U.S. patent application number 12/173515 was filed with the patent office on 2009-02-19 for microdevices for high-throughput screening of biomolecules.
This patent application is currently assigned to Zyomyx, Inc.. Invention is credited to Dana Ault-Riche, Christian Itin, Steffen Nock, Peter Wagner.
Application Number | 20090047695 12/173515 |
Document ID | / |
Family ID | 22361123 |
Filed Date | 2009-02-19 |
United States Patent
Application |
20090047695 |
Kind Code |
A1 |
Wagner; Peter ; et
al. |
February 19, 2009 |
MICRODEVICES FOR HIGH-THROUGHPUT SCREENING OF BIOMOLECULES
Abstract
Methods and devices for the parallel, in vitro screening of
biomolecular activity using miniaturized microfabricated devices
are provided. The biomolecules that can be immobilized on the
surface of the devices of the present invention include proteins,
polypeptides, nucleic acids, polysaccharides, phospholipids, and
related unnatural polymers of biological relevance. These devices
are useful in high-throughput drug screening and clinical
diagnostics and are preferably used for the parallel screening of
families of related proteins.
Inventors: |
Wagner; Peter; (Belmont,
CA) ; Ault-Riche; Dana; (Palo Alto, CA) ;
Nock; Steffen; (Redwood City, CA) ; Itin;
Christian; (Menlo Park, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Zyomyx, Inc.
Hayward
CA
|
Family ID: |
22361123 |
Appl. No.: |
12/173515 |
Filed: |
July 15, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10112847 |
Mar 29, 2002 |
|
|
|
12173515 |
|
|
|
|
09115397 |
Jul 14, 1998 |
6576478 |
|
|
10112847 |
|
|
|
|
Current U.S.
Class: |
435/15 ; 436/518;
436/86; 436/94 |
Current CPC
Class: |
Y10S 435/81 20130101;
Y10T 436/143333 20150115; B82Y 30/00 20130101; Y10S 435/805
20130101 |
Class at
Publication: |
435/15 ; 436/86;
436/94; 436/518 |
International
Class: |
C12Q 1/48 20060101
C12Q001/48; G01N 33/68 20060101 G01N033/68; G01N 33/50 20060101
G01N033/50; G01N 33/543 20060101 G01N033/543 |
Claims
1-56. (canceled)
57. A method comprising: providing a sample detection device, the
sample detection device comprising (i) a substrate having an upper
surface, (ii) a plurality of parallel channels formed in or on the
upper surface of the substrate, each channel extending between a
first and a second end and having a bottom wall surface, wherein
the channels are linearly aligned and can be loaded with fluids
containing one or more analytes, and (iii) a plurality of
immobilization molecules for immobilizing proteins, peptides, small
molecules or nucleic acids at immobilization regions in each
channel; (b) introducing the fluids into the channels so that the
fluids contact the immobilized proteins, peptides, small molecules
or nucleic acids; and (c) monitoring the interaction between the
analytes in the fluids and the immobilized proteins, peptides,
small molecules or nucleic acids in the channels.
58. The method of claim 57, wherein the plurality of parallel
channels includes at least 2 parallel microchannels.
59. The method of claim 57, wherein the plurality of parallel
channels Includes at least 10 parallel microchannels.
60. The method of claim 57, wherein the plurality of parallel
channels includes at least 100 parallel microchannels.
61. The method of claim 57, wherein each pair of adjacent channels
is separated a distance of between about 10 .mu.m and 5 mm.
62. The method of claim 57, wherein each channel has a cross
section width that is between about 10 .mu.m and 500 .mu.m.
63. The method of claim 57, wherein each channel has a cross
section length that is between about 1 mm and 20 mm.
64. The method of claim 57, wherein each channel has a trapezoidal
cross section.
65. The method of claim 57, wherein each channel has a rectangular
cross section.
66. The method of claim 57, wherein each channel has a
semi-circular cross section.
67. The method of claim 57, wherein the sample detection device
further comprises a cover over the channels.
68. The method of claim 57, wherein the sample detection device
further comprises a cover that is wholly or partly translucent or
transparent.
69. The method of claim 57, wherein the sample detection device
further comprises a cover that is wholly or partly non-translucent
or non-transparent.
70. The method of claim 57, wherein the sample detection device
further comprises a coating disposed between the bottom wall
surface and the immobilization molecules
71. The method of claim 57, wherein the immobilized proteins,
peptides, small molecules or nucleic acids are chemisorbed or
physisorbed to the immobilization regions.
72. A method for screening a plurality of biological moieties in
parallel for their ability to interact, react with or bind to a
component of a fluid sample, comprising: (a) delivering the fluid
sample to the reactive sites of a device for processing a fluid
sample, said device having at least 100 noncontiguous reactive
sites, each of said sites comprising: (i) a substrate; (ii) a
different immobilized protein, peptide, or nucleic acid; and (iii)
a monolayer chemisorbed or physisorbed on the surface of said
reactive sites, said monolayer comprising molecules of the formula
X-R-Y wherein R is a spacer, X is a functional group that binds R
to the surface and Y is a functional group for binding said
protein, peptide, or nucleic acid onto the monolayer; and (b)
detecting, either directly or indirectly, the interaction or
reaction of said component with the immobilized protein, peptide,
or nucleic acid, or the binding of said component with the protein,
peptide, or nucleic acid at a reactive site.
73. A method according to claim 72 wherein the device further
comprises: (iv) an affinity tag, wherein said affinity tag enhances
site-specific Immobilization of said protein, peptide, or nucleic
acid onto the monolayer.
74. A method according to claim 72 wherein each of said reactive
sites may independently react with a component of the fluid sample
and are separated from each other by a region of said substrate
that is free of molecules of the formula X-R-Y.
75. A method according to claim 72 in which a plurality of binding
candidates is screened in parallel for their ability to bind a
protein, peptide, or nucleic acid, and in which different fluid
samples, each containing at least one of the binding candidates,
are delivered to the reactive sites of the device, further
comprising (c) washing the reactive sites with fluid which does not
contain said binding candidate in order to elute unbound binding
candidates; and (d) detecting, either directly or indirectly, the
presence of said binding candidate retained at each reactive
site.
76. A method according to claim 72 in which a plurality of binding
candidates is screened in parallel for their ability to bind a
protein, peptide, or nucleic acid, and in which different fluid
samples, each containing at least one of the binding candidates,
are delivered to the reactive sites of the device, further
comprising (c) washing the reactive sites with fluid which does not
contain said binding candidate in order to elute unbound binding
candidates; and (d) detecting, either directly or indirectly, the
presence of said binding candidate retained at each reactive
site.
77. A method according to claim 72 in which a plurality of analytes
which bind said proteins, peptides, or nucleic acids is detected,
comprising: (a) delivering a fluid sample comprising said analytes
to the reactive sites of the device; (b) washing the reactive sites
with an analyte-free fluid to remove unbound analyte; and (c)
detecting, either directly or indirectly; the presence of analyte
retained at each reactive site.
78. A method according to claim 72 in which a plurality of proteins
is paired with their ligands, comprising: (a) delivering a fluid
sample comprising a ligand of a known protein family to the
reactive sites of the device, wherein each reactive site of the
device comprises a different protein; (b) washing the reactive
sites with fluid that does not contain said ligand to remove
unbound ligand; and (c) detecting, either directly or indirectly;
the presence of the ligand retained at each reactive site.
79. A method according to claim 72 wherein each reactive site
comprises an immobilized peptide.
80. A method according to claim 72 wherein each reactive site
comprises an immobilized nucleic acid.
81. A method according to claim 72 wherein each reactive site
comprises an immobilized protein.
82. A method according to claim 81 wherein the device comprises
immobilized proteins of the same family.
83. A method according to claim 81 wherein the device comprises
immobilized proteins that are functionally related.
84. A method according to claim 81 wherein the device comprises
immobilized proteins that are structurally related.
85. A method according to claim 81 wherein the device comprises
immobilized fusion proteins.
86. A method according to claim 85 wherein the device further
comprises an affinity tag, wherein said affinity tag enhances
site-specific immobilization of said protein onto the
monolayer.
87. A method according to claim 81 wherein the device comprises
immobilized recombinant proteins.
88. A method according to claim 81 wherein the device comprises
immobilized kinases.
89. A method according to claim 81 wherein the device comprises
immobilized antibodies.
90. A method according to claim 81 wherein the device comprises
immobilized capture agents.
91. A method according to claim 72, wherein said device comprises
from about 100 to about 500 microchannels.
92. A method according to claim 72, wherein said device comprises
from about 2 to about 500 parallel microchannels per cm.sup.2.
93. A method according to claim 81 wherein the device comprises
immobilized recombinant fusion proteins
Description
[0001] This application is a continuation of co-pending application
Ser. No. 09/115,397, filed Jul. 14, 1998, which is incorporated
herein by reference in its entirety for all purposes and the
specific purposes disclosed throughout this application.
BACKGROUND OF THE INVENTION
[0002] A vast number of new drug targets are now being identified
using a combination of genomics, bioinformaties, genetics, and
high-throughput (HTP) biochemistry. Genomics provides information
on the genetic composition and the activity of an organism's genes.
Bioinformatics uses computer algorithms to recognize and predict
structural patterns in DNA and proteins, defining families of
related genes and proteins. The information gained from the
combination of these approaches is expected to boost the number of
drug targets, usually proteins, from the current 500 to over 10,000
in the coming decade.
[0003] The number of chemical compounds available for screening as
potential drugs is also growing dramatically due to recent advances
in combinatorial chemistry, the production of large numbers of
organic compounds through rapid parallel and automated synthesis.
The compounds produced in the combinatorial libraries being
generated will far outnumber those compounds being prepared by
traditional, manual means, natural product extracts, or those in
the historical compound files of large pharmaceutical
companies.
[0004] Both the rapid increase of new drug targets and the
availability of vast libraries of chemical compounds creates an
enormous demand for new technologies which improve the screening
process. Current technological approaches which attempt to address
this need include multiwell-plate-based screening systems,
cell-based screening systems, microfluidics-based screening
systems, and screening of soluble targets against solid-phase
synthesized drug components.
[0005] Automated multiwell formats are the best developed
high-throughput screening systems. Automated 96-well plate-based
screening systems are the most widely used. The current trend in
plate-based screening systems is to reduce the volume of the
reaction wells further, thereby increasing the density of the wells
per plate (96-well to 384-, and 1536-well per plate). The reduction
in reaction volumes results in increased throughput, dramatically
decreased bioreagent costs, and a decrease in the number of plates
which need to be managed by automation.
[0006] However, although increases in well numbers per plate are
desirable for high throughput efficiency, the use of volumes
smaller than 1 microliter in the well format generates significant
problems with evaporation, dispensing times, protein inactivation,
and assay adaptation. Proteins are very sensitive to the physical
and chemical properties of the reaction chamber surfaces. Proteins
are prone to denaturation at the liquid/solid and liquid/air
interfaces. Miniaturization of assays to volumes smaller than 1
microliter increases the surface to volume ratio substantially.
(Changing volumes from 1 microliter to 10 nanoliter increases the
surface ratio by 460%, leading to increased protein inactivation.)
Furthermore, solutions of submicroliter volumes evaporate rapidly,
within seconds to a few minutes, when in contact with air.
Maintaining microscopic volumes in open systems is therefore very
difficult.
[0007] Other types of high-throughput assays, such as miniaturized
cell-based assays are also being developed. Miniaturized cell-based
assays have the potential to generate screening data of superior
quality and accuracy, due to their in vivo nature. However, the
interaction of drug compounds with proteins other than the desired
targets is a serious problem related to this approach which leads
to a high rate of false positive results.
