Classifications

• • 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

Definitions

• the present disclosure provides devices, systems, and methods related to electronic biosensors.
• the present disclosure provides devices, systems, and methods for fabricating nanometer-scale devices with external circuits using a combination of bottom-up and top-down integration techniques to create self-aligning single-molecule circuits.
• Embodiments of the present disclosure include a self-aligning molecular junction.
• the junction includes a first single-walled carbon nanotube (CNT) and a second CNT.
• each of the first and second CNTs comprise a coating.
• the junction also includes a single-molecule bridge functionally coupled to the first and second CNTs, thereby forming a single-molecule junction.
• the single-molecule junction is soluble in solution.
• the solution is an aqueous solution.
• the solution comprises a polar organic solvent.
• the solution comprises a non-polar organic solvent.
• the solution comprises a soluble coupling agent.
• the soluble coupling agent comprises O-(7-azabenzotriazole-l- yl)-N,N,N,N'-tctramcthyluronium hexafluorophosphate (HATU).
• the single-molecule bridge is functionally coupled to at least a third CNT.
• the coating comprises a polynucleotide.
• the polynucleotide is a single-stranded polynucleotide selected from DNA, RNA, PNA or any combinations or derivatives thereof.
• the single-stranded polynucleotide is a single-stranded DNA comprising guanine-thymine repeats.
• the single-molecule bridge comprises an organic molecule.
• the organic molecule comprises: a polynucleotide or a derivative or variant thereof; a coronene or a derivative or variant thereof; a norbornadiene or a derivative or variant thereof; a tetraphenyl or a derivative of variant thereof; and a scorpionate or a derivative or variant thereof.
• the polynucleotide comprises DNA, RNA, PNA, or any combinations or derivatives thereof. In some embodiments, the polynucleotide is functionally linked to at least one of the first and/or second CNT. In some embodiments, the polynucleotide is covalently linked to at least one of the first and/or second CNT.
• the single-molecule bridge comprises DNA origami. In some embodiments, the single-molecule bridge comprises an aptamer.
• the CNT is a single-walled CNT (SWNT) or a multi-walled CNT (MWNT). In some embodiments, the CNT is a metallic CNT (mCNT). In some embodiments, the CNT is a semiconducting CNT, such as a semiconducting single-walled CNT (sc-SWCNT).
• the single-molecule bridge comprises a peptide, polypeptide, or protein.
• the peptide, polypeptide, or protein comprises biotin-streptavidin.
• the protein comprises a polymerase, or a derivative or variant thereof.
• the single-molecule bridge comprises a scorpionate.
• the scorpionate comprises a pair of tri-dentate molecules and a metal ion ligated between the pair of tri-dentate molecules.
• the single-molecule bridge comprises a photochemically active molecule.
• the photochemically active molecule comprises a norbornadiene or a derivative or variant thereof.
• the single-molecule bridge comprises an electrically active molecule.
• the single-molecule bridge comprises an optically active molecule.
• the single-molecule bridge comprises a magnetically active molecule.
• the single-molecule bridge is from about 0.50 nm to about 250 nm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about 1 pm in length. In some embodiments, the single-molecule bridge is less than about 600 nm in length.
• At least one of the first and/or second CNT is functionalized with one or more linkers.
• the one or more linkers are configured to functionally couple at least one of the first and/or second CNT to the single-molecule bridge.
• the one or more linkers comprise biotin and/or streptavidin.
• Embodiments of the present disclosure also include a single-molecule electronic biosensor comprising at least one circuit that includes any of the junctions described herein.
• the first and the second CNTs are functionally coupled to pairs of microfabricated electrodes, thereby forming the at least one circuit.
• Embodiments of the present disclosure also include a method of detecting a target nucleic acid.
• the method includes introducing a sample comprising or suspected of comprising a target nucleic acid to any of the single-molecule electronic biosensors described herein.
• the single-molecule bridge is a polynucleotide (e.g., a DNA molecule, RNA molecule, aptamer, and the like) comprising a sequence that is at least partially complementary to at least a portion of the target nucleic acid.
• the method also includes detecting the presence of the target nucleic acid in the sample when the target nucleic acid binds to the complementary strand of the polynucleotide.
• detecting the presence of the target nucleic acid comprises detecting a change in conductance and/or current.
• Embodiments of the present disclosure also include an aqueous solution comprising a plurality of single-walled carbon nanotube (CNTs), each coated with a single-stranded polynucleotide, and a plurality of single-molecule bridges of varying lengths, each configured to bind at least one of the plurality of CNTs.
• CNTs single-walled carbon nanotube
• a plurality of single-molecule bridges of varying lengths each configured to bind at least one of the plurality of CNTs.
• incubating the plurality of CNTs with the plurality of single-molecule bridges produces a plurality of self-aligned, single-molecule junctions.
• Embodiments of the present disclosure also include a method of generating a plurality of self-aligned, single-molecule junctions.
• the method includes incubating any of the aqueous solutions described herein with a coupling agent.
• the coupling agent facilitates binding of a single-molecule bridge and two CNTs, thereby producing the plurality of self-aligned, single-molecule junctions.
• the method further comprises modifying pairs of micro fabricated electrodes to bind each of the plurality of self-aligned, single-molecule junctions, thereby forming a single-molecule electronic biosensor.
• the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least 1%.
• FIGS. 1A-1D Development of mCNT-DNA-mCNT junctions.
