CA2309450C - Nanoelectrode arrays - Google Patents
Nanoelectrode arrays Download PDFInfo
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- CA2309450C CA2309450C CA002309450A CA2309450A CA2309450C CA 2309450 C CA2309450 C CA 2309450C CA 002309450 A CA002309450 A CA 002309450A CA 2309450 A CA2309450 A CA 2309450A CA 2309450 C CA2309450 C CA 2309450C
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- electrodes
- microcantilever
- sensor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/60—Detection means characterised by use of a special device
- C12Q2565/607—Detection means characterised by use of a special device being a sensor, e.g. electrode
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/70—Nanostructure
- Y10S977/788—Of specified organic or carbon-based composition
- Y10S977/789—Of specified organic or carbon-based composition in array format
- Y10S977/79—Of specified organic or carbon-based composition in array format with heterogeneous nanostructures
- Y10S977/791—Molecular array
- Y10S977/792—Nucleic acid array, e.g. human genome array
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/70—Nanostructure
- Y10S977/788—Of specified organic or carbon-based composition
- Y10S977/789—Of specified organic or carbon-based composition in array format
- Y10S977/79—Of specified organic or carbon-based composition in array format with heterogeneous nanostructures
- Y10S977/791—Molecular array
- Y10S977/793—Protein array
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/84—Manufacture, treatment, or detection of nanostructure
- Y10S977/849—Manufacture, treatment, or detection of nanostructure with scanning probe
- Y10S977/852—Manufacture, treatment, or detection of nanostructure with scanning probe for detection of specific nanostructure sample or nanostructure-related property
- Y10S977/853—Biological sample
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/84—Manufacture, treatment, or detection of nanostructure
- Y10S977/849—Manufacture, treatment, or detection of nanostructure with scanning probe
- Y10S977/86—Scanning probe structure
- Y10S977/874—Probe tip array
Abstract
An array of electrodes (26a, 26b, and 26c) at the atomic or nano scale (nanoelectrodes) is built on a chip (22). The spatial distribution, height, width and electro-chemical composition of the nanoelectrodes is varied, such that protein-specific electronic receptors are built directly on the chip with the nanoelectrodes without the use of an y specific binding agents or molecules. Because of their size, a very large number of different receptors can be built as arrays on a single chip. The chip can be used to detect, characterize and quantify single molecules in solution such as individual proteins, complex protein mixtures, DNA or other molecules.
Description
~ANOELECTRODE ARRAYS
This application claims the benefit of U.S. Provisional Application No.
60/065,373 filed November 12, 1997.
The present invention relates generally to methods and apparatus for detecting and characterizing single biological molecules in solution and, more specifically, to detect and characterize individual proteins, protein mixtures, DNA or other molecules on a chip.
BACKGROUND OF THE INVENTION
The characterization and quantification of individual proteins or complex biological molecules is extremely important in fields as distant as medicine, forensics and the military.
For example in medicine the presence and concentration of given proteins can be used for disease or pre-disease diagnoses. In the military given proteins can be used to signal the presence or absence of given pathogens in the environment which is extremely important for example in potential germ warfare situations.
The detection of individual proteins or molecules in biological samples is currently complex and generally requires sophisticated and bulky equipment.
Several technologies have recently been disclosed to characterize given biological molecules. In particular success has been achieved in high density DNA chips build by Affymetrix as originally described in PCT International Publication No. WO
90/15070.
U.5. Patent 5,624,537, entitled "BIOSENSOR AND INTERFACE MEMBRANE," describes a protein-receiving matrix and a single electrode.
U.5. Patent 5,395,587, entitled "SURFACE PLASMON RESONANCE DETECTOR HAVING
COLLECTOR FOR ELUTED LIGATE," describes a system to measure immobilized ligands using a plasmon resonance detector.
U.5. Patent 5,607,567, entitled "PROTAMINE-RESPONSIVE POLYMERIC MEMBRANE
ELECTRODE," describes a membrane electrode.
U.5. Patent $,328,847, entitled "THIN MEMBRANE SENSOR WITH BIOCHEMICAL
SWITCH," describes a biosensor with a specific recognition biomolecule.
U.S. Patent 4,777,019, entitled "BIOSENSOR," describes a biosensor for biological monomers.
U.5. Patent 5,532,128, entitled "MULTI-SITE DETECTION APPARATUS," describes test wells combined with electrodes to detect given biological molecules.
U.5. Patent 4,983,510, entitled "ENZYMES IMMOBILIZED ON LATEX POLYMER
PARTICLES FOR USE WITH AN AMINO ACID ELECTROSENSOR," describes an electrosensor with a latex polymer trap.
U.S. Patent 5,384,02$, entitled "BIOSENSOR WITH A DATA MEMORY," describes a membrane biosensor with a memory module.
U.5. Patent 5,567,301, entitled "ANTIBODY COVALENTLY BOUND FILM IMMUNOBIO-sENSOR," describes an antibody biosensor.
U.S. Patent 5,310,469, entitled "BIOSENSOR WITH A MEMBRANE CONTAINING
BIOLOGICALLY ACTIVE MATERIAL," describes a membrane biosensor.
U.5. Patent 5,019,238, entitled "MEANS FOR QUANTITATIVE DETERMINATION OF
ANALYTE IN LIQUIDS," describes a means to sequentially test the ionic concentration of fluids.
This application claims the benefit of U.S. Provisional Application No.
60/065,373 filed November 12, 1997.
The present invention relates generally to methods and apparatus for detecting and characterizing single biological molecules in solution and, more specifically, to detect and characterize individual proteins, protein mixtures, DNA or other molecules on a chip.
BACKGROUND OF THE INVENTION
The characterization and quantification of individual proteins or complex biological molecules is extremely important in fields as distant as medicine, forensics and the military.
For example in medicine the presence and concentration of given proteins can be used for disease or pre-disease diagnoses. In the military given proteins can be used to signal the presence or absence of given pathogens in the environment which is extremely important for example in potential germ warfare situations.
The detection of individual proteins or molecules in biological samples is currently complex and generally requires sophisticated and bulky equipment.
Several technologies have recently been disclosed to characterize given biological molecules. In particular success has been achieved in high density DNA chips build by Affymetrix as originally described in PCT International Publication No. WO
90/15070.
U.5. Patent 5,624,537, entitled "BIOSENSOR AND INTERFACE MEMBRANE," describes a protein-receiving matrix and a single electrode.
U.5. Patent 5,395,587, entitled "SURFACE PLASMON RESONANCE DETECTOR HAVING
COLLECTOR FOR ELUTED LIGATE," describes a system to measure immobilized ligands using a plasmon resonance detector.
U.5. Patent 5,607,567, entitled "PROTAMINE-RESPONSIVE POLYMERIC MEMBRANE
ELECTRODE," describes a membrane electrode.
U.5. Patent $,328,847, entitled "THIN MEMBRANE SENSOR WITH BIOCHEMICAL
SWITCH," describes a biosensor with a specific recognition biomolecule.
U.S. Patent 4,777,019, entitled "BIOSENSOR," describes a biosensor for biological monomers.
U.5. Patent 5,532,128, entitled "MULTI-SITE DETECTION APPARATUS," describes test wells combined with electrodes to detect given biological molecules.
U.5. Patent 4,983,510, entitled "ENZYMES IMMOBILIZED ON LATEX POLYMER
PARTICLES FOR USE WITH AN AMINO ACID ELECTROSENSOR," describes an electrosensor with a latex polymer trap.
U.S. Patent 5,384,02$, entitled "BIOSENSOR WITH A DATA MEMORY," describes a membrane biosensor with a memory module.
U.5. Patent 5,567,301, entitled "ANTIBODY COVALENTLY BOUND FILM IMMUNOBIO-sENSOR," describes an antibody biosensor.
U.S. Patent 5,310,469, entitled "BIOSENSOR WITH A MEMBRANE CONTAINING
BIOLOGICALLY ACTIVE MATERIAL," describes a membrane biosensor.
U.5. Patent 5,019,238, entitled "MEANS FOR QUANTITATIVE DETERMINATION OF
ANALYTE IN LIQUIDS," describes a means to sequentially test the ionic concentration of fluids.
U.S. Patent 4,981,572, entitled "ELECTRODE UNIT AND PACKAGE FOR A BLOOD
ANALYZER," describes an electrode and apparatus to analyze blood.
U.5. Patent 4,452,682, entitled "APPARATUS FOR MEASURING CLINICAL EMERGENCY
CHECK ITEMS OF BLOOD," describes an apparatus to measure multiple elements in blood.
U.S. Patent 4,568,444, entitled "CHEMICAL SUBSTANCE MEASURING APPARATUS,"
describes an electrode to quantify chemical substances in a solution.
U.5. Patent 5,2$1,539, entitled "IMMUNOASSAY DEVICE FOR CONTINUOUS MONI-TORING," describes a two step immunoassay device.
U.5. Patent 5,192,507, entitled "RECEPTOR-BASED BIOSENSORS," describes a biosensor based on a polymeric film to detect opiates.
U.5. Patent 5,156,810, entitled "BIOSENSORS EMPLOYING ELECTRICAL, OPTICAL AND
MECHANICAL SIGNALS," describes a thin layer biosensor.
U.5. Patent 5,494,831, entitled "ELECTROCHEMICAL IMMUNOSENSOR SYSTEM AND
METHODS," describes an immunologic biosensor.