[0008] Microfluidics-based screening systems that measure in vitro
reactions in solution make use of ten to several-hundred micrometer
wide channels. Micropumps, electroosmotic flow, integrated valves
and mixing devices control liquid movement through the channel
network. Microfluidic networks prevent evaporation but, due to the
large surface to volume ratio, result in significant protein
inactivation. The successful use of microfluidic networks in
biomolecule screening remains to be shown.
[0009] Drug screening of soluble targets against solid-phase
synthesized drug components is intrinsically limited. The surfaces
required for solid state organic synthesis are chemically diverse
and often cause the inactivation or non-specific binding of
proteins, leading to a high rate of false-positive results.
Furthermore, the chemical diversity of drug compounds is limited by
the combinatorial synthesis approach that is used to generate the
compounds at the interface. Another major disadvantage of this
approach stems from the limited accessibility of the binding site
of the soluble target protein to the immobilized drug
candidates.
[0010] DNA microarray technology is not immediately transferable to
protein screening microdevices. To date, microarrays are
exclusively available for nucleic acid hybridization assays
(`DNA-chips`). Their underlying chemistry and materials are not
readily transferable to protein assays. Nucleic acids withstand
temperatures up to 100.degree. C., can be dried and re-hydrated
without loss of activity and bound directly to organic adhesion
layers absorbed on surfaces such as glass. In contrast, proteins
must remain hydrated, kept at ambient temperatures, and are very
sensitive to the physical and chemical properties of the support
materials. Therefore, maintaining protein activity at the
liquid-solid interface requires entirely different immobilization
strategies than those used for nucleic acids. Additionally, the
proper orientation of the protein at the interface is desirable to
ensure accessibility of their active sites with interacting
molecules.
[0011] In addition to the goal of achieving high-throughput
screening of compounds against targets to identify potential drug
leads, researchers also need to be able to identify a highly
specific lead compound early in the drug discovery process.
Analyzing a multitude of members of a protein family or forms of a
polymorphic protein in parallel enables quick identification of
highly specific lead compounds. Proteins within a structural family
share similar binding sites and catalytic mechanisms. Often, a
compound that effectively interferes with the activity of one
family member also interferes with other members of the same
family. Using standard technology to discover such additional
interactions requires a tremendous effort in time and costs and as
a consequence is simply not done.
[0012] However, cross-reactivity of a drug with related proteins
can be the cause of low efficacy or even side effects in patients.
For instance, AZT, a major treatment for AIDS, blocks not only
viral polymerases, but also human polymerases, causing deleterious
side effects. Cross-reactivity with closely related proteins is
also a problem with nonsteroidal anti-inflammatory drugs (NSAIDs)
and aspirin. These drugs inhibit cyclooxygenase-2, an enzyme which
promotes pain and inflammation. However, the same drugs also
strongly inhibit a related enzyme, cyclooxygenase-1, that is
responsible for keeping the stomach lining and kidneys healthy,
leading to common side-effects including stomach irritation.
[0013] For the foregoing reasons, there is a need for miniaturized
devices and for methods for the parallel, in vitro, high-throughput
screening of functionally and/or structurally related protein
targets against potential drug compounds in a manner that minimizes
reagent volumes and protein inactivation problems.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to a device and methods of
use of the device that satisfy the need for parallel, in vitro,
high-throughput screening of functionally or structurally related
protein targets against potential drug compounds in a manner that
minimizes reagent volumes and protein inactivation problems.
[0015] One embodiment of the present invention provides a device
that has a plurality of noncontiguous reactive sites and is useful
for processing fluid samples. On each of the reactive sites of the
device, biological moieties are immobilized on a monolayer via an
affinity tag which enhances the site-specific immobilization of the
biological moiety onto the monolayer. Each of the reactive sites is
separated from neighboring reactive sites by substrate that is free
of the monolayer. The monolayer is on a portion of a surface of a
substrate and comprises molecules of the formula X--R--Y where R is
a spacer, X is a functional group that binds R to the surface, and
Y is a functional group for binding a biological moiety onto the
monolayer via the affinity tag. Each of the reactive sites is
displayed on the device in a manner that allows it to react with a
component of a fluid sample.
[0016] In a preferred embodiment the device of the present
invention is a micromachined or microfabricated device.
[0017] In a particularly preferred embodiment of the device, the
plurality of reactive sites are contained within parallel
microchannels. These microchannels may be microfabricated into or
onto the substrate.
[0018] Optionally, at least one coating may be formed on the
substrate or applied to the substrate of a device of the present
invention such that the coating is positioned between the substrate
and the monolayer of each reactive site.
[0019] The monolayer of a device of the present invention may
optionally be a mixed monolayer of more than one type of organic
molecule.
[0020] In a preferred embodiment, an adaptor molecule is also
included in the device of the present invention to link the
affinity tag to the biological moiety.
[0021] The affinity tag, biological moiety, and the adaptor
molecule (if present) are preferably, but not necessarily, a fusion
protein.
[0022] The biological moiety immobilized on one reactive site can
either be the same as or different from the biological moiety
immobilized on a second reactive site. If the reactive sites are
different, the biological moieties of the different reactive sites
are preferably members of the same protein family or are otherwise
functionally or structurally related.
[0023] The present invention further provides for methods of using
the device to screen a plurality of biological moieties in parallel
for their ability to interact with a component of a fluid sample.
The interaction being assayed may be a binding interaction or a
catalytic one. Some embodiments of these methods first involve
delivering the fluid sample to the reactive sites of the device. If
binding between the biological moiety and the component is to be
detected, the reactive sites are then optionally washed to remove
any unbound component from the area. If binding interactions are
being monitored, the methods also involve detecting, either
directly or indirectly, the retention of the component at each
reactive site. If the interaction being assayed is catalytic, then
the presence, absence, or amount of reaction product is instead
detected.
[0024] In other embodiments of the present invention, similar
methods are used diagnostically to screen a fluid sample for the
presence, absence, or amount of a plurality of analytes (in
parallel).
[0025] In another method, the device may also be used to screen a
plurality of drug candidates in parallel for their ability to bind
or react with a biological moiety. In this method, different fluid
samples, each containing a different drug candidate (or a different
mixture of drug candidates) to be tested, is delivered to the
different reactive sites of the invention device.
[0026] The present invention also provides for methods of
determining in parallel whether or not a plurality of proteins
belong to a certain protein family based on either binding to a
common ligand or reactivity with a common substrate. These methods
involve delivering a fluid sample comprising a ligand or substrate
of a known protein family to the reactive sites of the invention
device that contain the different proteins and then detecting,
either directly or indirectly, for binding or reaction with the
known ligand that is characteristic of the protein family.
[0027] An alternative embodiment of the invention provides a device
for processing a fluid sample that comprises a substrate, a
plurality of parallel microchannels microfabricated into or onto
said substrates and a moiety immobilized within at least one of the
parallel microchannels, in such a way that the moiety interacts
with a component of the fluid sample. In a preferred embodiment,
the immobilized moiety is a biological moiety such as a
protein.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1 shows the top view of a covered microchannel array
device.
[0029] FIG. 2 shows a cross section of a covered microchannel array
fabricated by bulk micromachining.
[0030] FIG. 3 shows a cross section of a covered microchannel array
fabricated by sacrificial micromachining.
[0031] FIG. 4 shows aminoreactive monolayer molecules on a
substrate.
[0032] FIG. 5 shows aminoreactive monolayer molecules on a coated
substrate.
[0033] FIG. 6 shows a biological moiety immobilized on a
monolayer-coated substrate via an affinity tag.
[0034] FIG. 7 shows a cross section view of a biomolecule-coated
microchannel in a microchannel array device.
[0035] FIG. 8 shows a biological moiety immobilized on a
monolayer-coated substrate via an affinity tag and an adaptor
molecule.
[0036] FIG. 9 shows four possible expression vectors for expressing
fusion proteins of the desired protein and an affinity tag and,
optionally, an adaptor molecule.
[0037] FIG. 10 shows a schematic diagram of a fluorescence
detection unit which may be used to monitor interaction of the
immobilized biological moieties of a microchannel array with an
analyte.
DETAILED DESCRIPTION OF THE INVENTION
[0038] A variety of devices and methods useful for high-throughput
drug screening, clinical diagnostics, and related processes are
provided by the present invention.
(a) Definitions
[0039] The term "substrate" as used herein refers to the bulk,
underlying, and core material of the devices of the invention.
[0040] The terms "micromachining" and "microfabricating" are both
used herein to refer to any number of techniques which are useful
in the generation of microstructures (structures of sub-millimeter
scale). Such technologies include, but are not limited to, laser
ablation, sputtering, electrodeposition, low-pressure vapor
deposition, photolithography, and etching. Related technologies
such as LIGA (Lithographie, Galvanoformung und Abformtechnik, high
aspect ratio plating) are also included. Most of these techniques
were originally developed for use in semiconductors,
microelectronics, and microelectromechanical systems but are
applicable to the present invention as well.
[0041] The term "coating" is used herein to refer to a layer that
is either formed on or applied to the surface of the substrate. For
instance, exposure of a substrate, such as silicon, to air can
result in oxidation of the exposed surface. In the case of a
substrate made of silicon, a silicon oxide coating is formed on the
surface upon exposure to air. In other instances, the coating is in
no way derived from the substrate and may be placed upon the
surface via mechanical, electrical, or chemical means. An example
of this type of coating would be a metal coating that is applied to
a polymer substrate. Although a coating may be of any thickness,
typically the coating has a thickness smaller than that of the
substrate.
[0042] An "interlayer" is a second coating or layer that is
positioned between the first coating and the substrate. The primary
purpose of a typical interlayer is to aid adhesion between the
first coating and the substrate. One such example is the use of a
titanium interlayer to help attach a gold coating to a silicon
chip. However, other possible functions of an interlayer are also
anticipated. For instance, some interlayers may perform a role in
the detection system of the device.
[0043] The term "affinity tag" is used herein to refer to a
functional moiety capable of immobilizing a biological moiety onto
the exposed functionality of a monolayer. In some cases, the
affinity tag may be a simple chemical functional group. Other
possibilities include amino acids, polypeptides, proteins, lipid
bilayers, or a hydrogel. The affinity tag may be either covalently
or noncovalently attached to the biological moiety (via chemical
conjugation or as a fusion protein, for instance). In some cases,
an affinity tag may also be an internal part of the
biological-moiety, such as an amino acid. Likewise, the affinity
tag may bind to the monolayer either covalently or
noncovalently.
[0044] An "adaptor molecule", for purposes of this invention, is
any entity that links an affinity tag to a biological moiety. The
adaptor molecule need not necessarily be a discrete molecule that
is noncovalently attached to both the affinity tag and the
biological moiety. The adaptor molecule can be covalently attached
to the affinity tag or the biological moiety or both (via chemical
conjugation or as a fusion protein, for instance). Examples of
adaptor molecules include polypeptides, proteins, membrane anchors,
and biotin.
[0045] A "biological moiety" is any entity that either has, or is
suspected of having, a physiological function.
[0046] A "monolayer" is a single-molecule thick layer of organic
molecules on a surface. A monolayer may be disordered or ordered.
One face of the monolayer is composed of chemical functionalities
on the termini of the organic molecules that are chemisorbed or
physisorbed onto the surface material (headgroups). The other face
of the monolayer is exposed and may bear any number of chemical
functionalities (end groups). Preferably, the molecules of the
monolayer are highly ordered and tightly packed, largely due to
hydrophobic and van der Waals interactions between the
molecules.
[0047] The terms "polypeptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. These terms also apply to amino acid polymers in which
one or more amino acid residues is an artificial chemical analogue
of a corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. An amino acid polymer in
which one or more amino acid residues is an "unnatural" amino acid,
not corresponding to any naturally occurring amino acid, is also
encompassed by the use of the terms "protein" and "polypeptide"
herein.
