EP4649792A2 - Self-aligning molecular junctions for electronic biosensors - Google Patents

Abstract

The present disclosure provides devices, systems, and methods related to electronic biosensors. In particular, 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.

Description

SELF-ALIGNING MOLECULAR JUNCTIONS FOR ELECTRONIC BIOSENSORS

GOVERNMENT FUNDING

[0001] This invention was made with government support under 2036865 and 1807555 awarded by the National Science Foundation. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/479,881 filed January 13, 2023, which is incorporated herein by reference in its entirety and for all purposes.

FIELD

[0003] The present disclosure provides devices, systems, and methods related to electronic biosensors. In particular, 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.

BACKGROUND

[0004] The development of self-aligning transistors, where the gate electrode is used as a mask layer to pattern the source and drain, was a key breakthrough that greatly improved the manufacturing yield of semiconductor devices and enabled the continued scaling of electronic systems over many device generations. Now, in recent decades a variety of novel low-dimensional materials have been developed with unique electronic properties including carbon nanotubes (CNTs), semiconductor nanowires, graphene and other 2D materials, and even molecular electronic platforms. However, despite the promise of these materials, the ability to manufacture these systems at scale for useful applications, and to integrate them with larger-scale conventional lithography processes has remained difficult. This issue becomes increasingly prominent as the size-scale decreases to nanometer and molecular levels. For example, while an incredible variety of novel, molecularly-enabled electronic functions have been demonstrated, including quantum interference, neuromorphic and memristive activity, and optoelectronic control, the utility of these devices has been constrained to understanding physical and chemical processes at the nanoscale due to the difficulties with larger-scale integration. To realize the full utility of these systems they must be incorporated into stable, robust, and reliable electronic systems.

SUMMARY

[0005] Embodiments of the present disclosure include a self-aligning molecular junction. In accordance with these embodiments, the junction includes a first single-walled carbon nanotube (CNT) and a second CNT. In some embodiments, 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.

[0006] In some embodiments, the single-molecule junction is soluble in solution. In some embodiments, the solution is an aqueous solution. In some embodiments, the solution comprises a polar organic solvent. In some embodiments, the solution comprises a non-polar organic solvent. In some embodiments, the solution comprises a soluble coupling agent.

[0007] In some embodiments, the soluble coupling agent comprises O-(7-azabenzotriazole-l- yl)-N,N,N,N'-tctramcthyluronium hexafluorophosphate (HATU).

[0008] In some embodiments, the single-molecule bridge is functionally coupled to at least a third CNT.

[0009] In some embodiments, the coating comprises a polynucleotide. In some embodiments, the polynucleotide is a single-stranded polynucleotide selected from DNA, RNA, PNA or any combinations or derivatives thereof. In some embodiments, the single-stranded polynucleotide is a single-stranded DNA comprising guanine-thymine repeats.

[0010] In some embodiments, the single-molecule bridge comprises an organic molecule. In some embodiments, 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.

[0011] In some embodiments, 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.

[0012] In some embodiments, the single-molecule bridge comprises DNA origami. In some embodiments, the single-molecule bridge comprises an aptamer. [0013] In some embodiments, 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).

[0014] In some embodiments, the single-molecule bridge comprises a peptide, polypeptide, or protein. In some embodiments, the peptide, polypeptide, or protein comprises biotin-streptavidin. In some embodiments, the protein comprises a polymerase, or a derivative or variant thereof. In some embodiments, the single-molecule bridge comprises a scorpionate. In some embodiments, the scorpionate comprises a pair of tri-dentate molecules and a metal ion ligated between the pair of tri-dentate molecules.

[0015] In some embodiments, the single-molecule bridge comprises a photochemically active molecule. In some embodiments, the photochemically active molecule comprises a norbornadiene or a derivative or variant thereof. In some embodiments, the single-molecule bridge comprises an electrically active molecule. In some embodiments, the single-molecule bridge comprises an optically active molecule. In some embodiments, the single-molecule bridge comprises a magnetically active molecule.

[0016] In some embodiments, 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.

[0017] In some embodiments, at least one of the first and/or second CNT is functionalized with one or more linkers. In some embodiments, 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. In some embodiments, the one or more linkers comprise biotin and/or streptavidin.

[0018] 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. In some embodiments, the first and the second CNTs are functionally coupled to pairs of microfabricated electrodes, thereby forming the at least one circuit.

[0019] Embodiments of the present disclosure also include a method of detecting a target nucleic acid. In accordance with these embodiments, 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. In some embodiments, 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.

[0020] In some embodiments, detecting the presence of the target nucleic acid comprises detecting a change in conductance and/or current.

[0021] 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. In some embodiments, incubating the plurality of CNTs with the plurality of single-molecule bridges produces a plurality of self-aligned, single-molecule junctions.

[0022] Embodiments of the present disclosure also include a method of generating a plurality of self-aligned, single-molecule junctions. In accordance with these embodiments, the method includes incubating any of the aqueous solutions described herein with a coupling agent. In some embodiments, the coupling agent facilitates binding of a single-molecule bridge and two CNTs, thereby producing the plurality of self-aligned, single-molecule junctions.