U.5. Patent 5,332,479, entitled "BIOSENSOR AND METHOD OF QUANTITATIVE ANALYSIS
USING THE SAME," describes an electrode based sensor with a biologically active receptor.
U.5. Patent 5,582,697, entitled "BIOSENSOR, AND A METHOD AND A DEVICE FOR
QUANTIFYING A SUBSTRATE IN A SAMPLE LIQUID USING THE SAME," describes a biosensor based on the measure of reduction between a substrate and an oxidoreductase.
U.S. Patent 4,908,112, entitled "SILICON SEMICONDUCTOR WAFER FOR ANALYZING
MICRONIC BIOLOGICAL SAMPLES," describes a micro capillary separation device with detector capabilities.
U.5. Paterit 5,409,583, entitled "METHOD FOR MEASURING CONCENTRATIONS OF
ANALYZER," describes an electrode and apparatus to analyze blood.
U.5. Patent 4,452,682, entitled "APPARATUS FOR MEASURING CLINICAL EMERGENCY
CHECK ITEMS OF BLOOD," describes an apparatus to measure multiple elements in blood.
U.S. Patent 4,568,444, entitled "CHEMICAL SUBSTANCE MEASURING APPARATUS,"
describes an electrode to quantify chemical substances in a solution.
U.5. Patent 5,2$1,539, entitled "IMMUNOASSAY DEVICE FOR CONTINUOUS MONI-TORING," describes a two step immunoassay device.
U.5. Patent 5,192,507, entitled "RECEPTOR-BASED BIOSENSORS," describes a biosensor based on a polymeric film to detect opiates.
U.5. Patent 5,156,810, entitled "BIOSENSORS EMPLOYING ELECTRICAL, OPTICAL AND
MECHANICAL SIGNALS," describes a thin layer biosensor.
U.5. Patent 5,494,831, entitled "ELECTROCHEMICAL IMMUNOSENSOR SYSTEM AND
METHODS," describes an immunologic biosensor.
U.5. Patent 5,332,479, entitled "BIOSENSOR AND METHOD OF QUANTITATIVE ANALYSIS
USING THE SAME," describes an electrode based sensor with a biologically active receptor.
U.5. Patent 5,582,697, entitled "BIOSENSOR, AND A METHOD AND A DEVICE FOR
QUANTIFYING A SUBSTRATE IN A SAMPLE LIQUID USING THE SAME," describes a biosensor based on the measure of reduction between a substrate and an oxidoreductase.
U.S. Patent 4,908,112, entitled "SILICON SEMICONDUCTOR WAFER FOR ANALYZING
MICRONIC BIOLOGICAL SAMPLES," describes a micro capillary separation device with detector capabilities.
U.5. Paterit 5,409,583, entitled "METHOD FOR MEASURING CONCENTRATIONS OF
774J2-45,iS) SUBSTRATES IN A SAMPLE LIQUID BY USING A BIOSENSOR," describes a two step biosensor.
U.S. Statutory Invention H201, entitled "B10SENSORS FROM MEMBRANE PROTEINS
RECONSTITUTED IN POLYMERIZED LIPID B1LAYERS," describes a method for incorporating and using cell membrane proteins in biosensors.
The above described technologies are generally used for the detection of a single type or a few different types of molecules. None of these technologies are particularly adapted to allow a very large number of different types of proteins, protein variants or other biological molecules to be detected and quantified simultaneously on a single chip.
Furthermore, none of the prior art provides a suitable technology to directly build protein-specific electronic receptors on a chip without the use of any biological binding agents, synthetic probes or complex micro-structures such as test wells.
We disclose herein a novel, smaller, faster and more cost effective technique to detect, characterize and quantify individual proteins or other complex molecules on a chip. The technology described herein may also serve as a new method for DNA sequencing.
U.S. Statutory Invention H201, entitled "B10SENSORS FROM MEMBRANE PROTEINS
RECONSTITUTED IN POLYMERIZED LIPID B1LAYERS," describes a method for incorporating and using cell membrane proteins in biosensors.
The above described technologies are generally used for the detection of a single type or a few different types of molecules. None of these technologies are particularly adapted to allow a very large number of different types of proteins, protein variants or other biological molecules to be detected and quantified simultaneously on a single chip.
Furthermore, none of the prior art provides a suitable technology to directly build protein-specific electronic receptors on a chip without the use of any biological binding agents, synthetic probes or complex micro-structures such as test wells.
We disclose herein a novel, smaller, faster and more cost effective technique to detect, characterize and quantify individual proteins or other complex molecules on a chip. The technology described herein may also serve as a new method for DNA sequencing.
.r 7740'2-4~yS) SU~iARY OF THE INVENTION
According to one aspect of the present invention, there is provided a sensor for detecting biological molecules, said sensor comprising: a substrate; an electrode having the capacity to bind a preselected biological molecule, said electrode having between about 10-9 and 10-10 meters in height and width.
According to another aspect of the present invention, there is provided a sensor for detecting proteins, said sensor comprising: a micro-capillary tube; a plurality of electrodes disposed in said tube, said electrodes having the capacity to bind a preselected protein, said electrodes being between about 10-9 and 10-l0 meters in height and width.
According to still another aspect of the present invention, there is provided a sensor for detecting biological molecules, said sensor comprising: a substrate; a micro cantilever array on said substrate; at least one electrode disposed on at least one of said micro cantilevers.
According to yet another aspect of the present invention, there is provided a sensor for detecting a biological molecule, said sensor comprising: a microcantilever, wherein at least one electrode comprises a height and width and length disposed on the microcantilever, wherein the at least one electrode disposed on the microcantilever is adapted to interact with and bind to a concentration of a biological molecule; and further comprising at least one from the group consisting of a capacitive means, an electron tunneling means, a laser means, a piezoresistive means, a piezoelectric means, a 4a 774.0'2-4~'~S) resonance frequency shift means and a x-y positional fluorescence means for detecting the concentration of the biological molecule adapted to bind to the at least one electrode, and wherein the microcantilever has a plurality of electrodes disposed thereon forming a cluster, each electrode having varying dimensions adapted to bind the cluster with biological molecules and for detecting concentrations thereof.
According to a further aspect of the present invention, there is provided a sensor comprising: a base; a microcantilever integrally attached to the base; and at least one electrode disposed on the microcantilever, wherein the electrode extends from a principal surface of the microcantilever a distance of from about 2 Angstroms to about 5 nanometers, the electrode having a width of from about 2 Angstroms to about S nanometers.
According to yet a further aspect of the present invention, there is provided a method of sequencing nucleic acids, comprising the steps of: providing a sensor, said sensor having a substrate on which plurality of electrodes are disposed, said electrodes each being between about 10-9 and 10-1° meters in height and width; contacting said electrodes with a solution containing nucleic acids; said electrodes having the capacity to bind at least some of said nucleic acids.
According to still a further aspect of the present invention, there is provided a method for producing a sensor comprising: providing a microcantilever, the microcantilever having at least one electrode disposed on the microcantilever wherein the electrode extents from a principal surface of the microcantilever a distance of from 4b .r 774~0'2-45,f S) about 2 Angstroms to about 5 nanometers, and a width of from about 2 Angstroms to about 5 nanometers.
According to another aspect of the present invention, there is provided a silicon chip to detect individual proteins comprising at least one sensor manufactured with Angstrom level precision where the surface of the sensor complements exactly the three dimensional shape of a given protein.
In one aspect the present invention provides a sensor which is capable of distinguishing between different molecular structures in a mixture. The device includes a substrate on which nanoscale binding sites in the form of multiple electrode clusters are fabricated. Each binding site includes nanometer scale points which extend above the surface of a substrate. These points are preferably nanoelectrodes which are spatially configured to provide a three-4c dimensional electro-chemical binding profile which mimics a chemical binding site. Thus, the binding sites have selective affinity for a complementary binding site on a target molecule or for the target molecule itself.
In one aspect, the binding sites are arranged in an array on the substrate. In one aspect, the spatial and electro-chemical profiles of each site of the array are identical and provide an assay for a single target molecule. In another aspect, regions of the nanoelectrode array carry grouped arrays of electronically and/or spatially distinct binding sites for simultaneous detection and quantification of several molecular species.
In still another aspect, the materials used for the electrodes and surrounding surfaces are selected based on preferred intrinsic electrical and chemical properties.
The nanoelectrode array may be included in a chamber which can retain fluids.
Several arrays may be used in a single chamber and several different chambers may be used on a single chip.
In still another aspect, the nanoelectrode array and chamber are attached to at least one micro-fluidic delivery and separation system such as a micro-capillary which allows both the delivery and separation by size and electrical properties of the proteins or other molecules to be analyzed.
In another aspect, a microcontroller or microprocessor is provided to analyze signals from the nanoeLectrodes and/or to time and control the fluidics separation of the-molecules or proteins.
In another aspect, the chip with the nanoelectrode arrays is associated with an electronic temperature control system such as a thermoelectric device having a thermistor to vary the bonding kinetics or the electro-chemical affinity of the molecules with given nanoelectrodes, as well as the flow kinetics and separation of the molecules.
In another aspect, the nanoelectrodes are interspaced in a linear microtube to sequence DNA.
Thus, it is an object of the present invention to provide a novel and rapid method to analyze small biological molecules in solution such as proteins and to sequence DNA by using semiconductor chip technology with extremely high packing densities.
