WO2021237180A1 - Biocapteurs à pont de nanorubans semi-conducteurs amorphes pouvant être traités à température quasi-ambiante et dispositifs de mémoire - Google Patents

Biocapteurs à pont de nanorubans semi-conducteurs amorphes pouvant être traités à température quasi-ambiante et dispositifs de mémoire Download PDF

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WO2021237180A1
WO2021237180A1 PCT/US2021/033782 US2021033782W WO2021237180A1 WO 2021237180 A1 WO2021237180 A1 WO 2021237180A1 US 2021033782 W US2021033782 W US 2021033782W WO 2021237180 A1 WO2021237180 A1 WO 2021237180A1
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bridge
sensor
electrode
amorphous semiconductor
source
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Sungho Jin
Chulmin Choi
Paul MOLA
Barry Merriman
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Roswell Biotechnologies Inc
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Roswell Biotechnologies Inc
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    • 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
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
    • 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
    • C12Q1/001—Enzyme electrodes
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4146—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS involving nanosized elements, e.g. nanotubes, nanowires
    • 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
    • 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

Definitions

  • the present invention disclosure relates to biomolecular sensing and memory storage devices. More particularly, the present disclosure relates to the nanofabrication of molecular sensor bridge structures for analyzing DNA and related biomolecules.
  • elongated bridge structures include amorphous semiconductors of a- Si, a-Si:H or other amorphous nanobridges, which provide reliable DNA genome analysis performance and are amenable to scalable manufacturing.
  • the disclosed structures are also useful for DNA-based information storage devices including archival or randomly accessible memory and logic devices.
  • An exemplary sensor according to the invention includes a source electrode; a drain electrode spaced apart from the source electrode by a sensor gap; a gate electrode, wherein the source, drain and gate electrodes cooperate to form an electrode circuit; a bridge molecule bridging across said sensor gap coupling the source and drain electrodes, wherein the bridge molecule comprises an a-Si, a-Si:H or amorphous semiconductor nanoribbon film bridge; and a probe coupled to the bridge molecule, wherein interaction of the probe with a biomolecule is detectable by monitoring at least one parameter of the electrode circuit.
  • An exemplary the bridge molecule comprises an amorphous semiconductor nano-ribbon Field-Effect-Transistor (FET) bridge array.
  • the bridge molecule comprises a nano-ribbon semiconductor selected from the following: a-Si, a-Ge, a- Si:H, or In-Ga-Zn-O.
  • the bridge molecule comprises only one of a- Si, a-Ge, a-Si:H, or In-Ga-Zn-O.
  • a suitable probe molecule includes nucleic acids, peptides including alpha helixes, enzymes, and other biomolecules.
  • the probe comprises a nucleic acid polymerase enzyme such as a DNA or RNA polymerase.
  • the a-Si, a-Si:H or amorphous semiconductor nanoribbon bridges are attached to the source and drain electrodes by a molecular binding force, by metallization added near the bond location, or by using functionality complexes or ligands.
  • the invention includes methods of manufacturing a sensor described herein according to a method described herein. Such methods and suitable embodiments are also shown in the Figures.
  • the sensor is manufactured at about room temperature and comprises the use of a dielectric masking layer disposed on a conductor and comprising size-limited openings that define exposed conductive metals wherein each opening is less than ⁇ 10 nm and allow only a desired amount of bridge biomolecules to fit therein and to attach onto the exposed electrode surface region defined by each opening.
  • a sensor is manufactured as described herein wherein the a-Si, a-Si:H or amorphous semiconductor nanoribbon bridge molecules are aligned by microfluidic flow alignment, by electric field, or by stamp-transfer from separately prepared pre-aligned a-Si, a-Si:H or amorphous semiconductor nanoribbon arrays onto a final device surface, and where the bridge molecules are attached onto the ends of the source and drain electrodes.
  • a sensor is manufactured as described herein wherein the nanoribbon is split into two segments using a focused laser beam slicing, focused ion beam cutting, electron beam, neutron beam, or nanopatteming and etching.
  • the splitting of amorphous semiconductor into two separated parts produces a nanogap between the segments of between about 20 nm to about 100 nm.
  • a sensor is manufactured as described herein wherein a second molecular bridge comprising a nucleic acid or peptide is attached to the molecular bridge.
  • the nucleic acid or peptide is preferably attached by the use of electric field alignment, flow alignment in a microfluidic chamber, stamp transfer of pre-aligned DNA or peptide using PMMA, PDMS, or other polymer type soft stamps.
