CA2393766A1 - Column and row addressable high density biochip array - Google Patents
Column and row addressable high density biochip array Download PDFInfo
- Publication number
- CA2393766A1 CA2393766A1 CA002393766A CA2393766A CA2393766A1 CA 2393766 A1 CA2393766 A1 CA 2393766A1 CA 002393766 A CA002393766 A CA 002393766A CA 2393766 A CA2393766 A CA 2393766A CA 2393766 A1 CA2393766 A1 CA 2393766A1
- Authority
- CA
- Canada
- Prior art keywords
- conductive
- electrode
- probes
- target molecule
- impedance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00279—Features relating to reactor vessels
- B01J2219/00306—Reactor vessels in a multiple arrangement
- B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
- B01J2219/00315—Microtiter plates
- B01J2219/00317—Microwell devices, i.e. having large numbers of wells
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
- B01J2219/00511—Walls of reactor vessels
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00659—Two-dimensional arrays
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00698—Measurement and control of process parameters
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00702—Processes involving means for analysing and characterising the products
- B01J2219/00704—Processes involving means for analysing and characterising the products integrated with the reactor apparatus
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00718—Type of compounds synthesised
- B01J2219/0072—Organic compounds
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Electrochemistry (AREA)
- Composite Materials (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Materials Engineering (AREA)
- Crystallography & Structural Chemistry (AREA)
- Biotechnology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
- Physical Or Chemical Processes And Apparatus (AREA)
Abstract
The present invention provides a method and apparatus (9) comprising a platform for a column-and-row-addressable high-density biochip array. The apparatus (9) can be used as a high-density biochip array for electronic or electrochemical detection of molecular interactions between probe molecules bound to defined regions of the array and target molecules exposed to the array.
Description
COLUMN AND ROW ADDRESSABLE HIGH DENSITY BIOCHIP ARRAY
BACKGROUND OF THE INVENTION
1. Field of the Invention This invention relates to the detection of biomolecules. Specifically, the invention relates to electronic or electrochemical detection of biomolecules using biochip arrays. In particular, the invention provides an apparatus comprising a platform for a column-and-row addressable, high-density, enhanced-sensitivity biochip array, and methods of use thereof.
BACKGROUND OF THE INVENTION
1. Field of the Invention This invention relates to the detection of biomolecules. Specifically, the invention relates to electronic or electrochemical detection of biomolecules using biochip arrays. In particular, the invention provides an apparatus comprising a platform for a column-and-row addressable, high-density, enhanced-sensitivity biochip array, and methods of use thereof.
2. Background of the Invention A number of commonly utilized biological applications, including diagnoses of genetic disease, sequence-polymorphisms, analyses of gene expression, and studies of receptor-ligand interactions, rely on the ability to readily detect events related to probe-target interactions. In the past decades, autoradiography and fluorescence detection technologies have been used extensively in the molecular detection area.
The use of radioactivity to track molecules, however, presents serious health risks and requires adherence to burdensome regulatory procedures. Precautions must be taken by the user when using radiolabeled materials to avoid exposure to and contact with radioisotopes. Fluorescence technologies also require "labeling" to link the fluorescence marker to a biologically-relevant material, so that molecular interactions (such as nucleic acid hybridization or ligand/receptor binding) can be detected. Linkage of a fluorescent tag to a biomolecule inevitably increases the complexity of such molecules and can adversely affect probe/target interactions. In addition, fluorescence labeling is expensive, labor intensive and time consuming. This leads to increases in experimental cost (by requiring use of additional reagents, expensive hardware, and special equipment) and difficulty (resulting from the use of complicated procedures for handling and disposing of experimental byproducts). Furthermore, experimental reagents containing either radioactive or fluorescence tags often are of limited usefulness (for example, due to the radiochemical half life of the radioisotope, or due to light sensitivity of the fluorescence label).
In contrast, electronic or electrochemical detection processes are based on interactions between probe molecules on an electrode and target molecules in the detection solution that are detected as alterations in the electrical properties on the electrode.
Electronic or electrochemical detection eliminates many of the disadvantages inherent in using radioactive or fluorescent labels to discern molecular interactions.
More importantly, electronic or electrochemical detection devices can be made portable, as has been demonstrated in the case of widely available glucose sensors. Electrical and electrochemical detection devices thus provide an alternative molecular detection means that is safe, inexpensive, unobtrusive, and sensitive.
Electronic or electrochemical detection methods provide an attractive alternative to autoradiography or optical detection for identifying molecular interactions.
In the prior art, electrochemical detection of biological molecules (hereinafter, "biomolecules") has generally been achieved by one of two methods. The first is selective modification at specific sites of a biomolecule (such as a nucleic acid or protein) with redox active moieties such as transition metal complexes. The second approach is intercalation of redox-active moieties, e.g. into duplex DNA strands. In addition, in the prior art most detection schemes have been carried out using either a single electrode or (at most) a few electrodes (typically, more electrodes were used for experimental redundancy, i.e., in order to improve the accuracy of the result, rather than to increase experimental throughput).
Meade et al., in U.S. Patent Nos. 5,591,578, 5,705,348, 5,780,234 and 5,770,369, disclosed methods of detecting a target sequence in a nucleic acid by hybridizing a target sequence with a single stranded nucleic acid that was modified with redox active moieties such as transition metal complexes.
International Patent Application, Publication No. WO 97/01646 teaches a method of detecting a nucleic acid by oxidizing at least one preselected base (e.g., adenine or guanine) with a transition metal complex A significant disadvantage of the electronic or electrochemical detection devices known in the prior art is that these devices use low-density arrays. For example, in U.S.
Patent Nos. 5,670,322 and 5,532,128, Egger et. al. disclosed an apparatus for identifying biomolecular species within a sample substance using an array having a plurality of test sites upon which the sample was applied. Each test site had at least one electrode attached thereto for coupling with a second electrode surrounding the test site to form a capacitor in conjunction with the sample substance. Since the second electrode was preferably made of a ring located outside the array and also acted to contain the sample solution, Egger's array required a large amount of sample solution (i.e., enough to cover the area within the ring) in order for the array to function. More importantly, Egger's array could not be made row and column (x-y) addressable, limiting the density of the test sites in the array and thereby limiting the usefulness of this apparatus.
In U.S. Patent No. 5,653,939, Hollis et al. disclosed an x-y addressable array where test sites were composed of digitated electrodes located on a side bridge that was connected to both the x and y addressable conductive leads. However, the array of Hollis et al. is not practical to fabricate since the test sites are designed to bridge the x-and-y addressable conductive leads that are on two different planes with an insulating layer in-between.
Thus, there remains a need in this art for detecting molecular interactions by electronic or electrochemical means using high-density, row-and-column addressable arrays. In particular, the need is for an x-y addressable array that can be easily and cost-effectively fabricated, and that reduces the cost of performing various analyses, while increasing the effectiveness and utility thereof.
SUMMARY OF THE INVENTION
This invention provides an apparatus for electronic biomolecule detection using a column-and-row (x-y) addressable, high-density biochip array and methods of use thereof.
Specifically, the apparatus facilitates electronic or electrochemical detection of molecular interactions between probe molecules bound to defined regions of a high-density addressable array and target molecules in a solution that is exposed to the array. The apparatus comprises a multiplicity of individual well structures, each said well further comprising two electrodes that can be individually addressed by applying an electric signal specifically to a particular address (well) in the array. In preferred embodiments, the bottom of the well comprises one electrode surface, while the second electrode surrounds the top of the well. Probe molecules include but are not limited to oligonucleotides, nucleic acids (DNA, RNA, etc), proteins, antibodies and peptides that are immobilized at a specific address comprising a well in the array.
Immobilization of such species is accomplished by direct anchoring of the probe molecules on the electrode surface, preferably by attaching the probe molecules onto a supporting matrix on the surface of the electrodes. In the practice of the methods of the invention, the immobilized probe molecules are exposed to a solution containing an intended target molecule, for a time and under conditions sufficient for the probe molecules to bind to the target. An electrical signal is then applied to each of the individual well structures comprising the array. A change in the detected electrical signal in the presence of the solution (compared with the electrical signal detected in the absence of the solution) is used to determine whether a binding event between the probe and target has occurred at a particular address in the array.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.
DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates a schematic representation of a cross-section view of the device platform.
