U.S. patent application number 10/738465 was filed with the patent office on 2005-06-23 for sieving media from planar arrays of nanoscale grooves, method of making and method of using the same.
This patent application is currently assigned to INTEL CORPORATION. Invention is credited to Sibbett, Scott.
Application Number | 20050133437 10/738465 |
Document ID | / |
Family ID | 34677392 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050133437 |
Kind Code |
A1 |
Sibbett, Scott |
June 23, 2005 |
Sieving media from planar arrays of nanoscale grooves, method of
making and method of using the same
Abstract
Disclosed herein are an apparatus and a method for separating
molecules on the basis of size and or structure, and to a method of
making the apparatus. Generally, the separation method includes
passing a fluid comprising particles having different effective
molecular diameters through a plurality of open, nanoscale channels
disposed in surfaces of substrates. The method also includes
obtaining a plurality of fractions of the passed fluid such that
each of the fractions includes a major portion containing particles
having similar size and shape and substantially free of particles
having larger size and shape. The apparatus includes first and
second substrates each of which has a surface containing a
plurality of open, nanoscale channels disposed therein. The
surfaces are bonded together such that each of the channels of the
first substrate is in fluid communication with at least two of the
channels of the second substrate and is misaligned relative to the
channels of the second substrate. Interferometric lithography and
anodic bonding or flip-chip bonding techniques can be used to make
the apparatus.
Inventors: |
Sibbett, Scott; (Corrales,
NM) |
Correspondence
Address: |
Julia A Hodge
BLAKELY SOKOLOFF TAYLOR & ZAFMAN LLP
12400 Wilshire Boulevard
Seventh Floor
Los Angeles
CA
90025
US
|
Assignee: |
INTEL CORPORATION
2200 Mission College Blvd.
Santa Clara
CA
95052
|
Family ID: |
34677392 |
Appl. No.: |
10/738465 |
Filed: |
December 17, 2003 |
Current U.S.
Class: |
210/321.84 ;
210/500.22; 210/500.26; 210/650 |
Current CPC
Class: |
B01D 67/0062 20130101;
G01N 27/44756 20130101; G01N 30/0005 20130101; Y10S 977/701
20130101; Y10T 137/87249 20150401; Y10S 977/723 20130101; G01N
30/0005 20130101; G01N 30/6095 20130101; Y10S 977/72 20130101 |
Class at
Publication: |
210/321.84 ;
210/500.26; 210/650; 210/500.22 |
International
Class: |
B01D 063/00 |
Claims
What is claimed is:
1. An apparatus comprising first and second substrates, each of the
substrates having a surface containing a plurality of open,
nanoscale channels disposed therein, the surfaces bonded together
such that each of the channels of the first substrate is in fluid
communication with at least two of the channels of the second
substrate and is misaligned relative to the channels of the second
substrate.
2. The apparatus of claim 1, wherein the channels have equivalent
and constant cross-sectional diameters within a range of about 1
square nanometers (nm.sup.2) to about 10,000 nm.sup.2.
3. The apparatus of claim 1, wherein the channels have equivalent
and variable cross-sectional diameters within a range of about 1
nm.sup.2 to about 10,000 nm.sup.2.
4. The apparatus of claim 1, wherein each of said surfaces has at
least about 1000 channels to about ten million channels disposed
therein.
5. The apparatus of claim 1, wherein each of the channels traverses
an entire length of the surface.
6. The apparatus of claim 1, wherein the channels of the first
substrate are parallel to each other, and the channels of the
second substrate are parallel to each other.
7. The apparatus of claim 1, wherein the channels of the first
substrate are spaced equidistant from each other, and the channels
of the second substrate are spaced equidistant from each other.
8. The apparatus of claim 1, wherein the first and second
substrates comprise one or more materials selected from the group
consisting of quartz, silica, silicon, porous silicon, polysilicon,
and porous polysilicon.
9. The apparatus of claim 8, wherein the first and second
substrates comprise quartz.
10. The apparatus of claim 1, further comprising third and fourth
substrates bonded to edge surfaces of each of the first and second
substrates, the edge surfaces being substantially perpendicular to
the channels.
11. The apparatus of claim 10, wherein the third and fourth
substrates comprise one or more materials selected from the group
consisting of quartz, silica, silicon, porous silicon, polysilicon,
porous polysilicon, and silicon oxynitride.
12. The apparatus of claim 11, wherein the third and fourth
substrates comprise silicon oxynitride.