  • PMMA polymethyl methacrylate
  • PDMS polymer type soft stamps
  • Fig. 1 shows the schematics of fabrication steps for exemplary sequencing structure of DNA or RNA sensor comprising amorphous semiconductor nano-ribbon FET (Field-Effect -Transistor) bridge array prepared from near room temp deposited film
  • a-semiconductor e.g., a-Si, a-Ge, a-Si:H, In-Ga-Zn-O
  • a-semiconductor e.g., a-Si, a-Ge, a-Si:H, In-Ga-Zn-O
  • Processing steps to form a sequencing bridge with enzyme polymerase-DNA or polymerase-RNA Polymerase reaction of nucleotide attachment changes electrical properties of the a-Si nano ribbon FET bridge for sequencing.
  • Fig. 2 Illustrates an exemplary design of single molecule bridge of DNA or RNA sensor comprising size-limited, a-Si type semiconductor nano-ribbon region.
  • the polymerase reaction of nucleotide attachment alters the electrical current properties of the FET molecular bridge on pulsing for sequencing analysis.
  • Fig. 3. Shows a-Si, a-Si:H or other amorphous semiconductor nano-ribbon array patterned from deposited film is made transferable and releasable by a sequence of processes.
  • Fig. 4. Illustrates the Transfer of a-Si nano-ribbons by PDMSor PMAA type soft stamp.
  • the PDMS stamp optionally has protruding ridges for easier pick up of the nanoribbons.
  • PMMA stamp can be dissolved away by acetone type solvents.
  • Fig. 5. Is a top view of an array of Au electrode pairs (or other metal) with float- transferred or PDMS stamp-transferred a-Si nano-ribbon bridges, with redundant ribbons to ensure at least one bridge formation occurs.
  • a masking coating with blocking agent may be added to prevent biotin, streptavidin or polymerase adhesion, except for a local ⁇ 5 nm circle on a-Si nano-ribbon surface, so as to ensure only a single enzyme molecule is attached.
  • Fig. 6. Illustrates a-Si (or a-Si:H or other amorphous semiconductor) nano ribbon placement, (a) over nano gap, (b) on planarized surface, (c) a-Si surface size-limited by dielectric coating to allow preferably a single molecule enzyme (polymerase) to attach on exposed a-Si for genome sequencing, other protein sensing or DNA memory information reading. Electrical current signals are measured via complimentary nucleotide attaching events (A-T or G-C as adenine is the complementary base of thymine, and guanine is the complimentary base of cytosine in DNA and of uracil in RNA) for sequencing analysis.
  • the biosensor can be utilized for other protein or biomolecule sensing, or DNA memory reading.
  • Fig. 7 Cross-sectional view of producing diameter-reduced Au electrode tip for size-confined nano-ribbon bridge sensor formation.
  • Fig. 8 Sectional view showing an example process of creating 5-10 nm regime, size-limited a-Si (or a-Si:H) nanobridge structure to place a single molecule polymerase sensor.
  • Fig. 9 Sectional view of direct nanoimprinting shaping of a 5-10 nm regime, size-limiting funnel-shaped guiding structure in the PMMA or silica to place a single molecule polymerase sensor.
  • the protruding PMMA orsibca structure above also protects the attached polymerase from mechanically washed away by microfluidic solution flow or post-sequencing washing operations.
  • Fig. 10 Exemplary design of utilizing size-confined DNA assembly well to have only a ’’Single Streptavidin” immobilized on a-Si nano-ribbon bridge surface for sequencing or protein sensing.
  • FIG. 11 Top view of an array of a-Si (or a-Si:H or other amorphous semiconductor) nano-ribbon bridge FET sensors, with a size-limiting structure for single polymerase.
  • Massively parallel electronic sequencing analysis can be performed with many devices organized into a system, having as many as 10,000 or even at least 1 million devices.
  • Nanorazor blade mould can be utilized to form a sub-5 nm wide ribbons of inorganic semiconductors (such as amorphous, crystalline or 2D semiconductors), or conductors such as Au to enable nanobridge formation.
  • Such razor blade Si mould array (or SiCh, metallic or ceramic material) can be made, e.g., by DUV fabrication of Si line ridge array + repeated (gradient oxidation of Si mould surface at e.g., 800 - 1100°C/ 0.1 ⁇ 10 hrs + chemical etching of oxide skin) to sharpen the razor blade, which has a sharp tip radius of curvature of e.g., 3-5 nm.
