um.sup.2 holes) was wetted with medium. Fluorescein in
10% DMSO/PBS was spotted on platen using a FP9 floating pin
(VP-scientific.) Spots were examined under a confocal microscope at
5.times. magnification.
[0107] FIG. 30 shows cell uptake of Hoechst stain. PKC.beta.-GFP
cells were incubated overnight in 300 mesh gold platen (50 um.sup.2
holes) and washed with medium. Hoechst stain was then spotted on
the platen that was dried by blotting.
[0108] FIG. 31 shows microspotting on a gold platen. A 300 mesh
gold platen (50 um.sup.2 holes) was wetted with medium or briefly
dried by blotting. A drop of mineral oil was added to the platen
before adding Fluorescein in 10% DMSO/PBS using a FP9 floating pin.
Spots were examined under a confocal microscope at 5.times.
magnification.
[0109] FIG. 32 shows cell uptake of Hoechst stain by PKC.beta.-GFP
cells that were incubated overnight in a stainless steel cell-chip
and then washed with medium. Hoechst was spotted on a first platen
that was dried by blotting. The stainless steel platen (National
Jet Company, LaVale, M.D.) tested was a 1-inch square and 400-.mu.m
thick with pores 150 .mu.m in diameter. A second platen was
attached using adhesive sealing film with the center cut out. The
chip was placed in a cytospin sample chamber with the funnel
removed with a small gasket between the platen and the top of the
chamber.
[0110] FIG. 33 shows an anopore membrane sandwiched between two
stainless steel platens (as shown in FIG. 8). PKC.beta.-GFP cells
were added and incubated overnight. Note unequal distribution of
cells in the well, possibly due to leaks of media between platen
and membrane
[0111] FIG. 34 shows a cell microarray prototype based on a
200-.mu.m thick Tungsten platen. The platens (National Jet Company,
LaVale, M.D.) had pores of 300 .mu.m in diameter and were attached
with 4 screws.
[0112] FIG. 35 shows cell culture on an anopore membrane sandwiched
between two tungsten platens (as shown in FIG. 34). PKC.beta.-GFP
cells were added and incubated 48 hrs. Note more random cell
distribution with more secure attachment; however, the platen is
not yet optimized for cell attachment.
[0113] FIG. 36 shows an open array-based cell chip and delivery of
Cl2-resazurin to a single well on the array using a floating
pin.
[0114] FIG. 37 shows an open array-based cell chip with
PKC.beta.-GFP transfected cells, added at 5.times.10.sup.5/mL and
incubated 37.degree. C., 5% CO.sub.2. Images were acquired by
confocal microscopy.
[0115] FIG. 38 shows essentially that which is depicted in FIG. 12,
except here the platen is not shown.
[0116] FIG. 39 shows one particular embodiment of the
invention--specifically, an apparatus for in vitro and ex-vivo
analysis. A platen of rigid material such as metal or polystyrene
with pores of 10-300 .mu.m in diameter is attached to a porous
membrane or modified glass surface forming an array of microwells
with a porous bottom. Cells are added and allowed to adhere to the
membrane overnight before test compound is added with a micro
spotting pin. After incubation, high content image analysis is
performed. Membranes are processed for analysis of protein or mRNA
expression, or other assay or assay component of interest.
[0117] FIG. 40 is essentially that which is depicted in FIG. 15,
but here shows the cross-section of an individual well.
[0118] FIG. 41 shows structural enhancements to increase pressure
on the member and/or gaskets to seal off individual wells.
DETAILED DESCRIPTION OF THE INVENTION
[0119] The invention provides methods of creating, storing, and
screening diverse chemical and biological compositions, each
contained in a through-hole that traverses a platen, as well as
methods for making and using platens, particularly platens
containing cells. In certain embodiments, the methods include
transmitting reagents to a selected group of holes in a dense array
of through-holes. Additional rounds of reagent transmission are
provided as needed. The invention also provides for placing a
series of masks over a planar array of through-holes and flowing
reagents through the masks to build a defined pattern of probes or
reagents such that the contents of each through-hole can be known.
In an alternate embodiment, the invention provides distributing
probe-holding particles, such as beads or cells, into the array of
through-holes. Such probes include, but are not limited to, nucleic
acids, peptides, small molecules, and chemical sensing cells. Uses
of the arrays include screening of genetic libraries, producing and
screening compound libraries for discovery of pharmaceutical leads,
optimization of reaction conditions, gene expression analysis,
clinical diagnostics, genomics, functional genomics,
pharmacogenomics, structural genomics, proteomics, production and
optimization of industrial catalysts, chemical genetics,
identification of suitable conditions for reactions (e.g.,
conditions suitable for protein crystallization), genotyping,
polymorphism analysis, examination of RNA expression profiles in
cells or tissues, sequencing by hybridization, and recombinant
enzyme discovery.
[0120] A platen having a high-density array of through-holes in
accordance with one embodiment of the present invention is
illustrated in FIGS. 1 (top view) and 2 (cross-sectional side
view). The platen can be made of silicon or other rigid materials,
such as metal, glass, or plastic. The platen material can be
chemically inert, or can be rendered so by appropriate surface
treatments.
[0121] Referring to FIG. 1, each through-hole has a square
cross-section, although circular or rectangular cross-sections can
alternatively be used. The diameter of each through-hole is less
than 1 mm (e.g., less than about 600 .mu.m, 300 .mu.m, 100 .mu.m,
10 .mu.m, 1 .mu.m, or 100 nm), typically 200-250 .mu.m, and the
depth of the platen can be 10-2000 .mu.m or more, generally about
250-1000 .mu.m.
[0122] Greater depths can be achieved using a bundle of glass
capillaries. Alternatively, platens having greater depths can
achieved by bonding together multiple surfaces having parallel
grooves, creating a long three dimensional object having
through-holes running throughout the length of the object. This
object can be subsequently sliced horizontally, allowing
flexibility in the depth of the through-holes. The result is the
ability to use arrays with compatible positions of through-holes,
wherein the depths of the through-holes can vary from array to
array.
[0123] For spatial addressability, center-to-center spacing of
through-holes should be fairly precise. Hole-to-hole spacing
depends on the dimension of the through-holes within the platen.
The though-holes can be arranged in regular rows and columns,
hexagonal arrays, or other configurations (e.g., groupings of
through-holes into smaller sub-arrays). Multiple platens can be
fabricated with the same arrangement of through-holes so that the
pattern is reproducible, and each through-hole can be identified by
its own address within the array.
[0124] When three platens having through-holes are stacked, the
total volume of a single channel (i.e., three through-holes
stacked) is typically 100 nl. Using this volume as an example, if
the entire channel were filled from a dense yeast cell culture
(.about.10.sup.7/ml), each channel would thus contain approximately
10.sup.3 yeast cells. Based on a yeast cell volume of 70
.mu.m.sup.3, the maximum number of cells per 100 nl channel is on
the order of 10.sup.6. A minimum of 100 cells per microchannel can
be adequate to compensate for cell-to-cell variability of yeast
cell response to the bioassays. However, this volume can vary
depending not only on the diameter of the through-holes, but on the
depth of the through-holes. This ability to vary the volume of
samples allows flexibility, enabling the use of a wider variety of
materials, concentrations, and reaction conditions.
[0125] Optional features such as binary identification codes, or
holes and grooves for indexing and alignment, can also be
incorporated into each platen.
[0126] I. Methods of Making Devices having Arrays of
Through-Holes.
Fabrication of an Array of Through-Holes by Casting in Resin.
[0127] Conventional technologies for manufacturing high-density
through-hole arrays include micro-machining, electrospark discharge
machining (EDM), or chemical etching. Alternatively, the arrays can
be cast in a polymer or resin. A casting mold can be designed such
that the inner diameter of the mold will be equal to or larger than
the final outer diameter of the array device. The depth of the
casting mold can be as little as 0.5 mm for a single array, or 1
meter or longer. In the case where a long block of resin is cast,
the resin can be cross-sectioned into slices of desired thickness
and the surfaces can be polished or smoothed. The through-holes can
be defined in the cast by several methods. Solid wire, fiber, or an
array of pins of the desired geometry and diameter can be arranged
within the casting mold. If necessary, the wire, fiber or pins can
be immobilized in place with the use of one or more positioning
jigs within the casting mold. The chemistries of the wire, fiber,
or pins must be chosen such that they will not form a permanent
bond with the resin or polymer as it solidifies, so that they can
be pulled out to produce the through-holes. For example, the fibers
may be ethyleneterephthalate and the resin is
polymethylmethacrylate (PMMA). Alternatively, the surfaces of the
wire, fiber, or pins can be coated with a release agent such as an
oil, a fluoropolymer, water, or a polymerization inhibitor that
will facilitate the removal of the wire, fiber, or pins from the
cast resin or polymer once the final curing, setting, or
polymerization is complete.
[0128] In an alternate system, the through-holes can be defined by
positioning an array of hollow tubing or capillaries within the
casing mold. The hollow tubes can be immobilized within the casting
mold in a positioning jig. Use of tubing of different internal
diameter results in an array with through-holes of different
diameters. The chemistry of the hollow tubes and polymer will
ideally be chosen such that a permanent bond will form between the
outside hollow tube and the resin or polymer that is cast. The
inner surface of the hollow tubes will then make up the
through-holes of the array. The hollow tubes can be made of glass
or fused silica, a polymer, or a metal.
[0129] The chemistry of the resin or polymer that is cast can be
selected such that the surface of the array device is of a desired
hydrophobic or hydrophilic character. The chemistry of casting
resins, such as acrylate or polystyrene, can be modified with
hydrophobic groups to result in an array with the desired surface
chemistry. Alternatively, the surface chemistry of the array device
can be modified with standard techniques after slicing and
polishing. In addition, the chemical or physical properties of the
polymer can be modified by the addition of other materials. For
example, to control the electrical conductivity of the device,
particles of a conductive metal can be mixed into the resin or
polymer prior to casting the mold to confer conductivity to the
device. Generally, the more metal particles that are mixed with the
resin or polymer prior to casting, the greater the conductivity of
the devices will be.
[0130] Additives to the resin or polymer can be used to improve the
sensitivity of optical imaging of the array. For example, metal
particles can be added to make the material between the
through-holes. The metal particles enable light to scatter, causing
a fluorescent signal generated by a probe in a through-hole to
reflect toward the detector and to prevent cross-talk of signals.
Alternatively, carbon black may be added to make the material,
preventing cross-talk and minimizing signal from light scattered
off the surface of the array. More preferably, a combination of a
light scattering agent such as titanium dioxide and a light
absorbing agent, such as carbon black are added to the resin or
polymer to achieve maximum optical density between the holes.
[0131] Using hollow tubes in the casting mold to form through-holes
allows the chemical properties of the tubes to be varied according
to the needs of the application. Tubes manufactured from a
biologically inert polymer (e.g., polyetheretherketone (PEEK) or
poly(tetrafluoroethylene) (PTFE)) are desirable for some
applications. Alternatively, fused silica tubing can be used to
form the through-holes. The interior surface of the fused silica
can be derivatized prior to casting, allowing for virtually any
level of desired hydrophilicity or hydrophobicity. A metal or alloy
tubing can also be used to form the through-holes of the array.
Metal tubing can be coated to make it biocompatible. For example,
metals can be coated with thin layers of gold and the gold surfaces
can be readily coated with a variety of reagents possessing thiol
moieties.
[0132] The inner surfaces of the tubes or capillaries can, for
example, be coated with materials that facilitate the use of the
resulting slices as a probe array. For instance, each tube or
capillary can be coated with a different nucleic acid probe, so
that when the block of resin is sliced, the resulting platens can
be used as genosensors. Alternatively, the probes can be
immobilized on a porous material contained in the capillaries.
[0133] Once a block is cast from the desired resin or polymer, it
can be sliced to an appropriate thickness to form a platen having
an array of through-holes. In certain embodiments, the thickness of
the platen ranges from 0.2 to 25.0 mm. However, using this
technique, arrays of through-holes can be manufactured having the
same length as the casting mold that is used. If desired, casts
that are many meters in length can be prepared. Standard techniques
for producing silicon wafers by slicing and polishing silicon
ingots in the semiconductor industry can be directly applied to the
manufacture of an array of through-holes. Flowing a coolant through
the through-holes in the cast array while slicing can prevent heat
buildup that could otherwise melt the polymer or degrade coatings
or probes inside the holes. Examples of coolants include cold
water, cold aqueous ethylene glycol, and cold isopropanol.
[0134] If solid wires are arranged in the cast and the resulting
block is then sliced, the wires can be eroded by electrodeposition
onto a plate in an electrochemical cell or by chemical degradation
such as by placing the slice in concentrated nitric acid to give an
array of through-holes. The slices can be bonded to a metal sheet,
the polymer eroded, and the slices used as an electrode for
production of through-hole arrays by sink-EDM. The polymer can be
eroded by chemical means, melted, or burned off.
Method of Making an Array by Stacking and Bonding Multiple Grooved
Surfaces.
[0135] One method of manufacturing an array of through-holes is to
stack and bond together grooved plates having upper and lower
surfaces, creating a three-dimensional array. Any number of
materials can be used to manufacture the array of through-holes
including, but not limited to, silicon, glass, plastics, resins, or
metals. Depending on the material used, grooves can be machined
into the individual surfaces using a variety of techniques (for
example, micro-machining, chemical etching, embossing, or
stamping). The depth and width of each groove determines the
dimensions of the through-holes in the completed platens and can be
machined according to the desired specifications.
[0136] A precise layering or stacking of the grooved plates into a
three-dimensional array can be accomplished with the use of an
external or internal jig into which each surface is precisely
placed. Alternatively, registration devices, (for example notches,
posts, tongues, etc.) can be precisely integrated into each surface
to facilitate accurate stacking. After stacking the individual
grooved surfaces, they are bonded together in a permanent manner.
The use of traditional adhesives to bond the plates together is a
disfavored approach because excess adhesive can migrate into the
grooves and result in the blockage of some of the through-holes. A
preferred method for the bonding process is the use of a
combination of elevated temperature and pressure, resulting in a
fusion of the chosen materials. In some cases, one or both surfaces
of the grooved platens are coated with a material (for example,
gold) that, upon the application of an appropriate amount of
temperature and pressure, diffuses into the surfaces and results in
a permanent bond. The grooved plates can be made from materials
that include a thermoplastic, a ceramic, a glass or a metal such as
silicon.
[0137] In a preferred embodiment, the width of each of the grooved
plates is equivalent to the width of the final platen of
through-holes, but the length of each grooved plate is much larger
than the desired thickness of the final platen of through-holes.
This results in a three-dimensional array of through-holes that is
much thicker than required. Individual platens of through-holes can
then be precisely cut from this thick block to the desired
specification. The platens can further require polishing after they
have been cut in order to yield an optically flat surface. The
required surface chemistries are then be applied to the platens. An
advantage of this method of manufacturing is the creation of
platens with straight-walled through-holes that are much deeper
than those made using traditional micro-machining technology.
[0138] Minor misalignments in the stacking of the individual
grooved plates can result in small imperfections in the
registration of the through-holes in the final platens. This can
interfere with operations using the platens of through-holes (for
example, stacking of two or more platens to initiate massively
parallel reaction). However, even if minor errors in positional
registration exist, adjacent slices cut from each thick array of
through-holes will be a near-perfect match and the required
stacking operations can be accomplished.
Formation of a Silicon Oxide Layer.
[0139] In one embodiment, a dense array of through-holes is
produced in a silicon wafer. A silicon oxide layer is created
uniformly on all surfaces of the array by heating the silicon array
in a furnace to a temperature high enough to cause oxidation.
Oxygen and/or humidity levels in the furnace can be raised above
the ambient to speed oxidation process (see, e.g., Atalla et al.,
The Bell System Technical Journal, pp. 749-783, May 1959). The
silicon oxide layer is advantageous because it enables the
application of various chemical surface treatments to the array
surfaces. Examples of surface treatments are found in Immobilized
Affinity Ligand Techniques (Hermanson et al, Academic Press, San
Diego, 1992) and technical literature available from United
Chemical Technologies, Inc., Bristol, Pa. Use of silicon oxide
provides and additional advantage, allowing the optical
reflectivity of the surfaces to be controlled by adjusting the
thickness of the silicon oxide film (see, e.g., Principles of
Optics, M. Born & E. Wolf, Pergamon Press, 1980, pages
59-66).
[0140] II. Methods of Modifying the Surfaces of Array Devices and
Walls of Through-Holes.
[0141] The surfaces of the through-hole arrays can be modified in
various ways (e.g., to change their physical and chemical
properties). Types of surface modification can include, but are not
limited to, the application of polymer coatings, deposition of
metals, chemical derivitization, and mechanical polishing.
[0142] Selectively Modifying the Surface Chemistry of Array Faces:
In order to prevent aqueous solutions from adhering to array
surfaces and cross-communication between the various through-holes
during loading and other manipulations, it is desirable to coat the
surfaces of the platen with a hydrophobic coating. It is also
desirable to coat the inner surfaces of the through-holes with a
hydrophilic coating so that they retain fluids. The inner coating
can further be blocked, preventing non-specific binding, or
derivatized with affinity ligands. This combination of hydrophobic
surfaces and hydrophilic through-holes prevents aqueous solutions
from adhering to the surfaces of the array while allowing
instantaneous loading of the through-holes.
[0143] Generally, a platen having a dense array of through-holes is
produced in silicon and coated in silicon oxide by oxidation. The
platen is then cleaned, removing organic materials, by soaking in a
mixture of hydrogen peroxide and sulfuric acid, or other caustic
solvent cleaning solution. This treatment results in clean silicon
oxide with a high surface energy. Hydrophobic coatings produced
using this method are stable under high humidity and they can be
used repeatedly.
[0144] The top and bottom faces of the arrays are made hydrophobic
through exposure to vapor from a solution containing an appropriate
silanizing agent (such as polydimethylsiloxane sold as Glassclad
216 by United Chemical Technologies, Inc.) in a volatile solvent.
The silanizing agent reacts with the hydroxyl and silanol groups on
the array surface and creates covalent bonds to hydrophobic alkyl
groups. Selective modification of the surface chemistry can be
achieved by selection of alternative silanizing agents.
[0145] In one surface modification method, a positive pressure of
inert gas is applied to the surface of the platen opposite the
surface being treated. The positive pressure within the
through-holes prevents the silanizing vapor from reaching the
interior through-holes. Silanizing agents suitable for rendering
glassy surfaces hydrophobic include alkyltrichlorosilanes and
alkyltrimethoxysilanes, many of which are commercially
available.
[0146] In another method, a first platen is made from silicon, and
then oxidized. A second silicon platen is produced with identical
size and alignment holes, but without through-holes. The
through-hole array platen is placed on top of the solid platen on
the stacking jig such that each through-hole becomes a well. The
jig and plates are then treated with a surface-modifying chemical
reagent. The air trapped at the bottom of the wells prevents the
fluid from entering the wells. After sufficient time for the
reaction to occur, the plate is removed from solution, dried, and
heat-treated if appropriate. The backing is removed, the array is
flipped over, and the process repeated, coating the second
surface.
[0147] Yet another method for creating hydrophobic exterior
surfaces is to place the array on a matching array of pins such
that each single pin narrowly passes through its corresponding
through-hole. This array of pins can be the electrode used to
create the array of through-holes using sink EDM. Next a coating of
a hydrophobic polymer such as polypropylene or Teflon is deposited
on the exposed surfaces using gas phase deposition method such as
evaporative vapor deposition. The array of through-holes is removed
from the matching pin array, inverted, and the coating process is
repeated to cover the opposite surface.
[0148] Another method for producing hydrophobic coatings on the
platen surfaces involves coating platen surfaces with a metal such
as gold (then exposing the arrays to a chemical that selectively
reacts with the metal, but not with the uncoated through-hole
surfaces. For example electron beam vapor deposition can be used to
coat the outer surfaces of a platen containing a plurality of
through-holes with gold, other metal or semi-conductor. Electron
beam vapor deposition will preferentially deposit the gold on
surfaces normal to the beam direction. The gold-coated surface can
then react with alkane thiols to attach a hydrophobic alkyl groups
(Z. Hou et al., Langmuir, 14:3287-3297, 1998). The inner surfaces
of the through-holes that are not coated with gold will remain
hydrophilic. Alkane thiols are also reactive towards other
materials including silver, copper and gallium arsenide (Y Xia and
G. M. Whitesides, Annu. Rev. Mater. Sci., 28: 153-84, 1998).
Amphiphiles besides alkane thiols can be chemically reacted to
other inorganic solid materials to produce hydrophobic coatings.
Examples of such coatings include, but are not limited to,
alkanephosphates on metal oxides (D. Brovelli et al., Langmuir,
15:4324-4327, 1999 and R. Hofer et al., Langmuir, 17: 4014-4020,
2001), and alkane carboxylates on alumina (P. E. Laibinis et al.,
Science, 245: 845, 1989).
Selectively Modifying the Surface Chemistry of Through-Hole
Surfaces.
[0149] In one method, multiple, identical through-hole arrays are
prepared, aligned, and stacked. A chemical reagent is passed
through the continuous channels formed by the stacked arrays. The
reagent can be a solution, a suspension, a liquid, a vapor, or fine
powder. The stack of arrays is then washed, dried, and heated, as
appropriate for the particular coating. The stack of chips is then
physically separated from one another and the arrays on the top and
bottom of the stack ("sacrificial arrays") are discarded.
[0150] Another method for selectively coating through-hole surfaces
involves using a robot to position a fine needle or an array of
fine needles proximal to the entrance of each through-hole.
Chemical surface-modifying reagents can then be delivered through
this needle/capillary directly into individual holes
[0151] In another method, all surfaces of a silicon through-hole
array are chemically modified. The array faces are then
mechanically polished to restore the original surface character.
The array faces can then be coated again.
[0152] In still another method, the array faces are coated with a
material that is inert towards the desired surface-modifying
chemicals, and then the entire array is exposed to it. For example,
a gold coating can be applied to both faces of an oxidized silicon
through-hole array by electron beam deposition. The array can then
be submerged in a solution of chemical reagent such as a silanizing
reagent that reacts selectively with the siliceous through-hole
surfaces.
