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Claims  |
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What is claimed is:
1. An electronic device adapted to receive a solution comprising:
a substrate,
a plurality of selectively addressable electrodes, the electrodes being disposed upon the substrate and electrically insulated from one another prior to receipt of the solution,
a current source for providing a selective current for the electrodes, individual buffer reservoirs associated with said electrodes, and
individual permeation layers disposed adjacent said individual buffer reservoirs, forming addressable binding locations.
2. The electronic device of claim 1, further comprising a common reservoir for containing solutions including binding entities, reagents, and analytes.
3. The electronic device of claim 1, further comprising attachment layers disposed on said permeation layers, forming addressable binding locations.
4. The electronic device of claim 1, wherein said addressable binding locations are arranged in an array.
5. The electronic device of claim 1, wherein the permeation layer is selected from the group comprising: functionalized hydrophilic gels, membranes, and porous materials.
6. The electronic device of claim 1, further including specific binding entities bound to said addressable binding locations.
7. The electronic device of claim 1, wherein the width of the locations on the device is between 0.5 microns and 2 centimeters.
8. The electronic device of claim 7, wherein the width of the locations on the device is between 100 microns and 5 millimeters.
9. The electronic device of claim 1 wherein the electrodes are formed in part on a printed circuit board.
10. The electronic device of claim 1 wherein the addressable binding location is three dimensional.
11. The electronic device of claim 10 wherein the addressable binding location is in the shape of a tube.
12. The electronic device of claim 10 wherein the addressable binding location is in the shape of a cylinder.
13. The electronic device of claim 1 wherein the addressable binding location is in a hole in the substrate.
14. The electronic device of claim 1 wherein the electrodes are platinum.
15. The electronic device of claim 3 wherein the thickness of the combined permeation layer and attachment layer is in the range from 10 microns to 10 millimeters.
16. An electronic device adapted to receive a solution including electrolytes and one or more charged specific binding entities, the device being adapted to affect the electrophoretic transport of the specific binding entities in the solution to
selected locations on the device surface, the device comprising:
a substrate,
a first electrode, the electrode being supported by the substrate, the electrode being adapted to receive current,
a permeation layer having selective diffusion properties for the passage from the solution to the electrode of the electrolytes relative to the charged specific binding entities, permitting the free transport of counter-ions through the
permeation layer to the electrode, the permeation layer being disposed adjacent the first electrode, the permeation layer and the first electrode being separated by a buffer reservoir,
an attachment layer adjacent to the permeation layer with selective binding properties for the specific binding entities, and
a second electrode, the second electrode being electrically insulated from the first electrode prior to the application of the solution.
17. The electronic device of claim 16 further including a permeation layer disposed adjacent the second electrode, the permeation layer having selective diffusion properties for the passage from the solution to the second electrode of the
electrolytes relative to the charged specific binding entities, permitting the free transport of counter-ions through the permeation layer to the second electrode, and an attachment layer adjacent to the permeation layer adjacent to the second electrode
with selective binding properties for the specific binding entities.
18. The electronic device of claim 16, further including a current source.
19. The electronic device of claim 16 wherein the permeation layer is aminopropyltriethoxy silane.
20. The electronic device of claim 16, further comprising a common reservoir for containing solutions including binding entities, reagents, and analytes.
21. The electronic device of claim 16, wherein the permeation layer is selected from the group consisting of: functionalized hydrophilic gels, membranes and porous materials.
22. The electronic device of claim 16, further including specific binding entities bound to said addressable binding locations.
23. The electronic device of claim 16 wherein the substrate includes a printed circuit board.
24. An electronic device adapted to receive a solution including electrolytes and one or more charged specific binding entities, the device being adapted to affect the electrophoretic transport of the specific binding entities in the solution to
selected locations on the device surface, the device comprising:
a substrate,
a first electrode, the electrode being supported by the substrate, the electrode being adapted to receive current,
a permeation layer aminopropyltriethoxy silane, the permeation layer being disposed adjacent the first electrode,
an attachment layer adjacent to the permeation layer with selective binding properties for the specific binding entities, and
a second electrode, the second electrode being electrically insulated from the first electrode prior to the application of the solution.
25. The electronic device of claim 24, wherein the permeation layer has selective diffusion properties for the passage from the solution to the electrode of the electrolytes relative to the charged specific binding entities, permitting the free
transport of counter-ions through the permeation layer to the electrode.