  • Fig. 13 Example procedure of utilizing razor blade Si mould to produce sub-5 nm wide ribbons of amorphous semiconductor film bridge array for sequencing and DNA memory. Nanoimprinted valleys are RIE etched, and thin semiconductor film deposited and lift-off processed, with the nanoribbon array connected to the circuit by electrode lead wire deposition and patterning. A single enzyme type polymerase is added onto the bridge using binding conjugate functionality molecules (e.g., silane, biotin-streptavidin, antibody-antigen, azide-DBCO (Dibenzocyclooctyne-amine type click chemistry molecules).
  • binding conjugate functionality molecules e.g., silane, biotin-streptavidin, antibody-antigen, azide-DBCO (Dibenzocyclooctyne-amine type click chemistry molecules.
  • inorganic bridges can also be formed using the razor blade mould, e.g., to pattern underlying layer of M0S2, bandgap open narrow or porous graphene, other semoconductors.
  • Narrow, sub-5nm conductor bridges e.g., Au
  • FIB split to attach DNA or peptide molecular bridges to the gap created between the sliced Au nanowire leads.
  • Fig. 14 Example DNA memory device utilizing sequencing analysis biosensor bridge structure
  • Massively parallel DNA written information array in combination with massively parallel single- a-Si nanobridge reader array allows ultrafast, “random-access-enabled” DNA memory retrieval.
  • Several different configuration of tethered DNA memory array, with various methods of tethering and releasing, or nanosensor access schemes can be made to maximize the memory processing capability.
  • the term “molecular electronics” refers to electronic devices in which a single molecule or a single molecular complex is integrated as a component in an electronic circuit.
  • the term “molecular complex” refers to an assemblage of a small number of molecules, such as only two, held together by chemical conjugation, bioconjugation, or covalent or non-covalent bonds, such that the assembly retains an assembled configuration or affiliation during a process of incorporating the molecular complex into an electrical circuit to provide a sensor, and during use of the resulting sensor in assays.
  • a small assemblage of molecules may comprise just two molecules, such as a DNA oligonucleotide hybridization probe chemically bound to a bridge molecule.
  • a molecular complex may comprise a biomolecule conjugated to a bridge molecule, such as a polypeptide, enzyme or nucleic acid as the biomolecule.
  • a molecular complex for use in a molecular electronics sensor may comprise from 2 to 10, from 10 to 100, or from 100 to 1000 molecules in the complex.
  • nanoelectrode refers to an electrically conducting element having dimensions such as height, width and length of nanometer scale.
  • a length of a nanoelectrode may be substantially greater than both the height and width of the nanoelectrode such that an end portion of each nanoelectrode can be connected into a circuit.
  • nanoelectrodes are disposed in pairs, wherein in each pair of nanoelectrodes a first nanoelectrode and a second nanoelectrode are spaced-apart by a nanoscale gap referred to as a nanogap.
  • both nanoelectrodes and nanogaps may be called electrodes and gaps, respectively.
  • a nanoelectrode herein may comprise a metal such as Ag, Al, Au, Cr, Cu, Ni, Ga, Ti, Pt, Pd, Rb, Rh, Ru, or an alloy of these metals.
  • a contact may be disposed on a nanoelectrode, and the contact may be the same material as the nanoelectrode or a different material.
  • nanoelectrodes comprising Pt may each further comprise an Au nanoscale island in the form of nanopillars disposed at an end of the nanoelectrode.
  • bridge or “bridge molecule” includes any electrically conducting molecule than may be used to make a conducting connection across a gap between spaced-apart nanoelectrodes in a pair of electrodes.
  • Such molecules that function as bridges include all those described herein, including, but are not limited to, double stranded DNA, peptide alpha helices, polypeptides having particular amino acid sequences, graphene nanoribbons, pilin filaments or bacterial nano wires, other multi chain proteins or conjugates of multiple single-chain proteins, antibodies, carbon nanotubes e.g., single-walled carbon nanotubes (CNTs, SWCNTs), semiconductor layers such as transition metal dichalcogenides (TMD) or other semiconductor nanoribbons or nanowires, or conducting polymers such as polythiophene, poly(3,4-ethylenedioxythiophene (PEDOT) or other synthetic electrically conducting polymers.