[0153] In another method, the inner and outer surfaces of a
through-hole array are coated by filling the holes with a
removable, impermeable material. For example, the interior surfaces
of the through-holes in an array can be protected by filling them
with a solid that can later be removed. Examples of such a solid
include a wax, a plastic, a frozen oil, ice, dry ice, or a polymer
such as poly(ethylene-glycol). In one example, the through-holes
are filled, excess material removed from the surface of the platen,
and the surface coated, leaving the interiors uncoated. Methods for
removing excess materials include scraping, sanding, polishing,
dissolving with a solvent, melting or burning. The solid material
in the interior of the through-holes can be removed under similar
conditions. The interiors of the through-holes can then be
selectively coated or modified.
Derivatizing Through-Hole Surfaces.
[0154] In many cases it is desirable to immobilize probes on the
inner walls of all, some, or one of the through-holes. There are
many techniques for covalently attaching probes to glass or plastic
surfaces (see, e.g., Immobilized Affinity Ligand Techniques,
Hermanson et al., Academic Press, 1992). Those methods useful for
glass can also be used for the oxidized surfaces of a silicon
substrate. For example, the inner walls of the holes in an oxidized
silicon through-hole array can be reacted with g-glycidoxypropyl
trimethoxysilane in the presence of acid and heated to provide a
glycerol coating. This glycerol coating can then be covalently
linked to peptide or nucleic acid probes.
[0155] Rendering the Inner Walls Porous: The inner wall of a
through-hole can be made porous by chemical etching following the
procedure as outlined by Wei et al. (Nature, 399:243-246, 1999).
The larger area of the porous region increases surface area and
thus the amount of chemical reagent attached to the through-hole
inner wall. When used for synthetic transformation, the increased
reagent loading of porous through-holes increases yield. When used
for detection, the increased reagent loading increases sensitivity.
Furthermore, the material between adjacent through-holes can be
made porous allowing for communication between through-holes by
liquids or gases. This method can be useful for the controlled
delivery of reagents stored in adjacent through-holes, allowing
mixing of reagents and reactions to occur. All or part (e.g., just
the middle portion) of the through-hole can be made porous.
Polymer Scaffolding for Protein and Cell Immobilization in a
Platen
[0156] The interior walls of the through-holes of the platen can be
derivatized to allow covalent or non-covalent attachment of
proteins or cells. Any signal arising from the protein or cell thus
attached is then confined to the perimeter of the well, unless the
signal is enzymatically amplified, as in an ELISA assay. Even if
amplified in this way, however, the signal may be weakened, for
example, by low analyte concentration. In the case of cell
attachment, because the wells are bottomless, cells can attach only
to the interior walls and can grow upwards in two dimensions. An
alternative to passive protein adsorption and covalent attachment
to the walls of the array through-holes is the introduction of a
three-dimensional hydrophilic scaffold such as a hydrophilic
linear, gel or foam polymer filling activated for protein coupling
or capable of protein or cell entrapment within the well. Covalent
attachment of proteins to polymer-filled through-holes of the
platen
[0157] Because the interior surface of the through-holes is
hydrophilic, a hydrophilic or water soluble pre-polymer can easily
be loaded into the wells of the platen and the polymerization
reaction can be initiated by a change in temperature or pH or by
the addition of initiator. Protein coupling can be carried out
during polymerization, provided that the polymerization conditions
do not affect protein structure and function. Alternatively,
protein coupling can be carried out after polymerization. Proteins
can be coupled using any of a variety of reactions, including
reactions of free amines, free carboxylic acid groups, and free
sulfide groups. Reactions that form isourea linkages, diazo
linkages, or peptide bonds are among those typically used to couple
proteins to surfaces, but any aqueous based polymer reaction that
is easily controlled can be used. Examples of polymers scaffolds
include dextran and polyamides.
[0158] Dextran is a polysaccharide polymer that is very
hydrophilic. The sugar residues of dextran contain hydroxyl groups,
which can be chemically activated for covalent bond formation.
Hydroxyl groups also form hydrogen bonds with water molecules, and
thereby create an aqueous environment in the support. When
activated with an aldehyde, dextran can be easily coupled with
proteins via amine groups (e.g., using sodium cyanoborohydride).
When activated with hydrazide, dextran can be coupled with proteins
via aldehyde or carboxyl groups using
1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride) (EDC).
However, use of dextran for certain applications is limited by its
susceptibility to microbial attack.
[0159] An alternative method for covalent immobilization of
proteins or enzymes is through various derivatized polyamides, such
as Nylon.RTM.. Polyamides are best suited for the immobilization of
high molecular weight substrates. Chemically, many polyamides are
thermoplastic polymers with high mechanical strength, superficial
hardness, and resistance to abrasive conditions caused by the
intermolecular hydrogen bond interactions established between the
amide groups of parallel chains. These characteristics make
polyamides useful for immobilized enzymes, because they provide a
favorable hydrophilic microenvironment to support both catalytic
activity and enzyme structure. Proteins covalently attached to a
polymer scaffold are amenable to conventional biochemical assays
such as ELISAs, binding assays, and activity assays.
Encapsulation of Proteins (or Cells).
[0160] Both proteins and cells can be immobilized by encapsulation
within a web, matrix, or pores of semipermeable membranes, gels, or
foams. Encapsulation of cells requires special consideration of the
following factors: diffusion of materials within and through the
support; non-toxicity of the starting materials and of the polymer
to cells; and physical properties such as optical clarity,
temperature stability, flexibility, and resistance to chemical and
microbial attack. The support is preferably resilient and flexible,
capable of hydrogen bonding, and resistant to proteolysis and
hydrolysis.
[0161] Given an appropriate scaffold, mammalian cells can be
cultured in three dimensions in a platen through-hole, enabling
both the performance of many types of cell-based assays and the
potential for imaging of cells within the through-hole using
confocal techniques. Examples of types of assays that can be
carried out include reporter gene assays, cell growth assays,
apoptosis assays, and assays involving events occurring at the cell
surface or within the cytoplasmic region (e.g., measurements of
calcium efflux from the endoplasmic reticulum). Such assays are
useful for functional studies of chemical and biological libraries.
Examples of materials amenable to encapsulation include
Poly-(2-hydroxyethyl methacrylate) (poly-HEMA) gel, Poly(carbamoyl
sulfonate) (PCS) hydrogels, and Phosphorylated polyvinyl alcohol
(PVA) gel.
[0162] Poly-(2-hydroxyethyl methacrylate), "poly-HEMA," gel has
superior mechanical properties, temperature tolerance, and
resistance to microbial attack than many natural polymers (e.g.,
gelatin, agarose, carrageenan, cellulose, and albumin). Poly-HEMA
gels can be used for entrapment of both proteins and cells. The
process of biocatalyst immobilization in poly-HEMA hydrogels
generally allows for high retention of immobilized enzyme activity,
well-controlled porosity (e.g., to ensure sufficient mass transfer
of reactants and products), and good chemical resistance. Poly-HEMA
hydrogels also protect entrapped proteins and cells from bacterial
degradation, by resisting bacteria entry into the gel support.
Additionally, Poly-HEMA hydrogels can retain a large quantity of
water, providing a microenvironment that approximates in vivo
conditions.
[0163] Poly(carbamoyl sulfonate), "(PCS) hydrogels," afford
adjustable gelation time, high mechanical stability, and resistance
to microbial attack. PCS hydrogels also have a high degree of
flexibility, which can be exploited for filtering or expelling
samples.
[0164] Phosphorylated polyvinyl alcohol, " phosphorylated PVA" gels
can be used to immobilize cells of bacteria or yeast. These gels
are economical, nontoxic, durable and support a high cell
viability. Applications using phophorylated PVA gels are limited by
their poor gas permeability.
Growth of Cells on Membranes
[0165] The invention further methods for growing and analyzing
eukaryotic cells in a format where a porous membrane is sandwiched
between platens whose holes are aligned on both sides of the
membrane. Cells are grown in the wells formed by one of the platens
holes and the membrane. A removable gasket may be placed on top of
the device to allow cells to be added. Test compounds may be
introduced to the wells on either side of the membrane with
micro-spotting pins or by aligning a solid surface pre-spotted with
test compounds over the wells. The device may be covered on both
sides with cover slips separated by spacers to reduce evaporation.
A solid substrate, such as a glass microscope slide may be used to
support the device. Image analysis can be performed macroscopically
with conventional scanning devices or microscopically using light
or fluorescence detection. Biochemical analysis may be done on
supernatants or cell lysates using standard centrifugal or vacuum
filtration devices.
[0166] In particular embodiments, platens used to create
micro-wells are sufficiently rigid to make an even contact on the
membrane. The material is preferably biocompatible and may be
metal, plastic or ceramic. The holes formed by the platen may be
less than 0.5 mm in diameter spaced less than 0.1 mm apart, or any
other configuration and dimension that allows growth of adherent
eukaryotic cells. To prevent lateral diffusion, a watertight seal
may be formed between the membrane and the platen. This may be
accomplished by coating the platen where it contacts the membrane
with a hydrophobic substance or by using a secondary platen made of
a flexible biocompatible material such as silicon, rubber, or other
suitable material supported by a rigid platen on top. The two
platens may be attached by raised surfaces on one platen fitting
into recessed surfaces on the opposing platen. The total platen
thickness on either side of the membrane whether a single rigid
platen or a rigid platen on a flexible platen is typically less
than 1 mm.
[0167] In related embodiments, adherent cell growth is maintained
on commercially available membranes or any biocompatible porous
membrane that will allow cell attachment. Optimal cell growth for
adherent cells is maintained on membranes exposed to nutrient
containing medium on either side.
[0168] The construction of the chip with medium and cells begins by
filling the through-holes in a first platen with medium. This may
be done by placing the first platen on a solid support. A gasket
may be placed on the platen and cell medium is then overlayed on
the platen. After centrifugation, the air in the platen holes is
displaced with medium. Next, a pre-wetted porous membrane is place
on top of the first platen followed by placement of a second
platen(s). Guide holes in all of the platens will allow holes to be
aligned visually. Alternatively, pins attached to the bottom
support spaced in the same configuration as the guide holes on the
platens may be used to line up the platens. After platen assembly,
a gasket may be placed on top of the platen and medium containing
cells id added. After centrifugation to allow cells to enter
through-holes, excess medium may be removed. Spacers
(.about.<0.5 mm thick) may be placed on the edges of the top
platen followed by a cover slip. The device is then clamped
together and placed in a humidified chamber compatible with cell
growth.
[0169] Pre-spotted test compounds such as proteins, small molecular
weight compounds, RNAi or other nucleic acids may be applied to the
cells using a solid substrate with compounds arranged in the same
configuration as the platen through-holes. In such embodiments, the
compounds are spotted on a solid substrate in a medium with low
volatility such as glycerol to reduce evaporation. The compounds
are then applied to the solid substrate to have spatial orientation
as the platen through-holes. The solid substrate to which the
compounds are applied may also have a guide pin that fits into the
cell-chip so that each hole is aligned with a compound. In
addition, a layer of mineral oil may be applied to either or both
platens prior to compound contact to prevent lateral diffusion and
cross contamination of compounds. Contact of the compound into the
through-holes of the platen then allows diffusion into the
through-holes and dispersal to cells attached to the membrane.
Cells may then be incubated until analysis.
[0170] In other embodiments, analysis after addition of test
compound may include image and/or biochemical analysis. Image
analysis may be done with existing technologies such as visible
light, laser scanners, visible or fluorescence microscopy to detect
morphological changes in the cell or biochemical changes by visible
or fluorescent dyes. Biochemical components of the cell lysates or
supernatants such as RNA or protein may be analyzed by existing
technologies such as centrifugal or vacuum filtration to separate
components onto a membrane or module for analysis by qPCR, or SELDI
MS.
[0171] In other embodiments, compounds and other test substances
are delivered using a standard microarray-spotting pin. The micro
format allows high throughput screening using an array spotter.
Cell viability or morphological changes can be measured directly on
the membrane. Protein or mRNA expression can be determined after
membrane processing. Applications of this technology include lead
generation screening for drug discovery or gene function studies
using RNAi, cDNA and RNA.
Fiberglass Chip
[0172] A through-hole array having holes filled with fibers can be
created in a simple and inexpensive manner. Examples of useful
fibers include fiberglass filters, mesh glass fiber filters, and
polymer filters such as Nylon.RTM. and polyethersulfone. These
materials can be surface modified for specific interactions prior
to fusing.
[0173] In one method, a sheet of loose fiberglass mesh is fused
between through-holes of an array. The fusion can be achieved by
pressing the fiberglass against a through-hole array created from
silicon or other suitable metal that has been heated to a
temperature sufficient to fuse the fiberglass, and then removing
the array. Non-reactive lubricants such as graphite or molybdenum
grease can be used to aid in the separation of the heated array and
fiberglass arrays. Alternately, the fiberglass sheet can be pressed
between two, aligned, heated through-hole arrays.
[0174] The resulting fiberglass array can be treated to make the
regions between the porous holes hydrophobic. Such treatment can be
accomplished by blowing gas through the array while exposing the
opposing face to a silanizing vapor. The porous areas in the
resulting fiberglass array can be used for immobilizing probes such
as nucleic acids or peptides with the advantage of very high
surface areas for attachment, ease of flow through the array and
the optical transparency of glass.
[0175] An alternate method for producing the fiberglass arrays is
to inject or cause a polymerization of a thermoplastic material in
the space between the desired holes, for example, by using a
printing technique to deposit a polymer, epoxy, monomer, or
polymerization initiator, or by photo-initiating polymerization or
curing in the desired areas using one or more photomasks.
Growing Porous Glass in the Through-Holes.
[0176] Another method for producing an array of through-holes
containing a porous material is to machine an array by an
appropriate method such as EDM and then to grow material in the
array. Porous glass can be introduced into the array by using
pressure to force a mixture of potassium silicate mixed with
formamide into the array and then baking for several hours. An
array of capillaries that is fused or embedded in a binding agent
can be filled with porous glass by this method and then sectioned
with a cooled sectioning saw to create multiple, thin platens of
porous-glass filled through-hole arrays. By including particles
such as porous silica or polymer beads in the potassium silicate
mix, the affinity properties and porosity of the material can be
adjusted as desired.
[0177] III. Methods of Loading Array Devices with Samples.
Synthesis of an Array by Masking.
[0178] Certain methods feature applying at least one mask to a
platen, or an aligned stack of such platens, such that, when a
reagent is applied to the mask, the reagent communicates with only
the through-holes selected by the mask. In particular embodiments,
the reagent can be selected from an aqueous solution, an organic
solution, a dry powder, a gel, a gas, or an electromagnetic
radiation (e.g., heat, light, X-rays, ultraviolet radiation, a
magnetic field). Methods for introducing reagents through the mask
and into the array include applying a mechanical or optical
pressure to the reagent reservoir, diffusion, and electrophoresis.
Measures to prevent cross-contamination of neighboring wells
include placing a second, identical mask on the face of the platen
opposite the face to which the reagent is applied, and using a
blotter to absorb excess liquid flowing through the through-holes.
The masks and arrays can be held together by electrostatic,
magnetic, or gravitational forces, or by applied pressure (e.g., by
applying a clamp to the periphery of the stacked platens). Masks
and arrays can be separated when liquid fills the through-holes by
application of electrostatic, magnetic, or gravitational forces, or
by application of a negative pressure to the stacked platens.
Stacks of platens can optionally be dried to facilitate
separation.
[0179] The liquid samples in the through-holes can be subdivided by
placing an empty array beneath a filled array or array stack, and
then applying positive or negative pressure to force a portion of
the liquid into the empty array. A small (typically less than 100
.mu.m) air gap is maintained between the filled array and the empty
array to facilitate the physical separation of the two arrays after
the fluid transfer is complete.
[0180] Through repetitive cycles of adding masks, introducing
reagents, and optionally washing the array, a defined pattern of
chemicals can be created in the through-holes of the platen by
using solution phase chemistry.
[0181] By first derivatizing the inner surfaces of the
through-holes with a linker molecule that contains a free
functional group, the inner surface of each hole can be coated with
a member of a library of molecules. The patterned through-hole
array is then used to analyze chemical information. As described
above for solution phase systems, repeatedly adding masks,
introducing reagents, and washing the array, can result in a
defined pattern of chemicals attached to the linker molecules in
the through-holes. FIG. 1 is an illustration of this process. A
platen (1) having through-holes with derivatized inner surfaces (4)
is brought into contact with a mask (2) configured such that only
select through-holes in the platen communicate with the reagents to
which the masked platen is exposed. In this embodiment, two
identical masks (2,3) are placed in contact with the top and bottom
surfaces of the platen so as to prevent fluid from entering the
covered through-holes. Preferably, the masks and platen are flat
and polished (e.g., to an optical finish) so that they create an
airtight seal when contacted. Alternatively, the mask or platen, or
both, can be coated with a soft polymer or gasket-forming material
to facilitate sealing, or the mask and platen surfaces can be
manufactured with contoured surfaces such that one fits into the
other to form a large contacting surface area. The contoured
surfaces can also provide alignment features that can aid in
co-alignment of through-holes in the masks and platens. One example
of an interlocking array design is shown in cross-section in FIG.
2, where the through-hole arrays are contoured to have opposing and
matching geometrical features. The requisite geometrical features
can be obtained, for example, by patterned chemical etching or
micromilling.
[0182] Similar approaches can be applied to the manufacture of a
system having two masks with a platen sandwiched in between. The
mask-platen sandwich can then be loaded with a reagent, such that
the reagent enters into the open (i.e., non-masked) through-holes.
After the reagent has reacted with the linker molecules located
inside the through-holes, the excess reagent can be washed from the
sandwich, and the process can be repeated with a new reagent. The
mask can then be removed and a new mask applied, or the synthesized
material can be removed from each through-hole.
[0183] Another embodiment uses only one mask to block one end of
the through-hole with an airtight seal. The air trapped in the
through-hole prevents liquid from entering into the
through-hole.
[0184] In another embodiment, a porous material derivatized with a
linker molecule having a free functional group is in each
through-hole and members of a library of chemical probes are
attached to the linker molecules. Alternatively, the inner surfaces
of the through-holes are made porous (e.g., by etching with
hydrofluoric acid), and the library of probes is attached.
Containment of probes within the porous polymer or surface (i.e.,
as opposed to immobilization of library members on the inner
surface of the through-hole) increases the density of library
molecules in each through-hole.
[0185] For chemical synthesis, a porous polymer derivatized with a
linker molecule having a free functional group can be inserted into
the through-hole. By repeatedly adding masks, introducing reagents,
and, optionally, washing the array, a defined pattern of chemicals
attached to the linker molecules can be created in the
through-holes. Containment of linker molecules within the porous
polymer increases the density of synthesized molecules in each
through-hole.
[0186] A similar synthetic procedure can be used with a molecular
library immobilized inside a porous through-hole surface.
Mask Production Methods.
[0187] An embodiment of the invention provides for producing a mask
by use of a through-hole array substantially identical to the array
used for synthesis or analysis. In one embodiment, a mask can be
fabricated from metal, dielectric (glass, polymer) or semiconductor
(e.g., silicon, germanium, gallium arsenide). Inertial drilling is
a suitable manufacturing process for fabrication of masks in
polymers, glass or metals. Patterned chemical etching processes,
such as deep reactive ion etching (DRIE), provide another suitable
manufacturing process for fabrication of masks in semiconductors
and dielectrics such as glass. Electrospark discharge machining
(EDM) is another suitable manufacturing process for fabrication of
masks in conductive materials (e.g. conductive semiconductors and
metals).
[0188] Another embodiment of the invention, shown in FIG. 4,
provides for producing a mask by use of a through-hole array
substantially identical to the array used for synthesis or
analysis. A solution (1) is added to each hole of a through-hole
array (2) such that the solution contains a molecule or mixture of
molecules that polymerize upon irradiation (Step 1). The solution
could be for example, an aqueous solution of polymer and a
photo-reactive molecule that produces free-radical initiators of
polymerization when irradiated with ultraviolet light (3) (Step 2).
An example of a UV curable polymer suitable for mask fabrication
can be found in the class of UV-curable polyurethane epoxies. By
shining ultraviolet light onto each hole that the artisan wishes to
be blocked in a given step of adding reagent to a through-hole
array, a mask (4) is built (Step 3). The resulting polymer is
impervious to the fluids to which the mask is exposed. The
through-holes to be blocked can be illuminated through an optically
opaque mask or illuminated sequentially with focused light.
[0189] Another embodiment, shown in FIG. 5, uses an array of pins
or posts to make a mask. The external dimensions of the static pin
array are selected such that they have a precision fit into a
matching through-hole. Viewed in cross-section, a pre-fabricated
pin array (1) selectively blocks those through-holes from
communicating with a reagent as part of a synthesis sequence. A
through-hole array (2) is prepared with the inside surface of the
through-hole derivatized with a linker molecule having a free
functional group (Step 1). A pin array is inserted into the
through-hole array (Step 2) and by the process of introducing
reagent and optionally washing the array, a defined pattern of
chemicals in the open through-holes is created attached to the
linker molecules (Step 3). The pin array mask is removed and the
process is repeated with a different mask and reagents resulting in
a defined pattern of chemicals created in the through-hole array
(3) attached to the linker molecules (Step 4).
[0190] Pins in the array are fabricated to precisely fit into each
matching through-hole forming a hermetic seal. Polymer coatings
(e.g., Teflon) can be applied to each pin to facilitate sealing. An
advantage of this approach is that the post arrays are reusable and
need only to be made once. The large contact area between the pin
and through-hole interior surface ensures a viable hermetic seal.
As opposed to the plate masks, only the pins contact the array
plate, thereby facilitating decoupling of the mask from the array
plate after completion of a synthesis cycle. A further advantage is
gained by application of only one pin array to seal through-holes
in an array. This is achieved by physical blockage of the hole by
the inserted pin or by the pressure of air entrapped in the
through-hole. The pin array can be manufactured by a variety of
fabrication techniques. One example is to electro-spark discharge
machine (EDM) a regular array of pins having the precision cross
sections needed to hermetically seal a through-hole. With a
die-sinking EDM, selected posts could be machined away with a die
to form the spatial pattern of pins matching a particular mask
configuration. A second example is to start with a plate having
holes in the spatial pattern matching a mask configuration. The
pins are fitted into each through-hole and simultaneously soldered
in place.