13. The apparatus of claim 1, wherein the channels of the first
substrate are misaligned relative to the channels of the second
substrate by an angle of about 0.05.degree. to about 45.degree.,
the angle defined by an intersection of a channel of the first
substrate and a channel of the second substrate.
14. A method comprising: (a) passing a fluid comprising particles
having different effective molecular diameters through a plurality
of open, nanoscale channels disposed in surfaces of substrates, the
substrates bonded together such that each of the channels of a
first substrate is in fluid communication with at least two of the
channels of a second substrate and is misaligned relative to the
channels of the second substrate; (b) obtaining a plurality of
fractions of the passed fluid, each of the fractions comprising a
major portion comprising particles having similar size and shape
and substantially free of particles having larger size and
shape.
15. The method of claim 14, wherein the channels have equivalent
and constant cross-sectional diameters within a range of about 1
square nanometers (nm.sup.2) to about 10,000 nm.sup.2.
16. The method of claim 14, wherein the channels have equivalent
and variable cross-sectional diameters within a range of about 1
nm.sup.2 to about 10,000 nm.sup.2.
17. The method of claim 14, wherein each of said surfaces has at
least about 1000 channels to about ten million channels disposed
therein.
18. The method of claim 14, wherein each of the channels traverses
an entire length of the surface.
19. The method of claim 14, wherein the channels of the first
substrate are parallel to each other, and the channels of the
second substrate are parallel to each other.
20. The method of claim 14, wherein the channels of the first
substrate are spaced equidistant from each other, and the channels
of the second substrate are spaced equidistant from each other.
21. The method of claim 14, wherein the first and second substrates
comprise one or more materials selected from the group consisting
of quartz, silica, silicon, porous silicon, polysilicon, and porous
polysilicon.
22. The method of claim 21, wherein the first and second substrates
comprise quartz.
23. The method of claim 14, further comprising third and fourth
substrates bonded to edge surfaces of each of the first and second
substrates, the edge surfaces being substantially perpendicular to
the channels.
24. The method of claim 23, wherein the third and fourth substrates
comprise one or more materials selected from the group consisting
of quartz, silica, silicon, porous silicon, polysilicon, porous
polysilicon, and silicon oxynitride.
25. The method of claim 24, wherein the third and fourth substrates
comprise silicon oxynitride.
26. The method of claim 14, wherein the channels of the first
substrate are misaligned relative to the channels of the second
substrate by an angle of about 0.05.degree. to about 45.degree.,
the angle defined by an intersection of a channel of the first
substrate and a channel of the second substrate.
27. A method comprising: (a) patterning an array of open, nanoscale
channels on a major planar surface of each of a first substrate and
a second substrate; (b) bonding the channeled surfaces together
such that each of the channels of the first substrate is in fluid
communication with at least two of the channels of the second
substrate, and such that the each of the channels of the first
substrate is misaligned relative to the channels of the second
substrate; and, (c) bonding one or more cap substrates to one more
edge surfaces of each of the bonded first and second substrates,
the edge surfaces being substantially perpendicular to the
channels.
28. The method of claim 27, wherein said patterning comprises a
lithography method selected from the group consisting of
interferometric lithography, immersion interferometric lithography,
electron beam lithography, scanning probe lithography, nanoimprint,
extreme ultraviolet lithography, and X-ray lithography.
29. The method of claim 28, wherein said patterning comprises
interferometric lithography.
30. The method of claim 27, wherein the channeled surfaces are
bonded together by flip-chip bonding.
31. The method of claim 27, wherein the channeled surfaces are
bonded together by anodic bonding.
Description
BACKGROUND OF THE DISCLOSURE
[0001] 1. Field of the Invention
[0002] The invention relates generally to an apparatus and method
for separating molecules on the basis of size and/or structure, and
to a method of making the apparatus.
[0003] 2. Brief Description of Related Technology
[0004] It is important in the chemical and biological sciences to
be able to separate different molecules from one another. Accurate
and precise separation is especially important where the molecules
are present in only a small volume solution, such as, for example,
in the context of analytical and diagnostic testing. There remains
a need to improve the efficiencies of such separations and,
thereby, the convenience to researchers working in the chemical and
biological sciences.