  • Such molecules may include attachment groups, i. e.. functionality that
  • peptide refers to any contiguous single chain of amino acids, wherein the amino acids are standard, non-standard or modified, or amino-acid analogs that engage in a peptide bond. In various embodiments, peptides herein may be in the range of 10 to 300 amino acids long, or 20 to 200 amino acids long.
  • polymerase refers broadly to any of the enzymes that can synthesize DNA or RNA segments, of length one or more bases, starting from primed DNA or RNA templates. This includes, in contexts where it makes sense, DNA polymerases that synthesize a DNA strand from a primed DNA template. Well known examples of this include DNA polymerases such as Taq, Bst, Bsu, Klenow, Pfu, Vent, Phi29, and Nine Degrees North, and genetically modified forms of these naturally enzymes, which also occur under a variety of commercial trademark names. This also includes, in contexts where it makes sense, Reverse Transcriptases that take an RNA substrate and synthesis DNA.
  • RNA dependent RNA polymerases RdRp
  • polio virus 3DPol and COVID-19 RdRp.
  • RNA in cases involving RNA, the U base occurs instead of T, U forms Watson-Crick base pairing with T, and the synthesis of RNA utilizes dUTP instead of dTTP.
  • dUTP instead of dTTP.
  • reference to DNA, T and dTTP are meant to, in context where it would make sense, encompass RNA, U and dUTP.
  • aptamer means a DNA or RNA or other nucleic acid molecule that has specific binding to a target molecule, such as a specific target protein, small molecule, or other antigen or molecular target.
  • the “switch oligo” for an aptamer refers to a DNA or RNA oligo that binds part of the aptamer, and is displaced when the aptamer binds its specific target.
  • Such switch oligo once displaced, provides a means of detecting the primary aptamer-target binding event by detecting the displaced oligo, perhaps involving PCR or isothermal amplification.
  • Such a switch oligo may be separate from the aptamer, or may be tethered to it.
  • chip refers to a semiconductor chip or a CMOS chip.
  • semiconductor chip refers to an integrated circuit chip comprising semiconductor materials such as silicon or gallium, and which can be fabricated with techniques used in the semiconductor industry.
  • CMOS chip refers to an integrated circuit chip fabricated using CMOS process techniques from the semiconductor industry.
  • CMOS is an acronym for Complementary Metal-Oxide Semiconductor, and refers to a specific manufacturing process for making integrated circuit chips of the type most produced for processors, DRAM memory, and digital imager devices.
  • CMOS chip also refers to a device fabricated at the foundries that make such chips in industry, but which may also be postprocessed in accordance to the present disclosure, for example to add or to expose nanoelectrodes, suitably protection over such nanoelectrodes (e.g., dielectric layers), in order to configure the CMOS chip for use in molecule electronics sensor arrangements,
  • the technological foundation for the systems, devices and methods herein is molecular electronics. This is a general field of technology in which a single molecule is incorporated as a component within an electrical circuit, where the single molecule performs some useful electrical function such as transducing chemical or molecular events (such as binding/hybridization events) into an electrical signal. Such devices may be particularly powerful as biosensors.
  • molecular electronics sensors are formed by conjugating a molecular bridge between nanoelectrodes, applying a voltage, and measuring the resulting current, with the current vs. time trace then being the primary sensor signal output.
  • the bridge molecule interacts with molecules in its environment, corresponding current fluctuations in the current-time trace allow for the detection of such events by the sensor.
  • the bridge molecule is configured with suitable conjugation groups that allow it to bind to each of the nanoelectrodes to bridge the nanogap between electrodes.
  • suitable conjugation groups are known for binding molecules to metal electrodes, including use of a metal binding peptide, or a thiol, amine, carbene, or diazonium group.
  • the electrode surface may be functionalized with molecules exposing a conjugation group, and in this case, various conjugation pairs can be used, with one as the head group and the cognate partner configured on the bridge molecule.
  • a sensor device comprises: a first contact coupled to a first electrode; a second contact coupled to a second electrode; a sensor gap defined between one of the first contact and the first electrode and one of the second contact and the second electrode; a bridge molecule comprising a first end and a second end; wherein the bridge molecule comprises a a-Si, a-Si:H or amorphous semiconductor nanoribbon film bridge, and wherein the bridge molecule is coupled to the first contact at the first end and coupled to the second contact at the second end; and a probe molecule coupled to the biopolymer to form a sensor complex, wherein the probe molecule is coupled to the biopolymer by a precisely positioned linker comprising a streptavidin-biotin complex, wherein the sensor complex is configured to interact with at least one target molecule.