• Carboxyl-terminated metallic CNTs are first wrapped with (GT)2o, then coupled to aminofunctionalized DNA oligonucleotides in PBS buffer to form a self-aligning hybrid structure. Thereafter nanojunctions are drop-cast onto nanofabricated devices where cysteamine modified gold electrodes can anchor the nanojunction with an amide bond to create a robust single molecule junction.
• FIGS. 2A-2G Charge transport properties of mCNT-DNA-mCNT structures.
• A) 100 repeated I-V characteristics (gray background) for the 12 bp G:C sequence shown in the inset and a linear fitting to the data (R 2 0.989, red line) indicating ohmic transport.
• FIG. 3 Device Stability. Resistance of a single CNT-DNA-CNT junction measured over the course of 7 days to demonstrate device stability.
• FIGS. 4A-4B Measurement of Strand Dehybridization.
• A) A:T Rich DNA sequence measured.
• C) When heated the two strands will dehybridize resulting in the device returning to the single-stranded DNA conductance value.
• FIGS. 5A-5C Electronic oligonucleotide detection using a single-molecule biosensor.
• Phase 1 open-circuit, blank device, prior to junction deposition
• Phase 2 Conductance of 614G Probe/Target (PBS, 20°C)
• Phase 3 Replace solution with 20 pM 614G targets in PBS
• Phase 4 Dehybridize the duplex by heating the system at a rate of 0.5°C/s, a conductance drop of 3 orders of magnitude occurs when the solution temperature reaches 65°C indicating DNA dehybridization, with only the single-stranded 614G Probe remaining between the mCNTs resulting in a current below our detectable range
• Phase 5 During cooling the junction is reformed when a 614G target from solution binds to the probe sequence to reform a DNA duplex and conductive pathway.
• Phase 6 Replace solution with the mismatched 614D sequence (WT).
• Phase 7 Repeat dehybridization process by heating the sample, which again dehybridizes near 65 °C.
• Phase 8 upon cooling no device formation was observed.
• FIGS. 6A-6C CNT-DNA-CNT Junctions with metal ions in the DNA.
• A) Example of DNA with a metal ion (Ag+) in the DNA due to a mismatch in the DNA sequence.
• B) Statistics on device yield from the structure (low conductance values are open circuit; see also FIG. 7A).
• FIGS. 7A-7B Examples of metalated DNA Devices.
• FIG. 8 DNA Origami Structure. Example of a DNA origami structure. Six DNA strands are coupled together to form a 4-helix bundle of DNA that is ⁇ 30 basepairs in length.
• FIG. 9 DNA Origami Devices.
• Such Origami structures can be used to place a wide variety of other structures within the junction (e.g., nanoparticles, quantum dots, other molecules, etc.), which can yield a number of unique applications (e.g., sensing, optoelectronics, electronics, spintronics, etc.).
• FIGS. 10A-10B CNT-Protein-CNT Devices.
• FIGS. 11A-11C Small-Molecule Junctions - Spin-crossover molecules.
• FIG. 12 Spin Crossover Molecules. Conductance statistics for the two spin-crossover molecules shown in FIG. 11.
• FIGS. 13A-13B Optoelectronic Switches.
• NB Norbornadienene
• FIG. 14 Electronic Switching Molecules. This figure shows the same molecular system as FIG. 13, but it operates as a voltage-controlled switch, changing conductance values with the applied square-wave potential, again emphasizing its utility as a switch/memory device.
• Embodiments of the present disclosure include devices, systems, and methods related to electronic biosensors.
• the present disclosure provides devices, systems, and methods for fabricating nanometer-scale devices with external circuits using a combination of bottom-up and top-down integration techniques to create self-aligning single-molecule circuits.
• embodiments of the present disclosure include a method for combining controlled, bottom-up self-assembly processes, with top-down lithographic techniques to create self-aligning single-molecule devices with yields on the order of 45% at the chip-scale and demonstrate the utility of this approach by creating a dynamic, high signal-to-noise ratio (SNR), single-molecule electronic biosensor capable of identifying specific oligonucleotide sequences with high fidelity.
• SNR signal-to-noise ratio
• embodiments of the subject disclosure may include methods, compositions, systems and apparatuses/devices that may further include any and all elements from any other disclosed methods, compositions, systems, and devices. In other words, elements from one or another disclosed embodiments may be interchangeable with elements from other disclosed embodiments.
• some further embodiments may be realized by combining one and/or another feature disclosed herein with methods, compositions, systems and devices, and one or more features thereof, disclosed in materials incorporated by reference.
• one or more features/elements of disclosed embodiments may be removed and still result in patentable subject matter (and thus, resulting in yet more embodiments of the subject disclosure).
• some embodiments correspond to methods, compositions, systems, and devices which specifically lack one and/or another element, structure, and/or steps (as applicable), as compared to teachings of the prior art, and therefore represent patentable subject matter and are distinguishable therefrom (i.e. claims directed to such embodiments may contain negative limitations to note the lack of one or more features prior art teachings).
• a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
• the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
• This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
• “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
• the term “functionally coupled” generally refers to the binding, joining, or combining of two or more molecules such that the resultant combination comprises at least one functional property.
• the resultant combination of the two or more molecules maintains one or more functional properties of one or more of the uncoupled molecules.
• the resultant combination yields a functional property not previously present in the individual, uncoupled molecules.
• “functional coupling” includes the binding, joining, or combining of two or more molecules via chemical means (e.g., covalent or non-covalent interaction).