[0023] In some embodiments, 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.

[0024] In some embodiments, wherein the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least 1%.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIGS. 1A-1D: Development of mCNT-DNA-mCNT junctions. A) Reaction scheme for assembling the self-aligning CNT-DNA-CNT junctions and integrating them into the electronic circuit. 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. B) Fluorescence micrograph of CNT-DNA-CNT structures. C) Atomic force microscope image of CNT-DNA-CNT structures on a mica surface. D) Cryo-EM micrograph of a CNT-DNA- CNT junction with a ~6 nm gap structure.

[0026] 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 (R²= 0.989, red line) indicating ohmic transport. B) Current through the device shown in (A) over a 400 s time period. C) Aggregated data from 58 (300 nm) gap devices with the 12 bp sequence. There are 3 distinctive regimes: high conductance (> 10'¹ Go, yellow), medium conductance (lxl0'^4±1 Go, pink), and low conductance (< 10'⁶ Go, gray). 45% of the devices have a conductance value in the pink region representing single-molecule CNT-DNA- CNT junctions. D) SEM image of one of the devices in the pink region of (c) showing a single bridge between the electrodes. E) Plot of all devices with a conductance in the range of lxl0'^4±1 Go for all 4 G:C triplet sequences studied from 9-18 bp, and their average values (dashed lines). F) Conductance of the G:C triplet sequences vs. 1/L. Error bars are standard deviations calculated from all measurements in plot (E). G) Series of alternating G:C triplet DNA sequences studied.

[0027] FIG. 3: Device Stability. Resistance of a single CNT-DNA-CNT junction measured over the course of 7 days to demonstrate device stability.

[0028] FIGS. 4A-4B: Measurement of Strand Dehybridization. A) A:T Rich DNA sequence measured. B) If the two strands are dehybridized, then only a single-stranded DNA will bridge between the two CNT electrodes. In this case, the current is extremely low (single-strand has very high resistance). If the complementary strand is present in solution one of them will eventually rehybridize with the bound strand and the circuit will be completed causing an increase in the current. These measurements were done in solution phase. C) When heated the two strands will dehybridize resulting in the device returning to the single-stranded DNA conductance value.

[0029] FIGS. 5A-5C: Electronic oligonucleotide detection using a single-molecule biosensor. A) Schematic representation of the device with the 614G probe and target sequences (614G and 614D). B) Conductance as a function of time for a single mCNT-614G Probe-mCNT junction, with schematics above each phase indicating the process. 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. C) Similar procedure as in b) on a second device, but with sample injection order reversed (first mismatched 614D followed by perfectly matched 614G). Mismatched DNA does not rehybridize in the junction in either case, but the matched 614G sequence does.

[0030] 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). C) Examples of I-V curves demonstrating a clear non-linearity that was not observed with non- metalated DNA, suggesting the possibility of coloumb blockade, and the potential to create singleelectron transistor structures from such devices.

[0031] FIGS. 7A-7B: Examples of metalated DNA Devices. A) Devices with Ag+ ions and associated device yield (see also FIG. 6B). B) same sequence at a different pH binds Hg²⁺ ions and associated device conductance values. This suggests that devices can be used as ion-sensitive electrodes, for heavy-metal detection, and other sensing motifs.

[0032] 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.

[0033] FIG. 9: DNA Origami Devices. Example of IV characteristic from an exemplary DNA origami device, and associated yield/conductance values for the 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.).

[0034] FIGS. 10A-10B: CNT-Protein-CNT Devices. A) Schematic of CNT-protein-CNT device consisting of CNTs functionalized with biotin linkers and then bound to a streptavidin central unit. B) I-V characteristic associated with one CNT-Protein-CNT Device.

[0035] FIGS. 11A-11C: Small-Molecule Junctions - Spin-crossover molecules. A) Scorpionate derivative that forms a spin-crossover molecule with a high-spin and low-spin state that can be controlled via temperature, or potentially, electrically controlled. This derivative is predominantly in a high-spin state at room temperature and low-bias. B) Scorpionate derivative that is predominantly in a low-spin state at room temperature. C) Current- Voltage (I-V) characteristics for junctions from the two molecules. The low-spin system shows some spinswitching behavior.

[0036] FIG. 12: Spin Crossover Molecules. Conductance statistics for the two spin-crossover molecules shown in FIG. 11.

[0037] FIGS. 13A-13B: Optoelectronic Switches. A) Structure and functioning of Norbornadienene (NB) derivatives and how they can switch to quadricyclanes under certain conditions of light irradiation or applied bias/ current flow. B) Examples of device switching as the triangle wave on top is applied, 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.

[0038] 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.

DETAILED DESCRIPTION

[0039] Embodiments of the present disclosure include devices, systems, and methods related to electronic biosensors. In particular, 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. As described further herein, 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. This approach provides a basis for developing reliable methods for integrating single-molecule circuits with conventional electronic systems to allow the unique functionality of molecular systems to be harnessed at scale.