It is a further object of the present invention to ensure that the entire chip can be easily integrated into devices for automated analysis of proteins, DNA or other molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective diagrammatic view of a nanoelectrode array showing different nanoelectrode clusters.
FIG. 2 is a side elevational diagrammatic view of a protein-specific electronic receptor and its matching protein.
FIG. 2A is a side elevational cross-section of a protein-specific electronic receptor and its matching protein.
FIG. 3 is a side elevational cross-section of a nanoelectrode array inside a micro-fluidic tube, showing the trapping of a specific protein on its corresponding nanoelectrode receptor.
FIG. 4 is a diagrammatic side elevational cross-section of a microtube with a linear nanoelectrode array to detect DNA.
FIG. 5 is a cross-section of an integrated chip with nanoelectrode arrays, a micro-fluidic delivery system and associated electronics.
FIG. 6 is a side elevational cross-section of a nanoelectrode receptor showing the electrical field which is broken or modified upon binding of a specific molecule to said receptor.
FIG. 7 is a view of a cantilevered nanoplate with several identical nanoelectrode clusters.
D~T.~H~ED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is based in part on the fact that recent advances in technology WO 99/24823 PCTIUS98/2354~
such as the use_of scanning tunneling microscopy (STM) has demonstrated that ultra small structures of a single or a few atomic layers can be built on a semiconductor surface such as silicon. Because of the size of these structures, they are generally referred to as nanostructures (one nanometer or nm = 10'9m, 1 Angstrom or ~r = 10''°m). These structures can be as small as a few Angstroms in diameter which is well below the Stokes radius of a small protein (which is approximately 25-35~}. Since these structures can be built using different chemical elements (or the voltage applied to the structure can be selectively varied) and the spacial distribution, height width and shape of the structures can also be varied, these structures can be built in clusters to serve specifically as "molecular electrodes" whose electro-chemical properties and spacial distribution can be made to correspond precisely with the external three dimensional shape and electro-chemical properties of molecules, preferably biochemicals and most preferably proteins. Therefore each of these clusters can serve as individual electronic protein "receptors" (or detectors). Since a very large number of these molecular electrodes can be placed on a single chip, the resulting arrays, termed here "nanoelectrode arrays" can be used to detect, characterize and quantify many different proteins on a single chip. In a variation of the technology, the chip can also be used to sequence DNA.
Referring now to Figure 1 of the drawings, microelectronic molecular sensor 20 is seen having substrate 22 on which an array of binding sites or clusters 24 are formed. Substrate 22 may comprise any number of materials such as silicon, germanium, gallium arsenide, or other semiconductors. Referring now to Figure 2 of the drawings, one binding site 24 is shown in more detail having multiple electrodes 26a, 26b and 26c which are spatially distributed to form a pattern. Thus, it can be seen that each electrode 26a, 26b and 26c in this particular embodiment is spaced laterally from the adjacent electrode and is elevated at different heights off principal surface 28 of substrate 22.
It will be appreciated that through molecular modeling and empirical data, the topology of the binding sites and electrical charge are tailored to provide the required electrical and topographic properties to selectively recognize and bind a complementary region of a target molecule. As shown best in Figure 2, protein 30 having a defined shape specific to that protein attaches to a given nanoelectrode cluster composed of three nanoelectrodes 26a, 26b and 26c.
As will be explained more fully, each nanoelectrode may have slightly different electro-chemical properties because of differing charges and/or chemical compositions.
These individual electro-chemical properties match not only the electro-chemical affinities of the amino acids or atoms present on the grooves of the protein but also complement the shape of the groove itself. Thus, when a molecule having the proper complementary profile binds to "receptor" 24 bridging the gap between the electrodes, a change in electrical potential occurs which can be monitored through appropriate circuitry to provide an indication of the presence of the target molecule.
In the most preferred embodiments of the present invention binding sites 24 have nanoscale geometries. As illustrated in Figure 2, the distance from principal surface 28 to the top of electrode 26b is 1.9 nanometers, the width of electrode 26b is 0.7 nanometers and the distance between electrodes 26b and 26c is 1 nanometer. In general, each electrode will typically be between 0.2 and about 3 nanometers in height and from about 0.2 to about 2 nanometers in width. As used herein "nanoelectrode" shall include atomic scale as well as x'7402-45 (S) nanoscale structures, i.e. from 2A to 5 nanometers. There will also typically be.from about 2 to about 8 separate electrodes in a°.ac:h cluster 24, lrlectrodes 26x, 26b and 26c can be formed of a number of materials, either irnrinsic or doped, such as gold and platinum and copper and other electrometals. Gold is particularly preferred. Also it may be suitable to form the electrodes of one ma~~erial and coax the outer portion with a different material, e.g. gold coated with zinc oxide or gold coated with a thiol group.
The electrodes may be eaclo separately connected to a power source by small conductive regions or wires which may be for~~nc:d of gold. (n Figure ? A , individual conductive layers 34x, 34b and 34c are shown electrically connecting their respective electrodes 26x, 26b and 26c.
Dielectric layers 36 electrically isc:>late the individual conductive layers and dielectric sheaths 38 electrically isolate the individual electrodes. It will be appreciated that different potentials can be applied to the various indj~,-~idual electrodes and that electrodes from different clusters can be electrically linked to a single layer e.g., layer >4a. It will be appreciated that the various layers can be formed using conventional thin-filno fabrication techniques such as CVD, thermal growth and ion implantation.
It has been shown recentl~r tlhat electrical "wires" can be built of single atoms (see for example review by L.eo Kouwenh<:wf:n "Single-Molecule 7yransistors", Science, Vol. 275, pages 1896-1897,28 March 1997) .
The wires can be deposited in a m m~ber of different ways as pan of the microchip fabrication process, prior to the deposition o:(~ the nanoelectrodes. The nanoelectrodes can be deposited directly on the chip by Scanning ~l('unneling Microscope (as described in Kolb et al., Science, 77402-45(S) Vol. 275, pages 109?-1099, ? I F~b~ruary 19?7 >. A number of other chip fabrication methods are possible such as different Lithography techniques, etc.
In another aspect the naraoelectrodes are not corrrtected to any electrical wires or conductive layers. In this case the binding of the protein or other molecule is simply dependant on the shape and chemical properties of the individual nanoelectrode clusters.
Defection of the attachment of the given molecule to a given cluster can then be achieved by means other than electrical, for example by a highly precise x-y positionai fluorescence reader, similar to that used for the DNA chip technolog;~ ot' by resonance.
In case the nanoelectrodes are not connected to wires (i.e. are not "live"
electrodes), the nanoelectrodes may in some applications be intercoru'tected in a given cluster. In this case the clusters would comprise intercormected peaks and grooves and these would form a larger structure (i.e. from , to > 10 nanorr~eters). 'this structure could be tailored either to match precisely the actual biological re~:eptor of the target molecule or to allow the entire molecule to fit into a 3-dimensional "recelator" which would match at least a third of the overall 3-D
shape of the molecule. In some ir:~stances and depending on the overall shape of the molecule, the receptor that is built may not necessarily include a site corresponding to the actual biological receptor of the target molecule.
Several types of binding or adsorption of the molecule to the nanoelectrode receptor are possible, depending on the cherrtical composition of the nanoelectrodes, the voltage and the chemical to be measured. Binding forces may include covalent binding, electrostatic binding, hydrogen bonds and van der Waals bonds.
Depending on the type of detection that is required, the individual nanoelectrodes of individual clusters do not necessarily need to be composed of different electrometals since both the spacial distribution and the height of the nanoelectrodes can be varied and these two variables may be enough for specific molecule detection in given applications.
In some applications, each nanoelectrode can be selectively charged in a given cluster, allowing the electro-physical property of the nanoelectrode to be varied.
The entire sensor can be built using a computer controlled operation, where the spacing, height, width and the composition of the nanoelectrodes can be made to correspond exactly to the three dimensional shape and matching electro-chemical properties of a selected molecule.
Furthermore, since the position of the nanoelectrode clusters corresponding to a given receptor for a given molecule is determined during the fabrication process, this position information can be used to detect attachment or binding. For example, a large nanoelectrode array can be built with many different clusters, binding in a solution can be allowed, then the array be read using a highly accurate x-y reader in a way similar to the DNA chip. Computer control fabrication of the nanoelectrodes also allows for identical copies of the chip to be made.
It will be also be appreciated that the geometries that are built on the surface of the chip can be made to correspond exactly to the matching image of a crystallized protein surface taken from x-ray diffraction studies. Hence nanoelectrode array clusters can be built directly using crystallographic data and the resulting surfaces on the chip would favor protein-specific crystallization on given arrays.
In another aspect, since multiple identical receptors can be built on the same chip, this technology can be used not only to detect given molecules but also to precisely estimate the quantity of these molecules present in the sample by measuring binding rates in identical clusters.
Refernng to FIG. 3, two partial nanoelectrode arrays are shown facing each other and forming micro-channel or nanotube 60, which permits the flow of small molecules such as protein 70 therethrough. If protein 70 matches the shape of a receptor composed of electrodes 74, 78, and 82, the physical binding of the protein will cause a temporary minute change in the electrical signal which can be measured simultaneously in all said nanoelectrodes. The strength of the electrical signal can be modified for example by adding a conductant to the carrier solution for the molecules which need to be studied. Alternatively the nanoelectrodes themselves can be charged with a small current, which would change upon attachment of the given molecule. Depending on the electro-chemical properties of the nanoelectrodes and the analyte, the temperature and the flow rate, the binding may last only a fraction of a second or last longer. Time of retention in itself is another important variable which can be used in detecting and quantifying the types of molecules present in the sample.
