N-terminal polyfunctional conjugation of peptides and proteins for biosensing

Abstract

In various embodiments, an alpha-helical peptide-based bridge for "on-chip" molecular biosensing is disclosed. The peptide-based bridge serves as a common bridge for a wide variety of biosensing applications and targets, including nucleic acids, proteins, antigens, antibodies, and small molecules. The primary sensor element is preferably a "molecular wire," such as an alpha-helical peptide, integrated with current monitoring circuitry. The engineered peptide may contain a central conjugation site for attachment of various probe molecules, including nucleic acids, proteins, antigens, and antibodies. The probe-containing bridge enables the sensor to detect interactions with specific target molecules.

Description

[Technical Field]

[0001] Reference to Interrelated Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 404,944, filed September 8, 2022, by Jose Rizo et al., entitled "N-Terminal Multifunctional Conjugation of Proteins and Peptides for Biosensing," and International Patent Application No. PCT / US23 / 14291, filed March 1, 2023, by Prem Kumar Sinha et al., entitled "Peptide Based Bridges for Molecular Sensors and Methods for use thereof," the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0002] The present disclosure is generally directed to molecular sensors in which a probe is attached to an amino acid bridge molecule in a molecular circuit, and binding of a target or ligand to the probe is detectable by monitoring at least one parameter of the molecular circuit. [Background technology]

[0003] The following contains information that may be useful in understanding the present invention. None of the information provided herein is admitted to be prior art to or relevant to any invention described or claimed herein, or that any publication or document referenced specifically or implicitly is prior art.

[0004] The broad field of molecular electronics was introduced by Aviram and Ratner in the 1970s. Molecular electronics represents the ultimate scaling down of electrical circuits by using single molecules as circuit components. Molecular circuits containing single-molecule components can function diversely as switches, rectifiers, actuators, and sensors, depending on the properties of the molecules. Of particular interest is the application of such circuits as biosensors, where molecular interactions form the basis for single biomolecule sensing.

[0005] Biosensing is a rapidly evolving methodology for monitoring biochemical pathways and quantifying biomolecules, particularly those of medical relevance. As such, the need to develop synthetically accessible probes that are biologically representative of systems of interest has led to the emergence of the field of bioconjugation as a powerful tool at the intersection of biology, synthetic chemistry, and biochemistry. In the past, assessing synthetic suitability relied on the application of well-established synthetic techniques to link probes of interest. Probes, including but not limited to peptides, oligos, and other ligands of interest, have been incorporated into biosensing applications using synthetic techniques that target specific functional groups on the target.

[0006] These techniques have traditionally utilized activated esters targeting amine-containing residues, Michael acceptors targeting thiols, and, more recently, redox-mediated coupling targeting phenolic amino acid residues. While conferring a moderate to significant degree of chemoselectivity, these synthetic methods rarely provide regioselective ligation without considering secondary factors. Such considerations include protein engineering to remove or prevent the reactivity of residues that compete with the synthetic technique being utilized, or modification of these residues via synthetic or enzymatic techniques to adjust reactivity and selectivity. While nonselective ligation may be sufficient for certain applications, such as those simply probing binding events, nonspecific uptake of substrates, particularly enzymes, can introduce complications in systems where the activity of the enzyme may vary depending on the location of the modification. This is of particular concern in systems sensitive to kinetic variations caused by a population of probe-sensor complexes modified at various locations, such as single-molecule biosensor systems.

[0007] To this end, considerable effort has been devoted to developing methods that allow for chemoselective and regioselective conjugation of protein-based probes, providing a homogeneous population of probe-conjugate species. Two key factors were given great importance: first, the method must be minimally intrusive with respect to chemical modifications on the protein substrate, and second, the method should not require engineering the protein to attach any handles (chemically reactive moieties). With these constraints in mind, primary amines present on the N-terminus were identified as suitable targets for bioconjugation.

[0008] Although the literature is rich in previous methodologies with similar objectives, most rely on synthetic strategies incorporating moieties that are prone to cross-reactivity, thus resulting in poor to moderate yields and selectivity, or that have poor water solubility, thus necessitating the use of organic solvents and limiting their usefulness for applications utilizing larger, hydrophilic biomolecules. Similarly, a similar concern is the synthetic ease of the proposed reagents in question. While some of these synthetic strategies can be used to introduce simple modifications onto biomolecules of interest, their actual feasibility for introducing multifunctional molecules is greatly limited by the relative complexity of the synthesis of these linker reagents. However, some of these strategies are primary options for the application of chemoselective and regioselective probe ligation for biosensing applications. For example, both Mikkelson et al. and Ohana et al. reported two different moieties that provide N-terminal selective peptide modification. Importantly, however, the focus in these reports was to demonstrate the modification of small peptides with simple functionality.