  • a method of detecting an analyte comprising: exposing a closed circuit to the analyte in a fluid medium, the closed circuit comprising: a first electrode; a second electrode spaced apart from the first electrode by a nanogap; a first affinity probe bonded to the first electrode; a second affinity probe bonded to the second electrode; and a bridge molecule disclosed herein bonded at each end to the first and second affinity probes, bridging the nanogap; binding the analyte to the first affinity probe, thereby opening the closed circuit; and observing a resulting change in impedance from the opening of the closed circuit, the change in impedance indicating the presence of the analyte in the fluid medium.
  • a sensor in one exemplary embodiment, comprises a source electrode; a drain electrode spaced apart from the source electrode by a sensor gap;a gate electrode, wherein the source, drain and gate electrodes cooperate to form an electrode circuit; and a bridge molecule provided herein bridging across said sensor gap, connecting the source and drain electrodes; and a probe coupled to the bridge molecule, wherein interaction of the probe with a nucleic acid is detectable by monitoring at least one parameter of the electrode circuit.
  • a bridge-configured sensor structure comprising an elongated nano-dimension semiconductor wire or ribbon is one way of producing label-free molecular sensors for genome sequencing without introducing complicated fluorescence imaging methodologies.
  • semiconductor nanowires can be inorganic, or can be organic (such as DNA or peptide biomolecules). It is possible to atach a polymerase single molecule to such a nanobridge or other elongated biomolecules, e.g., using functionalities and ligands such as biotin-straptavidin, antibody-antigen or peptide complexes.
  • Inorganic nanobridges either van der Waals force connected or metallization connected to the device electrode leads can offer much higher electrical conductivity and substantially higher electrical sensor signals than organic nanobridges as the use of undesirably high electrical resistance ligands or ataching functionalities can be minimized.
  • such biosensor bridges between a pair of conducting electrodes can be accomplished by an active atachment route using electrical field (electrophoretic alignment and atachment using various AC or DC mode of electric fields, often at a level of 0.2 to 10 volts).
  • aligned nanobridge structures connecting the mating electrodes can also be achieved by stamp transfer of pre-aligned (or pre- patemed) inorganic nanobridge array from another substrate using PMMA (Polymethyl methacrylate), PDMS (Polydimethylsiloxane) or other polymer type stamp materials.
  • the nanobridge array in parallelly aligned configuration is prepared either by nanopateming by lithographic means, flow alignment in a microfluidic chamber, or by electrical field alignment (e.g., dielectrophoretic alignment using AC or DC electric field).
  • nanomaterials While a number of nanomaterials can be considered for such nanobridge structures, these nanomaterials need to be processed into nano-dimension wires or ribbons by nanolithography or other treatments, which tends to cause damage to crystallographic structures or disruption of atomic arrangements causing unintended changes or deterioration of physical or electronic properties. While a high temperature annealing process (e.g., 500 - 1,000°C) can sometimes repair or reduce such damages, the use of high temperature processing steps often damages electronic device structure and hence is to be avoided for ease of device manufacturing and reliability.
  • a high temperature annealing process e.g., 500 - 1,000°C
  • nucleotide atachment events or other biomolecule atachment events, e.g., to polymerase, to enable electrical signal detection for genome sequencing
  • a single elongated bridge made of inorganic or organic nanowires or nanoribbons
  • mating electrodes made of Au, Pd or other conducting lead wires. If multiple nanobridges are atached between the two mating electrodes, often clumped together, multiple signals may occur by the presence of parallel sensors, which makes the analysis of such complicated signals very difficult.
  • This invention discloses innovative approaches of producing a single nanobridge structure of amorphous nanoribbons, which can be made into a reliable nanobridge sensor structure since the material is already amorphous lacking crystallographic structure thus such a nanobridge material is less sensitive to nanopatteming-related damages of crystallographic structures or atomic arranges, and hence a need for high temperature processing is minimized.
  • Unique amorphous nanobridge structures, processing methods and applications of such a single-bridge amorphous nanoribbon biosensors are disclosed as described in the drawings (Fig. 1 - Fig. 14) and associated captions.