Figure 2 illustrates a schematic representation of a top view of the device platform.
Figure 3 is a schematic diagram of the row/column configuration of a high-density array useful in the practice of the invention.
Figure 4 is a photograph of an x-y addressable array of the invention.
Figures SA, SB and SC are masks for depositing electrode and insulating layers in the x-y addressable arrays of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides an apparatus for electronic or electrochemical detection of biomolecules. For the purposes of this invention, the term "biomolecule" is intended to encompass biologically-derived molecules that interact specifically with one another. Non-limiting examples of such biomolecules are complementary nucleic acid strands, ligand/receptor, agonist/receptor and antagonist/receptor pairs, antigens and their cognate antibodies, enzyme/substrate and enzyme/inhibitor combinations. In general, the biomolecules of the invention comprise a binding pair, whereby there is a specific interaction between each member of the pair. As used herein, one member of the pair is conveniently termed a "target" and the other a "probe." As used herein, "probe"
molecules are preferably bound to a solid substrate and "target" molecules comprise a sample to be tested for the presence, amount or concentration of the "target."
Target molecules can be any of these biomolecules, most preferably wherein at least one of the target molecules specifically interacts with one of the probe molecules.
In preferred embodiments, the probe molecules are oligonucleotides.
Oligonucleotide probes of length 5 to 1000 basepairs (bp), more preferably 5 to 100bp and most preferably about 5 to 40bp, can be attached to the attachment medium.
Targets include PCR amplicons, genomic DNA, cDNA and synthetic and cellular RNA. For protein binding devices, probes can be oligonucleotides such as aptamers or other oligonucleotides having well-defined secondary structure that will bind to proteins.
Alternatively, peptides, antibodies or antigens can be immobilized to perform binding assays.
The present invention provides an apparatus for electronic or electrochemical detection of biomolecules using a row-and-column ("x-y") addressable array having a plurality of addressable sites to which a target sample is applied, and methods of use thereof. Each addressable site comprises at least two electrodes that are connected to two conductive lead lines that can be addressed in a x-y coordination fashion. The addressable site is preferably a well structure as defined herein wherein the bottom of the well comprises the surface of one electrode, and the top of the well comprises the second electrode. In alternative embodiments, each said well structure further comprises at least one additional electrode, preferably a reference electrode, positioned between the top and bottom of the well. In more preferred embodiments, the devices of the invention comprise at least two electrodes, and a multiplicity of probe molecules immobilized in proximity to the electrodes, wherein the probe molecules are preferably immobilized at the surface of at least one of the electrodes.
Device embodiments of the invention are useful for performing methods for biomolecule detection by either electrochemical or electronic means. As used herein, the term "electrochemical detection" is intended to encompass methods based on oxidation/reduction (redox) processes induced by electron transfer between electrodes, most preferably mediated by an electrochemical reporter group attached to the probe moiety, the target moiety, or both. As used herein, the term "electronic detection" is intended to encompass methods that rely on impedance changes (such as resistance, capacitance and inductance) due to differences in electronic state occupancy in the biomolecules in the bound and unbound conformations.
An additional advantage of the devices of the invention is that both impedance and electrochemical measurements can be performed in the same assay using the same x-y addressable array to enhance the sensitivity and reduce system "noise"
resulting from nonspecific binding of biomolecules. For example, in probe arrays comprising nucleic acids, it is generally not possible to perform electrochemistry on the probe molecules themselves, since they cannot participate in redox reactions under readily-achievable voltage potentials unless they are linked to an electrochemical reporter group that can participate in such a redox reaction. However, an impedance measurement of the probe array can be performed in either the presence or absence of such electrochemical reporter groups to monitor the quality of probe attachment at each particular address prior to introduction of the target. This permits the user to set a reliable baseline for each x-y addressable position (or "pixel") in the array prior to performing an assay.
For electrochemical detection of nucleic acid hybridization of targets to oligonucleotide probes in a high-density, x-y addressable array, application of a low electric field (< 150 mV, less than the redox potential of most electrochemical reporter groups) can be used to concentrate target nucleic acid molecules in proximity of the array. This significantly enhances detection sensitivity and reduces probe-target interaction time; as a consequence, assay time is also reduced. After such electrically-enhanced hybridization is performed, electrochemistry can be performed on the molecular complex at or near the redox potential of the electrochemical reporter group where molecules tagged with an electrochemical reporter groups have hybridized to the immobilized probe. This provides an additive signal to be measured that distinguishes background binding from specific binding at each address in the x-y addressable array. This feature of the assay provides an increased assay sensitivity by reducing the baseline (noise or background) signal due to non-specific binding of the target to the probe. This feature is also a unique characteristic of the multielectrode device structure described here and is not found in the prior art.
In preferred embodiments, the electrochemical reporter groups comprise a transition metal complex, most preferably containing a transition metal ion that is ruthenium, cobalt, iron or osmium.
The use of radioactivity to track molecules, however, presents serious health risks and requires adherence to burdensome regulatory procedures. Precautions must be taken by the user when using radiolabeled materials to avoid exposure to and contact with radioisotopes. Fluorescence technologies also require "labeling" to link the fluorescence marker to a biologically-relevant material, so that molecular interactions (such as nucleic acid hybridization or ligand/receptor binding) can be detected. Linkage of a fluorescent tag to a biomolecule inevitably increases the complexity of such molecules and can adversely affect probe/target interactions. In addition, fluorescence labeling is expensive, labor intensive and time consuming. This leads to increases in experimental cost (by requiring use of additional reagents, expensive hardware, and special equipment) and difficulty (resulting from the use of complicated procedures for handling and disposing of experimental byproducts). Furthermore, experimental reagents containing either radioactive or fluorescence tags often are of limited usefulness (for example, due to the radiochemical half life of the radioisotope, or due to light sensitivity of the fluorescence label).
In contrast, electronic or electrochemical detection processes are based on interactions between probe molecules on an electrode and target molecules in the detection solution that are detected as alterations in the electrical properties on the electrode.
Electronic or electrochemical detection eliminates many of the disadvantages inherent in using radioactive or fluorescent labels to discern molecular interactions.
More importantly, electronic or electrochemical detection devices can be made portable, as has been demonstrated in the case of widely available glucose sensors. Electrical and electrochemical detection devices thus provide an alternative molecular detection means that is safe, inexpensive, unobtrusive, and sensitive.
Electronic or electrochemical detection methods provide an attractive alternative to autoradiography or optical detection for identifying molecular interactions.
In the prior art, electrochemical detection of biological molecules (hereinafter, "biomolecules") has generally been achieved by one of two methods. The first is selective modification at specific sites of a biomolecule (such as a nucleic acid or protein) with redox active moieties such as transition metal complexes. The second approach is intercalation of redox-active moieties, e.g. into duplex DNA strands. In addition, in the prior art most detection schemes have been carried out using either a single electrode or (at most) a few electrodes (typically, more electrodes were used for experimental redundancy, i.e., in order to improve the accuracy of the result, rather than to increase experimental throughput).
Meade et al., in U.S. Patent Nos. 5,591,578, 5,705,348, 5,780,234 and 5,770,369, disclosed methods of detecting a target sequence in a nucleic acid by hybridizing a target sequence with a single stranded nucleic acid that was modified with redox active moieties such as transition metal complexes.
International Patent Application, Publication No. WO 97/01646 teaches a method of detecting a nucleic acid by oxidizing at least one preselected base (e.g., adenine or guanine) with a transition metal complex A significant disadvantage of the electronic or electrochemical detection devices known in the prior art is that these devices use low-density arrays. For example, in U.S.
Patent Nos. 5,670,322 and 5,532,128, Egger et. al. disclosed an apparatus for identifying biomolecular species within a sample substance using an array having a plurality of test sites upon which the sample was applied. Each test site had at least one electrode attached thereto for coupling with a second electrode surrounding the test site to form a capacitor in conjunction with the sample substance. Since the second electrode was preferably made of a ring located outside the array and also acted to contain the sample solution, Egger's array required a large amount of sample solution (i.e., enough to cover the area within the ring) in order for the array to function. More importantly, Egger's array could not be made row and column (x-y) addressable, limiting the density of the test sites in the array and thereby limiting the usefulness of this apparatus.