In some applications, micro-channel 60 can form a part of a network of channels of different and specific sizes, matching the sizes of the proteins to measure.
Each of these channels can be_equipped with molecular sieves, allowing only proteins or molecules of certain size to pass through. The channels themselves can also serve as a means to separate molecules and deliver them to given detector chambers with nanoelectrode arrays which are specifically made to measure given classes of proteins or molecules of given molecular weights. In this case, each of the arrays would have nanoelectrodes with sizes corresponding to the sizes of the proteins to measure. As part of this network of channels, specific chambers can be added with specific functions such as a chamber to lyse cells. Other chambers can be filled with specific reagents which can be used as needed.
In another application, each of the micro-channels is equipped with only one or a few nanoelectrode clusters and the protein mix is flowed through each of the channels. With the help of a microcontroller or a microprocessor controlling the flow rate in each micro-channel, the signals from each of the nanoelectrode clusters is then measured combining the power of the following variables for detection: protein separation rates (based on the size and charge of the proteins) and retention time on each given cluster (based on th.e shape and electro-chemical properties of the molecule). Indeed, the more a given molecule matches a given receptor, the longer it will bind. It is obvious that the sophisticated control and measure of the electrical signals in each nanoelectrode (as well as the control of all other variables such as sample flow rates, temperature, etc.) can only be done with the help of a microcontroller or a microprocessor.
Refernng now to FIG. 4, a nanoarray of electrodes 90 is built in a linear microtube 100 with the spacing and electro-chemical composition of the nanoelectrodes varied in such a way *rB
to correspond exactly to the distance between given base pairs of a linear piece of DNA or RNA
110. In this case, the nanoelectrodes are built using only two variables:
precise spacing and electro-chemical composition (not height) favoring position-specific binding of specific base pairs of DNA or RNA to matching nanoelectrodes. The principle that is applied here is that DNA is known to behave as a linear molecule when flowed in a microtube and that this rate of flow can be controlled and measured with precision. Furthermore, the distance between 10 DNA base pairs being precisely 34 A, the nanoelectrodes can be spaced precisely in multiples of 3.4 t~ as shown in 120. By varying the spacing and charge and/or composition of the nanoelectrodes and by measuring the conductance changes over time in sequentially placed nanoelectrodes, an entire sequence is created, based on the timing of the signals of position-specific nanoelectrodes. The full DNA (or RNA) sequence is then reconstructed with the help of a microcontroller (or microprocessor) which can also control the flow rate in the microtube.
ANALYSIS OF PROTEIN VARIANTS
Mutations or other changes in the DNA result in amino-acid substitutions in the protein.
These substitutions in turn result in conformational shape changes in the protein and can result in proteins that are either non-functional or have different properties. Since the three-dimensional (3-D) structure of proteins can now be inferred with precision on the basis of x-ray crystallography or nuclear magnetic resonance (NMR), the 3-D shapes of the protein variants can also be generated using the same method. Hence the entire spectrum of protein variants for given classes of proteins can be measured and quantified using the nanotechnology described above. This is because the conformational changes of each protein variant can be represented by a given nanoelectrode cluster varying in the shape, distribution and electro-chemical properties of the nanoelectrodes. In fact, the building of the arrays can be_comput~er-controlled and link the information matching the putative 3-D structure of proteins of interest (and their variants} to the micro fabrication of all the matching receptors on the chip.
By measuring and quantifying these variants as described above, this approach represents a powerful alternative to direct DNA sequencing since all the possible mutation products of given genes which are expressed can be directly measured on a chip. Another advantage is that the chip would be fully reusable. Furthermore, given the extremely high density of the nanoelectrode arrays that can be built on a single chip, the entire spectra of protein variants for many genes can be measured at once on the same chip. In fact with a refinement in the technology, all existing human proteins and their variants could theoretically be measured on a single chip of 1 cm2 and the number of receptors that could be built on such a chip could theoretically exceed 1 billion which is a thousand fold improvement over any existing technology.
PROTEIN SEPARATION
As indicated above, the separation of molecules can be achieved by flowing said molecules in extremely small tubes (micro-capillaries, micro-channels or nanotubes) where smaller molecules travel faster than larger ones which are retained by friction and weak bonding interactions with the surfaces of the tubes. The result that is achieved is equivalent to electrophoresis but with the advantage of speed, cost and reusability of the micro-capillary.
Refernng now to FIG. 5, micro-channel 130 is shown with a sample input port 132 and a long loop flowing into an optional reagent micro-chamber 134, itself connected to an optional input port 136. Micro-channel 130 separates biological molecules by size and charge while micro-chamber 134 allows the selective input of an external reagent or solution. The flow and on/off position at each micro-channel juncture can be controlled electronically either by an external micro-pump (not shown), by thermocapillary action or by a change of electric potential. After entering micxo-chamber 134, the analyte then flows successfully into micro-chambers 138a, 138b, 138c, then 138d, each holding different nanoelectrode arrays with nanoelectrode clusters of varying sizes and densities. In this particular design, the nanoelectrode arrays are fabricated immediately adjacent to a micro-electronics multiplexing or control area 140, itself connected to an interface 142. After reacting with successive nanoelectrode arrays in successive micro-chambers, the sample exits via port 146. The micro-channels and micro-chambers can either be etched in the silicon surface itself or can be fabricated separately on a surface of a material like glass, diamond, plastic or the like, which is then attached to the silicon surface.
This design can be varied in many different ways and FIG. 5 illustrates just one of many possible combinations of micro-channels, nanoelectrode arrays and micro-electronics that can be fabricated on a chip. As indicated above, a chamber allowing the lysing of the cells or viruses to be analyzed can also be included on the chip. Also, it should be indicated that the directional flow in the micro-channels can be reversed and that each connecting micro-channel can be selectively opened or closed electrically. Hence, when the test is completed the entire system can be heated to allow protein denaturation (andlor the potential in the nanoelectrodes can be reversed), then the system can be flushed with a solution to clean the nanoelectrode arrays and allow reuse of the chip.
Hence a_complete and integrated protein separation and detection systerr~ can be built on a single chip. An important aspect of combining nanoelectrode arrays, micro-channels and a rnicrocontroller (or a microprocessor) is that the time of separation (from sample injection into port 132 to time of first detection) and the length of retention on given nanoelectrode receptors are important variables for characterizing individual protein or protein variants. For example, the system can be calibrated by injecting known proteins, then known mixes of proteins, prior to injecting the sample to be tested. The time taken to reach a given nanoelectrode receptor and the length of binding on different electronic receptors would be specific to specific proteins (or to protein variants) and the signal-specific profiles for each protein can then be stored in memory and compared to those of the sample to be tested.
While FIG. 5 shows an integrated design, it is obvious that the protein separation component and the electronic components can also be placed externally and that the chip can be as simple as having a single nanoelectrode array enclosed in a single chamber with an interface. This chip (which may be disposable) can then be inserted into a larger module with the above components. Also, as indicated below, other detection methods can be used and the design of the chip would change accordingly.
DETECTION
There are many ways in which the binding or adsorption of the analyte on the nanoelectrode array can be detected. Refernng now to FIG. 6, one way of detecting the signal due to adsorption on the nanoelectrode array is by electrical signal. In this case, at least one of the electrodes in each cluster of a given array is used as a "source" 160 while the rest of the cluster 165 is used as a "sink." When an analyte, say a protein, is adsorbed it changes the flow of the current (pico ampere) as shown in FIG. 6. The electrodes are isolated by a layer of oxide 170. The unwanted effects of the electrical current can be avoided by using an AC approach.
Referring now to FIG. 7, the second approach for detection of binding is by using a resonance approach. In this method, a nano structure is constructed. For example, nanoplate 180 of the dimension less than one micron is built. This structure can be free standing or it can be cantilevered. Identical sets of nanoelectrode receptors 24 are then fabricated on this surface.
The structure is designed to have resonance frequency in the MHz to low GHz region. As the analyte flows past these structures, they spend a longer time on the cantilever if they have a structure that is complementary to the nanoelectrode structure. In other words, the analyte molecules undergo collision with the nanoplate. If there exists any complimentary nature between the analyte and the substrate, the analyte will spend more time on the surface during collision. This can be detected optically by shining a laser diode on the structure and detecting the reflected signal using a position sensitive photodiode. The AC signal in the photodiode shows the resonance response of the structure. The greater the signal, the larger the concentration of bound biological molecules, i.e. the greater the concentration of the said molecule in the solution. Other detection techniques such as capacitive, piezoresistive, piezoelectric, electron tunneling, etc. could also be used.
The structure can be excited into resonance response by mechanical means using a piezoelectric element. In this technique, a nanoplate structure is attached to a piezoelectric material which can be vibrated using an AC signal. At resonance the structures oscillate with maximum amplitude. It can also be excited into resonance by modulating the diode laser using square wave power pulses. Since square waves contain all the Fourier components, there will be a component that corresponds to the resonance frequency of the structure.