[0009] Much more research and information is needed regarding larger polypeptides, particularly functional proteins, particularly regarding how their modifications can be implemented in useful applications. The inventions described herein address this need. Some embodiments herein are directed to the implementation of these selected moieties in rationally designed multifunctional linker molecules for use in a wider range of linking regimes, particularly for use in biosensing applications. Further novel embodiments of the invention are described herein, as described in detail herein below. Summary of the Invention

[0010] The invention described herein and claimed herein has many features and embodiments, including but not limited to those illustrated or described or referenced in this summary. The invention described herein and claimed herein is not limited to or by the features or embodiments identified in this summary, which is included merely for purposes of illustration and not limitation.

[0011] The present disclosure generally relates to biosensors, systems including biosensors, and methods of using biosensors and systems. In various embodiments, binding probe-based circuits are disclosed. Exemplary biosensors can be used, for example, to detect binding of a molecule of interest (embraced by the term "target" herein) to a probe, or a binding partner or ligand of the sensor's probe.

[0012] One particular embodiment of the present invention involves the use of modified phenolic esters to selectively modify polypeptides at the n-terminal residue. In another embodiment, conjugates of molecular wires and attached probe molecules, as well as methods for making these conjugates, are described herein. In another embodiment, methods are provided for making sensor devices (e.g., biosensors) that can incorporate and use these modified polypeptides. In another embodiment, biosensors incorporating such modified polypeptides are provided.

[0013] In some embodiments, the biosensor device comprises (i) a current-carrying molecular structure comprising metal contacts on the surface of an electrode, the metal contacts being attached to a molecular wire comprising a conjugation site, and (ii) a bound probe molecule connected to the molecular wire, forming a circuit capable of detecting and / or obtaining detailed information regarding the binding of the bound probe molecule to a target ligand or binding partner.

[0014] In some embodiments, the current-carrying molecular structure includes a positive electrode and a negative electrode. The positive electrode may have a metal contact on its surface, and the negative electrode may also have a metal contact on its surface.

[0015] In some embodiments of the biosensor device, the molecular wire is an alpha helical peptide.

[0016] In some embodiments of the biosensor device, the molecular wire is a polynucleotide, such as a polynucleotide with a modified backbone, single- or double-stranded DNA, RNA, or origami.

[0017] In some embodiments of the biosensor device, the molecular wire is a non-peptide bridging molecule such as graphene, indium oxide ribbon, carbon nanotube, DNA / polynucleotide, etc.

[0018] In some embodiments of the biosensor device, the alpha helical peptide comprises one or more conjugation sites for binding to a multifunctional linker molecule.

[0019] In some embodiments of the biosensor device, conjugation of the binding probe molecule to the molecular wire is via a multifunctional linker molecule that is covalently attached at one end to the N-terminus of the binding probe molecule and at the other end to a conjugation site on the molecular wire.

[0020] In further embodiments, the polyfunctional linker molecule comprises one or more chemically reactive moieties, handles, or surface modification / binding motifs. In some illustrative embodiments, the polyfunctional linker molecule comprises a vinyl boronate linker. In other illustrative embodiments, the polyfunctional linker molecule comprises an activated phenol linker. In certain embodiments, the one or more reactive moieties have a mixed anhydride, such as a sulfonamide or sulfonic acid, meta to the phenolic oxygen and adjacent to one or more electron-withdrawing groups, including, but not limited to, halogens, polyfluorinated hydrocarbons, nitriles, carboxylic acids and derivatives, and nitro groups. In certain embodiments, the polyfunctional linker molecule has a reactive moiety comprising an E-styrenyl with a para-electron-donating moiety, or an E-vinyl boronate derived from an α-nucleophile / electrophile alkyne, which reacts with a linker having one or more reactive groups that undergo selective "click"-type chemistry and can be utilized as a scaffold for further extension.