In U.S. Patent No. 5,653,939, Hollis et al. disclosed an x-y addressable array where test sites were composed of digitated electrodes located on a side bridge that was connected to both the x and y addressable conductive leads. However, the array of Hollis et al. is not practical to fabricate since the test sites are designed to bridge the x-and-y addressable conductive leads that are on two different planes with an insulating layer in-between.
Thus, there remains a need in this art for detecting molecular interactions by electronic or electrochemical means using high-density, row-and-column addressable arrays. In particular, the need is for an x-y addressable array that can be easily and cost-effectively fabricated, and that reduces the cost of performing various analyses, while increasing the effectiveness and utility thereof.
SUMMARY OF THE INVENTION
This invention provides an apparatus for electronic biomolecule detection using a column-and-row (x-y) addressable, high-density biochip array and methods of use thereof.
Specifically, the apparatus facilitates electronic or electrochemical detection of molecular interactions between probe molecules bound to defined regions of a high-density addressable array and target molecules in a solution that is exposed to the array. The apparatus comprises a multiplicity of individual well structures, each said well further comprising two electrodes that can be individually addressed by applying an electric signal specifically to a particular address (well) in the array. In preferred embodiments, the bottom of the well comprises one electrode surface, while the second electrode surrounds the top of the well. Probe molecules include but are not limited to oligonucleotides, nucleic acids (DNA, RNA, etc), proteins, antibodies and peptides that are immobilized at a specific address comprising a well in the array.
Immobilization of such species is accomplished by direct anchoring of the probe molecules on the electrode surface, preferably by attaching the probe molecules onto a supporting matrix on the surface of the electrodes. In the practice of the methods of the invention, the immobilized probe molecules are exposed to a solution containing an intended target molecule, for a time and under conditions sufficient for the probe molecules to bind to the target. An electrical signal is then applied to each of the individual well structures comprising the array. A change in the detected electrical signal in the presence of the solution (compared with the electrical signal detected in the absence of the solution) is used to determine whether a binding event between the probe and target has occurred at a particular address in the array.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.
DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates a schematic representation of a cross-section view of the device platform.
Figure 2 illustrates a schematic representation of a top view of the device platform.
Figure 3 is a schematic diagram of the row/column configuration of a high-density array useful in the practice of the invention.
Figure 4 is a photograph of an x-y addressable array of the invention.
Figures SA, SB and SC are masks for depositing electrode and insulating layers in the x-y addressable arrays of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides an apparatus for electronic or electrochemical detection of biomolecules. For the purposes of this invention, the term "biomolecule" is intended to encompass biologically-derived molecules that interact specifically with one another. Non-limiting examples of such biomolecules are complementary nucleic acid strands, ligand/receptor, agonist/receptor and antagonist/receptor pairs, antigens and their cognate antibodies, enzyme/substrate and enzyme/inhibitor combinations. In general, the biomolecules of the invention comprise a binding pair, whereby there is a specific interaction between each member of the pair. As used herein, one member of the pair is conveniently termed a "target" and the other a "probe." As used herein, "probe"
molecules are preferably bound to a solid substrate and "target" molecules comprise a sample to be tested for the presence, amount or concentration of the "target."
Target molecules can be any of these biomolecules, most preferably wherein at least one of the target molecules specifically interacts with one of the probe molecules.
In preferred embodiments, the probe molecules are oligonucleotides.
Oligonucleotide probes of length 5 to 1000 basepairs (bp), more preferably 5 to 100bp and most preferably about 5 to 40bp, can be attached to the attachment medium.
Targets include PCR amplicons, genomic DNA, cDNA and synthetic and cellular RNA. For protein binding devices, probes can be oligonucleotides such as aptamers or other oligonucleotides having well-defined secondary structure that will bind to proteins.
Alternatively, peptides, antibodies or antigens can be immobilized to perform binding assays.
The present invention provides an apparatus for electronic or electrochemical detection of biomolecules using a row-and-column ("x-y") addressable array having a plurality of addressable sites to which a target sample is applied, and methods of use thereof. Each addressable site comprises at least two electrodes that are connected to two conductive lead lines that can be addressed in a x-y coordination fashion. The addressable site is preferably a well structure as defined herein wherein the bottom of the well comprises the surface of one electrode, and the top of the well comprises the second electrode. In alternative embodiments, each said well structure further comprises at least one additional electrode, preferably a reference electrode, positioned between the top and bottom of the well. In more preferred embodiments, the devices of the invention comprise at least two electrodes, and a multiplicity of probe molecules immobilized in proximity to the electrodes, wherein the probe molecules are preferably immobilized at the surface of at least one of the electrodes.
Device embodiments of the invention are useful for performing methods for biomolecule detection by either electrochemical or electronic means. As used herein, the term "electrochemical detection" is intended to encompass methods based on oxidation/reduction (redox) processes induced by electron transfer between electrodes, most preferably mediated by an electrochemical reporter group attached to the probe moiety, the target moiety, or both. As used herein, the term "electronic detection" is intended to encompass methods that rely on impedance changes (such as resistance, capacitance and inductance) due to differences in electronic state occupancy in the biomolecules in the bound and unbound conformations.
An additional advantage of the devices of the invention is that both impedance and electrochemical measurements can be performed in the same assay using the same x-y addressable array to enhance the sensitivity and reduce system "noise"
resulting from nonspecific binding of biomolecules. For example, in probe arrays comprising nucleic acids, it is generally not possible to perform electrochemistry on the probe molecules themselves, since they cannot participate in redox reactions under readily-achievable voltage potentials unless they are linked to an electrochemical reporter group that can participate in such a redox reaction. However, an impedance measurement of the probe array can be performed in either the presence or absence of such electrochemical reporter groups to monitor the quality of probe attachment at each particular address prior to introduction of the target. This permits the user to set a reliable baseline for each x-y addressable position (or "pixel") in the array prior to performing an assay.
For electrochemical detection of nucleic acid hybridization of targets to oligonucleotide probes in a high-density, x-y addressable array, application of a low electric field (< 150 mV, less than the redox potential of most electrochemical reporter groups) can be used to concentrate target nucleic acid molecules in proximity of the array. This significantly enhances detection sensitivity and reduces probe-target interaction time; as a consequence, assay time is also reduced. After such electrically-enhanced hybridization is performed, electrochemistry can be performed on the molecular complex at or near the redox potential of the electrochemical reporter group where molecules tagged with an electrochemical reporter groups have hybridized to the immobilized probe. This provides an additive signal to be measured that distinguishes background binding from specific binding at each address in the x-y addressable array. This feature of the assay provides an increased assay sensitivity by reducing the baseline (noise or background) signal due to non-specific binding of the target to the probe. This feature is also a unique characteristic of the multielectrode device structure described here and is not found in the prior art.
In preferred embodiments, the electrochemical reporter groups comprise a transition metal complex, most preferably containing a transition metal ion that is ruthenium, cobalt, iron or osmium.
The preferred embodiment of the present invention and its advantages over previously investigated electronic or electrochemical detection devices are best understood by referring to Figures 1 and 2 and Example 1. Like numerals have been used in the drawings for like and corresponding parts.
Figure 1 illustrates a schematic representation of a cross-section view of the device platform 9 of the present invention. The device 9 is built on a solid supporting substrate 6, which can be made of any solid, non-porous substance, including and preferably glass, plastic, ceramic, semiconductor or printed circuit board (PCB). Patterned conductive electrodes 5 are fabricated on top of the solid supporting substrate. The patterned conductive electrodes 5 are fabricated of electrically-conductive metals (including but not limited to transition metals such as aluminum, gold, copper, silver, platinum, chromium, and titanium), transparent conductors (such as indium-tin-oxide and zinc oxide), conductive plastics (such as polymers like polythiophenes, polyanilines, polypyrroles, and metal impregnated polymers), or conductive carbon (such as graphite).
1 S The devices are advantageously formed by standard fabrication techniques used in semiconductor manufacturing. Non-limiting examples of methods for producing solid substrates comprising the device platforms of the invention include but are not limited to thermal evaporation, wire bonding, metallization (evaporation, plating, sputtering over a shadow mask), dielectric deposition (by plasma, chemical vapor deposition or sputtering ), wet or dry chemical etching, reactive ion etching, or liftoff after the desired pattern has been defined using conventional photolithography.