Since these nanoelectrodes can be constructed on geometrical structures with extremely small thermal mass (for example, nanoplates have a thermal mass of the order of many picograms or less), they can be heated and cooled in the micro to milliseconds time frame. This fact can be used to adsorb and desorb analytes in a periodic fashion. However, when there is a complimentary structure between surface and the analytes the desorption time scale will be different.
USE OF AN EXTERNAL DETECTOR
In another detection application the entire chip which has been allowed to react with the sample is placed in a x-y laser reader in a manner similar to the DNA chip. In this case, the chip is incorporated into a highly precise holder to ensure accurate position reading of each cluster. Detection may be done by fluorescence, for example after reaction of the bound samples to the clusters with a fluorescent molecule or with labeled antibodies.
Detection may also be done by other means such as laser-desorption-ionization mass spectrometry.
NANOELECTRODE CONSTRUCTION
Nanoelectrode arrays can be constructed on a doped semiconductor substrate by nanolithography using scanning probes. In this approach, metal clusters are deposited either from a solution or by field evaporation from a STM/AFM tip. Since the electric field between the tip and the substrate is very high (109V/m), many metals can be field evaporated. In solution many metals can be electrochemically deposited on a surface. The surface of the semiconductor can be oxidized to be an insulator.
Nanometer scale trenches and lines can be made on a semiconductor surface using STM
tip in an oxide etching solution producing a trench. The depth of the trench depends on the time spent by the tip at that location and the voltage on the tip. Hence, not only can the nanoelectrodes be built by deposition, but they can also be built by etching.
The trenches can also be used to make the channels to separate the proteins, as instructed above.
It should also be noted that nanotransistors can be built directly in the chip to facilitate detection and increase the density of the detectors. The nanotransistors can be built prior to the deposition of the nanoelectrodes as a sub-layer in the overall chip manufacturing process or be placed on an adjacent part of the chip.
The above-described principles illustrate the wide variety of applications that are possible in the micro fabrication and applications of the nanoelectrode arrays. For example, the entire system, from sample input to detection with output signals sent to an external device such as a monitor, can be built on a single chip, using micro-channels (for sample separation and delivery), miniature ionic pumps, sample detection, a built-in microcontroller, a method for temperature control, etc. This chip can be inserted into a measuring device, for example for use in a physician's office or into a field detector. If a very large nanosensor array is~ used, it may be preferable to use a microprocessor or several microcontrollers to control the above described functions. In some applications the large arrays can be used with an external laser reader. In this case, the array can be used in a way similar to the DNA chip, where the entire chip is allowed to react with the entire sample, washed and then inserted into an external reader. Using this approach the chip can be build into a convenient handling cassette.
While the invention has been described with respect to specific embodiment for complete and clear disclosure, the appended claims are not to be thus limited but are to be construed as embodying all modifications and alternative constructions that may occur to one skilled in the art which fairly fall within the basic teaching here set forth.
*rB
According to one aspect of the present invention, there is provided a sensor for detecting biological molecules, said sensor comprising: a substrate; an electrode having the capacity to bind a preselected biological molecule, said electrode having between about 10-9 and 10-10 meters in height and width.
According to another aspect of the present invention, there is provided a sensor for detecting proteins, said sensor comprising: a micro-capillary tube; a plurality of electrodes disposed in said tube, said electrodes having the capacity to bind a preselected protein, said electrodes being between about 10-9 and 10-l0 meters in height and width.
According to still another aspect of the present invention, there is provided a sensor for detecting biological molecules, said sensor comprising: a substrate; a micro cantilever array on said substrate; at least one electrode disposed on at least one of said micro cantilevers.
According to yet another aspect of the present invention, there is provided a sensor for detecting a biological molecule, said sensor comprising: a microcantilever, wherein at least one electrode comprises a height and width and length disposed on the microcantilever, wherein the at least one electrode disposed on the microcantilever is adapted to interact with and bind to a concentration of a biological molecule; and further comprising at least one from the group consisting of a capacitive means, an electron tunneling means, a laser means, a piezoresistive means, a piezoelectric means, a 4a 774.0'2-4~'~S) resonance frequency shift means and a x-y positional fluorescence means for detecting the concentration of the biological molecule adapted to bind to the at least one electrode, and wherein the microcantilever has a plurality of electrodes disposed thereon forming a cluster, each electrode having varying dimensions adapted to bind the cluster with biological molecules and for detecting concentrations thereof.
According to a further aspect of the present invention, there is provided a sensor comprising: a base; a microcantilever integrally attached to the base; and at least one electrode disposed on the microcantilever, wherein the electrode extends from a principal surface of the microcantilever a distance of from about 2 Angstroms to about 5 nanometers, the electrode having a width of from about 2 Angstroms to about S nanometers.
According to yet a further aspect of the present invention, there is provided a method of sequencing nucleic acids, comprising the steps of: providing a sensor, said sensor having a substrate on which plurality of electrodes are disposed, said electrodes each being between about 10-9 and 10-1° meters in height and width; contacting said electrodes with a solution containing nucleic acids; said electrodes having the capacity to bind at least some of said nucleic acids.
According to still a further aspect of the present invention, there is provided a method for producing a sensor comprising: providing a microcantilever, the microcantilever having at least one electrode disposed on the microcantilever wherein the electrode extents from a principal surface of the microcantilever a distance of from 4b .r 774~0'2-45,f S) about 2 Angstroms to about 5 nanometers, and a width of from about 2 Angstroms to about 5 nanometers.
According to another aspect of the present invention, there is provided a silicon chip to detect individual proteins comprising at least one sensor manufactured with Angstrom level precision where the surface of the sensor complements exactly the three dimensional shape of a given protein.
In one aspect the present invention provides a sensor which is capable of distinguishing between different molecular structures in a mixture. The device includes a substrate on which nanoscale binding sites in the form of multiple electrode clusters are fabricated. Each binding site includes nanometer scale points which extend above the surface of a substrate. These points are preferably nanoelectrodes which are spatially configured to provide a three-4c dimensional electro-chemical binding profile which mimics a chemical binding site. Thus, the binding sites have selective affinity for a complementary binding site on a target molecule or for the target molecule itself.
In one aspect, the binding sites are arranged in an array on the substrate. In one aspect, the spatial and electro-chemical profiles of each site of the array are identical and provide an assay for a single target molecule. In another aspect, regions of the nanoelectrode array carry grouped arrays of electronically and/or spatially distinct binding sites for simultaneous detection and quantification of several molecular species.
In still another aspect, the materials used for the electrodes and surrounding surfaces are selected based on preferred intrinsic electrical and chemical properties.
The nanoelectrode array may be included in a chamber which can retain fluids.
Several arrays may be used in a single chamber and several different chambers may be used on a single chip.
In still another aspect, the nanoelectrode array and chamber are attached to at least one micro-fluidic delivery and separation system such as a micro-capillary which allows both the delivery and separation by size and electrical properties of the proteins or other molecules to be analyzed.
In another aspect, a microcontroller or microprocessor is provided to analyze signals from the nanoeLectrodes and/or to time and control the fluidics separation of the-molecules or proteins.
In another aspect, the chip with the nanoelectrode arrays is associated with an electronic temperature control system such as a thermoelectric device having a thermistor to vary the bonding kinetics or the electro-chemical affinity of the molecules with given nanoelectrodes, as well as the flow kinetics and separation of the molecules.
In another aspect, the nanoelectrodes are interspaced in a linear microtube to sequence DNA.
Thus, it is an object of the present invention to provide a novel and rapid method to analyze small biological molecules in solution such as proteins and to sequence DNA by using semiconductor chip technology with extremely high packing densities.
It is a further object of the present invention to ensure that the entire chip can be easily integrated into devices for automated analysis of proteins, DNA or other molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective diagrammatic view of a nanoelectrode array showing different nanoelectrode clusters.
FIG. 2 is a side elevational diagrammatic view of a protein-specific electronic receptor and its matching protein.
FIG. 2A is a side elevational cross-section of a protein-specific electronic receptor and its matching protein.
FIG. 3 is a side elevational cross-section of a nanoelectrode array inside a micro-fluidic tube, showing the trapping of a specific protein on its corresponding nanoelectrode receptor.
FIG. 4 is a diagrammatic side elevational cross-section of a microtube with a linear nanoelectrode array to detect DNA.
FIG. 5 is a cross-section of an integrated chip with nanoelectrode arrays, a micro-fluidic delivery system and associated electronics.
FIG. 6 is a side elevational cross-section of a nanoelectrode receptor showing the electrical field which is broken or modified upon binding of a specific molecule to said receptor.
FIG. 7 is a view of a cantilevered nanoplate with several identical nanoelectrode clusters.
D~T.~H~ED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is based in part on the fact that recent advances in technology WO 99/24823 PCTIUS98/2354~
such as the use_of scanning tunneling microscopy (STM) has demonstrated that ultra small structures of a single or a few atomic layers can be built on a semiconductor surface such as silicon. Because of the size of these structures, they are generally referred to as nanostructures (one nanometer or nm = 10'9m, 1 Angstrom or ~r = 10''°m). These structures can be as small as a few Angstroms in diameter which is well below the Stokes radius of a small protein (which is approximately 25-35~}. Since these structures can be built using different chemical elements (or the voltage applied to the structure can be selectively varied) and the spacial distribution, height width and shape of the structures can also be varied, these structures can be built in clusters to serve specifically as "molecular electrodes" whose electro-chemical properties and spacial distribution can be made to correspond precisely with the external three dimensional shape and electro-chemical properties of molecules, preferably biochemicals and most preferably proteins. Therefore each of these clusters can serve as individual electronic protein "receptors" (or detectors). Since a very large number of these molecular electrodes can be placed on a single chip, the resulting arrays, termed here "nanoelectrode arrays" can be used to detect, characterize and quantify many different proteins on a single chip. In a variation of the technology, the chip can also be used to sequence DNA.