• the “functional coupling” of two or more molecules results in the formation of a junction having certain electrical and/or biochemical properties.
• covalent bond generally refers to a stable association between two atoms which share one or more electrons.
• covalent bonds include, without limitation, a peptide bond and a disulfide bond.
• Peptide bond refers to the covalent bond formed between the carboxyl group of an amino acid and the amine group of the adjacent amino acid.
• non-covalent bond generally refers to an attractive interaction between two molecules or two chemical groups that does not involve sharing of electrons.
• non-covalent bonds include, without limitation, a hydrogen bond, a hydrophobic bond, an ionic bond, and a Van der Waals bond.
• a “hydrogen bond” refers herein to attractive force between a hydrogen atom of a first molecule/group and an electronegative atom of a second molecule/group.
• a “hydrophobic bond” refers herein to a force that causes hydrophobic or non-polar molecules/ groups to aggregate or associate together in an aqueous environment.
• An “ionic bond” refers herein to an attraction between a positive ion and a negative ion.
• a “Van der Waals bond” refers herein to a non-specific attraction force between two adjacent molecules/ groups which have momentary random fluctuations in the distribution of electrons.
• linker generally refers to a molecule that is configured to facilitate the binding, joining, or combining of two or more molecules. In some cases, a linker connects a portion of a molecule to another portion of the same molecule or to a different molecule, moiety or solid support (e.g., electrode, CNT, bridge). Linkers can connect a molecule via a covalent bond or other means, such as ionic or hydrogen bond interactions.
• the single-molecule junctions and corresponding circuits and devices of the present disclosure can be generated using a CNT-molecule-CNT structure.
• CNTs can be wrapped with a molecule that protects the sidewalls from subsequent reaction steps. This process can be done with single-stranded DNA as a protecting group, but a range of other molecules can also be used, including but not limited to, various other biopolymers (e.g., peptide chains, proteins, RNA, etc.) and their analogs (e.g., PNA, LNA, etc.), other polymers (e.g., Polyethylene glycol), etc.), or small molecules (e.g., SDS, Triton, etc.).
• reactions can be performed without wrapping; however, this usually produces lower yields.
• a molecule with amine terminations is used to initiate a coupling reaction with the exposed carboxylic groups on the ends of the CNTs.
• a range of chemistries are available to initiate this reaction, as would be recognized by one of ordinary skill in the art based on the present disclosure. Reactions are generally performed in aqueous solutions; however, in some cases (e.g., with scorpionates), a polar organic solvent (e.g. acetone, dichloromethane) is used. Reactions can also be performed with non-polar organic solvents if the correct wrapping agents are used (also non-polar), and the reaction chemistry is designed with a soluble coupling agent (e.g., HATU).
• a soluble coupling agent e.g., HATU
• the process results in CNT-molecule-CNT junctions in solution phase with the separation between the CNTs separated by the length of the molecule. This provides a stable molecular junction. In the case metallic CNTs are used, this results in a metal-molecule-metal junction, though semiconducting CNTs can also be used, or a metal-molecule-semiconducting configuration, which can function as a type of tunnel-diode.
• the CNT-molecule-CNT junctions are then deposited on the surface of pre-fabricated electrodes. In some embodiments, this is achieved using drop-casting. However, this process can be aided by an electrical, mechanical, and/or fluid flow. In some embodiments, the CNT-molecule-CNT devices are deposited first, and lithography is then performed on top of them.
• the various embodiments of the present disclosure have been used to develop and study many different types of molecular junctions and circuits.
• the self-aligning molecular junctions of the present disclosure include the use of DNA, including DNA origami molecules, which comprises bundle of single-stranded DNA molecules that are cross-linked to produce DNA duplexes bound together (e.g., -lOnrn long).
• the self-aligning molecular junctions of the present disclosure comprise an aptamer. Aptamers are short sequences of artificial DNA, RNA, XNA, or peptides that bind a specific target molecule, or family of target molecules.
• the self-aligning molecular junctions of the present disclosure include the use of peptides, polypeptides, and proteins. Proteins can perform a wide range of mechanical, chemical and electrical functions. Results provided herein have demonstrated the ability to use a protein as the single-molecule in a self-aligning junction. For example, using a non-covalent interaction, CNTs were first biotinylated and then the biotinylated ends were bound to a streptavidin protein.
• a range of peptides, polypeptides, ad proteins can be used in the junctions, circuits, and devices of the present disclosure, including via binding to streptavidin with biotinylated tagging of specific proteins/targets (e.g., using biotin/ streptavidin as linkers), or by chemically binding the protein directly to the CNTs through a covalent bond (e.g., amidation reaction, reacting a malemide with cystines, etc.).
• self-aligning molecular junctions can also be generated using any modified nucleic acids and amino acids (e.g., RNA, PNA, LNA, cross-linked DNA, hybrids of different types of duplexes (e.g. DNA:RNA), DNA with non-canonical bases, modified bases or backbones, modifications to do sensing, enzymes, ribozymes, peptide chains, amino acids, etc.).
• modified nucleic acids and amino acids e.g., RNA, PNA, LNA, cross-linked DNA, hybrids of different types of duplexes (e.g. DNA:RNA), DNA with non-canonical bases, modified bases or backbones, modifications to do sensing, enzymes, ribozymes, peptide chains, amino acids, etc.
• the self-aligning molecular junctions of the present disclosure can be generated to include organic, non-biological molecules, including but not limited to, spincrossover molecule (e.g., scorpionates) and photochemically active molecules (e.g., norbomadienes).