A layer of insulative dielectric material 4, which can be made of polymers, metal oxide or nitrides such as SiO~, SiNX or AIOX is placed on top of the patterned conductive electrodes 5. An optional layer of conductive metal 3 is placed over the insulative dielectric material 4. This layer constitutes a reference electrode. In a preferred embodiment, the conductive metal layer 3 is silver, which is then advantageously converted to silver /silver chloride at a later stage in manufacturing. A
second layer of insulative dielectric material 2 is then placed on top of the conductive electrode layer 3. In embodiments not comprising a reference electrode 3, a continuous dielectric layer 2 comprising layers 2 and 4 as set forth herein are deposited. The second layer of insulative dielectric material 2 is optionally made of the same materials as the insulative layer 4.
Patterned conductive electrodes 1 constructed on top of the second layer of insulative dielectric material 2 constitute the final layer of each addressable site in the device 9. Well structures 7 are fabricated from this device by conventional photolithography or laser drilling methods used in the semiconductor industry for PCB
manufacturing. These wells can have rectangular, circular, trapezoidal or other polygonal openings. Additionally, the well walls may be either straight or curved, and may have an arbitrary angle with respect to the bottom electrode 5. An optional center electrode can alternatively protrude into the well area, as shown in Figure 3.
Figure 2 illustrates a schematic representation of a top view of the apparatus of the invention. The conductive electrodes 1 are preferred to be oriented in a direction orthogonal to the patterned conductive electrodes 5, generating row (i.e., patterned electrodes 5) and column (i.e., conductive electrodes 1) addressable high-density electronic or electrochemical mini-cells (i.e., well structures 7) with optional reference electrodes built in-between. The well structure is preferably produced wherein the bottom of the well structure comprises the top of electrode 5 surface, while the top of the well structure is surrounded by the second electrode 1.
The proposed device 9 can be used as an x-y addressable, high-density biochip array when biological probes 10 are immobilized on the patterned electrodes 5 inside each well structure 7. The apparatus is capable of detecting changes in the electrical properties of the probes 10 in each well structure arising from the interaction of the probes 10 with target molecules 11. Though the inventive apparatus is useful for single species detection, where only a few test wells (low density) are required, the advantages of the invention are more pronounced in a high density array where hundreds, thousands, or millions of test wells are integrated in one array.
In a preferred embodiment, the probe molecules may be oligonucleotides, nucleic acids (such as DNA or RNA), proteins, peptides, antibodies or small molecules such as ligands, wherein probe molecules are chemically modified to contain anchoring groups that permit immobilization. Preferred modifications to the oligonucleotides useful in the practice of the invention include but are not limited to -OH, -NH2, -SH, -COOR
(where R
= H, lower (C,_,2) alkyl, aryl, heterocyclic alkyl or aryl, or a metal ion), -CN, or -CHO.
Immobilization of such derivatized probes is accomplished by direct attaching of the probe molecules on the electrode surface through a functional group such -OH, -SH, -NH2.
Alternatively, probe molecules can be efficiently immobilized on the electrode surface through an intermediate species, termed a "spacer." In these embodiments, the surface of the electrode 5 is first modified with an intermediate species that carries -g_ functional groups such as hydroxyl (-OH), amino (-NHZ), thiol (-SH), carboxyl ester (-COOR, where R = H, lower (C,_,2) alkyl, aryl, heterocyclic alkyl or aryl, or a metal ion), nitrite (-CN), or aldehylde (-CHO), which can react with the probe molecules functionalized with complementary members of the aforementioned anchoring groups.
In another embodiment, the surface of the electrodes 5 is covered with a layer of polymer matrix. In these embodiments, probe molecules are attached onto a supporting matrix on the surface of the electrodes using the functional chemistry mentioned above.
The polymer matrix is preferably selected to be polypyrrole, polythiophene, polyaniline, polyacrylamide, agarose gel, polyethylene glycol, cellular, sot gels, dendrimers, metallic nanoparticles, carbon nanotubes, and their copolymers. To increase the probe loading capacity, porous matrix such as polyacrylamide, agarose, or sot gels are preferred.
Electronic or electrochemical detection of molecular interactions between probe and target molecules is achieved by devices having the structure, for example, as depicted in Figure 1. The electric or/and electrochemical methods used to interrogating the 1 S biomolecule targets may be selected from, but are not limited to, AC
impedance, cyclic voltammetry (CV), pulse voltammetry, square wave voltammetry, AC voltammetry (ACV), hydrodynamic modulation voltammetry, potential step method, potentiometric measurements, amperometric measurements, current step method, and combinations thereof.
In an alternative embodiment, an active driving circuit such as the one used in an active matrix liquid crystal display device can be built underneath or nearby each test well site to replace the electronic column and row drivers for x-y addressing such as the one used in the passive matrix liquid crystal display device.
In the practice of the invention, a high-density, x-y addressable probe array is exposed to an electrolyte solution containing a target molecule for a time and under conditions sufficient for the target to bind to a probe present in at least one of the particular addresses of the column-and-row addressable array. A voltage potential or other electric signal is applied to the each of the electrodes comprising each of the addressable sites through the x-y addressable column and row electrodes.
Changes in the electrical properties or electrical signals from a particular electrode at a particular site in the x-y addressable array arising from interactions between probe molecules on the electrode and target molecules in the solution are detected to determine the presence and concentration of the target molecules in the solution.
_9_ In certain advantageous embodiments, electrical cross-talk between electrodes is reduced or eliminated in the x-y addressable array during target interrogation with an external electrical source. In these embodiments, the electrodes at the top of the wells are covered with an array of microfluidic channels. These channels are designed to be independently isolated from each other, with each having its own isolatable liquid inlet and outlet port. In addition to functioning as an electrical isolator, the channels also act as containers or reaction chambers for liquid during probe-target hybridization, enzymatic reactions and target interrogation with the external electrical source. In an active driving array where the x-y addressable columns and rows are replaced by an active driving circuit built underneath or nearby each test well site, the microfluidic channels can be replaced by a single chamber that covers all the test sites with Electrolyte solutions useful in the apparatus and methods of the invention include any electrolyte solution at physiologically-relevant ionic strength (equivalent to about O.15M NaCI) and neutral pH. Nonlimiting examples of electrolyte solutions useful with the apparatus and methods of the invention include but are not limited to phosphate buffered saline, HEPES buffered solutions, and sodium bicarbonate buffered solutions. In alternative embodiments useful for electrical detection methods provided by the invention, the electrolyte solution comprises metal cations or polymerized cations that are ion conductive and capable of reacting with probes or probe-target complexes.
The Examples, which follow, are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
Fabrication of a linear microarray with four wells A linear test microarray with four wells was fabricated on a 3" inch silicon wafer as follows. A photograph of the array is shown in Figure 4.
The linear test array was fabricated by conventional photolithography in a class 100 clean room and fabrication was performed using three layers of masks as shown in Masks 12 (Figure SA), 14 (Figure SB) and 16 (Figure SC).
A three inch silicon wafer was cleaned using a solution of NH40H:H20 (1:10 v/v), rinsed with de-ionized water, and then dried using a stream of nitrogen at room temperature. On the top of the wafer, 2000 Si02 was deposited by conventional chemical vapor deposition technique.
The array was then prepared sequentially as follows.
1. Bottom Electrode Formation After cleaning the Si02-prepared substrate using a solution of NH40H:H20 (1:10 v/v), a de-ionized water rinse, and drying with a stream of nitrogen as described above, a thick (5 micron) photoresist (PR) layer was spin-coated on the wafer through a three stage process of spin-coating and softbaking. Using Mask 12 (shown in Figure SA) to protect the portion of the substrate that forms the bottom electrode, the surface was exposed to an ultraviolet light source using a wavelength of 365 nm and an intensity of 6 mW/cm3.
Following this treatment, the PR was hardbaked and developed. After removal of Mask 12, the following metals were deposited sequentially by evaporation: Ti (to a thickness of 1.0 Angstrom), Au (to a thickness of 21,000 Angstrom), and Ti (to a thickness of 500 Angstrom). After evaporative deposition of these metal layers, a liftoff protocol was used to produce the bottom patterned electrode.