  • Amorphous (or glassy) semiconductors can be made of different compositions and structures such as tetrahedrally bonded type amorphous semiconductors including amorphous Si (referred to as a-Si hereafter), a-Ge, and their alloys, hydrogenated amorphous silicon (a-Si:H), and chalcogenide type glasses including a- S, a-Se, a-Te, a-S * Sei- x , pnictogen-chalcogen (V-VI groups in the periodic table of elements) compounds such as a- As 2 Sey a-AsiS . a-PiSe . and tetragen-chalcogen (IV -VI groups) materials such as a-GeSe 2 ,a- SiS 2 ,a-SiSe 2 .
  • amorphous Si a-Si
  • hydrogenated amorphous silicon a-Si:H
  • amorphous InGaZnO amorphous InGaZnO
  • other related amorphous thin film semiconductors can be made into a desirably thin dimension at or near room temperature by combining sputtering, evaporation or other physical vapor deposition or chemical processes with nanopatteming (e.g., using e-beam lithography, nanoimprint lithography, template-induced nanopatteming and other means).
  • the thickness of the amorphous semiconductor thin film nanobridge is desirably made thinner (e.g., ⁇ 30 nm, preferably ⁇ 20 nm thick, more preferably ⁇ 10 nm thick layer, even more preferably ⁇ 5 nm), with the width ⁇ 40 nm, preferably ⁇ 20 nm, more preferably ⁇ 10 nm, even more preferably ⁇ 3 nm.
  • a smaller cross-section nanowire or nanoribbon bridge across two mating electrodes can provide higher signal to noise ratio on sequencing interaction.
  • a smaller cross-section nanowire or nanoribbon bridge of amorphous semiconductors ensures that a single (or at most a few) bridge can be formed due to size exclusion, instead of multiple bridges, with the latter introducing multiple, undesirably complicated signals from the parallel bridges.
  • a single and narrower sensor bridge will also prefer a single-molecule polymerase placement for each electrode pair.
  • Another approach to further ensure a single bridge formation of a- Si or hydrogenated amorphous silicon (a-Si:H), according to the invention, is to utilize a nano-mask array, for example, by placing an array of e.g., ⁇ 5 nm-tip-radius pointed graphene nanoribbon mask, or carbon, ceramic or metallic nanoribbon mask so that a- Si or a-Si:H underneath can be patterned and shaped to become narrow and optionally tip-pointed following the graphene mask shape.
  • Such pre-made narrowed bridge can be used as a sensor bridge on which a single enzyme molecule such as polymerase can be attached.
  • such amorphous semiconductor ribbon strip can be split into two segments between which another molecular bridge (such as a single DNA or peptide) can be attached for ease of polymerase bonding and nucleotide analysis.
  • another molecular bridge such as a single DNA or peptide
  • Such splitting of amorphous semiconductor into two separated parts with a nanogap in-between can be accomplished by e.g., focused laser beam slicing, focused ion beam cutting, electron beam, neutron beam, or nanopatteming and etching.
  • the ends of the split ribbons facing each other can desirably be sharpened to a pointed-tip geometry of e.g., 2-5 nm radius of curvature, so as to facilitate an attachment of a single DNA or a single peptide molecular bridge, using either electric field alignment, flow alignment in a microfluidic chamber, or stamp transfer of pre- aligned DNA or peptide.
  • Stamp transfer of pre-aligned DNA or peptide array can be made using PMMA, PDMS or other polymer type soft stamps.
  • Amorphous semiconductor films can be in situ nanopattemed as-deposited, or (substrate-removed + float transferred) into a nano-bridge array on the sequencer device itself, or a large-area film is separately processed into aligned nano-ribbon array, and made releasable from the substrate (e.g., sputtered on dissolvable metal, polymer, or SiCh substrate) so that a stamp transfer, or di electrophoretic (DEP) alignment or flow alignment is made possible in microfluidic chamber).
  • substrate e.g., sputtered on dissolvable metal, polymer, or SiCh substrate
  • the amorphous Si offers advantages of convenient lower processing temperatures, lower cost, and more convenient deposition on large area substrates for easier manufacturing.
  • a thin film of a-Si or a-Si:H (e.g., 3 -20 nm thick) can be deposited on SiCh or on crystalline Si substrates, or other dielectric-passivated flat surfaces.