[0005] Generally, molecular separation techniques can include the
use of a matrix (or membrane) where molecular transport and
filtration occur perpendicular to the surface of the matrix. In
such techniques, only those molecules having a precise,
pre-determined molecular weight and/or structure pass through the
matrix. These separation techniques, however, are limited. For
example, biomolecules may not be amenable to separation by such
techniques because, for example, they may undesirably react with,
or be rendered inactive by, the separating matrix. Even where
biomolecules are amenable to these techniques, the separation can
be imprecise, inaccurate, and/or difficult to reproduce due to
batch-to-batch variations in the manufacture of the matrices. Poor
separation efficiency and/or loss of sample volume also can be
encountered.
[0006] In the biological sciences, gel fractionation or
electrophoresis has been found to be a useful technique to separate
and identify biomolecules such as, for example, proteins.
Generally, in gel electrophoresis, the gel consists of a matrix of
entangled polymer chains; intermixed with a buffer solution. A
large number of interconnected pores are present within the matrix.
A solution of proteins having a net electrical charge are placed in
the matrix and travel through the pores under the influence of an
electric field. Typically, a charged protein will move towards the
pole with a charge opposite to that carried on the protein . The
free-solution mobilities of denatured proteins are identical. In
the presence of the gel matrix, however, protein mobilities tend to
differ because the larger the protein, the more likely it will
encounter a physical restriction in the matrix (either between or
within the pores), thus retarding the protein's progress through
the matrix relative to smaller proteins. The frictional force of
the gel material acts as a protein sieve (or, more generally, a
molecular sieve) separating the proteins by size. The rate at which
a protein migrates through the electric field and gel matrix
depends upon, for example, the strength of the field, size and
shape of the protein, relative hydrophobicity of the sample in
which the protein is present, and on the ionic strength and
temperature of the buffer in which the protein is moving. Thus, as
smaller proteins should move through the matrix faster than large
proteins, the proteins become separated with fast moving bands of
small proteins at the front and slow moving bands of larger
proteins trailing behind.
[0007] One particular type of gel fractionation is two-dimensional
(2-D) gel fractionation, which is useful for separating and
identifying proteins in a sample by displacement in two dimensions
oriented at right angles to one another. Two-dimensional gel
fractionation is generally used as a component of proteomics and is
a common step used to isolate proteins for further characterization
by, for example, mass spectroscopy. This fractionation technique
permits component proteins of the sample to separate over a larger
area, increasing the resolution of each component protein. IEF
(isoelectric focusing) and SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) comprise the two dimensions in
a 2-D gel fractionation. In a first dimension, IEF fractionates
biomolecules on the basis of pl values. In the subsequent, second
dimension, SDS-PAGE further fractionates the
previously-fractionated biomolecules based on size-charge ratios,
which roughly correspond to a fractionation based on molecular
weights.
[0008] Despite its widespread use, however, 2-D gel fractionation
has its limitations. For example, it is not particularly good at
resolving proteins or peptides having a low molecular mass as these
often migrate through a polyacrylamide gel too rapidly. 2-D gel
fractionation also is unsuitable for many proteins, such as
hydrophobic proteins, because the proteins often interact with the
gel matrix or otherwise undesirably react rendering subsequent
analysis of the proteins difficult or impossible. Even when and
where the proteins do not undesirably interact or react, it is
often difficult to remove them from the gel, thus compromising the
quality of any subsequent analysis of the protein. Another
particular limitation is that the fractionation takes a long time
to perform and requires extensive manual handling and attention,
which makes it a long and laborious process requiring a skilled
technician or scientist to master and perform. Performing the
fractionation is very much an art, requiring much experimentation
to find the correct conditions for sample preparation, focusing
times, etc. Moreover, it is often difficult to reproduce the exact
processing conditions under which multiple gels are made and,
therefore, there can be inconsistencies between the various gels.
Furthermore, gels can provide only limited resolution, which is
often inadequate for certain molecular separation and analytical
operations, and are often not re-usable. Still further, the gel
material can disadvantageously degrade--polyacrylamide gel is a
neurotoxin having a short shelf-life requiring that it be prepared
just prior to use, and having properties that vary from batch to
batch. Additionally, and given the foregoing limitations, the
technique is often inadequate and/or wholly inappropriate for use
in an integrated separation and analysis system. Though there have
been advances to improve on certain of the foregoing limitations,
many of the limitations still remain.