2. Top Electrode Formation After cleaning the bottom electrode-prepared substrate using a solution of NH40H:H20 (1:10 v/v), a de-ionized water rinse, and drying with a stream of nitrogen as described above, the wafer was coated with a thick (8 micron) layer of PR, as described above. Using Mask 14 (Figure SB) to protect the portion of the substrate that forms the top electrode, the surface was exposed to an ultraviolet light source using a wavelength of 365 nm and an intensity of 6 mW/cm3. Following this treatment, the PR was hardbaked and developed as described above After removal of Mask 14, the following metals were deposited sequentially by evaporation: Ti (to a thickness of 1.0 Angstrom) and Au (to a thickness of 21,000 Angstrom). After evaporative deposition of these metal layers, a liftoff protocol was used to produce the top patterned electrode, as described above.
3. Well Structure Formation After cleaning the top electrode-prepared substrate using a solution of NH40H:H20 (1:10 v/v), a de-ionized water rinse, and drying with a stream of nitrogen as described above, the wafer was then coated with a 4 micron layer of PR. The surface was exposed to an ultraviolet light source using a wavelength of 365 nm and an intensity of 6 mW/cm3. Following this treatment, the PR was hardbaked and developed as described above. The wafer was then subjected to buffer oxide etching solution (4:1) until each well opening was cleared. The PR was removed by placing in a Branson 4000 Sonicator.
It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.
Figure 1 illustrates a schematic representation of a cross-section view of the device platform 9 of the present invention. The device 9 is built on a solid supporting substrate 6, which can be made of any solid, non-porous substance, including and preferably glass, plastic, ceramic, semiconductor or printed circuit board (PCB). Patterned conductive electrodes 5 are fabricated on top of the solid supporting substrate. The patterned conductive electrodes 5 are fabricated of electrically-conductive metals (including but not limited to transition metals such as aluminum, gold, copper, silver, platinum, chromium, and titanium), transparent conductors (such as indium-tin-oxide and zinc oxide), conductive plastics (such as polymers like polythiophenes, polyanilines, polypyrroles, and metal impregnated polymers), or conductive carbon (such as graphite).
1 S The devices are advantageously formed by standard fabrication techniques used in semiconductor manufacturing. Non-limiting examples of methods for producing solid substrates comprising the device platforms of the invention include but are not limited to thermal evaporation, wire bonding, metallization (evaporation, plating, sputtering over a shadow mask), dielectric deposition (by plasma, chemical vapor deposition or sputtering ), wet or dry chemical etching, reactive ion etching, or liftoff after the desired pattern has been defined using conventional photolithography.
A layer of insulative dielectric material 4, which can be made of polymers, metal oxide or nitrides such as SiO~, SiNX or AIOX is placed on top of the patterned conductive electrodes 5. An optional layer of conductive metal 3 is placed over the insulative dielectric material 4. This layer constitutes a reference electrode. In a preferred embodiment, the conductive metal layer 3 is silver, which is then advantageously converted to silver /silver chloride at a later stage in manufacturing. A
second layer of insulative dielectric material 2 is then placed on top of the conductive electrode layer 3. In embodiments not comprising a reference electrode 3, a continuous dielectric layer 2 comprising layers 2 and 4 as set forth herein are deposited. The second layer of insulative dielectric material 2 is optionally made of the same materials as the insulative layer 4.
Patterned conductive electrodes 1 constructed on top of the second layer of insulative dielectric material 2 constitute the final layer of each addressable site in the device 9. Well structures 7 are fabricated from this device by conventional photolithography or laser drilling methods used in the semiconductor industry for PCB
manufacturing. These wells can have rectangular, circular, trapezoidal or other polygonal openings. Additionally, the well walls may be either straight or curved, and may have an arbitrary angle with respect to the bottom electrode 5. An optional center electrode can alternatively protrude into the well area, as shown in Figure 3.
Figure 2 illustrates a schematic representation of a top view of the apparatus of the invention. The conductive electrodes 1 are preferred to be oriented in a direction orthogonal to the patterned conductive electrodes 5, generating row (i.e., patterned electrodes 5) and column (i.e., conductive electrodes 1) addressable high-density electronic or electrochemical mini-cells (i.e., well structures 7) with optional reference electrodes built in-between. The well structure is preferably produced wherein the bottom of the well structure comprises the top of electrode 5 surface, while the top of the well structure is surrounded by the second electrode 1.
The proposed device 9 can be used as an x-y addressable, high-density biochip array when biological probes 10 are immobilized on the patterned electrodes 5 inside each well structure 7. The apparatus is capable of detecting changes in the electrical properties of the probes 10 in each well structure arising from the interaction of the probes 10 with target molecules 11. Though the inventive apparatus is useful for single species detection, where only a few test wells (low density) are required, the advantages of the invention are more pronounced in a high density array where hundreds, thousands, or millions of test wells are integrated in one array.
In a preferred embodiment, the probe molecules may be oligonucleotides, nucleic acids (such as DNA or RNA), proteins, peptides, antibodies or small molecules such as ligands, wherein probe molecules are chemically modified to contain anchoring groups that permit immobilization. Preferred modifications to the oligonucleotides useful in the practice of the invention include but are not limited to -OH, -NH2, -SH, -COOR
(where R
= H, lower (C,_,2) alkyl, aryl, heterocyclic alkyl or aryl, or a metal ion), -CN, or -CHO.
Immobilization of such derivatized probes is accomplished by direct attaching of the probe molecules on the electrode surface through a functional group such -OH, -SH, -NH2.
Alternatively, probe molecules can be efficiently immobilized on the electrode surface through an intermediate species, termed a "spacer." In these embodiments, the surface of the electrode 5 is first modified with an intermediate species that carries -g_ functional groups such as hydroxyl (-OH), amino (-NHZ), thiol (-SH), carboxyl ester (-COOR, where R = H, lower (C,_,2) alkyl, aryl, heterocyclic alkyl or aryl, or a metal ion), nitrite (-CN), or aldehylde (-CHO), which can react with the probe molecules functionalized with complementary members of the aforementioned anchoring groups.
In another embodiment, the surface of the electrodes 5 is covered with a layer of polymer matrix. In these embodiments, probe molecules are attached onto a supporting matrix on the surface of the electrodes using the functional chemistry mentioned above.
The polymer matrix is preferably selected to be polypyrrole, polythiophene, polyaniline, polyacrylamide, agarose gel, polyethylene glycol, cellular, sot gels, dendrimers, metallic nanoparticles, carbon nanotubes, and their copolymers. To increase the probe loading capacity, porous matrix such as polyacrylamide, agarose, or sot gels are preferred.
Electronic or electrochemical detection of molecular interactions between probe and target molecules is achieved by devices having the structure, for example, as depicted in Figure 1. The electric or/and electrochemical methods used to interrogating the 1 S biomolecule targets may be selected from, but are not limited to, AC
impedance, cyclic voltammetry (CV), pulse voltammetry, square wave voltammetry, AC voltammetry (ACV), hydrodynamic modulation voltammetry, potential step method, potentiometric measurements, amperometric measurements, current step method, and combinations thereof.
In an alternative embodiment, an active driving circuit such as the one used in an active matrix liquid crystal display device can be built underneath or nearby each test well site to replace the electronic column and row drivers for x-y addressing such as the one used in the passive matrix liquid crystal display device.
In the practice of the invention, a high-density, x-y addressable probe array is exposed to an electrolyte solution containing a target molecule for a time and under conditions sufficient for the target to bind to a probe present in at least one of the particular addresses of the column-and-row addressable array. A voltage potential or other electric signal is applied to the each of the electrodes comprising each of the addressable sites through the x-y addressable column and row electrodes.
Changes in the electrical properties or electrical signals from a particular electrode at a particular site in the x-y addressable array arising from interactions between probe molecules on the electrode and target molecules in the solution are detected to determine the presence and concentration of the target molecules in the solution.
_9_ In certain advantageous embodiments, electrical cross-talk between electrodes is reduced or eliminated in the x-y addressable array during target interrogation with an external electrical source. In these embodiments, the electrodes at the top of the wells are covered with an array of microfluidic channels. These channels are designed to be independently isolated from each other, with each having its own isolatable liquid inlet and outlet port. In addition to functioning as an electrical isolator, the channels also act as containers or reaction chambers for liquid during probe-target hybridization, enzymatic reactions and target interrogation with the external electrical source. In an active driving array where the x-y addressable columns and rows are replaced by an active driving circuit built underneath or nearby each test well site, the microfluidic channels can be replaced by a single chamber that covers all the test sites with Electrolyte solutions useful in the apparatus and methods of the invention include any electrolyte solution at physiologically-relevant ionic strength (equivalent to about O.15M NaCI) and neutral pH. Nonlimiting examples of electrolyte solutions useful with the apparatus and methods of the invention include but are not limited to phosphate buffered saline, HEPES buffered solutions, and sodium bicarbonate buffered solutions. In alternative embodiments useful for electrical detection methods provided by the invention, the electrolyte solution comprises metal cations or polymerized cations that are ion conductive and capable of reacting with probes or probe-target complexes.
