Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
  • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
  • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00306—Reactor vessels in a multiple arrangement
  • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
  • B01J2219/00315—Microtiter plates
  • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00497—Features relating to the solid phase supports
  • B01J2219/00511—Walls of reactor vessels
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00659—Two-dimensional arrays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/0068—Means for controlling the apparatus of the process
  • B01J2219/00698—Measurement and control of process parameters
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/0068—Means for controlling the apparatus of the process
  • B01J2219/00702—Processes involving means for analysing and characterising the products
  • B01J2219/00704—Processes involving means for analysing and characterising the products integrated with the reactor apparatus
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds

Definitions

• This invention relates to the detection of biomolecules. Specifically, the invention relates to electronic or electrochemical detection of biomolecules using biochip arrays. In particular, the invention provides an apparatus comprising a platform for a column-and- row addressable, high-density, enhanced-sensitivity biochip array, and methods of use thereof.
• Fluorescence technologies also require "labeling" to link the fluorescence marker to a biologically-relevant material, so that molecular interactions (such as nucleic acid hybridization or ligand/receptor binding) can be detected. Linkage of a fluorescent tag to a biomolecule inevitably increases the complexity of such molecules and can adversely affect probe/target interactions. In addition, fluorescence labeling is expensive, labor intensive and time consuming.
• experimental reagents containing either radioactive or fluorescence tags often are of limited usefulness (for example, due to the radiochemical half-life of the radioisotope, or due to light sensitivity of the fluorescence label).
• electronic or electrochemical detection processes are based on interactions between probe molecules on an electrode and target molecules in the detection solution that are detected as alterations in the electrical properties on the electrode.
• Electronic or electrochemical detection eliminates many of the disadvantages inherent in using radioactive or fluorescent labels to discern molecular interactions. More importantly, electronic or electrochemical detection devices can be made portable, as has been demonstrated in the case of widely available glucose sensors. Electrical and electrochemical detection devices thus provide an alternative molecular detection means that is safe, inexpensive, unobtrusive, and sensitive.
• biomolecules Electronic or electrochemical detection methods provide an attractive alternative to autoradiography or optical detection for identifying molecular interactions.
• electrochemical detection of biological molecules has generally been achieved by one of two methods. The first is selective modification at specific sites of a biomolecule (such as a nucleic acid or protein) with redox active moieties such as transition metal complexes. The second approach is intercalation of redox-active moieties, e.g. into duplex DNA strands.
• a significant disadvantage of the electronic or electrochemical detection devices known in the prior art is that these devices use low-density arrays.
• Egger et. al. disclosed an apparatus for identifying biomolecular species within a sample substance using an array having a plurality of test sites upon which the sample was applied. Each test site had at least one electrode attached thereto for coupling with a second electrode surrounding the test site to form a capacitor in conjunction with the sample substance.
• the second electrode was preferably made of a ring located outside the array and also acted to contain the sample solution
• Egger's array required a large amount of sample solution (i.e., enough to cover the area within the ring) in order for the array to function. More importantly, Egger's array could not be made row and column (x-y) addressable, limiting the density of the test sites in the array and thereby limiting the usefulness of this apparatus.
• Hollis et al. disclosed an x-y addressable array where test sites were composed of digitated electrodes located on a side bridge that was connected to both the x and y addressable conductive leads.
• the array of Hollis et al. is not practical to fabricate since the test sites are designed to bridge the x-and-y addressable conductive leads that are on two different planes with an insulating layer in- between.
• This invention provides an apparatus for electronic biomolecule detection using a column-and-row (x-y) addressable, high-density biochip array and methods of use thereof. Specifically, the apparatus facilitates electronic or electrochemical detection of molecular interactions between probe molecules bound to defined regions of a high-density addressable array and target molecules in a solution that is exposed to the array.
• the apparatus comprises a multiplicity of individual well structures, each said well further comprising two electrodes that can be individually addressed by applying an electric signal specifically to a particular address (well) in the array.
• the bottom of the well comprises one electrode surface, while the second electrode surrounds the top of the well.
• Probe molecules include but are not limited to oligonucleotides, nucleic acids (DNA, RNA, etc), proteins, antibodies and peptides that are immobilized at a specific address comprising a well in the array.
• Immobilization of such species is accomplished by direct anchoring of the probe molecules on the electrode surface, preferably by attaching the probe molecules onto a supporting matrix on the surface of the electrodes.
