Bioelectronic circuits, systems, and methods of making and using the same

文档序号：474507 发布日期：2021-12-31 浏览：6次中文

阅读说明：本技术 生物电子电路、系统及其制备和使用方法 (Bioelectronic circuits, systems, and methods of making and using the same ) 是由 S.林德赛 B.张 H.邓于 2020-01-30 设计创作，主要内容包括：用于组装和电连接蛋白质以制造生物电子检测器和逻辑电路、探索配体-受体相互作用的电子性质和蛋白质内部的准金属性质的通用连接系统。(A universal linkage system for assembling and electrically connecting proteins to make bioelectronic detectors and logic circuits, exploring the electronic properties of ligand-receptor interactions and metalloids properties inside proteins.)

1. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) first and second ligands specific for a protein, wherein the ligands are modified such that the first ligand is attached to the first electrode and the second ligand is attached to the second electrode, and

(c) a protein modified to bind to the first and second ligands, wherein binding of the protein to the first and second ligands forms an electronic contact between the electrode and the protein.

2. The bioelectronic circuit of claim 1 wherein said electrode comprises a metal selected from the group consisting of: palladium, platinum or gold.

3. The bioelectronic circuit according to claim 1 or claim 2 wherein said ligand is selected from the group consisting of: HSCH₂CH₂Dinitrophenol, CALDRWEKIRLR (SEQ ID NO:1), CHNTPVYKLDISEATQV (SEQ ID NO:2), cyclic RGDfC, sulfurized streptavidin, and HSCH₂CH₂-biotin.

4. The bioelectronic circuit according to claim 1 or claim 2 wherein said protein is selected from the group consisting of: IgE anti-DNP, IgG anti-HIV, IgG anti-Ebola, Fab anti-Ebola, alpha_Vβ₃Integrin, and streptavidin.

5. The bioelectronic circuit according to claim 1 or claim 2 further comprising wherein the protein is a polymerase, endonuclease, helicase or proteasome.

6. The bioelectronic circuit according to claim 1 wherein at least one ligand is modified to comprise a thiol, amine, disulfide or cyanide moiety.

7. The bioelectronic circuit of claim 1 wherein said ligand is sulfurized streptavidin.

8. The bioelectronic circuit of claim 1 or claim 7 wherein said protein is modified to contain biotin.

9. The bioelectronic circuit of claim 1 or claim 2 wherein said protein is a bis-biotinylated polymerase.

10. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) a first ligand specific for a protein, wherein the first ligand is modified such that it is attached to the first electrode,

(d) a second ligand bound to the protein and modified to attach it to the second electrode, thereby forming electronic contact between the first and second electrodes and the protein.

11. The bioelectronic circuit of claim 10 wherein said binding site on said protein to said first ligand is proximal to a flexible linker sequence.

12. The bioelectronic circuit of claim 10 wherein said second binding site on said protein for said second ligand is adjacent to a flexible linker sequence.

13. The bioelectronic circuit of claim 11 or 12 wherein said flexible linker sequence comprises GNSTNGTSNGSS (SEQ ID NO: 3).

14. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) a first protein, wherein the first protein is attached to the first electrode via a biotin-streptavidin interaction,

(c) a second protein, wherein the second protein is attached to the first protein via a biotin-streptavidin interaction and is modified to bind to a ligand,

(d) a ligand that binds to the second protein and is modified so that it is attached to the second electrode, thereby forming electronic contact between the first and second electrodes and the first and second proteins.

15. The bioelectronic circuit of claim 14 wherein said first protein comprises two biotins.

16. The bioelectronic circuit of claim 15, wherein said second protein comprises two biotinylated Avitag sequences.

17. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) first and second proteins, wherein one or both of the first and second proteins are attached to the first and second electrodes through a biotin-streptavidin interaction, thereby forming an electronic contact between the first and second electrodes and one or both of the first and second proteins.

18. The bioelectronic circuit of claim 17, wherein the first binding site comprises one or more residues that can be biotinylated.

19. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) a first protein, wherein the first protein is attached to the first electrode via a biotin-streptavidin interaction,

(c) a second protein, wherein the second protein is attached to the first protein via a biotin-streptavidin interaction and to the second electrode via a biotin-streptavidin interaction,

(d) a third protein, wherein the third protein is attached to the first and second proteins via biotin-streptavidin interaction, thereby forming electronic contact between the first and second electrodes and the first, second, and third proteins.

20. A bioelectronic circuit comprising:

(a) a first electrode for forming a first electrode layer on a substrate,

(b) a first protein, wherein the first protein is attached to the first electrode via a biotin-streptavidin interaction,

(c) a second protein, wherein the second protein is attached to the first protein via a biotin-streptavidin interaction and to the second electrode via a biotin-streptavidin interaction,

(d) a second electrode in contact with an electrolyte and connected to the first electrode,

(e) means for applying a bias voltage to the substrate,

(f) means for sensing current, thereby forming electronic contact between the first and second electrodes and the first and second proteins.

21. A method of making a bioelectronic circuit, the method comprising:

(a) attaching at least one streptavidin molecule to at least one electrode,

(b) introducing a biotinylated protein to the streptavidin-electrode from step (a) to form a complex comprising the at least one electrode, the at least one streptavidin molecule, and the biotinylated protein.

22. A method for detecting polymerase activity, the method comprising introducing a solution of a DNA template and a nuclear triphosphate (nucleotphosphate) into the bioelectronic circuit of claim 1, wherein a polymerization product indicates that the polymerase is active.

23. A method for detecting the activity of a protein, the method comprising introducing a substrate for the protein into the bioelectronic circuit of claim 1 and detecting an electrical change.

24. A system for electrical measurement of protein activity, comprising:

(a) the bioelectronic circuit of claim 1,

(b) means for applying a bias voltage between said first and second electrodes, and

25. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(d) a third protein modified to bind to the first and second proteins, wherein binding of the third protein to the first and second proteins forms an electronic contact between the electrode and the protein.

26. The bioelectronic circuit of claim 25, wherein said first and second proteins are streptavidin.

27. The bioelectronic circuit of claim 25, wherein said first and second ligands are biotin.

28. The bioelectronic circuit of claim 25, wherein said third protein is biotinylated.

29. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(c) at least a first protein and a second protein, which bind to the first ligand and the second ligand and can be coupled to other proteins via the ligands, thereby expanding the range in which conduction is obtained.

Background

The use of streptavidin-biotin linkages to construct molecular assemblies is well known in the art and many reagents are commercially available. It is also well known that chemical coupling of proteins to electrodes enhances electron transfer between metal electrodes and proteins. This may take the form of specific covalent linkages¹Or functionalizing the electrode with small molecules (e.g., amino acids) that make the electrode hydrophilic and thus capable of forming hydrogen bonds with proteins.²

Although some proteins are known to have conductivity over very long distances^2-4However, it has recently become clear that such conductivity is only apparent when a specific chemical contact is made between the electrode and the protein, in particular a linkage based on the interaction of a cognate ligand with the hydrophobic interior of the protein that has evolved to interact with it.⁵In addition, this recent work shows that high electron conductivity (nS over multiple (2-20) nm distances) appears to be a common property of proteins, not just proteins that have evolved to perform electron transport functions. Such behavior is referred to herein as metalloid conduction. Finally, this work also shows that binding of a specific ligand molecule (itself bound to the electrode by a thiol linkage) to the protein results in better contact with the protein than if the protein itself was directly attached to the electrode via surface thiol modification of the protein. Thus, a ligand that can bind via a weak and reversible bond but in so doing contacts the hydrophobic interior of a protein is a better electrical connector than a strong irreversible covalent bond to a residue on the hydrophilic exterior of a protein. The most robust method of making electrical contact between a protein and an electrode, or between one protein and another, is to take advantage of the specific chemical contact that proteins evolve to form: i.e., ligand-receptor interactions. In addition, there is a theoretical basis⁶It is believed that the protein internally exhibits metalloid properties as an electron transport material. This is consistent with the discovery by Zhang et al that is, in non-electronic deliveryIn a series of proteins, the electrical conductance is too high to measure (i.e., contact limited).⁵The same properties are present in electron transport proteins (where they may be desirable depending on biological needs). Interestingly, all the tested functional proteins have this metalloid property, although the evolutionary role of this property in proteins that do not perform electron transport functions is not known at present.

Disclosure of Invention

The present disclosure provides bioelectronic circuits, systems, and methods of making and using the same.