[0048] Proteins are considered herein to be members of the same
"protein family" or to be "related" if they show significant
similarities in structure and/or function, as would be recognized
by one of ordinary skill in the art. Related proteins can be
identified by sequence homology searches of DNA and protein
databases using standard bioinformatics resources and software
packages (examples of public databases: NCBI, NIH, EMBL, SwissProt,
Brookhaven database, Washington University--Merck; private
databases: Incyte, Hyseq, Hunan Genome Science; examples of
software packages include EMOTIF, Blast, Fasta, Multalign, GCG
Wisconsin University). Enzymatically related proteins of
non-homologous sequence can be identified by one of ordinary skill
in the art by screening the scientific literature (example: Medline
database).
[0049] The term "fusion protein" refers to a protein composed of
two or more polypeptides that, although typically unjoined in their
native state, are joined by their respective amino and carboxyl
termini through a peptide linkage to form a single continuous
polypeptide. It is understood that the two or more polypeptide
components can either be directly joined or indirectly joined
through a peptide linker/spacer.
[0050] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide monomer or polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogs of natural nucleotides that can function in a manner
similar to naturally occurring nucleotides. The nucleic acid may be
obtained from a natural source, an in vitro reaction enzymatic or
chemical synthesis. No distinction is made herein between a nucleic
acid, a polynucleotide, and an oligonucleotide.
[0051] The term "normal physiological condition" is used herein to
refer to conditions that are typical inside a living organism or a
cell. While it is recognized that some organs or organisms provide
extreme conditions, the intra-organismal and intra-cellular
environment normally varies around pH 7 (i.e. from pH 6.5 to pH
7.5), contains water as the predominant solvent, and exists at a
temperature above 0.degree. C. and below 50.degree. C. It will be
recognized that the concentration of various salts depends on the
organ, organism, cell, or cellular compartment used as a
reference.
(b) Devices with a Plurality of Bioreactive Sites
[0052] In one aspect, the present invention provides a device for
processing a fluid sample. This device has a plurality of
noncontiguous reactive sites, each of the sites comprising the
following: a substrate; a monolayer on a portion of a surface of
the substrate, comprising molecules of the formula X--R--Y where R
is a spacer, X is a functional group that binds R to the surface,
and Y is a functional group for binding a biological moiety onto
the monolayer; an affinity tag that enhances site-specific
immobilization of a biological moiety onto the monolayer; and a
biological moiety immobilized on the monolayer through Y via the
affinity tag.
[0053] Each of the sites of the device may independently react with
a component of the fluid sample and are separated from each other
by substrate that is free of monolayer molecules of the formula
X--R--Y.
[0054] Numerous different materials may be used as the substrate of
the invention device. The substrate may be organic or inorganic,
biological or non-biological, or any combination of these
materials. The substrate can optionally be transparent or
translucent. Substrates suitable for micromachining or
microfabrication are preferred. The substrate of the invention can
optionally comprise a material selected from a group consisting of
silicon, silica, quartz, glass, controlled pore glass, carbon,
alumina, titanium dioxide, germanium, silicon nitride, zeolites,
and gallium arsenide. Many metals such as gold, platinum, aluminum,
copper, titanium, and their alloys are also options for substrates.
In addition, many ceramics and polymers may be used as substrates,
either in planar or bead form. Polymers which may be used as
substrates include, but are not limited to, the following:
polystyrene; poly(tetra)fluorethylene; (poly)vinylidenedifluoride;
polycarbonate; polymethylmethacrylate; polyvinylethylene;
polyethyleneimine; poly(etherether)ketone; polyoxymethylene (POM);
polyvinylphenol; polylactides; polymethacrylimide (PMI);
polyalkenesulfone (PAS); polyhydroxyethylmethacrylate;
polydimethylsiloxane; polyacrylamide; polyimide; co-block-polymers;
and Eupergit.RTM.. Photoresists, polymerized Langmuir-Blodgett
films, and LIGA structures may also serve as substrates in the
present invention. The preferred substrates of the present
invention comprise silicon, silica, glass, or a polymer.
[0055] In a preferred embodiment of the present invention the
device comprises a micromachined or microfabricated device. The
device is optionally a microdevice with dimensions on the
millimeter to centimeter scale.
[0056] In a preferred embodiment of the invention, each of the
reactive sites of the device is in a microchannel oriented parallel
to microchannels of other reactive sites on the device. The
microchannels of such a device have optionally been microfabricated
or micromachined into or onto the substrate of the device. FIG. 1
illustrates one embodiment of the invention showing an array of
microchannels 1 that have been fabricated into a bulk substrate
material. In the particular device shown, eighty parallel
microchannels 1 have been microfabricated into a substrate 3.
[0057] In one embodiment of the invention, the device comprises at
least 2 parallel microchannel reactive sites. In another embodiment
of the invention, the device comprises at least 10 parallel
microchannel reactive sites. In a preferred embodiment of the
invention, the device comprises at least 100 parallel microchannel
reactive sites. In a particularly preferred embodiment, the device
comprises from about 100 to about 500 parallel microchannels. The
microchannels are typically separated from one another by from
about 10 .mu.m to about 5 mm. The device may optionally comprise
from about 2 to about 500 parallel microchannels per cm.sup.2 of
substrate.
[0058] The dimensions of the microchannels may vary. However, in
preferred embodiments the scale is small enough so as to only
require minute fluid sample volumes. The width and depth of each
microchannel of the invention device is typically between about 10
.mu.m and about 500 .mu.m. In a preferred embodiment of the device,
the width and depth of each microchannel is between about 50 and
200 .mu.m. The length of each microchannel is from about 1 to about
20 mm in length. In a preferred embodiment, the length of each
microchannel is from about 2 to about 8 mm long. Any one of a
variety of channel cross-section geometries (trapezoidal,
rectangular, v-shaped, semicircular, etc.) may be employed in the
device. The geometry is determined by the type of microfabrication
or micromachining process used to generate the microchannels, as is
known in the art. Trapezoidal or rectangular cross-section
geometries are preferred for the microchannels, since they readily
accommodate standard fluorescence detection methods.
[0059] In order to generate a plurality of reactive sites, such as
a parallel array of microchannels, the substrate material first has
to be cleaned to remove contaminants such as solvent stains, dust,
or organic residues. A variety of cleaning procedures can be used
depending on the substrate material and origin of contaminants.
These include wet immersion processes (for example, RCA1+2,
"pyranha", solvents), dry vapor phase cleaning, thermal treatment,
plasma or glow discharge techniques, polishing with abrasive
compounds, short-wavelength light exposure, ultrasonic agitation
and treatment with supercritical fluids.
[0060] Channels can then be formed on the surface of the substrate
by either (1) bulk micromachining, (2) sacrificial micromachining,
(3) LIGA (high aspect ratio plating) or (4) other techniques, or
any combination thereof. Such techniques are well known in the
semiconductor and microelectronics industries and are described in,
for example, Ghandi, VLSI Fabrication Principles, Wiley (1983) and
Sze, VLSI Technology, 2nd. Ed., McGraw-Hill (1988); Wolf and Taube,
Silicon Processing for the VLSI Era, Vol. 1, Lattice Press (1986),
and Madou, Fundamentals of Microfabrication, CRC Press (1997).
[0061] In bulk micromachining, large portions of the substrate are
removed to form rectangular or v-shaped grooves comprising the
final dimensions of the microchannels. This process is usually
carried out with standard photolithographic techniques involving
spin-coating of resist materials, illumination through lithography
masks followed by wet-chemical development and posttreatment steps
such as descumming and post-baking. The resulting resist pattern is
then used as an etch resist material for subsequent wet or dry
etching of the bulk material to form the desired topographical
structures. Typical resist materials include positive and negative
organic resists (such as Kodak 747, PR102), inorganic materials
(such as polysilicon, silicon nitride) and biological etch resists
(for example Langmuir-Blodgett films and two-dimensional protein
crystals such as the S-layer of Sulfolobus acidocladarius). Pattern
transfer into the substrate and resist stripping occurs via
wet-chemical and dry etching techniques including plasma etching,
reactive ion etching, sputtering, ion-beam-assisted chemical
etching and reactive ion beam etching.
[0062] In one embodiment of the invention, for instance, a
photoresist may be spincoated onto a cleaned 4 inch Si(110) wafer.
Ultraviolet light exposure through a photomask onto the photoresist
then results in a pattern of channels in the photoresist, exposing
a pattern of strips of the silicon underneath. Wet-chemical etching
techniques can then be applied to etch the channel pattern into the
silicon. Next, a thin layer of titanium can be coated on the
surface. A thin layer of gold is then coated on the surface via
thermal or electron beam evaporation. Standard resist stripping
follows. (Alternatively, the gold-coating could be carried out
after the strip resist.)
[0063] FIG. 2 shows a cross section view of one example of a
microchannel array fabricated by bulk micromachining. A
microchannel 1 in substrate 3 is covered by a glass cover 2. At the
bottom of the microchannel, the surface of the substrate 3 is
covered with a coating 5.
[0064] In sacrificial micromachining, the substrate is left
essentially untouched. Various thick layers of other materials are
built up by vapor deposition, plasma-enhanced chemical vapor
deposition (PECVD) or spin coating and selectively remain behind or
are removed by subsequent processing steps. Thus, the resulting
channel walls are chemically different from the bottom of the
channels and the resist material remains as part of the
microdevice. Typical resist materials for sacrificial
micromachining are silicon nitride (Si.sub.3N.sub.4), polysilicon,
thermally grown silicon oxide and organic resists such as SU-8 and
polyimides allowing the formation of high aspect-ratio features
with straight sidewalls.
[0065] FIG. 3 shows a cross section view of one example of a
microchannel array that has been fabricated by sacrificial
micromachining. Microchannel 1 has walls that consist of
photoresist 4 and a floor that comprises a substrate 3 that is
covered with a coating 5.
[0066] In high-aspect ratio plating or LIGA, three-dimensional
metal structures are made by high-energy X-ray radiation exposures
on materials coated with X-ray resists. Subsequent
electrodeposition and resist removal result in metal structures
that can be used for precision plastic injection molding. These
injection-molded plastic parts can be used either as the final
microdevice or as lost molds. The LIGA process has been described
by Becker et al., Microelectron Engineering (1986) 4:35-56 and
Becker et al., Naturwissenschaften (1982) 69:520-523.
[0067] Alternative techniques for the fabrication of microchannel
arrays include focused ion-beam (FIB) milling, electrostatic
discharge machining (EDM), ultrasonic drilling, laser ablation
(U.S. Pat. No. 5,571,410), mechanical milling and thermal molding
techniques. One skilled in the art will recognize that many
variations in microfabrication or micromachining techniques may be
used to construct the device of the present invention.
[0068] In one embodiment, transparent or translucent covers are
attached to the substrate via anodic bonding or adhesive coatings,
resulting in microchannel arrays with inlet and outlet ports. In a
preferred embodiment, the microchannel covers are glass, especially
Pyrex or quartz glass. In alternative embodiments, a cover which is
neither transparent nor translucent may be bonded or otherwise
attached to the substrate to enclose the microchannels. In other
embodiments the cover may be part of a detection system to monitor
the interaction between biological moieties immobilized within the
channel and an analyte. Alternatively, a polymeric cover may be
attached to a polymeric substrate channel array by other means,
such as by the application of heat with pressure or through
solvent-based bonding.
[0069] One particular embodiment of a covered microchannel array is
illustrated by FIG. 1. In this device, a transparent glass cover 2
covers most of the length, although not all, of each of the
parallel microchannels of the array. Since in this particular
embodiment the microchannels do not extend fully to the edge of the
substrate, the incomplete coverage of the channel length provides
an inlet and outlet port for each of the microchannels.
[0070] Attachment of the covers to the microchannel array can
precede monolayer formation. If this is the case, then the
monolayer component containing solution (typically an organic
solvent) can be applied to the interior of the channels via
microfabricated dispensing systems that have integrated
microcapillaries and suitable entry/exit ports. Alternatively, the
monolayers can be deposited in the microchannels prior to enclosure
of the microchannels. For these embodiments, monolayers can
optionally be transferred to the inner microchannel surfaces via
simple immersion or through microcontact printing (see PCT
Publication WO 96/29629).