• the immobilized probe molecules are exposed to a solution containing an intended target molecule, for a time and under conditions sufficient for the probe molecules to bind to the target.
• An electrical signal is then applied to each of the individual well structures comprising the array.
• a change in the detected electrical signal in the presence of the solution (compared with the electrical signal detected in the absence of the solution) is used to determine whether a binding event between the probe and target has occurred at a particular address in the array.
• Figure 1 illustrates a schematic representation of a cross-section view of the device platform.
• Figure 2 illustrates a schematic representation of a top view of the device platform.
• Figure 3 is a schematic diagram of the row/column configuration of a high-density array useful in the practice of the invention.
• Figure 4 is a photograph of an x-y addressable array of the invention.
• Figures 5A, 5B and 5C are masks for depositing electrode and insulating layers in the x-y addressable arrays of the invention.
• biomolecule is intended to encompass biologically-derived molecules that interact specifically with one another.
• biomolecules are complementary nucleic acid strands, ligand/receptor, agonist/receptor and antagonist/receptor pairs, antigens and their cognate antibodies, enzyme/substrate and enzyme/inhibitor combinations.
• the biomolecules of the invention comprise a binding pair, whereby there is a specific interaction between each member of the pair.
• target one member of the pair is conveniently termed a “target” and the other a “probe.”
• probe molecules are preferably bound to a solid substrate and "target” molecules comprise a sample to be tested for the presence, amount or concentration of the "target.”
• Target molecules can be any of these biomolecules, most preferably wherein at least one of the target molecules specifically interacts with one of the probe molecules.
• the probe molecules are oligonucleotides.
• Oligonucleotide probes of length 5 to 1000 basepairs (bp), more preferably 5 to 1 OObp and most preferably about 5 to 40bp, can be attached to the attachment medium.
• Targets include PCR amplicons, genomic DNA, cDNA and synthetic and cellular RNA.
• probes can be oligonucleotides such as aptamers or other oligonucleotides having well-defined secondary structure that will bind to proteins.
• peptides, antibodies or antigens can be immobilized to perform binding assays.
• the present invention provides an apparatus for electronic or electrochemical detection of biomolecules using a row-and-column ("x-y") addressable array having a plurality of addressable sites to which a target sample is applied, and methods of use thereof.
• Each addressable site comprises at least two electrodes that are connected to two conductive lead lines that can be addressed in a x-y coordination fashion.
• the addressable site is preferably a well structure as defined herein wherein the bottom of the well comprises the surface of one electrode, and the top of the well comprises the second electrode.
• each said well structure further comprises at least one additional electrode, preferably a reference electrode, positioned between the top and bottom of the well.
• the devices of the invention comprise at least two electrodes, and a multiplicity of probe molecules immobilized in proximity to the electrodes, wherein the probe molecules are preferably immobilized at the surface of at least one of the electrodes.
• Device embodiments of the invention are useful for performing methods for biomolecule detection by either electrochemical or electronic means.
• electrochemical detection is intended to encompass methods based on oxidation/reduction (redox) processes induced by electron transfer between electrodes, most preferably mediated by an electrochemical reporter group attached to the probe moiety, the target moiety, or both.
• electrochemical detection is intended to encompass methods that rely on impedance changes (such as resistance, capacitance and inductance) due to differences in electronic state occupancy in the biomolecules in the bound and unbound conformations.
• An additional advantage of the devices of the invention is that both impedance and electrochemical measurements can be performed in the same assay using the same x-y addressable array to enhance the sensitivity and reduce system "noise" resulting from nonspecific binding of biomolecules.
• probe arrays comprising nucleic acids
• electrochemistry it is generally not possible to perform electrochemistry on the probe molecules themselves, since they cannot participate in redox reactions under readily-achievable voltage potentials unless they are linked to an electrochemical reporter group that can participate in such a redox reaction.
• an impedance measurement of the probe array can be performed in either the presence or absence of such electrochemical reporter groups to monitor the quality of probe attachment at each particular address prior to introduction of the target.
• electrochemistry can be performed on the molecular complex at or near the redox potential of the electrochemical reporter group where molecules tagged with an electrochemical reporter groups have hybridized to the immobilized probe.
• This provides an additive signal to be measured that distinguishes background binding from specific binding at each address in the x-y addressable array.