In one embodiment, a bioelectronic circuit is provided. In one aspect of this embodiment, the bioelectronic circuit comprises (a) at least one electrode, (b) at least one ligand specific for a protein, wherein the ligand is modified such that it is attached to the at least one electrode, and (c) at least one protein that binds to the ligand, thereby forming an electronic contact between the electrode and the protein.

In a second aspect, a bioelectronic circuit comprises (a) first and second electrodes, (b) first and second ligands specific for a protein, wherein the ligands are modified such that the first ligand is attached to the first electrode and the second ligand is attached to the second electrode, and (c) a protein modified to bind to the first and second ligands, wherein the binding between the protein and the first and second ligands forms an electronic contact between the electrodes and the protein.

In a third aspect, a bioelectronic circuit comprises (a) first and second electrodes, (b) a first ligand specific for a protein, wherein the first ligand is modified such that it is attached to the first electrode, (c) a protein that binds to the first ligand and is modified such that it binds to the second ligand, and (d) a second ligand that binds to the protein and is modified such that it is attached to the second electrode, thereby forming an electronic contact between the first and second electrodes and the protein.

In a fourth aspect, a bioelectronic circuit comprises (a) first and second electrodes, (b) a first protein, wherein the first protein is attached to the first electrode via a biotin-streptavidin interaction, (c) a second protein, wherein the second protein is attached to the first protein via a biotin-streptavidin interaction and is modified to bind to a ligand, (d) a ligand that binds to the second protein and is modified to attach it to the second electrode, thereby forming electronic contact between the first and second electrodes and the first and second proteins.

In a fifth aspect, a bioelectronic circuit comprises (a) first and second electrodes, (b) first and second proteins, wherein one or both of the first and second proteins are attached to the first and second electrodes via biotin-streptavidin interaction, thereby forming an electronic contact between the first and second electrodes and one or both of the first and second proteins.

In a sixth aspect, a bioelectronic circuit comprises (a) first and second electrodes, (b) a first protein, wherein the first protein is attached to the first electrode via a biotin-streptavidin interaction, (c) a second protein, wherein the second protein is attached to the first protein via a biotin-streptavidin interaction and to the second electrode via a biotin-streptavidin interaction, (d) a third protein, wherein the third protein is attached to the first and second proteins via a biotin-streptavidin interaction, thereby forming electronic contacts between the first and second electrodes and the first, second and third proteins.

In a seventh aspect, a bioelectronic circuit comprises (a) a first electrode, (b) a first protein, wherein the first protein is attached to the first electrode via biotin-streptavidin interaction, (c) a second protein, wherein the second protein is attached to the first protein via biotin-streptavidin interaction and to the second electrode via biotin-streptavidin interaction, (d) a second electrode in contact with an electrolyte and connected to the first electrode, (e) means for applying a bias voltage, (f) means for sensing a current, thereby forming electronic contact between the first and second electrodes and the first and second proteins.

In an eighth aspect, the bioelectronic circuit is as described herein, wherein the protein comprises two or more Avitag sequences located on the surface of the protein and no more than 10 amino acid residues from tyrosine, tryptophan, or histidine within the protein.

In a ninth aspect, a bioelectronic circuit comprises (a) first and second electrodes, (b) first and second ligands specific for a protein, wherein the ligands are modified such that the first ligand is attached to the first electrode and the second ligand is attached to the second electrode, and (c) first and second proteins that bind to the first and second ligands, and (d) a third protein modified to bind to the first and second proteins, wherein binding of the third protein to the first and second proteins forms an electronic contact between the electrodes and the protein.

In a tenth aspect, a bioelectronic circuit comprises (a) first and second electrodes, (b) first and second ligands specific for a protein, wherein the ligands are modified such that the first ligand is attached to the first electrode and the second ligand is attached to the second electrode, (c) at least a first protein and a second protein that bind to the first ligand and the second ligand and that can be coupled to other proteins via the ligands, thereby enlarging the range over which conduction is obtained.

In another embodiment, a system for electrical measurement of protein activity is provided. The system comprises (a) a bioelectronic circuit as described herein, (b) means for applying a bias voltage between the first and second electrodes, and (c) means for detecting a current through the bioelectronic circuit.

In another embodiment, a method of making a bioelectronic circuit is provided. In one aspect, the method comprises (a) attaching at least one streptavidin molecule to at least one electrode, (b) introducing a biotinylated protein into the streptavidin-electrode from step (a) to form a complex comprising the at least one electrode, the at least one streptavidin molecule, and the biotinylated protein.

In another embodiment, a method for detecting polymerase activity, the method comprising introducing a solution of a DNA template and a nuclear triphosphate (nucleotphosphate) into any of the bioelectronic circuits described herein, wherein a polymerization product indicates that the polymerase is active.

In another embodiment, a method for detecting protein activity. The method comprises introducing a substrate for the protein into any of the bioelectronic circuits described herein and detecting the electrical change.

Drawings

FIG. 1 shows the chemical structure of a biotin cystamine molecule.

FIG. 2 shows the chemical structure of the reduced form of the molecule shown in FIG. 1.

FIG. 3 shows a schematic of a bioelectronic circuit for wiring a biotin-modified polymerase (wire) into the circuit using a streptavidin wire.

FIG. 4 shows a gel assay demonstrating that biotinylated polymerase bound to two streptavidin molecules is active and able to effectively perform an enzymatic action.

Fig. 5A and 5B show a comparison of the measured conductance profiles of the singly-biotinylated and doubly-biotinylated polymerases.

FIGS. 6A and 6B show a log of current versus time for a polymerase molecule wired into a bioelectronic circuit with streptavidin.

FIGS. 7A and 7B show a comparison of the measured conductance profile of sulfurized streptavidin bound to biotin and coupled directly to the electrode with the measured conductance profile of wild-type streptavidin bound to the electrode by the sulfurized biotin shown in FIG. 2. Here, biotin is shown in an enlarged scale for clarity.

FIG. 8 shows a schematic of a directional circuit element using more than one selective ligand to form a biomolecule AND gate (AND gate).

FIGS. 9A and 9B show selected cloning constraints on protein sequence modifications.

Fig. 10A and 10B show an example of a protein sequence of phi9 polymerase modified to include two Avitag linkers, showing how the incorporation of charged residues can lead to misfolding.

FIGS. 11A and 11B show the conductance profiles measured for two polymerases with and without flexible linker sequences when they were wired into a circuit through a biotin-streptavidin linker.

FIG. 12 shows a schematic of a biomolecule OR gate (OR gate).

FIG. 13 shows a schematic diagram of a biomolecule tri-state gate (three-state gate).

Figure 14 shows a schematic of a single electrode circuit in which the second contact is formed by a redox active protein soaked in an electrolyte solution.

FIG. 15 shows a bioelectronic circuit in which unmodified streptavidin is tethered to an electrode by the sulfurized biotin of FIG. 2.

FIG. 16 shows a bioelectronic circuit in which coupling is extended in distance by incorporation of a second streptavidin and the bisbiotin molecule of FIG. 1.

Detailed Description

The invention comprises the following contents:

1. a bioelectronic circuit as shown and described.

2. A system for the electrical measurement of protein activity as shown and described.

3. Methods for detecting protein activity as shown and described.

4. A bioelectronic circuit comprising:

(a) at least one of the electrodes is provided with a plurality of electrodes,

(b) at least one ligand specific for a protein, wherein the ligand is modified such that it is attached to at least one electrode, and

(c) at least one protein that binds to the ligand, thereby forming an electronic contact between the electrode and the protein.

5. The bioelectronic circuit according to 4 above wherein the electrode comprises a metal selected from the group consisting of: palladium, platinum or gold.

6. The bioelectronic circuit according to 4 or 5 above, wherein the ligand is selected from the group consisting of: HSCH₂CH₂Di-nitro (E) -dinitratePhenylphenol, CALDRWEKIRLR (SEQ ID NO:1), CHNTPVYKLDISEATQV (SEQ ID NO:2), cyclic RGDfC, sulfurized streptavidin, and HSCH₂CH₂-biotin.

7. The bioelectronic circuit according to any of the above 4-6, wherein the protein is selected from the group consisting of: IgE anti-DNP, IgG anti-HIV, IgG anti-Ebola, Fab anti-Ebola, alpha_Vβ₃Integrin, and streptavidin.

8. The bioelectronic circuit according to any one of claims 4-6 above, further comprising wherein the protein is a polymerase, endonuclease, helicase or proteasome.