The Examples, which follow, are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
Fabrication of a linear microarray with four wells A linear test microarray with four wells was fabricated on a 3" inch silicon wafer as follows. A photograph of the array is shown in Figure 4.
The linear test array was fabricated by conventional photolithography in a class 100 clean room and fabrication was performed using three layers of masks as shown in Masks 12 (Figure SA), 14 (Figure SB) and 16 (Figure SC).
A three inch silicon wafer was cleaned using a solution of NH40H:H20 (1:10 v/v), rinsed with de-ionized water, and then dried using a stream of nitrogen at room temperature. On the top of the wafer, 2000 Si02 was deposited by conventional chemical vapor deposition technique.
The array was then prepared sequentially as follows.
1. Bottom Electrode Formation After cleaning the Si02-prepared substrate using a solution of NH40H:H20 (1:10 v/v), a de-ionized water rinse, and drying with a stream of nitrogen as described above, a thick (5 micron) photoresist (PR) layer was spin-coated on the wafer through a three stage process of spin-coating and softbaking. Using Mask 12 (shown in Figure SA) to protect the portion of the substrate that forms the bottom electrode, the surface was exposed to an ultraviolet light source using a wavelength of 365 nm and an intensity of 6 mW/cm3.
Following this treatment, the PR was hardbaked and developed. After removal of Mask 12, the following metals were deposited sequentially by evaporation: Ti (to a thickness of 1.0 Angstrom), Au (to a thickness of 21,000 Angstrom), and Ti (to a thickness of 500 Angstrom). After evaporative deposition of these metal layers, a liftoff protocol was used to produce the bottom patterned electrode.
2. Top Electrode Formation After cleaning the bottom electrode-prepared substrate using a solution of NH40H:H20 (1:10 v/v), a de-ionized water rinse, and drying with a stream of nitrogen as described above, the wafer was coated with a thick (8 micron) layer of PR, as described above. Using Mask 14 (Figure SB) to protect the portion of the substrate that forms the top electrode, the surface was exposed to an ultraviolet light source using a wavelength of 365 nm and an intensity of 6 mW/cm3. Following this treatment, the PR was hardbaked and developed as described above After removal of Mask 14, the following metals were deposited sequentially by evaporation: Ti (to a thickness of 1.0 Angstrom) and Au (to a thickness of 21,000 Angstrom). After evaporative deposition of these metal layers, a liftoff protocol was used to produce the top patterned electrode, as described above.
3. Well Structure Formation After cleaning the top electrode-prepared substrate using a solution of NH40H:H20 (1:10 v/v), a de-ionized water rinse, and drying with a stream of nitrogen as described above, the wafer was then coated with a 4 micron layer of PR. The surface was exposed to an ultraviolet light source using a wavelength of 365 nm and an intensity of 6 mW/cm3. Following this treatment, the PR was hardbaked and developed as described above. The wafer was then subjected to buffer oxide etching solution (4:1) until each well opening was cleared. The PR was removed by placing in a Branson 4000 Sonicator.
It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.
Claims (36)
1. An apparatus for electronic or electrochemical detection of molecular interactions between an immobilized probe and a target molecule, comprising:
(a) a solid, non-porous supporting substrate, (b) a first conductive electrode layer comprising a plurality of patterned first conductive electrodes placed on top of the supporting substrate, (c) a first insulative dielectric layer placed on top of the first conductive electrode layer, (d) a first conductive metal layer placed on top of the first insulative dielectric layer, (e) a second insulative dielectric layer placed on top of the first conductive metal layer, (f) a second conductive electrode layer comprising a plurality of patterned second conductive electrodes placed on top of the second insulative dielectric layer, wherein said second conductive electrodes overlie at least a portion of said first conductive electrodes in said first conductive electrode layer, (g) a plurality of wells, each defined through a second conductive electrode in said second conductive layer, second insulative dielectric layer, first conductive metal layer, and first insulative dielectric layer, wherein the bottom of each of said wells are formed by a first conductive electrode in said first conductive electrode layer, (h) probes immobilized on at least one of said first or second conductive electrodes in the wells.
(a) a solid, non-porous supporting substrate, (b) a first conductive electrode layer comprising a plurality of patterned first conductive electrodes placed on top of the supporting substrate, (c) a first insulative dielectric layer placed on top of the first conductive electrode layer, (d) a first conductive metal layer placed on top of the first insulative dielectric layer, (e) a second insulative dielectric layer placed on top of the first conductive metal layer, (f) a second conductive electrode layer comprising a plurality of patterned second conductive electrodes placed on top of the second insulative dielectric layer, wherein said second conductive electrodes overlie at least a portion of said first conductive electrodes in said first conductive electrode layer, (g) a plurality of wells, each defined through a second conductive electrode in said second conductive layer, second insulative dielectric layer, first conductive metal layer, and first insulative dielectric layer, wherein the bottom of each of said wells are formed by a first conductive electrode in said first conductive electrode layer, (h) probes immobilized on at least one of said first or second conductive electrodes in the wells.
2. The apparatus of claim 1, wherein the supporting substrate comprises glass, plastic, ceramic, silicon, or printed circuit board.
3. The apparatus according to claim 1, wherein said first electrode comprises a metal selected from the group consisting of aluminum, gold, copper, silver, platinum, chromium and titanium.
4. The apparatus according to claim 1, wherein said first electrode comprises a transparent conductor.
5. The apparatus according to claim 1, wherein said first electrode comprises a conductive plastic ar conductive carbon.
6. The apparatus of claim 1, wherein the first insulative dielectric layer comprises a polymer, metal oxide or nitride.
7. The apparatus of claim 1, wherein the second insulative dielectric layer comprises a polymer, metal oxide or nitride.
8. The apparatus according to claim 1, wherein said second electrode comprises a metal selected from the group consisting of aluminum, gold, copper, silver, platinum, chromium and titanium.
9. The apparatus of claim 1, comprising a reference electrode comprising a conductive electrode layer.
10. The apparatus according to claim 1, wherein said conductive metal layer comprises a metal selected from the group consisting of aluminum, gold, copper, silver, platinum, chromium and titanium.
11. The apparatus according to claim 1, wherein the conductive metal layer of the reference electrode comprises silver or a mixture of metallic silver and silver chloride.
12. The apparatus of claim 1, wherein the probes are nucleic acids.
13. The apparatus of claim 12, wherein the probes are oligonucleotide probes.
14. The apparatus of claim 1, wherein the probes are peptides.
15. The apparatus according to claims 12, 13 or 14, wherein the probes are covalently attached to the first or the second electrode..
16. The apparatus of claim 1, wherein the well structures are fabricated by photolithography or laser drilling.
17. The apparatus of claim 15, wherein the probes are attached to the first conductive electrode through a spacer or a polymer matrix.
18. A method for electric or electrochemical detection of molecular interactions between an immobilized probe and an electrochemically active reporter-labeled target molecule comprising:
(a) exposing a plurality of well structures in an x-y addressable array to an electrolyte solution containing an electrochemically-labeled target molecule in order to generate probe-target complexes (b) detecting an electrical signal in at least one of the plurality of well structures in the x-y addressable array to which probes have been attached.
(a) exposing a plurality of well structures in an x-y addressable array to an electrolyte solution containing an electrochemically-labeled target molecule in order to generate probe-target complexes (b) detecting an electrical signal in at least one of the plurality of well structures in the x-y addressable array to which probes have been attached.
19. The method of Claim 18, wherein molecular interactions between an immobilized probe and an electrochemically active reporter-labeled target molecule are detected by AC
impedance, cyclic voltammetry, stripping voltammetry, pulse voltammetry, square wave voltammetry, AC voltammetry, hydrodynamic modulation voltammetry, potential step method, potentiometric measurements, amperometric measurements, current step method, or combinations thereof.
impedance, cyclic voltammetry, stripping voltammetry, pulse voltammetry, square wave voltammetry, AC voltammetry, hydrodynamic modulation voltammetry, potential step method, potentiometric measurements, amperometric measurements, current step method, or combinations thereof.
