Ohmic contacts for bioelectronic devices and systems

Abstract

The present disclosure provides devices, systems, and methods related to bioelectronic devices. In particular, the present disclosure provides bioelectronic devices, and methods of making bioelectronic devices, comprising conductive anchors and conductive anchor binding moieties to minimize contact resistance of a protein-of-interest coupled to electrodes in the bioelectronic devices. The bioelectronic devices and methods of the present disclosure are useful for a variety of applications, including the direct measurement of protein activity.

Description

OHMIC CONTACTS FOR BIOELECTRONIC DEVICES AND SYSTEMSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 581,436 filed September 8, 2023, which is incorporated herein by reference in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R01 HG011079 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporation by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 3,569 bytes Byte XML file named “RAX-42227-601” created on September 6, 2024.FIELD

[0004] The present disclosure provides devices, systems, and methods related to bioelectronic devices. In particular, the present disclosure provides bioelectronic devices, and methods of making bioelectronic devices, comprising conductive anchors and conductive anchor binding moieties to minimize contact resistance of a protein-of-interest coupled to electrodes in the bioelectronic devices. The bioelectronic devices and methods of the present disclosure are useful for a variety of applications, including the direct measurement of protein activity.BACKGROUND

[0005] Recent investigations have indicated that proteins can function as electronic conductors, including proteins that do not contain redox cofactors. In some cases, proteins transport current with less resistance per unit distance than is the case for conventional molecular wires. However, in some cases, proteins exhibit a high contact resistance due to the difference in Fermi energy between the electrodes and the solvent-exposed amino acid residues. Therefore, the need exists for bioelectronic devices, and methods of building such devices, that include functional protein molecular wires (e.g., a fully-folded polypeptide witha hydrophobic core surrounded by a hydrophilic exterior) with features designed to minimize the contact resistance for proteins that form part of a bioelectronic circuit.SUMMARY

[0006] Embodiments of the present disclosure include a bioelectronic device comprising a first electrode and a second electrode separated by a gap, a protein attached to the first and second electrodes via a linker, and at least one conductive anchor coupled to the protein, thereby minimizing contact resistance of the protein.

[0007] In some embodiments, the conductive anchor comprises one or more small molecules comprising an aromatic moiety (e.g., an aromatic group including aryl diazonium salts and porphyrins). In some embodiments, the conductive anchor comprises a protein-based redox system comprising at least one metalloprotein (e.g., proteinaceous redox systems based on metalloproteins including, but not limited to, [2Fe-2S] Reiske clusters, [4Fe-4S] high potential clusters, 6-type cytochromes , c-type cytochromes (class IV, class lib, class Ila, class I), / -type cytochromes, Cuacenters, and blue copper proteins (excluding rusticyanin)).

[0008] In some embodiments, at least one conductive anchor is coupled to the outside of the protein. In some embodiments, at least one conductive anchor is coupled to the N-terminal or C-terminal of the protein. In some embodiments, at least one conductive anchor is coupled to the protein via a covalent chemical bond.

[0009] In some embodiments, at least one conductive anchor is coupled to the protein via at least one conductive anchor binding moiety that is coupled to the protein. In some embodiments, at least one conductive anchor binding moiety comprises a cysteine-XX- cysteine-histidine (CXXCH) motif, wherein X is any amino acid. In some embodiments, at least one conductive anchor binding moiety comprises a protein maquette comprising at least one alpha helix. In some embodiments, at least one conductive anchor comprises a heme moiety.

[0010] In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned from about 1 nm to about 2 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned about 1.4 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned no greater than about 1.4 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, atleast one conductive anchor is coupled to the protein such that it is positioned no greater than about 1.4 nm from another conductive anchor.

[0011] In some embodiments, at least one conductive anchor comprises a redox potential from about 100 mV to about 300 mV on the standard hydrogen electrode (SHE) scale.

[0012] In some embodiments, the first and / or the second electrode comprises gold, palladium, platinum, silver, copper, carbon, indium tin oxide (ITO), or any derivatives or alloys thereof.

[0013] In some embodiments, at least one conductive anchor comprises an energy level that matches the energy level of the at least one electrode.

[0014] In some embodiments, the device comprises a dielectric layer at least partially covering a surface of the first and / or second electrode.

[0015] In some embodiments, the first electrode and second electrode are positioned so that about a 1 nm to about a 50 nm gap is formed between the two electrodes.

[0016] In some embodiments, the protein is attached to the first electrode via a first linker, and the second electrode is attached to the second electrode via a second linker. In some embodiments, each of the first and the second linkers comprises at least one chemical bond and is attached to a region of the protein that is inactive.

[0017] In some embodiments, the protein and the first and / or second electrodes are biotinylated, and the first and / or the second linker comprises a streptavidin molecule. In some embodiments, the first and / or the second linker is attached to the N-terminal region of the protein, or the C-terminal region of the protein. In some embodiments, the first or the second linker is attached to the exonuclease region of a polymerase.

[0018] In some embodiments, each of the first and the second linkers comprises a covalent chemical bond. In some embodiments, the first and / or the second linker comprises a genetically modified streptavidin variant containing at least one point mutation resulting in a cysteine residue. In some embodiments, the protein is biotinylated.

[0019] In some embodiments, the protein is selected from the group consisting of a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase, and an endonuclease. In some embodiments, the protein is a polymerase. In some embodiments, exonuclease activity of the polymerase is disabled.

[0020] In some embodiments, the bioelectronic device further comprises at least one adaptor polypeptide. In some embodiments, the at least one adaptor polypeptide comprises a consensus tetratricopeptide repeat (CTPR) adaptor polypeptide. In some embodiments, the CTPR is selected from the group consisting of CTPR1, CTPR2, CTPR3, CTPR4, CTPR5, CTPR6,CTPR7, CTPR8, CTPR9, CTPR10, CTPR11, CTPR12, CTPR13, CTPR14, CTPR15, CTPR16, CTPR17, CTPR18, CTPR19, CTPR20, and any derivatives or variants thereof.

[0021] In some embodiments, the at least one adaptor polypeptide is directly coupled to at least one of the first and second electrodes. In some embodiments, the at least one adaptor polypeptide is coupled to at least one of the first and second electrodes via a linker. In some embodiments, the linker is selected from the group consisting of a biotin-streptavidin linker, a spytag-spycatcher linker, a halotag linker, a thiolated linker, a thiolated TPR linker, and a thiolated CTPR linker.

[0022] In some embodiments, the at least one adaptor polypeptide is directly coupled to the protein. In some embodiments, the at least one adaptor polypeptide is coupled to the protein electrodes via a linker. In some embodiments, the linker is selected from the group consisting of a biotin-streptavidin linker, a spytag-spycatcher linker, a halotag linker, a thiolated linker, a thiolated TPR linker, and a thiolated CTPR linker.

[0023] Embodiments of the present disclosure also include a system comprising a plurality of any of the devices described herein.

[0024] Embodiments of the present disclosure also include a method for direct electrical measurement of protein activity. In accordance with these embodiments, the method comprises introducing an analyte capable of interacting with the protein of any of the devices described herein, applying a voltage bias between the first and second electrodes that is 100 mV or less, and observing fluctuations in current between the first and second electrodes that occur when the analyte interacts with the protein. In some embodiments, the analyte is a biopolymer selected from the group consisting of a DNA molecule, an RNA molecule, a peptide, a polypeptide, and / or a glycan.

[0025] Embodiments of the present disclosure also include a method of manufacturing any of the devices described herein using a cell-based system. In accordance with these embodiments, the protein is a polymerase, and the cell is engineered to deliver the polymerase to the periplasm using a sacrificial peptide leader sequence to activate secretion mechanisms. In some embodiments, the sacrificial leader sequence is PelB.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 : Representative data of the conductivity of various molecular wires as a function of their length, according to one embodiment of the present disclosure.