9. The bioelectronic circuit according to 4 above wherein at least one ligand is modified to comprise a thiol, amine, disulfide or cyanide moiety.

10. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) first and second ligands specific for the protein, wherein the ligands are modified such that the first ligand is attached to the first electrode and the second ligand is attached to the second electrode, and

(c) a protein modified to bind to the first and second ligands, wherein binding of the protein to the first and second ligands forms an electronic contact between the electrode and the protein.

11. The bioelectronic circuit of above 10, wherein the ligand is sulfurized streptavidin.

12. The bioelectronic circuit of 10 or 11 above, wherein the protein is modified to comprise biotin.

13. The bioelectronic circuit of any of the above 10-12, wherein the protein is a bis-biotinylated polymerase.

14. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) a first ligand specific for a protein, wherein the first ligand is modified such that it is attached to a first electrode,

(d) a second ligand that binds to the protein and is modified such that it is attached to the second electrode, thereby forming electronic contact between the first and second electrodes and the protein.

15. The bioelectronic circuit of above 14 wherein the binding site on the protein to the first ligand is proximal to the flexible linker sequence.

16. The bioelectronic circuit of above 14 wherein the second binding site on the protein for the second ligand is proximal to the flexible linker sequence.

17. The bioelectronic circuit of claim 15 or 16 above, wherein the flexible linker sequence comprises GNSTNGTSNGSS (SEQ ID NO: 3).

18. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) a first protein, wherein the first protein is attached to the first electrode via a biotin-streptavidin interaction,

(c) a second protein, wherein the second protein is attached to the first protein via a biotin-streptavidin interaction and is modified to bind to a ligand,

(d) a ligand that binds to the second protein and is modified such that it is attached to the second electrode, thereby forming electronic contact between the first and second electrodes and the first and second proteins.

19. The bioelectronic circuit of above 18 wherein the first protein comprises two biotins.

20. The bioelectronic circuit of above 19, wherein the second protein comprises two biotinylated Avitag sequences.

21. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) a first and a second protein, wherein one or both of the first and second proteins are attached to the first and second electrodes by a biotin-streptavidin interaction, thereby forming an electronic contact between the first and second electrodes and one or both of the first and second proteins.

22. The bioelectronic circuit of above 21, wherein the first binding site comprises one or more residues that can be biotinylated.

23. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(b) a first protein, wherein the first protein is attached to the first electrode via a biotin-streptavidin interaction,

(c) a second protein, wherein the second protein is attached to the first protein via a biotin-streptavidin interaction and to the second electrode via a biotin-streptavidin interaction,

24. A bioelectronic circuit comprising:

(a) a first electrode for forming a first electrode layer on a substrate,

(b) a first protein, wherein the first protein is attached to the first electrode via a biotin-streptavidin interaction,

(c) a second protein, wherein the second protein is attached to the first protein via a biotin-streptavidin interaction and to the second electrode via a biotin-streptavidin interaction,

(d) a second electrode in contact with the electrolyte and connected to the first electrode,

(e) means for applying a bias voltage to the substrate,

(f) means for sensing the current, thereby forming electronic contact between the first and second electrodes and the first and second proteins.

25. A method of making a bioelectronic circuit, the method comprising:

(a) attaching at least one streptavidin molecule to at least one electrode,

(b) introducing a biotinylated protein to the streptavidin-electrode from step (a) to form a complex comprising at least one electrode, at least one streptavidin molecule, and the biotinylated protein.

26. A method for detecting polymerase activity, the method comprising introducing a solution of a DNA template and a triphosphate nucleus into the bioelectronic circuit of above 4, wherein a polymerization product indicates that the polymerase is active.

27. A method for detecting the activity of a protein, the method comprising introducing a substrate for the protein into the bioelectronic circuit of 4 above and detecting an electrical change.

28. A system for electrical measurement of protein activity, comprising:

(a) as described herein with respect to the bioelectronic circuit,

(b) means for applying a bias voltage between the first and second electrodes, and

29. The bioelectronic circuit as described herein, wherein a protein comprises two or more Avitag sequences located at the surface of the protein and no more than 10 amino acid residues from tyrosine, tryptophan, or histidine within the protein.

30. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

31. The bioelectronic circuit of above 30, wherein the first and second proteins are streptavidin.

32. The bioelectronic circuit of above 30, wherein the first and second ligands are biotin.

33. The bioelectronic circuit of above 30, wherein the third protein is biotinylated.

34. A bioelectronic circuit comprising:

(a) a first and a second electrode, wherein the first and the second electrodes are arranged in a matrix,

(c) at least a first protein and a second protein, which are bound to a first ligand and a second ligand and can be coupled to other proteins via the ligands, thereby expanding the range in which conduction is obtained.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The term "an item" may denote more than one item.

The terms "and" or "may refer to conjunctions or disjunctions and mean" and/or ".

The term "about" means within ± 10% of the stated value. For example, "about 100" would mean any number between 90-110.

Bioelectronic circuit

The present disclosure provides a bioelectronic circuit. The bioelectronic circuit comprises (a) at least one electrode, (b) at least one ligand specific for a protein, wherein the ligand is modified such that it is attached to the at least one electrode, and (c) at least one protein that binds to the at least one ligand, thereby forming an electronic contact between the electrode and the protein.

Depending on the application, the bioelectronic circuit comprises one or two electrodes in direct contact with the protein. In the case where only one electrode is in contact with the protein, the circuit is completed by an ionic current flowing between the redox-active protein and the remote electrode. In one embodiment, the bioelectronic circuit comprises one electrode. In another embodiment, the bioelectronic circuit comprises two electrodes. In some aspects of this embodiment, the second electrode is not separated from the first electrode by the electrolyte.

At least one of the electrodes comprises a noble metal. In one embodiment, at least one electrode comprises a noble metal selected from the group consisting of: palladium, gold and platinum. In another embodiment, at least one electrode is palladium. In another embodiment, at least one electrode is gold. In another embodiment, at least one electrode is platinum. In addition to the at least one electrode, the bioelectronic circuit further comprises a ligand specific for the protein and modified to attach it to the at least one electrode.

The ligand may be modified to contain a thiol terminus at one end for coupling to a metal as described by Zhang et al.⁵Examples of ligands are peptide epitopes of antibodies comprising cysteine residues at one end, recognition peptides (e.g. RGD peptides comprising cysteine for binding to integrins) and small molecules to which selected proteins bind (e.g. IgE molecules comprising thiols binding to dinitrophenyl).

Exemplary ligands specific for proteins and modified to attach to electrodes include, but are not limited to, HSCH₂CH₂Dinitrophenol (target protein IgE anti-DNP), CALDRWEKIRLR (target protein IgG anti-HIV) (SEQ ID NO:1), CHNTPVYKLDISEATQV (target protein IgG anti-Ebola) (SEQ ID NO:2), CHNTPVYKLDISEATQV (target protein Fab anti-Ebola) (SEQ ID NO:2), cyclic RGDfC (target protein alpha_Vβ₃Integrin), thiostreptavidin (biotin of interest), and HSCH₂CH₂Biotin (target streptavidin). A summary of these different binding arrangements is given in Table 1 below, adapted from Zhang et al.⁵

Table 1.

Proteins and ligands used in this study. Cysteine or thiol for electrode attachment is shown in bold. Linear dimensions are from RSCB PDB, either across the smallest diameter, or, for antibodies, from binding head to binding head:¹the IgE structure 4GRG is shown in the specification,²IgG, Structure 4NHH, Fab fragment Structure 1YUH,⁴the integrin structure 1L5G was,⁵streptavidin structure 1 VWA.

The bioelectronic circuit further comprises at least one protein bound to at least one ligand. The protein may be any protein capable of being expressed in a medium that allows modification of the native protein sequence. Thus, any protein function can be incorporated into the circuit such that changes induced by ligand or substrate binding or enzyme activity can be subsequently electrically measured.

In one embodiment, the bioelectronic circuit comprises (a) two palladium electrodes, (b) sulfurized streptavidin attached to the palladium electrodes, and (c) a biotinylated polymerase, thereby forming an electronic contact between the electrodes and the polymerase. It should be understood that although the bioelectronic circuit described below includes a palladium electrode, sulfurized streptavidin, and a biotinylated polymerase, this embodiment is illustrative of the present disclosure and the scope of the present disclosure is not limited to this one embodiment.