Referring now to Figure 1 of the drawings, microelectronic molecular sensor 20 is seen having substrate 22 on which an array of binding sites or clusters 24 are formed. Substrate 22 may comprise any number of materials such as silicon, germanium, gallium arsenide, or other semiconductors. Referring now to Figure 2 of the drawings, one binding site 24 is shown in more detail having multiple electrodes 26a, 26b and 26c which are spatially distributed to form a pattern. Thus, it can be seen that each electrode 26a, 26b and 26c in this particular embodiment is spaced laterally from the adjacent electrode and is elevated at different heights off principal surface 28 of substrate 22.
It will be appreciated that through molecular modeling and empirical data, the topology of the binding sites and electrical charge are tailored to provide the required electrical and topographic properties to selectively recognize and bind a complementary region of a target molecule. As shown best in Figure 2, protein 30 having a defined shape specific to that protein attaches to a given nanoelectrode cluster composed of three nanoelectrodes 26a, 26b and 26c.
As will be explained more fully, each nanoelectrode may have slightly different electro-chemical properties because of differing charges and/or chemical compositions.
These individual electro-chemical properties match not only the electro-chemical affinities of the amino acids or atoms present on the grooves of the protein but also complement the shape of the groove itself. Thus, when a molecule having the proper complementary profile binds to "receptor" 24 bridging the gap between the electrodes, a change in electrical potential occurs which can be monitored through appropriate circuitry to provide an indication of the presence of the target molecule.
In the most preferred embodiments of the present invention binding sites 24 have nanoscale geometries. As illustrated in Figure 2, the distance from principal surface 28 to the top of electrode 26b is 1.9 nanometers, the width of electrode 26b is 0.7 nanometers and the distance between electrodes 26b and 26c is 1 nanometer. In general, each electrode will typically be between 0.2 and about 3 nanometers in height and from about 0.2 to about 2 nanometers in width. As used herein "nanoelectrode" shall include atomic scale as well as x'7402-45 (S) nanoscale structures, i.e. from 2A to 5 nanometers. There will also typically be.from about 2 to about 8 separate electrodes in a°.ac:h cluster 24, lrlectrodes 26x, 26b and 26c can be formed of a number of materials, either irnrinsic or doped, such as gold and platinum and copper and other electrometals. Gold is particularly preferred. Also it may be suitable to form the electrodes of one ma~~erial and coax the outer portion with a different material, e.g. gold coated with zinc oxide or gold coated with a thiol group.
The electrodes may be eaclo separately connected to a power source by small conductive regions or wires which may be for~~nc:d of gold. (n Figure ? A , individual conductive layers 34x, 34b and 34c are shown electrically connecting their respective electrodes 26x, 26b and 26c.
Dielectric layers 36 electrically isc:>late the individual conductive layers and dielectric sheaths 38 electrically isolate the individual electrodes. It will be appreciated that different potentials can be applied to the various indj~,-~idual electrodes and that electrodes from different clusters can be electrically linked to a single layer e.g., layer >4a. It will be appreciated that the various layers can be formed using conventional thin-filno fabrication techniques such as CVD, thermal growth and ion implantation.
It has been shown recentl~r tlhat electrical "wires" can be built of single atoms (see for example review by L.eo Kouwenh<:wf:n "Single-Molecule 7yransistors", Science, Vol. 275, pages 1896-1897,28 March 1997) .
The wires can be deposited in a m m~ber of different ways as pan of the microchip fabrication process, prior to the deposition o:(~ the nanoelectrodes. The nanoelectrodes can be deposited directly on the chip by Scanning ~l('unneling Microscope (as described in Kolb et al., Science, 77402-45(S) Vol. 275, pages 109?-1099, ? I F~b~ruary 19?7 >. A number of other chip fabrication methods are possible such as different Lithography techniques, etc.
In another aspect the naraoelectrodes are not corrrtected to any electrical wires or conductive layers. In this case the binding of the protein or other molecule is simply dependant on the shape and chemical properties of the individual nanoelectrode clusters.
Defection of the attachment of the given molecule to a given cluster can then be achieved by means other than electrical, for example by a highly precise x-y positionai fluorescence reader, similar to that used for the DNA chip technolog;~ ot' by resonance.
In case the nanoelectrodes are not connected to wires (i.e. are not "live"
electrodes), the nanoelectrodes may in some applications be intercoru'tected in a given cluster. In this case the clusters would comprise intercormected peaks and grooves and these would form a larger structure (i.e. from , to > 10 nanorr~eters). 'this structure could be tailored either to match precisely the actual biological re~:eptor of the target molecule or to allow the entire molecule to fit into a 3-dimensional "recelator" which would match at least a third of the overall 3-D
shape of the molecule. In some ir:~stances and depending on the overall shape of the molecule, the receptor that is built may not necessarily include a site corresponding to the actual biological receptor of the target molecule.
Several types of binding or adsorption of the molecule to the nanoelectrode receptor are possible, depending on the cherrtical composition of the nanoelectrodes, the voltage and the chemical to be measured. Binding forces may include covalent binding, electrostatic binding, hydrogen bonds and van der Waals bonds.
Depending on the type of detection that is required, the individual nanoelectrodes of individual clusters do not necessarily need to be composed of different electrometals since both the spacial distribution and the height of the nanoelectrodes can be varied and these two variables may be enough for specific molecule detection in given applications.
In some applications, each nanoelectrode can be selectively charged in a given cluster, allowing the electro-physical property of the nanoelectrode to be varied.
The entire sensor can be built using a computer controlled operation, where the spacing, height, width and the composition of the nanoelectrodes can be made to correspond exactly to the three dimensional shape and matching electro-chemical properties of a selected molecule.
Furthermore, since the position of the nanoelectrode clusters corresponding to a given receptor for a given molecule is determined during the fabrication process, this position information can be used to detect attachment or binding. For example, a large nanoelectrode array can be built with many different clusters, binding in a solution can be allowed, then the array be read using a highly accurate x-y reader in a way similar to the DNA chip. Computer control fabrication of the nanoelectrodes also allows for identical copies of the chip to be made.
It will be also be appreciated that the geometries that are built on the surface of the chip can be made to correspond exactly to the matching image of a crystallized protein surface taken from x-ray diffraction studies. Hence nanoelectrode array clusters can be built directly using crystallographic data and the resulting surfaces on the chip would favor protein-specific crystallization on given arrays.
In another aspect, since multiple identical receptors can be built on the same chip, this technology can be used not only to detect given molecules but also to precisely estimate the quantity of these molecules present in the sample by measuring binding rates in identical clusters.
Refernng to FIG. 3, two partial nanoelectrode arrays are shown facing each other and forming micro-channel or nanotube 60, which permits the flow of small molecules such as protein 70 therethrough. If protein 70 matches the shape of a receptor composed of electrodes 74, 78, and 82, the physical binding of the protein will cause a temporary minute change in the electrical signal which can be measured simultaneously in all said nanoelectrodes. The strength of the electrical signal can be modified for example by adding a conductant to the carrier solution for the molecules which need to be studied. Alternatively the nanoelectrodes themselves can be charged with a small current, which would change upon attachment of the given molecule. Depending on the electro-chemical properties of the nanoelectrodes and the analyte, the temperature and the flow rate, the binding may last only a fraction of a second or last longer. Time of retention in itself is another important variable which can be used in detecting and quantifying the types of molecules present in the sample.
In some applications, micro-channel 60 can form a part of a network of channels of different and specific sizes, matching the sizes of the proteins to measure.
Each of these channels can be_equipped with molecular sieves, allowing only proteins or molecules of certain size to pass through. The channels themselves can also serve as a means to separate molecules and deliver them to given detector chambers with nanoelectrode arrays which are specifically made to measure given classes of proteins or molecules of given molecular weights. In this case, each of the arrays would have nanoelectrodes with sizes corresponding to the sizes of the proteins to measure. As part of this network of channels, specific chambers can be added with specific functions such as a chamber to lyse cells. Other chambers can be filled with specific reagents which can be used as needed.
In another application, each of the micro-channels is equipped with only one or a few nanoelectrode clusters and the protein mix is flowed through each of the channels. With the help of a microcontroller or a microprocessor controlling the flow rate in each micro-channel, the signals from each of the nanoelectrode clusters is then measured combining the power of the following variables for detection: protein separation rates (based on the size and charge of the proteins) and retention time on each given cluster (based on th.e shape and electro-chemical properties of the molecule). Indeed, the more a given molecule matches a given receptor, the longer it will bind. It is obvious that the sophisticated control and measure of the electrical signals in each nanoelectrode (as well as the control of all other variables such as sample flow rates, temperature, etc.) can only be done with the help of a microcontroller or a microprocessor.