  • a method of manufacturing a structure usable in a molecular sensor device comprising providing a substrate that defines a substrate plane and further comprises a protrusion protruding from the substrate at an angle to the substrate plane; depositing a first electrode layer on the substrate to form a first electrode sheet; depositing an inner dielectric layer on the first electrode sheet to form an inner dielectric sheet; depositing a second electrode layer on the inner dielectric sheet to form a second electrode sheet; depositing an outer dielectric layer on the second electrode sheet to form an outer dielectric sheet, planarizing the first and second electrode sheets, the inner dielectric sheet, and the outer dielectric sheet; removing an exposed end portion of the inner dielectric sheet to form a groove located on an exposed end portion of the inner dielectric sheet; and connecting first and second lead conductors to the first and second electrode sheets, wherein the steps of depositing the first electrode layer, depositing the inner dielectric layer, depositing the second electrode layer, and depositing the outer dielectric layer are
  • the electrode layer can be deposited by physical vapor deposition such as sputtering, thermal evaporation, e-beam evaporation, ion beam deposition, laser ablation, molecular beam epitaxy, or chemical vapor deposition, followed by using nanopatteming techniques well known in the prior art and as described above (e.g., using photolithography, e-beam lithography, nanoimprint lithography, EUV (extreme UV) lithography, involving nano features exposed to light or electron beam, developed, etched or lift-off processed).
  • An alternative processing route is to utilize a pre-made layer of amorphous semiconductors can be stamp transferred onto the sensor device during the sensor structure assembly/fabrication processes.
  • a thin film of amorphous semiconductor layer can conveniently be deposited at or near room temperature in a nanopattemed way on a separate etchable substrate (e.g., by sputtering, evaporation, and so forth) and released by etching removal of the substrate, or deposited as a peelable layer on a substrate such as PDMS (polydimethylsiloxane) or PTFE (polytetrafluoroethylene, also known as Teflon) from which the thin flexible polymer substrate can be peeled off.
  • a separate etchable substrate e.g., by sputtering, evaporation, and so forth
  • a substrate such as PDMS (polydimethylsiloxane) or PTFE (polytetrafluoroethylene, also known as Teflon) from which the thin flexible polymer substrate can be peeled off.
  • PDMS polydimethylsiloxane
  • PTFE polytetrafluoro
  • a transfer of the amorphous semiconductor layer may be carried out first onto the sensor device with the polymer carrier substrate still on, with the polymer carrier removed afterwards once the transfer to the sensor structure is completed.
  • the adhesion of transferred amorphous semiconductor to the base sensor structure is often enhanced by van der Waals force commonly present in nanostructures.
  • anchoring structure such as dielectric layer deposition near the ends or sides of the amorphous nanoribbons can be utilized.
  • Another exemplary illustrative embodiment of a method of manufacturing a structure usable in a molecular sensor device comprises forming a stack comprising: providing a first outer dielectric layer; depositing a first electrode layer on the first outer dielectric layer; depositing an inner dielectric layer having a first thickness on the first electrode layer; depositing a second electrode layer on the inner dielectric layer; and depositing a second outer dielectric layer having a second thickness on the second electrode layer, wherein the second thickness of the second outer dielectric layer is at least one order of magnitude greater than the firs thickness of the inner dielectric layer; slicing through the stack at least once at an angle to the layers in the stack to form a plurality of chips from the sliced portions of the stack; attaching the plurality of chips to a substrate so that the sliced portions of the first electrode layer and the second electrode layer form a plurality of pairs of electrode sheets at an angle to a substrate plane defined by the substrate, and so that the sliced portions of the inner dielectric layer forms a plurality
  • a sequencing device comprising:
  • a single or a few direction-aligned and two-ends bonded a-Si, a-Si:H or amorphous semiconductor nanoribbon film bridge (preferably ⁇ 20 nm wide, ⁇ 10 nm thick) conveniently prepared at or near room temperature, connected to two-electrode or three- electrode-gated Field Effect Transistors, various methods to achieve such useful bridge configurations for genome sequencing device or DNA memory device, as described or implied in Fig. 1 to Fig. 14 and associated descriptions in the specification, and various applications for biomolecular sensing and information storage.
  • a dielectric masking layer disposed on the conductor and comprising size-limited openings (with optional funnel-like guiding structure) that define exposed Au or other related noble metals/alloys, each opening sized to be less than ⁇ 10 nm, preferably ⁇ 5 nm, so as to allow only a single (or a few) enzyme biomolecules to fit therein and to attach onto the exposed electrode surface region defined by each opening;
  • a microfluidic system encasing the electrode array, wherein attachment or detachment of a biomolecule selected from the group consisting of a nucleotide monomer, a protein, and a DNA segment or other related biomolecules, onto the enzyme molecule, one at a time, can be monitored as a uniquely identifiable electrical signal pulse to determine the specific nature of the biomolecule attaching or detaching.