26. The electronic device of claim 25 wherein the aminopropyltriethoxy silane forms a combined permeation layer and attachment layer.
27. The electronic device of claim 25 or 26 wherein the permeation layer is an aminopropyltriethoxy silane derivitized metal oxide.
28. The electronic device of claim 25 or 26 wherein the permeation layer is an aminopropyltriethoxy silane derivitized silicon oxide.
29. The electronic device of claim 25 or 26 wherein the permeation layer is an aminopropyltriethoxy silane derivitized aluminum oxide.
30. The electronic device of claim 25 or 26 wherein the permeation layer includes metal with hydroxyl groups.
31. The electronic device of claim 25 or 26 wherein the permeation layer includes silicon with hydroxyl groups.
32. The electronic device of claim 24, further including a permeation layer disposed adjacent the second electrode, the permeation layer having selective diffusion properties for the passage from the solution to the second electrode of the
electrolytes relative to the charged specific binding entities, permitting the free transport of counter-ions through the permeation layer to the second electrode, and an attachment layer adjacent to the permeation layer adjacent to the second electrode
with selective binding properties for the specific binding entities.
33. The electronic device of claim 24, wherein the substrate comprises a circuit board.
34. The electronic device of claim 24, further including a current source.
35. The electronic device of claim 24 wherein the second electrode is an unaddressed micro-location.
36. An electronic device adapted to receive a solution including electrolytes and one or more charged specific binding entities, the device being adapted to affect the electrophoretic transport of the specific binding entities in the solution to
selected locations on the device surface, the device comprising:
a substrate,
a first electrode, the electrode being supported by the substrate, the electrode being adapted to receive current,
a permeation layer having selective diffusion properties for the passage from the solution to the electrode of the electrolytes relative to the charged specific binding entities, permitting the free transport of counter-ions through the
permeation layer to the electrode, the permeation layer being disposed adjacent the first electrode,
an attachment layer adjacent to the permeation layer with selective binding properties for the specific binding entities, and
an unaddressed microlocation, the first electrode being electrically insulated from the unaddressed microlocation prior to the application of the solution.
37. The electronic device of claim 36 wherein the unaddressed microlocation functions to store reagents.
38. The electronic device of claim 36 wherein the unaddressed microlocation functions to hold reactants.
39. The electronic device of claim 36 wherein the unaddressed microlocation functions to hold analytes.
40. The electronic device of claim 36 wherein the unaddressed microlocation functions to dispose of one or more of the group comprising: excess reactants, analytes or interfering components in the solution.
41. The electronic device of claim 36 wherein the unaddressed microlocation is a plain microlocation.
42. The electronic device of claim 41 wherein the plain microlocation functions to dispose of one or more of the group comprising: excess reactants, analytes or interfering components in the solution.
43. The electronic device of claim 36 further including a second electronic device, the second electronic device including at least a second electrode.
44. The electronic device of claim 36 wherein the second electrode of the second electronic device further includes a permeation layer having selective diffusion properties for the passage from the solution to the electrode of the electrolytes
relative to the charged specific binding entities, permitting the free transport of counter-ions through the permeation layer to the electrode, the permeation layer being disposed adjacent the second electrode.
45. The electronic device of claim 43 wherein the second electrode of the second electronic device functions to store reagents.
46. The electronic device of claim 43 wherein the second electrode of the second electronic device functions to hold reactants.
47. The electronic device of claim 43 wherein the second electrode of the second electronic device functions to hold analytes. |
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Claims |
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Description |
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FIELD OF THE INVENTION
This invention pertains to the design, fabrication, and uses of a self-addressable, self-assembling microelectronic system which can actively carry out and control multi-step and multiplex reactions in microscopic formats. In particular, these
reactions include molecular biological reactions, such as nucleic acid hybridizations, antibody/antigen reactions, clinical diagnostics, and biopolymer synthesis.
BACKGROUND OF THE INVENTION
Molecular biology comprises a wide variety of techniques for the analysis of nucleic acid and protein, many of which form the basis of clinical diagnostic assays. These techniques include nucleic acid hybridization analysis, restriction enzyme
analysis, genetic sequence analysis, and separation and purification of nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York, 1989).
Most molecular biology techniques involve carrying out numerous operations (e.g., pipetting) on a large number of samples. They are often complex and time consuming, and generally require a high degree of accuracy. Many a technique is limited
in its application by a lack of sensitivity, specificity, or reproducibility. For example, problems with sensitivity and specificity have so far limited the application of nucleic acid hybridization.