[0071] The volume of each enclosed microchannel may optionally be
from about 5 nanoliters to about 300 nanoliters. In a preferred
embodiment, the volume of an enclosed microchannel of the invention
device is between 10 and 50 nanoliters.
[0072] Volumes of fluid may be moved through each microchannel by a
number of standard means known to those skilled in the art. The
sophisticated means required for moving fluids through microfluidic
devices and mixing in microtiter plates are not needed for the
microchannel array of the present invention. Simple liquid exchange
techniques commonly used with capillary technologies will suffice.
For instance, fluid may be moved through the channel using standard
pumps. Alternatively, more sophisticated methods of fluid movement
through the microchannels such as electro-osmosis may be employed
(for example, see U.S. Pat. No. 4,908,112).
[0073] In one embodiment of the present invention, bulk-loading
dispensing devices can be used to load all microchannels of the
device at once with the same fluid. Alternatively, integrated
microcapillary dispensing devices may be microfabricated out of
glass or other substrates to load fluids separately to each
microchannel of the device.
[0074] After formation of a microchannel, the sides, bottom, or
cover of the microchannel or any combination thereof, can then be
further chemically modified to achieve the desired bioreactive and
biocompatible properties.
[0075] The reactive sites of the device may optionally further
comprise a coating between a substrate and its monolayer. This
coating may either be formed on the substrate or applied to the
substrate. The substrate can be modified with a coating using
thin-film technology based on either physical vapor deposition
(PVD) or plasma-enhanced chemical vapor deposition (PECVD). Thin
layers of metals or the exposure of hydroxylated surfaces on a
substrate are often necessary to allow chemisorption or
physisorption of bioreactive organic monolayer systems.
Alternatively, plasma exposure can be used to directly activate the
substrate. For instance, plasma etch procedures can be used to
oxidize a polymeric surface (i.e. polystyrene or polyethylene) to
expose polar functionalities such as hydroxyls, carboxylic acids,
aldehydes, or the like on the surface.
[0076] The coating on the substrate may comprise a metal film.
Possible metal films include, but are not limited to aluminum,
chromium, titanium, nickel, stainless steel, zinc, lead, iron,
magnesium, manganese, cadmium, tungsten, cobalt, and alloys or
oxides thereof. In a preferred embodiment, the metal film is a
noble metal film. Noble metals that may be used for a noble metal
film include, but are not limited to gold, platinum, silver,
copper, and palladium. In an especially preferred embodiment, the
coating comprises gold or a gold alloy. In a preferred embodiment,
the metal film is from about 50 nm to about 500 nm in
thickness.
[0077] In alternative embodiments, the coating on the substrate
comprises a composition such as silicon, silicon oxide, silicon
nitride, silicon hydride, indium tin oxide, magnesium oxide,
alumina, glass, hydroxylated surfaces, or a polymer.
[0078] If the reactive site comprises a coating between the
substrate and the monolayer, then it is understood that the coating
must be composed of a material for which a suitable functional
group X is available (see below). If no such coating is present,
then it is understood that the substrate must be composed of a
material for which a suitable functional group X is available.
[0079] It is contemplated that many coatings will require the
addition of at least one adhesion layer or mediator between said
coating and said substrate. For instance, a layer of titanium may
be desirable between a silicon wafer substrate and a gold coating.
In an alternative embodiment an epoxy glue such as Epo-tek 377.RTM.
and Epo-tek 301-2.RTM. (Epoxy Technology Inc., Billerica, Mass.)
may be preferred to aid adherence of the coating to the substrate.
Determinations as to what material should be used for the adhesion
layer would be obvious to one skilled in the art once materials
were chosen for the substrate and coating. In other embodiments,
additional adhesion mediators or interlayers may be necessary to
improve the mechanical or optical properties of the device, for
example, for detection purposes in waveguides.
[0080] Deposition or formation of the coating on the substrate (if
such coatings are desired) must occur prior to the formation of
bioreactive monolayers thereon.
[0081] The monolayer of each reactive site is comprised of
molecules of the general formula X--R--Y where R is a spacer, X is
a functional group that binds R to the surface of a portion of the
substrate, and Y is a functional group for binding a biological
moiety onto the monolayer. Three major classes of monolayer
formation are preferably used to expose high densities of
bioreactive omega-functionalities on the substrate: (i)
alkylsiloxane monolayers ("silanes") on hydroxylated surfaces (as
taught in, for example, U.S. Pat. No. 5,405,766, PCT Publication WO
96/38726, U.S. Pat. No. 5,412,087, and U.S. Pat. No. 5,688,642);
(ii) alkyl-thiol/dialkyldisulfide monolayers on noble metals
(preferably Au(111)) (as, for example, described in Allara et al.,
U.S. Pat. No. 4,690,715; Bamdad et al., U.S. Pat. No. 5,620,850;
Wagner et al., Biophysical Journal, 1996, 70:2052-2066); and (iii)
alkyl monolayer formation on oxide-free passivated silicon (as
taught in, for example, Linford et al., J. Am. Chem. Soc., 1995,
117:3145-3155, Wagner et al., Journal of Structural Biology, 1997,
119:189-201, and U.S. Pat. No. 5,429,708). One of ordinary skill in
the art however, will recognize that many possible moieties may be
substituted for X, R, and/or Y, dependent largely upon the choice
of substrate, coating, and affinity tag. Many examples of
monolayers are described in Ulman, An Introduction to Ultrathin
Organic Films From Langmuir-Blodgett to Self-Assembly, Academic
Press (1991), herein incorporated by reference.
[0082] R of the monolayer molecule may comprise a hydrocarbon chain
from about 1 to about 200 carbons long. The hydrocarbon chain may
comprise an alkyl, aryl, alkenyl, alkynyl, cycloalkyl, alkaryl,
aralkyl group, or any combination thereof. In a preferred
embodiment, R is a chemical moiety that promotes formation of a
self-assembled monolayer. In a preferred embodiment, R is an
alkylchain from about 8 to about 22 carbons long. In a further
preferred embodiment, R is a straight alkane from about 8 to about
22 carbons long. However, it is also contemplated that in an
alternative embodiment, R may readily comprise a hydrocarbon chain
from about 2 to about 200 carbons long and interrupted by at least
one hetero atom. The interrupting hetero groups can include --O--,
--CONH--, --CONHCO--, --NH--, --CSNH--, --CO--, --CS--, --S--,
--SO--, --(OCH.sub.2CH.sub.2).sub.n-(where n=1-20),
--(CF.sub.2).sub.n-(where n=1-22), and the like. Alternatively, one
or more of the hydrogen moieties of R can optionally be substituted
with deuterium.
[0083] X may be chosen as any group which affords chemisorption or
physisorption of the monolayer onto the surface of the substrate
(or the coating, if present). For instance, if the substrate or
coating is a metal or metal alloy, X, at least prior to
incorporation into the monolayer, is preferably an asymmetrical or
symmetrical disulfide, sulfide, diselenide, selenide, thiol,
isonitrile, selenol, a trivalent phosphorus compound,
isothiocyanate, isocyanate, xanthanate, thiocarbamate, a phosphine,
a amine, thio acid or dithio acid. This embodiment is especially
preferred when the substrate, or coating if used, is a noble metal
such as gold, silver, or platinum.
[0084] If the substrate of the device is a material such as
silicon, silicon oxide, indium tin oxide, magnesium oxide, alumina,
quartz, glass, or silica, then a preferred embodiment of the
invention comprises an X that, prior to incorporation into said
monolayer, is a monohalosilane, dihalosilane, trihalosilane,
trialkoxysilane, dialkoxysilane, or a monoalkoxysilane. Among these
silanes, trichlorosilane and trialkoxysilane are particularly
preferred.
[0085] In other embodiments, the surface of the substrate (or
coating thereon) is composed of a metal oxide such as titanium
oxide, tantalum oxide, indium tin oxide, magnesium oxide, or
alumina and X is a carboxylic acid. Alternatively, if the surface
of the substrate (or coating thereon) of the device is copper, then
X may optionally be a hydroxamic acid.
[0086] If the substrate used in the invention is a polymer, then in
many cases a coating on the substrate such as a copper coating will
be included in the device. An appropriate functional group X for
the coating would then be chosen for use in the device. In an
alternative embodiment comprising a polymer substrate, the surface
of the polymer may be plasma-modified to expose desirable surface
functionalities for monolayer formation. For instance, EP 780423
describes the use of a monolayer molecule that has an alkene X
functionality on a plasma exposed surface. Still another
possibility for the invention device comprised of a polymer is that
the surface of the polymer on which the monolayer is formed is
functionalized due to copolymerization of appropriately
functionalized precursor molecules.
[0087] Another possibility is that prior to incorporation into the
monolayer, X can be a free-radical-producing moiety. This
functional group is especially appropriate when the surface on
which the monolayer is formed is a hydrogenated silicon surface.
Possible free-radical producing moieties include, but are not
limited to, diacylperoxides, peroxides, and azo compounds.
Alternatively, unsaturated moieties such as unsubstituted alkenes,
alkynes, cyano compounds and isonitrile compounds can be used for
X, if the reaction with X is accompanied by ultraviolet, infrared,
visible, or microwave radiation.
[0088] In alternative embodiments, X, prior to incorporation into
the monolayer, may be a hydroxyl, carboxyl, vinyl, sulfonyl,
phosphoryl, silicon hydride, or an amino group.
[0089] The component, Y, of the monolayer is responsible for
binding a biological moiety onto the monolayer. In a preferred
embodiment of the invention, the Y group is either highly reactive
(activated) towards the biological moiety or is easily converted
into such an activated form. In a preferred embodiment, the
coupling of Y with the biological moiety occurs readily under
normal physiological conditions not detrimental to the biological
activity of the biological moiety. One skilled in the art will also
appreciate that in many cases, the functional group Y will be
altered upon interacting with the biological moiety. Although the
functional group Y may either form a covalent linkage or a
noncovalent linkage with the biological moiety, a covalent linkage
is preferred.
[0090] In one embodiment of the present invention, Y is a
functional group that is activated in situ before attachment of the
biological moiety's affinity tag. Possibilities for this type of
functional group include, but are not limited to, such simple
moieties such as a hydroxyl, carboxyl, amino, aldehyde, carbonyl,
methyl, methylene, alkene, alkyne, carbonate, aryliodide, or a
vinyl group. Appropriate modes of activation for these simple
functional groups would be known by one of ordinary skill in the
art. Alternatively, Y can comprise a functional group that requires
photoactivation prior to becoming activated enough to trap the
biological moiety.
[0091] In an especially preferred embodiment of the device of the
present invention, Y is a highly reactive functional moiety
compatible with monolayer formation and needs no in situ activation
prior to reaction with the biological moiety or its affinity tag.
Such possibilities for Y include, but are not limited to,
maleimide, N-hydroxysuccimide (Wagner et al., Biophysical Journal,
1996, 70:2052-2066), nitrilotriacetic acid (U.S. Pat. No.
5,620,850), activated hydroxyl, haloacetyl, bromoacetyl,
iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine,
trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole,
imidazolecarbamate, succinimidylcarbonate, arylazide, anhydride,
diazoacetate, benzophenone, isothiocyanate, isocyanate, imidoester,
fluorobenzene, and biotin.
[0092] FIG. 4 shows one example of a monolayer on a substrate 3. In
this example, substrate 3 comprises silicon (having a silicon oxide
surface). The monolayer is aminoreactive because it bears a
functional group Y that is N-hydroxysuccinimide.