In one aspect of this embodiment, sulfurized streptavidin having an average of 2.5 thiols per tetramer is obtained from ProteinMods (Madison, Wisconsin). Streptavidin (31 in fig. 3) was incubated in 1 μ M aqueous solution with a pair of noble metal (palladium) electrodes (the pair of electrodes formed nanoscale nodules with approximately 5nm gaps, 32, 33 in fig. 3). Overnight incubation produced a dense coating of streptavidin attached to the metal electrode by surface thiols (34).

To complete the bioelectronic circuit, a double biotinylated φ 29 polymerase (35 in FIG. 3) was constructed. The Avitag peptide sequence, GLNDIFEAQKIEWHE (SEQ ID NO:4), was inserted between residues G111 and K112, and a second Avitag peptide sequence was inserted between E279 and D280 of the φ 29 polymerase sequence (whose exonuclease activity was deleted by mutating D12 and E14 to alanine). This produced a polymerase with two Avitags about 5nm apart. Avitag was biotinylated using BirA enzyme, as is well known in the art. Thus, the resulting molecule (35) comprises two biotin molecules (36 in fig. 3). The biotinylated polymerase was incubated with streptavidin-functionalized knots for two hours (in a 1. mu.M aqueous solution of φ 29 polymerase). The electrical properties of the bioelectronic circuit are measured by applying a bias voltage V (37) thereacross and recording the current (38) through the circuit. As noted by Zhang et al,⁵this bias must be less than 100mV to avoid noise from the touch, and the data shown here is collected using a 50mV bias.

To confirm that the modified polymerase is still active, rolling circle amplification of the DNA template is performed with molecules 35 bound to two streptavidin molecules. The polymerase activity monitored by the DNA gel of the polymerization product (42 in FIG. 4) was comparable to the native enzyme (41 in FIG. 4) from which the exonuclease activity had been deleted.

To confirm that the double biotinylated molecule 35 bridges the junction, a single biotinylated φ 29 polymerase was prepared. To this end, the following sequence was added to the N-terminus of the WT (but exonuclease inactive) enzyme: MGSSHHHHHHSSGLVPRGSGLNDIFEAQKIEWHEGASS (SEQ ID NO:5), where five histidines are his-tag for protein purification and GLNDIFEAQKIEWHE (SEQ ID NO:4) is Avitag.

To characterize the conductance of all possible binding geometries of molecular circuits, the following methods are providedRepeated measurements were taken on different molecular junctions and the frequency at which a particular conductance value was recorded on a logarithmic conductance scale was plotted (as described by Zhang et al)⁵). Fig. 5 shows such distributions for a single biotinylated polymerase (51) and a double biotinylated polymerase (52). The bisbiotinylated molecules showed a new high conductance profile (53), which was not present in the distribution of the single biotinylated molecules measured. This characteristic corresponds to a conductance of about 4nS, with a significant increase relative to the highest conductance characteristic observed for biotinylated molecules (about 0.7 nS). This is a similar increase to that observed for molecules like Zhang et al⁵The antibody linked by one or two specific contacts. Note that: these very high conductances were obtained using streptavidin molecules forming part of the circuit, indicating that the assembly of several proteins in the sequence retains its metalloid properties if correctly linked.

FIG. 6 shows the electrical signals obtained in the resting state 61 and the active state 62 of a bis-biotinylated polymerase molecule, wherein in the active state it actively polymerizes the DNA template in the presence of dNTPs and Mg. In each case, the current data was recorded about 90s after the electrode was moved into contact with the complex. The formation of the streptavidin- φ 29 polymerase contact takes about 20s, after which the current (at 50mV bias) jumps from 0 to about 60 pA. In the case of the control molecule 61, the current remained fairly quiet and constant during the remainder of the run. For the active molecules 62, a plurality of noise bursts 63 were observed, each lasting several seconds. It is also shown that the 50ms portion (64) of one of these bursts-the burst itself is composed of a number of sub-bursts (65), the duration and interval of which is in accordance with the following explanation: each sub-burst (65) marks the incorporation of a single nucleic acid (given the known φ 29 polymerase kinetics). This illustrates the use of the invention in the construction of a circuit capable of electrically monitoring enzyme activity.

Recognizing the importance of placing contacts as close as possible to the hydrophobic interior of the protein while still allowing the contact itself to appear on the surface of the protein, the insertion site for avitag should be placed as close as possible to the aromatic residue while still exposing the biotinylation site outside the protein. Therefore, Avitag should be placed as close as possible to tyrosine, tryptophan, or histidine near the surface of the protein.

In a second embodiment, the bioelectronic circuit comprises (a) two palladium electrodes, (b) biotin sulfide attached to the palladium electrodes, and (c) streptavidin. This embodiment allows for uniform coating of small molecules on the electrode.

In one aspect of this embodiment, biotinylated cystamine is used as the thiobiotin. N, N' -biotinyl-cystamine 11 (chemical structure shown in fig. 1) was synthesized as described in the examples. 11 are stable in an oxidizing environment (e.g., air) due to the presence of the S-S linkage (12 in FIG. 1). In the presence of a strong reducing agent, it can be reduced to monothiol 20 (the chemical shown in fig. 2). Reducing agents are known in the art, and any variety may be used. In some embodiments, the reducing agent is an immobilized TCEP (tris [ 2-carboxyethyl ] phosphine hydrochloride) disulfide reduction gel from Thermo Scientific (cat # 77712).

In this second embodiment, the electrode is functionalized with thiobiotin, and the junction is subsequently exposed to wild-type (i.e., streptavidin lacking surface thiols). The result is a strong electrically conductive bridge. This is illustrated in fig. 7. In the control experiment (71), the gap was bridged by a thiostreptavidin molecule that had been incubated with biotin (because, as described by Zhang et al⁵Biotin binding changes the conductance of streptavidin, so it is necessary to compare biotin-bound streptavidin molecules to see the inherent poor contact in these experiments). In a second measurement (72), the electrode is first functionalized with monosulfided biotin (20), followed by the introduction of wild-type streptavidin to bind and complete the circuit. The current distribution of sulfurized streptavidin (biotin-bound) directly attached to the electrode is shown at 73. The distribution of the captured wild-type streptavidin between the biotinylated electrodes is shown at 74. In the case of streptavidin attached to the electrode using a thiobiotin molecule (20), a new high conductance characteristic (75) was observed near 7 nS. It is worth noting thatThe linkage via a specific binding ligand (biotin), which is itself attached to the electrode via a thiol, may form a better contact than the direct attachment of the protein itself via a surface thiol on the protein.

In the embodiments described above, the protein comprises one ligand contact. In embodiments described in the following paragraphs, the protein comprises a second ligand contact.

For example, the integrin molecules listed in table 1 have a small ligand binding site with good electrical contact (e.g., cysteine-incorporating cyclic RGD peptides listed in table 1). But not the second site via another ligand because the integrin has only one RGD binding site. However, by incorporating the Avitag sequence into the region near the N-terminus of the integrin (near), a clear circuit can be completed, so it now has two specific binding sites: (1) an RGD peptide binding site and (2) a biotin binding site. Such heterogeneous contacts (peptide binding at one site and biotinylation at the other) have the following advantages: proteins can be oriented in the assembly by utilizing the selective attachment now incorporated into the protein. For example, one electrode may be functionalized with streptavidin and the second with a peptide (i.e., cyclic RGD), so that the modified protein will then always bind in a well-defined orientation with respect to the two electrodes. The same technique enables sequential assembly of protein circuits. This is shown by the protein AND gate ("AND" gate) shown in FIG. 8.

More specifically, protein a (81), which is designed or selected such that binding of ligand a causes an increase in conductivity, AND protein B (82), which is designed or selected such that binding of ligand B causes an increase in conductivity, are used in series between the electrodes, the pairing comprising a chemo-AND gate (AND gate) because only a high conductance state is obtained when both ligand a AND ligand B are present. Clearly, one way to ensure that the proteins are linked in the desired order is to use selective contacts. Thus, the first electrode, 84, is modified with a thiobiotin molecule 86 that binds to streptavidin 87. This in turn binds to protein a 81, which protein a 81 has been modified to have two biotinylated Avitag sequences 83 incorporated. Further incubation with streptavidin places a second streptavidin 84 on protein a. At this time, protein B82 can be bound using biotinylated Avitag sequence 83. If the same biotin-streptavidin coupling is used to complete the electrical contact with the second electrode 85, the undesirable likelihood that protein A will be incorporated at the second location where protein B is desired increases. To overcome this possibility, a heterogeneous linkage was used, in this case employing a peptide binding site 88 on protein B. The corresponding peptide ligand 89 bound to the second electrode 85 completes the circuit by binding to the cognate site on protein B. It will be appreciated that other useful building blocks may be created by linking ligands to each other. For example, by attaching a peptide ligand (such as one of those shown in table 1) to a biotin molecule, a first protein having a peptide-specific binding site can be linked to streptavidin, e.g., for subsequent incorporation into a circuit via the remaining unoccupied biotin binding sites on the streptavidin.