[0191] Another embodiment uses an actuated pin array forming a mask
to hermetically seal selected through-holes in an array. The
actuated pin array is similar in design to the static pin array
except that each pin can be extended or retracted such as to
reconfigure the pin array to make a different mask. This is
different from the static pin array in that each different mask
requires a different pin array. Extended pins are inserted into
through-holes whilst retracted pins are not. Individual pins in the
array are electronically addressable to be actuated by one of
several types of methods including: piezoelectric, electromagnetic
(solenoid), magnetostrictive, shape memory alloy or conducting
polymer. One advantage of this approach is a single pin array can
be reconfigured to produce n!/(n-2)! number of different masks
where n is the number of pins in the array. A second advantage is
generation of different masks in an automated manner. This is
important when the processes requiring masking of the through-hole
array are also automated.
[0192] In still another embodiment, the mask has holes that allow
reagents to flow to selected positions in the through-hole plate.
In other positions, the mask includes raised features (e.g., pins
or bumps) that fit into the holes to be blocked. This approach aids
in alignment of the mask and platen, allows a single mask to be
used, and ensures a good seal between the mask and the platen.
[0193] The through-hole array can also be fabricated with valves on
one side of the through-hole array. Each through-hole thus has a
valve that either blocks or unblocks one end of the through-hole.
Pressure of air entrapped in the through-hole prevents liquid from
entering the open end of the blocked through-hole. The valve can be
formed as a bilayer actuated by shape memory alloy,
electrostrictive, electroporous, piezoelectric, magnetostrictive,
or conducting polymer materials. Microsolenoid activated valves can
also be used to perform a similar function.
[0194] Another method for producing a flow-mask is to laminate a
platen on at least one side with a non-permeable membrane such as
an adhesive tape. The mask is then created by selectively
perforating the laminate material. Methods by which the laminate
can be perforated are: by an actuated pin array, by laser
machining, by contacting with a platen that allow heat to be
applied in a localized manner, thus melting or burning a hole in
the laminate. Serial dilutions can be performed during loading.
[0195] This operation can be performed to fill a series of
through-holes with different concentrations of the same solute. In
a typical example, a microsyringe, or other fluid transfer device,
is positioned over the first through-hole and used to fill the
first and second through-holes with a 16.times. solution of the
solute. The outer surface of the syringe tip and the faces of the
array must be nonwetting toward the solution being dispensed. For
each hole, a sufficient volume of solution, referred to below as "Y
nl," is dispensed to overfill the hole enough to create positive
menisci. The microsyringe tip is then rinsed three times and filled
with solvent. The syringe tip is positioned above the second
through-hole and Y nl of solvent is expelled such that it forms a
droplet at the end of the syringe tip. The syringe tip is lowered
until the solvent droplet contacts the solution surface, causing
the two liquids to mix and produce an 8.times. solution. The
syringe plunger is then withdrawn to suck up Y nl of 8.times.
solution and dispense it into the next through-hole. Another
droplet of solvent is then formed and the process can be repeated
to dispense 4.times., 2.times., 1.times., etc. into individual
array through-holes.
Serial Array of Masks on a Flexible Sheet
[0196] Since synthesis of an array of probes in through-holes can
involve the use of many masks, a rapid and automated method for
interchanging masks is desirable. One method involves creating the
multiple masks in a single, flexible tape, such as a metal or
plastic tape with a width greater than that of the array to be
synthesized. The first mask can be aligned with the array and
reagents can be transferred to the array. The tape can then be
advanced to reveal the next mask prior to addition of the second
reagent. This process can be repeated until the solid-phase
synthesis is complete. For steps that require washing the entire
array, a single large hole, or holes corresponding to each position
in the array, can be produced on the tape. For additional
through-put and customization of the synthesis, the tape can be
produced concurrently with the synthesis, for example, by having an
array of punches or a micro-positioned laser drilling system to
create holes as the tape advances. A second tape or blotter can be
used on the opposite side of the array (e.g., to prevent
cross-talk). Various methods for aligning the masks with the arrays
can be used, including placing precise alignment notches in the
tape, and using optical or amperometric detection to determine mask
position relative to the array.
Synthesis of Arrays Using Masks and a Membrane.
[0197] The mask-synthesis methods described here can also be used
with non-addressable porous membranes (e.g., a filter), instead of
with the rigid platen.
Capillary Tube Array
[0198] Viewed in cross-section, a capillary tube array (FIG. 6) is
constructed from capillary tubing (1) with an external diameter
that fits precisely into the through-holes of a second array. The
tubing array (3) is designed such that tubing at one end has a
center-to-center spacing equal to the spacing between holes in a
through-hole array and tubing at the opposite end has a
center-to-center spacing equal to the center-to-center spacing of
wells in a microtiter plate (2). Plates with through-holes having
these separations serve as jigs (4) to hold the tubing in a regular
array. Additional through-hole plates placed between the two ends
are spacer jigs providing additional support for the tubing array
as the center-to-center spacing is changed over the tubing
length.
[0199] The internal volume of each tube in the array is slightly
greater than the total volume of a column of aligned holes in the
array stack. For example, if the through-hole dimensions in the
array are 250 .mu.m.times.250 .mu.m.times.1000 .mu.m giving a
volume per through-hole equal to 62.5 nl, then the volume of one
set of holes in a stack of 100 arrays is 6.25 .mu.l (100.times.62.5
nl). Capillary tubing with an internal diameter of 200 .mu.m and an
external diameter of 245 .mu.m is readily available; thus a minimum
tube length of 200 mm stores the volume of fluid needed to fill
this set of through-holes.
[0200] One end of the tubing array is inserted into the wells of a
microtiter plate where each tube is inserted into a matching well.
A negative pressure is applied across the length of tubing, drawing
liquid from each well into its corresponding tube. Negative
pressure can be applied to each tube individually or as shown in
FIG. 6, the ends of the tube array can terminate in a chamber that
can be partially evacuated. After filling each tube of the array,
the microtiter plate is removed. The liquid can be stored in the
tubing array for an indefinite period of time, either frozen or in
a humidified environment. Multiple tubing arrays can be filled from
the same microtiter plate (assuming there is sufficient volume of
liquid per well) or different tubing arrays can be filled from
different microtiter plates.
Transfer from a Microtiter Plate with an Array of Flexible
Members
[0201] As illustrated in FIG. 7, fluid can be transferred from
individual wells of a microtiter plate (3) with an array of
flexible members (2) (e.g., shape memory alloy fibers). The fiber
diameter is equal to or less than the inside dimension of the
through-holes in the array (1) into which fluid will be
transferred. The number of fibers in the bundle can, for example,
be equal to the number of wells in the microtiter plate. The ends
of the fibers at one end of the bundle can have a center-to-center
spacing equal to the spacing of the holes in the through-hole
array, while the ends of the fibers at the opposite end can have a
center-to-center spacing equal to the spacing of wells in the
microtiter plate. The fibers can be held in place with a series of
through-hole jigs designed to increase the spacing between fibers
from one end of the bundle to another. Once fixed in place, shape
memory alloy fibers can be heated above their critical transition
temperature to make the imposed fiber curvature permanent. After
they are cooled to room temperature, the fibers can be removed from
the holding jig, with the change in fiber center-to-center spacing
intact. The close packed end of the fiber bundle can then be
inserted into the through-hole array into which fluid from each
well in the plate is to be placed. The opposite end can be arranged
such that each fiber is positioned above a well in the microtiter
plate, and the ends of the fibers can be immersed in the fluid
contained in each well. On retraction, a small volume drop (4) can
remain attached (e.g., by surface tension) to the end of each
fiber. A force can be applied to the opposite end of the fiber
bundle to pull the bundle through the holes of the through-hole
array, such that the fluid is brought into contact with the
corresponding through-holes. As the fibers are pulled through the
hole, surface tension can act to hold the liquid in the
through-hole as the fiber is removed.
Pressure Loading
[0202] The array can also be loaded by applying a pressure across
the platen, thereby causing a dilute solution of reagent and/or
sample to flow through the array of through-holes. This method can
be advantageous if the through-holes are already loaded with
reagents, and a reaction with a second set of reagents is
desired.
Bead Loading
[0203] Bead loading can be used to load an entire combinatorial
library immobilized on microscopic polymer spheres of uniform size.
In this method, the through-holes in an array can be shaped so as
to hold only one microsphere per through-hole. The through-holes
can additionally have a tapered cross-section, such that the
microspheres sits in the holes either at or below the array surface
(FIG. 8).
Transferring Contents from a Second Array
[0204] Replicating a platen containing through-holes reproduce an
array wherein each channel contains a colony of cells having a
unique genetic profile. The master plate is prepared from a
suspension of cells of diverse genetic characters. The suspension
can be diluted such that when an array is loaded from the dilute
solution, an average of one cell is transferred into each channel.
The array is then incubated in an enclosed humidity chamber with
the appropriate temperature and agitation for the cell type until
the cells have reached mid log phase. The cell number density can
be estimated by observing select channels under an optical
microscope, measuring the amount of scattering when light is
incident on the arrays, or if the cells also contain a gene for
green fluorescence protein production, by measuring the intensity
of fluorescence from each channel. Various methods can be used to
transfer a portion of the cells from each channel into
corresponding channels in a second array.
[0205] One method of transfer involves freeze-drying the contents
of the through-holes. A master through-hole array is prepared from
a suspension of cells of diverse genetic characters as described
above. A second identical through-hole array is filled with growth
media. The two through-hole arrays are aligned and stacked to mix
contents of corresponding through-holes. The stacked through-hole
arrays are freeze-dried and separated. The colonies are
reconstituted by filling the array with media. In the case of
robust cells such as bacteria and yeast cells, dehydration by
evaporation can be sufficient to remove the liquid without
significantly compromising cell viability. This method is also
useful for storing compound libraries such as small molecule
libraries in a dry form. For example, the crystalline compounds can
adhere to the walls of the channels. Compounds can then be stored
for long periods of time and reconstituted by the addition of
solvent. Compound libraries can also be stored in a powdered or
crystalline form while frozen in an inert matrix. A suspension of
crystalline compounds can be made in a low molecular weight
perfluorinated hydrocarbon and stored frozen. An example of a
perfluorinated hydrocarbon that can be used for this purpose is
perfluorohexane, which has a melting point of -4.degree. C. and a
boiling point of about 59.degree. C. Upon retrieval of the sample,
the hydrocarbon can readily evaporate at atmospheric pressure or
under an applied vacuum; the samples can then be reconstituted with
DMSO, water, or other solvent.
Transfering/Mixing Samples in a Through-Hole Array with Samples on
a Flat Surface
[0206] Liquid samples contained in a through-hole array can be
transferred in part or in whole to a flat surface having a pattern
of hydrophilic and hydrophobic regions. The hydrophilic regions
must be spatially isolated from one another and must match the
spacing of the through-holes such that when the array is contacted
with the surface, the contents of each through-hole contacts at
most one hydrophilic region. The hydrophobic regions on the surface
also serve to isolate the transferred fluids from one another.
[0207] The surface may support an array of samples that can be
registered with an array of probes contained in a through-hole
array. The samples must be spatially isolated from one another and
must match the spacing of the through-holes such that when the
array is contacted with this surface, the contents of each
through-hole contacts at most one sample. The surface can also
contain a hydrophobic pattern matching the pattern of the
through-hole array to prevent cross-contamination after the surface
and array are contacted. If a hydrophobic pattern is not provided,
the platen and surface can be pressed tightly against one another
to form a hermetic seal and thus prevent mixing between adjacent
samples.
[0208] Alternatively the flat surface can support an array of
probes, such as fluorescently labeled oligonucleotides, chemical
substrates, or cells, matched to a through-hole array containing
samples. The probes can be attached to the surface in a variety of
methods: they can be chemically or physically absorbed on the
surface, trapped in a porous matrix, attached with an adhesion
layer, or contained in a drop of liquid. The probes can be used to
generate a change in a detectable physical property of the sample
(such as fluorescence, optical absorption or mass) in response to a
chemical or biological characteristic of the sample as binding
activity or enzyme activity.
Plunger Sterilization
[0209] Plunger sterilization can be an important aspect of a serial
sampling scheme. One approach is to have at least two
plungers--while one plunger is sampling, the other is being
sterilized. The two plungers can be located in a common mechanical
housing, for example, mounted to rotate about an axis parallel to
the plunger axis. Plunger sterilization can be accomplished by heat
or exposure to sterilizing agent (e.g., 70% ethanol). A wire (e.g.,
platinum) loop inside a ceramic sheath is an example of a suitable
plunger design. The ceramic sheath imparts mechanical rigidity and
is an electrical insulator whereas the wire loop permits heating
with an electrical current. As an example, assume that a platinum
wire loop is used, having a specific heat of 4 J/kg-.degree. C. To
electrically heat a 10.sup.-4 kg wire to 1000.degree. C. in 0.2 s
requires a maximum current of 4.5 A, which is easily achieved with
medium power thyristors. Rapid cooling can be achieved from the
spray of a volatile sterilizing agent (e.g., ethanol), the high
latent heat of vaporization of which can aid in cooling the heated
wire. Alternatively, the wire can be rapidly cooled by a spray of
gas or liquefied gas such as liquid nitrogen.
[0210] Sequential sampling with a single sampling device can be
extended to a linear array of sampling devices as, for example, a
linear array of mechanical plungers. There can be a substantial
time saving (e.g., 1/M for an M.times.M array of through-holes),
since motion along one orthogonal direction can be avoided. Similar
time saving considerations are applied to two-dimensional sampling
techniques. In an alternate approach, pressure generated by
spatially localized jets of liquid, solid, or gas can be used in
place of the plungers.
Loading with High Protein/Surfactant Media
[0211] Loading the platen by submerging the platen into a reagent
of interest when the reagent or media is high in protein or
surfactant and therefore low in surface tension, can be a
challenge. Droplets of the low surface tension fluid can remain on
the surface of the platen after removal from the fluid to be
loaded. If droplets or a surface coating of protein rich media
remains on the surface, it can result in contamination of the assay
or crosstalk between through-holes. This problem is significantly
lessened by pulling the platen up through a layer of a hydrophobic
fluid that is immiscible with the fluid to be loaded. This provides
a wiping or "liquid squeegee" effect, removing the proteins or
surfactants adhering to the surface of the platen. The wiping fluid
setup can be generated one of at least three methods.
[0212] Submerging the platen in the fluid to be loaded, ensuring
the through-holes are filled. The platen is withdrawn from the
fluid to be loaded, and submerged in a hydrophobic fluid that has a
greater affinity for the proteins or surfactants than the protein
or surfactant has for the surface of the platen. Such fluids can
include but are not limited to perfluorodecalin, silicone oil and
mineral oil.
[0213] Alternatively, the platen is first submerged into the fluid
to be loaded. Then a small amount of a less dense, hydrophobic
fluid such as but not limited to mineral oil and silicone oil is
gently layered on the surface so that the surface of the loading
fluid is completely covered with this wiping fluid. Then the platen
is slowly removed from the loading fluid, up through the wiping
fluid.
[0214] Additionally, a container can be used as in FIG. 23. The
container has a baffle that extends from one side if the container
to another, but does not extend to the bottom of the container. The
container is filled to a level midway up the baffle with the fluid
to be loaded. This creates two open surfaces of loading fluid
connected by a channel underneath. Wiping fluid is gently layered
on one of the surfaces, and is kept from the other surface by the
baffle. The platen is submerged into the fluid to be loaded,
underneath the baffle, and removed through the wiping fluid. This
method for employing the wiping fluid is well suited for high
throughput and automation methods.
Synthesis of Arrays by Selective Loading of Fluids into
Through-Holes.
[0215] An array of pins (1) can be fabricated with pins arranged to
be co-registered and co-aligned with a second regular array of
through-holes (2) (FIG. 3). Each through-hole can be prepared such
that linker molecules suitable for chemical synthesis are
immobilized on the interior surface of each through-hole. The ends
or tips of the pins can be made hydrophilic over a pre-determined
surface area whilst the remainder of the array surface area is made
hydrophobic. In one embodiment, the tips of the pins in the pin
array are brought into contact with the fluid to be loaded into the
through-holes of the through-hole array (3). When retracted, a
small volume liquid drop adheres to the hydrophilic region of each
pin (e.g., by virtue of surface tension). The volume of liquid
adhering to the pin is determined by the relative surface energies
between the liquid and solid surface and the depth of immersion of
the pin into the liquid relative to the hydrophobic surface area.
The pin array with the adherent liquid drops can be arranged
relative to the through-hole array such that the through-holes into
which liquid is to be placed are aligned relative to each pin with
an adherent liquid drop. The two arrays can be brought into contact
such that the drops enter into the corresponding through-holes
(e.g., by capillary pressure). Removal of the pin array leaves
behind the fluid placed into the through-holes of the array (4). A
chemical reaction can thus be initiated between the linker
molecules immobilized on the interior surface of the through-hole
and the liquid placed in the through-hole. The chemical reaction
rate can be increased by raising temperature and/or changing the
partial pressure and/or composition of gas in the atmosphere
surround the through-hole array. After the reaction is complete,
the array can be washed to remove unreacted components, and dried
to remove excess solvent. A second pin array with either the same
or different pin configuration can be loaded with fluid and the
synthesis process can be repeated. Because the array loading
process can rely on simple mechanical motions, the array loading
can be quite rapid. The rate-limiting step, therefore, would be the
synthesis step itself.
[0216] An alternative embodiment features the use of a pin array
sparsely populated with pins aligned with respect to the
through-holes of a regular array. The pins can be fabricated such
that their length is at least twice the thickness of the
through-hole array platen. Each through-hole can be prepared such
that linker molecules suitable for chemical synthesis can be
immobilized onto the interior surface of each through-hole. The end
of each pin can, for example, by made hydrophilic and the remainder
of the array can be made hydrophobic. The lateral dimension of the
pins can be set such that the pins can be inserted into the
matching through-holes in a regular array. The pin array can then
be inserted through the second through-hole array such that the
pins extend through to the opposite side of the platen. This
assembly can be arranged relative to the surface of a fluid that is
to be placed into the through-holes through which pins have been
inserted. The tips of the pins can be brought into contact with the
fluid surface, and, on retraction, small volume drops can adhere to
the end of each pin. As the pin array is retracted relative to the
through-hole array, the liquid drops can come into contact with the
through-hole into which the pin has been inserted. As the pin
leaves the through-hole, surface tension can keep the fluid volume
inside the through-hole.
[0217] An advantage of both embodiments is that they provide a
rapid, simple, and precise method by which fluid can be loaded into
through-holes of an array. Fluids containing surfactants can, for
example, be easily transferred into the array with minimal
contamination between adjacent through-holes because of the long
path along the array surface separating the ends of adjacent
pins.
Synthesis of a Stochastic Array.
[0218] A stochastic array can be created using a nozzle moving
randomly to different through-holes on the array. Loading the
through-holes in a stochastic method, wherein one variable of the
sample is varied in a random manner has many applications. For
example, the stochastic loading can vary with respect to the
concentration of a particular reagent in the sample loaded. The
difference in concentration of the reagent allows simultaneous
sampling, in a controlled manner, of many different reaction
conditions. For example, this sort of application of samples can be
used to optimize reaction conditions in chemical synthesis or can
optimize parameters of a crystallization experiment.
[0219] IV. Reactions/Experiments in the Platens.
Synthesis of Combinatorial Libraries.
[0220] The present invention provides new methods for producing a
combinatorial library in a platen. The types of combinatorial
libraries that can be produced using the new methods include, but
are not limited to, nucleic acid arrays, peptide arrays, protein
arrays, polymer arrays, and arrays of small molecules.
[0221] Certain of the new methods include immobilizing a linker
molecule on the inner walls of the platen's through-holes, or in a
porous material located inside the through-holes, and sequentially
flowing reagents through masks to build a pattern of chemicals. For
example, to create a nucleic acid array, phosphoramidite monomers
can be sequentially placed in the through-holes in defined patterns
with activation, reaction, washing, and deprotection steps in
between each addition of monomer. To create an array of small
molecules using solid or liquid phase synthetic chemistry, linker
molecules with, for example, protected amide groups can be
sequentially placed in the through-holes in defined patterns with
washing and deprotection steps between each synthetic reagent
addition step. Chemical synthesis with solid phase chemistry can be
carried out on core molecules linked to the interior surface of a
through-hole, inside a porous material placed in a through-hole, or
on a polymer bead placed in the through-hole. Core molecules with
chemically active side groups can also be prepared in the
through-holes using solution phase chemistry.
Chemical and Physical Process Optimization
[0222] Optimization of a chemical or physical process requires
searching a multivariate space of experimental conditions for a
subset of those parameters producing the desired outcome. The
search strategy can be either systematic or stochastic; either or
both strategies can be implemented as embodiments of the present
invention. Systematic optimization is aided by producing an array
of chemical or physical conditions from one through-hole to the
next in a known and regular way.
[0223] Stochastic variation of reagent concentrations from one
through-hole to the next can be accomplished, for example, by first
uniformly loading an array with a first reagent. A container
holding a second reagent can then be positioned above the array,
for example, on a motorized two-axis mount, and the second reagent
can be dispensed through a nozzle with an electronically controlled
valve. The nozzle can be moved to different randomly selected array
positions (or to positions determined by an algorithm), and the
amount of liquid dispensed through the nozzle can be determined by
a randomly selected (or algorithm-selected) time duration less than
a pre-selected maximum. In this manner, different amounts of the
second reagent are dispensed into through-holes containing the
first reagent. This process can be repeated with additional
reagents as needed. The reactions with optimal outcomes can be
identified by analyzing the contents of the through-holes. If the
second reagent is distributed randomly, rather than according to an
algorithm, lack of specific knowledge regarding the starting
conditions for these reactions can make duplication difficult.