[0093] FIG. 5 shows another example of a monolayer on a substrate
3. In this case, however, a thin film coating 5 comprised of gold
covers the surface of the substrate 3. Also, in this embodiment, an
adhesion interlayer 6 is used to adhere the coating 5 to the
substrate 3 and is comprised of titanium. This monolayer is also
aminoreactive because it bears a functional group Y that is
N-hydroxysuccinimide.
[0094] In an alternative embodiment, Y is selected from the group
of simple functional moieties. Possible Y functional groups
include, but are not limited to, --OH, --NH.sub.2, --COOH, --COOR,
--RSR, --PO.sub.4.sup.-3, OSO.sub.3.sup.-2, --SO.sub.3--, --COO--,
--SOO--, --CONR.sub.2, --CN, --NR.sub.2, and the like. Simple
groups such as these are only preferred for X when the affinity tag
of the invention composes a layer of affinity tag molecules (such
as poly-lysine) that coats the exposed portion of the monolayer
prior to immobilization of the biological moiety (see below).
[0095] The monolayer molecules of the present invention can
optionally be assembled on the surface in parts. In other words,
the monolayer need not necessarily be constructed by chemisorption
or physisorption of molecules of the formula X--R--Y to the surface
of the substrate (or coating). Instead, in one embodiment, X may be
chemisorbed or physisorbed to the surface of the substrate (or
coating) alone first. Then, R or even just individual components of
R can be attached to X through a suitable chemical reaction. Upon
completion of addition of the spacer R to the X moiety already
immobilized on the surface, Y can be attached to the ends of the
monolayer molecule through a suitable covalent linkage.
[0096] Not all monolayer molecules at a given reactive site need to
be identical. Some may consist of mixed monolayers. For instance,
the monolayer of an individual reactive site may optionally
comprise at least two different X--R--Y molecules. This second
X--R--Y molecule may immobilize the same or a different biological
moiety. In addition, many of the monolayer molecules, X--R--Y, of a
reactive site may have failed to attach any biological moiety.
[0097] As another alternative of the invention, the monolayer of an
individual reactive site can comprise a second organic molecule
that is of the formula, X--R--V where R is the spacer, X is the
functional group that binds R to the surface, and V is a moiety
resistant to the non-specific binding of biomolecules. One of
ordinary skill in the art will recognize that the possibilities for
V will vary depending upon the nature of the biological moiety
chosen for the sites of the device. For instance, functional groups
V which are resistant to non-specific protein binding are used if
the immobilized biological moiety of the device comprises protein.
The nature of V will be somewhat dependent upon the type of
proteins and solutions used. However, V will most typically
comprise a hydroxyl, saccharide, or polyethylene glycol moiety (EP
Publication 780423).
[0098] As a still further alternative of the invention device, the
device may further comprise at least one unreactive site devoid of
any biological moiety that comprises a monolayer of molecules of
the formula X--R--V, where R is the spacer, X is the functional
group that binds R to the surface, and V is the moiety resistant to
the non-specific binding of biomolecules. In this embodiment, the
unreactive site does not comprise any monolayers of molecules of
the formula X--R--Y.
[0099] Regardless of the nature of the monolayer molecules, in some
cases it can be desirable to provide crosslinking between molecules
of the monolayer. In general, this confers additional stability to
the monolayer. Methods of crosslinking such monolayers are known to
those skilled in the art (see Ullman, An Introduction to Ultrathin
Organic Films: From Langmuir-Blodgett to Self-Assembly, Academic
Press (1991).
[0100] In addition to facilitating binding of the biological moiety
to the substrate, functionalization of the substrate with
monolayers is necessary for other reasons as well. Many biological
moieties and protein, in particular, are susceptible to disruption
of their bioactivities at surface interfaces. Proteins are prone to
both denaturation and undesirable, non-specific binding at the
solid/liquid interface. Other biological moieties such as small
molecule ligands may have less problematic interactions with the
substrate surface interface, but upon approach of the biological
binding partner, presumably a protein, to the small molecule in an
assay, problems of inactivation become highly relevant. A
highly-ordered organic monolayer effectively "carpets" the surface
of the substrate or coating, protecting the biological moiety from
contact with the surface. These highly-ordered, self-assembled
monolayers are preferred in the present invention. Additionally,
the spacer R creates distance between the immobilized biological
moiety and the surface.
[0101] Following formation of monolayers on the reactive sites of
the invention device, the biological moieties are immobilized on
the monolayers via the affinity tags. A solution containing the
biological moiety to be immobilized can be exposed to the
bioreactive units of the microdevice by either dispensing the
solution by means of microfabricated adapter systems with
integrated microcapillaries and entry/exit ports or alternatively
by transferring the biological moieties via noncontact printing
using dispensing microdevices with ball-point pen-type of
mechanisms. The use of other printing techniques is also
anticipated. Following attachment of the biological moieties to the
monolayer, unreacted Y-functional groups are preferably quenched
prior to use of the device.
[0102] The affinity tag is of critical importance to the present
invention. The use of an affinity tag on the biological moiety of
interest to be immobilized, allows for at least one of two
advantages. An affinity tag can confer enhanced binding or reaction
with Y. This enhancement effect may be either kinetic or
thermodynamic. In general, affinity tag/Y-group combination used in
the present invention preferably allows for immobilization of the
biological molecule in a manner which does not require harsh
conditions. This helps ensure that the biological reactivity of the
biological moiety remains intact. Aqueous, biological buffer
conditions are ideal. An affinity tag can also offer immobilization
that is specific to a designated site or location on the biological
moiety. This site-specific immobilization will help ensure that the
reactive site of the biological moiety is accessible to ligands in
solution.
[0103] In a preferred embodiment, especially when the biological
moiety of the invention device is a protein or a polypeptide, the
affinity tag comprises at least one amino acid. The affinity tag
may be a polypeptide comprising at least one monolayer-reactive
amino acid. Alternatively, the affinity tag may be a lone,
monolayer-reactive amino acid. Examples of possible
monolayer-reactive amino acids include cysteine, lysine, histidine,
arginine, tyrosine, and glutamine. A polypeptide or single amino
acid affinity tag is preferably expressed as a fusion protein along
with the biological moiety. Amino acid tags provide either a single
amino acid or a series of amino acids that can interact with the
Y-functionalities of the monolayer. Amino acid affinity tags can
also be introduced to a specific site on a recombinant protein to
facilitate oriented immobilization by covalent binding to the
bioreactive Y-functional group of the monolayer.
[0104] The affinity tag may comprise a poly(amino acid) tag. A
poly(amino acid) tag is a polypeptide that comprises from about 2
to about 100 residues of a single amino acid. Other amino acid
residues may also be present in the affinity tag. For instance, the
affinity tag may comprise a poly-cysteine, poly-lysine,
poly-arginine, or poly-histidine. Amino acid tags are preferably
composed of two to twenty residues of a single amino acid, such as,
for example, histidines, lysines, arginines, cysteines, glutamines,
tyrosines, or any combination of these. According to a preferred
embodiment an amino acid tag of one to twenty amino acids includes
at least one to ten cysteines for thioether linkage; or one to ten
lysines for amide linkage; or one to ten arginines for coupling to
vicinal dicarbonyl groups. One of ordinary skill in the art can
readily pair suitable affinity tags with a given
Y-functionality.
[0105] The position of the amino acid tag can be at the amino-, or
carboxy-terminus of the protein or anywhere in-between. Where
compatible with protein function, affinity tags introduced for
protein purification are preferentially located at the C-terminus
of the recombinant protein to ensure that only full-length proteins
are isolated during protein purification.
[0106] Affinity tags may also contain one or more unnatural amino
acids. Unnatural amino acids can be introduced using suppressor
tRNAs that recognize stop codons (i.e. amber), as described in
Noren et al., Science, 1989, 244:182-188, Ellman et al., Methods
Enzym., 1991, 202:301-336, and Cload et al., Chem. Biol., 1996,
3:1033-1038, herein incorporated by reference. The tRNAs are
chemically amino-acylated to contain chemically altered
("unnatural") amino acids for use with specific coupling
chemistries (i.e. ketone modifications, photoreactive groups).
[0107] In an alternative embodiment the affinity tag can comprise a
whole protein, such as, but not limited to, glutathione
S-transferase, an antibody, avidin, or streptavidin.
[0108] Other bioconjugation and immobilization techniques known in
the art may be adapted for the purpose of immobilizing biomolecules
on activated monolayers. For instance, in an alternative
embodiment, the affinity tag may be an organic bioconjugate which
is chemically coupled to the biomolecule of interest. Biotin or an
antigen may be chemically crosslinked to the biomolecule.
Alternatively, a chemical crosslinker may be used that attaches a
simple functional moiety such as a thiol or an amine to the
biomolecule.
[0109] FIG. 6 shows a biological moiety 10 immobilized on a
monolayer 7 on a substrate 3. An affinity tag 8 connects the
biological moiety 10 to the monolayer 7. The monolayer 7 is formed
on a coating 5 separated from the surface of the substrate 3 by an
interlayer 6.
[0110] FIG. 7 shows a cross section of a biomolecule-coated
microchannel of one embodiment of a microchannel array device. The
microchannel 1 is covered by a glass cover 2. The walls of the
microchannel are comprised of substrate 3, coated first with an
interlayer 6, then with a coating 5, then with an organic monolayer
7 and finally, with the biological moiety 10 via the affinity tag
8.
[0111] In an alternative embodiment, the affinity tag is a
component of a layer of affinity tag molecules immobilized on the
monolayer. A hydrogel can serve as a suitable layer of affinity tag
molecules. Dextran is one possible hydrogel material useful for the
present invention. Alternatively, other polysaccharides or
water-swellable organic polymers may also suffice. Uses of such
hydrogels to immobilize biological moieties are described in U.S.
Pat. No. 5,242,828. Poly-lysine is another option. An example of a
use of poly-lysine layer on an 11-mercaptoundecanoic acid monolayer
can be found in U.S. Pat. No. 5,629,213. The layer of affinity tag
molecules can optionally instead constitute a phospholipid
monolayer or a phospholipid bilayer, as described in PCT
Publication WO 96/38726. Use of a phospholipid monolayer or bilayer
as an affinity tag would be suitable if the biological moiety to be
immobilized is a membrane protein, such as an ion channel
protein.
[0112] Another major embodiment of devices of the present invention
comprises an adaptor molecule that links the affinity tag to the
immobilized biological moiety. The additional spacing of the
biological moiety from the surface of the substrate (or coating)
that is afforded by the use of an adaptor molecule is particularly
advantageous if the biological moiety is a type of molecule which
is known to be prone to surface inactivation, as is the case with
proteins. One of ordinary skill in the art will be able to choose
an adaptor molecule which is appropriate for a given affinity tag
and/or biological moiety. For instance, if the affinity tag is
streptavidin, then the adaptor molecule could be a biotin molecule
that is chemically conjugated to the biological moiety.
Alternatively, if the affinity tag is a phospholipid bilayer or
monolayer then a membrane anchor could be chosen as a suitable
adaptor molecule.
[0113] In one embodiment, the adaptor is a polypeptide, such as
protein G or protein A. In a preferred embodiment, the affinity
tag, adaptor molecule, and biological moiety together compose a
fusion protein. Such a fusion protein may be readily expressed
using standard recombinant DNA technology. Adaptor proteins are
especially useful to increase the solubility of the protein of
interest and to increase the distance between the surface of the
substrate or coating and the protein of interest. Use of an adaptor
protein or polypeptide can also be very useful in facilitating the
preparative steps of protein purification by affinity binding.
Examples of possible adaptor proteins include
glutathione-S-transferase (GST), maltose-binding protein,
chitin-binding protein, thioredoxin, green-fluorescent protein
(GFP). GFP can also be used for quantification of surface
binding.
[0114] FIG. 8 shows a biological moiety 10 immobilized on a
monolayer 7 via both an affinity tag 8 and an adaptor molecule 9.