Refernng now to FIG. 4, a nanoarray of electrodes 90 is built in a linear microtube 100 with the spacing and electro-chemical composition of the nanoelectrodes varied in such a way *rB
to correspond exactly to the distance between given base pairs of a linear piece of DNA or RNA
110. In this case, the nanoelectrodes are built using only two variables:
precise spacing and electro-chemical composition (not height) favoring position-specific binding of specific base pairs of DNA or RNA to matching nanoelectrodes. The principle that is applied here is that DNA is known to behave as a linear molecule when flowed in a microtube and that this rate of flow can be controlled and measured with precision. Furthermore, the distance between 10 DNA base pairs being precisely 34 A, the nanoelectrodes can be spaced precisely in multiples of 3.4 t~ as shown in 120. By varying the spacing and charge and/or composition of the nanoelectrodes and by measuring the conductance changes over time in sequentially placed nanoelectrodes, an entire sequence is created, based on the timing of the signals of position-specific nanoelectrodes. The full DNA (or RNA) sequence is then reconstructed with the help of a microcontroller (or microprocessor) which can also control the flow rate in the microtube.
ANALYSIS OF PROTEIN VARIANTS
Mutations or other changes in the DNA result in amino-acid substitutions in the protein.
These substitutions in turn result in conformational shape changes in the protein and can result in proteins that are either non-functional or have different properties. Since the three-dimensional (3-D) structure of proteins can now be inferred with precision on the basis of x-ray crystallography or nuclear magnetic resonance (NMR), the 3-D shapes of the protein variants can also be generated using the same method. Hence the entire spectrum of protein variants for given classes of proteins can be measured and quantified using the nanotechnology described above. This is because the conformational changes of each protein variant can be represented by a given nanoelectrode cluster varying in the shape, distribution and electro-chemical properties of the nanoelectrodes. In fact, the building of the arrays can be_comput~er-controlled and link the information matching the putative 3-D structure of proteins of interest (and their variants} to the micro fabrication of all the matching receptors on the chip.
By measuring and quantifying these variants as described above, this approach represents a powerful alternative to direct DNA sequencing since all the possible mutation products of given genes which are expressed can be directly measured on a chip. Another advantage is that the chip would be fully reusable. Furthermore, given the extremely high density of the nanoelectrode arrays that can be built on a single chip, the entire spectra of protein variants for many genes can be measured at once on the same chip. In fact with a refinement in the technology, all existing human proteins and their variants could theoretically be measured on a single chip of 1 cm2 and the number of receptors that could be built on such a chip could theoretically exceed 1 billion which is a thousand fold improvement over any existing technology.
PROTEIN SEPARATION
As indicated above, the separation of molecules can be achieved by flowing said molecules in extremely small tubes (micro-capillaries, micro-channels or nanotubes) where smaller molecules travel faster than larger ones which are retained by friction and weak bonding interactions with the surfaces of the tubes. The result that is achieved is equivalent to electrophoresis but with the advantage of speed, cost and reusability of the micro-capillary.
Refernng now to FIG. 5, micro-channel 130 is shown with a sample input port 132 and a long loop flowing into an optional reagent micro-chamber 134, itself connected to an optional input port 136. Micro-channel 130 separates biological molecules by size and charge while micro-chamber 134 allows the selective input of an external reagent or solution. The flow and on/off position at each micro-channel juncture can be controlled electronically either by an external micro-pump (not shown), by thermocapillary action or by a change of electric potential. After entering micxo-chamber 134, the analyte then flows successfully into micro-chambers 138a, 138b, 138c, then 138d, each holding different nanoelectrode arrays with nanoelectrode clusters of varying sizes and densities. In this particular design, the nanoelectrode arrays are fabricated immediately adjacent to a micro-electronics multiplexing or control area 140, itself connected to an interface 142. After reacting with successive nanoelectrode arrays in successive micro-chambers, the sample exits via port 146. The micro-channels and micro-chambers can either be etched in the silicon surface itself or can be fabricated separately on a surface of a material like glass, diamond, plastic or the like, which is then attached to the silicon surface.
This design can be varied in many different ways and FIG. 5 illustrates just one of many possible combinations of micro-channels, nanoelectrode arrays and micro-electronics that can be fabricated on a chip. As indicated above, a chamber allowing the lysing of the cells or viruses to be analyzed can also be included on the chip. Also, it should be indicated that the directional flow in the micro-channels can be reversed and that each connecting micro-channel can be selectively opened or closed electrically. Hence, when the test is completed the entire system can be heated to allow protein denaturation (andlor the potential in the nanoelectrodes can be reversed), then the system can be flushed with a solution to clean the nanoelectrode arrays and allow reuse of the chip.
Hence a_complete and integrated protein separation and detection systerr~ can be built on a single chip. An important aspect of combining nanoelectrode arrays, micro-channels and a rnicrocontroller (or a microprocessor) is that the time of separation (from sample injection into port 132 to time of first detection) and the length of retention on given nanoelectrode receptors are important variables for characterizing individual protein or protein variants. For example, the system can be calibrated by injecting known proteins, then known mixes of proteins, prior to injecting the sample to be tested. The time taken to reach a given nanoelectrode receptor and the length of binding on different electronic receptors would be specific to specific proteins (or to protein variants) and the signal-specific profiles for each protein can then be stored in memory and compared to those of the sample to be tested.
While FIG. 5 shows an integrated design, it is obvious that the protein separation component and the electronic components can also be placed externally and that the chip can be as simple as having a single nanoelectrode array enclosed in a single chamber with an interface. This chip (which may be disposable) can then be inserted into a larger module with the above components. Also, as indicated below, other detection methods can be used and the design of the chip would change accordingly.
DETECTION
There are many ways in which the binding or adsorption of the analyte on the nanoelectrode array can be detected. Refernng now to FIG. 6, one way of detecting the signal due to adsorption on the nanoelectrode array is by electrical signal. In this case, at least one of the electrodes in each cluster of a given array is used as a "source" 160 while the rest of the cluster 165 is used as a "sink." When an analyte, say a protein, is adsorbed it changes the flow of the current (pico ampere) as shown in FIG. 6. The electrodes are isolated by a layer of oxide 170. The unwanted effects of the electrical current can be avoided by using an AC approach.
Referring now to FIG. 7, the second approach for detection of binding is by using a resonance approach. In this method, a nano structure is constructed. For example, nanoplate 180 of the dimension less than one micron is built. This structure can be free standing or it can be cantilevered. Identical sets of nanoelectrode receptors 24 are then fabricated on this surface.
The structure is designed to have resonance frequency in the MHz to low GHz region. As the analyte flows past these structures, they spend a longer time on the cantilever if they have a structure that is complementary to the nanoelectrode structure. In other words, the analyte molecules undergo collision with the nanoplate. If there exists any complimentary nature between the analyte and the substrate, the analyte will spend more time on the surface during collision. This can be detected optically by shining a laser diode on the structure and detecting the reflected signal using a position sensitive photodiode. The AC signal in the photodiode shows the resonance response of the structure. The greater the signal, the larger the concentration of bound biological molecules, i.e. the greater the concentration of the said molecule in the solution. Other detection techniques such as capacitive, piezoresistive, piezoelectric, electron tunneling, etc. could also be used.
The structure can be excited into resonance response by mechanical means using a piezoelectric element. In this technique, a nanoplate structure is attached to a piezoelectric material which can be vibrated using an AC signal. At resonance the structures oscillate with maximum amplitude. It can also be excited into resonance by modulating the diode laser using square wave power pulses. Since square waves contain all the Fourier components, there will be a component that corresponds to the resonance frequency of the structure.
Since these nanoelectrodes can be constructed on geometrical structures with extremely small thermal mass (for example, nanoplates have a thermal mass of the order of many picograms or less), they can be heated and cooled in the micro to milliseconds time frame. This fact can be used to adsorb and desorb analytes in a periodic fashion. However, when there is a complimentary structure between surface and the analytes the desorption time scale will be different.
USE OF AN EXTERNAL DETECTOR
In another detection application the entire chip which has been allowed to react with the sample is placed in a x-y laser reader in a manner similar to the DNA chip. In this case, the chip is incorporated into a highly precise holder to ensure accurate position reading of each cluster. Detection may be done by fluorescence, for example after reaction of the bound samples to the clusters with a fluorescent molecule or with labeled antibodies.
Detection may also be done by other means such as laser-desorption-ionization mass spectrometry.
NANOELECTRODE CONSTRUCTION
Nanoelectrode arrays can be constructed on a doped semiconductor substrate by nanolithography using scanning probes. In this approach, metal clusters are deposited either from a solution or by field evaporation from a STM/AFM tip. Since the electric field between the tip and the substrate is very high (109V/m), many metals can be field evaporated. In solution many metals can be electrochemically deposited on a surface. The surface of the semiconductor can be oxidized to be an insulator.
Nanometer scale trenches and lines can be made on a semiconductor surface using STM
tip in an oxide etching solution producing a trench. The depth of the trench depends on the time spent by the tip at that location and the voltage on the tip. Hence, not only can the nanoelectrodes be built by deposition, but they can also be built by etching.
The trenches can also be used to make the channels to separate the proteins, as instructed above.
It should also be noted that nanotransistors can be built directly in the chip to facilitate detection and increase the density of the detectors. The nanotransistors can be built prior to the deposition of the nanoelectrodes as a sub-layer in the overall chip manufacturing process or be placed on an adjacent part of the chip.