[0009] Alternatives to 2-D gel fractionation include techniques
that utilize artificial gel media. In contrast to polyacrylamide
gels where the sieving matrix is defined by random arrangement of
long-chain polymers, the sieving matrix in artificial gels is
defined by microfabrication and/or nanofabrication. Thus, the
dimensions and topology of the sieving matrix in an artificial gel
can be controlled and measured more precisely, and can be
mass-produced more easily. For example, conventional
photoresist-based lithography can be used to etch a pattern of
obstacles on a silicon substrate (floor), which can be sealed with
a glass or elastomeric ceiling layer to form a sieve through which
a solution of molecules can be electrophoresed. Similarly,
monolithic structures can be prepared with a sacrificial layer
sandwiched between a dielectric floor and ceiling layers to define
a working gap, wherein the sacrificial layer represents what will
be the open space in the finished structure and, thus, the negative
of the desired pattern of obstacles is etched into it. After the
floor, ceiling, and retarding obstacles have been put in place, the
sacrificial layer is removed by a wet chemical etch, leaving a
working gap whose vertical dimensions are defined by the thickness
of the removed layer. Due presumably to critical dimension
limitations, however, only nucleic acid separations have been
reported with these structures. Protein albumin is about four
nanometers (nm) wide and about fifteen nanometers in length and,
therefore, is too small to interact physically with patterned
structures having 100 nm diameter pillars on a 200 nm pitch. Other
techniques contemplate the use of electrochromatography, in situ
casting of sieving media within preformed channels of a substrate,
and the use of porous materials such as porous silicon as a porous
media. Notwithstanding these advances, there remain limitations not
adequately addressed in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] For a more complete understanding of the disclosure,
reference should be made to the following detailed description and
accompanying drawings wherein:
[0011] FIG. 1 is an enlarged, top view of a surface of a substrate
having a plurality of nanoscale channels disposed therein;
[0012] FIG. 2 is an enlarged, cross-sectional view of the substrate
taken along line 2-2 in FIG. 1;
[0013] FIG. 3 is an enlarged, exploded view of a portion of an
apparatus showing its constituent parts;
[0014] FIG. 4 is an enlarged, fragmentary plan view of the
apparatus with the nanoscale channels disposed in each substrate
shown in phantom; and,
[0015] FIG. 5 is an enlarged, cut-away view of the apparatus
showing the path of a material traversing the nanoscale
channels.
[0016] While the disclosed apparatus and methods are susceptible of
embodiments in various forms, there are illustrated in the drawings
(and will hereafter be described) specific embodiments of the
invention, with the understanding that the disclosure is intended
to be illustrative, and is not intended to limit the invention to
the specific embodiments described and illustrated herein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] As used herein the term "nanoscale channel" refers to any
void space in a surface of a substrate having a diameter in at
least one direction of about one to about 500 nm. When referring to
the channel, the term "diameter" is used in its ordinary sense,
i.e., the distance across and through the middle of the channel,
perpendicular to the axis of the channel, and parallel to the plane
of the substrate in which the channel is disposed. When referring
to a channel(s), however, the term "diameter" is not intended to
limit the cross-sectional shape of the channel(s) to a circle, as
any channel shape can be employed. Thus, the term "diameter" as
used herein also includes "equivalent diameter" as defined in Table
5-8 of "Perry's Chemical Engineers' Handbook," at p. 5-25 (6.sup.th
Ed., 1984) (see also 7.sup.th Ed., 1997, at pp. 6-12 to 6-13). As
used herein, the term "array" refers to any arrangement of
nanoscale structures or channels.
[0018] Disclosed herein is an apparatus comprising first and second
substrates, each of the substrates having a surface containing a
plurality of open, nanoscale channels disposed therein. The
surfaces are bonded together such that each of the channels of the
first substrate is in fluid communication with at least two of the
channels of the second substrate and is misaligned relative to the
channels of the second substrate.
[0019] The channels can have equivalent and constant
cross-sectional diameters within a range of about one square
nanometer (nm.sup.2) to about 10,000 nm.sup.2, and more preferably,
about 10 nm.sup.2 to about 1000 nm.sup.2. Alternatively, the
channels can have equivalent and variable cross-sectional diameters
within a range of about 1 nm.sup.2 to about 10,000 nm.sup.2, and
more preferably, about 10 nm.sup.2 to about 1000 nm.sup.2. Thus, in
such an embodiment, a first portion of the channel can have a
cross-section diameter of about 10,000 nm.sup.2, for example, while
a second portion of the channel can have a cross-sectional diameter
of about 1 nm.sup.2, for example. Benefits of such variable
cross-sectional diameters within the same channel can be realized
when resolution of various, different-sized particles is desired.