However, if one is interested in the reaction products alone, then
a stochastic approach can provide a facile method for rapidly
searching a large experimental parameter space for a desired
reaction outcome. Moreover, the reaction conditions can sometimes
be inferred, for example, by examining the contents of other
through-holes in the array where little or no reaction occurred,
and then combining the results in a multivariate plot. Optimal
reaction conditions can be inferred from domains containing little
or no data. Another approach to assess initial reaction conditions
is to produce a replica plate using the same dispensing protocol
but into an array uniformly loaded with solvent without any of the
first reagent. Comparison of through-holes showing the desired
chemical activity with the contents of the corresponding
through-hole in the replica plate would provide information as to
the most likely starting conditions of the observed reaction.
[0224] If large numbers of conditions need to be tested, the
multiple reagents can be randomly sprayed onto the array until all
of the through-holes have been filled. The surface can then be
wiped with a rubber spatula to remove excess fluid. A reaction can
be initiated by stacking the array with a second array, and the
result can be probed optically. Because the contents of each
address in the array will be unknown, one can either chose
promising addresses, and then analyze the contents to determine
what was in the hole, or else replicate the plate prior to
initiating the reaction, and then use the replicate plate to
determine the optimal conditions.
[0225] These examples also allow for physical parameters to be
either systematically or randomly varied from one through-hole to
the next. For example, a temperature gradient can be imposed across
one or two-dimensions of an array fabricated from thermally
conductive material, for example, by holding the edges at different
temperatures. If the temperature of a heating/cooling source is
changed with time, then the temperature distribution across the
array can be varied. The rate of temperature change is generally
proportional to temperature; thus, both the temperature and the
rate of temperature change can vary from one through-hole to the
next. Alternatively, a focused laser beam can be directed to heat
each through-hole independently, thus enabling control of the
liquid in each through-hole with time as described, for example, in
U.S. Pat. No. 5,998,768, incorporated by reference in its
entirety.
Protein Crystallization
[0226] X-ray diffraction from crystallized proteins is an important
analytical tool for determination of protein structure and
function. Proteins can be difficult to crystallize because they
generally include a multiplicity of hydrophobic and hydrophilic
molecular groups. As a consequence, proteins often crystallize only
under a specific set of solvent, pH, salt concentration, and
temperature conditions. A high throughput method for protein
crystallization is enabled by the present invention.
Screening Methods
[0227] The parameters that determine whether or not a biochemically
efficacious compound is suitable for further development as a
pharmaceutical compound include absorption, distribution,
metabolism, excretion, and toxicology ("ADMET"). As the number of
potential drug leads increases due to advances in primary high
throughput screening, ADMET testing can become increasingly rate
limiting in the drug discovery process.
Adsorption.
[0228] Oral administration is the preferred route of administration
for small molecule drugs. For an orally administered drug to have
biological efficacy it must be bio-available (i.e. it must have the
ability to pass through the gut and into the bloodstream). The
ability to assess the ability of a drug candidate to pass through
the lining of the gut in an in vitro assay is highly desirable.
Typically, such absorption assays utilize a monolayer of cells
grown on a semipermeable membrane that separates two liquid-filled
chambers. The drug candidate is added to one of the chambers and
after sufficient time for diffusion or transport, the concentration
of the molecule in the other chamber is quantitatively
analyzed.
[0229] One such absorption assay commonly used in the
bio-pharmaceutical industry is the CaCo-2 absorption assay. The
CaCo-2 assay interrogates the ability of a molecule to pass through
a single layer of a colon cell line, known as CaCo-2. Typically,
the rate of both apical to basal and basal to apical diffusion
across the cell layer is determined. The CaCo2 assay is usually
performed in an apparatus that provides two chambers of fluid
separated by a porous membrane. The membrane is permeable to cell
growth products, but acts as a support and impermeable barrier for
the cells. A monolayer of cells is grown across the surface of the
membrane, and the active or passive transport of molecules from one
chamber to the other across this cell barrier is assayed.
[0230] The platens containing arrays of through-holes can be
configured several ways to provide an array of absorption assays
(e.g. using CaCo-2 or other cells), thus increasing throughput and
minimizing reagent volumes. In one embodiment, a isotropically
porous membrane (such as, but not limited, to a PTFE filter)
treated to provide a biologically compatible surface for growing
adherent cells. The membrane has dimensions at least the dimensions
of the array of through-holes, and is placed on one platen so that
it covers the through-holes of the platen. A second platen with
matching through-holes is placed on the membrane so that the
membrane is sandwiched between the two platens. Pressure is applied
to the platens so that the membrane is collapsed between adjacent
through-holes and no chemical crosstalk may occur between
non-opposing through-holes, but allowing chemical communication
between opposing through-holes for the assay.
[0231] In a second embodiment, the membrane is anisotropically
porous, consisting of parallel pores through the membrane. Examples
of this type of membrane are Isopore and Nucleopore filters sold by
Millipore Corporation. As in the previous embodiment, the membrane
is sandwiched between two platens, and pressure is applied. In this
embodiment, pressure is not required to collapse the membrane
between adjacent through-holes, but only to seal between adjacent
through-holes. The parallel pores of the membrane allow chemical
communication between opposing through-holes but not adjacent or
non-opposing through-holes.
[0232] In a third embodiment, the membrane is patterned with
regions of porosity spaced and sized like the pattern of
through-holes in the platen. The membrane is sandwiched between two
platens so that the areas of porosity match up with the
through-holes, allowing chemical communication between opposing
through-holes but not adjacent or non-opposing through-holes.
[0233] In a fourth embodiment, the membrane is made from a platen
of through-holes, in which the through-holes contain a porous
material, such as but not limited to porous silica. In this
embodiment, the membrane platen is sandwiched between two platens
of though holes, allowing chemical communication between opposing
through-holes but not adjacent or non-opposing through-holes.
Metabolism.
[0234] For metabolism studies, compounds can be tested for their
propensity to be degraded by various cytochrome P-450 (CYP-450)
enzymes or by liver microsome preparations. Propensity for causing
drug-drug interactions can be estimated by assaying inhibition of
various CYP450 enzymes by each drug or drug candidate.
[0235] An embodiment of this invention provides for measurement of
cellular metabolism of compounds from a library. As described above
in connection with adsorption assays, the compounds can be small
organic molecules, peptides, oligonucleotides, or oligosaccharides.
The cells can either be suspended in liquid, or can be grown as a
monolayer of cells inside the through-holes or on a membrane having
high longitudinal permeability and low lateral permeability. The
membrane can include, for example, polymerized monomers in the
through-holes of a plate, or hydrophilic/hydrophobic domains in a
flexible membrane with domain size and center-to-center spacing
equal to that of the through-hole array. Volumes of known
concentrations can be loaded from the compound library into one
through-hole array. The cell array or layer can be placed in
contact with the library array and incubated, and the array
composition can be analyzed to determine the change in compound
composition or amount with cellular metabolism.
Toxicity.
[0236] An embodiment of this invention provides for measurement of
cellular toxicity of compounds from a library. As described above
in connection with adsorption and metabolism assays, the compounds
can be small organic molecules, peptides, oligonucleotides or
oligosaccharides, and can either be suspended in liquid or grown as
a monolayer of cells inside the through-holes or on a membrane
having high longitudinal permeability and low lateral permeability.
The membrane can include, for example, polymerized monomers in the
through-holes of a plate or hydrophilic/hydrophobic domains in a
flexible membrane with domain size and center-to-center spacing
equal to that of a through-hole array. Volumes of known
concentrations are loaded from the compound library into one
through-hole array. The cell layer can be placed in contact with
the library array and incubated, and the cells in each through-hole
can be analyzed for viability.
Ligand Screening by Affinity.
[0237] It can be desirable to measure or rank the affinity of
various members of a compound library toward a particular target
macromolecule, or to measure the affinity of an analyte toward
various members of a probe array. Such screening can be carried out
using the new methods described herein. For example, affinity
experiments can be carried out by immobilizing a target in many
holes of the through-hole array and probing with a library of
potential ligands, or by immobilizing a ligand library in an array
and probing with a target.
Thermal Denaturation Ranking.
[0238] As new drug targets are rapidly being discovered, methods
are needed to find molecules with affinity to these targets in the
absence of a functional assay. Fulfillment of this goal can be
accomplished by immobilizing the target biomolecule on the inner
surfaces of an array, incubating the holes of the array with a
library of compounds, and detecting those members of the array that
retain a compound. Bound compounds also stabilize target molecules
to thermal denaturation to a degree that can correlates with the
degree of affinity. By detecting unfolding of protein as a function
of temperature or denaturing solvent condition, affinities can be
ranked, as described, for example, in U.S. Pat. No. 6,020,141 to
Pantoliano et al.
Gene Probes.
[0239] An embodiment of the invention provides for the production
of a through-hole array containing numerous, known nucleic acid
sequences, adding a nucleic acid solution that has at least some
unknown sequences to each of the holes in the array, providing
sufficient time, temperature, and solution conditions for the
unknown nucleic acid to bind specifically to complementary nucleic
acids in the through-holes, and analyzing the degree of
hybridization between the nucleic acids of known and unknown
sequence in each through-hole. After binding, the array can be
washed with a solution of the desired stringency. Often, the
unknown nucleic acid has a fluorescent probe attached. An advantage
of the invention is that amplification of the signal can be
achieved by using an enzyme reaction that is associated with the
hybridized nucleic acids. For example, the unknown nucleic acid can
be labeled with horseradish peroxidase and incubated with a
substrate that produces a luminescent, fluorescent or chromogenic
signal upon reaction with the enzyme following the binding and
washing steps. Such amplification techniques can be incompatible
with conventional nucleic acid arrays on planar surfaces, since the
activated substrate in solution generally cannot be assigned to a
particular point on the array due to diffusion. PCR or other
thermal-cycling reactions may be performed in the array by
submerging the array in a water-immiscible liquid such as an oil,
alkane, or perfluorinated solvent. The array and water-immiscible
liquid may be contained in a thermally conducting container such as
a metal box, and then inserted into a thermal cycler adapted to
receive the box.
Long-Term or High Temperature Culture of Cells
[0240] In order to minimize evaporation, it is known in the art to
layer a small amount of a low volatility, immiscible liquid on top
of a small volume of aqueous reaction media. For example, a small
amount of mineral oil can be layered on a reaction vial containing
a PCR (Polymerase Chain Reaction) reaction, in order to minimize
evaporation during heating cycles. It is also known that fluids
with high oxygen solubility contents can be used to enable oxygen
transport to systems that require oxygen. It is a novel aspect of
this invention that an immiscible fluid with a high oxygen
solubility content can be used to eliminate evaporation from the
array of sub-microliter samples, while facilitating oxygen
transport to maintain cell viability.
[0241] In order to culture non-adherent cells in a nano-volume
format for long periods (e.g., greater than 12 hours), it can be
desirable to reduce evaporation by containing the cells in a
hydrophobic, low volatility fluid. To allow for aerobic respiration
or other gas exchange process to occur, an oxygenated emulsion of
perfluorinated compounds can be used. These compounds have been the
subject of clinical testing as artificial blood substitutes.
Examples include perfluorodecalin, which is sold as Fluosol-DA.TM.
by Green Cross Corp. of Japan; Oxycyte.TM., which is being
developed by Synthetic Blood International; and Oxygent.TM.-brand
perfluorooctylbromide, which is being tested by Alliance
Pharmaceuticals. In a typical application of these methods and
materials using the through-hole array, cells are grown in a platen
that is submerged in perfluorodecalin, while oxygen or air is
bubbled through the medium in a manner that does not disturb the
cells.
[0242] Culture of cells that adhere to the walls of the
through-holes or to porous substances immobilized in the
through-holes is comparatively simpler, as oxygenated aqueous media
can be perfused through or around the platen as required. Culturing
thermophilic organisms under aerobic conditions at low volumes and
high temperature can be particularly problematic, since evaporation
tends to act quickly at temperatures such as 90.degree. C. By
submerging the array of through-holes in an appropriate fluorinated
solvent, these temperatures can be used without significant
evaporation, while maintaining a supply of oxygen to the cells.
[0243] Often the act of assay detection or sample "picking" may
require that a platen be exposed to non-humidified environments or
bright lights for a period of time. These are conditions under
which evaporation from the through-holes can be problematic. In a
typical experiment, sample evaporation is minimized by performing
operations under a layer of immiscible fluid such as but not
limited to perfluorodecalin, and samples may be added to or removed
from the through-holes with a microsyringe while submerged under
such a fluid.
[0244] Other operations, such as imaging and platen manipulation
such as platen stacking may be performed under the immiscible fluid
as well.
[0245] V. Methods of Analyzing and Manipulating Output from Array
Devices.
Methods of Transferring Samples from Through-Holes.
[0246] Still another method features transfer into microtiter
plates. In order to recover samples giving a positive response to a
test, there is often a need to transfer fluid from selected
through-holes in a high-density array plate to a microtiter plate
having a lower density of wells. Often, this transfer process must
be performed with sterile technique. This will allow for sampling
of materials held in through-holes with selected properties from a
larger collection of samples. There are three general methods for
transferring fluids from the high density array plate to the wells
of a microtiter plate: transfer with a single sampling device,
transfer with a linear array of sampling devices and transfer with
a two-dimensional array of sampling devices. Samples from
proscribed through-holes can be removed by spatially localized
mechanical action. One general embodiment is to insert a member
through the hole to mechanically displace the material out the
opposite side and into a receptacle positioned beneath the hole. A
second general embodiment is to apply a localized gas or liquid jet
to cause material in the hole to be displaced out the opposite end
and into a receptacle positioned beneath the hole. A third, but
slower, method is to transfer liquid from the hole to a waiting
receptacle by transferring liquid onto a pin or into a syringe,
moving the pin or syringe to the receptacle and dispensing the
liquid.
[0247] In order to maximize throughput of the transfer system, the
number of times that the low-density plate is moved should be
minimized. If the high-density array is imaged and the imaged
stored on a computer, the coordinates of the desired through-holes
is available for input into the transfer apparatus. By aligning the
high-density array above the low-density plate, and calculating
which desired samples sit above an empty well in the low-density
plate, a maximum number of samples can be transferred without
re-positioning the low-density plate. The low-density plate can
then be moved to a position that allows the greatest possible
number of samples to be transferred in the next step. In order to
minimize the cost of the transfer apparatus, the high-density
through-hole array should remain stationary to avoid use of a
high-precision alignment system. Some high-throughput systems that
can be used in this way are described below.
[0248] Another method features transfer with a single sampling
device. This method can be accomplished, for example, by fast
sequential positioning of a mechanical plunger over the
through-holes to be sampled and pushing the plunger through the
hole to transfer the hole's contents to the well of a microtiter
plate located at a small distance below the through-hole array (6)
(FIG. 9). The mechanical plunger (1) can be actuated by, for
example, a linear electromagnetic motor (2). This process is
repeated until each well (3) of the microtiter plate (4) contains
the contents of a different through-hole. Once complete, the filled
plate is replaced with an empty plate and the process is repeated.
A servo-controlled, linear motor-actuated two axis stages (5) with
magnetic or air bearings can position a mechanical plunger whose
diameter is slightly less than a through-hole diameter to within
< 1/100 of a hole diameter (.about.1 .mu.m) with velocities up
to 1 m/s. Thus if 1% of a 100,000 hole array is to be transferred
to microtiter plates and the time to sample each hole is on average
0.2 s, then 1% of the array can be sampled in about 200 s. This
time excludes, of course, the time required to change microliter
plates, the time needed to sterilize the plunger between samples
and, if needed, the time to position a well below the sampled
through-hole.
Spatially Localized Gas, Liquid, or Solid Jets
[0249] With reference to FIG. 10, a beam from a laser (1) passes
through a shutter (2) and can be directed by a beam scanning system
(3) to be focused onto a specified through-hole (4) in a
high-density array of through-holes (5). The laser wavelength can
be chosen to coincide with an absorption band of a fluid in a
targeted through-hole. The corresponding absorption coefficient can
be such that a large percentage of the incident laser radiation is
absorbed in a thin layer at the top of the liquid column. The
shutter can control the length of time the liquid in the
through-hole is exposed to laser radiation. When the shutter opens,
laser light illuminates the liquid, and sufficient energy is
absorbed during the exposure time to rapidly heat a thin liquid
layer to vaporization, causing the rapid build-up of pressure at
one end of the through-hole. The resulting force from the expanding
vapor causes ejection of liquid from the opposite end of the hole
(6) into a well of a microtiter plate located below the
through-hole array (7). A further increase in force is possible if
the volume above the heated surface is hermetically sealed thus
increasing the pressure applied to the liquid. Rapid vaporization
and expulsion of liquid from the column requires the laser energy
to be deposited in a time less than the thermalization time. Rapid
expulsion is needed to increase throughput and to prevent
substantial degradation of the cells or reagents contained in the
liquid.
[0250] The case of a water-filled through-hole provides an
illustrative example. Indeed, in many cases, the analytical
substance is in water. The absorption coefficient of water at 10.6
.mu.m is .about.1000 cm.sup.-1 indicating 99% of the incident
radiation will be absorbed within 46 .mu.m of the surface--a small
fraction of the water column's length assuming a length of 0.5 mm
or greater. The thermalization time, .tau., is the time required
for the water column to reach thermal equilibrium and is given by
r=l.sup.2/4.alpha. where l is the distance from the source of
thermal energy and a is thermal diffusivity (=.kappa./c.rho.) in
which the thermal conductivity is .kappa., c is the specific heat
and .rho. is the density. Inputting appropriate values for water,
the thermalization time for a column of water 1 mm in length is
1.75 s while for a column 0.5 mm long, .tau. is 0.44 s. Adiabatic
heating with the focused laser beam will take place if the laser
pulse length .DELTA.t is less than .tau..
[0251] The peak pressure generated by the instantaneous
vaporization of a volume of water 46 .mu.m thick by 200 .mu.m in
extent can be estimated assuming the water vapor is an ideal gas.
The pressure, P, in this volume, V (=1.4.times.10.sup.-12 m.sup.3)
, when the liquid is vaporized is P=nRT/V where n is the moles of
water (=88 nanomoles), T is the gas temperature (=373 K) and R is
the ideal gas constant (=8.2 m.sup.3-Pa/mole-.degree. K.).
Inputting these values gives P.sub.max=186.times.10.sup.6 Pa equal
to a force of 5.8 N on the water column; sufficient to expel the
liquid from the through-hole.
[0252] The laser power to vaporize a 46 .mu.m thick layer of water
in a 200 .mu.m diameter through-hole can be found by computing the
energy, Q, to vaporize this volume of water: The thermal energy is
found for Q=m (c.DELTA.T+.DELTA.H.sub.vap) where m is the mass of
the water in this volume (1.6.times.10.sup.-9 kg), c is the
specific heat of water (=4184 J/kg/.degree. K.), .DELTA.T is the
temperature change (353.degree. K.) and .DELTA.H.sub.vap is water's
latent heat of vaporization (=2.3 MJ/kg). Inputting these values
gives Q equal to 6 mJ. Assuming 99% absorption of the incident
laser energy, 6 mJ is deposited in the sample by a 10 W laser
illuminating the liquid surface for 0.6 ms. For random-access
scanning, typical settling times for galvanometer-steered mirrors
is 10 ms and for 1000 through-holes in an array to be individually
addressed, it will take approximately 10.6 seconds.
[0253] In alternative embodiments, solids (e.g., powders) or
liquids can be used to displace or force out (e.g., under pressure)
the contents of specific through-holes or the contents of the
through-holes in general. (See FIG. 22) Examples of liquids that
can be used include liquids that are miscible with the contents of
the through-holes (e.g., water when the contents of the
through-holes are aqueous) or liquids that are immiscible with the
contents of the through-holes (such as oils or organic solutions
when the contents of the through-holes are aqueous). Application of
pressurized liquid to an array can be used to wash contents of each
hole into, for example, a common container.
Explosive Charge.
[0254] An extension of the previous embodiment is the expulsion of
the liquid from a through-hole by a pressure wave generated by
rapidly expanding gas on ignition of an explosive charge located in
proximity to one end of the through-hole. With reference to FIG.
10, an array of discrete slow-burning explosive charges (1) is
co-registered with respect to the through-hole array such that one
charge is located above one through-hole. Each charge is placed in
a chamber having a thin membrane as a common wall with the
through-hole. The charge array is bonded (or tightly attached to)
the through-hole array. Examples of explosive material for this
application include plastic explosive sheets such as "C4", or
trinitrotoluene (TNT) embedded in plastic. The charge array is
addressable, for example, electrically or optically. An individual
charge can be ignited by passing an electrical current through a
resistive element (2) located in the chamber or by the thermal
energy deposited in the chamber by a focused laser beam. Once
ignited, the expanding gas from the explosion generates sufficient
pressure to burst the separating membrane and drive liquid (3) from
the through-hole (4) into the well (5) of a microtiter plate (6)
located below the through-hole array (7).
[0255] Alternatively, as shown in FIG. 11, the explosive charge can
be embedded as a uniform stochastic distribution in a thin plastic
sheet (1). Conversely, the explosive chemicals could be printed
onto the sheet in the same pattern as the through-hole array, as
shown in FIG. 12. Printed onto the sheet are resistive elements (2)
at discrete spatial locations with the same pattern as the
through-hole array. Alternatively, spatial locations on the sheet
are addressable by a focused laser beam providing the energy
required to ignite the explosive chemicals. The sheet is bonded to
the through-hole array (3) and the charges ignited above the
through-holes whose contents (4) are to be transferred into a well
(5) of a microtiter plate (6) located below the through-hole
array.
[0256] To increase the inertia imparted to the liquid column from
the expanding gas charge, metal or ceramic microspheres are mixed
with the explosive charge and are accelerated by the explosion.
Alternatively, a plug of material between the explosive charge and
liquid column accelerated by the explosion will act as a mechanical
plunger to expel liquid from the through-hole.
[0257] Forming the exit of the chamber containing the explosive
charge into a nozzle will increase the spatial localization and
inertia of the exiting gas before impacting the liquid column. The
nozzle exit either is fluid with the chamber surface or protrudes
slightly to insert into the opposing through-hole.
Sample Aspiration.
[0258] Liquid samples in a through-hole or group of through-holes
can be transferred out of the through-holes by aspiration into a
tube or channel. The tip of a piece of flexible or rigid tubing,
generally having an outer diameter narrower than the inner diameter
of the through-hole, can be aligned within the through-hole.