Another consideration that arises in connecting a functional protein to a circuit with two contact points is the possible disruption of function due to mechanical constraints created by tethering the protein at two points. The second attachment point, especially one chosen to be remote from the first point (in order to sense the movement of the protein), can obviously disrupt protein function when the connection at one point leaves the protein free to move as it would in solution. Therefore, it is highly desirable to incorporate a flexible linker in the region where the second contact site is incorporated. Of course, this may be at either of the two contact sites.

Amino acid sequences that form flexible linkers are well known in the art, and examples are listed on the world wide web bmrb. There are three important design considerations when selecting a particular flexible joint.

The first consideration is that the incorporated sequence should not consist of a short repetitive sequence, as this would complicate cloning. To illustrate this, consider the well-known flexible linker amino acid sequence: GGSGGSGGSGGS (SEQ ID NO: 6). The corresponding DNA template is shown in fig. 9A, which also shows dimers that may form between the primer sequences. When cloned into an expression vector, the results are shown in fig. 9B, which shows the sequence of the resulting plasmid with the product of tagged undesired primer dimer.

The second consideration is that the isoelectric point of the novel protein should not be significantly altered and therefore the residues in the inserted linker should be chosen to be neutral or near neutral. This is illustrated by the synthesis of phi29 polymerase containing two avitags to be wired into the circuit. In this case, the flexible linker is placed next to (next to) the Avitag sequence located near the N-terminus. In the first case (sequence 101 in fig. 10), the flexible joints (circled, 103) have the following sequence: GDSTDGTSDGSS (SEQ ID NO:7), the result was that the protein product had a calculated pI of 7.25. This small deviation from the pI of the native protein (which is a pI of about 8) is sufficient to cause misfolding which results in the protein appearing as a shorter product 105 in gel 106. When aspartic acid (D) in the linker sequence was changed to asparagine (N) (now GNSTNGTSNGSS (SEQ ID NO:3) -104 in sequence 102), the native pI was restored (pI 8.13), and the product now ran at the position of the correctly folded native protein (107 in gel 108).

The third consideration occurs when two identical sequences (i.e., two Avitag sequences) are inserted into the same clonal expression system because the corresponding repetitive DNA sequences result in primer dimers. To overcome this, cloning was performed in two steps. A clone with one Avitag sequence was first generated, followed by the generation of a second clone from the first clone, and the insertion of a second Avitag sequence.

In view of the flexibility of the GNSTNGTSNGSS (SEQ ID NO:3) linker and the factors that may require a specific protein fold to support this metalloid state inside the protein, a critical issue is whether the conductive properties of the protein are maintained when the linker is inserted. FIG. 11 shows a reference conductance profile 1101 for two Avitag polymerases, previously shown as 52 in FIG. 5 (lacking the flexible linker sequence). Here, the high conductance characteristic resulting from binding to the two biotinylation sites is labeled 1103. 1102 shows the conductance profile of phi29 incorporating a flexible linker (protein sequence 102 in FIG. 101). The high conductance features are retained (1104), indicating that a flexible peptide linker can be incorporated while maintaining the metalloids state of the protein.

Streptavidin is tetravalent (it binds to up to four biotins), and therefore, combining these remarkable and unexpected electronic properties, including the coupling scheme and the protein itself, a completely new approach to building a bioelectronic circuit becomes feasible.

FIG. 12 shows an OR gate (OR gate) based on the same assembly rules (numbered components as described in FIG. 8). If ligand A or ligand B is present, this will enter a high conductance state.

The tetravalent nature of streptavidin allows for the assembly of even more complex circuits. One way of this possibility is shown in fig. 13, where the numbering is also like in fig. 8). Here, the third protein "C" (1300) is attached to the knot of protein a and protein B at a site not occupied by the coupled streptavidin 87. If, for example, protein C is an electrochemically active protein that changes its oxidation state, e.g., in the presence of an oxidizing molecule, then a change in charge on protein C will modulate electron transport through the A-B chain. Thus, the "a" and "B" functions may result in a third state that depends on the electronic state of "C".

The embodiments described so far all make use of both the first and second electrodes. However, the metallo-conductance inside the proteins coupled with ligand-based linkages as described herein will allow for extremely efficient electron transfer at the sensor electrode surface immersed in a solution contained within the sensing protein (e.g., glucose receptors)¹) To produce redox active molecules. Thus, the protein or proteins of interest will only be linked directly to one electrode, as shown in fig. 14. Here, protein D1400 is a redox active protein (e.g., glucose oxidase) soaked in an electrolyte 1401, the electrolyte 1401 comprising a redox couple 1402, 1403 (e.g., oxidized and reduced glucose), and a second (distal) electrode 1404 in contact with the electrolyte 1401And is also connected to the first electrode 84 by means for applying a voltage bias 1405 and means for sensing a current 1406 flowing between the first electrode 84 and the second electrode 1404. This is an example of the use of the links of the present invention in applications to improve existing electrochemical sensing technology.

The bioelectronic circuit of fig. 3 requires the direct attachment of the modified conjugated protein to the electrode. However, as shown by Zhang et al, coupling would be more efficient if coupling was performed using an unmodified coupled protein with a small ligand attached to the electrode. This has the following advantages: functionalization with small ligands can be used, which makes it easier to obtain uniform coverage. Thus, fig. 15 shows the binding of unmodified streptavidin 1531 to the first electrode 1532 modified with thiobiotin 1534 of fig. 2, which unmodified streptavidin 1531 in turn binds to the biotinylated protein 1535 at two sites 1536. Similar functionalization completes coupling to the second electrode 1533.

As shown in fig. 16, by forming a daisy chain of coupled proteins with each electrode, the gap size for monitoring protein conduction can be significantly increased at very low conductivity cost. Here, a second conjugated protein 1602 is added to the circuit, coupled to the first 1531 by a divalent linker 1601 (e.g., the bis-biotin shown in fig. 1).

Examples

The following examples are set forth for illustrative purposes and should not be used to limit the scope of the disclosed subject matter.

Sources of materials

RGD peptide (cyclo (Arg-Gly-Asp-D-Phe-Cys)) was purchased from Peptides International (Louisville, Kentucky). Peptide ligands for anti-HIV antibodies and anti-Ebola antibodies were synthesized by CPC Scientific (Sunnyvale, California) with > 95% purity. DNP and biotin disulphide were synthesized in our laboratory (SI appendix S10 and S13) and reduced by immobilised TCEP (tris [ 2-carboxyethyl ] phosphine hydrochloride) disulphide reduction gel from Thermo Scientific (cat #77712) for 2 hours prior to use according to the manufacturing instructions. The preparation of the solutions used in this disclosure is described in the SI appendix. anti-DNP antibodies (mouse monoclonal IgE antibodies), wild-type streptavidin and all other chemicals were purchased from Sigma Aldrich (Saint Louis, Missouri). anti-HIV antibodies (anti-HIV 1p 17 antibody [32/1.24.89]) and all isotype controls were purchased from Abcam (Cambridge, MA). anti-Ebola antibodies were cultured from plants as described below. The binding affinity of all three antibodies was measured by Surface Plasmon Resonance (SPR). On average 2.5 thiols per tetramer of thiostreptavidin was from protein modules (Madison, Wisconsin). Ag/AgCl reference electrodes bridged by 3M KCl or 10mM KCl salt were prepared as previously described. Zhang et al, Nano Futures 1 (2017). The complete details of cyclic voltammetry are provided in the SI appendix. anti-Ebola antibodies and corresponding monomeric Fab fragments were prepared and purified as described in example 7.

Example 1 Synthesis of N, N' -bis-biotinyl-cystamine 11 (chemical structure shown in FIG. 1).