The channels within each substrate should be parallel to each other
and should traverse an entire length of the surface in which they
are disposed. Preferably, the channels within each substrate are
spaced equidistant from each other, though they need not be. Given
the cross-sectional diameters of the channels and the intended use
of the apparatus, each of the surfaces in which the channels are
disposed preferably contains at least about 1000 channels to about
ten million channels.
[0020] Generally, the substrates can be constructed of any material
that is amenable to patterning of nanoscale channels and capable of
being bonded together. Preferably, however, the first and second
substrates can be made from one or more materials selected from the
group consisting of quartz, silica, silicon, porous silicon,
polysilicon, and porous polysilicon. More preferably, one of the
materials of construction is quartz. Suitable silicon materials
include porous silicon.
[0021] The apparatus preferably also includes third and fourth
substrates bonded to edge surfaces of each of the first and second
substrates. The edge surfaces should be substantially perpendicular
to the channels.
[0022] Preferably, the third and fourth substrates can be made from
one or more materials selected from the group consisting of quartz,
silica, silicon, porous silicon, polysilicon, porous polysilicon,
and silicon oxynitride. More preferably, one of the materials of
construction is silicon oxynitride.
[0023] As previously noted, each of the channels of the first
substrate is misaligned relative to the channels of the second
substrate. The misalignment can be defined by an angle, which
itself is defined by an intersection of a channel of the first
substrate and a channel of the second substrate. Preferably, the
channels of the first substrate are misaligned relative to the
channels of the second substrate by an angle of about 0.05.degree.
to about 45.degree., more preferably about 0.05.degree. to about
15.degree., highly preferably, about 0.1.degree. to about
10.degree., and even more highly preferably about 0.5.degree. to
about 5.degree..
[0024] Generally, the apparatus can be made by patterning an array
of open, nanoscale channels on a major planar surface of each of a
first substrate and a second substrate. The channeled surfaces can
be bonded together such that each of the channels of the first
substrate is in fluid communication with at least two of the
channels of the second substrate, and such that the each of the
channels of the first substrate is misaligned relative to the
channels of the second substrate. A preferred method of bonding
includes suitable flip-chip bonding methods. Edge surfaces of each
of the bonded first and second substrates can be capped with one or
more cap substrates bonded to each edge surface, wherein the edge
surfaces are substantially perpendicular to the channels.
[0025] There are numerous suitable methods of patterning an array
of open, nanoscale channels on a surface of a substrate. Examples
of such suitable methods include lithography methods such as, for
example, interferometric lithography ("IL"), immersion
interferometric lithography, electron beam lithography, scanning
probe lithography, nanoimprint, extreme ultraviolet lithography,
and X-ray lithography. Generally, IL is a preferred method of
patterning the nanoscale channels.
[0026] Generally, lithography is a highly-specialized printing
process used to create detailed patterns on a substrate, such as a
silicon wafer. An image containing a desired pattern is projected
onto the wafer, which is coated by a thin layer of photosensitive
material called "resist". The bright parts of the image pattern
cause chemical reactions which, in turn, render the resist material
soluble, and, thus, dissolve away in a developer liquid, whereas
the dark portions of the image remain insoluble. After development,
the resist forms a stenciled pattern across the wafer surface,
which accurately matches the desired pattern. Finally, the pattern
is permanently transferred into the wafer surface, for example by a
chemical etchant, which etches those parts of the surface
unprotected by the resist.
[0027] Interferometric lithography ("IL") generally refers to a
process of lithography where two or more mutually coherent light
waves (or beams) interfere to produce a standing wave, which can be
recorded in a photoresist. More specifically, in IL, a sinusoidal
standing wave pattern of light intensity is produced by
interference at the region of intersection of two coherent light
beams. A photoresist-coated substrate positioned at the point of
intersection undergoes exposure, printing a periodic, line-space
pattern whose period (P) is determined by the wavelength of light
(.lambda.) and the angle of intersection (A) of the light beams
(i.e., P=.lambda./2 sin(A)). The angle (A) should be sufficiently
large to produce an interference pattern that has a high spatial
frequency. The resulting interference pattern should have nanoscale
dimensions.