Application of negative pressure to the distal end of the tubing
can then be used to aspirate fluid from the through-hole into the
tubing. The amount of fluid to be aspirated can be accurately
controlled by manipulating several variables. For example, the
length and internal diameter of the tube can be determinative of
the pressure drop across that piece of tubing, which can in turn
affect the rate of flow through that piece of tubing for a given
amount of applied negative pressure. A metered amount of fluid can
be aspirated from the through-holes into a valve assembly, from
which the fluid can be moved by positive or negative pressure to
any type of fluidic circuit that is required by a given
application. In one embodiment, fluid is aspirated from a
through-hole into a fluidic valve. Actuation of that valve
introduces the fluid via positive pressure to a mass spectrometer
for analysis of that fluid. Alternatively, further sample
preparation or characterization (e.g., chromatography,
spectroscopy) can be performed on the fluid once it has been
aspirated from the through-hole.
[0259] Electrophoresis and Electroblotting: The invention also
provides methods for introducing an ionic sample into a
through-hole array containing chemical probes, for modulating the
stringency of the binding between the probes and certain ions in
the sample, and for removing an ionic sample from the through-hole
array. The method includes placing the through-hole array
containing chemical probes localized in the holes into a buffer in
an electrophoresis apparatus. A sample containing ions is
introduced into the electrophoresis apparatus on one side of the
planar through-hole array, such that when an electric field is
applied, the ions of the appropriate charge will migrate in the
direction of the through-hole array. If a particular ion does not
bind to the array and the electric field is applied for sufficient
time, that ion will migrate through the hole to the opposite side
of the through-hole array. By periodically changing the direction
of the electric field, approach to equilibrium in the binding
between the charged species and the chemical probes in the
through-holes can be accelerated. Partial purification of the
sample to be analyzed can optionally be achieved by electrophoresis
of the sample through a gel prior to its migration to the
through-hole array. Once the analytes of interest in the sample
have associated with the chemical probes, the field can be applied
for sufficient time and with sufficient strength to dissociate
non-specifically bound ions from the chemical probes. The
through-hole array can then be taken from the electrophoresis
apparatus, and the pattern of binding can be analyzed. The bound
analytes can also be removed for further analysis, for example, by
electroblotting onto a membrane, and then removing the membrane for
further analysis.
[0260] Chromatic analysis of samples in an array of
through-holes.
[0261] For many applications, samples must be isolated, purified,
or concentrated by a chromatographic step. Many different types of
chromatography can be performed on liquid samples. Examples of
well-known chromatographic methods include, but are not limited to,
ion exchange, reversed phase, size exclusion, bio-affinity, and gel
permeation chromatography. The chromatography matrix can be in the
form of an insoluble bead, gel, resin, polymer, or slurry. The
matrix can alternatively be a micro-machined structure. One
embodiment of such a micromachined structure is a grouping of
square through-holes or channels with sides of dimension on the
order of 0.01 to 10 gm. The walls of these through-holes can be
coated with a surface having a desired affinity such that a
separation is achieved as sample is flowed through the group of
through-holes. The analyte mixture of interest is then introduced
to this matrix and selective binding of components of the analyte
mixture to the matrix takes place. Analytes of interest or
contaminants can then be selectively eluted from the matrix by
changing the physical or chemical environment of the matrix. Liquid
chromatography is typically performed in a column in which the
chromatography matrix is immobilized and the analyte is flowed
through the column, allowing chemical and/or physical interaction
between the sample and the chromatography medium to take place. The
length and internal diameter of the array of capillaries generally
determines the amount of chromatography matrix that can be loaded
into each column and, therefore, is directly related to the loading
capacity of each column.
[0262] Immobilizing a chromatography matrix inside an array of
through-holes can create an array of miniature liquid
chromatography columns. A suitable length-to-diameter ratio of the
array of through-holes can be selected. Typically, a minimum
length-to-diameter ratio of at least about 10 is required to form
an effective chromatography column. In certain embodiments, the
internal diameter of the columns formed in the through-holes is
less than a millimeter, allowing for precise and accurate
manipulation of very small amounts of sample. The chromatography
matrix can be immobilized within the chromatography columns by
positioning a porous frit at the exit end of the column or by
chemically binding a porous polymeric ceramic or glass substrate to
the inside of the column. The porous ceramic or glass substrate can
either act as a frit to immobilize a bead or resin chromatography
media or as a method to increase the total surface area within a
column. In surface-effect driven chromatographic methods (e.g., ion
exchange, affinity, or reversed phase chromatography), chemical
derivatization of the interior surface of the columns can provide
the necessary separation.
[0263] In one embodiment, samples are loaded into the array of
columns with syringes. A submicroliter volume of sample can be
drawn into the needle of the syringe or a bank of syringes with a
spacing co-registered to the spacing of the array of columns, and
then transferred to the array of columns. The barrels of the
syringes can contain a larger quantity of liquid for performing
wash and/or elution steps in the bundle of capillaries. The sample
in the needle of the syringes and the liquid in the barrel of the
syringes can be isolated from one another by drawing up a small
amount of air into the syringes. The needles of syringes can be
docked into the array of capillaries with a liquid-tight
compression fitting. As the content of the syringes are ejected
into the array of columns the samples in the needles of the
syringes initially elute onto the column bed. The chromatography
media used and buffer drawn into the barrel of the syringe will
dictate the chromatographic separation. Once the samples have been
loaded onto the columns, the syringes can be removed from their
docking ports and a second aliquot of a similar or different buffer
can be drawn into the syringes. As many wash and/or elution steps
as necessary can be performed by re-docking the syringes to the
array of capillaries by tightening the compression fitting and
ejecting the liquid from the syringes. A first array of columns can
also be mated to a second array of columns for further
separation.
[0264] It is often desirable to analyze the eluate from a
chromatography column in real time using a variety of spectroscopic
methods. Spectroscopic devices for interrogating the eluate from a
chromatography column are well known. If required by the specific
application, the eluate from each column in a bundle of columns can
be analyzed on-line in real time, spectroscopically (e.g., by
absorption, fluorescence, or Raman spectroscopy),
electrochemically, or otherwise. The light from the spectroscopic
light source can be delivered to and recovered from the eluate of
each column in a bundle of columns as it passes through an
observation window machined into the exit capillary of that column
with a fiber-optic cable.
Mating an Array of Through-Holes to an Array of Liquid
Chromatography Channels
[0265] A device can be manufactured to include an array of
chromatography columns with a chromatography matrix immobilized
within the array of columns, for example, with spacing such that it
can be co-registered with the through-holes in an array of
through-holes that do not contain a chromatography matrix. The
physical size of the array of through-holes can determine the size
of the array of columns. Preferably, the internal diameter of each
column in an array of columns will be similar to the internal
diameter of the corresponding co-registered through-hole in an
array of through-holes.
[0266] The array of columns should be mated to the array of
through-holes in a manner that allows the application of a positive
or negative pressure across the device and does not result in cross
talk between the individual samples. The number of columns need not
be in a one-to-one ration with the through holes. Radial diffusion
of samples between the layers of a stack of arrays of through-holes
in response to an applied external pressure may result in cross
talk and must be avoided. Inter-sample cross talk can be eliminated
by forming a liquid-tight seal hermetic seal between the
through-holes to eliminate radial diffusion. An elastomer sheet
with holes co-registered with the through-holes in the array can be
compressed between the layers of a stack to form such a seal.
Alternatively, a thin, inert, porous polymer sheet can be placed
between the two arrays such that, when the two columns are pressed
together, liquid can flow through the pores in the sheet, but
cannot flow laterally. Another approach entails manufacturing the
array of through-holes such that one side of the immediate area
around each through hole is raised relative to the rest of the
array. An o-ring can then be placed around this raised area. When
two or more arrays of through-holes are compressed together, the
o-rings will form a hermetic seal, thereby eliminating radial
diffusion and sample cross talk. The cross-section of any pair of
mating arrays can also be fabricated to be interlocking with an
elastomer gasket or coating between the mating surfaces, to provide
a leak-tight fluidic seal.
[0267] One or more arrays of through-holes can be mated to an array
of columns in a similar manner. A top plate can then be affixed to
the assembly that allows for the necessary chromatographic wash
and/or elution buffers to be applied to the each through-hole. The
liquid can be forced through the device in such a manner that the
sample will be pushed through the array of through-holes into the
array of columns upon which the chromatography will take place. If
required by a specific application, the wash and/or elution buffers
from the chromatography columns can be transferred via capillaries
to another chromatography device, chromatographic fraction
collector, or another array of through-holes. An illustration of
such a device is shown in FIG. 14. The fluidic connections that
lead to and from the device can be machined for easy coupling to
standard fluidic connections using standard fluidic components such
as ferules and compression fittings.
Device for Fraction Collection
[0268] Chromatography generally entails separation of a mixture of
compounds on the basis of differential chemical and/or physical
interactions of the individual components of that mixture with a
chromatographic matrix. A sudden or gradual change in the physical
and/or chemical environment can affect the interactions between the
components of a mixture and the chromatographic matrix. Typically,
each component of a mixture elutes individually from the
chromatographic matrix as the physical and/or chemical conditions
are varied. It can be desirable to isolate a given component of a
mixture of compounds for further analysis or chromatography. In
some cases, a component of interest can be identified by online
spectroscopic analysis.
[0269] Devices for fractionating the eluant of a chromatographic
column and storing individual fractions are well known. One
embodiment of the present invention features a system for
collecting chromatographic fractions from an array of columns. The
column eluate can be spotted dropwise onto another array of
through-holes. By controlling the speed at which the array of
through-holes is moved with respect to the array of columns, the
volume of each fraction can be controlled. A fluid bridge can be
formed between the exit capillary from the array of columns and a
through-hole in a through-holes array if the interior surface of
the through-hole is coated with a material with the appropriate
affinity for the eluant. The maximum number of fractions that can
be collected from a given column will depend on the size of each
fraction, on the speed at which the collection array of
through-holes is moved, and on the density of the array of columns.
If a large number of fractions must be collected from each column,
a linear array of columns can be used, its output being collected
in a two-dimensional array of through-holes. If the fraction
collection array of through-holes is then moved perpendicularly to
the linear array of columns, the number of fractions that can be
collected is limited only by the physical size of the collection
array.
[0270] For some applications, a single chromatographic separation
can take several minutes or longer to complete. If long periods are
required, it is possible that evaporation of liquid from the array
of through-holes in the fraction collection device will occur. To
avoid evaporative loss from the through-holes in the fraction
collection device, the entire array of through-holes used to
collect fractions can be placed within an environmentally
controlled enclosure. If a high-humidity environment is maintained
within the enclosure, evaporative losses can be minimized.
Additionally, it can be desirable to maintain a certain temperature
within the enclosure (e.g., 4.degree. C.) to maintain compound
stability. The elution capillaries from the array of columns can
enter the enclosure through a series of precision-machined holes to
maintain the integrity of the enclosure while allowing for
introduction of the eluant from the array of columns. Elastomer
gaskets may be used to ensure a good seal around the enclosure.
[0271] An array of through-holes containing the fractions collected
from an array of columns can be stacked with another array of
through-holes to initiate a second mixing operation to initiate a
chemical reaction. A second chromatography application can be
initiated by stacking the array of through-holes into which the
fractions were collected with a second array of columns.
Alternatively, further spectroscopic or spectrometric analyses can
be performed on the collected fractions at this time.
Spectrometric Analysis of Compounds in an Array of
Through-Holes
[0272] Atmospheric Pressure Ionization Mass Spectrometry
(API-MS)
[0273] Samples in an array of through-holes can be analyzed by a
spectrometric technique such as atmospheric pressure ionization
mass spectrometry (API-MS). The spectrometric analyses are
typically performed serially. Therefore, the chips should be
environmentally isolated in a controlled temperature and humidity
environment to avoid loss of sample due to evaporation. In API-MS,
one simple method for introducing the sample to the mass
spectrometer features aspirating a selected sample directly from a
particular through-hole into a valve using a length of capillary
tubing (e.g., as described herein). A metered volume of sample can
then be introduced into a mass spectrometer using standard API-MS
protocols.
[0274] Matrix Assisted Laser Desorption Ionization Time of Flight
Mass Spectrometry (MALDI TOF-MS)
[0275] In MALDI TOF-MS analysis, a sample of interest is generally
mixed with one or more matrix-forming compounds. Typically, a
saturated solution of an organic matrix material (e.g., derivatives
of hydroxycinnamic acid) is mixed with an equal volume of sample.
In some applications of MALDI TOF-MS, the organic matrix compound
is replaced by inorganic nanoparticles (e.g., colloidal gold,
quantum dots, or porous silica). The mixture is then spotted in the
form of a regular and addressable array on a flat plate and allowed
to evaporate completely. The sample plate is then positioned in the
mass spectrometer, and the samples are ionized by irradiation from
a pulsed laser.
[0276] Samples in an array of through-holes are well suited for
analysis using MALDI TOF-MS and related applications, since the
necessary sample preparations steps can easily be accomplished in a
parallel fashion. For example, a second array of through-holes can
be loaded with a saturated solution of an organic matrix or a
slurry of an inorganic matrix compound. The array of through-holes
can either be dip-loaded uniformly, or, if desired, any number of
different matrix compounds can be loaded into individual
through-holes in an addressable fashion. The sample and matrix
arrays of through-holes can be mixed together by bringing the chips
together (e.g., as described herein). After allowing the solvent to
completely evaporate, the array of through-holes can be placed in a
slightly modified receptacle in most commercially available MALDI
TOF mass spectrometers. The conventional flat metal MALDI plate can
be machined down to compensate for the thickness of the array of
through-holes. The array of through-holes can be affixed with a
temporary adhesive within the recessed area of the standard sample
holder.
[0277] The laser used for sample ionization in the MALDI TOF mass
spectrometer can be focused within the through-hole to provide the
necessary irradiance for sample ionization.
[0278] Internal reflection of the laser beam within the
through-hole can possibly increase the amount of laser energy
absorbed by the matrix and transferred to the sample, thereby
increasing the amount of sample ionization. Additionally, an array
of through-holes can allow for a very high density of samples to be
spatially located in a small footprint without inter-sample
contamination.
[0279] Typical MALDI-MS sample plates are solid surfaces onto which
samples are spotted. The laser used to ionize the samples must be
on the same side of the plate as the inlet of the flight tube of
the mass spectrometer, since the sample plate is opaque to the
laser energy. The use of an array of through-holes as the sample
plate allows for the source of laser irradiation and the inlet to
the TOF mass spectrometer to be located on opposite faces of the
sample plate. A scheme of this linear MALDI TOF mass spectrometer
is shown in FIG. 15. Translocation of the sample plate in front of
the inlet of the flight tube allows for the laser ionization of a
selected sample.
[0280] Alternatively, an array of posts or pins, precision-machined
to fit into an array of through-holes, can be coated with the MALDI
matrix material by dipping the array into a bulk matrix solution.
After the solvent has evaporated, the pin array can be inserted
into the through-hole array. Fluid contained in each through-hole
is transferred to the corresponding pin surface. After the solvent
has evaporated, the pin array can be placed at the input to a TOF
mass spectrometer and the pins can be illuminated sequentially with
a focused laser beam. In such a pin array, a portion of the sample
from each through-hole can be held isolated from its neighbor by
the air gap between each pin.
Through-Hole Array/Surface Method.
[0281] When placed into an electric field or driven by pressure,
the through-hole array can be used as a parallel capillary
electrophoresis, electrokinetic chromatography or chromatography
device. An array of samples in one through-hole array can be
introduced into a second, typically longer, through-hole array. The
second through-hole array can be filled with a gel (e.g. silica), a
polymer (e.g., polyacrylamide) or a resin and can have a coating on
its walls to prevent or enhance electro-osmosis or protein binding.
However, if electrophoresis or chromatography is performed in such
an array, it will be difficult to analyze the output of each column
as molecules emerge from it. One way to alleviate this problem is
to pass the output through a moving surface (e.g, nitrocellulose
sheet) with an affinity for the analyte molecules and then move the
web to an imaging detector. For example, fluorescently labeled DNA
or protein could be eluted onto a moving nitrocellulose membrane
and passed to a fluorescent imager to analyze. A continuously
moving surface, moving in the manner of a tape, would cause
smearing of the samples, therefore it is advantageous to reduce or
reverse the polarity of the electric field or pressure during those
periods of time when the surface is moving. An increase in
sensitivity of the detection system is can be achieved by further
delaying the movement of the surface and the voltage or pressure
while the detector is acquiring an image. Alternately, the imager
could be a line-scanner such as a fluorescence laser line-scanner.
The surface could be fed from a long spool if desired (e.g., in a
tape like manner). The surface can further be taken up on a second
spool.
Readout Methods.
A Method of Using Wetting Properties of the Array to Detect
Chemical Binding to the Walls of the Through-Holes.
[0282] The binding between two proteins, such as between an
antibody and an antigen is detected via its affect on the surface
energy of the channel interiors.
[0283] In a preferred embodiment, a library (e.g., a library of
antibodies, receptors, macromolecules, or molecular probes) is
bound to the interior walls of a through-hole array having
hydrophilic channel walls and hydrophobic faces. The array is
rinsed with a blocking agent, which binds to non-specific protein
absorption sites on the channel walls but does not change the
hydrophilic character of the wall surface. Such blocking agents
include bovine serum albumin (BSA), powdered milk, and gelatin. The
array is then immersed in a solution that contains antigen and is
incubated for sufficient time to allow binding of antigen to
complementary antibody. The arrays are then removed from the
antigen solution and washed with buffer. The array is dried and
dipped into an aqueous solution containing a chromophore or
fluorophore. The presence of the antigen on the surfaces lowers the
surface energy sufficiently such that the liquid is prevented from
entering the through-holes. The empty through-holes can be
identified by imaging the array. The empty through-holes then
correspond to the antibodies in the library that effectively bind
the ligand.
[0284] In another embodiment, the interior walls of a set of small
array devices, each having roughly 1-100 through-holes, are coated
such that each array has a different antibody. The arrays are
placed together in a solution of antigen and incubated for
sufficient time to allow binding of antigen to complementary
antibody. The arrays are then removed from the antigen solution and
washed with buffer. All arrays are then placed together in a
separation bath that contains a liquid that is non-destructive to
the proteins and can be adjusted in density in some manner (e.g.,
by addition of a higher density liquid, or by adding a thickening
agent).
[0285] When the holes remain empty, the density of the array in the
bath is reduced and, under appropriate conditions, the array
containing bound antigen can float to the surface. The presence of
the antigen on the surfaces lowers the surface energy sufficiently
such that the liquid is prevented from entering the through-holes.
These floating arrays are removed and the identity of the antibody
bound to each array is determined by mass spectroscopy, or from a
code on each array, or by some other means such as a bar code or
radio transponder built into the arrays. For this method the array
can be a porous structure such as an aerogel or a porous bead.
A Device for the Analysis of an Array of Through-Holes by Mass
Spectrometry.
[0286] Samples or aliquots of samples can be removed from an array
of through-holes for analysis by mass spectrometry by one of
several different methods. One such method features drawing the
sample or an aliquot thereof into a tube with the application of
negative pressure. In one example of this approach, the tip of a
syringe is inserted into a selected through-hole in an array and a
metered amount of sample is drawn into the syringe. Alternatively a
vacuum could be used to aspirate the samples into a length of
tubing, a valve, or a container for storage.
[0287] For certain applications, it can be desirable to assay each
sample in an array of through-holes by a serial process such as
mass spectrometry. Application of a serial process to a large
number of samples in an array of through-holes, even if done very
rapidly, can still require a significant amount of time. If
humidity conditions and temperature are not strictly regulated
during this time, evaporation of samples from the through-holes can
occur and artificially bias assay results.
[0288] One approach to controlling evaporation and facilitating the
aspirating of individual samples from an array of through-holes is
to design an additional array of through-holes in which each of the
through-holes is coregistered with a sample through-hole in the
assay. This additional array of through-holes can be placed on top
of the arrays of through-holes used in the assay to create a top
plate. The through-holes in this top plate can be designed such
that the diameter of each through-hole can be made to be much
larger at the outer surface than the diameter in the surface that
contacts the arrays of through-holes used in the assay. The conical
shape formed by such a through-hole will act as a guide for a
syringe needle into a selected through-hole and facilitate
efficient sampling. The outer surface of this top plate can also be
coated with a thin film of polymer similar to that used in
lamination. The sealed surface will act to retard evaporation. The
syringe needle used for aspirating the sample out of the
through-holes can easily perforate this thin film and will not
hinder efficient sampling.
[0289] Once the sample is aspirated into a syringe, it can be
delivered into a mass spectrometer for analysis by any one of many
techniques known by those skilled in the art. These can include
atmospheric pressure ionization techniques such as electrospray
ionization (ESI) or atmospheric pressure chemical ionization
(APCI).
[0290] In another embodiment of the invention, a metal plate can be
used as a bottom plate for the array of through-holes. The solvent
used in the assay can be allowed to evaporate and a solid sample
can form in a footprint on the bottom plate that corresponds to the
internal diameter of the through-hole. If desired, a matrix can be
added to the samples before complete evaporation. Alternatively, a
matrix can be added to the surface of the metal bottom plate before
it is stacked with the arrays of through-holes used in the assay.
Once the samples have completely evaporated the metal bottom plate
can be removed from the arrays of through-holes and each sample can
be analyzed by matrix assisted laser-desorption ionization (MALDI)
or a similar surface based ionization mass spectrometry technique
generally practiced by those skilled in the art.
[0291] In another embodiment of the invention, an array of pins
coregistered with the array of through-holes can be dipped into the
array of through-holes and removed. Sample that is residually
removed with the array of through-holes can be allowed to evaporate
on the tips of the array of pins. As in the previous embodiment,
this evaporated sample can be used for a surface based mass
spectrometry method.