Nucleic acid hybridization analysis generally involves the detection of a very small numbers of specific target nucleic acids (DNA or RNA) with probes among a large amount of non-target nucleic acids. In order to keep high specificity,
hybridization is normally carried out under the most stringent condition, achieved through a combination of temperature, salts, detergents, solvents, chaotropic agents, and denaturants.
Multiple sample nucleic acid hybridization analysis has been conducted on a variety of filter and solid support formats (see G. A. Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu, L. Grossmam, K. Moldave, Eds., Academic Press, New
York, Chapter 19, pp. 266-308, 1985). One format, the so-called "dot blot" hybridization, involves the non-covalent attachment of target DNAs to a filter, which are subsequently hybridized with a radioisotope labeled probe(s). "Dot blot" hybridization
gained wide-spread use, and many versions were developed (see M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridization--A Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington DC, Chapter 4, pp. 73-111, 1985). It has
been developed for multiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for the detection of overlapping clones and the construction of genomic maps (G. A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15,
1993).
Another format, the so-called "sandwich" hybridization, involves attaching oligonucleotide probes covalently to a solid support and using them to capture and detect multiple nucleic acid targets. (M. Ranki et al., Gene, 21, pp. 77-85, 1983; A.
M. Palva, T. M. Ranki, and H. E. Soderlund, in UK Patent Application GB 2156074A, Oct. 2, 1985; T. M. Ranki and H. E. Soderlund in U.S. Pat. No. 4,563,419, Jan. 7, 1986; A. D. B. Malcolm and J. A. Langdale, in PCT WO 86/03782, Jul. 3, 1986; Y.
Stabinsky, in U.S. Pat. No. 4,751,177, Jan. 14, 1988; T. H. Adams et al., in PCT WO 90/01564, Feb. 22, 1990; R. B. Wallace et al. 6 Nucleic Acid Res. 11, p. 3543, 1979; and B. J. Connor et al., 80 Proc. Natl. Acad. Sci. USA pp. 278-282, 1983).
Using the current nucleic acid hybridization formats and stringency control methods, it remains difficult to detect low copy number (i.e., 1-100,000) nucleic acid targets even with the most sensitive reporter groups (enzyme, fluorophores,
radioisotopes, etc.) and associated detection systems (fluorometers, luminometers, photon counters, scintillation counters, etc.).
This difficulty is caused by several underlying problems associated with direct probe hybridization. The first and the most serious problem relates to the stringency control of hybridization reactions. Hybridization reactions are usually
carried out under the most stringent conditions in order to achieve the highest degree of specificity. Methods of stringency control involve primarily the optimization of temperature, ionic strength, and denaturants in hybridization and subsequent
washing procedures. Unfortunately, the application of these stringency conditions causes a significant decrease in the number of hybridized probe/target complexes for detection.
The second problem relates to the high complexity of DNA in most samples, particularly in human genomic DNA samples. When a sample is composed of an enormous number of sequences which are closely related to the specific target sequence, even the
most unique probe sequence has a large number of partial hybridizations with non-target sequences.
The third problem relates to the unfavorable hybridization dynamics between a probe and its specific target. Even under the best conditions, most hybridization reactions are conducted with relatively low concentrations of probes and target
molecules. In addition, a probe often has to compete with the complementary strand for the target nucleic acid.
The fourth problem for most present hybridization formats is the high level of non-specific background signal. This is caused by the affinity of DNA probes to almost any material.
These problems, either individually or in combination, lead to a loss of sensitivity and/or specificity for nucleic acid hybridization in the above described formats. This is unfortunate because the detection of low copy number nucleic acid
targets is necessary for most nucleic acid-based clinical diagnostic assays.
Because of the difficulty in detecting low copy number nucleic acid targets, the research community relies heavily on the polymerase chain reaction (PCR) for the amplification of target nucleic acid sequences (see M. A. Innis et al., PCR
Protocols: A Guide to Methods and Applications, Academic Press, 1990). The enormous number of target nucleic acid sequences produced by the PCR reaction improves the subsequent direct nucleic acid probe techniques, albeit at the cost of a lengthy and
cumbersome procedure.