The monolayer 7 has been formed on a coating 5 on a substrate 3. An
interlayer 6 is also used between the coating 5 and the substrate
3.
[0115] In preparation for immobilization to the devices and arrays
of the present invention, fusion proteins can be expressed from
recombinant DNA either in vivo or in vitro. Amino acid affinity
tags are introduced by polymerase chain reaction. Expression in
vivo is in either bacteria (Escherichia coli), lower eukaryotes
(Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris) or
higher eukaryotes (bacculo-infected insect cells, insect cells
mammalian cells), or in vitro (Escherichia coli lysates, wheat germ
extracts, reticulocyte lysates). Proteins are purified by affinity
chromatography using commercially available resins.
[0116] DNA sequences encoding amino acid affinity tags and adaptor
proteins are engineered into the expression vectors such that the
genes of interest can be cloned in frame either 5' or 3' of the DNA
sequence encoding the affinity tag and adaptor protein.
[0117] FIG. 9 shows four possible expression vectors useful for
expressing a protein of interest, a polypeptide affinity tag, and a
polypeptide adaptor molecule as a fusion protein. The vector
contains an origin of replication sequence 35 and a gene 36 capable
of conferring antibiotic resistance to a host cell. The insert of
the vector contains a promoter sequence 30 and a termination signal
sequence 34. Between the sequences 30 and 34, the insert also
contains a gene 33 encoding the protein of interest and sequence
31, encoding the polypeptide affinity tag. Sequence 32 which codes
for a polypeptide adaptor molecule may also be included in the
plasmid construct and is positioned between the protein and
affinity-tag coding regions (33 and 31, respectively).
[0118] Preferably, production of families of related proteins
involves parallel processing from cloning to protein expression and
protein purification. cDNAs for the protein of interest will be
amplified by PCR using cDNA libraries or EST (expressed sequence
tag) clones as templates. For in vivo expression of the proteins,
cDNAs can be cloned into commercial expression vectors (Qiagen,
Novagen, Clontech) and introduced into the appropriate organism for
expression (organisms include: Escherichia coli, Saccharomyces
cerevisiae, Saccharomyces pombe, Pichia pastoris,
bacculovirus/insect cells, insect cells, mammalian cells). For in
vitro expression PCR-amplified DNA sequences are directly used in
coupled in vitro transcription/translation systems (Escherichia
coli S30 lysates from T7 RNA polymerase expressing, preferably
protease-deficient strains, wheat germ lysates, reticulocyte
lysates with and without microsomes (Promega, Pharmacia, Panvera)).
The choice of organism for optimal expression depends on the extent
of post-translational modifications (i.e. glycosylation,
lipid-modifications).
[0119] Escherichia coli based protein expression will be the method
of choice for soluble proteins that do not require extensive
post-translational modifications for activity. Extracellular or
intracellular domains of membrane proteins will be fused to protein
adaptors for expression and purification.
[0120] The entire approach can be performed using 96-well assay
plates. PCR reactions are carried out under standard conditions.
Oligonucleotide primers contain unique restriction sites for facile
cloning into the expression vectors. Alternatively, the TA cloning
system (Clontech) can be used. Expression vectors contain the
sequences for affinity tags and the protein adaptors. PCR products
are ligated into the expression vectors (under inducible promoters)
and introduced into the appropriate competent Escherichia coli
strain by calcium-dependent transformation (strains include: XL-1
blue, BL21, SG13009(lon-)). Transformed Escherichia coli cells are
plated and individual colonies transferred into 96-array blocks.
Cultures are grown to mid-log phase, induced for expression, and
cells collected by centrifugation. Cells are resuspended containing
lysozyme and the membranes broken by rapid freeze/thaw cycles, or
by sonication. Cell debris is removed by centrifugation and the
supernatants transferred to 96-tube arrays. The appropriate
affinity matrix is added, protein of interest bound and
nonspecifically bound proteins removed by repeated washing steps
using 12-96 pin suction devices and centrifugation. Alternatively,
magnetic affinity beads and filtration devices can be used
(Qiagen). The proteins are eluted and transferred to a new 96-well
array. Protein concentrations are determined and an aliquot of each
protein is spotted onto a nitrocellulose filter and verified by
Western analysis using an antibody directed against the affinity
tag. The purity of each sample is assessed by SDS-PAGE and
Coomassie staining or mass spectroscopy. Proteins are snap-frozen
and stored at -80.degree. C.
[0121] Saccharomyces cerevisiae allows for core glycosylation and
lipid modifications of proteins. The approach described above for
Escherichia coli can be used with slight modifications for
transformation and cell lysis. Transformation of Saccharomyces
cerevisiae is by lithium-acetate and cell lysis is either by
lyticase digestion of the cell walls followed by freeze-thaw,
sonication or glass-bead extraction. Variations of
post-translational modifications can be obtained by different yeast
strains (i.e. Saccharomyces pombe, Pichia pastoris).
[0122] The advantage of the bacculovirus system or mammalian cells
are the wealth of post-translational modifications that can be
obtained. The bacculo-system requires cloning of viruses, obtaining
high titer stocks and infection of liquid insect cell suspensions
(cells are SF9, SF21). Mammalian cell-based expression requires
transfection and cloning of cell lines. Soluble proteins are
collected from the medium while intracellular or membrane bound
proteins require cell lysis (either detergent solubilization,
freeze-thaw). Proteins can then be purified analogous to the
procedure described for Escherichia coli.
[0123] For in vitro translation the system of choice is Escherichia
coli lysates obtained from protease-deficient and T7 RNA polymerase
overexpressing strains. Escherichia coli lysates provide efficient
protein expression (30-50 .mu.g/ml lysate). The entire process is
carried out in 96-well arrays. Genes of interest are amplified by
PCR using oligonucleotides that contain the gene-specific sequences
containing a T7 RNA polymerase promoter and binding site and a
sequence encoding the affinity tag. Alternatively, an adaptor
protein can be fused to the gene of interest by PCR. Amplified DNAs
can be directly transcribed and translated in the Escherichia coli
lysates without prior cloning for fast analysis. The proteins are
then isolated by binding to an affinity matrix and processed as
described above.
[0124] Alternative systems which may be used include wheat germ
extracts and reticulocyte extracts. In vitro synthesis of membrane
proteins and or post-translationally modified proteins will require
reticulocyte lysates in combination with microsomes.
[0125] In one embodiment of the present invention, the biological
moiety of one reactive site differs from the biological moiety of a
second reactive site on the same device.
[0126] In another embodiment of the present invention, although the
biological moiety of one reactive site is different from that of
another, the two biological moieties are related. In a preferred
embodiment the biological moieties are members of the same protein
family. The different biological moieties may be functionally
related or just suspected of being functionally related. In another
embodiment, however, the function of the biological moieties may
not be entirely known. In these cases, the different biological
moieties may either share a similarity in structure or sequence or
be suspected of sharing a similarity in structure or sequence. In
one embodiment, the different immobilized biological moieties may
simply be fragments of different members of the same protein
family. In another embodiment, the biological moieties may be known
isozymes.
[0127] Examples of protein families preferred in the present
invention include, but are not limited to, receptor families
(examples: growth factor receptors, catecholamine receptors, amino
acid derivative receptors, cytokine receptors, lectins), ligand
families (examples: cytokines, serpins), enzyme families (examples:
proteases, kinases, phosphatases, ras-like GTPases, hydrolases),
and transcription factors (examples: steroid hormone receptors,
heat-shock transcription factors, zinc-finger, leucine-zipper,
homeodomain). In one embodiment, the different biological moieties
are all HIV proteases. In another embodiment, the different
biological moieties are all hepatitis C virus (HCV) proteases.
Other examples of biological moieties of interest include
antibodies and fragments thereof (such as Fab).
[0128] In an alternative embodiment of the invention device, the
biological moieties of different reactive sites are identical to
one another.
[0129] Although proteins, or fragments thereof, are the preferred
biological moieties of the present invention, the immobilized
biological moiety may optionally instead comprise a nucleic acid, a
peptide nucleic acid, a hormone, an antigen, an epitope, or any
small organic molecule which either has or is suspected of having a
physiological function.
[0130] Methods for using the devices of the present invention are
provided by other aspects of the invention. The devices of the
present invention are particularly well-suited for use in
high-throughput drug screening. Other uses include medical
diagnostics and biosensors. In each case a plurality of biological
moieties or drug candidates or analytes can be screened in
parallel.
[0131] In one aspect of the invention, a method for screening a
plurality of biological moieties in parallel for their ability to
interact with a component of a fluid sample is provided. This
method comprises delivering the fluid sample to the reactive sites
of one of the invention devices having a different biological
moiety immobilized on each reactive site and then detecting the
interaction of said component with the immobilized biological
moiety at each reactive site. The invention device is suitable for
assaying both a catalytic reaction of an enzyme, a binding event,
or a translocation by a membrane protein through a lipid
bilayer.
[0132] Possible interactions towards which the present invention
may be directed include, but are not limited to, antibody/antigen,
antibody/hapten, enzyme/substrate, carrier protein/substrate,
lectin/carbohydrate, receptor/hormone, receptor/effector,
protein/DNA, protein/RNA, complementary strands of nucleic acid,
repressor/inducer, or the like.
[0133] One embodiment of the present invention provides for a
method of screening a plurality of biological moieties in parallel
for their ability to react with a component of a fluid sample,
comprising delivering the fluid sample to the reactive sites of a
device of the present invention, wherein each reactive site of the
device comprises a different biological moiety and detecting,
either directly or indirectly, formation of product of the reaction
of the component with the immobilized biological moiety at each
reactive site.
[0134] Another embodiment of the invention provides a method for
screening a plurality of biological moieties in parallel for their
ability to bind a component of a fluid sample. This method
comprises the following steps: delivering the fluid sample to the
reactive sites of the invention device; washing the reactive site
with fluid which does not contain the component in order to elute
unbound component from the reactive sites; and detecting, either
directly or indirectly, the presence, absence, or amount of the
component retained at each reactive site.
[0135] An alternative method for screening a plurality of
biological moieties for their ability to bind a component of a
fluid sample comprises adding a known ligand of the biological
moieties to the fluid sample, delivering the fluid sample to the
reactive sites of the invention device, washing said reactive sites
with fluid that does not contain either the known ligand or the
component in order to elute unbound molecules of the known ligand
and the component, detecting the presence of the known ligand
retained at each reactive site, and comparing retention of the
known ligand detected with retention of the known ligand in the
absence of the component.
[0136] A wide range of detection methods are applicable to this and
other methods of the invention. The invention device can be
interfaced with a means for detection of absorption in the visible
range, chemiluminescence, or fluorescence (including lifetime,
polarization, fluorescence correlation spectroscopy (FCS), and
fluorescence-resonance energy transfer (FRET)). Furthermore,
built-in detectors such as optical waveguides (PCT Publication WO
96/26432 and U.S. Pat. No. 5,677,196), surface plasmons, and
surface charge sensors are compatible with many embodiments of the
invention.
[0137] FIG. 10 shows a schematic diagram of one type of
fluorescence detection unit which may be used to monitor the
interaction of immobilized biological moieties of a microchannel
array with an analyte. In the illustrated detection unit, the
microchannel array device 21 is positioned on a base plate 20.
Light from a 100 W mercury arc lamp 25 is directed through an
excitation filter 24 and onto a beam splitter 23. The light is then
directed through a lens 22, such as a Micro Nikkor 55 mm 1:2:8
lens, and onto the microchannels of the device 21. Fluorescence
emission from the device returns through the lens 22 and the beam
splitter 23. After also passing through an emission filter 26, the
emission is received by a cooled CCD camera 27, such as the
Slowscan TE/CCD-1024SF&SB (Princeton Instruments). The camera
is operably connected to a CPU 28, which is, in turn, operably
connected to a VCR/monitor 29.