[0027] FIG. 2: Representative schematic diagram of energy levels in a system of two electrodes spanned by a protein, according to one embodiment of the present disclosure.Electron energy is shown increasing on the vertical axis with distance plotted on the horizontal axis.

[0028] FIG. 3 : Representative chart of the redox potentials of various conductive anchors (e.g., biological cofactors), according to one embodiment of the present disclosure. The box marks the approximate range of Fermi energies for palladium and gold.

[0029] FIG. 4: Representative diagram of a bioelectronic device of the present disclosure comprising a conductive anchor coupled to a protein-of-interest to minimize contact resistance.

[0030] FIG. 5: Representative diagram of a small cytochrome (i.e., conductive anchor binding moiety) suitable for incorporation into a protein-of-interest and for binding a heme molecule (i.e., conductive anchor), according to one embodiment of the present disclosure.

[0031] FIG. 6: Representative diagram of a protein-of-interest expressed with cytochrome contacts (i.e., conductive anchor binding moiety), according to one embodiment of the present disclosure.

[0032] FIG. 7 : Representative diagram of a protein-of-interest expressed with bare heme contacts (i.e., conductive anchor), according to one embodiment of the present disclosure.

[0033] FIG. 8: Representative diagram of a process for engineering heme-containing proteins-of-interest, according to one embodiment of the present disclosure.

[0034] FIG. 9: Representative diagram of a fusion protein comprised of a Phi29 polymerase (light blue, 901) and a heme-binding three helix bundle “maquette” (gold, 902), according to one embodiment of the present disclosure.

[0035] FIG. 10: Representative diagram of a fusion protein (“protein maquette”) comprised of a Phi29 polymerase (light blue, 901) and a heme-binding three helix bundle maquette (gold, 902), with the location heme groups shown (i.e., 1001 and 1002), according to one embodiment of the present disclosure.

[0036] FIG. 11 : Representative diagram of a fusion protein (“protein maquette”) comprised of a Phi29 polymerase (light blue, 901) and a heme-binding three helix bundle maquette (gold, 902), with the location of exemplary contact points on the maquette protein (i.e., 1101 and 1102), according to one embodiment of the present disclosure.DETAILED DESCRIPTION

[0037] In the following detailed description, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration embodiments that may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope. Therefore, thefollowing detailed description is not to be taken in a limiting sense, and the scope of embodiments is defined by the appended claims and their equivalents.

[0038] Various operations may be described as multiple discrete operations in turn, in a manner that may be helpful in understanding embodiments; however, the order of description should not be construed to imply that these operations are order dependent.

[0039] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); and other similar references.

[0040] Suitable methods and materials for the practice or testing of this disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which this disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0041] 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

[0042] To facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided below. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0043] 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.”

[0044] 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.

[0045] 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.

[0046] 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 noB 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.

[0047] 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.

[0048] As used herein, “biological sample” generally refers to a biological specimen containing genomic DNA, RNA (such as mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, saliva, peripheral blood, urine, tissue biopsy, surgical specimen, and autopsy material. In embodiments, the biological sample is a bodily fluid, such as blood, or a component thereof, such as plasma or serum.

[0049] As used herein, “biopolymer” generally refers to polymers (e.g., produced by living organisms or synthetically generated). Biopolymers contain monomeric units that are covalently bonded to form larger structures. There are three main classes of biopolymers, classified according to the monomeric units used and the structure of the biopolymer formed: polynucleotides (RNA and DNA), which are long polymers composed of 13 or more nucleotide monomers; polypeptides, which are short polymers of amino acids; and polysaccharides, which are often linear bonded polymeric carbohydrate structures. Other examples of biopolymers include rubber, suberin, melanin and lignin.

[0050] As used herein, an “isolated” biological component (e.g., such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been “isolated” may be understood to have been purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.

[0051] As used herein, “modification,” “chemical modification,” or “chemically modified” generally refers to any of a number of various processes involving the alteration of the chemicalconstituent or structure of molecules. For example, a polymerase can be chemically modified to form a chemical bond with a first electrode and a second electrode. In one example, a chemically-modified electrode is an electrode that has a surface chemically converted to change the electrode’s properties, such as its physical, chemical, electrochemical, optical, electrical, and / or transport characteristics. As provided herein, the chemical modification can also involve chemically altering a polymerase so that it is compatible with a linker that binds to an electrode (e.g., SypCatcher / SpyTag, biotin / streptavidin, HaloTag, and the like). In other embodiments, a modification can be generated via protein synthesis. For example, a polymerase can be designed to comprise one or more modifications (e.g., a linker) when synthesized from a polynucleotide that encodes the protein and the modification (e.g. linker).

[0052] As used herein, “contact” and “contacting” can include placement in direct physical association, including both a solid and liquid form. “Contacting” can include a specific chemical contact between two different substances (e.g., covalent bond, or non-covalent bond having specific ligand interaction with specific amino acid residues).

[0053] As used herein, “complementarity” or “complementary” generally refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” generally indicates that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0054] As used herein, “current stream” generally refers to a current signal generated over time, such as from the bioelectronic devices of the present disclosure.

[0055] As used herein, a “label” generally refers to an agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a nucleic acid molecule or protein (indirectly or directly), thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents,fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

[0056] As used herein, the term “linker” or “linked” means joined together, either directly or indirectly. For example, a first moiety may be covalently or noncovalently (e.g., electrostatically) linked to a second moiety. This includes, but is not limited to, covalently bonding one molecule to another molecule, noncovalently bonding one molecule to another (e.g., electrostatically bonding), non- covalently bonding one molecule to another molecule by hydrogen bonding, non-covalently bonding one molecule to another molecule by van der Waals forces, and any and all combinations of such couplings. Indirect attachment is possible, such as by using a “linker” (a molecule or group of atoms positioned between two moieties). In several embodiments, linked components are associated in a chemical or physical manner so that the components are not freely dispersible from one another. For example, two components may be covalently bound to one another so that the two components are incapable of separately dispersing or diffusing. In several embodiments, linked components are associated in a chemical or physical manner so that the components are not freely dispersible from one another. For example, two components may be covalently bound to one another so that the two components are incapable of separately dispersing or diffusing.

[0057] As used herein, the terms “non-naturally occurring” and “engineered” interchangeably indicate the involvement of the hand of man. These terms, when referring to nucleic acid molecules or polypeptides, generally indicate that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0058] As used herein, “nucleic acid” generally refers to a deoxyribonucleotide or ribonucleotide polymer, which can include analogues of natural nucleotides that hybridize to nucleic acid molecules in a manner similar to naturally occurring nucleotides. In one example, a nucleic acid molecule is a single stranded (ss) DNA or RNA molecule, such as a probe or primer. In another example, a nucleic acid molecule is a double stranded (ds) nucleic acid. In another example, a nucleic acid is a modified DNA or RNA molecule, such as a xenonucleic acid (XNA). The term “nucleotide” generally refers to a base-sugar-phosphate combination and includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof.

[0059] As used herein, “polypeptide,” “peptide,” and “protein” generally refer to a polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred in nature. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A substantially purified polypeptide as used herein refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In one embodiment, the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.

[0060] Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0061] A non-conservative amino acid substitution can result from changes in: (a) the structure of the amino acid backbone in the area of the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl. Variant amino acid sequences may, for example, be 80, 90 or even 95 or 98% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI website.

[0062] As used herein, “probe” generally refers to a short sequence of nucleotides, such as at least 8, at least 10, at least 15, at least 20, or at least 21 nucleotides in length, which can be used to detect the presence of a complementary sequence by molecular hybridization. In particular examples, oligonucleotide probes include a label that permits detection ofoligonucleotide probe:target sequence hybridization complexes. Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.