Cystamine dihydrochloride (60mg, 0.27mmol) was added to DMF (2mL) followed by triethylamine (0.44mL, 3.19 mmol). The mixture was stirred for 30min, biotin NHS ester (0.27g, 0.80mmol) was added thereto, and stirred at room temperature for 16 h. TLC indicated depletion of cystamine, yielding R in 20% methanol/DCM_fProduct with a value of 0.63. The mixture was co-evaporated with dichloromethane until most of the DMF and TEA were removed. The residue was separated on a column in an automated flash chromatograph Teledyne Isco using a gradient of 0-20% methanol in DCM over 120mins at a flow rate of 3 mL/min. The product was obtained as a white solid (0.11g and 67.1%).¹HNMR(400MHz，DMSO-d₆): δ 1.20-1.60(m, 12H), 2.07(t, 7.6Hz, 4H), 2.57(d, 12.4Hz, 2H), 2.77(t, 6.6Hz, 4H), 2.83(dd, 5.1 and 12.6, 2H), 3.07-3.12(m, 2H), 3.31(t, 6.4Hz, 4H), 4.11-4.15(m, 2H), 4.31(t, 5.2Hz, 2H);¹³CNMR(100MHz，DMSO-d₆)：δ25.70，28.50，28.65，35.62，37.83，38.37，40.32，55.92，59.68，61.52，163.18，172.69。

EXAMPLE 2 Synthesis of Compound 20

Compound 20 was produced by: n, N' -bis-biotinyl-cystamine 11 was exposed to an immobilized TCEP (tris [ 2-carboxyethyl ] phosphine hydrochloride) disulfide reduction gel from Thermo Scientific (cat #77712) for 2 hours (immediately prior to use in the equipment) according to the manufacturing instructions. The product (chemical structure shown in figure 2) was dissolved in 20% methanol/DCM, and this solution was subsequently used to modify the electrode.

Example 3 functionalization of substrates and STM probes

The palladium substrate for STM measurement was prepared as follows: a200 nm palladium film was evaporated onto silicon wafers using an electron beam evaporator (Lesker PVD 75) with a 10nm titanium adhesion layer. The substrate is functionalized immediately after treatment with hydrogen flame, followed by overnight soaking in a solution of sulfurized DNP biotin, streptavidin, or a peptide containing cysteine residues. Substrate functionalization with small ligands was characterized by Fourier Transform Infrared (FTIR) spectroscopy (fig. S9) and ellipsometry. Substrate coverage was monitored by STM and AFM imaging.

STM probes were etched from 0.25mm Pd Wires (California Fine Wires) by AC electrochemical methods. To avoid current leakage, the probes were insulated with high density polyethylene according to the method described previously for gold probes. M. tuchband et al, Rev Sci Instrum 83, 015102 (2012). Each probe was passed the STM test in 1mM PB buffer at +0.5V bias to ensure a leakage current <1 pA. For functionalization, the probe is soaked in the ligand solution for 4h or overnight. After this time, it was taken out, rinsed with water, blown dry lightly with nitrogen, and used immediately. Further details of STM measurements are given in example 4.

Example 4 STM measurement

STM measurements were performed on a PicoSPM scanning probe microscope (Agilent Technologies) coupled with a DAQ card (PCI-6821 or PCIE-7842R, National Instruments) for data acquisition. The Teflon cell, to which the buffer solution and analyte were added, was cleaned with Piranha solution followed by three sonications in Milli-Q water to remove the residue (note that Piranha solution is highly corrosive and extra care must be taken). To better control the surface potential, an Ag/AgCl reference electrode with a 10mM KCl salt bridge was attached to the substrate. The probe was first engaged with a-0.2V biased 4pA set point current and then allowed to stabilize for 2 hours before measurement. For STM IV scanning, the servo is first turned off and the probe is retracted Δ Z nm at a speed of 1 nm/s. The probe was then hovered at this height for 1 minute, during which time the current change was monitored using a custom Labview program. Once the current exceeds the 50pA threshold, a binding event is considered and a sweep from-0.2V to +0.2V is initiated, followed by a return, with a sweep rate of 1V/s, and then a 0.2s rest is performed. Subsequently, the current is checked again. If the current is still above twice the noise level (6pA), the IV curve is recorded continuously until the bound protein molecule escapes. After 1 minute of measurement, the servo was turned on to re-bind the probe, and the whole process was repeated. At least 1000 IV curves were collected in each measurement, from which curves with overlapping sweeps up and down (80% of the total) were selected to construct a conductance distribution histogram. The current versus time trace was recorded by another Labview program with a similar procedure except that the bias voltage was held constant during the probe hold. The analog-to-digital sampling rate is 50 KHz. The conductance measurement process was the same for all analytes but with different efficiencies due to differences in binding affinity and functionalization efficiency (table S1).

Table s1. the frequency at which the sweep generates the IV curve varies with gap size and functionalization. Z₀～2.5nm。

No curve was obtained using the control sample (table 1).

Repeated measurements were performed for each analyte with the newly functionalized substrate and probe (table S4). The potential relative to the reference electrode is set using a battery powered voltage source connected between the substrate and the reference electrode.

TABLE S4 number of replicates per experiment

EXAMPLE 5 solution preparation

Sulfurized DNP and biotin were prepared in fresh degassed pure ethanol at a final concentration of 0.1 mM. The peptide was first dissolved as such in degassed water, then aliquoted and frozen at-80 ℃. Before each use, an aliquot is removed and diluted with degassed water to the desired concentration, typically 0.1mM for substrate and probe functionalization. To reduce the DNP and peptide functionalization density on the substrate, 2-Mercaptoethanol (MCE) was added at a concentration of 2 mM. Antibodies and isotype controls were aliquoted and stored at-80 ℃ before dilution in 1mM PB buffer (pH = 7.4) prior to use. For STM and solid-state chip measurements, 100nM antibody or isotype control in 1mM Phosphate Buffer (PB) was used. For substrate functionalization, 1 μ M thiostreptavidin in 1mM PB buffer was prepared. 1mM free biotin in 1mM Pb buffer and 100nM wild-type streptavidin were used for conductivity measurements by STM. All buffers and solutions were prepared in Milli-Q water with a conductivity of 18.2m Ω. For all measurements, 1mM PB buffer (pH 7.4) was degassed with argon to avoid oxygen interference.

Example 6 Cyclic voltammetry

The Pd substrate was cut to a size of 0.5cm x 4.0cm and used as a working electrode, with an active cell area of about 0.5cm x 1.0 cm. Prior to functionalization, the substrate is treated with a hydrogen flame. Cyclic voltammetry was performed on a potentiostat (Model AFCBP1, Pine Instruments) using a Pt wire as counter electrode and Ag/AgCl (3M KCl) as reference electrode. Unless otherwise stated, the sweep ranged from-0.5V to +0.5V, with a sweep rate of 10 mV/s.

Example 7 expression and purification of anti-Ebola (EBOV) mAb 6D8 from Nicotiana benthamiana plants

The coding sequences for the heavy and light chains of 6D8 (Lai, H. et al, Plant Biotechnol J10, 95-104, doi: 10.1111/j.1467-7652.2011.00649.x (2012) were cloned into a Magnicon-based expression vector Lai, H. et al, Proc Natl Acad Sci U S A107, 2419. sup. 2424, doi: 10.1073/pnas.0914503107 (2010). 6D8 was then transiently expressed in the previously described native tobacco (N.benthamiana) plants.Yang, M. et al, Plant Biotechnol J16, 572. sup. 580, doi: 10.1111/pbi.12796 (2018). 6D8 was isolated from native tobacco (N.benthamiana) leaves by protein A affinity chromatography and purified to > 95% homogeneity A. 95. sup. 132. homogeneity, mAb. 132. g. 9. sup. 132).

Example 8.6 production of monomeric Fab of D8

Monomeric Fab fragments were prepared from 6D8 using the Pierce Fab preparation kit (Thermo Scientific) according to the manufacturing instructions (Thermo Scientific pub.no. man0011651). Briefly, purified 6D8 was first incubated with papain immobilized to agarose beads for 6-12hr at 37 ℃. The digested mAb mixture was then recovered by centrifugation at 5000xg for 1 minute and separated on a protein a chromatography column. The Fab fragments were collected in the flowthrough fraction while capturing the Fc fragment and undigested mAb on a protein a column. The successful production of monomeric Fab was confirmed by SDS-PAGE analysis under reducing and non-reducing conditions.