[0028] Examples of suitable IL techniques that can be used to
pattern the array of channels are described in, for example, Brueck
et al. U.S. Pat. No. 5,705,321. Generally, the photoresist-coated
substrate is prepared by depositing a thin, etch-mask layer on a
silicon substrate (wafer); then depositing a thin, photoresist
layer on top of the etch-mask layer. Thereafter, the photoresist
layer is exposed to the periodic pattern of lines using fine-line
IL optimized to yield the appropriate nanoscale dimension of
unexposed photoresist. The photoresist is developed to remove the
exposed photoresist. The photoresist pattern is next transferred
into the etch-mask using an etching process (over-etching the
etch-mask at this point can undesirably undercut the etch mask and
further narrow the etch mask pattern). The remaining photoresist is
then removed. A highly anisotropic etching process, such as with
potassium hydroxide, for example, can be used to etch the exposed
substrate (e.g., a silicon (Si) substrate), in which case the lines
of the periodic pattern should be aligned with the {111} Si
directions prior to photoresist exposure. In this process, the
{111} Si surfaces are almost totally unetched and, thereby leave
very narrow, quantum-sized Si walls with a very high aspect ratio.
If reactive-ion or ion-milling etch processes are used instead of
postassium hydroxide, then it is not necessary to pre-align the
pattern with the {111} Si directions. The remaining etch mask then
can be removed, leaving an all Si surface, which can be oxcized.
The same basic method can be used to fabricate more complex
structures by the use of multiple-exposure IL and/or combining IL
with conventional optical lithography either during the photoresist
exposure step or within multiple iterations of portions of the
process.
[0029] The complex interference pattern produced on the photoresist
layer or layers can be varied by rotating and/or translating the
substrate, changing the angle (A), varying the number of exposures
and/or the optical intensity, using a phase-amplitude mask in one
or both illuminating beams of coherent radiation, and any
combination of the foregoing. Further flexibility can be attained
by a combination of any of the foregoing variations along with
suitable optical imaging lithography techniques.
[0030] Though there are many suitable methods of patterning the
nanoscale channels, IL represents one of the more convenient and
preferred methods of patterning nanostructured features because it
can be used to generate the entire pattern in one, parallel step
and is not a serial writing technique. Other parallel techniques
(e.g., imprint lithography) rely upon a primary patterning
technique to generate a master that subsequently can be used to
produce replicas of nanostructured features in a parallel fashion.
The use of IL to pattern an array of nanoscale channels has
additional advantages over other techniques (such as traditional
acrylamide gel polymerization) since it is capable of creating
highly-ordered structures, provides the possibility of creating
macroscopic arrays of continually varying size or chemistry across
one dimension, is highly reproducible, can be carried out rapidly
over larger macroscopic areas at low cost (low relative to
electron-beam lithography, for example), and can be more easily
implemented in the creation of complex, integrated separation
systems that are disposable or reusable. Furthermore, the use of
lithographically-defined-separation matrices lends itself to simple
implementation of these matrices into multi-level, 3-dimensional
separation devices in which different screening mechanisms allow
enhanced separations. Additionally, IL can be used to easily
generate arrays of nanostructures (protrusions or channels) whose
dimensions vary semi-continuously in the plane of surface of the
material being patterned. Once the surface of the substrate has
been patterned with the desired pattern of nanoscale channels, the
patterned surfaces is bonded with another similarly patterned
surface. Thus, the formed apparatus aims to eliminate some of the
current limitations by the fabrication of highly-uniform and
accurately-reproducible nanoscale separation systems prepared by
nano- and microlithography.
[0031] A purpose in bonding the substrates together is to create
intimate, physical contact between the surfaces of the substrates
such that the solution and any particles therein that are
traversing the channels remain confined to the void space defined
by the channels. Suitable methods of bonding the patterned (or
channeled) surfaces together include, but are not limited to,
anodic bonding methods and flip-chip bonding methods capable of
mating the surfaces such that each of the channels of the first
substrate is in fluid communication with at least two of the
channels of the second substrate, and such that the each of the
channels of the first substrate is misaligned relative to the
channels of the second substrate. Both anodic bonding and flip-chip
bonding methods are known by those skilled in the art. Generally,
flip-chip bonding (also known in the art as direct chip attach
(DCA)) is a direct electrical connection of face-down ("flipped")
electronic components onto substrates, circuit boards, or carriers,
by mean of conductive bumps on chip bond-pads. In contrast, wire
bonding uses face-up chips with a wire connection to each chip
bond-pad.
[0032] Flip-chip bonding typically comprises bumping a first
substrate (or wafer), attaching the bumped substrate to a second
substrate, and filling any remaining void space between surfaces of
the substrate with a filler material, such as an
electrically-non-conductive material, keeping in mind that the
filler material should not fill the channels of the substrate. In
the electronics arts, the bump can serve many purposes, such as
providing a path for transferring an electric charge or heat from
one substrate to another. Here, however, the bump advantageously
provides a mechanical mounting to assist in attaching one substrate
to another. The bump can be prepared by a variety of methods
including, for example, those using solder or stud-bumping
techniques, vacuum deposition, electroplating, and adhesives.