[0021] In some embodiments, the binding probe molecule comprises a protein, peptide, polypeptide, or protein complex of biological or synthetic origin. In selected embodiments, the binding probe molecule is a polypeptide or protein, including, but not limited to, a polymerase, a viral antigen, or an antibody. Examples of binding probe molecules useful herein include DNA polymerase, HIV-1 p24 viral antigen, an anti-IL-6 antibody or binding fragment thereof, or protein A.

[0022] In other embodiments, described herein are composites of molecular wires and binding probe molecules for use in biosensors. In an exemplary embodiment, the molecular wire comprises an alpha-helical peptide having a conjugation moiety, the binding probe molecule is a protein having an N-terminus, and the conjugation of the binding probe molecule to the alpha-helical peptide is via a multifunctional linker molecule that is covalently attached at one end to the N-terminus of the binding probe molecule and at the other end to the conjugation moiety in the alpha-helical peptide. Suitable binding probe molecules to be used in these composites include, but are not limited to, enzymes, antibodies or binding portions thereof, selected antigens, and protein A. Further examples of binding probe molecules for use in the above embodiments include DNA polymerase, HIV-1 p24 viral antigen, or anti-IL-6 antibodies or binding fragments thereof.

[0023] In another aspect, a method for preparing a conjugate of a molecular wire and a binding probe molecule as described above is provided. An example of preparing such a conjugate includes the steps of synthesizing a multifunctional linker molecule containing one or more chemically reactive moieties / handles; selecting a binding probe molecule of interest; reacting the binding probe molecule with a bifunctional linker reagent under selected conditions to generate a clickable intermediate binding probe molecule suitable for carbon-heterobond-forming reactions or click chemistry; reacting the clickable intermediate binding probe molecule with a preselected molecular wire under appropriate reaction conditions; and purifying the conjugate of the molecular wire and the binding probe molecule. These conjugates can then be used to prepare specific biosensors by reacting a sensor device with the conjugate of the molecular wire and the binding probe to generate a biosensor for detecting a binding target molecule or ligand that binds to the binding probe molecule.

[0024] In yet another embodiment, a method for detecting a target is provided, the method including selecting a suitable biosensor such as those described herein, initiating at least one of a voltage or a current through the biosensor, exposing the biosensor to a sample suspected of containing the target of interest, applying a voltage to the sensor, and measuring an electrical change in the circuit. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 shows a schematic of a generalized strategy for synthesizing primary multifunctional linkers. The synthesis shown utilizes an N-capping strategy to enable a diverse set of bioconjugations. The first step (A) illustrates the synthesis of an activated phenol, which leads to a linker with a reactive handle. The second step (B) shows the synthesis of a bifunctional vinyl boronate linker, which leads to a further refined linker with a reactive handle. The dotted area indicates the reactive handle, and the solid-lined area indicates a generalizable scaffold for further bioconjugation reactions. [Figure 2] Figure 1 shows a generalized bioconjugation strategy for regio- and chemoselectively linking a protein of interest and conjugating it onto a surface-bound substrate, such as, but not limited to, a molecular wire for biosensing purposes. The first step illustrates the synthesis via an activated phenol, and the second step shows the synthesis via an activated vinyl boronate. [Figure 3] FIG. 1 depicts surface modifications or conjugated anchors for biosensing on various electrode surfaces. [Figure 4] FIG. 1 depicts two representative strategies for on-chip attachment of N-terminally linked probes. [Figure 5]This figure shows an example of the selective conjugation of an enzyme, in this case Bst polymerase with a modified short linker A, to a molecular wire for biosensing purposes. It includes quality and purification data, including an FPLC chromatogram shown on the left and an SDS-PAGE gel shown on the right. In the SDS-PAGE gel, lane 1 is the mw ladder / marker, lane 2 is the starting bridge material, lane 3 is the starting Bst polymerase, lane 4 is the reaction mixture, and lanes 5-9 represent different FPLC fractions. [Figure 6] Quality and purification data (FPLC chromatogram on the left, SDS-PAGE gel on the right) for the conjugated enzyme product, in this case a polymerase and molecular wire with a modified long linker B. In the SDS-PAGE gel, lane 1 is the mw protein ladder, lanes 2-4 are the starting bridge material, and lanes 5-8 represent the different FPLC fractions C9. [Figure 7] Figure 1 shows the results of a polymerase activity assay highlighting the relative rates of incorporation relative to dATP. The enzymes used in this case are the starting Bst polymerase (left) and two N-terminally coupled Bst polymerases to the bridge peptide via a short linker A (compound A, center) and a long linker (compound B, right). [Figure 8] Figure 1 shows the isolation of bridges conjugated to HIV-1 p24 antigen. In the SDS-PAGE gel, lane 1 is HIV p24; lane 2 is a protein ladder / marker; and lanes 3-10 represent FPLC fractions C9-D3. [Figure 9] Figure 1 shows a schematic diagram of the HIV-1 p24 antigen attached to a bridge-peptide. The left panel shows a schematic diagram of the bridge-probe construct for the HIV-1 p24 antibody target. Binding response curves from the various chips are also shown (right panel) by plotting the fraction of the time bound versus antibody concentration. [Figure 10]FIG. 1 shows the isolation of a bridging peptide conjugated to the N-terminus of an anti-IL-6 antibody. [Figure 11] Schematic showing an anti-IL-6 antibody attached to a bridging peptide (left panel). A binding response curve from a typical sensor is shown by plotting the percentage of time bound versus the IL-6 antigen concentration, along with a dose-response curve (right panel). [Figure 12] Schematic showing protein A attached to a bridge peptide (left panel). A binding response curve from a typical sensor is shown by plotting the percentage of time bound versus IgG antibody concentration, along with a dose-response curve (right panel). [Figure 13] 1 is a schematic illustrating the general concept of attaching a binding probe molecule onto a molecular wire within an electronic circuit to act as a sensor capable of detecting a binding event between the probe and its target, according to various embodiments. As shown, a multifunctional linker molecule is covalently attached at one end to the N-terminus of the binding probe molecule and at the other end to a conjugation site on the molecular wire spanning the positive and negative electrodes. DETAILED DESCRIPTION OF THE INVENTION