Single molecule conductance measurement

Reproducible two-point measurement of the conductance of a molecule requires reproducible contact,⁷it was therefore a surprising finding that large (nS scale) fluctuations in conductance were reproducibly observed in a single integrin molecule through which the cognate ligand bound to only one of the two electrodes. (8) This previous work did not detect the low bias region (where there was no fluctuation) because the leakage current masked any dc current through the protein. Here, Scanning Tunneling Microscopy (STM) was used to perform single molecule measurements in solution, systematically exploring the role of both specific and non-specific contacts. STM probes using appropriate insulation⁹And potential control of the electrodes to reduce background leakage current to less than 1pA over the entire bias range. By sufficiently stabilizing, the STM gap remains constant for one minute, so the gap control servo can be disabled, the tip retracted, and the current-voltage (IV) curve recorded. Up to 60 such curves (up-sweep and down-sweep) were recorded before re-engaging the servo and repeating the process on another area of the substrate. To obtain two specific contacts, bivalent antibodies (IgE and two IgG's), each of which presents two binding sites, are used, and up to four antibodies are usedThe biotin molecule binds to streptavidin, so that the epitope or biotin-functionalized motor can be bridged by a specific bond. In the case of a bare metal electrode, the contact was made with a surface thiol on streptavidin modified with an average of 2.5 surface thiols of the enzyme molecule. Furthermore, repeated measurements using integrins can form specific bonds with only one of the two peptide functionalized electrodes. The proteins and ligands are listed in table 1.

The measured conductance depends on the contact

The current is only observed when the protein specifically binds to at least one of the two electrodes. Typically, no current is recorded for a few seconds after retraction, and then the current jumps to a larger (and variable) value in the presence of bound protein. Although the current fluctuates on the minute time scale, it is generally stable for several seconds, so 80% of the curve recorded at the time of the up-sweep can be reproduced at the time of the down-sweep (data not shown). Controls (buffer only or non-homologous protein only in solution) gave no signal. For anti-DNP (data not shown) and all other protein studies above 0.1V, a rapidly fluctuating (ms-time scale) Telegraph Noise (TN) for integrin reporting was also observed. This is a ubiquitous protein capture signal, indicating the same two-stage switch in all cases. These fluctuations were initially observed for proteins captured in the immobilized biochip⁸Although the work today uses an STM, TN measurements were repeated in the chip as well as in the STM to show that there was no some artifact in the measurement method (data not shown). The voltage threshold of the TN is not dependent on the gap until the contact is almost broken (data not shown), which means that it is related to the fluctuation of the contact driven by the potential drop, which occurs mostly at the contact, as previously proposed⁸And discussed in more detail below.

In addition to TN, the response is linear, so each IV trace can be characterized by a single conductance value G. The distribution of measured G follows a log-normal distribution that is typically observed in single molecule measurements (data not shown).¹⁰The distribution is similar to recording current pairs at a fixed gap and biasThe distribution of current values obtained over time (data not shown) is therefore due to the different types of contact between the electrodes and the molecules. The distribution of integrin (gap-4.5 nm) and thiostreptavidin (gap-2.5 nm) had a single peak at about 0.3nS (data not shown). Capturing thiostreptavidin using a bare metal electrode, wherein thiol-mediated contact displaces contaminants on the electrode surface,¹¹direct metal-molecule contact is formed. The integrin was captured only by the cyclic RGD peptide at one of the two electrodes and no signal was observed unless both electrodes were functionalized. Functionalization with peptides allows non-specific contact with hydrophilic sites on proteins at non-specifically coupled electrodes. The three antibodies produced two conductance peaks (. about.0.3 nS and. about.2 nS), indicating that there are two binding modes: nS-S for integrin, and S-S required when both antigen binding sites specifically bind (data not shown). This interpretation was tested in the following way: the peptide on one electrode was replaced with mercaptoethanol, making it hydrophilic and capable of forming an NS-S bridge. Only a single peak was observed (data not shown). As a further test, Fab fragments with only monovalent binding heads were prepared from anti-Ebola IgG. The fragment was too small to bridge the 4.5nm gap, so data in the 2.5nm gap was recorded. Only a single peak in the conductance distribution, reflecting a single NS-S contact (data not shown). Thus, a higher conductance peak must correspond to conduction through both antigen binding sites. To see this effect, the data set must be dominated by single molecule contacts. Strikingly, the conductance of the single Fab fragment across the 2.5nm gap was much less than the conductance of the antibody across the 4.5nm gap (data not shown). This indicates that the intrinsic internal conductance of the protein is much higher than the measured (contact limited) value. This finding explains the reports of similar conductances for previously measured proteins of greatly differing size (12, 13).

Conductance is not dependent on gap size

The presence of an internal (through the (14) molecular) high conductance path is illustrated by a series of measurements made at different gap sizes using the technique described above but increasing the amount of initial tip retraction (data not shown). Strikingly, the conductance peak did not change with the gap size despite the decrease in frequency of data accumulation (data not shown). This effect reflects the probe area available for contact at a given height. When the gap is highly comparable to the protein, few sites are available (listed for similar structures visible in the protein database in table 1). Gap-independent conductance between azurin (azurin) (see SI of Ruiz et al (15)) and the rod-shaped molecules captured between the probe and the substrate has been previously reported. (14) As noted above, the contact points change over the course of the measurement (. about.min), which reflects the angstrom scale change in the position of the STM probe. It was confirmed that these different contact geometries resulted in the overall shape of the conductance distribution (data not shown). Since the distribution maintained the same peak position and shape at different gap sizes, the data showed no indication that the protein was "squeezed" at smaller gap sizes.

The voltage threshold versus gap size plot for TN turn-on shows that the voltage threshold does not vary significantly with the gap size. Therefore, the TN fluctuations must be driven by the local electric field at the metal-molecule interface, with a relatively small potential drop across the protein interior. This is also consistent with our finding that the lifetime of a TN is exponential with the current peak, an observation that can be explained by a single "weak chain" tunnel junction of the dominant conductance in the circuit. (8)

Conductance is sensitive to changes in protein structure

Since the conductance path follows the protein geometry, either internally or along the surface contour, changes in the protein geometry and hence the conduction path, affect which contact points control conductance. This would enable direct sensing of structural changes in the protein. This effect is demonstrated in fig. 7A. Biotin complexation in the sulfurized streptavidin sample significantly altered the conductance profile (fig. 7A). The streptavidin molecule has four biotin binding sites, (16) so that the unsulfurized apolipoprotein can be crosslinked by two biotins. Biotin sulfide was synthesized and used to functionalize both the probe and the substrate (FIG. 7B), followed by the flow of apolipoprotein-streptavidin into the sample cell. The resulting G distribution has three peaks with a maximum value close to 7 nS. Thus, the measured conductance is sensitive to both the local change in protein structure and the chemistry of the contact, indicating how the contact is affected by the change in protein structure. Similar results (data not shown) with thiostreptavidin linked to the substrate and detection with biotinylated probe indicate that a single biotin-mediated contact is sufficient to produce a high conductance state at 7 nS.

Possible mechanisms

The electron tunneling decays too quickly to allow long distance transport. Tunneling conductance can be from G to G₀exp (-betax) estimation, where G₀Is in the range of 77 mu S,(24) for small proteins at-4 nm, this gives G<10^-21S, 12 orders of magnitude less than observed. To explain the observation of nS conductance at a distance of 10nm, it is necessary toIn the case of well-studied DNA, when the distance between easily ionizable guanines exceeds three nucleotides, hopping of thermal activation (17) results in transport that is nearly independent of distance. (18) Similar transport (via easily oxidizable amino acids) has been observed in peptides. (19) In these cases, transport is limited by charge injection, overcoming a charge injection barrier of 1.5eV with chromophores that are photo-excited at 630nm (2 eV). If a similar transport mechanism is working in the case of charge injection from the electrodes, the potential barrier will be determined by the energy gap between the fermi energy of Pd (work function 5.2eV) and the absolute redox potential of the easily oxidizable residues tyrosine and tryptophan. Relative to NHE, these potentials are ± +1 to +1.2V (20) (21), so using 4.4eV as the work function (22) of NHE yields an absolute potential of about 5.4-5.6eV under vacuum, or a +0.2 to 0.4eV barrier corresponding to the Fermi energy of Pd. Therefore, this magnitude barrier must be overcome by the polarization of the bonds associated with protein and electrode binding. This is consistent with the range of work function variations observed for small molecules attached to noble metal surfaces through thiol linkages. (23) Significant current was obtained in three cases: (a) when passing throughDirect thiol-mediated binding when in contact with both electrodes; (b) when one bond is formed by specific binding to an epitope or ligand and the other is formed by non-specific interaction between a hydrophilic molecule attached to an electrode and the hydrophilic exterior of the protein; and (c) the maximum current is observed when the protein is bound by the recognition ligand at both electrodes (in all cases the ligand is linked to the electrode via the thiol). Weak non-specific bonds do not result in significant current flow: at least one attachment must be via a covalent or ligand-mediated linkage. Thus, if the protein itself is not covalently modified to bind directly to the electrode, the charge injection barrier is overcome by binding to at least one specific ligand.