A distinctive exception to the general difficulty in detecting low copy number target nucleic acid with a direct probe is the in-situ hybridization technique. This technique allows low copy number unique nucleic acid sequences to be detected in
individual cells. In the in situ format, target nucleic acid is naturally confined to the area of a cell (.about.20-50 .mu.m.sup.2) or a nucleus (.about.10 .mu.m.sup.2) at a relatively high local concentration. Furthermore, the probe/target
hybridization signal is confined to a morphologically distinct area; this makes it easier to distinguish a positive signal from artificial or non-specific signals than hybridization on a solid support.
Mimicking the in-situ hybridization, new techniques are being developed for carrying out multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp.
1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. These hybridization formats are micro-scale versions
of the conventional "dot blot" and "sandwich" hybridization systems.
The micro-formatted hybridization can be used to carry out "sequencing by hybridization" (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of all possible n-nucleotide oligomers
(n-mers) to identify n-mers in an unknown DNA sample, which are subsequently aligned by algorithm analysis to produce the DNA sequence (R. Drmanac and R. Crkvenjakov, Yugoslav Patent Application #570/87, 1987; R. Drmanac et al., 4 Genomics, 114, 1989;
Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1991; and R. Drmanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13, 1993).
There are two formats for carrying out SBH. The first format involves creating an array of all possible n-mers on a support, which is then hybridized with the target sequence. The second format involves attaching the target sequence to a
support, which is sequentially probed with all possible n-mers. Both formats have the fundamental problems of direct probe hybridizations and additional difficulties related to multiplex hybridizations.
Southern, United Kingdom Patent Application GB 8810400, 1988; E. M. Southern et al., 13 Genomics 1008, 1992, proposed using the first format to analyze or sequence DNA. Southern identified a known single point mutation using PCR amplified
genomic DNA. Southern also described a method for synthesizing an array of oligonucleotides on a solid support for SBH. However, Southern did not address how to achieve optimal stringency condition for each oligonucleotide on an array.
Fodor et al., 364 Nature, pp. 555-556, 1993, used an array of 1,024 8-mer oligonucleotides on a solid support to sequence DNA. In this case, the target DNA was a fluorescently labeled single-stranded 12-mer oligonucleotide containing only
nucleotides A and C. 1 pmol (.about.6.times.10.sup.11 molecules) of the 12-mer target sequence was necessary for the hybridization with the 8-mer oligomers on the array. The results showed many mismatches. Like Southern, Fodor et al., did not address
the underlying problems of direct probe hybridization, such as stringency control for multiplex hybridizations. These problems, together with the requirement of a large quantity of the simple 12-mer target, indicate severe limitations to this SBH
format.
Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used the second format to sequence several short (116 bp) DNA sequences. Target DNAs were attached to membrane supports ("dot blot" format). Each filter was sequentially hybridized with
272 labeled 10-mer and 11-mer oligonucleotides. A wide range of stringency condition was used to achieve specific hybridization for each n-mer probe; washing times varied from 5 minutes to overnight, and temperatures from 0.degree. C. to 16.degree. C.
Most probes required 3 hours of washing at 16.degree. C. The filters had to be exposed for 2 to 18 hours in order to detect hybridization signals. The overall false positive hybridization rate was 5% in spite of the simple target sequences, the reduced
set of oligomer probes, and the use of the most stringent conditions available.
Fodor et al., 251 Science 767-773, 1991, used photolithographic techniques to synthesize oligonucleotides on a matrix. Pirrung et al., in U.S. Pat. No. 5,143,854, Sep. 1, 1992, teach large scale photolithographic solid phase synthesis of
polypeptides in an array fashion on silicon substrates.
In another approach of matrix hybridization, Beattie et al., in The 1992 San Diego Conference: Genetic Recognition, November, 1992, used a microrobotic system to deposit micro-droplets containing specific DNA sequences into individual
microfabricated sample wells on a glass substrate. The hybridization in each sample well is detected by interrogating miniature electrode test fixtures, which surround each individual microwell with an alternating current (AC) electric field.
Regardless of the format, current micro-scale DNA hybridization and SBH approaches do not overcome the underlying physical problems associated with direct probe hybridization reactions. They require very high levels of relatively short
single-stranded target sequences or PCR amplified DNA, and produce a high level of false positive hybridization signals even under the most stringent conditions. In the case of multiplex formats using arrays of short oligonucleotide sequences, it is not
possible to optimize the stringency condition for each individual sequence with any conventional approach because the arrays or devices used for these formats can not change or adjust the temperature, ionic strength, or denaturants at an individual
location, relative to other locations. Therefore, a common stringency condition must be used for all the sequences on the device. This results in a large number of non-specific and partial hybridizations and severely limits the application of the
device. The problem becomes more compounded as the number of different sequences on the array increases, and as the length of the sequences decreases. This is particularly troublesome for SBH, which requires a large number of short oligonucleotide
probes.