• spincrossover molecule e.g., scorpionates
• photochemically active molecules e.g., norbomadienes
• embodiments of the present disclosure include a selfaligned molecular junction.
• the junction includes a first single-walled carbon nanotube (CNT) and a second CNT.
• each of the first and second CNTs comprise a coating.
• the self-aligned molecular junction includes a single-molecule bridge functionally coupled to the first and second CNTs, thereby forming a single-molecule junction.
• the CNT is a single-walled CNT (SWNT) or a multi-walled CNT (MWNT).
• the CNT is a metallic CNT (mCNT).
• the CNT is a semiconducting CNT, such as a semiconducting single-walled CNT (sc-SWCNT).
• the single-molecule junction is soluble in solution.
• the solution is an aqueous solution.
• the solution comprises a polar organic solvent.
• the solution comprises a non-polar organic solvent.
• the solution comprises a soluble coupling agent.
• the soluble coupling agent comprises O-(7-azabenzotriazole-l-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate (HATU).
• HATU O-(7-azabenzotriazole-l-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate
• the single-molecule bridge of the self-aligning molecular junctions of the present disclosure is covalently linked to at least a third CNT. In some embodiments, the single-molecule bridge is covalently linked to at least a fourth CNT. In some embodiments, the single-molecule bridge is covalently linked to at least a fifth CNT. In some embodiments, the single-molecule bridge is covalently linked to a plurality of CNTs (e.g., transistor or cross-wire memory systems).
• CNTs e.g., transistor or cross-wire memory systems
• the coating for the CNTs of the present disclosure comprises a biopolymer, including but not limited to, a polynucleotide.
• the polynucleotide is a single-stranded polynucleotide selected from DNA, RNA, PNA or any combinations or derivatives thereof.
• the single-stranded polynucleotide is a single-stranded DNA comprising guanine-thymine repeats (e.g., (GT) n DNA).
• the single-stranded polynucleotide is single-stranded (GT) n DNA, wherein n corresponds to an integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or higher).
• the coating comprises another material, such as a surfactant or polymer.
• a self-aligned, single-molecule junction is assembled in non-aqueous solutions (e.g., organics, ionic liquids, etc.), semiconducting CNTs can be used.
• the self-aligned, single-molecule junction includes a singlemolecule bridge covalently linked to the first and second CNTs.
• the singlemolecule bridge is an organic molecule.
• the organic molecule is selected from the group consisting of a polynucleotide or a derivative or variant thereof; a coronene or a derivative or variant thereof; a norbornadiene or a derivative or variant thereof; a tetraphenyl or a derivative of variant thereof; and a scorpionate or a derivative or variant thereof.
• the polynucleotide is DNA, RNA, PNA, or any combinations or derivatives thereof.
• click chemistry is used to covalently link the polynucleotide to the first and second CNTs.
• the polynucleotide is covalently linked to the first and second CNTs (e.g., via an amidation reaction).
• the single-molecule bridge comprises DNA origami.
• the single-molecule bridge comprises a peptide, polypeptide, or protein.
• the peptide, polypeptide, or protein comprises biotin-streptavidin.
• the protein comprises a polymerase, or a derivative or variant thereof.
• the single-molecule bridge comprises a scorpionate.
• the scorpionate comprises a pair of tri-dentate molecules and a metal ion ligated between the pair of tri-dentate molecules.
• the single-molecule bridge comprises a photochemically active molecule.
• the photochemically active molecule comprises a norbornadiene or a derivative or variant thereof.
• other examples of peptide, polypeptide, proteins, scorpionates, and photochemically active small molecules can use used in the selfaligning molecular junctions described herein.
• the single-molecule bridge comprises an electrically active molecule.
• the single-molecule bridge comprises an optically active molecule.
• the single-molecule bridge comprises a magnetically active molecule.
• the single-molecule bridge is from about 0.50 nm to about 250 nm in length. In some embodiments, the single-molecule bridge is from about 10 nm to about 250 nm in length. In some embodiments, the single-molecule bridge is from about 25 nm to about 250 nm in length. In some embodiments, the single-molecule bridge is from about 50 nm to about 250 nm in length. In some embodiments, the single-molecule bridge is from about 100 nm to about 250 nm in length. In some embodiments, the single-molecule bridge is from about 200 nm to about 250 nm in length. In some embodiments, the single-molecule bridge is from about 0.50 nm to about 200 nm in length.
• the single-molecule bridge is from about 0.50 nm to about 100 nm in length. In some embodiments, the single-molecule bridge is from about 0.50 nm to about 50 nm in length. In some embodiments, the single-molecule bridge is from about 0.50 nm to about 25 nm in length. In some embodiments, the single-molecule bridge is from about 0.50 nm to about 10 nm in length. In some embodiments, the single-molecule bridge is from about 10 nm to about 200 nm in length. In some embodiments, the single-molecule bridge is from about 10 nm to about 100 nm in length. In some embodiments, the single-molecule bridge is from about 50 nm to about 200 nm in length.
• the single-molecule bridge is from about 250 nm to about 1 pm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about 900 nm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about
• the single-molecule bridge is from about 250 nm to about
• the single-molecule bridge is from about 250 nm to about 600 nm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about 500 nm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about 400 nm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about
• the single-molecule bridge is from about 300 nm to about 1 pm in length. In some embodiments, the single-molecule bridge is from about 400 nm to about
• the single-molecule bridge is from about 500 nm to about
• the single-molecule bridge is from about 600 nm to about
• the single-molecule bridge is from about 700 nm to about
• the single-molecule bridge is from about 800 nm to about
• the single-molecule bridge is from about 900 nm to about
• the single-molecule bridge is from about 250 nm to about
• the single-molecule bridge is from about 400 nm to about
• the single-molecule bridge is less than about 600 nm in length.