[0026] Various aspects of the present invention will now be described with reference to the following sections, which will be understood as being provided by way of example only and not as constituting a limitation on the scope of the invention.

[0027] As used herein, the terms "bridge molecule" or "molecular wire" or "nanowire" can be used interchangeably herein and further refer to molecular wires or other conductive molecules that can be used to form conductive connections. Numerous molecular wires are described in detail herein. Molecules that function as molecular wires include, but are not limited to, peptide alpha helices, long peptides or polypeptides, modified versions thereof, graphene nanoribbons, pilin filaments, or bacterial nanowires, nucleic acids (natural or modified), double-stranded DNA, other multi-chain proteins, or composites of multiple single-chain proteins, antibodies, semiconducting layers such as carbon nanotubes, e.g., single-walled carbon nanotubes (CNTs, SWCNTs), transition metal dichalcogenides (TMDs), or other semiconducting nanoribbons or nanowires, or conducting polymers such as polythiophenes, poly(3,4-ethylenedioxythiophene (PEDOT)), or other synthetic conducting polymers. Such molecules may contain binding groups, i.e., functional groups that provide specific binding and / or self-assembly to nanoelectrodes or contacts (e.g., metals), such as islands or deposits thereon. Various embodiments described in more detail herein are directed to specific conjugation of binding probe molecules to bridge molecules or molecular wires.

[0028] As used herein, "clickable" or click chemistry-compatible intermediates, structures, functional groups, monomers, oligomers, etc., should be understood to refer to compounds, materials, etc. that are structurally characterized by including one or more chemical moieties suitable for participating in a click chemistry reaction. In click chemistry embodiments in which copper-catalyzed azide-alkyne cycloaddition (CuAAC) is utilized to functionalize materials as disclosed herein, "clickable" compounds may include terminal alkyne and / or terminal azide functional groups. "Click chemistry" is further described in U.S. Pat. No. 7,375,234, which is incorporated herein by reference in its entirety.