[0040] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. 1. Definitions

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0042] As noted herein, the disclosed embodiments have been presented for illustrative purposes only and are not limiting. Other embodiments are possible and are covered by the disclosure, which will be apparent from the teachings contained herein. Thus, the breadth and scope of the disclosure should not be limited by any of the above-described embodiments but should be defined only in accordance with claims supported by the present disclosure and their equivalents. Moreover, 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. Moreover, 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. In addition, 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). Furthermore, 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).

[0043] When describing the molecular detecting methods, systems and devices, terms such as linked, bound, connect, attach, interact, and so forth should be understood as referring to linkages that result in the joining of the elements being referred to, whether such joining is permanent or potentially reversible. These terms should not be read as requiring a specific bond type except as expressly stated.

[0044] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0045] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, 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.

[0046] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of’ or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of’ “only one of’ or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0047] As used herein in the specification and in the claims, 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. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or 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.

[0048] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0049] As used herein, 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. In some cases, the resultant combination of the two or more molecules maintains one or more functional properties of one or more of the uncoupled molecules. In other cases, the resultant combination yields a functional property not previously present in the individual, uncoupled molecules. In some cases, “functional coupling” includes the binding, joining, or combining of two or more molecules via chemical means (e.g., covalent or non-covalent interaction). As described further herein, in some cases, the “functional coupling” of two or more molecules results in the formation of a junction having certain electrical and/or biochemical properties.

[0050] As used herein, the term “covalent bond” generally refers to a stable association between two atoms which share one or more electrons. Examples of the covalent bonds include, without limitation, a peptide bond and a disulfide bond. “Peptide bond” as used herein refers to the covalent bond formed between the carboxyl group of an amino acid and the amine group of the adjacent amino acid.

[0051] As used herein, the term “non-covalent bond” generally refers to an attractive interaction between two molecules or two chemical groups that does not involve sharing of electrons. Examples of 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.

[0052] As used here, the term “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.

2. Devices, Systems, and Methods

[0053] Fabricating and integrating nanometer-scale devices with external circuits has been an ongoing challenge in nanoscience and nanotechnology since its inception. As described further herein, a combination of bottom-up and top-down integration techniques were used to create selfaligning single -molecule circuits. This approach covalently binds metallic single-walled carbon nano tubes (CNTs) to single DNA duplexes in solution to act as electrodes and create singlemolecule CNT-DNA-CNT junctions. These nanoscale hybrid devices are then integrated with lithographically fabricated microelectrodes to create stable single-molecule circuits with a yield of at least 1%. The electronic properties of these circuits are dominated by the DNA duplex within junction, have a small conductance dispersion, and enable the development of dynamic detection specific of oligonucleotides sequences with high specificity at the single-molecule level.

[0054] As described further herein, the single-molecule junctions and corresponding circuits and devices of the present disclosure can be generated using a CNT-molecule-CNT structure. For example, 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.). In some embodiments, reactions can be performed without wrapping; however, this usually produces lower yields.

[0055] In some embodiments, 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).

[0056] 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.

[0057] As described further herein, the various embodiments of the present disclosure have been used to develop and study many different types of molecular junctions and circuits. For example, 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). In some embodiments, 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. In other embodiments, 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. This demonstrates that 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.).

[0058] As would be recognized by one of ordinary skill in the art based on the present disclosure, 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.). In addition, and as described further herein, 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).

[0059] In accordance with the above, embodiments of the present disclosure include a selfaligned molecular junction. In some embodiments, the junction includes a first single-walled carbon nanotube (CNT) and a second CNT. In some embodiments, each of the first and second CNTs comprise a coating. In some embodiments, the self-aligned molecular junction includes a single-molecule bridge functionally coupled to the first and second CNTs, thereby forming a single-molecule junction. In some embodiments, 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).

[0060] In some embodiments, the single-molecule junction is soluble in solution. In some embodiments, the solution is an aqueous solution. In some embodiments, the solution comprises a polar organic solvent. In some embodiments, the solution comprises a non-polar organic solvent. In some embodiments, the solution comprises a soluble coupling agent. In some embodiments, the soluble coupling agent comprises O-(7-azabenzotriazole-l-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate (HATU). In accordance with these embodiments, the solutions and/or coupling agents of the present disclosure facilitate and/or enhance the functional coupling of the single-molecule bridge to at least one of the first and/or second CNT.

[0061] In some embodiments, 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).

[0062] In some embodiments, the coating for the CNTs of the present disclosure comprises a biopolymer, including but not limited to, a polynucleotide. In some embodiments, the polynucleotide is a single-stranded polynucleotide selected from DNA, RNA, PNA or any combinations or derivatives thereof. In some embodiments, the single-stranded polynucleotide is a single-stranded DNA comprising guanine-thymine repeats (e.g., (GT)_n DNA). In some embodiments, 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). In other embodiments, the coating comprises another material, such as a surfactant or polymer. And in cases where a self-aligned, single-molecule junction is assembled in non-aqueous solutions (e.g., organics, ionic liquids, etc.), semiconducting CNTs can be used.