• the single-molecule bridge is less than about 550 nm in length. In some embodiments, the single-molecule bridge is less than about 500 nm in length. In some embodiments, the single-molecule bridge is less than about 450 nm in length. In some embodiments, the single-molecule bridge is less than about 400 nm in length. In some embodiments, the single-molecule bridge is less than about 350 nm in length. In some embodiments, the single-molecule bridge is less than about 300 nm in length. In some embodiments, the single-molecule bridge is less than about 250 nm in length. In some embodiments, the single-molecule bridge is less than about 200 nm in length. In some embodiments, the single-molecule bridge is less than about 150 nm in length. In some embodiments, the single-molecule bridge is less than about 100 nm in length. In some embodiments, the single-molecule bridge is less than about 50 nm in length.
• the self-aligning molecular junctions of the present disclosure include one or more linkers that can facilitate or enhance the binding of a single-molecule to one or more CNTs.
• at least one of the first and/or second CNT is functionalized with one or more linkers.
• the one or more linkers are configured to functionally couple at least one of the first and/or second CNT to the single-molecule bridge.
• the one or more linkers comprise biotin and/or streptavidin.
• Embodiments of the present disclosure also include a single-molecule electronic biosensor comprising at least one circuit comprising any of the junctions described herein.
• the first and the second CNTs are coupled to pairs of micro fabricated electrodes, thereby forming at least one circuit.
• a plurality of CNTs are coupled to pluralities of microfabricated electrodes to form a plurality of circuits.
• the present disclosure provides a method of detecting a target nucleic acid using the self-aligned, single-molecule circuits and biosensors described herein.
• the method includes introducing a sample comprising or suspected of comprising a target nucleic acid to a single-molecule electronic biosensors.
• the biosensor comprises at least one self-aligned, single-molecule junction that includes a single-molecule bridge covalently linked to at least a first and a second CNT.
• the single-molecule bridge is a DNA molecule comprising a sequence that is at least partially complementary to at least a portion of the target nucleic acid.
• detecting the presence of the target nucleic acid in the sample occurs when the target nucleic acid binds to the complementary strand of the double-stranded DNA molecule. In some embodiments, detecting the presence of the target nucleic acid comprises detecting a change in conductance and/or current.
• Embodiments of the present disclosure also include an aqueous solution comprising a plurality of metallic, single-walled carbon nanotubes (CNTs).
• CNTs metallic, single-walled carbon nanotubes
• each CNT is coated with a single-stranded polynucleotide.
• a plurality of single -molecule bridges of varying lengths are each configured to form covalent bonds with two or more CNTs.
• incubating the plurality of CNTs with the plurality of single-molecule bridges produces a plurality of self-aligned, single-molecule junctions.
• Embodiments of the present disclosure also include a method of generating a plurality of self-aligned, single-molecule junctions comprising incubating any of the aqueous solutions described herein with a coupling agent.
• the coupling agent facilitates covalent binding between a single-molecule bridge and two CNTs, thereby producing the plurality of self-aligned, single-molecule junctions.
• the method further comprises modifying pairs of micro fabricated electrodes to bind each of the plurality of self-aligned, single-molecule junctions, thereby forming a single-molecule electronic biosensor.
• the method generates the plurality of self-aligned, singlemoleculejunctions with a yield of at least about 1%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least about 5%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least about 10%. In some embodiments, the method generates the plurality of selfaligned, single-molecule junctions with a yield of at least about 15%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least about 20%.
• the method generates the plurality of self-aligned, singlemoleculejunctions with a yield of at least about 25%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least about 30%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least about 35%. In some embodiments, the method generates the plurality of selfaligned, single-molecule junctions with a yield of at least about 40%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least about 45%. In some embodiments, the method generates the plurality of self-aligned, singlemoleculejunctions with a yield of at least about 50%.
• the method generates the plurality of self-aligned, singlemoleculejunctions with a yield from about 1% to about 50%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield from about 1% to about 40%. In some embodiments, the method generates the plurality of self-aligned, singlemoleculejunctions with a yield from about 1% to about 30%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield from about 1% to about 20%. In some embodiments, the method generates the plurality of self-aligned, singlemoleculejunctions with a yield from about 1% to about 10%.
• the method generates the plurality of self-aligned, single-molecule junctions with a yield from about 10% to about 50%. In some embodiments, the method generates the plurality of self-aligned, singlemoleculejunctions with a yield from about 20% to about 50%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield from about 30% to about 50%. In some embodiments, the method generates the plurality of self-aligned, singlemoleculejunctions with a yield from about 40% to about 50%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield from about 5% to about 45%.
• the method generates the plurality of self-aligned, singlemoleculejunctions with a yield from about 10% to about 40%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield from about 15% to about 35%. In some embodiments, the method generates the plurality of self-aligned, singlemoleculejunctions with a yield from about 10% to about 25%. In some embodiments, the method generates the plurality of self-aligned, single-molecule junctions with a yield from about 25% to about 45%.
• mCNTs Metallic single wall carbon nanotubes
• PBS Phosphate buffer solution
• HATU O-(7- azabenzotriazole-l-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate
• All other analytical-grade chemicals were purchased from Sigma Aldrich. All DNAs were obtained from Alpha DNA.