Flip-chip bonding is advantageous because if offers a high-speed,
low-cost assembly method and results in a suitably rugged bond.
Given the flip-chip bonding processing conditions, and the intended
use of the formed apparatus, one skilled in the art can
appropriately select the materials of construction for use in
bonding the various substrates together.
[0033] As noted herein, cap substrates can be bonded to edge
surfaces of the already-bonded first and second substrates to
prevent the sample solution from entering or exiting the exposed
edges of the apparatus in a direction parallel to the nanoscale
channels, and to constrain the solution into and out of the
apparatus in a direction roughly perpendicular to the direction of
the channels. Generally, the cap surface is constructed of silicon
oxynitride. The cap surface can be bonded with the aid of an
adhesive suitable for attaching a silicon oxynitride surface, for
example, to the edge surfaces of the already-bonded first and
second substrates.
[0034] Once the cap substrates have been appropriately bonded to
the edge surfaces, the apparatus can be suitably attached to or
otherwise incorporated into a device capable of introducing an
electric field and a solution of molecules.
[0035] The end result of these manufacturing steps is an apparatus
whose nanoscale channels are freely accessible to solution added
along the uncapped edges of the 2-chip stack. Hence, conventional
electrophoresis and other forms of chromatography can be performed
within the nanochannels. The apparatus can be used by filling the
interior space (e.g., via capillary action) with a solution
containing the molecules to be separated, and then applying an
electric field along a direction roughly perpendicular to the
channels. As previously noted, a charged molecule will migrate with
(or against) the general direction of this applied field. The
actual path of any given charged molecule will be quite torturous,
as explained hereinafter.
[0036] Referring now to the drawing figures, wherein like reference
numbers refer to the identical or similar elements in the various
figures, FIG. 1 is an enlarged, top view of a surface of a
substrate having a plurality of nanoscale channels disposed
therein. More specifically, FIG. 1 shows a substrate 10 having a
surface 12 on or within which are disposed nanoscale channels 14.
Edge surfaces (not shown) of the substrate 10 are capped with cap
substrates 16 and 18. As shown in FIG. 1, each of the channels 14
has a constant cross-sectional diameter and each appears to be
spaced equidistant from one another. as previously noted, the
channels need not have a constant cross-sectional diameter or be
spaced equidistant from one another. FIG. 2 is an enlarged,
cross-sectional view of the substrate 10 taken along line 2-2 in
FIG. 1.
[0037] FIG. 3 is an enlarged, exploded view of a portion of an
apparatus 30 showing its constituent parts. As shown, the apparatus
30 includes the substrate 10 and a second substrate 20 having a
surface 22 on or within which are disposed nanoscale channels 24.
Edge surfaces 26 and 28 of the substrates 10 and 20, respectively,
are capped with cap substrates 16 and 18. When the substrates 10
and 20 are mated, the apparatus 30 is formed as edges X-X' and Z-Z'
meet and cap substrates 16 and 18 are bonded to the edge surfaces
26 and 28.
[0038] FIG. 4 is an enlarged, fragmentary plan view of a portion of
the formed apparatus 30 with the nanoscale channels 14 and 24
disposed in each substrate shown in phantom. As shown, each of the
channels 14 of the first substrate 10 is misaligned relative to
each channel 24 of the second substrate 20. The misalignment is
defined in FIG. 4 by an angle (.alpha.), which itself is defined by
the intersection of a channel 14 of the first substrate 10 and a
channel 24 of the second substrate 20.
[0039] FIG. 5 is an enlarged, cut-away view of the apparatus
showing the path of a material traversing the nanoscale channels 14
and 24, as depicted by the arrows. More specifically, shown in FIG.
5 is the apparatus 30 comprising the mated substrates 10 and 20,
and the nanoscale channels 14 and 24 disposed therein. With the
application of a force, such as a pressure or an electric field,
molecules within a solution (depicted by the arrows in FIG. 5) can
traverse the tortuous path created by the nanoscale channels 14 and
24. Moreover, the molecules are constantly zig-zagging through an
interior space comprised almost entirely of wedge-shaped cracks.
These cracks are the molecular-scale physical constrictions that
impart a sieving capability to the 2-chip stack. The speed at which
a particular molecule traverses from one end of the apparatus 30 to
the other will, of course, depend upon the molecular weight and
structure, as described above.