[0063] In some embodiments, the self-aligned, single-molecule junction includes a singlemolecule bridge covalently linked to the first and second CNTs. In some embodiments, the singlemolecule bridge is an organic molecule. In some embodiments, 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. In some embodiments, the polynucleotide is DNA, RNA, PNA, or any combinations or derivatives thereof. In some embodiments, click chemistry is used to covalently link the polynucleotide to the first and second CNTs. In some embodiments, the polynucleotide is covalently linked to the first and second CNTs (e.g., via an amidation reaction). In some embodiments, the single-molecule bridge comprises DNA origami. [0064] In some embodiments, the single-molecule bridge comprises a peptide, polypeptide, or protein. In some embodiments, the peptide, polypeptide, or protein comprises biotin-streptavidin. In some embodiments, the protein comprises a polymerase, or a derivative or variant thereof. In some embodiments, the single-molecule bridge comprises a scorpionate. In some embodiments, the scorpionate comprises a pair of tri-dentate molecules and a metal ion ligated between the pair of tri-dentate molecules. In some embodiments, the single-molecule bridge comprises a photochemically active molecule. In some embodiments, the photochemically active molecule comprises a norbornadiene or a derivative or variant thereof. As would be appreciated by one of ordinary skill in the art based on the present disclosure, other examples of peptide, polypeptide, proteins, scorpionates, and photochemically active small molecules can use used in the selfaligning molecular junctions described herein. In some embodiments, the single-molecule bridge comprises an electrically active molecule. In some embodiments, the single-molecule bridge comprises an optically active molecule. In some embodiments, the single-molecule bridge comprises a magnetically active molecule.

[0065] In some embodiments, 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. In some embodiments, 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.

[0066] 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 from about 250 nm to about 900 nm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about

800 nm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about

700 nm in length. In some embodiments, 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

300 nm in length. In some embodiments, 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

1 pm in length. In some embodiments, the single-molecule bridge is from about 500 nm to about

1 pm in length. In some embodiments, the single-molecule bridge is from about 600 nm to about

1 pm in length. In some embodiments, the single-molecule bridge is from about 700 nm to about

1 pm in length. In some embodiments, the single-molecule bridge is from about 800 nm to about

1 pm in length. In some embodiments, the single-molecule bridge is from about 900 nm to about

1 pm in length. In some embodiments, the single-molecule bridge is from about 250 nm to about

750 nm in length. In some embodiments, the single-molecule bridge is from about 400 nm to about

600 nm in length.

[0067] In some embodiments, the single-molecule bridge is less than about 600 nm in length.

In some embodiments, 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.

[0068] In some embodiments, 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. In some embodiments, at least one of the first and/or second CNT is functionalized with one or more linkers. In some embodiments, 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. In some embodiments, the one or more linkers comprise biotin and/or streptavidin.

[0069] Embodiments of the present disclosure also include a single-molecule electronic biosensor comprising at least one circuit comprising any of the junctions described herein. In some embodiments, the first and the second CNTs are coupled to pairs of micro fabricated electrodes, thereby forming at least one circuit. In some embodiments, a plurality of CNTs are coupled to pluralities of microfabricated electrodes to form a plurality of circuits.

[0070] In one exemplary embodiment, the present disclosure provides a method of detecting a target nucleic acid using the self-aligned, single-molecule circuits and biosensors described herein. In accordance with these embodiments, the method includes introducing a sample comprising or suspected of comprising a target nucleic acid to a single-molecule electronic biosensors. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0071] Embodiments of the present disclosure also include an aqueous solution comprising a plurality of metallic, single-walled carbon nanotubes (CNTs). In some embodiments, each CNT is coated with a single-stranded polynucleotide. In some embodiments, a plurality of single -molecule bridges of varying lengths are each configured to form covalent bonds with two or more CNTs. In some embodiments, incubating the plurality of CNTs with the plurality of single-molecule bridges produces a plurality of self-aligned, single-molecule junctions.

[0072] 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. In some embodiments, the coupling agent facilitates covalent binding between a single-molecule bridge and two CNTs, thereby producing the plurality of self-aligned, single-molecule junctions. [0073] In some embodiments, 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.

[0074] In some embodiments, 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%. In some embodiments, 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%.

[0075] In some embodiments, 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%. In some embodiments, 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%. In some embodiments, 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%.

3. Materials and Methods

[0076] Preparation ofMCNT-aptamer nanostructures. Metallic single wall carbon nanotubes (mCNTs) were purchased from Nano Integris, Inc. Phosphate buffer solution (PBS) and O-(7- azabenzotriazole-l-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate (HATU) were purchased from Thermo Scientific. All other analytical-grade chemicals were purchased from Sigma Aldrich. All DNAs were obtained from Alpha DNA.

[0077] The final sequence of single-stranded DNA (ssDNA) used to wrap MCNTs is 5 ’-(GT)2o- 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.