Nucleic acids of different size, charge, or conformation are routinely separated by electrophoresis techniques which can distinguish hybridization species by their differential mobility in an electric field. Pulse field electrophoresis uses an
arrangement of multiple electrodes around a medium (e.g., a gel) to separate very large DNA fragments which cannot be resolved by conventional gel electrophoresis systems (see R. Anand and E. M. Southern in Gel Electrophoresis of Nucleic Acids--A
Practical Approach, 2 ed., D. Rickwood and B. D. Hames Eds., IRL Press, New York, pp. 101-122, 1990).
Pace, U.S. Pat. No. 4,908,112, Mar. 13, 1990, teaches using micro-fabrication techniques to produce a capillary gel electrophoresis system on a silicon substrate. Multiple electrodes are incorporated into the system to move molecules through
the separation medium within the device.
Soane and Soane, U.S. Pat. No. 5,126,022, Jun. 30, 1992, teach that a number of electrodes can be used to control the linear movement of charged molecules in a mixture through a gel separation medium contained in a tube. Electrodes have to be
installed within the tube to control the movement and position of molecules in the separation medium.
Washizu, M. and Kurosawa, O., 26 IEEE Transactions on Industry Applications 6, pp. 1165-1172, 1990, used high-frequency alternating current (AC) fields to orient DNA molecules in electric field lines produced between microfabricated electrodes.
However, the use of direct current (DC) fields is prohibitive for their work. Washizu 25 Journal of Electrostatics 109-123, 1990, describes the manipulation of cells and biological molecules using dielectrophoresis. Cells can be fused and biological
molecules can be oriented along the electric fields lines produced by AC voltages between the microelectrode structures. However, the dielectrophoresis process requires a very high frequency AC (1 MHz) voltage and a low conductivity medium. While these
techniques can orient DNA molecules of different sizes along the AC field lines, they cannot distinguish between hybridization complexes of the same size.
As is apparent from the preceding discussion, numerous attempts have been made to provide effective techniques to conduct multi-step, multiplex molecular biological reactions. However, for the reasons stated above, these techniques have been
proved deficient. Despite the long-recognized need for effective technique, no satisfactory solution has been proposed previously.
SUMMARY OF THE INVENTION
The present invention relates to the design, fabrication, and uses of a self-addressable self-assembling microelectronic system and device which can actively carry out controlled multi-step and multiplex reactions in microscopic formats. These
reactions include, but are not limited to, most molecular biological procedures, such as nucleic acid hybridization, antibody/antigen reaction, and related clinical diagnostics. In addition, the device is able to carry out multi-step combinational
biopolymer synthesis, including, but not limited to, the synthesis of different oligonucleotides or peptides at specific micro-locations.
The device is fabricated using both microlithographic and micro-machining techniques. The device has a matrix of addressable microscopic locations on its surface; each individual micro-location is able to electronically control and direct the
transport and attachment of specific binding entities (e.g., nucleic acids, antibodies) to itself. All micro-locations can be addressed with their specific binding entities. Using this device, the system can be self-assembled with minimal outside
intervention.
The device is able to control and actively carry out a variety of assays and reactions. Analytes or reactants can be transported by free field electrophoresis to any specific micro-location where the analytes or reactants are effectively
concentrated and reacted with the specific binding entity at said micro-location. The sensitivity for detecting a specific analyte or reactant is improved because of the concentrating effect. Any un-bound analytes or reactants can be removed by
reversing the polarity of a micro-location. Thus, the device also improves the specificity of assays and reactions.
The device provides independent stringency control for hybridization reactions at specific micro-locations. Thus all the micro-locations on the matrix can have different stringency conditions at the same time, allowing multiple hybridizations to
be conducted at optimal conditions.
The device also facilitates the detection of hybridized complexes at each micro-location by using an associated optical (fluorescent or spectrophotometric) imaging detector system or an integrated sensing component.