[0063] As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is purer than the protein in its natural environment within a cell. For example, a preparation of a protein is purified such that the protein represents at least 50% of the total protein content of the preparation. Similarly, a purified oligonucleotide preparation is one in which the oligonucleotide is purer than in an environment including a complex mixture of oligonucleotides. Purity of a compound may be determined, for example, by high performance liquid chromatography (HPLC) or other conventional methods.

[0064] As used herein, “recombinant” generally refers to recombinant nucleic acid or protein that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. The term recombinant includes nucleic acids and proteins that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein.

[0065] As used herein, the terms, “substantial identity” or “substantially identical” generally refer to a nucleic acid or fragment thereof, that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), refers to a nucleotide sequence having at least about 95% sequence identity, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0066] As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur- containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine - leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamateaspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0067] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutant thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6. 1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.

[0068] As used herein, “variant” generally refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. “SNP” refers to a variant that is a single nucleotide polymorphism. Representative examples of “biological activity” include the ability to be bound by a specific antibody or to promote an immune response. Variant is also used herein to describe a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e. replacing an amino acid with a different amino acid of similar properties (<?.g., hydrophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0069] As used herein, “pause” generally refers to a section of a current stream in which the fluctuations in measured current are interrupted by a slower feature of about twice the duration of the neighboring features. Typically, a pause is observed before and after a nucleotide has been incorporated into a template sequence, and the duration of the pause relative to the neighboring pulses of current increases as the concentration of nucleotide triphosphates is lowered.

[0070] As used herein, a “polymerase” generally refers to an enzyme that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying a DNA template strand using base-pairing interactions or RNA by half ladder replication.

[0071] 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.

[0072] 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 which may further include any and all elements from any other disclosed methods, compositions, systems, and devices, including any and all elements corresponding to detecting protein activity. 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).2. Bioelectronic Devices and Systems

[0073] Proteins are quite good electronic conductors, even those containing no redox cofactors. The mechanism of this conductivity has recently been described in a theoretical analysis that predicts experimental data quantitatively (Krishnan, Aksimentiev et al. 2023). This work calculates the conductivity by deriving a diffusion constant for charge carriers, D, from an all-atom-simulation. A model for the charge-injection process is used to calculate the density of carriers injected into the protein by an electrode, n. These two quantities, n and D suffice to describe the resistance of a protein electronic component. Modeling a linear protein as a rod of length 1 and cross sectional area A with resistivity p, the resistance of the protein in Ohms is given

[0074] R =1 1

[0075] where we have used the Einstein relation to convert carrier mobility to a diffusion constant and kT is the available thermal energy. Thus, for a given A and 1, the resistance is determined entirely by the quantity nD. D is an intrinsic property of the protein wire. However, since there are no intrinsic free carriers in a protein (in contrast to a metal where n~1026 / m3) the determining factor in the conductivity of a protein is the number of charges injected at a contact. This is evident in experimental data for the conductivity of various molecular wires shown in FIG. 1. This plots the logarithm of the conductance (in units of the quantum of conductance Go) against the lengths of oligomers of the various molecules. The data points are for two types of DNA wires of various lengths (labeled DNA), a synthetic molecular wire, oligo phenyleneimide (OPI) again of several lengths, and a series of linear proteins, the consensus tetratricopeptide proteins (CTPR). The slopes of these plots are a measure of the reciprocal of the diffusion constant, 1 / D, and all of these molecules have broadly similar diffusion constants in that the slopes 1, 2, and 3 are similar. However, the contact conductance (1 / (contact resistance)) varies drastically. These are given by the value of G / Go at L=0 (4, 5, 6). These vary over more than two orders of magnitude, showing that charge injection (as reflected in the contact resistance) is the limiting factor in the conductivity of molecular wires.

[0076] In particular, the conductance of CTPR proteins decays more slowly than that of the other molecules, so much so, that after some 5 nm, it becomes a better conductor than the molecular wire (OPI). However, the CTPR proteins have a much higher contact resistance than the OPI. Therefore, experiments were conducted to examine the energetics of hopping conductivity in the CTPR wires. A schematic diagram of energy levels in a system of two electrodes spanned by a protein is shown in FIG. 2. In order to relate the various energies toelectrochemical data, energies are labeled both with respect to the vacuum 23 and the standard hydrogen electrode (SHE) 24. A first electrode 21 is attached to one end of a protein wire 22. Electrons from the Fermi energy of the first electrode 26, once injected into the protein hop between aromatic residues 25. These have states that are nearly aligned with the Fermi level of gold or palladium electrodes and this fact is what enables long-range conductance in these proteins.

[0077] One important factor is that these energy levels are not significantly perturbed when charged by removal of an electron. This is because the polarization relaxation time inside the protein is much longer than the time the charge spends on a given site. When the charge diffuses to the residue at the far end of the protein 28, it is captured by the second electrode 29 if this is held at a positive potential with respect to the first electrode 21. This, however, does not necessarily apply to residues exposed to the surface where solvent polarization is fast. It is these aromatic residues at the edge of the protein 27 that mediate charge injection. However, the energy of their radical cation state are no longer aligned with the Fermi energy of the electrode, instead the levels lie closer to the thermodynamic equilibrium value of +1 V vs SHE. It is this mismatch that generates the large contact resistance. Accordingly, the various embodiments of the present disclosure provide novel contact chemistry (e.g., conductive anchors and associated binding moieties) such that the energy of an aromatic entity at the charge entry site is matched to the Fermi energy of the metal, thereby minimizing contact resistance of a protein.

[0078] In accordance with this, embodiments of the present disclosure are based on the existence of redox cofactors with redox potentials (when at least partially solvated) that are close to the Fermi energies of gold or palladium. There are many such factors, as shown by those that fall within the shaded box in FIG. 3 (data are from (Hosseinzadeh and Lu 2016)). As described further herein, these cofactors can be referred to as conductive anchors, and the associated moieties that couple to these conductive anchors to a protein-of-interest can be referred to as conductive anchor binding moieties.

[0079] One embodiment of the present disclosure is shown in FIG. 4, where the residue chosen for one exemplary embodiment of a conductive anchor is the heme group from cytochromes. As would be recognized by one of ordinary skill in the art based on the present disclosure, any other suitable conductive anchor can be used, including but not limited to, the cofactors listed in FIG. 3. The use of a cytochrome heme is not intended to be limiting.

[0080] Furthermore, FIG. 4 shows the same arrangement of electrodes and a protein as in FIG. 2 but with a heme group now incorporated at the end of each of the contact points to theprotein 41, 42 by means to be described below. The reduction potentials of these hemes are now aligned approximately with the Fermi energy of the electrodes, eliminating the barrier to charge injection posed by the hydrated aromatic residue shown in FIG. 2.

[0081] In one embodiment of the present disclosure, the contacts are formed by incorporating a small cytochrome protein (FIG. 5) into the N- and C- terminals of the protein of interest. This process is illustrated here for the case of a Phi29 DNA polymerase, though the methods described here can be applied to any other protein that can be expressed in an appropriate bacterial system. FIG. 6 shows a Phi29 DNA polymerase 61 in which cytochromes 62, 63 have been incorporated at the N- (64) and C- (65) termini of the Phi29 sequence. The cytochromes incorporate exposed cysteine residues 66, 67 that are within 1.4 nm of the hemes, and the cysteines form chemical contacts to the electrodes via sulfur-metal bonds. That the heme or similar redox cofactor can be within 1.4 nm of the contact point is based on the observation that electron tunneling in proteins becomes negligible at distances larger than this (Page, Moser et al. 1999).