The above-described principles illustrate the wide variety of applications that are possible in the micro fabrication and applications of the nanoelectrode arrays. For example, the entire system, from sample input to detection with output signals sent to an external device such as a monitor, can be built on a single chip, using micro-channels (for sample separation and delivery), miniature ionic pumps, sample detection, a built-in microcontroller, a method for temperature control, etc. This chip can be inserted into a measuring device, for example for use in a physician's office or into a field detector. If a very large nanosensor array is~ used, it may be preferable to use a microprocessor or several microcontrollers to control the above described functions. In some applications the large arrays can be used with an external laser reader. In this case, the array can be used in a way similar to the DNA chip, where the entire chip is allowed to react with the entire sample, washed and then inserted into an external reader. Using this approach the chip can be build into a convenient handling cassette.
While the invention has been described with respect to specific embodiment for complete and clear disclosure, the appended claims are not to be thus limited but are to be construed as embodying all modifications and alternative constructions that may occur to one skilled in the art which fairly fall within the basic teaching here set forth.
*rB
Claims (45)
1. A sensor for detecting a biological molecule, said sensor comprising:
a microcantilever, wherein at least one electrode comprises a height and width and length disposed on the microcantilever, wherein the at least one electrode disposed on the microcantilever is adapted to interact with and bind to a concentration of a biological molecule; and further comprising at least one from the group consisting of a capacitive means, an electron tunneling means, a laser means, a piezoresistive means, a piezoelectric means, a resonance frequency shift means and a x-y positional fluorescence means for detecting the concentration of the biological molecule adapted to bind to the at least one electrode, and wherein the microcantilever has a plurality of electrodes disposed thereon forming a cluster, each electrode having varying dimensions adapted to bind the cluster with biological molecules and for detecting concentrations thereof.
a microcantilever, wherein at least one electrode comprises a height and width and length disposed on the microcantilever, wherein the at least one electrode disposed on the microcantilever is adapted to interact with and bind to a concentration of a biological molecule; and further comprising at least one from the group consisting of a capacitive means, an electron tunneling means, a laser means, a piezoresistive means, a piezoelectric means, a resonance frequency shift means and a x-y positional fluorescence means for detecting the concentration of the biological molecule adapted to bind to the at least one electrode, and wherein the microcantilever has a plurality of electrodes disposed thereon forming a cluster, each electrode having varying dimensions adapted to bind the cluster with biological molecules and for detecting concentrations thereof.
2. The sensor recited in claim 1, wherein each of the electrodes has a similar chemical composition.
3. The sensor recited in claim 1, wherein at least one of the electrodes has a chemical composition different than another of the plurality of electrodes.
4. The sensor recited in claim 3, wherein the electrode extends from a principal surface of the microcantilever a distance of from and about 2 Angstroms to about 5 nanometers, the electrode having a width of from about 2 Angstroms to about 5 nanometers.
5. The sensor recited in claim 2, further comprising a base integral with the microcantilever.
6. The sensor recited in claim 1, wherein the sensor further comprises electro-chemical properties, some of the plurality of electrodes being spaced a distance away from each other, and wherein the plurality of electrodes complement and bind a site of the biological molecules.
7. The sensor recited in claim 6, wherein the biological molecules are at least one from the group consisting of proteins, DNA and RNA.
8. The sensor recited in claim 7, wherein the electrode extends from a principal surface of the microcantilever a distance of from about 2 Angstroms to about 5 nanometers, the electrode having a width of from about 2 Angstroms to about 5 nanometers.
9. The sensor recited in claim 8, further comprising at least one from the group consisting of a capacitive means, a resonance frequency shift means and x-y positional fluorescence means for detecting the concentration of the biological molecules bound to the at least one electrode.
10. The sensor recited in claim 9, wherein the cluster forms a three-dimensional electro-chemical binding profile which mimics a chemical binding site.
11. The sensor recited in claim 10, wherein each microcantilever and base comprise one piece.
12. The sensor recited in claim 11, wherein each microcantilever is connectable to a heat source to adsorb or desorb the bound biological molecules.
13. A sensor comprising:
a base;
a microcantilever integrally attached to the base;
and at least one electrode disposed on the microcantilever, wherein the electrode extends from a principal surface of the microcantilever a distance of from about 2 Angstroms to about 5 nanometers, the electrode having a width of from about 2 Angstroms to about 5 nanometers.
a base;
a microcantilever integrally attached to the base;
and at least one electrode disposed on the microcantilever, wherein the electrode extends from a principal surface of the microcantilever a distance of from about 2 Angstroms to about 5 nanometers, the electrode having a width of from about 2 Angstroms to about 5 nanometers.
14. The sensor recited in claim 13, wherein the microcantilever has a plurality of electrodes disposed thereon, and at least one electrode may have a different size and shape from at least one of the other plurality of electrodes.
15. The sensor recited in claim 14, wherein the microcantilever is connectable to a heat source to adsorb and desorb the bound biological molecules.
16. The sensor recited in claim 15, wherein the electrodes are made of a chemical element.
17. The sensor recited in claim 15, wherein the electrodes are made of a metal,
18. The sensor recited in claim 17, further comprising a laser for determining the concentration of biological molecules bound to the sensor.
19. The sensor recited in claim 18, wherein the electrodes are coated with a specific chemical.
20. The sensor recited in claim 19, wherein the biological molecules are at least one from the group consisting of proteins, DNA and RNA.
21. The sensor recited in claim 20, further comprising at least one from the group consisting of a capacitive means, an electron tunneling means, a piezoresistive means, a piezoelectric means, a resonance frequency shift means and a x-y positional fluorescence means for detecting the concentration of the biological molecules bound to the at least one electrode.
22. The sensor recited in claim 21, wherein the biological molecules are at least one from the group consisting of proteins, DNA and/or RNA.
23. A method for producing a sensor comprising:
providing a microcantilever, the microcantilever having at least one electrode disposed on the microcantilever wherein the electrode extends from a principal surface of the microcantilever a distance of from about 2 Angstroms to about 5 nanometers, and a width of from about 2 Angstroms to about 5 nanometers.
providing a microcantilever, the microcantilever having at least one electrode disposed on the microcantilever wherein the electrode extends from a principal surface of the microcantilever a distance of from about 2 Angstroms to about 5 nanometers, and a width of from about 2 Angstroms to about 5 nanometers.
24. The method for producing a sensor recited in claim 23, further comprising a base attached to the microcantilever.
25. The method for producing a sensor recited in claim 24, further providing a plurality of cluster shapes formed from a plurality of electrodes, wherein the cluster shapes form a three-dimensional electro-chemical binding profile which mimics a chemical binding site.
26. The method for producing a sensor recited in claim 25, wherein the electrodes are fabricated by lithography.
27. The method for producing a sensor recited in claim 26, wherein the base and the microcantilever are fabricated from one piece.
28. The method for producing a sensor recited in claim 25, wherein the electrodes are deposited onto the microcantilever by a scanning tunneling microscope.
29. The method for producing a sensor recited in claim 28, further comprising the base attached to the microcantilever to be fabricated from one piece.
30. The method for producing a sensor recited in claim 25, further providing a heat source connectable to the microcantilever to adsorb or desorb biological molecules.
31. The method for producing a sensor recited in claim 23, further providing a plurality of cluster shapes formed from a plurality of electrodes; wherein the cluster shapes are derived from x-ray diffraction data for given proteins.
32. The method for producing a sensor recited in claim 23, wherein each of the electrodes has a similar chemical composition.
33. The method for producing a sensor recited in claim 23, wherein each of the electrodes has a chemical composition which is different than another of said electrodes.
34. The method for producing a sensor recited in claim 23, wherein the plurality of the electrodes extend from a principal surface of the microcantilever and wherein at least one of the electrodes extends farther from the principal surface than another of the electrodes.
35. The method for producing a sensor recited in claim 34, wherein the width of at least one of the electrodes is greater than the width of another of the electrodes.
36. The method for producing a sensor recited in claim 23, wherein the electrodes are spaced laterally from one another on the microcantilever.
37. The method for producing a sensor recited in claim 36, wherein clusters are formed from the spaced apart electrodes to form a cluster array.
38. The method for producing a sensor recited in claim 23, wherein electro-chemical properties, width and spacing of the electrodes complement and bind a site of biological molecules.
39. The method for producing a sensor recited in claim 23, wherein the biological molecules are at least one from the group consisting of proteins, DNA and RNA.
40. The method for producing a sensor recited in claim 23, wherein the electrodes are made of a metal.
41. The method for producing a sensor recited in claim 40, further providing a laser for determining the concentration of biological molecules bound to the sensor.
42. The method for producing a sensor recited in claim 23, further providing the electrodes being made of a chemical element.
43. The method for producing a sensor recited in claim 42, further providing a laser for determining the concentration of biological molecules bound to the sensor.
44. The method for producing a sensor recited in claim 23, further comprising at least one from the group consisting of a capacitive means, an electron tunneling means, a piezoresistive means, a piezoelectric means, a resonance frequency shift means and a x-y positional fluorescence means for detecting the concentration of the biological molecules bound to the at least one electrode.
45. The method for producing a sensor recited in claim 23, further providing a computer controlled operation wherein the spacing, height and composition of the electrodes may correspond with the three dimensional shape and electromechanical properties of a selected biological molecule adapted to interact with the microcantilever.
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| US60/065,373 | 1998-03-19 | ||
| PCT/US1998/023547 WO1999024823A1 (en) | 1997-11-12 | 1998-11-04 | Nanoelectrode arrays |
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