20. The method of Claim 18, wherein molecular interactions between an immobilized probe and an electrochemically active reporter-labeled target molecule are detected by AC
impedance measured over a range of frequencies before and after exposing the plurality of well structures in the x-y addressable array to an electrolyte solution containing the electrochemically active reporter-labeled target molecule.
impedance measured over a range of frequencies before and after exposing the plurality of well structures in the x-y addressable array to an electrolyte solution containing the electrochemically active reporter-labeled target molecule.
21. The method of Claim 18, wherein molecular interactions between an immobilized probe and an electrochemically active reporter-labeled target molecule are detected by AC
impedance measured by transient methods with AC signal perturbation superimposed upon a DC potential applied to an electrochemical cell.
impedance measured by transient methods with AC signal perturbation superimposed upon a DC potential applied to an electrochemical cell.
22. The method of Claim 18, wherein molecular interactions between an immobilized probe acid an electrochemically active reporter-labeled target molecule are detected by AC
impedance measured by impedance analyzer, lock-in amplifier, AC bridge, AC
voltammetry, or combinations thereof.
impedance measured by impedance analyzer, lock-in amplifier, AC bridge, AC
voltammetry, or combinations thereof.
23. The method of Claim 18, wherein the molecular interactions detected thereby are single base mismatches within nucleic acid probe-target complexes.
24. The method of Claim 18, wherein the molecular interaction detected is quantification of electrochemically active reporter-labeled target molecules in a reaction mixture for gene expression analyses.
25. The method of Claim 18, wherein the electrochemically active reporter labeled target molecules are labeled with electrochemical reporter groups comprising a transition metal complex.
26. The method of Claim 25, wherein the transition metal ion is ruthenium, cobalt, iron, or osmium.
27. A method for electrical detection of molecular interactions between an immobilized probe and a target molecule, comprising:
(a) contacting a plurality of well structures in an x-y addressable array to which probes have been attached with an electrolyte solution, (b) measuring the impedance in the well structures, (c) exposing the well structures to an electrolyte solution containing a target molecule to generate probe-target complexes, (d) measuring the impedance at the well structures, and (e) detecting molecular interactions between the immobilized probe and the target molecule by detecting a change in the measured impedance in the well structures before and after exposing the probes in the well structures to the electrolyte solution containing the target molecule.
(a) contacting a plurality of well structures in an x-y addressable array to which probes have been attached with an electrolyte solution, (b) measuring the impedance in the well structures, (c) exposing the well structures to an electrolyte solution containing a target molecule to generate probe-target complexes, (d) measuring the impedance at the well structures, and (e) detecting molecular interactions between the immobilized probe and the target molecule by detecting a change in the measured impedance in the well structures before and after exposing the probes in the well structures to the electrolyte solution containing the target molecule.
28. The method of Claim 27, wherein the electrolyte solution comprises metal, non metal or polymerized cations that are ion-conductive and capable of reacting with probes or probe-target complexes.
29. The method of Claim 27, wherein impedance is measured over a range of frequencies prior to and after exposing the well structures to a reaction mixture containing the target molecule.
30. The method of Claim 27, wherein impedance is measured by transient methods with AC signal perturbation superimposed upon a DC potential applied to an electrochemical cell.
31. The method of Claim 27, wherein impedance is measured by impedance analyzer, lock-in amplifier, AC bridge, AC voltammetry, or combinations thereof.
32. The method of Claim 27, wherein the molecular interactions detected thereby are single base mismatches within nucleic acid probe-target complexes.
33. The method of Claim 27, wherein the molecular interaction detected is quantification of target molecules in a reaction mixture for gene expression analyses.
34. An apparatus according to claim 1, wherein said first conductive electrodes are arranged in a plurality of rows, each row of said first conductive electrode sharing a row interconnect, and said second conductive electrodes are arranged in columns oriented orthoganally to the rows of said first conductive electrodes, each column of said conductive electrode sharing a column interconnect.
35. An apparatus according to claim 1, wherein each of said well being addressable by said first conductive electrode and said second conductive electrode.
36. An apparatus according to claim 34, wherein each of said wells can be uniquely addressed.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US46450099A | 1999-12-15 | 1999-12-15 | |
US09/464,500 | 1999-12-15 | ||
PCT/US2000/034222 WO2001043870A2 (en) | 1999-12-15 | 2000-12-14 | Column and row addressable high density biochip array |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2393766A1 true CA2393766A1 (en) | 2001-06-21 |
Family
ID=23844193
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002393766A Abandoned CA2393766A1 (en) | 1999-12-15 | 2000-12-14 | Column and row addressable high density biochip array |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1251955A2 (en) |
JP (1) | JP2003517149A (en) |
AU (1) | AU2108901A (en) |
CA (1) | CA2393766A1 (en) |
WO (1) | WO2001043870A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2492674A1 (en) | 2011-02-28 | 2012-08-29 | Lexogen GmbH | Biosensor array formed by junctions of functionalized electrodes |
Families Citing this family (46)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6824669B1 (en) | 2000-02-17 | 2004-11-30 | Motorola, Inc. | Protein and peptide sensors using electrical detection methods |
US6824974B2 (en) | 2001-06-11 | 2004-11-30 | Genorx, Inc. | Electronic detection of biological molecules using thin layers |
CA2450109A1 (en) * | 2001-06-11 | 2003-05-22 | Genorx, Inc. | Electronic detection of biological molecules using thin layers |
GB2377026A (en) * | 2001-06-29 | 2002-12-31 | Imp College Innovations Ltd | Electrically addressable electrochemical cell array |
DE10145700A1 (en) * | 2001-09-17 | 2003-04-10 | Infineon Technologies Ag | Biochip arrangement, sensor arrangement and method for operating a biochip arrangement |
US7074519B2 (en) * | 2001-10-26 | 2006-07-11 | The Regents Of The University Of California | Molehole embedded 3-D crossbar architecture used in electrochemical molecular memory device |
DE10211358B4 (en) * | 2002-03-14 | 2006-10-26 | Siemens Ag | Vertical impedance sensor assembly and method of fabricating a vertical impedance sensor assembly |
FR2839783B1 (en) * | 2002-05-17 | 2004-08-06 | Apibio | NEW LAYOUT OF BIOCHIP LINK SITES |
FR2840072B1 (en) * | 2002-05-24 | 2004-08-06 | Apibio | BIOPUCE WITH IMPROVED ORGANIZATION |
JP4101801B2 (en) | 2002-06-24 | 2008-06-18 | シーメンス アクチエンゲゼルシャフト | Biosensor array and method of use thereof |
DE10228124A1 (en) * | 2002-06-24 | 2004-01-29 | Infineon Technologies Ag | Biosensor array comprising a substrate on which biosensors are mounted which have two connectors, one of which is connected to control lead while other is connected to detector lead |
DE10228125A1 (en) * | 2002-06-24 | 2004-01-22 | Infineon Technologies Ag | Biosensor array comprising a substrate on which biosensors are mounted which have two connectors, one of which is connected to control lead while other is connected to detector lead |
US6962822B2 (en) | 2002-08-07 | 2005-11-08 | International Business Machines Corporation | Discrete nano-textured structures in biomolecular arrays, and method of use |