[0082] In one embodiment, bare heme groups are covalently attached to two exposed points on a protein surface via insertion of a cysteine-xx-cysteine-histidine (CXXCH) motif (where X = any amino acid residue). FIG. 7 shows an example of a Phi29 polyemerase 70 incorporating exposed hemes at the N- (71) and C-termini (72). The hemes naturally form strong attachments to metal electrodes and can serve the dual purpose of serving as anchor groups to the electrodes.

[0083] In some embodiments of the present disclosure, assembly of a c-type cytochrome expression system for Escherichia coli includes the introduction and uptake of two circular plasmids encoding for an operon to enable c-type cytochrome production under aerobic conditions, the Ccm pathway which contains nine essential components: A, B, C, D, E, F, G, H, and I (Sanders, Turkarslan S Fau - Lee et al.). In some embodiments, an open reading frame containing: 1) the necessary genetic mutations to add a CxxCH motif to both the N and C termini of Phi29 (to express the construct shown in FIG. 7), or 2) the genetic encoding of a full cytochrome protein sequence (to express the construct shown in FIG. 6). Although E. coli natively contains endogenous machinery necessary to produce c-type cytochromes, these genetic components are only activated under anaerobic conditions, which results in slow growth rates and extended growth duration. The Ccm pathway is important for synthesis of sufficient quantities of Fe-protoporphyrin IX, the protein machinery required to translocate the pre-cytochrome polypeptides to the periplasmic space, and finally to facilitate covalent attachment the heme group to the Phi29 protein scaffold within the periplasmic space. The incorporation of hemes in the periplasmic space is illustrated in FIG. 8 for the example of anapo c-type cytochrome (81). This cytochrome is translocated into the periplasmic space by a membrane translocation protein 82 where a maturation protein 83 facilitates covalent bond formation between the protein 81 and the heme 84 via the formation of thioether bonds between the cysteines on the protein and vinyl groups on the heme. Prior to covalent attachment, the heme group is translocated into the periplasmic space by a heme translocation protein 85.

[0084] Expression of Phi29 containing two covalently attached heme groups can be completed via introduction of amino acids with the sequence CxxCH, a motif commonly found in c-type cytochromes, where x is any amino acid, C is a cysteine residue, and H is a histidine residue. At the most basic level, the plasmid introduced to E. coli for uptake and further to episomal expression must contain the DNA coding sequence for the CxxCH amino acid motif. Introduction of this sequence is completed via cloning techniques to produce an in frame mutation of the codons to code for each amino acid. An RNA polymerase will then transcribe the Phi29 open reading frame, harbored within the plasmid DNA, to mRNA to act as a template for the ribosomal machinery to produce a full length polypeptide. The initiation of this nascent apo-protein must contain a leader peptide sequence that the cell will use to direct the fully assembled apo-protein to membrane bound proteins that facilitate transfer across the bilipid membrane to the periplasmic space. Transport across this membrane results in cleavage of the leader peptide sequence and expulsion of the apo-protein to the periplasmic space. Once the apo-protein has been delivered to the periplasmic space the apo-protein will have the heme group covalently attached via chaperone proteins from the Ccm pathway. The final holoprotein for Phi29 containing two covalent heme groups will be located in the periplasmic space which enables a facile purification procedure as the entire protein contents of E. coli within the cytoplasmic space do not interfere with purification since the entire cell does not need to be opened to retrieve the fully matured protein products.

[0085] In accordance with the above, embodiments of the present disclosure include a bioelectronic device comprising a first electrode and a second electrode separated by a gap, a protein attached to the first and second electrodes via a linker, and at least one conductive anchor coupled to the protein, thereby minimizing contact resistance of the protein. In some embodiments, the conductive anchor comprises one or more small molecules comprising an aromatic moiety (e.g., an aromatic group including aryl diazonium salts and porphyrins). In some embodiments, the conductive anchor comprises a protein-based redox system comprising at least one metalloprotein (e.g., proteinaceous redox systems based on metalloproteins including, but not limited to, [2Fe-2S] Reiske clusters, [4Fe-4S] high potential clusters, / vtypccytochromes , c-type cytochromes (class IV, class lib, class Ila, class I), / -type cytochromes, Cua centers, and blue copper proteins (excluding rusticyanin)).

[0086] In some embodiments, at least one conductive anchor is coupled to the outside of the protein. In some embodiments, at least one conductive anchor is coupled to the N-terminal or C-terminal of the protein. In some embodiments, at least one conductive anchor is coupled to the protein via a covalent chemical bond. In some embodiments, at least one conductive anchor is coupled to the protein via at least one conductive anchor binding moiety that is coupled to the protein. In some embodiments, at least one conductive anchor binding moiety comprises a cysteine -XX-cysteine-histidine (CXXCH) motif, wherein X is any amino acid. In some embodiments, at least one conducive anchor comprises a heme moiety.

[0087] In some embodiments, the present disclosure includes an engineered protein-of- interest capable of incorporating heme molecules (“protein maquettes;” see, e.g., Lichtenstein, et al., “Engineering oxidoreductases: maquette proteins designed from scratch;” Biochem Soc Trans 40(3): 561-566 (2012); and Hutchins, et al., “An expandable, modular de novo protein platform for precision redox engineering;” Proc Natl Acad Sci U S A 120(31): e2306046120 (2023)). Referred to as a protein maquette, these proteins serve as conductive anchor binding moieties because of their ability to incorporate a conductive anchor, such as a heme molecule, into a protein-of-interest.

[0088] In some embodiments, the protein maquette is expressed as a fusion protein with a protein-of-interest, such as a polymerase. For example, FIG. 9 provides a representative diagram of a fusion protein comprised of a Phi29 polymerase (light blue, 901) and a hemebinding three helix bundle “maquette” (gold, 902), according to one embodiment of the present disclosure. The amino acid sequence of this fusion protein is provided here: MGSSHHHHHHSSGLVPRGSHGLNDIFEAQKIEWHEGASSMPRKMYSCDFETTTKVE DCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLE RNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKI AKDFKLTVLKGDIDYHKERPVGYKITGSPELREKHRALAEQVYATHQEMLKNTSNS PELREKHRALAEQVYATGQEMLKNGSVSPSPELREKHRALAEQVYATGQEMLKNTS NGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVFDEDYPLHIQHIRCEFELKEG YIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKA TTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGAL GFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVI KDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGASSGNST NGTSNGSSGLNDIFEAQKIEWHE (SEQ ID NO: 1). The three helix maquette sequence is directly encoded into the Phi29 polymerase sequence between the Threonine at position 195 and the Glycine at position 283, and the amino acid sequence is provided here: GSPELREKHRALAEQVYATHQEMLKNTSNSPELREKHRALAEQVYATGQEMLKNG SVSPSPELREKHRALAEQVYATGQEMLKNTSN (SEQ ID NO: 2). Therefore, the connection of the three helix maquette to the polymerase is through insertion of its amino acid sequence into the amino acid sequence of the polymerase, creating a single polypeptide chain that will fold into the resulting fusion construct. In accordance with these embodiments, the protein maquette can include at least one alpha helix. In some embodiments, the protein maquette can include at least two alpha helices. In some embodiments, the protein maquette can include at least three alpha helices (an alpha helix bundle). And as would be understood by one of ordinary skill in the art based on the present disclosure, a maquette sequence can be inserted into a protein-of-interest at various locations, such as locations which provide one or more sites for conductive anchors in order to minimize contact resistance of the protein-of- interest when coupled to electrodes in the bioelectronic devices of the present disclosure.