[0040] The formed apparatus is useful to resolve the various
molecules present in a solution. Higher resolution can be obtained
where the apparatus is used in combination with any one or more of
the following mechanisms: affinity interaction (molecular
recognition), asymmetric diffusion, electrophoretic mobility,
entropic trapping, hydrophobic interaction, isoelectric point, and
size exclusion.
[0041] The disclosed apparatus is useful to separate particles
within a fluid having different effective molecular diameters into
discrete portions characterized by common effective molecular
diameter. Such particles are separated on the basis of the ability
of particles having a smaller effective molecular diameter to pass
through the apparatus channels more quickly than those having
larger effective molecular diameters. Where particles have
substantially equivalent molecular diameters, those molecules that
are shorter in length should pass through the apparatus more
quickly than those molecules that are longer in length. Higher
resolution can be obtained where the apparatus is used in
combination with any one or more of the following mechanisms:
affinity interaction (molecular recognition), asymmetric diffusion,
electrophoretic mobility, entropic trapping, hydrophobic
interaction, isoelectric point, and size exclusion.
[0042] Suitable fluids that can pass through the apparatus include
biologically derived materials such as, for example, peptides,
polypeptides, proteins, antigens, antibodies, nucleotides,
oligonucleotides, polynucleotides, aptamers, DNA, RNA,
carbohydrates, complexes thereof, and suitable buffers. Fluids also
can include non-biologically-derived materials such as, for
example, synthetic polymers.
[0043] A buffer is a defined solution that resists change in pH
when a small amount of an acid of base is added or when the
solution is diluted. For example, the pH of the blood in a healthy
individual remains remarkably constant at 7.35 to 7.45 because the
blood contains a number of buffers that protect against pH change
due to the presence of acidic or basic metabolities. From a
physiological viewpoint, a change of +0.3 or -0.3 pH unit can be
considered to be extreme. Many biological reactions of interest
occur in the pH range of 6 to 8. Specific enzyme reactions that
might be used for analyses may occur in the pH range of 4 to 19 or
even greater. Thus, buffers are very useful for maintaining the pH
at an optimum value. The proper selection of buffers for the study
of biological reactions or for use in clinical analyses can be
critical in determining whether of not they influence the
reaction.
[0044] Proteins are amphoteric compounds; their net charge
therefore is determined by the pH of the medium in which they are
suspended. In a solution having a pH above the protein's
isoelectric point, a protein has a net negative charge and migrates
towards the anode in an electrical field. Below its isoelectric
point, the protein is positively charged and migrates towards the
cathode. The net charge carried by a protein is independent of its
size, meaning that the charge carried per unit mass (or length,
given proteins and nucleic acids are linear macromolecules) of
molecule differs from protein to protein. Thus, at a given pH and
under non-denaturing conditions, the electrophoretic separation of
proteins is determined by both size and charge of the molecules. In
contrast to proteins, nucleic acids remain negative at any pH used
for electrophoresis and carry a fixed negative charge per unit
length of molecule, provided by the phosphate group of each
nucleotide of the nucleic acid. Thus, electrophoretic separation of
nucleic acids proceeds strictly according to size.
[0045] Sodium dodecyl sulphate (SDS) is an anionic detergent that
is used to denature proteins by "wrapping around" the polypeptide
backbone of proteins--and SDS binds to proteins fairly specifically
in a mass ratio of about 1.4:1. In so doing, SDS confers a negative
charge to the polypeptide in proportion to its length (the
denatured polypeptides become "rods" of negative charge cloud with
equal charge or charge densities per unit length). It is usually
necessary to reduce disulphide bridges in proteins before they
adopt the random-coil configuration necessary for separation by
size, such as, for example, with 2-mercaptoethanol or
dithiothreitol. In denaturing SDS-based separations, therefore,
migration is determined not by intrinsic electrical charge of the
polypeptide, but by molecular weight.
[0046] Thus, if a mixture of SDS-complexed proteins in a suitable
buffer is electrophoresed through the 2-chip stack, the larger the
protein, the more likely it will encounter a restriction, and hence
be retarded relative to a smaller protein. Proteins will elute from
the chip in the order of size, the smallest first, and the largest
last. The separated proteins can be further analyzed as
desired.
[0047] The foregoing description is given for clearness of
understanding only, and no unnecessary limitations should be
understood therefrom, as modifications within the scope of the
disclosure may be apparent to those having ordinary skill in the
art.
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