[0138] To test the specificity of a drug candidate, its interaction
with multiple members of a protein family is determined. Members of
the protein family are separately immobilized in microchannels. The
drug candidate's ability to interfere with protein activity, such
as binding, catalytic conversion, or translocation of a ligand
through a lipid bilayer, is then determined.
[0139] For instance, to test a drug candidate's ability to
interfere with a protein binding event, the drug candidate and a
known ligand of a member of the protein family that is labeled by a
chemically-conjugated fluorescent moiety, are delivered in a fluid
sample into each microchannel. After a short incubation period, the
microchannels are flushed with fluid which lacks both the drug
candidate and the ligand. The amount of fluorescent ligand
remaining in each of the microchannels (and presumably bound to the
protein molecules of that microchannel) can be detected by using a
fluorescence detector/quantifier with optical access to the
reactive site, either through a transparent or translucent cover or
substrate.
[0140] To test a drug candidate's ability to interfere with a
catalytic conversion of a ligand, drug candidate and ligand are
delivered into the microchannel in a fluid sample and changes in
the chromogenic or fluorescent properties can be detected by using
an optical detector/quantifier with optical access to the reactive
site, either through a transparent or translucent cover or
substrate.
[0141] In a more general sense, the present invention provides for
a method of screening the ability of a drug candidate to inhibit
the reaction of a plurality of members of a protein family with
their substrate, comprising the following steps: combining the drug
candidate and the substrate in a fluid sample; delivering the fluid
sample to the reactive sites of a device of the present invention,
wherein each reactive site of the device comprises a different
member of the protein family; and detecting, either directly or
indirectly, for the inhibition of product formation at each
reactive site.
[0142] To test a drug candidate's ability to interfere with the
translocation of a ligand through a lipid bilayer, drug candidate
and ligand are delivered in a fluid sample to each microchannel.
After a short incubation period the microchannels may be flushed
with fluid lacking ligand and the ligand accumulated between lipid
bilayer and the device is determined by measuring changes in
fluorescence, absorption, or electrical charge.
[0143] An alternative embodiment of the invention provides for a
method for screening a plurality of drug candidates in parallel for
their ability to inhibit a reaction of an enzyme with its
substrate. This method first involves adding the enzyme's substrate
to a plurality of fluid samples, each of which contains at least
one of the drug candidates of interest. Next, each of the fluid
samples is delivered to a reactive site of an invention device,
such as the microchannel array. In this embodiment, each reactive
site bears the immobilized enzyme. Finally, any inhibition of
product formation at each reactive site (due to the presence of the
drug candidate in the solution) is monitored.
[0144] Another aspect of the invention provides a method for
screening a plurality of binding candidates in parallel for their
ability to bind a biological moiety. This method comprises
delivering different fluid samples, each containing at least one of
the binding candidates, to the reactive sites of the invention
device, washing the reactive sites with fluid which does not
contain the binding candidate in order to elute unbound binding
candidates, and detecting, either directly or indirectly, the
presence of said binding candidate retained at each reactive
site.
[0145] An alternative method for screening a plurality of binding
candidates in parallel for their ability to bind a biological
moiety is also provided by the present invention comprises the
following: adding a known ligand of the biological moiety to a
plurality of fluid samples, each of the fluid samples containing at
least one of the binding candidates; delivering a different fluid
sample to each of the reactive sites of the invention device,
wherein multiple reactive sites comprise the same biological
moiety; washing said reactive sites with fluid that contains
neither the known ligand nor a binding candidate in order to elute
unbound molecules of each; detecting the presence of the known
ligand retained at each reactive site; and comparing retention of
the known ligand detected with retention of the known ligand in the
absence of the binding candidate.
[0146] The present invention also provides for a method of pairing
a plurality of proteins with their substrates. In this method, a
fluid sample comprising a substrate of a known enzyme family is
first delivered to the reactive sites of the invention device where
each reactive site of the device comprises a different protein.
Next, any suitable detection means may be used to detect, either
directly or indirectly, for the presence of product formed by the
reaction of the substrate with the protein of each reactive
site.
[0147] In another aspect of the invention, a method for pairing a
plurality of proteins with their ligands is provided. This method
comprises delivering a fluid sample comprising a ligand of a known
protein family to the reactive sites of the invention device;
washing the reactive sites with fluid that does not contain the
ligand to remove unbound ligand; and detecting, either directly or
indirectly, the presence of the ligand retained at each reactive
site.
[0148] The device of the present invention may also be used in a
diagnostic manner. In the diagnostic embodiments of the invention,
the different biological moieties of the reactive sites are not
preferably members of the same protein family. One diagnostic use
of the invention device is a method for detecting in a fluid sample
the presence of a plurality of analytes which react with said
biological moieties. The steps of this method comprise delivering
the fluid sample to the reactive sites of the invention device; and
detecting the interaction of the analyte with the immobilized
biological moiety at each reactive site.
[0149] Another diagnostic method for detecting in a fluid sample
the presence of a plurality of analytes which bind said biological
moieties, comprises the following: delivering the fluid sample to
the reactive sites of the invention device; washing said reactive
sites with an analyte-free fluid to remove unbound analyte; and
detecting, either directly or indirectly, the presence of analyte
retained at each reactive site.
[0150] An alternative embodiment of the invention provides a device
for processing a fluid sample that comprises a substrate, a
plurality of parallel microchannels microfabricated into or onto
said substrate; and a moiety immobilized within at least one of the
parallel microchannels. In the device, the immobilized moiety is
free to interact with a component of the fluid sample. In a
preferred embodiment of this invention, the immobilized moiety is
an immobilized biological moiety. In an especially preferred
embodiment, the immobilized biological moiety is a protein.
[0151] The device may optionally comprise at least 10 parallel
microchannels. In a preferred embodiment, the device comprises from
about 100 to about 500 microchannels. In one embodiment, the device
comprises from about 2 to about 500 parallel microchannels per
cm.sup.2.
[0152] The width of each of the microchannels is optionally between
about 10 .mu.m and about 500 .mu.m. The depth of each of the
microchannels is also optionally between about 10 .mu.m and about
500 .mu.m.
[0153] One embodiment of the device further comprises a cover over
at least a part of each of the microchannels. In one embodiment,
the cover is a glass cover. The volume of each of the covered
microchannels may range from about 5 nanoliters to about 300
nanoliters. In a preferred embodiment, the volume of each of the
covered microchannels is between about 10 nanoliters and about 50
nanoliters.
(c) Examples
[0154] The following specific examples are intended to illustrate
the invention and should not be construed as limiting the scope of
the claims:
Example 1
Fabrication of a Microchannel Array by Bulk Micromachining
[0155] In a preferred embodiment microchannel arrays are fabricated
via standard microstereolithography into the device material (bulk
micromachining). Alternative techniques include
surface-micromachining and LIGA (injection molding). Usually, a
computer-aided design pattern (reflecting the final channel
geometries) is transferred to a photomask using standard
techniques, which is then used to transfer the pattern onto a
silicon wafer coated with photoresist.
[0156] In a typical example, the device ("chip"), with lateral
dimensions of 50.times.15 mm, contains a series of 100 parallel
channels separated with a spacing of 250 .mu.m. Each channel is 5
mm long and has a cross-section of 100.times.100 .mu.m. The channel
volume is 50 nl. 4'' diameter Si(100) wafers (Virginia
Semiconductor) or 4'' diameter Corning 7740 glass wafers are used
as bulk materials. Si(100) wafers are first cleaned in a 5:1:1 DI
water:NH.sub.3:H.sub.2O.sub.2 bath (RCA1, 90.degree. C., 10 min),
followed by a 5:1:1 deionized (DI) water:HCl:H.sub.2O.sub.2 bath
(RCA2, 90.degree. C., 10 min) and finally passivated in 1% aqueous
HF and singed at 150.degree. C. for 30 min. After the wafer has
been spincoated with polymethyl methacrylate PMMA as positive
photoresist and prebaked for 25 minutes at 90.degree. C., it is
exposed using a Karl Suss contact printer and developed according
to standard protocols. The wafer is then dried and postbaked at
110.degree. C. for 25 min. Deep silicon reactive ion etching (RIE)
is used to anisotropically dry-etch the channel features into the
bulk material resulting in high aspect ratio, vertical sidewall
features in the silicon (etch rate 2.5 .mu.m/min). In the next
step, the wafer is primed with a 20 nm thin titanium layer,
followed by a 200 nm thin gold layer both layers deposited using
electron-beam evaporation (5 .ANG./s, Thermionics). After resist
stripping (acetone) and a short plasma treatment, the device is
covered and sealed with a 50 .mu.m thin glass cover (pyrex 7740)
using low-temperature field assisted glass-silicon bonding
resulting in a multichannel array with inlet and outlet ports. The
gold-coated channel walls can then be further chemically modified
to achieve the desired bioreactive and biocompatible properties
(see Example 3, below).
[0157] Additional details of these procedures can be found in the
following references: Madou, Fundamentals of Microfabrication, CRC
Press (1997); Wolf and Tauber, Silicon Processing for the VLSI Era,
Vol. 1: Process Technology, Lattice Press, (1986); and Thomson et
al., Introduction to Microlithography, American Chemical Society,
(1994).
Example 2
Fabrication of a Microchannel Array by Sacrificial
Micromachining
[0158] In sacrificial micromachining, the bulk material is left
essentially untouched. Various thick layers of other materials are
built up by either physical vapor deposition (PVD), plasma-enhanced
chemical vapor deposition (PECVD) or spin coating and selectively
remain behind or are removed by subsequent processing steps. Thus,
the resulting channel walls are chemically different from the
bottom of the channels and the resist material remains as part of
the microdevice. Typical resist materials for sacrificial
micromachining are silicon nitride (Si.sub.3N.sub.4), polysilicon,
thermally grown silicon oxide and organic resists such as
epoxy-based SU-8 and polyimides allowing the formation of high
aspect-ratio features with straight sidewalls.
[0159] In a typical example, the device ("chip"), with lateral
dimensions of 50.times.15 mm, contains a series of 100 parallel
channels separated with a spacing of 250 .mu.m. Each channel is 5
mm long and has a cross-section of 100.times.100 .mu.m. The channel
volume is 50 nl. 4'' diameter Si(100) wafers (Virginia
Semiconductor) or 4'' diameter Corning 7740 glass wafers are used
as bulk materials. Si(100) wafers are first cleaned in a 5:1:1 DI
water:NH.sub.3:H.sub.2O.sub.2 bath (RCA1, 90.degree. C., 10 min),
followed by a 5:1:1 deionized (DI) water:HCl:H.sub.2O.sub.2 bath
(RCA2, 90.degree. C., 10 min) and finally passivated in 1% aqueous
HF and singed at 150.degree. C. for 30 min. Spincoating of the
wafer with EPON SU-8 results in a 100 .mu.m thick film that is
exposed similar to Example 1, above, and developed in a
propyleneglycol-monomethyletheracetate (PGMEA) solution resulting
in a multi-channel structure with high-aspect ratio vertical
sidewalls. Deposition of metal films (20 nm Ti, 200 nm Au) is
carried out as described in Example 1, above. The device is covered
with a 50 .mu.m thin adhesive glass cover. The gold-coated channel
walls can then be further chemically modified to achieve the
desired bioreactive and biocompatible properties (see Example 3,
below).
[0160] Additional details on sacrificial micromachining processes
can be found in Lorenz, et al., Proceedings of MME'96 (Micro
Mechanics Europe), Barcelona, Spain, October 1996, p. 32-35.