[0089] In accordance with these embodiments, heme molecules, either expressed endogenously or added in vitro, are captured by histidine residues. In some embodiments, the protein maquette captures two hemes, 1001 and 1002, as shown in FIG. 10. In the case of the exemplary three-helix maquette shown in FIG. 10, these are His232 (1003), His215 (1004), and His262 (1005). In this arrangement, the closest approach of the two tetrapyrrole rings of the hemes (1006) is 0.33 nm, ensuring rapid charge transport between the rings.

[0090] This protein maquette forms a highly conductive molecular wire, capable of charge transport over a 4.3 nm distance with contacts at the positions 1101 and 1102, as shown in FIG. 11 , which are comprised in the loops that connect the helices in the bundle and are located at Ser223 and Ser251. In order to form contacts at these points, either non-specific binding residues (such as cysteines) or specific binding residues (such as azido phenylalanine), can be substituted for these serine residues allowing for direct binding or click- chemistry anchoring to electrode contacts. As described further herein, the electrodes include noble metals or metallic carbon nanotubes, functionalized for click chemistry, as is well known in the art based on the present disclosure.

[0091] The entire protein maquette wire can be contained within a loop of the Phi29 polymerase; therefore, motions of the polymerase are communicated to the wire, modulating its conductivity so that conformational fluctuations of the polymerase transduce currentfluctuations in the electrical current passing through the maquette wire. Alternative construct designs may include, but are not limited to, alterations of the Phi29 sequence via deletion, insertion, or mutagenesis of native residues, as well as alterations of the protein maquette sequence or structural assembly by the same methods. Rational design of the protein maquette feature may include addition or deletion of helices to satisfy structural or charge transport requirements. Placement of the protein maquette within the Phi29 sequence as part of a fusion is not limited to flexible loop insertion, but may also be integrated with preexisting structural elements of the native sequence. In some embodiments, placement of the protein maquette within a protein-of-interest can be done to ensure that each conductive anchor(s) (e.g., heme molecule or an aromatic amino acid residue) is positioned no more than 1.4 nm from a contact point to an electrode or to another conductive anchor. This configuration helps to minimize the contact resistance for a protein-of-interest in the bioelectronic circuits of the present disclosure.

[0092] In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned from about 1.0 nm to about 2 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned from about 1.2 nm to about 2 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned from about 1.4 nm to about 2 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned from about 1.0 nm to about 1.8 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned from about 1.0 nm to about 1.6 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned from about 1.0 nm to about 1.4 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned from about 1.2 nm to about 1.6 nm from an attachment point linking the protein to the first or second electrode. In some embodiments, at least one conductive anchor is coupled to the protein such that it is positioned about 1.4 nm from an attachment point linking the protein to the first or second electrode.

[0093] In some embodiments, and as described further herein, at least one conductive anchor comprises a redox potential from about 100 mV to about 300 mV on the standard hydrogen electrode (SHE) scale (see, e.g., FIG. 3). In some embodiments, and as describedfurther herein, at least one conductive anchor comprises an energy level that matches the energy level of the at least one electrode (see, e.g., FIG. 4). In some embodiments, the first and / or the second electrode comprises gold, palladium, platinum, silver, copper, carbon, indium tin oxide (ITO), or any derivatives or alloys thereof. In some embodiments, the device comprises a dielectric layer at least partially covering a surface of the first and / or second electrode. In some embodiments, the first electrode and second electrode are positioned so that about a 1 nm to about a 50 nm gap is formed between the two electrodes.

[0094] In some embodiments, the protein is attached to the first electrode via a first linker and attached to a second electrode via a second linker. In some embodiments, each of the first and the second linkers comprises at least one chemical bond and is attached to a region of the protein that is inactive. In some embodiments, the protein and the first and / or second electrodes are biotinylated, and the first and / or the second linker comprises a streptavidin molecule. In some embodiments, the first and / or the second linker is attached to the N-terminal region of the protein, or the C-terminal region of the protein. In some embodiments, the first or the second linker is attached to the exonuclease region of the polymerase. In some embodiments, each of the first and the second linkers comprises a covalent chemical bond. In some embodiments, the first and / or the second linker comprises a genetically modified streptavidin variant containing at least one point mutation resulting in a cysteine residue. In some embodiments, the protein is biotinylated.

[0095] In some embodiments, the bioelectronic device further comprises at least one adaptor polypeptide. In some embodiments, the at least one adaptor polypeptide comprises a consensus tetratricopeptide repeat (CTPR) adaptor polypeptide. In some embodiments, the CTPR is selected from the group consisting of CTPR1, CTPR2, CTPR3, CTPR4, CTPR5, CTPR6, CTPR7, CTPR8, CTPR9, CTPR10, CTPR11, CTPR12, CTPR13, CTPR14, CTPR15, CTPR16, CTPR17, CTPR18, CTPR19, CTPR20, and any derivatives or variants thereof. Adjustable adaptor polypeptides with repeatable motifs and enhanced conductive properties can be found in U.S. Patent Appln. Serial No. 17 / 333,087, which is herein incorporated by reference in its entirety and for all purposes.

[0096] In some embodiments, the at least one adaptor polypeptide is directly coupled to at least one of the first and second electrodes. In some embodiments, the at least one adaptor polypeptide is coupled to at least one of the first and second electrodes via a linker. In some embodiments, the linker is selected from the group consisting of a biotin-streptavidin linker, a spytag-spycatcher linker, a halotag linker, a thiolated linker, a thiolated TPR linker, and a thiolated CTPR linker.

[0097] In some embodiments, the at least one adaptor polypeptide is directly coupled to the protein. In some embodiments, the at least one adaptor polypeptide is coupled to the protein electrodes via a linker. In some embodiments, the linker is selected from the group consisting of a biotin-streptavidin linker, a spytag-spycatcher linker, a halotag linker, a thiolated linker, a thiolated TPR linker, and a thiolated CTPR linker.

[0098] Embodiments of the present disclosure also include a system comprising a plurality of any of the devices described herein.

[0099] Embodiments of the present disclosure also include a method for direct electrical measurement of protein activity. In accordance with these embodiments, the method comprises introducing an analyte capable of interacting with the protein of any of the devices described herein, applying a voltage bias between the first and second electrodes that is 100 mV or less, and observing fluctuations in current between the first and second electrodes that occur when the analyte interacts with the protein. In some embodiments, the analyte is a biopolymer selected from the group consisting of a DNA molecule, an RNA molecule, a peptide, a polypeptide, and a glycan.

[0100] Embodiments of the present disclosure also include a method of manufacturing any of the devices described herein using a cell-based system. In accordance with these embodiments, the protein is a polymerase, and the cell is engineered to deliver the polymerase to the periplasm using a sacrificial peptide leader sequence to activate secretion mechanisms. In some embodiments, the sacrificial leader sequence is PelB.

[0101] The bioelectronic devices and systems of the present disclosure also include a first electrode and a second electrode that are configured for contact with a sample (e.g., biopolymer sample) to be analyzed. In some embodiments, such as when the electrodes are planar, the first and / or second electrode do not require a dielectric layer. In other embodiments, the first and / or second electrode can have a dielectric layer. In some embodiments, the first and / or the second electrode comprises gold, palladium, platinum, silver, copper, carbon, indium tin oxide (ITO), or any derivatives or alloys thereof. In some embodiments, the metal is palladium. In some embodiments, the methods of the present disclosure include applying a voltage bias between the first and second electrodes that is lOOmV or less. It will be recognized by one of ordinary skill in the art based on the present disclosure that the bioelectronic devices and systems of the present disclosure, according to the various methods described herein, can be used to sequence DNA and RNA polymers by similar techniques (e.g., sequence RNA polymers using an RNA dependent RNA polymerase and four different ribonucleotides each carrying a distinctive charge).