Time-Gated Fluorescence Imaging of a Through-Hole Array
[0292] Many biological assays are configured to give a fluorescent
readout that can be acquired from an array of through-holes by
fluorescence imaging. Typically, light from an excitation lamp or
laser is passed through an excitation filter, through the array,
through an emission filter and then to a CCD camera. In many cases,
the sensitivity of the signal is limited by background light due to
imperfect performance of the filters, and by inelastic and elastic
scattering of light by the sample and optical components. Whereas
the fluorophores of interest have fluorescence lifetimes of about 1
ns to 1 ms, scattering occurs at much shorter timescales. Thus
removal of background light can be accomplished by the technique of
time-gating. Time-gating the process of illuminating the sample
while preventing the camera from acquiring data, quickly removing
the excitation light, then waiting for a delay time before
acquiring the fluorescence emission image. By not collecting
photons emitted during the first 1 to 100 ps of after excitation,
background noise is significantly reduced and signal to noise is
improved. A similar apparatus can be used to repeat the data
acquisition with varying delay times, thus yielding fluorescence
lifetime information for each of the through-holes in the
array.
[0293] Various strategies can be used to construct a time-gated
fluorescence imaging system. A pulsed excitation source is needed
and can be either a flash lamp or laser such as a passive or active
mode-locked or Q-switched laser. If a laser is used, a beam
expander and diffuser plate will give uniform irradiation of the
platen. A continuous excitation source can also be used with a
means for rapidly blocking and un-blocking the light such as an
electro-optical, an acousto-optical cell or a rapidly rotating disk
with slits. A pulse generator can be used to trigger the
illumination source and detector at a given delay. The CCD camera
can be electronically shuttered or physically shuttered as with a
rotating disk with slits that is out of phase with the excitation
pulsing.
Optical Readout Based on Insertion of a Through-Hole Array into an
Optical Resonator or Interferometer
[0294] A through-hole array can be inserted into either an optical
resonator or an optical interferometer for simultaneous and
parallel interaction of an optical field with material contained in
each through-hole. (See FIG. 21) Such a method can be advantageous
when the through-hole array (2) is placed in an optical resonator
(Irradiation (1) is shined and amplified between mirrors (2) and
(3).) so that the optical path length is increased over the length
of the through-hole array, to increase absorption (5). The optical
path length is increased as a multiple of the through-length, to
increase optical absorption. For example, for simultaneous
initiation of chemical reactions by the enhanced optical field
characteristic of an optical resonator. It can also be advantageous
as a means for simultaneous analysis of materials and interactions
between materials contained within the through-holes, for example,
by recording changes in incident optical field intensity, phase,
polarization, or frequency, or by recording of these parameters, of
light emitted from as a result of interaction between an incident
optical field (e.g. fluorescence, phosphorescence), the materials
contained in a through-hole, or a change in the material itself
(e.g. luminescence).
[0295] One advantage of measuring these parameters with the chip as
part of an optical resonator is that the resonator's resonance
condition will change on interaction of the optical field contained
within the resonator structure with the materials contained within
each through-hole. These changes (e.g., phase, intensity,
polarization, frequency) are intimately related to the composition
and physical state of the material contained in the through-hole.
The change in optical field parameters changes the resonant
condition of the cavity, which, in turn, changes the intensity of
light passed through or reflected from the resonator..
[0296] One can also take advantage of the increased optical field
strength characteristic of optical resonant structures, for
example, to initiate photochemical reactions or non-linear optical
effects (multi photon absorption, harmonic conversion, etc.) as a
probe to measure properties of the materials contained in the
through-holes. The optical field incident on the resonator can be
either continuous in time or it can vary with time as in an optical
pulse.
[0297] Examples of optical resonator structures that can be used in
this embodiment include Fabry-Perot-style interferometers with two
planar mirrors and confocal Fabry-Perot-style interferometers
having two curved mirrors or one planar and one curved minor.
[0298] The through-hole array can either be part of or be inserted
into a two-beam interferometer. Examples of such interferometers
are many, and include Michelson, Twyman-Green, Sagnac, and
Mach-Zhender-type interferometers. In the embodiment where the
array is inserted into one of the optical paths of a two-beam
interferometer, the phase of the light is delayed according to the
complex refractive index (refractive index and absorption) as a
function of wavelength. The interferometer can be illuminated with
a beam of white light of sufficient width to also illuminate the
through-hole array. For each optical path length difference between
the two arms of the interferometer, a camera at the interferometer
output can record the pattern of light exiting the interferometer
and corresponding to light that has passed through each hole in the
array. A series of images can thus be acquired for each optical
path length difference, and taking the Fourier transform of each
pixel of the image as a function of optical path length can
generate an optical absorption or emission spectrum for the
materials in each through-hole of the array.
[0299] Another embodiment uses a two-beam interferometer to analyze
light passed through or emitted from a through-hole array. A camera
records the light pattern from the interferometer for each optical
path length difference imposed between the two plane mirrors that
make up the interferometer. After recording a sequence of images
corresponding to each path length difference, individual
interferograms can be generated from each set of pixels
co-registered across the image sequence. Application of the Fourier
transform to each interferogram can generate an absorption or
emission spectrum at each spatial position in the image. In this
way, the spectral content of light interacting with material
contained in each through-hole of the array can be determined.
[0300] Application of the approach described in U.S. Pat. No.
6,088,100, incorporated herein by reference in its entirety,
adapted for full-field imaging, can also allow for capture of
absorption spectroscopic information from each through-hole in a
stack of through-hole arrays.
Image Center of Array as a Function of Thermal Perturbation.
[0301] The invention is compatible with many systems for detecting
the output of the arrays of chemical probes. Commercially available
fluorescence scanners can be used if desired. Because each position
in the array has two apertures (i.e., on the top and bottom faces
of the platens), the array can be exposed to electromagnetic
radiation on one face of the platen, and the optical properties of
the samples in the array can be measured via detection at the
opposite face of the platen. The positions of the array can be
imaged in parallel or by serial scanning techniques. Both static
and kinetic analysis of reactions can be utilized.
[0302] One method of detecting binding between an analyte and
probes immobilized in the walls of the through-holes includes
observing the distribution of analyte within each through-hole as a
function of a perturbation. For example, a ligand of interest can
be covalently attached to the inner walls of each of 10,000
through-holes in a 2 sq cm. platen. The chip can then be stacked
with another chip containing, for example, a peptide library such
that each member of the library occupies its own through-hole and
has a fluorescent tag. After allowing sufficient time for
non-covalent binding reactions to reach equilibrium, the chip can
be rinsed with buffer to remove unbound material. By observing the
fluorescence distribution in each hole as a function of a
perturbation such as increasing temperature or increasing formamide
concentration, the members of the library can be ranked as to
binding energy. Binding kinetics can be determined by following the
fluorescence distribution as a function of time following a rapid
perturbation such as a temperature jump. The sensitivity of the
assay can be improved by using a mask that includes of an array of
through-holes complementary to the chip, but of smaller diameter,
that spatially filters the light such that only the interior of
each through-hole is observed. Another method of increasing
signal-to-noise ratio includes applying a periodic or stochastic
perturbation such as temperature, and then observing only signal
correlated with the perturbation.
[0303] An increasingly common technique in drug discovery is
millisecond time-scale fluorescence analysis of cell populations.
Existing commercially available devices utilize a bank of syringes
that add reagents from one 384-well microplate containing drug
candidates to a second 384-well microplate containing cells loaded
with a fluorescent indicator of calcium such as Fura-2, followed by
laser scanning of the underside of the second plate to generate
millisecond kinetic measurements of calcium release from the
endoplasmic reticulum of the cells. It would be advantageous to use
a white-light excitation source and a digital camera to acquire
such kinetic data due to lower cost and greater choice of
excitation wavelengths. This was previously difficult because the
syringe bank would obstruct the light path in such a system and
would hayed to be moved on a millisecond time scale, which is not
typically possible. Use of a white-light source is possible by
initiating mixing of samples stored in an array of through-holes
with cells growing in a second array, both arrays being in the
camera system. An additional benefit of this method is that the
throughput of samples collected is greatly increased by the use of
through-hole arrays containing as many as 20,000 or more samples.
Typically, the system is automated and will collect data that
begins at the moment of mixing or otherwise indexes the times
associated with collected data points to the moment of stacking.
The types of assays possible with this method are not limited to
cell or fluorescence assays, any assay with an optical read-out
that occurs on a time scale of less than minutes can benefit from
the invention.
[0304] A preferred embodiment of the invention includes at least
two stacked and co-aligned platens containing through-hole arrays
consists of (i) a detection device; (ii) a means for introducing
platens into the detection device (iii) a means to register the
platens to cause fluid to communicate between at least some of the
co-registered through-holes and (iv) a means of contacting the
platens to initiate mixing of reagents simultaneously in at least
some of the through-holes. The detection device (FIG. 24) can be an
imaging device, such as a CCD camera, with optical filters and a
light source for illumination. Light from an optical source (1)
illuminates parabolic collimation mirrors (3) after passage through
a flexible, bifurcated fiber bundle (2). The light then illuminates
the stack of arrays at an oblique angle such that light rays
transmitted through the array through-holes does not enter into the
camera lens (5). This optical arrangement is desirable as a simple
means to decrease optical background for increased optical
sensitivity.
Separation Methods.
[0305] A Method of Separating a Stack of Two or More Arrays Filled
with Liquid.
[0306] It can be desirable to separate arrays once the contents of
individual through-holes have been combined by stacking. For
example, in order to perform a dilution by two an array filled with
a chemical library is stacked onto an array containing solvent
buffer. After sufficient time for mixing of the two sets of liquids
(approximately 15 seconds for 100 nl), the plates are separated to
produce two identical arrays of libraries members at half the
initial concentration. This process can be repeated to produce a
dilution series.
[0307] When two through-hole arrays are stacked such that their
contents mix, the two plates are not readily separated with out
cross-contamination between neighboring through-holes.
[0308] Pulling one plate against the additive surface tension
created by many microscopic columns of fluid invariably introduces
shear forces that mixes the contents of individual through-holes
together as the chips are separated.
[0309] A method is proposed to separate each fluid column into two
shorter columns separated by a small vapor phase. Small electrodes
at the interface between the two stacked arrays produce a small
volume of gas in each through-hole. As the bubble grows it
recreates the liquid vapor interfaces between the two arrays. After
each column is cleaved in two the arrays are separated
mechanically.
[0310] Alternatively an inert, humid gas is introduced into the
atmosphere above and/or below the stacked arrays and nucleated at
the interface between the two stacked arrays.
[0311] Alternatively the gas can be pumped into the center of each
through a matching array of very fine hollow tubes.
Centrifuging (Performing Gravimetric Separation in) an Array of
Through-Holes.
[0312] Many biological or chemical assays require centrifugation
and/or filtration of samples. In many it can be desirable to filter
or centrifuge samples in a biological or chemical assay that is
performed in array of through-holes. The following invention
pertains to a device for the centrifugation and or filtration of an
array of through-holes.
[0313] A metal jig with two flat surfaces larger than the array of
through-holes can be built. A single or a stack of arrays of
through-holes are placed between the two flat surfaces and evenly
compressed together with the application of force on the metal
plates. The metal jig will be machined such that the amount of
compression can be adjusted as desired, preferably by a simple
tightening of several screws holding the jig together.
[0314] In some applications the filtrate or pellet will need to be
recovered from the centrifuged sample. In other cases the pellet
formed after centrifugation will need to be removed. A simple
solution for this is to machine a plate that contains an array of
wells or dimples of a metered volume that are coregistered to the
array of through-holes. The array(s) of through-holes can be
stacked atop this bottom plate with coregistered wells. If desired,
a filter can be placed between the bottom plate and the array(s) of
through-holes. After the centrifugation is complete, the bottom
plate can be removed and the filtrate or contaminating precipitate
can be removed. Alternatively, if the supernatant is the desired
fraction, it can be aspirated directly from the array of
through-holes without the need for removal of the bottom plate.
[0315] In certain applications a large centrifugal force can need
to be applied to the array(s) of through-holes. Even with a bottom
plate and/or a filter application of large amounts of centrifugal
force a stack of arrays of through-holes can result in a lateral
displacement of samples that are forced into the spaces between the
individual layers of the arrays of through-holes. Coating of the
contacting surfaces of the array of through-holes with a
hydrophobic material will inhibit lateral diffusion between layers
at relatively low centrifugal forces. When high levels of
centrifugal forces are required a material can be stacked within
each individual array of through-holes such that when the array of
through-holes are compressed together by the metal jig a leak-tight
seal will be formed between the two arrays of through-holes. If the
material used to form the seal is porous it will not impede the
flow of liquid between the layers of through-holes, yet it will
form a tight seal against lateral flow.
[0316] VI. Miscellaneous Uses.
[0317] To fully realize the potential of reagent volume savings and
increased throughput provided by nanoliter volume fluid handling,
means and methods of storing chemical and biological samples at
high density in low volumes are necessary. These storage systems
must be easily access by microfluidic screening
instrumentation.
[0318] One embodiment of the invention provides the a method to
store chemical or biochemical samples at high density and in low
volumes. Additionally, samples stored using the devices and methods
of the invention can be assayed with a minimum of liquid handling
steps or other manipulations. The method includes placing a small
volume of compounds dissolved in a solvent in an array of
through-holes and adding a second solvent to the array of
through-holes without causing substantial migration of the
compounds out of the through-holes. The result is an array of
compounds dissolved in a liquid that is primarily comprised of the
second solvent.
[0319] The invention further provides a method of storing compounds
in a manner wherein the samples can be readily introduced into
aqueous medium for performance of an assay.
[0320] The step include dispensing a volume of compound in a
solvent that is much less than the volume of the container that it
is dispensed in, storing for some time, and adding an aqueous
medium to fill the remaining volume of the container in preparation
for an assay.
[0321] In a preferred embodiment, integration of low-volume
compound library storage and screening includes the following
steps: (1) Dispensing volumes of compound dissolved in a
non-aqueous solvent that are smaller than the total volume of the
containers to be used for screening. Usually, the volume dispensed
will be less than half and could be 1/10th of, 1/40th of, or less
than the total capacity of the container. Usually, the containers
will be part of an array of containers. The container is preferably
a hydrophilic area surrounded by hydrophobic areas, such as a
channel of a through-hole array, or a spot on a glass slide with
spots of hydrophobic areas in a hydrophilic background. (2) Storing
the compounds for some period of time. Storage conditions may
consist of a low temperature such as 4.degree. C., -20.degree. C.,
-80.degree. C., submerged in liquid nitrogen, or a lower
temperature. Desiccation is usually desirable. (3) Removing the
compounds from storage, and elevating them to above the freezing
point of the aqueous media to be loaded into the containers, but
above the freezing. (4) Adding aqueous medium to the containers.
Preferably, the aqueous medium is chilled to above its freezing
point and below the freezing point of the non-aqueous medium. The
addition could be done by dispensers, but is more rapidly and
inexpensively done by dipping the containers into a bath of the
aqueous medium. It is advantageous to have the non-aqueous solvent
be in the solid form so as to minimize loss of the compounds into
the aqueous medium and to prevent communication of compounds in
adjacent or nearby channels. (5) Waiting for a time sufficient to
allow mixing of the compound with the aqueous medium. And (6)
Performing an assay or measurement upon the samples.
Methods for Dispensing Small Volumes into Large Holes.
[0322] It is advantageous to screen samples such as drug candidates
in very small volumes in order to conserve reagents and increase
through-put. A typical through-hole array format will have channels
that hold 60 nl of fluid and a typical assay will be done with two
stacked chips, holding a total of 120 nl. If the maximum
concentration of DMSO acceptable in the assay is 2%, then a total
of 2.4 nl must be dispensed into one of the channels and this is
extremely difficult to do with conventional liquid handling
systems. One way to solve this problem is to dissolve the compounds
in a volatile solvent such as ethanol, DMSO, water, dispense the
compounds and allow the solvent to evaporate, leaving a spot of
dried compound in the container. This has some major disadvantages
in that the compound may crystallize into a form that does not
easily re-solubalize, and is not sufficiently immobilized. Another
approach would be to dispense the compound in a volatile solvent
such as DMSO and allow the DMSO to evaporate to leave the desired
compound in a lower volume of DMSO. In this case, it may be
difficult to evenly evaporate the solvent, which may interfere with
the assay to be performed on the samples.
[0323] A more robust approach is to dissolve the compound in a
first volatile solvent such as DMSO and a second, more volatile
solvent, such as ethanol or methanol, dispense the mixture into the
containers and allow the second solvent to evaporate, leaving
compound dissolved in to first solvent. Ethanol and methanol are
good choices since they will dissolve most drug-like compounds and
are hydrophilic enough to remain contained in a hydrophilic
container surrounded by hydrophobic barriers, although other
solvents could be used. DMSO is a good solvent for use as the first
solvent since it dissolves most compounds, will stay in place due
to its hydrophilicity and is not very volatile, evaporating more
slowly than water.
[0324] The invention also features a method for storing compounds
dry in an array of sub-microliter containers separated by
hydrophobic barrier. Compounds are dissolved in a volatile solvent
or combination of solvents, introduced into the array of containers
and the solvent is allowed to dry, leaving a thin film of solid
compound. If necessary, a biocompatible adhesive is added to keep
the film attached to the walls of the container.
[0325] A solvent mixture containing a first volatile solvent and a
second less-volatile solvent is added to substantially all of the
containers in the array. In a preferred embodiment, the solvent
mixture comprises an alcohol such as ethanol and DMSO. The more
volatile solvent is allowed to evaporate, leaving a residue of the
less-volatile solvent that dissolves at least most of the compounds
in the array. Typically, the less-volatile solvent will comprise
the minority of the solvent mixture, is usually less than 20%, and
is often less than 5% of the solvent mixture. The array is then
prepared for assay by introducing an aqueous media that is
compatible with the assay such as water or a buffer. The aqueous
media may be dispensed by the methods described above.
Examples
[0326] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
DNA Probe Array
[0327] A 50,000-channel through-hole array is fabricated from
silicon. Using the gene database, series of 80 spatial filter top
masks with another 80 identical bottom masks are fabricated. Arrays
are aligned and derivatized with 3-glycidoxypropyltrimethoxysilane
in order to provide a free functional group coating on the interior
surfaces of the through-holes.
[0328] A stack of ten coated platens is aligned. Alignment is
verified by observing the optical transmission of the various
channels. Mask #1 is aligned with the top of the stack and an
identical mask aligned in the same orientation and the bottom of
the stack. Mask #1 is constructed such that the positions in the
mask that corresponded to a gene in the database with an adenosine
in the first position are open and allow flow through the
through-holes in those addressable positions. The stack is rinsed
with dry acetonitrile. A phosphoramidite monomer at a concentration
of 0.1 M acetonitrile and tetrazole is added by a pressurized flow
against the mask. The coupling reaction is allowed to proceed for 3
minutes. An oxidizing solution of
iodine/lutidine/acetonitrile/water is introduced into the chip
stack and incubated for 2 minutes, followed by rinses with
acetonitrile and dichloromethane. The chip is dried by vacuum.
[0329] The masks are removed and replaced with a top and bottom
Mask #2 corresponding to positions to which a T monomer is to be
added. A deprotection agent is added, and the T coupling reaction
is commenced.
[0330] This process is repeated with Mask #3 for G in the first
position, and Mask #4 for C in the first position. Mask #5
corresponds to A in the second position, and so on. After the
synthesis is complete the chip is stored in 30% ammonia for 12
hours.
[0331] To test the array, a fluorescein end-labeled 20-mer
corresponding to one of the intended probes in the array is
synthesized by standard methods, dissolved in a hybridizing
solution of 6.times.SSC/0.5% SDS, and introduced into all
through-holes of the array simultaneously. The array is washed with
0.5.times.SSC and the chip is imaged fluorescently. Only the
position in the array corresponding to the probe complementary to
the test oligonucleotide shows a significant increase in
fluorescent intensity over the background level determined by
averaging the signal from the remaining positions in the array.
Example 2
Catalyst Screening
[0332] A recombinant enzyme library is screened in a dense array of
through-holes against a fluorogenic substrate. Genetically diverse
E. coli containing the gene for the protease subtilisin with a poly
histidine tag is created by mutagenesis. A dilute solution of the
bacteria is added to a nickel-coated array such that there is an
average of 1 to 2 bacteria per through-hole. The bacteria are
allowed to grow to the log phase and replicate plated (as described
above). The bacteria in the array are lysed by heating, allowing
the tagged subtilisin to attach to the nickel coated walls of the
array. Another array containing the fluorogenic substrate and
reaction buffer (Boehringer) in each well is stacked with the first
array. The stack is immediately placed in a CCD camera-based
fluorescent imaging system, and the rate of increase in
fluorescence intensity is measured for each through-hole. The
enzyme with the fastest rate is selected and the corresponding
bacteria in the replica plate are grown for further studies.
Example 3
High Throughput Screening with Beads
[0333] A 100,000 member combinatorial library immobilized via a
photocleavable linker on 10 micron diameter beads is purchased from
Affymax. A platen is prepared with 100,000 through-holes of a
diameter such that only one bead fits in each hole. The beads are
washed in PBS, suspended in a solution containing a fluorogenic
substrate for the enzyme to be screened, and spread over the platen
with a rubber spatula. An ultraviolet lamp is used to decouple the
members of the library from the beads. A second platen filled with
a solution of the enzyme in a reaction buffer is aligned and
stacked on top of the first chip. The stack is immediately imaged
by epi-fluorescence to determine the rate of increase in
fluorescence intensity as a function of channel position. Those
holes that exhibit decreased enzyme rates are selected for further
analysis as drug leads.
Example 4
Absorption Assay
[0334] A single-cell layer of CaCo-2 cells is grown on two
identical anisotropic membranes coated with collagen slightly
larger than the array of through-holes. The cell culture
conditions, media, and membrane coatings used in the in vitro
growth and maintenance of CaCo-2 cells are well known by those
skilled in the art. Once a uniform layer of cells is established,
each membrane is sandwiched between two identical arrays of through
holes dip-loaded with culture maintenance media. The array and
membrane assemblies are cultured an additional day to allow for the
CaCo-2 cells to equilibrate and form an intact layer within the
through-holes. Two additional arrays with through-holes
co-registered with the CaCo-2 arrays are loaded with a chemical
diversity library of small molecules. A compound known not to pass
through CaCo-2 cells (e.g. mannitol) is used as a negative control
to assess the integrity of the CaCo-2 cell monolayer, while a
compound known to easily diffuse through CaCo-2 cell monolayers is
used as a positive control. One of the arrays containing the
chemical library is stacked on the apical side of one of the CaCo-2
cell monolayer arrays to assess apical to basal absorption while
the second identical chemical library array is placed on the basal
side of the other CaCo-2 cell monolayer array to assess basal to
apical absorption. The completed arrays are incubated for 1 hour,
allowing time for transport or permeation of the library compounds.