• ssDNA single-stranded DNA
• GT GT2o- 3
• This sequence was employed for the wrapping of mCNTs as it was shown to be able to work efficiently for blocking unwanted carboxyl groups on mCNT’s sidewall.
• SARS-CoV-2 sequences [0089] 5’-amino-(CH 2 ) 6 -ATA GTC CCA CAA TTG ACG-(CH 2 )6-amino-3’ Probe (SEQ ID NO: 9);
• the mCNT wrapping process is driven primarily by favorable interactions between the ssDNA bases and the mCNT due to van der Waals and hydrophobic interactions.
• 0.1 mL of the mCNT (Img/mL) solution was first diluted with 2.5 mL PBS, then 2.5 mL of 5 pM (GT) 2 o was added. After overnight incubation at room temperature, the mixture was placed in a clean tube surrounded by ice for at least 2 hours under sonication (at the power level of 3W) and then centrifuged at 5000 rpm (Eppendorf 5415C) for 2 hours to remove insoluble material. Then supernatant was collected and diluted with DI water (MilliQ), the volume of (GT) 2 o-dispersed mCNT solutions was kept to 2mL.
• amino -terminated dsDNA was prepared at a concentration of 5 ./M in 2mL of PBS buffer and mixed with 2 mL (GT) 2 o wrapped mCNTs and ⁇ 0.13mM of Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU). The mixture was left to react at room temperature for 3 hours under weak sonication. Subsequently, any insoluble material was removed via 15 mins centrifugation at 12000rpm. The mCNT-DNA-mCNT nanohybrids were then extracted from the supernatant.
• microelectrode platform used to electrically characterize the nanostructures was fabricated using optical lithography.
• a Si/Si3N4 wafer was first spin-coated with HMDS as an adhesive layer, followed by PID controlled baking at 110°C for 1 min.
• microelectrodes were patterned on the wafer using photolithography with KL5302 Hi-Res Photoresist (Kem Lab).
• TMAH High-Res Photoresist
• the wafer was gently rinsed with DI water and blow-dried with nitrogen to stop the development process.
• a PETS-RIE plasma etcher was employed to etch ⁇ 65 nm trenches into the silicon nitride.
• a Cr(adhesive)/Au multilayer of thickness 10nm/55 nm was deposited by E-Beam evaporator.
• the sample was then immersed in MICROPOSITTM Remover 1165 for 90 minutes, and ultrasonicated to lift off residual resist.
• lOOnm Si 2 N4 insulating layer the was deposited using PECVD.
• Micron-sized windows for the probing and sensor areas were patterned on using the same photolithography process.
• a -100 nm etch was added into the PECVD-cover layer using the Plasma-Therma RIE to open the desired windows.
• the freshly made devices were immersed in a 10 mM coupling agent, cysteamine, overnight.
• the devices were then removed from the solution, gently rinsed with DI water and dried with a stream of nitrogen gas. After device characterization (blank test), a silicone ring was mounted and sealed to sensor area as sample reservoir. Thereafter, the modified Au electrodes were incubated with the nanostructures in PBS solution to covalently bridge the gaps. In cases where the electrodes were not coated with cysteamine, it was also possible to obtain singlemolecule circuits, but the devices were not as stable. In general, linking molecules capable of binding to both the Au electrode and the mCNT will be useful for improving the stability of the device.
• Alignment and binding to the electrodes can also be obtained by using external forces to drive the mCNT-DNA-mCNT junctions into the gap. Dielectrophoresis, where an alternating high-frequency field is applied between the electrodes to generate a force that pushes the nanostructure into the gap, has been shown to be useful to create the final circuit configuration. In addition, hydrodynamic, and fluidic controls can also be used to help position the nanostructure in the gap.
• ssDNA 1 NH 2 -C 6 Hi2-ACG GCC GCG GCA GCA CGC GCG CGG CCC TGC GG- H12C6-NH2 (SEQ ID NO: 12);
• ssDNA 2 TGC CGG CGG CTC GAG CGG CCG GCA CGA GCT GGA GGA GAC CCC GCC AGG CGC GCG CCG GGA CGC C (SEQ ID NO: 13);
• ssDNA 3 GAC CGA CGC GGC GTT GGT GCT GCC CTA GGA CCG CGG TCA GAC GGT CGG CCA CAA TCG TGC GAG T (SEQ ID NO: 14);
• ssDNA 4 CTG GCT GCG CCG CAA CCC TGG CGG CAC GCT CA (SEQ ID NO:
• ssDNA 5 CCG GCC GTG CTC GAC CTC CTC TGG GAT TGT GG (SEQ ID NO:
• ssDNA 6 GCT CGA GCG ATC CTG GCG CCA GTC TGC CAG CC (SEQ ID NO:
• the present disclosure provides devices, systems, and methods related to electronic biosensors.
• the present disclosure provides devices, systems, and methods for fabricating nanometer-scale devices with external circuits using a combination of bottom-up and top-down integration techniques to create self-aligning single-molecule circuits.
• mCNTs single-walled carbon nanotubes
• cryogenic transmission electron microscopy was used to examine the structures.
• FIG. ID shows specific cases where gaps appear between mCNT structures.
• FIG. 2A shows a set of 100 I-V characteristics from a single, bridged 300 nm gap measured in a lOOmM sodium phosphate buffer solution (PBS) at room temperature.