[0078] Alternating G:C triplet series sequences:

[0079] 5’-amino-(CH2)6-GGG CCC GGG-(CH2)₆-amino-3 ’ (SEQ ID NO: 1);

[0080] 5’-amino-(CH2)6-GGG CCC GGG CCC-(CH2)6-amino-3’ (SEQ ID NO: 2);

[0081] 5’-amino-(CH2)6-GGG CCC GGG CCC GGG-(CH2)₆-amino-3 ’ (SEQ ID NO: 3); and [0082] 5’-amino-(CH2)6-GGG CCC GGG CCC GGG CCC-(CH2)6-amino-3’ (SEQ ID NO: 4). [0083] The sequence of the DNA complementary to (G3:C3)n series:

[0084] 3 ’-CCC GGG CCC-5’ (SEQ ID NO: 5);

[0085] 3 ’-CCC GGG CCC GGG-5’(SEQ ID NO: 6);

[0086] 3 ’-CCC GGG CCC GGG CCC-5’ (SEQ ID NO: 7); and

[0087] 3 ’-CCC GGG CCC GGG CCC GGG-5’ (SEQ ID NO: 8).

[0088] SARS-CoV-2 sequences: [0089] 5’-amino-(CH₂)₆-ATA GTC CCA CAA TTG ACG-(CH₂)6-amino-3’ Probe (SEQ ID NO: 9);

[0090] 3’-TAT CAG GGT GTT AAC TGC-5’ 614G (SEQ ID NO: 10); and

[0091] 3’-TAT CAG GAT GTT AAC TGC-5’ D614G (SEQ ID NO: 11).

[0092] 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)₂o 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)₂o-dispersed mCNT solutions was kept to 2mL.

[0093] To covalently attach oligonucleotides to (GT)₂o wrapped mCNTs, amino -terminated dsDNA was prepared at a concentration of 5 ./M in 2mL of PBS buffer and mixed with 2 mL (GT)₂o 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.

[0094] Single-molecule device fabrication. The microelectrode platform used to electrically characterize the nanostructures was fabricated using optical lithography. In brief, 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. Then microelectrodes were patterned on the wafer using photolithography with KL5302 Hi-Res Photoresist (Kem Lab). After ~15 s development in CD-26 (TMAH), the wafer was gently rinsed with DI water and blow-dried with nitrogen to stop the development process. Then 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 MICROPOSIT™ Remover 1165 for 90 minutes, and ultrasonicated to lift off residual resist. After that, lOOnm Si₂N4 insulating layer the was deposited using PECVD. Micron-sized windows for the probing and sensor areas were patterned on using the same photolithography process. Finally, a -100 nm etch was added into the PECVD-cover layer using the Plasma-Therma RIE to open the desired windows. [0095] 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.

[0096] 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.

[0097] Electrical Characterization. All the device characterizations were performed by using a Cascade probe station equipped with the semiconducting parameter analyzer (Keithley 4200- SCS) at room temperature with a consistent moisture content (30%). For thermal cycling measurements, a PID-controlled heater stage (±0.01 °C) was employed to adjust the temperature (20°C-100°C) at a constant rate. Sample surface temperatures were monitored via infrared thermometer (Fluke-561).

[0098] Current sampling measurement was conducted with 0.5V bias, and at least 3 minutes of monitoring for each electrode. Voltammetry scans (-0.5V~0.5V) were conducted simultaneously. [0099] DNA Nanostructures. An exemplary DNA origami structure for use in the devices described further herein was developed (FIG. 8 and FIG. 9). Six DNA strands were coupled together to form a 4-helix bundle of DNA that is ~30 basepairs in length. The nucleic acid sequences of each of the six strands are provided below:

[0100] ssDNA 1: NH₂-C₆Hi2-ACG GCC GCG GCA GCA CGC GCG CGG CCC TGC GG- H12C6-NH2 (SEQ ID NO: 12);

[0101] 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); [0102] 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);

[0103] ssDNA 4: CTG GCT GCG CCG CAA CCC TGG CGG CAC GCT CA (SEQ ID NO:

15);

[0104] ssDNA 5: CCG GCC GTG CTC GAC CTC CTC TGG GAT TGT GG (SEQ ID NO:

16); and

[0105] ssDNA 6: GCT CGA GCG ATC CTG GCG CCA GTC TGC CAG CC (SEQ ID NO:

17).

[0106] These sequences were placed in a phosphate buffer solution with lOmM MgCl, and heated to 95°C and cooled to room temperature slowly to form the DNA nanostructure.

4. Examples

[0107] The present disclosure provides devices, systems, and methods related to electronic biosensors. In particular, 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.

[0108] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

[0109] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

Example 1

[0110] To create robust, self-aligned single-molecule junctions metallic, single-walled carbon nanotubes (mCNTs) were used to create mCNT-molecule-mCNT nanohybrids in solution phase. This step ensures that the gap between the two mCNTs is atomically matched to the length of the molecular system. Such a feat is exceedingly difficult with conventional lithography, but by harnessing the inherent strengths of molecular systems, this solution-phase chemical approach allows facile junction self-alignment. To create these structures, mCNTs were used to improve the reproducibility and decrease the dispersion of the conductance properties, and DNA was selected as an initial molecular target due to its extensively studied electronic properties, and its potential as a biosensing element. After these mCNT-DNA-mCNT nanohybrid junctions were formed and characterized, they were integrated with a microelectrode platform that was fabricated using conventional photolithographic approaches. The conductance of the system was systematically examined with the DNA length (9-18 base pairs, bps) to inspect the self-alignment capabilities and the measured resistance by the DNA within the nanohybrid. To verify the single-molecule nature of the system and demonstrate the utility of this approach for electronic applications, the system was used for label-free detection of short COVID-relevant sequences.