[0102] In some embodiments, the gap between the first and second electrode is a width of about 1.0 nm to about 50.0 nm. In some embodiments, the gap between the first and second electrode is a width of about 1.0 nm to about 40.0 nm. In some embodiments, the gap between the first and second electrode is a width of about 1.0 nm to about 30.0 nm. In some embodiments, the gap between the first and second electrode is a width of about 1.0 nm to about 20.0 nm. In some embodiments, the gap has a width of about 1.0 nm to about 10.0 nm. In some embodiments, the gap has a width of about 1.0 nm to about 7.5 nm. In some embodiments, the gap has a width of about 1.0 nm to about 5.0 nm. In some embodiments, the gap has a width of about 4.0 nm to about 5.0 nm. As provided herein, bioelectronic devices and systems of the present disclosure include an adaptor polypeptide(s) that stabilize a protein-of- interest within the gap between a first and second electrode.

[0103] In some embodiments, the protein-of-interest can include, but is not limited to, a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase and an endonuclease. In some embodiments, the protein-of-interest is a polymerase. In some embodiments, the polymerase can be attached to one electrode in one embodiment and to both electrodes in another embodiment. The polymerase can be attached to the electrode(s) either directly or indirectly. In some embodiments, the polymerase is attached to the electrode(s) via a linker, including but not limited to, a biotin-streptavidin linker, a SpyTag- Spy Catcher linker, a halotag linker, and a thiolated CTPR linker. In some embodiments, the polymerase is attached to the electrode indirectly via interactions with a ligand attached to the electrode. In some embodiments, the polymerase is modified to incorporate a ligand-binding site. In some embodiments, the polymerase is a biotinylated polymerase. In some embodiments, the polymerase comprises an Avitag. In some embodiments, the polymerase is a biotinylated polymerase and is attached to the electrode via streptavidin. In some embodiments, the polymerase is modified to incorporate an amino acid residue that allows for click-chemistry attachment of other chemical groups to the electrodes (e.g., 4-Azido-L-phenylalanine.). In some embodiments, the exonuclease activity of the polymerase is disabled. In some embodiments, linker is attached to a region of the polymerase that is inactive. When the polymerase is attached to both electrodes, the two attachment points must not move relative to each other when the polymerase undergoes open-to-closed conformational changes. The crystal structures of many polymerases are currently available (see, e.g., www.rcsb.org) in both open and closed forms.

[0104] In accordance with these embodiments, the present disclosure also provides bioelectronic devices and systems in which the charge on a protein-of-interest is altered in orderto modulate the overall conductance of a protein complex (e.g., a protein-of-interest and corresponding linker). For example, the conductance of a given protein complex in a bioelectronic device configured with platinum electrodes can be modulated (e.g., increased) to be similar to the conductance provided by gold electrodes by altering the charge of the protein complex (e.g., shifting the potential). Thus, embodiments of the present disclosure include a bioelectronic device that includes a first electrode and a second electrode separated by a gap, and a protein (e.g., a polymerase) attached to the first and second electrodes via a linker comprising a distinctive electrical charge. In some embodiments, the distinctive electrical charge modulates conductance through the protein in a manner that enhances the function of the bioelectronic device (e.g., sequencing of a biopolymer).

[0105] In some embodiments, the linker used to generate a bioelectronic device of the present disclosure includes a peptide or polypeptide. In some embodiments, the linker comprises streptavidin. In some embodiments, the linker comprises streptavidin that has been modified to have a positive or negative charge (e.g., with polyglutamate). In some embodiments, the protein-of-interest is biotinylated. In some embodiments, the linker comprises a distinctive negative charge. In some embodiments, the distinctive negative charge is conferred by addition of a glutamate moiety, an aspartate moiety, or a combination thereof (including derivatives, variants, and polymers), coupled to the streptavidin. In some embodiments, the distinctive negative charge increases the conductance through the protein. In some embodiments, the linker comprises a distinctive positive charge. In some embodiments, the distinctive positive charge is conferred by addition of an arginine moiety, a histidine moiety, a lysine moiety, or a combination thereof (including derivatives, variants, and polymers), coupled to the streptavidin. In some embodiments, the distinctive positive charge increases or decreases the conductance through the protein.

[0106] Charge modulation of conductance confers several advantages, including but not limited to, increasing the conductance of a complex using electrodes whose rest potentials place them far from the peak of the conductance vs. potential curve. Peak conductances can be obtained with gold or gold-palladium combinations of electrodes, but gold has various limitations as an electrode material in many semiconductor processes. Therefore, it can be advantageous to use other metals, such as Pt or Pd or other noble metals, but doing so can lead to a reduced signal. Thus, as provided herein, the signal can be restored or enhanced by altering the charge of the protein complex (e.g., using a modified streptavidin as a linker). While this effect applies to streptavidin linker molecules, it readily can be applied to any other type ofprotein molecule used as a connector / linker, as would be recognized by one of ordinary skill in the art based on the present disclosure.

[0107] As persons of ordinary skill in the art will readily recognize and appreciate after having benefited from the teachings of the present disclosure, the methods described herein can be used with any bioelectronic device that senses the duration of the open and closed states of an enzyme (e.g., polymerase). Exemplary devices include, but are not limited to, the bioelectronic devices and systems disclosed in U.S. Patent No. 10,422,787 and PCT Appln. No. PCT / US2019 / 032707, both of which are herein incorporated by reference in their entirety and for all purposes.

[0108] In accordance with these embodiments, the devices and systems of the present disclosure include a bioelectronic device comprising a first electrode and a second electrode separated by a gap, a protein attached to the first and second electrodes via a linker, and at least one adaptor polypeptide coupled to the protein, thereby stabilizing the protein within the gap. In some embodiments, the bioelectronic device comprises a single adaptor polypeptide coupled to the protein and at least one electrode. In some embodiments, the bioelectronic device comprises two adaptor polypeptides coupled to the protein and the two electrodes. In some embodiments, the bioelectronic device comprises more than two adaptor polypeptides coupled to the two electrodes. The precise configuration of the protein and adaptor polypeptides with respect to the first and second electrodes can vary, as described further herein. The precise configuration used in a particular bioelectronic device depends on a variety of factors, including but not limited to, the specific protein-of- interest, the presence / absence of linkers, the size of the gap between the electrodes, the amount of conductance required, and the like.

[0109] In some embodiments, the adaptor polypeptide comprises a hydrophobic core. In some embodiments, the adaptor polypeptide comprises a hydrophilic exterior. In some embodiments, the adaptor polypeptide is capable of folding into a stable conformation such that the hydrophobic core faces the inside of the polypeptide and the hydrophilic exterior faces the outside of the polypeptide.

[0110] In some embodiments, the adaptor polypeptide(s) comprises a repeatable motif, such that the size or length of the adaptor polypeptide(s) can be adjusted or engineered based on the size of an electrode gap and / or a protein-of-interest, among other factors. In some embodiments, the number of repeatable motifs is adjustable based on the size of the gap between the first and second electrodes. In some embodiments, the at least one repeatable motif comprises an a-helix and / or a [3-sheet, and any combination thereof. In some embodiments, the repeatable motif includes at least two or more a-helices. In some embodiments, the repeatableT1motif includes at least two or more [3-sheets. In some embodiments, the repeatable motif includes one or more [3-barrels.

[0111] In some embodiments, the adaptor polypeptide includes at least one aromatic amino acid positioned within its hydrophobic core. In some embodiments, the adaptor polypeptide includes at least one amino acid substitution from a non-aromatic amino acid to an aromatic amino acid positioned within its hydrophobic core. In some embodiments, the adaptor polypeptide includes at least one amino acid substitution from an aromatic amino acid to a non- aromatic amino acid positioned within its hydrophobic core. In some embodiments, the at least one aromatic amino acid is tyrosine, tryptophan, or phenylalanine. In some embodiments, modulating the number of aromatic amino acids within the hydrophobic core of the adaptor polypeptide alters the conductance of a bioelectronic device.