• This feature of the assay provides an increased assay sensitivity by reducing the baseline (noise or background) signal due to non-specific binding of the target to the probe.
• This feature is also a unique characteristic of the multielectrode device structure described here and is not found in the prior art.
• the electrochemical reporter groups comprise a transition metal complex, most preferably containing a transition metal ion that is ruthenium, cobalt, iron or osmium.
• a transition metal complex most preferably containing a transition metal ion that is ruthenium, cobalt, iron or osmium.
• the patterned conductive electrodes 5 are fabricated of electrically-conductive metals (including but not limited to transition metals such as aluminum, gold, copper, silver, platinum, chromium, and titanium), transparent conductors (such as indium-tin-oxide and zinc oxide), conductive plastics (such as polymers like polythiophenes, polyanilines, polypyrroles, and metal impregnated polymers), or conductive carbon (such as graphite).
• electrically-conductive metals including but not limited to transition metals such as aluminum, gold, copper, silver, platinum, chromium, and titanium
• transparent conductors such as indium-tin-oxide and zinc oxide
• conductive plastics such as polymers like polythiophenes, polyanilines, polypyrroles, and metal impregnated polymers
• conductive carbon such as graphite
• Non-limiting examples of methods for producing solid substrates comprising the device platforms of the invention include but are not limited to thermal evaporation, wire bonding, metallization (evaporation, plating, sputtering over a shadow mask), dielectric deposition (by plasma, chemical vapor deposition or sputtering ), wet or dry chemical etching, reactive ion etching, or liftoff after the desired pattern has been defined using conventional photolithography.
• An optional layer of conductive metal 3 is placed over the insulative dielectric material 4. This layer constitutes a reference electrode.
• the conductive metal layer 3 is silver, which is then advantageously converted to silver /silver chloride at a later stage in manufacturing.
• a second layer of insulative dielectric material 2 is then placed on top of the conductive electrode layer 3.
• a continuous dielectric layer 2 comprising layers 2 and 4 as set forth herein are deposited.
• the second layer of insulative dielectric material 2 is optionally made of the same materials as the insulative layer 4.
• Patterned conductive electrodes 1 constructed on top of the second layer of insulative dielectric material 2 constitute the final layer of each addressable site in the device 9.
• Well structures 7 are fabricated from this device by conventional photolithography or laser drilling methods used in the semiconductor industry for PCB manufacturing. These wells can have rectangular, circular, trapezoidal or other polygonal openings. Additionally, the well walls may be either straight or curved, and may have an arbitrary angle with respect to the bottom electrode 5. An optional center electrode can alternatively protrude into the well area, as shown in Figure 3.
• Figure 2 illustrates a schematic representation of a top view of the apparatus of the invention.
• the conductive electrodes 1 are preferred to be oriented in a direction orthogonal to the patterned conductive electrodes 5, generating row (i.e., patterned electrodes 5) and column (i.e., conductive electrodes 1) addressable high-density electronic or electrochemical mini-cells (i.e., well structures 7) with optional reference electrodes built in-between.
• the well structure is preferably produced wherein the bottom of the well structure comprises the top of electrode 5 surface, while the top of the well structure is surrounded by the second electrode 1.
• the proposed device 9 can be used as an x-y addressable, high-density biochip array when biological probes 10 are immobilized on the patterned electrodes 5 inside each well structure 7.
• the apparatus is capable of detecting changes in the electrical properties of the probes 10 in each well structure arising from the interaction of the probes 10 with target molecules 11.
• the inventive apparatus is useful for single species detection, where only a few test wells (low density) are required, the advantages of the invention are more pronounced in a high density array where hundreds, thousands, or millions of test wells are integrated in one array.
• the probe molecules may be oligonucleotides, nucleic acids (such as DNA or RNA), proteins, peptides, antibodies or small molecules such as ligands, wherein probe molecules are chemically modified to contain anchoring groups that permit immobilization.
• probe molecules can be efficiently immobilized on the electrode surface through an intermediate species, termed a "spacer.”
• the surface of the electrodes 5 is covered with a layer of polymer matrix.
• probe molecules are attached onto a supporting matrix on the surface of the electrodes using the functional chemistry mentioned above.
• the polymer matrix is preferably selected to be polypyrrole, polythiophene, polyaniline, polyacrylamide, agarose gel, polyethylene glycol, cellular, sol gels, dendrimers, metallic nanoparticles, carbon nanotubes, and their copolymers.