It has been shown that small changes in interface charge at the contact strongly affect transport (24), a variable that we can use potential control to change if faraday currents are to be avoided, although only over a small range. Presumably, the smaller changes are dominated by the larger fields due to the bond polarization at the interface. In addition to the redox potential of amino acid residues, the three-dimensional folding of proteins must also play an important role. This is because the small peptides stretched in the broken junction are not conductive (25), but the small peptides folded on the electrode surface are conductive. (26) This may be the result of some special geometry (27) or hydrogen bonding arrangements. (28)

Finally, we turned to the fluctuations set above the applied bias of ± 100 mV. This small dependence of the threshold voltage on the gap size (data not shown) is consistent with the assumption that the protein internal conductance is much higher than at the contact, which means that these signals come from voltage driven fluctuations of the contact itself. In our early integrin studies (8), we proposed a mechanism in which we demonstrate the lifetime of the "on" state, τ, and the peak current of the telegraphic noise peak, i_pCorrelation is via the following equation: τ. varies.. ln (i)_p) The relationship can be explained by a single barrier that determines both current and binding strength. Once open, the current increases linearly with voltage, indicating that a new ohmic conduction channel is open (data not shown). This opening process is indicated by the following formNumber to describe:wherein V_cIs the activation voltage. Fitting yield V_c0.25V, which is a characteristic value of hydrogen bond strength in water (29), indicates that the hydrogen bond may be a "weak link" in the circuit.

Notably, as described above, this 0.25V barrier is similar to the charge injection barrier deduced from the redox potential of amino acids. If the rate of charge injection is limited by thermally activated hopping over a potential barrier of 0.22 to 0.47V, and it is this rate that determines conductance, we would expect to observe the following conductance:wherein 0.22<V<0.47 volts from 12nS to 0.5pS, with the range encompassing the values reported herein.

Role of specific binding in electronic conductance

Our conclusion is that specific ligand-receptor interactions form good electrical connections to proteins. This is illustrated by the data shown in fig. 7A and 7B. Attachment via covalent (thiol) modification of surface lysines directly bound to the metal electrode resulted in a lower maximum conductance (0.56nS) compared to non-covalent streptavidin-biotin coupling (6.8nS) via an ethane linkage at the thiol terminus to the electrode. When only one biotinylated linker was used, there was little change in conductance (data not shown). Thus, weaker coupling to the hydrophobic interior of the protein is more effective than stronger coupling to the hydrophilic exterior, even when only one such coupling is performed. If, once injected, the electrons easily move inside the protein, the second (non-specific) contact will only act as a barrier to the hydrophilic surface of the protein. This mechanism may explain the high conductance of the integrin when bound by the ligand at only one site, as well as the complete absence of conductance when both contact points are non-covalent and non-specific.

Reference to the literature

The following references are hereby incorporated by reference in their entirety:

U.S. patent application 62/673,080 (filed 5/17/2018).

U.S. patent application 62,682,991 (filed 6/10 in 2018).

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2.Bostick，C.D.，S.Mukhopadhyay，I.Pecht，M.Sheves，D.Cahen,and D.Lederman，Protein bioelectronics：a review of what we do and do not know.Reports on Progress in Physics，2018.81：p.026601.

3.Adhikari，R.Y.，N.S.Malvankar，M.T.Tuominen,and D.R.Lovley，Conductivity of individual Geobacter pili.RSC Advances，2016.6：p.8354-8357.

4.Malvankar，N.S.，M.Vargas，K.P.Nevin，A.E.Franks，C.Leang，B.C.Kim，K.Inoue，T.Mester，S.F.Covalla，J.P.Johnson，V.M.Rotello，M.T.Tuominen,and D.R.Lovley，Tunable metallic-like conductivity in microbial nanowire networks.Nat Nanotechnol，2011.6(9)：p.573-9.

5.Zhang，B.，W.Song，P.Pang，H.Lai，Q.Chen，P.Zhang,and S.Lindsay，The Role of Contacts in Long-Range Protein Conductance.Proc Natl Acad Sci U S A，2019.submitted.

6.Vattay，G.a.，D.Salahub，I.a.Csabai，A.Nassimi,and S.A.Kaufmann，Quantum Criticality at the Origin of Life.Journal of Physics：Conference Series 2015.626：p.012023.

7.Cui XD,et al.(2001)Reproducible measurement of single-molecule conductivity.Science(New York，N.Y.)294(5542):571-574.

8.Nitzan A(2006)Chemical dynamics in condensed phases(Oxford University Press.，Oxford).

9.Tuchband M，He J，Huang S，&Lindsay S(2012)Insulated gold scanning tunneling microscopy probes for recognition tunneling in an aqueous environment.Rev Sci Instrum 83(1):015102.

10.Chang S,et al.(2012)Chemical recognition and binding kinetics in a functionalized tunnel junction.Nanotechnology 23(23):235101

11.Smith T(1980)The hydrophilic nature of a clean gold surface.J.Colloid Interface Science 75:51-55.

12.Bostick CD,et al.(2018)Protein bioelectronics：a review of what we do and do not know.Reports on Progress in Physics 81:026601.

13.Nadav Amdursky,et al.(2014)Electronic Transport via Proteins.Advanced Materials 26:7142–7161.

14.Leary E,et al.(2011)Unambiguous one-molecule conductance measurements under ambient conditions.Nano letters 11(6):2236-2241.

15.Ruiz MP,et al.(2017)Bioengineering a Single-Protein Junction.Journal of the American Chemical Society 139(43):15337-15346.

16.Sano T&Cantor CR(1990)Cooperative biotin binding by streptavidin.Journal of Biological Chemistry 25:3369-3373.

17.Giese B&Spichty M(2000)Long distance charge transport through DNA：quantification and extension of the hopping model.Chemphyschem 1(4):195-198.

18.Giese B，Amaudrut J，Kohler AK，Spormann M，&Wessely S(2001)Direct observation of hole transfer through DNA by hopping between adenine bases and by tunnelling.Nature 412(6844):318-320.

19.Aubert C，Vos MH，Mathis P，Eker AP，&Brettel K(2000)Intraprotein radical transfer during photoactivation of DNA photolyase.Nature 405(6786):586-590.

20.Harriman A(1987)Further comments on the redox potentials of tryptophan and tyrosine.Journal of Physical Chemistry 91:6102-6104.

21.Odella E,et al.(2018)Controlling Proton-Coupled Electron Transfer in Bioinspired Artificial Photosynthetic Relays.Journal of the American Chemical Society 140(45):15450-15460.

22.Tripkovic V，Skúlason E，&Rossmeisl J(2011)Standard hydrogen electrode and potential of zero charge in density functional calculations.Phys.Rev.B 84:115452.

23.Alloway DM,et al.(2003)Interface Dipoles Arising from Self-Assembled Monolayers on Gold：UV-Photoemission Studies of Alkanethiols and Partially Fluorinated Alkanethiols.J.Phys.Chem.B 107:11690-11699.

24.Garg K,et al.(2018)Interface Electrostatics Dictates the Electron Transport via Bioelectronic Junctions.ACS Appl Mater Interfaces.

25.Xiao X，Xu B，&Tao N(2004)Conductance titration of single-peptide molecules.Journal of the American Chemical Society 126(17):5370-5371.

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It is obvious to a person skilled in the art that particular embodiments of the present disclosure may relate to one or more of the above and below described embodiments in any combination.

Although specific materials, formulations, sequences of operations, process parameters and end products have been set forth to illustrate and describe the invention, they are not intended to be limiting. On the contrary, it should be noted by those skilled in the art that the written disclosure is merely exemplary and that various other substitutions, alterations, and modifications may be made within the scope of the disclosure. Accordingly, the present disclosure is not limited to the specific embodiments described herein, but only by the claims that follow.

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