[0112] In some embodiments, one or more amino acid residues on the adaptor polypeptide can be functionalized. In some embodiments, the adaptor polypeptide comprises a C-terminal and / or N-terminal cysteine residue. In some embodiments, the adaptor polypeptide is directly coupled to at least one of the first and second electrodes. In some embodiments, the adaptor polypeptide is coupled to at least one of the first and second electrodes via a linker. In some embodiments, the linker is selected from the group consisting of a biotin-streptavidin linker, a SpyTag-SpyCatcher linker, a halotag linker, and a thiolated CTPR linker. In some embodiments, the adaptor polypeptide is directly coupled to the protein. In some embodiments, the adaptor polypeptide is coupled to the protein electrodes via a linker. In some embodiments, the linker is selected from the group consisting of a biotin-streptavidin linker, a SpyTag- SpyCatcher linker, a halotag linker, and a thiolated CTPR linker.

[0113] In some embodiments, the presence of the adaptor polypeptide in a bioelectronic device reduces conductance decay as the gap between the first and second electrodes increases. As would be recognized by one of ordinary skill in the art based on the present disclosure, many different polypeptides and proteins can be used as an adaptor polypeptide in the context of the bioelectronic devices described herein. Generally, an adaptor polypeptide comprises a hydrophobic core, a hydrophilic exterior, at least one repeatable motif to facilitate size adjustment, and can form a stable conformation. In some embodiments, the adaptor polypeptide comprises a consensus tetratricopeptide repeat (CTPR) adaptor polypeptide. In some embodiments, the CTPR is selected from the group consisting of CTPR1, CTPR2, CTPR3, CTPR4, CTPR5, CTPR6, CTPR7, CTPR8, CTPR9, CTPR10, CTPR11, CTPR12, CTPR13, CTPR14, CTPR15, CTPR16, CTPR17, CTPR18, CTPR19, CTPR20, and any derivatives or variants thereof. In some embodiments, the device comprises two CTPR adaptor polypeptides,and the two CTPR adaptor polypeptides are the same size. In some embodiments, the device comprises two CTPR adaptor polypeptides, and each CTPR adaptor polypeptide is a different size. In some embodiments, the linker and / or the at least one adaptor polypeptide is coupled to an inactive region of the protein-of-interest.

[0114] In some embodiments, the linker comprises a distinctive negative charge. In some embodiments, the distinctive negative charge is conferred by addition of a glutamate moiety, an aspartate moiety, or a combination thereof, coupled to the linker. In some embodiments, the distinctive negative charge increases conductance through the protein. In some embodiments, the linker comprises a distinctive positive charge. In some embodiments, the distinctive positive charge is conferred by addition of an arginine moiety, a histidine moiety, a lysine moiety, or a combination thereof, coupled to the linker. In some embodiments, the distinctive positive charge increases or decreases conductance through the protein.

[0115] Embodiments of the present disclosure also include a system comprising a plurality of the devices described herein. In some embodiments, the systems of the present disclosure are used for direct electrical measurement of protein activity. In accordance with these embodiments, the system includes any of the bioelectronic devices described herein, a means for introducing an analyte capable of interacting with the protein, a means for applying a voltage bias between the first and second electrodes that is 1 OOmV or less, and a means for monitoring fluctuations that occur as the chemical entity interacts with the protein.

[0116] Embodiments of the present disclosure also include an array comprising a plurality of any of the bioelectronic devices described herein. In some embodiments, the array includes a means for introducing an analyte capable of interacting with the protein, a means for applying a voltage bias between the first and second electrodes that is lOOmV or less, and a means for monitoring fluctuations that occur as the chemical entity interacts with the protein. The array can be configured in a variety of ways, as would be appreciated by one of ordinary skill in the art based on the present disclosure.

[0117] Embodiments of the present disclosure also include a method for direct electrical measurement of protein activity. In accordance with these embodiments, the method includes introducing an analyte capable of interacting with the protein to any of the bioelectronic devices described herein, applying a voltage bias between the first and second electrodes that is 1 OOmV or less, and observing fluctuations in current between the first and second electrodes that occur when the analyte interacts with the protein. In some embodiments, the analyte is a biopolymer, such as, but not limited to, a DNA molecule, an RNA molecule, a peptide, a polypeptide, or a glycan.3. Methods of Use

[0118] Embodiments of the present disclosure also include methods of measuring electronic conductance through a protein using any of the devices and systems described herein. In accordance with these embodiments, the present disclosure includes methods for direct electrical measurement of protein activity. In some embodiments, the method includes introducing an analyte capable of interacting with the protein to any of the bioelectronic devices described herein, applying a voltage bias between the first and second electrodes that is 100 mV or less, and observing fluctuations in current between the first and second electrodes that occur when the analyte interacts with the protein. In some embodiments, the analyte is a biopolymer selected from the group consisting of a DNA molecule, an RNA molecule, a peptide, a polypeptide, and a glycan. In some embodiments, methods of the present disclosure include use of the devices and systems described herein to sequence a biopolymer. In some embodiments, the present disclosure includes methods for sequencing a polynucleotide using a bioelectronic device that obtains a bioelectronic signature of polymerase activity based on current fluctuations as complementary nucleotidepolyphosphate monomers are incorporated into the template polynucleotide.

[0119] As described further herein, the devices, systems, and methods of the present disclosure can be used to generate a bioelectronic signature of an enzyme-of-interest, which can be used to determine the sequence of any biopolymer (e.g., polynucleotide). In some embodiments, the enzyme-of-interest can be a polymerase, and various aspects of a bioelectronic signature of a polymerase as it adds nucleotide monomers to a template polynucleotide strand can be used to determine the sequence of that template polynucleotide. For example, a bioelectronic signature of polymerase activity can be based on current fluctuations as each complementary nucleotide monomer is incorporated into the template polynucleotide. In some embodiments, the bioelectronic device used to generate a bioelectronic signature comprises a polymerase functionally coupled to both a first electrode and a second electrode using the adaptor polypeptides of the present disclosure. The term “nucleotide” generally refers to a base-sugar-phosphate combination and includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof.

[0120] As one of ordinary skill in the art will readily recognize and appreciate after having benefited from the teachings of the present disclosure, the methods described herein can be used with any bioelectronic device that senses the duration of the open and closed states of anenzyme (e.g., polymerase). Exemplary devices include, but are not limited to, the bioelectronic devices and systems disclosed in U.S. Patent No. 10,422,787 and PCT Appln. No. PCT / US2019 / 032707, both of which are herein incorporated by reference in their entirety and for all purposes. Additionally, it will be readily recognized and appreciated by those of ordinary skill in the art based on the present disclosure that the forgoing embodiments apply equally to (and include) sequencing RNAs with the substitution of rNTPs for dNTPs and the use of an RNA polymerase.