Example 3
Synthesis of an Aminoreactive Monolayer Molecule (Following the
Procedure Outlined in Wagner et al., Biophys. J., 1996,
70:2052-2066)
[0161] General. .sup.1H- and .sup.13C-NMR spectra are recorded on
Bruker instruments (100 to 400 MHz). Chemical shifts (.delta.) are
reported in ppm relative to internal standard ((CH.sub.3).sub.4Si,
.delta.=0.00 (1H- and .sup.13C-NMR)). FAB-mass spectra are recorded
on a VG-SABSEQ instrument (Cs.sup.+, 20 keV). Transmission infrared
spectra are obtained as dispersions in KBr on an FTIR Perkin-Elmer
1600 Series instrument. Tin-layer chromatography (TLC) is performed
on precoated silica gel 60 F254 plates (MERCK, Darmstadt, FRG), and
detection was done using Cl.sub.2/toluidine, PdCl.sub.2 and
UV-detection under NH.sub.3-vapor. Medium pressure liquid
chromatography (MPLC) is performed on a Labomatic MD-80 (LABOMATIC
INSTR. AG, Allschwil, Switzerland) using a Buechi column
(460.times.36 mm; BUECHI, Flawil, Switzerland), filled with silica
gel 60 (particle size 15-40 .mu.m) from Merck.
[0162] Synthesis of 11,11'-dithiobis(succinimidylundecanoate)
(DSU). Sodium thiosulfate (55.3 g, 350 mmol) is added to a
suspension of 11-bromo-undecanoic acid (92.8 g, 350 mmol) in 50%
aqueous 1,4-dioxane (1000 ml). The mixture is heated at reflux
(90.degree. C.) for 2 h until the reaction to the intermediate
Bunte salt was complete (clear solution). The oxidation to the
corresponding disulfide is carried out in situ by adding iodine in
portions until the solution retained with a yellow to brown colour.
The surplus of iodine is retitrated with 15% sodium pyrosulfite in
water. After removal of 1,4-dioxane by rotary evaporation the
creamy suspension is filtered to yield product
11,11'-dithiobis(undecanoic acid). Recrystallization from ethyl
acetate/THF provides a white solid (73.4 g, 96.5%): mp 94.degree.
C.; .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.3OD 95: 5): .delta.
2.69 (t, 2H, J=7.3 Hz), 2.29 (t, 2H, J=7.5 Hz), 1.76-1.57 (m, 4H),
and 1.40-1.29 (m, 12H); FAB-MS (Cs.sup.+, 20 keV): m/z (relative
intensity) 434 (100, M.sup.+). Anal. Calcd. for
C.sub.22H.sub.42O.sub.4S.sub.2: C, 60.79; H, 9.74; S, 14.75. Found:
C, 60.95; H, 9.82; S, 14.74. To a solution of
11,11'-dithiobis(undecanoic acid). (1.0 g, 2.3 mmol) in THF (50 ml)
is added N-hydroxysuccinimide (0.575 g, 5 mmol) followed by DCC
(1.03 g, 5 mmol) at 0.degree. C. After the reaction mixture is
allowed to warm to 23.degree. C. and is stirred for 36 h at room
temperature, the dicyclohexylurea (DCU) is filtered. Removal of the
solvent under reduced pressure and recrystallization from
acetone/hexane provides 11,11'-dithiobis(succinimidylundecanoate)
as a white solid. Final purification is achieved by medium pressure
liquid chromatography (9 bar) using silica gel and a 2:1 mixture of
ethyl acetate and hexane. The organic phase is concentrated and
dried in vacuum to afford 11,11'-dithiobis(succinimidylundecanoate)
(1.12 g, 78%): mp 95.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 2.83 (s, 4H), 2.68 (t, 2H, J=7.3 Hz), 2.60 (t, 2H, J=7.5
Hz), 1.78-1.63 (m, 4H), and 1.43-1.29 (m, 12H); FAB-MS (Cs.sup.+,
20 keV): m/z (relative intensity) 514 (100), 628 (86, M.sup.+).
Anal. Calcd. for C.sub.30H.sub.48N.sub.2O.sub.8S.sub.2: C, 57.30;
H, 7.69; N, 4.45; S, 10.20. Found: C, 57.32; H, 7.60; N, 4.39; S,
10.25.
Example 4
Formation of an Aminoreactive Monolayer on Gold (Following the
Procedure of Wagner et al., Biophys. J., 1996, 70:2052-2066)
[0163] Monolayers based on
11,11'-dithiobis(succinimidylundecanoate) (DSU) are deposited on
Au(111) surfaces of microdevices described under Examples 1 and 2
by immersing them into a 1 mM solution of DSU in chloroform at room
temperature for 1 hour. After rinsing with 10 volumes of solvent,
the N-hydroxysuccinimide-terminated monolayer are dried under a
stream of nitrogen and immediately used for protein
immobilization.
Example 5
Expression and Purification of HIV Protease Variants
[0164] The HIV protease (Genebank HIVHXB2CG) is an essential
component of the HIV life cycle, and a major target in anti-viral
therapy. HIV protease is required for the proteolytic processing of
the gag and gag-pol gene products into functional proteins.
Inhibition of HIV protease prevents the production of infectious
viral progeny, and hence further rounds of infection. HIV protease
belongs to the family of aspartic proteases and is a symmetric
homodimer with an active site formed at the interface of the two 99
amino acids long subunits. The core residues in the active site
consist of a conserved tripeptide motif (Asp-Thr-Gly) (Roberts et
al., Science, 1990, 248:358). Resistant variants of HIV protease
have emerged against all inhibitors currently used. Most prevalent
mutations causing resistance individually or in combination are:
L10R, D30N, M46I, L63P, A71V, V82F (Kaplan et al., Proc. Natl.
Acad. Sci., 1994, 91:5597; Ho et al., J. Virol. 1994, 68: 2016;
Condra et al., Nature, 1995, 374:569; Schock et al., J. Biol.
Chem., 1996, 271:31957; Korant and Rizzo, Adv. Exp. Med. Biol.,
1997, 421:279). Additional mutations that preserve protease
activity are systematically generated (Loeb et al., Nature, 1989,
340:397).
[0165] Mutant proteases are generated by PCR mutagenesis (Weiner et
al., Gene, 1994, 151:119) and expressed in Escherichia coli using
two approaches: (i) mutant and wild-type protease cDNAs are cloned
into a Escherichia coli expression vector containing a N-terminal
histidine tag (H.sub.6; Hochuli et al., Biotechnology 1988, 6:1321)
followed by a factor Xa cleavage site, while the stop codon of HIV
protease is replaced by a sequence encoding a lysine tag (K.sub.6)
followed by a stop codon. The resulting fusion protein is purified
from inclusion bodies as described in Wondrak and Louis,
Biochemistry, 1996, 35:12957, and the histidine tag removed by
factor Xa as described in Wu et al., Biochemistry, 1998, 37:4518;
or (ii) mutant and wild-type protease cDNAs are cloned into an
Escherichia coli expression vector creating a fusion between HIV
protease, a tri-glycine linker, glutathione S-transferase (GST) and
a lysine-tag (HIV-GST-K.sub.6). The autoprocessing site F*P at the
carboxy terminus of the HIV protease is changed to F*I to prevent
self-cleavage of the fusion proteins (Louis et al., Eur. J.
Biochem., 1991, 199:36.1). The resulting proteins HIV-GST-K.sub.6
are purified from Escherichia coli lysates by standard
chromatography on glutathione agarose beads and stored in an
amine-free buffer at -80.degree. C. (25 mM HEPES, pH 7.5, 150 mM
NaCl).
Example 6
Immobilization of Fusion Proteins on an Aminoreactive Monolayer
[0166] HIV protease variants, in the form of HIV-GST-K.sub.6, and
GST-K.sub.6 are immobilized to the aminoreactive monolayer surface
of the microchannel device (see Example 4, above). HIV-GST-K.sub.6
and GST-K.sub.6 are diluted to concentrations of 1 .mu.g/ml in 25
mM HEPES buffer (pH 7.5) containing 150 mM NaCl. First, 50 .mu.l of
protein-free buffer is transferred through the channels to hydrate
the monolayer surface. After 5 min of incubation, 10 .mu.l of the
corresponding protein solutions are flushed through the channels to
guarantee total replacement with protein-containing solution.
Immobilization is finished after 30 min at room temperature. The
channels are rinsed with 50 .mu.l immobilization buffer and
subjected to analysis. Each microchannel displays a different
HIV-GST-K.sub.6 variant or control (GST-K.sub.6). Ultrapure water
with a resistance of 18 M.OMEGA.cm is generally used for all
aqueous buffers (purified by passage through a Barnstead
Nanopure.RTM. system).
Example 7
Assay of Protease Activity in Microchannels
[0167] HIV protease requires at least a heptapeptide substrate
(Moore et al., Biochem. Biophys. Res. Commun., 1989, 159:420). To
analyze the activity of the different HIV variants, a continuous
assay based on intra-molecular fluorescence resonance energy
transfer (FRET) is used. A peptide substrate corresponding to the
p17-p24 cleavage site of the viral gag protein (Skalka, Cell, 1989,
56:911) is modified by the addition of an energy-transfer pair
(Geoghegan et al., FEBS Lett., 1990, 262:119): In
Dns-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Trp (Dns-SSQNYPIVW), the Dns
(dansyl) and Trp groups are the N- and C-terminal extensions,
respectively (Geoghegan et al.). Excitation of Trp is at 290 nm,
and emission of Dns is at 575 nm. Cleavage of the peptide at the
Tyr-Pro bond reduces the Dns emission and increases Trp emission at
360 nm. The modified heptapeptide Dns-SSQNYPIVW is prepared as
described (Geoghegan et al.) and analyzed by amino acid analysis,
nuclear magnetic resonance and mass spectrometry. The purity is
checked by HPLC analysis using a Vydac C-4 column and an
acetonitrile gradient in 0.1% TFA. In order to test the activity of
all the HIV variants described above, each microchannel with an
immobilized HIV variant (see Example 6) is filled with 20 .mu.M of
Dns-SSQNYPIVW in 50 mM sodium acetate, pH 5.5, 13% glycerol, 10 mM
DTT. Addition of the substrate to the immobilized proteins leads to
time-dependent intensity changes in the fluorescence emission
spectrum. The 360 nm Trp emission peak progressively will increase
to about 2.5 times its initial intensity, while the Dns group's
emission band (575 nm) will decline in intensity. This intensity
change will be observed in all the channels containing active forms
of the HIV variants. To control for changes in background
fluorescence, GST-K.sub.6 fusion protein is measured in
parallel.
[0168] Competition assays can be carried out to test the
specificity of the proteolysis by the HIV variants. In one assay,
both Dns-SSQNYPIVW and a small organic molecule that is to be
tested for its potential as a drug, is delivered in a 50 mM sodium
acetate, pH 5.5, 13% glycerol, 10 mM DTT solution to each channel.
An organic molecule which acts as an inhibitor for a wide range of
HIV protease variants will diminish the Trp emission peak increase
and the Dns emission band decrease associated with reaction of the
protease with the peptide substrate in a number of the
microchannels. A less desirable drug candidate, on the other hand,
will inhibit the reaction of the HIV protease with the peptide
substrate only in selected microchannels (or none at all).
[0169] Protease inhibitors Sequinavir (Roche), Ritonavir (Abbot) or
Indinavir (Merck) can also be added to the reaction buffer and used
as positive controls for the specificity of inhibition. Sequinavir
will inhibit all the HIV variants except those containing either
the G48V or the L90M mutation. Ritonavir in contrast is unable to
block the activity of the M46I, L63P, A71V, V82F and the 184V
variants. Indinavir has a similar inhibition pattern like Ritonavir
except that the A71V variant is not affected. In addition Indinavir
is not able to decrease the activity of the L10R HIV protease
variant.
[0170] These experiments demonstrate how an HIV-variant
microchannel device may be used to test the inhibitory effect of
small organic molecules on the activity of the HIV protease.
[0171] All documents cited in the above specification are herein
incorporated by reference. Various modifications and variations of
the present invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention which are obvious to those skilled
in the art are intended to be within the scope of the following
claims.
* * * * *