The oral bio-availability of the chemical library is assessed by
quantifying the amount of library compound diffused through the
CaCo-2 cell monolayer (e.g. by mass spectrometry).
Example 5
Ligand Fishing by Blotting from a 2-D Gel
[0335] Cellular proteins exhibiting an affinity for a ligand are
identified using a 2-D gel. A platen having 500,000 through-holes
is derivatized in order to covalently link a segment of the human
epidermal growth factor receptor to the inside of each
through-hole. Each hole is filled with a buffer solution. A
cellular extract of a human cell line is then separated on a 2-D
gel of a size similar to that of the platen, and then aligned with
the platen. The proteins in the 2-D gel are then blotted onto the
chip by applying a buffer above the chip and drawing fluid through
the chip by placing the chip on a blotter. The proteins are thus
transferred through the chip and those that have an affinity for
the epidermal growth factor receptor are retained in the chip. A
denaturing solution composed of 1M formamide in 100 mM Tris buffer
at pH 8 is used to blot the contents of the chip onto a platen for
mass spectrometric analysis. The location of those holes that
contain a protein with affinity for the receptor and the mass of
the proteins contained in those holes are used to determine the
identity of the binding proteins. These proteins can be targets for
drugs that block the EGF signaling pathway as a treatment for
certain cancers.
Example 6
Screening for Antibiotics
[0336] A 500,000 member combinatorial peptide library is prepared
in a platen that includes 500,000 through-holes such that the
peptides are dissolved in a sterile cell culture medium within the
holes. The library is prepared such that the identity of the
peptide present in each through-hole is known. A dilute culture of
Enterococcus faecium bacteria is prepared such that each
through-hole will receive on average 10 bacteria in cell culture
medium. The bacteria platen is stacked with the peptide library
platen to achieve mixing then incubated at 30.degree. C. for 5
hours. The stacked platens are then imaged by light scattering
measurement to determine the degree of bacterial growth in each
through-hole.
[0337] The holes showing a greater than 99% reduction in growth are
identified, and larger quantities of the corresponding peptides are
synthesized for further analysis.
Example 7
Finding the Peptide Target of a Kinase by Radiolabeling
[0338] A protein suspected of being a protein kinase is isolated.
The protein is incubated with radiolabeled ATP substrate in the
presence of 100,000 different proteins, all occupying unique
positions in a platen having through-holes. After incubation for a
sufficient time (e.g., about 20 minutes), the platen containing
through-holes is washed with water and the presence of radiolabeled
proteins is detected by a phosphor-imaging system. The protein
target for the kinase is thus identified.
Example 8
Cytochrome P450 Inhibition Assays by Fluorescence
[0339] The purpose of this experiment is to examine the potential
of a library of compounds to inhibit a specific CYP450 enzyme. The
protocol is adapted from Crespi et al., Anal. Biochem. 248:188-190,
1997. The fluorometric substrate is 3-cyano-7-ethoxycoumarin for
CYP1A2, CYP2C9, CYP2C19 and CYP2D6, and resoufin benzyl ether
(BzRes) for CYP3A4. These reagents are obtained from Pharmazyme. A
compound library is loaded into one platen array device for each of
the CYP450 enzymes to be tested at a concentration equivalent to
the Km of each enzyme. The appropriate substrate containing
reaction mixture is added to a second platen array device and the
enzyme is added to a third platen array device. The chips are
stacked to initiate the reaction, and the increase in fluorescent
signal is monitored continuously by fluorescent imaging. The
relative rates of P450 inhibition are used to select a drug lead
from the candidate compound library.
Example 9
High Throughput Protein Crystallization
[0340] A protein in a solvent is uniformly loaded into a
through-hole array. The array through-holes are randomly filled
with solutions containing different salts, randomly changing the
concentration and relative abundance of the salts. Acidic and basic
solutions are subsequently loaded into the through-holes, varying
the pH. A temperature gradient is applied to the array device (or
the array can alternatively be held at a constant temperature),
causing the solvent to evaporate at a given rate. Additionally, the
partial pressure of solvent can also be changed in the container in
which the array is placed to change the evaporation rate. Those
through-holes in which protein crystallization is observed are
exposed to a beam of X-rays or electrons and the diffraction
pattern recorded for analysis. An important benefit is the rapid
and efficient discovery of experimental conditions leading to
crystallization. Furthermore, protein crystals can be analyzed
directly in the through-hole array.
[0341] Sixteen different crystallant solutions, and eleven
different buffers, are obtained from Emerald Biostructures
(Bainbridge Island, Wash.), and randomly loaded into the platen
through-holes together with lysozyme. A temperature gradient is
applied across the platen, and the excess liquid is removed with a
rubber spatula. The system is sealed in a container with 20 ml of
precipitant solution. Optimal solution conditions are determined
for crystallization using LC-MS (liquid chromatography-mass
spectrometry).
Example 10
Ultra High Throughput Mixture Separation and Screening in Dense
Arrays of Through-Holes
[0342] In many situations such as screening of natural products for
pharmacologically active molecules, complex mixtures need to be
rapidly and efficiently separated and screened against protein
targets. The purpose of this experiment is to separate and screen
in a dense array of through-holes a complex mixture of natural
products against a fluorogenic substrate. The natural product
sample is first prepared in the normal manner for high pressure
liquid chromatography (HPLC). As liquid elutes from the
chromatographic column, equi-volume samples are acquired and stored
sequentially in the array through-holes. A replicate plate can be
generated simultaneously. Fluorogenic substrate is then loaded
uniformly into a second through-hole array. After completion of
chromatographic separation, each sample in the array is exposed to
the substrate by stacking the sample plate onto the substrate
plate. Optical monitoring the fluorescence signal from the assayed
fractions selects samples for further evaluation either from the
assayed plate or the replicate plate.
[0343] An extension of this method to applications where multiple
mixtures are stored in a dense array of through-holes is also
described (e.g., separation and identification of the active
component in a mixture). In one embodiment, a capillary tube array
having the same center-to-center spacing as through-holes in the
platen is brought into contact with the sample array. Each tube is
located to spatially address one hole in the array. The opposite
end of the tubing array is inserted into a stack of arrays where
each capillary tube addresses a single column of through-holes on
spatially co-registered arrays. The number of stacked arrays equals
the number of elution samples to be captured and analyzed. Each
tube is pre-filled with a porous gel suitable for chromatographic
separation of the mixtures. An array plate filled with buffer is
stacked onto the sample plate and a pressure is applied to drive
the buffer through each through-hole and into the corresponding
capillary tube. As liquid exits from each tube, the through-hole in
which the tube resides is filled. As the tube array is slowly
withdrawn from the array stack the liquid sample is retained in the
through-hole. One advantage of this scheme is that fractions eluted
from the array of capillary tubes are simultaneously collected.
Once an array is filled, it can be removed from the stack and
assayed, (e.g., contacting with a platen array containing a
fluorogenic substrate). Active compounds revealed by fluorescence
emission are then removed from the plate for analysis.
Alternatively, the flow rate of liquid from the tubing array and
withdrawal velocity can be chosen such that two plates are filled
with essentially the same fraction eluted from the column. The two
plates are removed; one is assayed while the other serves as a
replicate plate.
[0344] A further extension of this method to interface parallel
HPLC separation with inherently serial analytical methods such as
mass spectrometry is now described. FIG. 13 depicts, an array of
capillary tubing (1) is interfaced with a sample filled
through-hole array (2). However the opposite end of the tubing
array is splayed in such a manner so as to increase the distance
between consecutive rows (or columns) of the array whilst keeping
the others intact. A series of spacers (3) through which the tubing
is inserted to form the array provides structural integrity. The
sample array is brought into contact and co-registered with the
capillary tubing array. Each capillary tube in the array is
pre-filled with porous gel suitable for chromatographic separation
of the mixtures contained in the array through-holes. Pressurized
carrier solvent is forced through the holes in the array and
carries one sample into one capillary tube. Lengths of the tubing
in the array are chosen so as to give the desired separation
efficiency for the components in the mixtures analyzed. A fiber or
thin tape runs just below and parallel to a row or column of the
capillary tubing array. Lateral motion of the tubing array relative
to the fiber brings the tubing ends in one column/row in the array
into contact with the fiber (or tape) (4). Fluid from each tube is
transferred to discrete spatial locations along the fiber. After
the fluid is transferred, the fiber is advanced through a vacuum
interface (5) and the fluid drops are sequentially presented to the
mass spectrometer (6) for analysis. After one set of drops is
deposited, the surface (e.g., nitrocellulose fiber) is advanced a
distance sufficient for next set of drops to be deposited. Note
there are only two displacements required, the surface in one
direction and the array in the orthogonal direction.
[0345] With a combined time to make a mass spectral measurement and
move the fiber of about 300 ms, a row of 100 drops is transported
and analyzed in 30 seconds. The entire 10,000-tube array is
analyzed in 3000 seconds (50 minutes). As shown in U.S. Pat. No.
6,005,664, stochastic sampling can substantially reduce the number
of data points (by up to a factor of 10) needed to reconstruct a
signal compared with equi-spaced sampling. Implementation of a
stochastic sampling protocol could greatly reduce the analysis
time.
Example 11
Optimization of Chemical Synthesis Conditions
[0346] Optimization of a chemical synthesis by changing one or more
process parameters and recording the amount of material synthesized
for a set of reaction conditions. Modified process parameters
include types of reagents, reagent concentration, sequence of
addition/mixing, temperature, and time.
[0347] A series of hydantoin compounds, pharmaceutical drugs useful
for treatment of epilepsy, are prepared using the new methods in
solid-phase synthesis. Each step in the synthesis process consists
of reaction with a solvated reagent under a given set of time and
temperature conditions and then a wash to remove the excess
(unreacted) reagents. Microspheres made from resins having
different functional groups (e.g., hydrogen, phenyl, methyl,
benzyl, and s-butyl) and a protected amide group are either
purchased or synthesized. An array is manufactured with tapered
holes such that as the beads are spread across the surface, only
one resin bead fits into each through-hole. Microspheres made from
different resins are mixed together and spread across the array in
a dilute solution such that all the holes are filled, each with a
microsphere made from a different resin. Next, a mask is placed
over the regular array of through-holes and the amine groups of the
exposed resin beads are deprotected by exposure to a strong acid
(e.g. trifluoroacetic acid) or base. After a wash step to remove
the acid or base, the same resins are exposed to one member from an
isocyanate group library consisting of different chemical and
structural moieties. Reaction between the isocyanates and amide
groups from a urea with a variable moiety. Such chemical units
include hydrogen, butyl, allyl, 2-trifluorotoyl and
4-methoxyphenyl. The reaction proceeds at an elevated temperature
for a certain time after which the exposed holes are washed to
remove unreacted reagents. This process is repeated with a new mask
exposing some of the original holes as well as new ones to expose
these microspheres to a new isocyanate library group. Upon
completion, the hydantoins are screened against targets to find
those potent compounds. Varying the reagents, their concentrations,
the reagent sequences, the reaction temperature and reaction time
provides a rapid and efficient method to optimize the synthetic
rout.
Example 12
Selection of Phage Antibody Libraries (Multi-Chip Method)
[0348] A library of phage antibodies against a target antigen in a
dense array of through-holes is screened. The protocol was adapted
from (Winter,
http://aximtl.imt.uni-marburg.de/.about.rek/AEPStart.html).
[0349] Through application of recombinant DNA technology a large
(10.sup.9 to 10.sup.10) and diverse monoclonal antibody library is
produced and stored as DNA plasmids in bacterial (E. coli)
colonies. A single round of conventional affinity selection is
performed in an immunotube coated with the target antigen. After
incubating, blocking, and washing, the specifically bound phage are
eluted and used to reinfect additional E. coli.
[0350] Two 10,000-through-hole silicon arrays are fabricated. Both
array devices are loaded with a solution of purified target
antigen. The array devices are then incubated at 4.degree. C. for
approximately 12 hours, washed and filled with a blocking solution
consisting of 4% dry milk powder in phosphate buffered saline
(PBS). Adler one hour the blocking solution is removed, the array
devices are rinsed and refilled with a buffered solution of IPTG to
induce phage expression in the bacteria.
[0351] A sufficiently dilute solution of the phage-infected
bacteria is added to a third 10,000 through-hole array such that
there is an average of 1-10 bacteria per through-hole. The
resulting through-hole array, henceforth referred to as the Library
Expression Chip, is incubated until the bacteria reach log phase.
The Library Expression Chip is stacked between the two antigen
coated through-hole arrays. The stack is then incubated for two
hours in a high humidity chamber to allow binding of the phage
antibodies to the antigen. The antigen through-hole arrays are
washed to remove bacteria and unbound phage and separated from the
Library Expression Chip.
[0352] The bound phage from one of the two antigen through-hole
arrays, henceforth referred to as the Phage Inoculation Chip, is
eluted by filling the through-holes with a solution of 100 mM
triethylamine, incubated for approximately 10 minutes, then
neutralized by stacking onto another 10,000 through-hole array
containing 2.times. Tris-HCl buffer. The eluted phage is used to
inoculate uninfected bacteria contained in a fourth 10,000 channel
array. This array, henceforth referred to as the Positive
Expression Chip, is grown to log phase and stored.
[0353] Meanwhile, the second antigen-phage chip, the Antibody
Selection Chip, is analyzed for bound phage antibody via an
indirect ELISA assay. The protocol is adapted from Current
Protocols in Immunology, Supplement 15, pages 11.2.2-11.2.5. This
protocol is an indirect ELISA to detect specific antibodies. Other
forms of ELISA assays, such as direct competitive ELISA assays can
be implemented in the through-hole arrays with minor modifications
to the procedure described in this example.
[0354] The Antibody Selection Chip is filled with a solution of the
developing reagent, an-M13 conjugated to horseradish peroxidase in
blocking solution. After incubating for thirty minutes at room
temperature, the array is washed, blocked, and washed again. The
array is then filled with a solution of fluorescent substrate, and
incubated for 30 minutes at room temperature. A fluorescent image
is then collected every 5 minutes for one hour. All positive
through-holes are identified by an increase in fluorescent
intensity with time. Each corresponding bacterial culture is then
extracted from the Positive Expression Chip and dispersed onto agar
(containing ampicilin) in a separate cell culture well. These
selected bacterial cultures constitute a source of additional phage
antibody for subsequent analysis.
[0355] In an alternative method, the Library Expression plate,
rather than the chip is replica plated. Only a single antigen
through-hole array is exposed to the antibody library. After
identifying positive interactions in the Antigen Selection chip,
the bacteria from the corresponding through-holes of the replicated
Library Expression plate are diluted and dispersed across another
10,000 through-hole array. The assay is repeated until to ensure
that all selected bacterial colonies are monocultures.
Example 13
Selection of Phage Antibodies (Single Plate Method)
[0356] A library of phage display antibodies is screened against a
target antigen using a single 10,000 channel array of
through-holes. By performing the selection in a single array
simplifies the screening process, the phage must be reconstructed
from their DNA, ecause the ELISA assay renders selected phage
incapable of reinfecting bacteria.
[0357] The target antigen is immobilized on the walls of the
through-holes by filling an array with antigen solution and
incubating in the environmental chamber. Non-specific binding sites
are blocked by washing the array with blocking solution.
[0358] A solution of the phage-infected bacteria is added to all
through-holes in the array such that there is an average of 1-10
bacteria per through-hole. The assay device is then incubated in a
humidified chamber until the bacteria reach log phase. The assay
device is submerged in a solution of IPTG for a period of time
sufficient for diffusion of IPTG into through-holes, but
insufficient to permit bacteria to diffuse out of the array. The
assay device is incubated to allow expression of phage antibody,
and antibody-antigen binding. The expression of phage antibody in
the presence of the target antibody is counter to current methods
in the art, and can reduce the number of antibodies selected in the
screen.
[0359] Next, the array is washed to remove bacteria, supernatant,
and unbound phage from through-holes. Bound phage antibody is then
detected by an indirect ELISA assay. After collecting a fluorescent
image, the plate is washed and filled with elution reagent to elute
bound phage. Solutions in through-holes that are identified
corresponding to all positive through-holes by cherry picking to
individual wells of a 96-well or higher density microtiter plate.
Phage DNA is then amplified by PCR and sequenced. The DNA sequences
are used to identify the structure of selected antibodies and to
reproduce more phage antibody for further experimentation.
Example 14
Protein Chip
[0360] One hundred thousand protein-binding probes are produced
using phage display technology according to the methods described
in Sheets et al., Proc. Natl. Acad. Sci. USA, 95:6157-6162, 1998,
incorporated by reference in its entirety, such that each probe
selectively binds a particular human protein with high affinity and
specificity. The probes are transferred into a platen, such that
different protein-binding probe in each of its through-holes. The
platen has a TEFLON.RTM. surface to prevent wetting and protein
absorption.
[0361] Tissue samples are homogenized, and the protein portions are
extracted and equilibrated with the platen. The platen is washed to
remove non-specifically bound molecules. The contents of each
through-holes are analyzed by heating the platen from ambient
temperature to 100.degree. C. over a two minute period while using
Raman imaging to detect protein desorption from the walls of the
through-holes into the centers of the through-holes.
Example 15
Construction of an Array of Micro-HPLC Columns for Rapid Parallel
Sample Separation and Purification
[0362] An array of twelve through-holes in a linear arrangement is
machined in a block of material (e.g., metal, ceramic, or plastic).
The through-holes have a diameter of 250 .mu.m, a total length of
20 mm, and a center-to-center spacing of 9 millimeters. The
internal diameter of the distal end of the array of through-holes
is increased and threaded to accept standard 1/16'' outer diameter
HPLC tubing using a standard nut-and-ferule compression fitting. A
2 cm long piece of 50 .mu.m internal diameter, 1/16'' outer
diameter tubing is mated to each of the through-holes in the array
through machined compression fittings. A 1/16'' outer diameter
stainless steel frit is placed inside the through-hole and is held
in place with the compression fitting. Chromatography media is then
packed into the each through-hole in the array in the form of a
slurry. The chromatography media is immobilized within the
through-hole due to the stainless steel frit at the distal end, and
creates a micro-HPLC column. When pressurized, sample, wash, and
elution buffers will flow through the chromatography media and the
frit, and elute from the through-holes through the mated 50 .mu.m
internal diameter tubing at the distal end.
[0363] Samples are loaded onto the array of micro-HPLC columns by
mating a bank of syringes to the micro-HPLC array as detailed in
examples 2 and 3. Wash and elution buffers are flowed through the
micro-column array using the same syringe bank. A flow rate of 1 to
20 .mu.l of liquid per minute is used to wash and elute the samples
from the micro-columns. The use of narrow bore tubing at the distal
end of micro-HPLC columns results in the liquid eluting from the
column to form small droplets. The column eluate is collected in an
array of wells (e.g., a microtiter plate). The eluate is
fractionated by collecting individual droplets as they elute from
the micro-HPLC columns in different wells of the microtiter plate.
Chemical and physical analysis (e.g., spectroscopy or spectrometry)
of the samples can be performed on the samples within the
wells.
Example 16
Fluidic Seals for Parallel HPLC in Microcapillary Channels
[0364] With reference to FIG. 16, an array of through-holes is
machined in a block of material (e.g., metal, ceramic, or plastic).
The through-holes have a diameter of less than 1 min and an aspect
ratio greater than 10. The through-holes are chamfered to accept an
o-ring gasket. A syringe bank is fabricated with the same
center-to-center spacing as the through-hole array. The syringe
needles pass through a metal block that is attached to the syringe
bank holder by pneumatically actuated, spring-loaded pins. O-ring
gaskets are placed onto the syringe needles protruding through the
block. The syringes are loaded with fluid and inserted into the
capillary tubes. The pins are pneumatically actuated to bring the
metal block in contact with the top surface of the capillary tube
block and press the o-ring gaskets into the hole chamfer and around
the syringe needle. This makes a leak tight seal between the needle
and capillary channel thus allowing the capillary channels to be
pressurized by the syringes (FIG. 117). FIGS. 18 and 19 illustrate
a similar approach, except that the syringe bank in those figures
is shown bolted to the capillary tube array to result in a rigid
structure for easy handling.
Example 17
Identifying a Ligand to a Biomolecular Target
[0365] The purpose of this experiment is to use low volume
chromatography using through-hole arrays to identify ligands in a
chemical, biochemical or biological mixture that bind to a
biomolecular target, in this case a protein. Two linear
through-hole arrays are constructed such that each through-hole
holds 50 nl of liquid when filled. The exterior surfaces of the
arrays are treated to be hydrophobic and the interiors are treated
to be hydrophilic. A center to center spacing of 9 mm between each
of the through-holes is used and registration holes are includes to
ensure alignment and co-registration of the through-holes upon
stacking of the arrays using pins on a precision jig. A bank of
syringes is used to dispense 50 nl of target protein solution into
the first array and 50 nl of different compound libraries into each
through-hole of a second array. The arrays are stacked using a
precision jig and the fluids are allowed to mix in each of the
co-registered through-hole positions. A bank of syringes with
compression fittings is filled with 1 ml of eluant, followed by a
small air bubble and the used to draw up the 100 nl of the
protein-library reaction mixture. The reaction mixture is held in
the tip of the syringe and kept separate from the eluant reservoir
by the air gap. The syringe tips are the placed into the orifice of
an array of size exclusion columns and the compression fittings are
tightened to ensure a seal. The samples are dispensed into the
column, followed by the eluant. By monitoring the UV absorbance of
the sample exiting at least one column, one may determine when
protein bound to ligand is exiting the column. This protein is then
recovered onto an array of reverse phase columns by coupling the
two column arrays. The ligands are removed from the protein by
reverse phase chromatography, recovered, and analyzed to determine
their identity.