• the structures and conductance values were very stable over time.
• 58 pairs of 300 nm gap devices were examined across three different substrates, and the data from each of these gaps was plotted in FIG. 2C. There are primarily three different conductance regimes visible in this plot. The grey region shows gaps that are open circuits, indicating no bridging occurred. The yellow region includes all high-conductance values.
• the third, pink region which spans a conductance range of (lxlO' 4 ⁇ o 5 Go), shows a series of devices with a narrow conductance distribution at 1.02x1 O' 4 ⁇ 0.17 X 10' 4 Go (where Go is the conductance quantum, Go 77.48 pS).
• Scanning electron microscopy (SEM) images of the devices from this conductance range verify that they are bridged by a single entity (FIG. 2D). Notably, of the 60 300 nm gap structures measured, 45% had a conductance value in this window. These devices are stable for extended periods of time (FIG. 3).
• This sequence modification was an early mutation that occurred and greatly impacted the virulence of SARS-CoV-2, and by focusing on this sequence with a known point mutation difference between the wild type and the variant, experiments can be conducted to examine the specificity of this detection approach, and its resilience when challenged with potentially interfering targets.
• an 18 bp sequence was extracted from the original SARS- CoV-2 genome that included several bases on either side of the point mutation, which were referred to as the 614G sequence (FIG. 5 A), and a complementary DNA strand with amine linkers on either end was used as a probe molecule.
• the probe was hybridized with the perfectly matched 614G target and linked to mCNTs following the procedures outlined above. This structure was incubated on a microelectrode chip for 30 minutes before probing the chip and identifying a bridged gap. The conductance was stable at ⁇ 6x 1 O' 6 Go (FIG.
• DNA-based systems can be designed with mismatches or non-canonical bases that will bind metal ions and integrated into the CNT-molecule-CNT structures.
• Ag FIG. 6 and FIG. 7A
• Hg FIG. 7B
• a T-T mismatch can bind an Ag + ion (FIG. 6A), which yields a conductance value near 7xlO' 4 Go (FIG. 6B, 7A), and nonlinear IV curves (FIG. 6C).
• a pH of 8.0 the system preferentially binds to a Hg ion (FIG. 7B).
• Scorpionates are molecules that use 3 -donor site ligands (tridentate system) to bind a central atom.
• a molecule was used that has a central iron (Fe) atom.
• the shape and binding of the ligands controls the spin-state of the Fe ion. It can have either a high net spin or a low net spin depending on the electron filling of the atomic orbitals.
• the transition between high and low spin states can be controlled by the types of ligands, the temperature, and external parameters such as the applied electric field, current passing through the device, or a magnetic field.
• FIG. 12 shows several example junctions generated using these small-molecule, spincrossover molecules.
• a junction was created using a scorpionate derivative that forms a spin-crossover molecule with a high-spin and low-spin state, which can be controlled via temperature, and/or via electrical means. This derivative is predominantly in a high-spin state at room temperature and low-bias.
• a junction was created using a scorpionate derivative that is predominantly in a low-spin state at room temperature.
• These molecules can be used to generate a wide range of an electronic devices, including but not limited to, a switch, a transistor, a memory device, a spin filter, a spin polarizer, and a spin-controlled switch.
• Norbomadienes are photo chemically active molecules that switch to the quadricyclane (QC) form when irradiated with the proper wavelength of light (UV range). These molecules can also be electrically switched using the single-molecule break junction technique, and operate as memristor.
• a CNT-NB-CNT molecular junction was generated, using a similar process as described for the scorpionate molecules.
• the CNTs were wrapped with DNA, and then diluted in a water/acetone solution at a concentration that would allow the norbomadiene derivatives to dissolve in solution. After this, the amidation reaction between the CNTs and the amino endgroups on the NB derivative was activated using HATU as described above.
• the molecule can be both electrically and optically switched in the CNT junctions thus opening opportunities for this device to operate as an optoelectronic device such as a switch photocell, light-capture device, or optical receiver, or to operate as an electrical component such as a switch, memory, or memristor for applications in computing, neuromorphic computing, or communications.
• an optoelectronic device such as a switch photocell, light-capture device, or optical receiver
• an electrical component such as a switch, memory, or memristor for applications in computing, neuromorphic computing, or communications.
• the self-aligning molecular junction generated using norbomadienene (NB) derivatives were electrically and optically tested and controlled.
• NB and NB derivatives can switch to quadricyclanes under certain conditions of light irradiation or applied bias/current flow. Examples of device switching is shown in FIG. 13B; the device is irradiated with UV light, causing a switch to the QC form and a change in the resistance, as is evidenced by the IV traces in the lower panel.
• These devices hold promise as electronic switches, photodetectors, memories, etc.
• the results of FIG. 14 demonstrate that the same molecular junction can operate as a voltage-controlled switch, changing conductance values with the applied square-wave potential. This demonstrates the utility of the self-aligning junctions of the present disclosure as a switch/memory device.
• a self-aligning molecular junction comprising: a first single-walled carbon nanotube (CNT) and a second CNT, wherein each of the first and second CNTs comprise a coating; and a single-molecule bridge functionally coupled to the first and second CNTs, thereby forming a single-molecule junction.
• CNT single-walled carbon nanotube
• Clause 2 The junction of clause 1, wherein the single-molecule junction is soluble in solution.
• Clause 3 The junction of clause 1 or clause 2, wherein the solution is an aqueous solution.