EP1548437A4 (en) * | 2002-09-17 | 2006-06-07 | Olympus Corp | Method and apparatus for arranging liquid reaction components on substrate surface for detecting target substance by reaction among plural reaction components on substrate and article utilized in the method |
DE10257604A1 (en) * | 2002-12-09 | 2004-07-22 | Axaron Bioscience Ag | Capacitive detection of bound molecules |
WO2005016115A2 (en) | 2003-01-23 | 2005-02-24 | Montana State University | Biosensors utilizing dendrimer-immobilized ligands and their use thereof |
GB2398751A (en) * | 2003-02-28 | 2004-09-01 | Univ Surrey | A dielectrophoretic separation device |
JP2004298783A (en) * | 2003-03-31 | 2004-10-28 | Kitakyushu Foundation For The Advancement Of Industry Science & Technology | Membrane for separating chemical substance, its manufacturing method, method for detecting chemical substance and apparatus for detecting chemical substance |
US7399400B2 (en) | 2003-09-30 | 2008-07-15 | Nano-Proprietary, Inc. | Nanobiosensor and carbon nanotube thin film transistors |
EP1682880A1 (en) | 2003-10-08 | 2006-07-26 | Philips Intellectual Property & Standards GmbH | Bulk acoustic wave sensor |
US20080070803A1 (en) * | 2003-10-15 | 2008-03-20 | Egeland Ryan D | Electrochemical Treatment Of Substrates |
US7390622B2 (en) * | 2003-10-16 | 2008-06-24 | Hai Kang Life Corporation Limited | Apparatus and methods for detecting nucleic acid in biological samples |
KR101165860B1 (en) | 2003-10-16 | 2012-07-13 | 하이 캉 라이프 코포레이션 리미티드 | Apparatus and methods for detecting nucleic acid in biological samples |
US20050244811A1 (en) * | 2003-12-15 | 2005-11-03 | Nano-Proprietary, Inc. | Matrix array nanobiosensor |
DE102004031672A1 (en) | 2004-06-30 | 2006-01-19 | Infineon Technologies Ag | Planar sensor array, sensor array, and method of fabricating a planar sensor array |
JP5041680B2 (en) * | 2005-06-17 | 2012-10-03 | 株式会社ハイペップ研究所 | Biochip substrate and biochip |
JP4635258B2 (en) * | 2006-03-02 | 2011-02-23 | 独立行政法人産業技術総合研究所 | Biosensor |
EP2002248A1 (en) | 2006-03-17 | 2008-12-17 | Element Six Limited | Microelectrode array |
US9550185B2 (en) | 2006-11-09 | 2017-01-24 | Asmag-Holding Gmbh | Titer plate with thin-film-light sensor |
AT505688A1 (en) | 2007-09-13 | 2009-03-15 | Nanoident Technologies Ag | SENSOR MATRIX FROM SEMICONDUCTOR COMPONENTS |
JP5176235B2 (en) * | 2008-07-03 | 2013-04-03 | 国立大学法人東北大学 | Electrochemical measuring device |
GB0821810D0 (en) * | 2008-11-28 | 2009-01-07 | Nanoflex Ltd | Electrode assembly |
DE102011010767A1 (en) * | 2011-02-09 | 2012-08-09 | Forschungszentrum Jülich GmbH | Method for producing a device for detecting an analyte, and device and their use |
EP2812344A4 (en) | 2012-02-07 | 2015-10-28 | Vibrant Holdings Llc | Substrates, peptide arrays, and methods |
US10006909B2 (en) | 2012-09-28 | 2018-06-26 | Vibrant Holdings, Llc | Methods, systems, and arrays for biomolecular analysis |
DE102013207779A1 (en) * | 2012-10-23 | 2014-05-15 | Heraeus Precious Metals Gmbh & Co. Kg | Multilayer structure with alternating conductive and non-conductive layers |
US9523642B2 (en) * | 2012-11-09 | 2016-12-20 | Taiwan Semiconductor Manufacturing Company, Ltd. | Integrated electro-microfluidic probe card, system and method for using the same |
US10286376B2 (en) | 2012-11-14 | 2019-05-14 | Vibrant Holdings, Llc | Substrates, systems, and methods for array synthesis and biomolecular analysis |
WO2014127328A2 (en) * | 2013-02-15 | 2014-08-21 | Vibrant Holdings, Llc. | Methods and compositions for amplified electrochemiluminescence detection |
CN103627788A (en) * | 2013-08-21 | 2014-03-12 | 中山大学达安基因股份有限公司 | Preparation method for biochip of electrochemical gene sensor |
US9498142B2 (en) | 2014-07-03 | 2016-11-22 | Heraeus Deutschland GmbH & Co. KG | Multi-layered structure and method |
EP3237879B1 (en) | 2014-12-23 | 2021-02-03 | Heraeus Nexensos GmbH | Sensor for detecting soot particles and method for adjusting such a sensor |
US20180024023A1 (en) * | 2015-01-29 | 2018-01-25 | Aram Corporation | Liquid sensor |
JP6116080B1 (en) * | 2016-04-26 | 2017-04-19 | 日本航空電子工業株式会社 | Electrochemical measurement method, electrochemical measurement apparatus and transducer |
US10538808B2 (en) | 2017-05-26 | 2020-01-21 | Vibrant Holdings, Llc | Photoactive compounds and methods for biomolecule detection and sequencing |
KR102423723B1 (en) * | 2020-08-27 | 2022-07-21 | 성균관대학교산학협력단 | Apparatus for detecting nucleic acid based on plasmonic well and method for fabricating the same using Roll-to-Roll processing |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6440662B1 (en) * | 1995-12-01 | 2002-08-27 | Innogenetics N.V. | Impedimetric detection system and method of production thereof |
US5727977A (en) * | 1996-03-04 | 1998-03-17 | Motorola, Inc. | Process for manufacturing a field-emission device |
CA2310622A1 (en) * | 1997-08-08 | 1999-02-18 | California Institute Of Technology | Techniques and systems for analyte detection |
GB9810568D0 (en) * | 1998-05-18 | 1998-07-15 | Imco 1097 Limited | Electrode system |
-
2000
- 2000-12-14 WO PCT/US2000/034222 patent/WO2001043870A2/en not_active Application Discontinuation
- 2000-12-14 AU AU21089/01A patent/AU2108901A/en not_active Abandoned
- 2000-12-14 CA CA002393766A patent/CA2393766A1/en not_active Abandoned
- 2000-12-14 EP EP00984476A patent/EP1251955A2/en not_active Withdrawn
- 2000-12-14 JP JP2001544994A patent/JP2003517149A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2492674A1 (en) | 2011-02-28 | 2012-08-29 | Lexogen GmbH | Biosensor array formed by junctions of functionalized electrodes |
WO2012116386A1 (en) | 2011-02-28 | 2012-09-07 | Lexogen Gmbh | Biosensor array formed by junctions of functionalized electrodes |
Also Published As
Publication number | Publication date |
---|---|
JP2003517149A (en) | 2003-05-20 |
WO2001043870A2 (en) | 2001-06-21 |
WO2001043870A3 (en) | 2002-02-21 |
AU2108901A (en) | 2001-06-25 |
EP1251955A2 (en) | 2002-10-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2393766A1 (en) | Column and row addressable high density biochip array | |
US6602400B1 (en) | Method for enhanced bio-conjugation events | |
JP4562914B2 (en) | Chemical or biological analysis multipoint microsystem | |
Kelley et al. | Electrochemistry of methylene blue bound to a DNA-modified electrode | |
Drummond et al. | Electrochemical DNA sensors | |
US8097134B2 (en) | Addressable chem/bio chip array | |
E Ferapontova | Electrochemical indicators for DNA electroanalysis | |
EP2681545B1 (en) | Biosensor array formed by junctions of functionalized electrodes | |
US20020028441A1 (en) | Detection of molecules and molecule complexes | |
US7488607B2 (en) | Electronically readable microarray with electronic addressing function | |
WO1999067628A1 (en) | Multi-sensor array for electrochemical recognition of nucleotide sequences and methods | |
AU2907201A (en) | Methods and compositions relating to electrical detection of nucleic acid reactions | |
US9400278B2 (en) | Biochip | |
US20040157263A1 (en) | Method for impedimetric detection of one or more analytes in a sample, and device for use therin | |
JP2004524534A (en) | Detection method of giant biopolymer using electrode structure | |
EP1190253A1 (en) | Biosensors which utilize charge neutral conjugated polymers | |
de Lumley-Woodyear et al. | Reactive electrophoretic activation of a microelectrode for enzyme-amplified recognition and for melting-temperature determination of 105 copies of a simple oligonucleotide | |
JP2003090815A (en) | Method for electrochemically detecting gene, and nucleic acid tip | |
Gajovic-Eichelmann et al. | Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer | |
US20020051975A1 (en) | Reporterless genosensors using electrical detection methods | |
US20050069905A1 (en) | Detection of molecular binding events | |
CA2471339A1 (en) | Method and device for the quantitative electrical detection of analytes | |
WONG | Recent trends in DNA biosensing technologies | |
KR20050080544A (en) | Method of manufacturing microelectrode -array type dna chip | |
KR100633048B1 (en) | Electrochemical detection method for indicator- free gene |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Discontinued |