Example 18
Protection of Through-holes by Coating with Wax
[0366] Paraffin wax with a melting point of 54.degree. C. is heated
above its melting point. A through-hole array is submerged in a
thin layer of molten poly(ethylene glycol) wax with an average
molecular weight of 1500. The through-hole array and wax are cooled
to cause the wax to harden. Excess wax is removed from the surface
by scraping with a sharp blade and then polishing. The wax-filled
array is then exposed for 10 seconds to vapor from a solution of
(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane diluted
1:20 in xylenes. The coating is then cured by baking the platen at
110.degree. C. for 30 minutes while allowing the wax to melt and
drip out of the platen. Wax residues can be removed from the
channel interiors by washing with water. The platen is then rinsed
for 10 seconds in sulfuric acid/ hydrogen peroxide (2:1), and then
with water, to remove residues and ensure oxidation of the channel
interiors. The resulting through-hole array has a hydrophobic
exterior and a hydrophilic interior.
Example 19
Photo-Initiation Method of Fiberglass Through-Hole Array
Production
[0367] A sheet of fiberglass filter such as that available from
Millipore (Bedford, Mass.) is soaked in a polymerizable solution
(e.g., a solution containing methyl methacylate monomer and a
photoinitiator such as benzoin methyl ether) and placed between two
quartz plates, each having an array of dots that serve to mask the
areas that will remain open and porous Polymerization of the
monomer solution is effected by illuminating the photomasks with
ultraviolet light.
Example 20
Application of an Assay to an Array of Through-Holes
[0368] A common assay used in drug discovery efforts is the
cytochrome P450 inhibition assay. Compounds that inhibit the
activity of P450 enzymes are generally undesirable as
pharmaceuticals as they have the potential to cause serious
side-effects and have the potential for negative reactions if taken
in conjunction with other pharmaceuticals. A common assay that is
used to screen compounds that are potential candidates for becoming
pharmaceuticals is to incubate the compounds in a cellular,
extra-cellular, or recombinant system than expresses P450 enzymes
and to assay the activity of the enzymes in the presence of the
candidate compound. Such an assay could be modified for analysis in
a massively parallel manner in an array of through-holes.
[0369] Four co-registered arrays of through-holes in which the
through-holes are used. The enzyme or cellular compartment (e.g.,
microsomal compartment of a liver cell) that expresses P450 enzymes
can be loaded into one array of through-holes such that each
through-hole contains an equivalent volume and concentration of
P450s. A known substrate for a chosen P450 enzyme along with a
source of NADPH (a source of energy) can be loaded onto a second
array of through-holes such that each through-hole contains an
equivalent volume and concentration of substrate and NADPH. A third
plate can be loaded with known concentrations (e.g., serial
dilutions) of the compounds that are to be assayed along with
positive and negative controls. An invention for the rapid loading
of multiple arrays of through-holes has been described elsewhere in
this document. The assay is started when these 3 arrays of
through-holes are brought in to contact with one another. As has
been described elsewhere in this document, by controlling the size,
density, and surface chemistry of the through-holes and the arrays
themselves the contents of the 3 arrays can be made to rapidly mix
with one another while inter-through-hole contamination between two
or more adjacent through-holes can be avoided.
[0370] The stacked array of through-holes is then incubated at
37.degree. C. in a controlled humidity environment to allow the
reaction to proceed. By maintaining a high humidity environment
during this incubation evaporative loss from the through-holes can
be minimized. After 30 minutes a stop solution (e.g., organic
solvent, urea, high salt solution etc.) is added to the each
through-hole by stacking a fourth array filled with the stop
solution with the assay arrays. The stop solution works to stop the
assay by causing the P450 enzymes to denature and/or precipitate
out of solution. This precipitate can be pelleted by centrifugation
using an invention described elsewhere in this document. The top
array of through-holes can then be removed from the others and used
for analysis. In many assays, when the substrate is metabolized by
the P450 enzymes it is transformed from a non-fluorescent compound
into a fluorescent one. The amount of fluorescence will be directly
proportional to the amount of P450 activity. If the compound being
assayed inhibits the P450 enzyme, the less the metabolism that will
occur and less fluorescent material will be generated. The
concentration at which the P450 enzyme is inhibited by 50%
(IC.sub.50) can be determined if multiple concentrations of
inhibitors were used in the assay. Fluorometric and/or mass
spectrometric analysis of the samples can be performed using
inventions described elsewhere in this document.
Example 21
Washing Protein from a Surface of a Through-Hole Array
[0371] A through-hole array is prepared from silicon with a
fluoro-chloro-alkane coating on its surface and is treated with an
oxidizing solution that renders the interiors of the holes
hydrophilic. The array is dipped in water and frozen to -80.degree.
C., then quickly dipped into a solution of the fluoropolymer
FluoroPel.RTM. (Cytonix Corp., Beltsville, Md.). The array is baked
at 200.degree. C. for 20 minutes, and then dipped into cell media
containing 10% fetal calf serum. Dipping the through-hole array
into the medium fills the holes in the array and wets the surface.
By then dipping the through-hole array into perfluorooctane and
withdrawing slowly, the surface is cleaned of all aqueous
media.
Example 22
DNA Sequencing
[0372] Fluorescently labeled DNA fragments are prepared by Sanger
sequencing and arrayed in an array of 2500 through-holes in a
platen of 0.5 mm thickness. This platen is then stacked on a second
platen of 80 mm thickness containing a gel and inserted into an
actively cooled electrophoresis tank. As fluorescent oligos emerge
from the column array they bind to a nitrocellulose membrane
unwound from a spool. A computer controls both the movement of the
membrane and turns off the electric field while the nitrocellulose
membrane (in the form of a tape) is moving. The membrane is then
moved to cause exposure to a light source and CCD camera, analyzing
the images and creating elution profiles for each of the
through-holes. This achieves reconstruction of the DNA
sequence.
Example 23
Method of Manufacturing a Platen Using a Plurality of Grooved
Plates
[0373] Platens having through-holes are to be manufactured from
silicon plates having parallel grooves. See FIG. 20. The resulting
array has 5000 through-holes, wherein the through-holes are
approximately square with each side 0.25 mm in length and the
platens are about 1 mm deep.
[0374] Nine inch silicon wafers (0.5 millimeter thickness) are
used. The circular wafers are precisely cut to yield rectangular
pieces (60 in total) that are 55.times.160 millimeters (a total of
three surfaces per wafer are obtained). Grooves of 250 microns
width and 250 micron depth are then etched lengthwise into each
piece of silicon. A total of 100 grooves are etched into 50 of the
silicon pieces and the distance between each groove is adjusted to
about 250 microns. The remaining 10 pieces of silicon are not
etched. Chemical etching of silicon is a process known to those
skilled in the art, and involves the masking the appropriate areas
of silicon and treating with acid. The unmasked areas are then
etched away in a controlled manner. A total of 25 millimeters from
each side is not etched, providing a solid border to the finished
platens.
[0375] Once the chemical etching of the surfaces is complete, the
mask used in the etching process is removed and the non-grooved
surface of each piece of silicon is sputter-coated with a thin
layer of gold. The pieces of silicon are then stacked together in a
jig having a flat surface with a right-angle bracket. Five pieces
of silicon without grooves are first stacked, followed by the 50
grooved surfaces, and another 5 pieces without grooves. The entire
jig containing the pieces of silicon is moved to a press and
pressure is applied to the stacked pieces of silicon. The assembly
is heated and allowed to remain under elevated pressure and
temperature for about 16 hours. This treatment results in permanent
bonding of the platens into a single large piece of silicon with
55.times.30.times.160 mm dimensions having 5000 through-holes (in a
100.times.50 array) and a 25 mm solid border of silicon around the
through-holes. A wire saw is used to cut the individual platens to
a thickness of 0.5 millimeter. The platens are cut by slicing in
the plane orthogonal to the through-holes. See FIG. 20. The wire
used in the saw has a thickness of 150 microns and as a result this
amount of material is lost during the sawing process. Including the
removal of uneven platens created at either end, a total of 230
platens fitting the required specifications are cut from this large
piece of silicon.
[0376] Both surfaces of the platens are polished in a lapping
machine to ensure a flat surface, resulting in an insignificant
amount of silicon being removed. Finally, surface chemistry is
applied to the platens to provide the finished product, having the
desired physical characteristics.
Example 24
Storing and Screening a Nonoliter Volume Compound Library
[0377] Compounds are reformatted from 96 well plates into a
through-hole array as follows: Compounds in 96 well plates are
dissolved in DMSO to 100 times the concentration that they willed
be assayed at--for example, 100 uM for a luM final concentration in
the assay. The total volume of DMSO sample solution in each well of
the 96 well plate is 10 ul. An additional 90 ul of ethanol is mixed
into each well and the samples are immediately drawn into a syringe
bank for dispensing. The automated syringe bank the dispensed 60 nl
volume into each stack of a through-hole array having the same
footprint as a 96 well plate. This process is repeated with new
96-well plates until the through-hole arrays are substantially
full. The alcohol is then allowed to evaporate, leaving a residue
of approximately 600 pl of DMSO dissolved compound in each channel
of each through-hole array. After storage of the compounds for the
desired time under desiccation at -80.degree. C., a through-hole
array containing the arrayed compounds is removed, brought to
10.degree. C. so that the DMSO remains frozen and dipped into a
beaker containing aqueous assay solution that has been chilled to
10.degree. C. Removal of the through-hole array from the beaker
under a humidified environment results in the through-holes of the
chip being filled with the aqueous medium. The through-hole array
is warmed to ambient temperature to allow mixing of the solvents. A
second through-hole array is uniformly filled with a freshly
prepared, chilled assay buffer containing an enzyme and a
fluorogenic substrate; in this case a Matrix Metalloprotease assay.
Stacking of the two chips, warming to ambient temperature and
fluorescent imaging at several time points over 30 minutes gives a
primary screen for enzyme inhibition.
Example 24
Selecting Membranes for Cell Culture
[0378] The invention provides methods for the growth of various
cell types, including eukaryotic cells, including but not limited
to adult stem cells, chondrocytes, embryonic stem cells,
endothelial cells, epithelial cells, fibroblasts, hematopoietic
cells, muscle cells, including cardiac muscle cells of the heart,
neurons, osteocytes). A schematic of this method is shown in FIG.
25. To facilitate the growth of these cells, porous membranes were
assayed for their ability to support the attachment, survival,
growth, or proliferation of an exemplary cell type, HEK 293 cells
transfected with a PKCb-GFP expression vector (FIG. 26). The cells
were plated into wells of a 24-well plate (BD-Biocoat 24 well) that
contained inserts to be assayed. 3 .mu.m pore inserts that were
uncoated or that were coated with fibronectin, laminin, and
collagen were tested, as were membranes fabricated from aluminum
oxide having a uniform capillary pore structure, ANOPORE tissue
culture inserts, of 0.2-.mu.m pore size (Nunc Inc.). The membranes
were incubated overnight with 200,000 cells. Following this
incubation, the ANOPORE membranes were completely confluent (see
FIG. 25). The laminin coated membrane was about 20% confluent. The
other membranes were less than 10% confluent. Cells seeded at
75,000 per well on an Anopore insert continued to grow until
confluency at day 6 (see FIG. 27).
Example 25
Cell Chip Construction Methods
[0379] For cell chip construction, anodisc membranes, which are the
membranes in the ANOPORE inserts, were used. First, membranes were
placed on a glass slide and a 400 .mu.m thick stainless steel
platen with 150 .mu.m diameter pores was placed over the membrane.
The device was placed in a the chamber of a Cytospin slide
centrifuge and cells were added to the top of the platen. After a
twenty-four hour incubation the platen was washed three times with
cell culture medium. Although there were cells adhering to the
membrane, the cells were rounded and there were no firm attachments
or spreading as seen in previous experiments using the anopore
insert. With the inserts, the membrane was not placed on a solid
support, but was suspended within the platen, which allow the
membrane to be in contact with medium on both surfaces.
[0380] A double platen was constructed with a anodisc membrane in
between the platens as shown in FIG. 28. The bottom platen was
pre-wetted by placing a drop of medium over the platen and moving a
glass cover slip perpendicular to the platen over the surface to
push the liquid into the pores. This was repeated on the top platen
using a drop of cell suspension at 1.times.10.sup.6 cells/mL. A
pre-wetted membrane was then placed between the platens, and the
platens were adjusted under a microscope so that the pores of both
platens were brought into register or alignment. The device was
clamped and incubated overnight. Examination of the membrane after
washing showed that the cells had a characteristic adherent
morphology similar to that seen with the inserts (FIG. 28). Cells
were examined every 24 hours and proliferated until confluent (FIG.
28).
Example 26
Array Spotting
[0381] To assess the potential of the cell-chip to be used with an
array spotter for compound testing, single pin spotting was
characterized (FIG. 29). FIG. 30 shows microspotting on gold platen
using Hoechst dye. FIG. 36 shows microspotting of C12 resazurin to
determine cell viability. C12 resazurin is a detection agent used
to study cellular metabolism. The reduction product of
C12-resazurin is C12-resorufin, which exhibits enhanced cellular
retention and detection relative to the reduction product of
resazurin. Metabolically active cells reduce C12 resazurin to C12
resorufin which fluoresces red. Hoechst stain was included as a
counter stain. After briefly spotting on the platen, the cell-chip
was incubated for 15 minutes at 37.degree. C., 5% CO.sub.2. The
chip was examined using fluorescence microscopy. Several wells in
the area of the spot showed Hoechst staining and contained
metabolically active cells. This area was about 300 uM in diameter,
about 1.5 times the diameter of the pin (see FIG. 29). FIG. 36
shows an open array-based cell chip and delivery of C12-resazurin
to a single well on the array using a floating pin.
[0382] The technology for microarray spotting allows the generation
of high density microarrays by spotting cDNAs and/or
oligonucleotides on a solid chip surface. In this report, the chip
surface is modified providing for improved performance of an
ultra-high throughput cell-based screening assay. This novel chip
technology includes an ANODISC membrane selected for its ability to
support cell attachment and viability and a microwell stainless
steel platen.
[0383] Membranes with either 3 um or 0.2 .mu.m pore diameter were
tested for their ability to support PKCb-GFP cell attachment and
growth. An ANODISC 0.2 .mu.m membranes was covered with a confluent
layer of cells after an overnight incubation. In contrast, less
than 10% confluency was present when a polycarbonate membrane
having a 3 .mu.m pore size and treated with laminin, collagen,
fibronectin or untreated. These experiments suggest that membranes
having a smaller pore size have an enhanced ability to support cell
attachment, differences in membrane material cannot be excluded.
Alternatively, difference in the fabrication material can not be
excluded.
[0384] When cells were cultured with a cell-chip consisting of a
membrane on a glass slide covered by a stainless steel platen, the
cells appeared round and did not attach well. In contrast, cells
cultured on membranes in framed inserts (i.e., on a membrane
between two platens) that provided contact with culture medium on
both sides solved this problem. This configuration could can be
adapted for use in a variety of analystical or culture systems. In
one embodiment, a chip including cells on a membrane is placed over
another chip having a membrane designed for protein or RNA
attachment. Cells are lysed and the lysed contents is spotted onto
the second membrane by vacuum filtration for dot blot analysis.
[0385] These experiments described herein further demonstrated that
a compound could be spotted directly onto the cell-chip and
processed by metabolically active cells with minimal diffusion on
the platen. In order to decrease diffusion further and achieve high
density spotting, the platen surface may be treated with a
hydrophobic agent. Mineral oil and silicone coating dramatically
limited diffusion and created a small spot in studies carried out
using membranes having 50 .mu.m pore in a gold platen (FIG.
31).
[0386] Because of the small well size, this novel technology
provides for the culture and analysis of rare cell types and
further provides ultra high throughput single cell screening
(uHTS). Single cell uHTS may have a transformational effect on
antibody engineering as activated B cells may be tested without
fusion (variable regions amplified from positive cells).
[0387] The present invention overcomes limitations present in the
prior art. In particular, using the cell-chip configuration
described herein, living cells can be analyzed using fluorescence
markers for cell function. Further more, cultured cells may be
lysed and the contents transferred through a membrane for further
biochemical analysis, such as protein analysis or gene
expression.
Example 27
Hoechst Staining of Cells
[0388] Cells were incubated overnight on a stainless steel
cell-chip and then washed with medium. Hoechst was spotted on a
first platen that was dried by blotting. The stainless steel platen
(National Jet Company, LaVale, M.D.) tested was a 1-inch square,
400-.mu.m thick with pores 150 .mu.m in diameter. A second platen
was attached to the first platen using adhesive sealing film with
the center cut out. The chip was placed in a cytospin sample
chamber with a small gasket between the platen and the top of the
chamber. No funnel was used. The results of this experiment are
shown in FIG. 32. FIG. 33 shows an anopore membrane sandwiched
between two stainless steel platens. PKC.beta.-GFP cells were added
and incubated overnight.
Example 28
Tungsten Platens
[0389] A cell microarray prototype was constructed on a 200-.mu.m
thick Tungsten platen. The platens (National Jet Company, LaVale,
M.D.) had pores of 300 .mu.m in diameter and were attached with 4
screws. The prototype is shown in FIG. 34 . An Anopore membrane of
aluminum oxide was sandwiched between two tungsten platens (as
shown in FIG. 34). PKC.beta.-GFP cells were added and incubated for
48 hours (FIG. 35). In this experiment, random cell distribution
was observed and the cells were more securely attached to the
substrate. Metal platens having biocompatibility may be used in
such methods. Metals that are not biocompatible may be coated with
a biocompatible polymer (PEG) or metal.
Example 29
Confocal Images of Cultured Cells
[0390] FIG. 37 shows an open array-based cell chip with
PKC.beta.-GFP transfected cells, added at 5.times.10.sup.5/mL and
incubated 37.degree. C., 5% CO.sub.2. Images were acquired by
confocal microscopy. FIG. 38 shows essentially that which is
depicted in FIG. 37, except here the platen is not shown.
Example 30
Rigid Materials may be Attached to the Porous Membrane
[0391] A platen of rigid material, such as metal or polystyrene
having pores between 10-300 .mu.m in diameter is attached to a
porous membrane or modified glass surface to form an array of
microwells having a porous bottom (FIG. 39). Cells are added and
allowed to adhere to the membrane overnight. Subsequently, one or
more test compounds is added using a micro spotting pin. After
incubation, high content image analysis is performed. Membranes are
processed to analyse protein, mRNA expression, or to detect changes
in a biological function of interest. FIG. 40 shows a cross-section
of an individual well depicted in FIG. 39. The present invention
can be adapted as shown in FIG. 41. FIG. 41 depicts structural
enhancements to increase pressure on the member and/or gaskets to
seal off individual wells.
[0392] Cell chip assays were carried out using the following
methods and materials.
Cell Culture Media
[0393] DMEM supplemented with 10% FCS and 0.22 ug/mL hygromycin was
used to maintain PKCb-GFP cells. In some experiments, DMEM without
phenol red supplemented with 10% FCS and 100 IU/mL penicillin, 100
ug/mL streptomycin was used.
Cells
[0394] Human PKC.beta.II cDNA (GenBank Accession No.: X07109) was
isolated by PCR from reverse transcribed human spleen
marathon-ready cDNA (Clontech) and subcloned into the pCDNA3 vector
containing a hygromycin resistant gene (Invitrogen). The gene was
inserted downstream of sequences encoding a green Xuorescent
protein (ZsGFP) (Clontech). A stable cell line expressing
GFP-PKC_II was obtained by transfecting pcDNA3/GFP-PKC_II vector
into human embryonic kidney (HEK) 293 cells (QBI, Montreal, Quebec,
Canada) followed by selection with 600 .mu.g/ml hygromycin B. Drug
resistant colonies were picked and screened for PKCBII protein
expression by immunoblotting using an anti-PKCBII antibody (Santa
Cruz Biotechnology, Santa Cruz, Calif.). The expression of GFP was
tested by Xuorescence microscopy. Positive clones were maintained
in the growth medium containing 300 .mu.g/ml of hygromycin B.
HEK293 cells transfected with GFP-tagged PKC-.beta.II protein were
maintained in DMEM containing 10% FBS, glutamine, antibiotics, and
hygromycin B at 37.degree. C. See, Ilyin et al., Methods 37:
280-288, 2005, which is hereby incorporated by reference.
[0395] Cell Culture on Membranes
[0396] Cell culture inserts from BD-Biocoat membranes were obtained
in 3 uM pores size (BD Biosciences, San Jose, Calif.). The inserts
were pre-coated with lamalin, collagen or fibronectin. Anopore
inserts having a 0.2 uM pore size (Nunc) membranes were also
tested. Inserts were placed in a 24-well culture plate. Cell
culture medium was added until the level reached the membrane.
Cells were added to the inserts in 200 uL of medium. Plates were
incubated overnight at 37.degree. C., 5% CO.sub.2. In subsequent
experiments using membranes without framed inserts, anodisc
membranes having 0.2 or 0.1 .mu.m (Whatman, Clifton, N.J.) pores
were used.
Platens
[0397] Stainless steel shims 400 .mu.m thick were obtained and sent
to National Jet Company (LaVale, M.D.) for manufacture of a
10.times.10 platen of 150 uM diameter holes spaced 50 .mu.m apart
using micro electro-discharge machining. Each shim was cut to about
1 inch square with the platen in the center.
Spotting Pins
[0398] An FP9 0.229 mm diameter floating tube pin with a volume
delivery range of 5-15 nL (V&P Scientific, Inc., San Diego,
Calif.) was used in all experiments. Spotting solutions were made
in an eppendorf tube. The spotting pin was dipped in solution and
then spotted on the platen by briefly touching perpendicular to the
platen.
[0399] Cell Viability Assay
[0400] C12-Resazurin (molecular probes) was diluted in DMEM 10% FCS
w/o phenol red. Hoechst 33258 (molecular probes) was included to
control for spotting efficiency. Solution was spotted onto platen
on top of cells as described above. The cell chamber was incubated
at 37.degree. C., 5% CO.sub.2 for 15 minutes. After incubation, the
chamber was examined by fluorescence microscopy for Hoechst DNA
staining. This area was then analyzed using confocal microscopy for
conversion of C12-Resazurin to red-fluorescent resorufin by viable
cells (Abs/Em 563/587).
Other Embodiments
[0401] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0402] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof. All patents and publications
mentioned in this specification are herein incorporated by
reference to the same extent as if each independent patent and
publication was specifically and individually indicated to be
incorporated by reference.
* * * * *
References