• Clause 4 The junction of any one of clauses 1 to 3, wherein the solution comprises a polar organic solvent.
• Clause 5 The junction of any one of clauses 1 to 4, wherein the solution comprises a non-polar organic solvent.
• Clause 6 The junction of any one of clauses 1 to 5, wherein the solution comprises a soluble coupling agent.
• Clause 7 The junction of any one of clauses 1 to 6, wherein the soluble coupling agent comprises O-(7-azabenzotriazole-l-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate (HATU).
• Clause 8 The junction of any one of clauses 1 to 7, wherein the single-molecule bridge is functionally coupled to at least a third CNT.
• Clause 9 The junction of any one of clauses 1 to 8, wherein the coating comprises a polynucleotide.
• Clause 10 The junction of any one of clauses 1 to 9, wherein the polynucleotide is a single-stranded polynucleotide selected from DNA, RNA, PNA or any combinations or derivatives thereof.
• Clause 11 The junction of any one of clauses 1 to 10, wherein the single-stranded polynucleotide is a single-stranded DNA comprising guanine-thymine repeats.
• Clause 12 The junction of any one of clauses 1 to 11, wherein the single-molecule bridge comprises an organic molecule.
• Clause 13 The junction of any one of clauses 1 to 12, wherein the organic molecule comprises: a polynucleotide or a derivative or variant thereof; a coronene or a derivative or variant thereof; a norbornadiene or a derivative or variant thereof; a tetraphenyl or a derivative of variant thereof; and a scorpionate or a derivative or variant thereof.
• Clause 14 The junction of any one of clauses 1 to 13, wherein the polynucleotide comprises DNA, RNA, PNA, or any combinations or derivatives thereof.
• Clause 15 The junction of any one of clauses 1 to 14, wherein the polynucleotide is covalently linked to at least one of the first and/or second CNT.
• Clause 16 The junction of any one of clauses 1 to 15, wherein the single-molecule bridge comprises DNA origami.
• Clause 17 The junction of any one of clauses 1 to 16, wherein the single-molecule bridge comprises a peptide, polypeptide, or protein.
• Clause 18 The junction of any one of clauses 1 to 17, wherein the peptide, polypeptide, or protein comprises biotin-streptavidin.
• Clause 19 The junction of any one of clauses 1 to 18, wherein the single-molecule bridge comprises a scorpionate.
• Clause 20 The junction of any one of clauses 1 to 19, wherein the scorpionate comprises a PRRR053 scorpionate and/or a PRRR057 scorpionate.
• Clause 21 The junction of any one of clauses 1 to 20, wherein the single-molecule bridge comprises a photochemically active molecule, an electrically active molecule, an optically active molecule, and/or a magnetically active molecule.
• Clause 22 The junction of any one of clauses 1 to 21, wherein the photochemically active molecule comprises a norbomadiene or a derivative or variant thereof.
• Clause 23 The junction of any one of clauses 1 to 22, wherein the single-molecule bridge is from about 0.50 nm to about 250 nm in length.
• Clause 24 The junction of any one of clauses 1 to 23, wherein the single-molecule bridge is from about 250 nm to about 1 pm in length.
• Clause 25 The junction of any one of clauses 1 to 24, wherein the single-molecule bridge is less than about 600 nm in length.
• Clause 26 The junction of any one of clauses 1 to 25, wherein at least one of the first and/or second CNT is functionalized with one or more linkers.
• Clause 27 The junction of any one of clauses 1 to 26, wherein the one or more linkers are configured to functionally couple at least one of the first and/or second CNT to the singlemolecule bridge.
• Clause 28 The junction of any one of clauses 1 to 27, wherein the one or more linkers comprise biotin and/or streptavidin.
• Clause 29 A single-molecule electronic biosensor comprising at least one circuit comprising the junction of any one of clauses 1 to 28, wherein the first and the second CNTs are functionally coupled to pairs of micro fabricated electrodes, thereby forming the at least one circuit.
• Clause 30 A method of detecting a target nucleic acid, the method comprising: introducing a sample comprising or suspected of comprising a target nucleic acid to the singlemolecule electronic biosensor of clause 29, wherein the single-molecule bridge is a DNA molecule comprising a sequence that is at least partially complementary to at least a portion of the target nucleic acid; and detecting the presence of the target nucleic acid in the sample when the target nucleic acid binds to the complementary strand of the DNA molecule.
• Clause 31 The method of clause 30, wherein detecting the presence of the target nucleic acid comprises detecting a change in conductance and/or current.
• Clause 32 An aqueous solution comprising: a plurality of single-walled carbon nanotube (CNTs), each coated with a single-stranded polynucleotide; and a plurality of single- molecule bridges of varying lengths, each configured to bind at least one of the plurality of CNTs; wherein incubating the plurality of CNTs with the plurality of single-molecule bridges produces a plurality of self-aligned, single-molecule junctions.
• CNTs single-walled carbon nanotube
• Clause 33 A method of generating a plurality of self-aligned, single-molecule junctions comprising incubating the aqueous solution of clause 32 with a coupling agent, wherein the coupling agent facilitates binding of a single-molecule bridge and two CNTs, thereby producing the plurality of self-aligned, single-molecule junctions.
• Clause 34 The method of clause 33, wherein the method further comprises modifying pairs of microfabricated electrodes to bind each of the plurality of self-aligned, single-molecule junctions, thereby forming a single-molecule electronic biosensor.
• Clause 35 The method of clause 33 or clause 34, wherein the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least 1%.

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