• porous matrix such as polyacrylamide, agarose, or sol gels are preferred.
• Electronic or electrochemical detection of molecular interactions between probe and target molecules is achieved by devices having the structure, for example, as depicted in Figure 1.
• the electric or/and electrochemical methods used to interrogating the biomolecule targets may be selected from, but are not limited to, AC impedance, cyclic voltammetry (CN), pulse voltammetry, square wave voltammetry, AC voltammetry (ACN), hydrodynamic modulation voltammetry, potential step method, potentiometric measurements, amperometric measurements, current step method, and combinations thereof.
• an active driving circuit such as the one used in an active matrix liquid crystal display device can be built underneath or nearby each test well site to replace the electronic column and row drivers for x-y addressing such as the one used in the passive matrix liquid crystal display device.
• a high-density, x-y addressable probe array is exposed to an electrolyte solution containing a target molecule for a time and under conditions sufficient for the target to bind to a probe present in at least one of the particular addresses of the column-and-row addressable array.
• a voltage potential or other electric signal is applied to the each of the electrodes comprising each of the addressable sites through the x-y addressable column and row electrodes. Changes in the electrical properties or electrical signals from a particular electrode at a particular site in the x-y addressable array arising from interactions between probe molecules on the electrode and target molecules in the solution are detected to determine the presence and concentration of the target molecules in the solution.
• electrical cross-talk between electrodes is reduced or eliminated in the x-y addressable array during target interrogation with an external electrical source.
• the electrodes at the top of the wells are covered with an array of microfluidic channels. These channels are designed to be independently isolated from each other, with each having its own isolatable liquid inlet and outlet port. In addition to functioning as an electrical isolator, the channels also act as containers or reaction chambers for liquid during probe-target hybridization, enzymatic reactions and target interrogation with the external electrical source.
• the microfluidic channels can be replaced by a single chamber that covers all the test sites with
• Electrolyte solutions useful in the apparatus and methods of the invention include any electrolyte solution at physiologically-relevant ionic strength (equivalent to about 0.15M NaCl) and neutral pH.
• Nonlimiting examples of electrolyte solutions useful with the apparatus and methods of the invention include but are not limited to phosphate buffered saline, HEPES buffered solutions, and sodium bicarbonate buffered solutions.
• the electrolyte solution comprises metal cations or polymerized cations that are ion conductive and capable of reacting with probes or probe-target complexes.
• the Examples, which follow, are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
• a linear test microarray with four wells was fabricated on a 3" inch silicon wafer as follows. A photograph of the array is shown in Figure 4.
• the linear test array was fabricated by conventional photolithography in a class 100 clean room and fabrication was performed using three layers of masks as shown in Masks 12 ( Figure 5 A), 14 ( Figure 5B) and 16 ( Figure 5C).
• a three inch silicon wafer was cleaned using a solution of NH OH:H 2 0 (1 : 10 v/v), rinsed with de-ionized water, and then dried using a stream of nitrogen at room temperature.
• 2000A Si0 2 was deposited by conventional chemical vapor deposition technique.
• the array was then prepared sequentially as follows.
• the PR was hardbaked and developed.
• the following metals were deposited sequentially by evaporation: Ti (to a thickness of 1.0 Angstrom), Au (to a thickness of 21,000 Angstrom), and Ti (to a thickness of 500 Angstrom). After evaporative deposition of these metal layers, a liftoff protocol was used to produce the bottom patterned electrode.
• the wafer was coated with a thick (8 micron) layer of PR, as described above.
• Mask 14 Figure 5B to protect the portion of the substrate that forms the top electrode, the surface was exposed to an ultraviolet light source using a wavelength of 365 nm and an intensity of 6 mW/cm 3 .
• the PR was hardbaked and developed as described above
• the following metals were deposited sequentially by evaporation: Ti (to a thickness of 1.0 Angstrom) and Au (to a thickness of 21,000 Angstrom). After evaporative deposition of these metal layers, a liftoff protocol was used to produce the top patterned electrode, as described above.
• the surface was exposed to an ultraviolet light source using a wavelength of 365 nm and an intensity of 6 mW/cm 3 .
• the PR was hardbaked and developed as described above.
• the wafer was then subjected to buffer oxide etching solution (4: 1) until each well opening was cleared.
• the PR was removed by placing in a Branson 4000 Sonicator.

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