[0121] Further, one of ordinary skill in the art would readily recognize and appreciate that the methods described herein can be used in conjunction with other methods involving the sequencing of a biopolymer. In particular, the various embodiments disclosed in PCT Application No. PCT / US21 / 19428, which is herein incorporated by reference in its entirety, describes the interpretation of current fluctuations generated by a DNA polymerase as it actively extends a template, and how signal features (e.g., bioelectronic signature) may be interpreted in terms of the nucleotide being incorporated, and thus, how these signals can read the sequence of the template. This approach utilizes features of the signal that vary in time. For example, the time that the polymerase stays in a low current state reflects the concentration of the nucleotidetriphosphate in solution. If the concentration of a particular nucleotidetriphosphate is low, then the polymerase must stay open for a longer time in order to capture the correct nucleotide, and since the open conformation of the polymerase corresponds to a lower current, the dip in current associated with the open state lasts for longer. Additionally, the various embodiments disclosed in PCT Application No. PCT / US20 / 38740, which is herein incorporated by reference in its entirety, describes how the base-stacking polymerization rate constant differences are reflected in the closed-state (high current states) so that the duration of these states may also be used as an indication of which one of the four nucleotides is being incorporated. It can be desirable to be able to use the amplitude of the signal as yet an additional contribution to determining sequence. Further, the various embodiments disclosed in PCT Application No. PCT / US21 / 17583, which is herein incorporated by reference in its entirety, describes methods that utilize a defined electrical potential to maximize electrical conductance of a protein-of-interest (e.g., polymerase), which can serve as a basis for the fabrication of enhanced bioelectronic devices for the direct measurement of protein activity. Additionally, the various embodiments disclosed in PCT Application No. PCT / US21 / 30239, which is herein incorporated by reference in its entirety, describes methods for sequencing a polynucleotide using a bioelectronic device that obtains a bioelectronic signature of polymerase activity based on current fluctuations as complementarynucleotidepolyphosphate monomers having distinctive charges are incorporated into the template polynucleotide.

[0122] Although certain embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a wide variety of alternate and / or equivalent embodiments or implementations calculated to achieve the same purposes may be substituted for the embodiments shown and described without departing from the scope. Those with skill in the art will readily appreciate that embodiments may be implemented in a very wide variety of ways. This application is intended to cover any adaptations or variations of the embodiments discussed herein. Therefore, it is manifestly intended that embodiments be limited only by the claims and the equivalents thereof.

Claims

CLAIMSWhat is claimed is:

1. A bioelectronic device comprising: a first electrode and a second electrode separated by a gap; a protein attached to the first and second electrodes via a linker; and at least one conductive anchor coupled to the protein, thereby minimizing contact resistance of the protein.

2. The device of claim 1, wherein the at least one conductive anchor comprises one or more of the following:(i) a small molecule comprising an aromatic moiety; and / or(ii) a protein-based redox system comprising at least one metalloprotein.

3. The device of claim 1 or claim 2, wherein at least one conductive anchor is coupled to the outside of the protein.

4. The device of any one of claims 1 to 3, wherein at least one conductive anchor is coupled to the N-terminal or C-terminal of the protein.

5. The device of any one of claims 1 to 4, wherein at least one conductive anchor is coupled to the protein via a covalent chemical bond.

6. The device of any one of claims 1 to 5, wherein at least one conductive anchor is coupled to the protein via at least one conductive anchor binding moiety that is coupled to the protein.

7. The device of claim 6, wherein at least one conductive anchor binding moiety comprises a cysteine-XX-cysteine-histidine (CXXCH) motif, wherein X is any amino acid.

8. The device of claim 6, wherein at least one conductive anchor binding moiety comprises a protein maquette comprising at least one alpha helix.

9. The device of claim 7 or claim 8, wherein the at least one conducive anchor comprises a heme moiety.

10. The device of any one of claims 1 to 9, wherein the at least one conductive anchor is coupled to the protein such that it is positioned from about 1 nm to about 2 nm from an attachment point linking the protein to the first or second electrode.

11. The device of any one of claims 1 to 10, wherein the at least one conductive anchor is coupled to the protein such that it is positioned no greater than about 1.4 nm from an attachment point linking the protein to the first or second electrode.

12. The device of any one of claims 1 to 10, wherein the at least one conductive anchor comprises a redox potential from about 100 mV to about 300 mV on the standard hydrogen electrode (SHE) scale.

13. The device of any one of claims 1 to 12, wherein the first and / or the second electrode comprises gold, palladium, platinum, silver, copper, carbon, indium tin oxide (ITO), or any derivatives or alloys thereof.

14. The device of any one of claims 1 to 13, wherein at least one conductive anchor comprises an energy level that matches the energy level of the at least one electrode.

15. The device of any one of claims 1 to 14, wherein the device comprises a dielectric layer at least partially covering a surface of the first and / or second electrode.

16. The device of any one of claims 1 to 15, wherein the first electrode and second electrode are positioned so that about a 1 nm to about a 50 nm gap is formed between the two electrodes.

17. The device of any one of claims 1 to 16, wherein the protein is attached to the first electrode via a first linker, and the second electrode is attached to the second electrode via a second linker, wherein each of the first and the second linkers comprises at least one chemical bond and is attached to a region of the protein that is inactive.

18. The device of any one of claims 1 to 17, wherein the protein is selected from the group consisting of a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase, and an endonuclease.

19. The device of claim 18, wherein the protein is a polymerase.

20. The device of claim 19, wherein exonuclease activity of the polymerase is disabled.

21. The device of any one of claims 17 to 20, wherein each of the first and the second linkers comprises a covalent chemical bond.

22. The device of claim 21, wherein the first and / or the second linker comprises a genetically modified streptavidin variant containing at least one point mutation resulting in a cysteine residue, and wherein the protein is biotinylated.

23. The device of claim 21, wherein the protein and the first and / or second electrodes are biotinylated, and wherein the first and / or the second linker comprises a streptavidin molecule.

24. The device of any one of claims 17 to 23, wherein the first and / or the second linker is attached to the N-terminal region of the protein, or the C-terminal region of the protein.

25. The device of claim 21, wherein the first or the second linker is attached to the exonuclease region of the polymerase.

26. The device of any one of claims 1 to 25, wherein the bioelectronic device further comprises at least one adaptor polypeptide.

27. The device of claim 26, wherein the at least one adaptor polypeptide comprises a consensus tetratricopeptide repeat (CTPR) adaptor polypeptide.

28. The device of claim 27, wherein the CTPR is selected from the group consisting of CTPR1, CTPR2, CTPR3, CTPR4, CTPR5, CTPR6, CTPR7, CTPR8, CTPR9, CTPR10, CTPR11, CTPR12, CTPR13, CTPR14, CTPR15, CTPR16, CTPR17, CTPR18, CTPR19, CTPR20, and any derivatives or variants thereof.

29. The device of claim 26, wherein the at least one adaptor polypeptide is directly coupled to at least one of the first and second electrodes.

30. The device of claim 26, wherein the at least one adaptor polypeptide is coupled to at least one of the first and second electrodes via a linker.

31. The device of claim 30, wherein the linker is selected from the group consisting of a biotin-streptavidin linker, a spytag-spycatcher linker, a halotag linker, a thiolated linker, a thiolated TPR linker, and a thiolated CTPR linker.

32. The device of claim 26, wherein the at least one adaptor polypeptide is directly coupled to the protein.

33. The device of claim 32, wherein the at least one adaptor polypeptide is coupled to the protein electrodes via a linker.

34. The device of claim 33, wherein the linker is selected from the group consisting of a biotin-streptavidin linker, a spytag-spycatcher linker, a halotag linker, a thiolated linker, a thiolated TPR linker, and a thiolated CTPR linker.

35. A system comprising a plurality of the devices of any one of claims 1 to 34.

36. A method for direct electrical measurement of protein activity, the method comprising: introducing an analyte capable of interacting with the protein of the device of any one of claims 1 to 34; applying a voltage bias between the first and second electrodes that is 100 mV or less; and observing fluctuations in current between the first and second electrodes that occur when the analyte interacts with the protein.

37. The method of claim 35, wherein the analyte is a biopolymer selected from the group consisting of a DNA molecule, an RNA molecule, a peptide, a polypeptide, and a glycan.

38. A method of manufacturing the device of any one of claims 1 to 34 using a cell-based system, wherein the protein is a polymerase, and wherein the cell is engineered to deliver the polymerase to the periplasm using a sacrificial peptide leader sequence to activate secretion mechanisms.

39. The method of claim 38, wherein the sacrificial leader sequence is PelB.

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