  • Embodiment 2 The device of embodiment 1, wherein the a-Si, a-Si:H or amorphous semiconductor nanoribbon bridge are aligned by microfluidic flow alignment, by electric field, or by stamp-transfer from separately prepared pre-aligned a-Si, a-Si:H or amorphous semiconductor nanoribbon arrays onto the final device surface, and are attached onto the ends of two mating electrodes.
  • Embodiment 3 The device claim of embodiment 1, wherein the a-Si, a-Si:H or amorphous semiconductor nanoribbon bridges are attached to the electrodes by molecular binding force like van der Waals force, by metallization added near the bond location, or by using functionality complexes or ligands.
  • Embodiment 4 The device of embodiment 1, wherein the polymerase type enzyme molecule is attached to the DNA bridge using molecular connection functionalities and ligands including biotin-streptavin complexes, antibody-antigen complex, peptide complex, etc. for more accurate detection of nucleotide attachment event and type of nucleotides.
  • Embodiment 5 Any variations of structures, materials involved, methods of bridge formation, and biological applications beyond the description in the specification and Figs 1-14. Some examples include a splitting of amorphous film semiconductor nano-ribbon into two (e.g., by focused laser, focused ion beam or nano patterning), between which a molecular bridge such as DNA or peptide is attached. A tip sharpening of the split amorphous semiconductor nano-ribbon is optional to ensure an attachment of only a single (or a few) DNA type bridges. [0070] Embodiment 6. Use of the DNA or RNA sequencing devices and systems of embodiments 1-5 to perform a whole genome sequencing, diagnosis of various diseases including cancer.
  • Embodiment 7 Application of the biosensor device for encoded DNA memory storage array devices including tethered memory block array or continuously feed memory wire DNA that can be sliced mechanically or chemically, and subsequently read by a-Si, a- Si:H or amorphous semiconductor nanoribbon bridge array structures comprising polymerase sensor array.
  • Embodiment 8 DNA memory storage that can be written in a massively parallel way, and can be read in a massively parallel manner using high-density a-Si, a-Si:H or amorphous semiconductor nanoribbon bridge structures comprising polymerase sensor array, for ultrafast read capability, and also “random-access-enabled” reading configurations.
  • Embodiment 9 The device and structure of Embodiments 1-8 wherein the amorphous semiconductor ribbon strip is split into two segments between which another molecular bridge (such as a single DNA or peptide) can be attached for ease of polymerase bonding and nucleotide analysis.
  • another molecular bridge such as a single DNA or peptide
  • Embodiment 10 The device of Embodiment 9 wherein the splitting of amorphous semiconductor into two separated parts with a nanogap of e.g., 20 - 100 nm is accomplished by e.g., focused laser beam slicing, focused ion beam cutting, electron beam, neutron beam, or nanopatteming and etching.
  • Embodiment 11 The device of Embodiment 9 wherein the attachment of a single DNA or a single peptide molecular bridge (or a few bridges), is accomplished by using either electric field alignment, flow alignment in a microfluidic chamber, or stamp transfer of pre-aligned DNA or peptide using PMMA, PDMS or other polymer type soft stamps.

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Abstract

L'invention concerne des capteurs biomoléculaires et des procédés de fabrication de ces capteurs. Un capteur donné à titre d'exemple comporte une électrode de source ; une électrode de drain espacée vis-à-vis de l'électrode de source par un espace de capteur ; une électrode de grille, les électrodes de source, de drain et de grille coopérant de sorte à former un circuit d'électrode ; une molécule de pont formant un pont à travers ledit espace de capteur couplant les électrodes de source et de drain, la molécule de pont comprenant un pont en film de nanoruban semi-conducteur a-Si, a-Si:H ou amorphe ; et une sonde couplée à la molécule de pont, l'interaction de la sonde avec une biomolécule étant détectable par surveillance d'au moins un paramètre du circuit d'électrode. L'invention concerne en outre des procédés de fabrication du capteur à température ambiante.
PCT/US2021/033782 2020-05-22 2021-05-21 Biocapteurs à pont de nanorubans semi-conducteurs amorphes pouvant être traités à température quasi-ambiante et dispositifs de mémoire Ceased WO2021237180A1 (fr)

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WO2023191858A3 (fr) * 2021-12-08 2024-02-15 Twist Bioscience Corporation Dispositifs nanoélectriques et leur utilisation

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