[01H] To create the mCNT-DNA-mCNT hybrids, commercially available mCNTs were used that are soluble in aqueous solutions. To protect the mCNT sidewalls during the impending chemical processes, they are coated with single-stranded (GT)2o DNA (FIG. 1 A). This sequence has previously been demonstrated to bind strongly to DNA through noncovalent interactions and ensures the continued solubility of the mCNTs in aqueous phases. After this step, the DNA wrapped-mCNTs are incubated in the presence of synthetic DNA duplexes that are terminated on the 5’ ends with an amine group (-(CFhje-NFh). By adding 0.13 mM Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU) to this solution during sonication (30 mins), an amidation reaction was initiated between the terminal amine groups of DNA and the exposed carboxylic acid groups on the ends of the mCNTs.

Example 2

[0112] To examine the morphological properties of these potential mCNT-DNA-mCNT structures, fluorescence microscopy was used. By incubating the samples with ethidium bromide (EtBr) and comparing the resulting fluorescence images (FIG. IB) with control experiments, it was found that the ssDNA wrapping process of mCNTs proceeds successfully. Moreover, it was noted that many of the structures visualized in the images have either single or multiple bends in the backbone, indicative of a linked structure with differing persistence lengths. These results were further corroborated by atomic force microscopy (AFM) images where V-shaped features commonly appear (FIG. 1C). The AFM images reveal that the resulting structures are typically on the order of 250-350 nm and that there are often locations within the structures that possess a smaller cross-section than the rest of the structures (0.4 nm vs. 1 nm). However, it was challenging to determine whether these features are due to the presence of single-molecules between two mCNTs or due to defects in the single-stranded DNA wrapping process. Thus, to further investigate this possibility, cryogenic transmission electron microscopy (cryo-EM) was used to examine the structures. In the cryo-EM images, it was possible to discern specific cases where gaps appear between mCNT structures (FIG. ID), which is again indicative of the formation of mCNT-DNA-mCNTs. Taken together these characterization approaches consistently demonstrated molecularly aligned mCNT-DNA-mCNT structures were formed using the above procedure.

[0113] Given the difficulty in the single-molecule level characterization of the structures using the conventional approaches above, electrical characterization techniques were used to examine the formation of the targeted structures. The electrical properties of a 12 bp sequence with alternating G:C triplets (5’-GGG CCC GGG CCC-3’) with amine linkers on each end was first examined. To create the circuits for electrical characterization, a microfabricated substrate containing 40 electrode pairs with 300 nm gaps was incubated in the mCNT-DNA-mCNT solution. 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. For bias voltages swept over a ±0.5 V range, the I-V curves of this 12 bp device are nearly linear (R² from fitting = 0.989) indicating ohmic transport behavior. And, as can be seen by the reproducibility of the I-V characteristics and the time series measurements on these devices (FIG. 2B), the structures and conductance values were very stable over time. To examine the reproducibility of the measured conductance values, 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. These electrode pairs are either short-circuited during fabrication or directly bridged by mCNTs or bundled structures. 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).

Example 3

|0114] Upon verifying that the measured conductance values stem from individual bridges between the electrodes, the next step was to determine whether these conductance values are determined by mCNT-DNA-mCNT-based single-molecule junctions. To do so, advantage was taken of the self-alignment capabilities of this approach, and the conductance of these alternating G:C triplet sequences with lengths of 9, 12, 15, and 18 base pairs was systematically measured (FIG. 2G). As seen in FIG. 2E, the conductance of each of these molecules fell in a narrow region around 1x1 O'⁴ Go, which is consistent with a number of measurements on GC-rich DNA using Au electrodes. Moreover, it was also apparent that there is a systematic decrease in the conductance with increasing length (L), and it was found that this change in conductance is directly proportional to 1/L (FIG. 2F). This linear dependence of the conductance on length is indicative of a hoppingbased transport mechanism, which has been widely attributed to GC rich sequences. If the DNA was not playing a role in the conductance of these systems, no length dependence of the conductance would be expected because the nominal gap size is 300 nm in all cases. Thus, these measurements provided a clear indication that the circuits are derived from single-molecule junction. Altogether, from the four molecules, 197 300 nm gap were measured devices and an overall yield of 44.6% (88 bridged gaps) was obtained.

Example 4

[0115] To fully demonstrate both the single-molecule nature of these circuits and their potential utility as active electronic components, the system was utilized as an electronic, single-molecule biosensor (see FIG. 4 and FIG. 5 for distinct examples with different DNA sequences). For the experiments in FIG. 4, a generalized, Adenine: Thymine rich duplex was used as a test case. The experiments in FIG. 5 were conducted to test whether the presence of a specific oligonucleotide sequence related to the SARS-CoV-2 virus can be detected. A sequence related to the D614G mutation (aspartic acid swapped for glycine at amino acid residue 614 on the spike protein) was selected for detection. 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.