In addition, the active nature of the device allows complex multi-step reactions to be carried out with minimal outside physical manipulations. If desired, a master device addressed with specific binding entities can be electronically replicated
or copied to another base device.
Thus, the disclosed device can carry out multi-step and multiplex reactions with complete and precise electronic control, preferably with a micro-processor. The rate, specificity, and sensitivity of multi-step and multiplex reactions are greatly
improved at specific micro-locations of the disclosed device.
The present invention overcomes the limitations of the arrays and devices for multi-sample hybridizations described in the background of the invention. Previous methods and devices are functionally passive regarding the actual hybridization
process. While sophisticated photolithographic techniques were used to make an array, or microelectronic sensing elements were incorporated for detection, previous devices did not control or influence the actual hybridization process. They are not
designed to actively overcome any of the underlying physical problems associated with hybridization reactions.
This invention may utilize micro-locations of any size or shape consistent with the objective of the invention. In the preferred embodiment of the invention, micro-locations in the sub-millimeter range are used.
By "specific binding entity" is generally meant a biological or synthetic molecule that has specific affinity to another molecule, through covalent bonding or non-covalent bonding. Preferably, a specific binding entity contains (either by nature
or by modification) a functional chemical group (primary amine, sulfhydryl, aldehyde, etc.), a common sequence (nucleic acids), an epitope (antibodies), a hapten, or a ligand, that allows it to covalently react or non-covalently bind to a common
functional group on the surface of a micro-location. Specific binding entities include, but are not limited to: deoxyribonucleic acids (DNA), ribonucleic acids (RNA), synthetic oligonucleotides, antibodies, proteins, peptides, lectins, modified
polysaccharides, synthetic composite macromolecules, functionalized nanostructures, synthetic polymers, modified/blocked nucleotides/nucleosides, modified/blocked amino acids, fluorophores, chromophores, ligands, chelates and haptens.
By "stringency control" is meant the ability to discriminate specific and non-specific binding interactions.
Thus, in a first aspect, the present invention features a device with an array of electronically self-addressable microscopic locations. Each microscopic location contains an underlying working direct current (DC) micro-electrode supported by a
substrate. The surface of each micro-location has a permeation layer for the free transport of small counter-ions, and an attachment layer for the covalent coupling of specific binding entities.
By "array" or "matrix" is meant an arrangement of locations on the device. The locations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats. The number of locations can range from several to at least
hundreds of thousands.
In a second aspect, this invention features a method for transporting the binding entity to any specific microlocation on the device. When activated, a micro-location can affect the free field electrophoretic transport of any charged
functionalized specific binding entity directly to itself. Upon contacting the specific micro-location, the functionalized specific binding entity immediately becomes covalently attached to the attachment layer surface of that specific micro-location.
Other micro-locations can be simultaneously protected by maintaining them at the opposite potential to the charged molecules. The process can be rapidly repeated until all the micro-locations are addressed with their specific binding entities.
By "charged functionalized specific binding entity" is meant a specific binding entity that is chemically reactive (i.e., capable of covalent attachment to a location) and carrying a net change (either positive or negative).
In a third aspect, this inventions features a method for concentrating and reacting analytes or reactants at any specific micro-location on the device. After the attachment of the specific binding entities, the underlying microelectrode at each
micro-location continues to function in a direct current (DC) mode. This unique feature allows relatively dilute charged analytes or reactant molecules free in solution to be rapidly transported, concentrated, and reacted in a serial or parallel manner
at any specific micro-locations which are maintained at the opposite charge to the analyte or reactant molecules. Specific micro-locations can be protected or shielded by maintaining them at the same charge as the analytes or reactants molecules. This
ability to concentrate dilute analyte or reactant molecules at selected micro-locations greatly accelerates the reaction rates at these micro-locations.
When the desired reaction is complete, the microelectrode potential can be reversed to remove non-specific analytes or unreacted molecules from the micro-locations.
Specific analytes or reaction products may be released from any micro-location and transported to other locations for further analysis; or stored at other addressable locations; or removed completely from the system.
The subsequent analysis of the analytes at the specific micro-locations is also greatly improved by the ability to repulse non-specific entities from these locations.
In a fourth aspect, this invention features a method for improving stringency control of nucleic acid hybridization reactions, comprising the steps of:
rapidly concentrating dilute target DNA and/or probe DNA sequences at specific micro-location(s) where hybridization is to occur;
rapidly removing non-specifically bound ta | | |