[0116] For the detection process, 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. 5B, Phase 2), then to decrease the possibility of additional mCNT-DNA-mCNT junctions bridging the electrodes during the sensing process, the CNT solution was replaced with 20 pM 614G targets in PBS; the conductance remained close to the original value during this buffer exchange process (FIG. 5B, Phase 3). To complete the sensor preparation, the system was heated at a rate of 0.5°C/s to dehybridize the duplex within the junction. When the temperature reached ~65°C, which corresponded to the calculated melting temperature for this sequence (T_m = 64.4°C), the conductance suddenly dropped to the resolution of the current amplifier, indicating a breakdown of the device (FIG. 5B, Phase 4). With the conductance at effectively zero, experiments were conducted to detect the target sequence in the solution. To do so, the temperature of the cell was decreased back toward room temperature, and within several minutes of cooling, there was a sharp increase in conductance (FIG. 5B, Phase 5), which returned to 2.3x1 O'⁶ Go. This binary change in the conductance with the formation and breakdown of the system was a clear indicator of a single-molecule binding process.

Example 5

[0117] To gauge the specificity of the system and determine whether the binary conductance change occurs due to the molecule or due to changes in the overall bridged structure upon temperature cycling, this experimental procedure was repeated after replacing the 614G solution in the cell with a 20 pM concentration of the mismatched D614G sequence in PBS. Upon heating, the device again broke down at ~65°C, but this time, upon cooling no clear device formation was observed (FIG. 5B, Phase 6-8). This difference in conductance response between 614G and D614G indicated that the sensing platform is extremely sensitive to single nucleotide polymorphisms. To confirm this result, the complementary experiment starting with mismatched D614G bound to the probe in a 20 pM D614G solution was performed (FIG. 5C). In this case, the junction conductance still decreased to zero upon heating, and again no junction formation occurred when cooling with an excess of D614G present in the solution (FIG. 5C, Phase 1-5). However, when the D614G solution was replaced with the perfectly matched 614G sequence, (FIG. 5C, Phase 6-8), junction formation was again observed with a conductance of 4.8x1 O'⁶ Go.

[0118] Taken together, these data demonstrate that this self-aligning approach for creating single-molecule electronic sensors provides an ability to create dynamic yet stable single-molecule devices that are integrated with conventional lithographic systems with yields that are orders of magnitude higher than conventional single-molecule approaches, and opens the door for creating manufacturable single-molecule devices. These data demonstrated that the resulting sensors were able to reliably detect targeted sequences with limited possibilities for false positives from interfering sequences. This target-agnostic platform will allow DNA and RNA sequence detection to be implemented for a wide range of applications in biology, health-care, and security, examining the presence of and identifying pathogens, performing liquid biopsies, and tracking disease progression. Moreover, the straightforward chemistry for creating these self-aligning structures provides new opportunities for integrating other molecular systems with electronic platforms including proteins, and more conventional molecular electronic devices such as switches, memory units, and diodes.

Example 6

[0119] A variety of additional DNA structures can be integrated and measured including DNA nanostructures (FIG. 8 and FIG. 9), and metalated DNA (FIG. 6 and FIG. 7). 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. We have tested two instances of these systems with Ag (FIG. 6 and FIG. 7A) and Hg (FIG. 7B) ions. At pH I l a T-T mismatch can bind an Ag⁺ ion (FIG. 6A), which yields a conductance value near 7xlO'⁴Go (FIG. 6B, 7A), and nonlinear IV curves (FIG. 6C). With a pH of 8.0 the system preferentially binds to a Hg ion (FIG. 7B).

Example 7

[0120] Spin-crossover compounds, due to their switchable spin state, are targets of high interest for the development of switchable molecular electronic and spintronic devices. Scorpionates are molecules that use 3 -donor site ligands (tridentate system) to bind a central atom. In this example, 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.

[0121] In accordance with this, experiments were conducted to examine two different scorpionates. One is in the high-spin state at room temperature (FIG. 11 A) and the other is in the predominantly low-spin state at room temperature (-90% occupation, FIG. 1 IB). Current-Voltage (I-V) characteristics were obtained for junctions created using these two molecules (FIG. 11C). The high spin state molecule demonstrated stochastic switching behavior at room temperature indicating in situ probing of the spin state of the molecule.

[0122] FIG. 12 shows several example junctions generated using these small-molecule, spincrossover molecules. In one example, 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. In another example, 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.

Example 8

[0123] Norbomadienes (NBs) 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. In this example, a CNT-NB-CNT molecular junction was generated, using a similar process as described for the scorpionate molecules. Here 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 results, described below demonstrate that 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.

[0124] As shown in FIGS. 13 and 14, 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.

[0125] Embodiments of the present disclosure are set forth in the following clauses:

[0126] Clause 1: 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.

[0127] Clause 2: The junction of clause 1, wherein the single-molecule junction is soluble in solution.

[0128] Clause 3: The junction of clause 1 or clause 2, wherein the solution is an aqueous solution.

[0129] Clause 4: The junction of any one of clauses 1 to 3, wherein the solution comprises a polar organic solvent.

[0130] Clause 5: The junction of any one of clauses 1 to 4, wherein the solution comprises a non-polar organic solvent.

[0131] Clause 6: The junction of any one of clauses 1 to 5, wherein the solution comprises a soluble coupling agent.

[0132] 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). [0133] 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.

[0134] Clause 9: The junction of any one of clauses 1 to 8, wherein the coating comprises a polynucleotide.

[0135] 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.

[0136] 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.

[0137] Clause 12: The junction of any one of clauses 1 to 11, wherein the single-molecule bridge comprises an organic molecule.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] Clause 16: The junction of any one of clauses 1 to 15, wherein the single-molecule bridge comprises DNA origami.

[0142] Clause 17: The junction of any one of clauses 1 to 16, wherein the single-molecule bridge comprises a peptide, polypeptide, or protein.

[0143] Clause 18: The junction of any one of clauses 1 to 17, wherein the peptide, polypeptide, or protein comprises biotin-streptavidin.

[0144] Clause 19: The junction of any one of clauses 1 to 18, wherein the single-molecule bridge comprises a scorpionate.

[0145] Clause 20: The junction of any one of clauses 1 to 19, wherein the scorpionate comprises a PRRR053 scorpionate and/or a PRRR057 scorpionate. [0146] 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.

[0147] 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.

10148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] Clause 28: The junction of any one of clauses 1 to 27, wherein the one or more linkers comprise biotin and/or streptavidin.

[0154] 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. [0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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%.

Claims

CLAIMS What is claimed is:

1. 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.

2. The junction of claim 1, wherein the single-molecule junction is soluble in solution.

3. The junction of claim 2, wherein the solution is an aqueous solution.

4. The junction of claim 2, wherein the solution comprises a polar organic solvent.

5. The junction of claim 2, wherein the solution comprises a non-polar organic solvent.

6. The junction of claim 2, wherein the solution comprises a soluble coupling agent.

7. The junction of claim 6, wherein the soluble coupling agent comprises O-(7- azabenzotriazole- 1 -yl )-N,N,N,N'-tctramcthyluronium hexafluorophosphate (HATU).

8. The junction of claim 1, wherein the single-molecule bridge is functionally coupled to at least a third CNT.

9. The junction of claim 1, wherein the coating comprises a polynucleotide.

10. The junction of claim 9, wherein the polynucleotide is a single-stranded polynucleotide selected from DNA, RNA, PNA or any combinations or derivatives thereof.

11. The junction of claim 10, wherein the single-stranded polynucleotide is a single-stranded DNA comprising guanine-thymine repeats.

12. The junction of claim 1, wherein the single-molecule bridge comprises an organic molecule.

13. The junction of claim 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.

14. The junction of claim 13, wherein the polynucleotide comprises DNA, RNA, PNA, or any combinations or derivatives thereof.

15. The junction of claim 13, wherein the polynucleotide is covalently linked to at least one of the first and/or second CNT.

16. The junction of claim 1, wherein the single-molecule bridge comprises DNA origami.

17. The junction of claim 1, wherein the single-molecule bridge comprises a peptide, polypeptide, or protein.

18. The junction of claim 17, wherein the peptide, polypeptide, or protein comprises biotinstreptavidin.

19. The junction of claim 1, wherein the single-molecule bridge comprises a scorpionate.

20. The junction of claim 19, wherein the scorpionate comprises a pair of tri-dentate molecules and a metal ion ligated between the pair of tri-dentate molecules.

21. The junction of claim 1 , wherein the single-molecule bridge comprises a photochemically active molecule.

22. The junction of claim 21, wherein the photochemically active molecule comprises a norbomadiene or a derivative or variant thereof.

23. The junction of claim 1, wherein the single-molecule bridge is from about 0.50 nm to about 250 nm in length.

24. The junction of claim 1, wherein the single-molecule bridge is from about 250 nm to about 1 pm in length.

25. The junction of claim 1, wherein the single-molecule bridge is less than about 600 nm in length.

26. The junction of claim 1, wherein at least one of the first and/or second CNT is functionalized with one or more linkers.

27. The junction of claim 26, wherein 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.

28. The junction of claim 1, wherein the one or more linkers comprise biotin and/or streptavidin.

29. A single-molecule electronic biosensor comprising at least one circuit comprising the junction of claim 1, wherein the first and the second CNTs are functionally coupled to pairs of microfabricated electrodes, thereby forming the at least one circuit.

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 single-molecule electronic biosensor of claim 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.

31. The method of claim 30, wherein detecting the presence of the target nucleic acid comprises detecting a change in conductance and/or current.

32. An aqueous solution comprising: a plurality of single-walled carbon nano tube (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.

33. A method of generating a plurality of self-aligned, single-molecule junctions comprising incubating the aqueous solution of claim 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.

34. The method of claim 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.

35. The method of claim 33, wherein the method generates the plurality of self-aligned, single-molecule junctions with a yield of at least 1%.