[0029] While the illustrative click chemistry reaction described herein is CuAAC, those skilled in the art will appreciate that other click chemistry-compatible reactions, which would be appreciated upon reading these descriptions as being equivalent to CuAAC, can be utilized without departing from the scope of the inventive concepts described herein. For example, in various embodiments, click chemistry-compatible reactions can include strained alkene reactions, such as CuAAC, strain-promoted azide-alkyne cycloaddition (SPAAC), strain-promoted alkyne-nitrone cycloaddition (SPANC), alkene-azide cycloaddition, and the like. Click chemistry-compatible reactions may also be considered to include alkene-tetrazine inverse demand Diers-Alder reactions, alkene-tetrazole photoclick reactions, Michael addition of thiols, nucleophilic substitution of thiols with amines, and certain Diels-Alder reactions, such as those disclosed by Becer, et al. "Click chemistry beyond metal-catalyzed cycloaddition," Angew. Chem. Int. Ed. 2009, 48: pp. 4900-4908, and equivalents thereof, as would be understood by one of skill in the art upon reading this disclosure. Accordingly, click chemistry-compatible groups, compounds, and the like, in various embodiments, should be understood to include one or more suitable chemical moieties that convey the ability to participate in any combination of the exemplary click chemistries described above.

[0030] In various embodiments of the present disclosure, molecular sensors include binding probe molecules connected to current-carrying structures (e.g., electrodes) by bridge molecules or nanowires to complete a circuit. Interaction of the binding probe molecules with ligands or binding target molecules is detectable as a change in current or other electrical parameter measured across the circuit (see FIG. 11). The binding probes may be, for example, enzymes (e.g., polymerases), antibodies, antigens, or any other polypeptides or proteins conjugated to the molecular wires or bridge molecules described above. Another illustrative embodiment (not shown) differs from the general concept of molecular electronic circuits in that the enzymes are directly conjugated or "tethered" to both the positive and negative electrodes, rather than being attached to molecular wires spanning the gap between the electrodes to complete the circuit.

[0031] In various embodiments of the present disclosure, at least one of a voltage or a current is initiated in the probe-based molecular circuit. When a target interacts with the probe, an electrical change in the circuit is sensed. These electrical changes or informative electrical signals may include current, voltage, impedance, conductivity, resistance, capacitance, or the like. In some examples, a voltage is initiated in the circuit, and then a change in current through the circuit is measured when a substrate interacts with a bound probe. In other examples, a current is initiated in the circuit, and a change to the voltage in the circuit is measured when a substrate interacts with an enzyme. In other examples, impedance, conductivity, or resistance is measured. In examples where the circuit further includes a gate electrode, such as one positioned under the gap between the positive and negative electrodes, at least one of a voltage or a current may be applied to the gate electrode, and further, a voltage, current, impedance, conductivity, resistance, or other electrical change in the circuit may be measured when a substrate interacts with a bound probe. Suitable circuits are described in Applicant's prior related patent applications and patent documents, including U.S. Patent No. 10,036,064, U.S. Patent No. 10,508,296, U.S. Patent No. 10,648,941, U.S. Patent No. 10,584,410, U.S. Patent No. 10,913,966, U.S. Patent No. 11,143,617, and WO / 2020 / 146823 A9, all of which are incorporated herein by reference in their entirety.

[0032] Certain methods provided herein are directed to the use of modified phenolic esters to selectively modify polypeptides at the n-terminal residue. As noted above, Mikkelson et al. demonstrated the ability of modified phenolic esters to selectively modify short peptides at the n-terminal residue. This selectivity is the culmination of several factors that have been considered. As they report, 3,5-dichloro-2-hydroxybenzenesulfonic acid esters (PSEs) address the problems associated with traditional activated esters, such as NHS esters. A fundamental drawback of utilizing traditional activated esters is their poor regioselectivity and chemoselectivity, due in part to their high reactivity with competing nucleophiles, including other nucleophilic residues and solvents. PSEs circumvent this in a twofold manner. This moiety provides stability through an intramolecular hydrogen bond formed between the carbonyl group of the reagent and the sulfonyl group of the phenol. Similarly, the electrophilicity of the reagent is matched with that of the amine at the n-terminal residue through the addition of an electron-withdrawing group. Therefore, when reactions are performed at near-neutral pH, the HOMO and LUMO of the respective nucleophiles and electrophiles favor reaction at the carbonyl group of the activated ester. This is particularly due to the difference in pKa between the N-terminal amine and the ε-amino group on lysine, ~9 and 10.5, respectively. Other factors include the improved water solubility afforded by the inclusion of a sulfonyl moiety and the relative ease of synthesis of multifunctional bioconjugate reagents.

[0033] Bioconjugation targeted to the N-terminus of a protein of interest: The process of bioconjugating the N-terminus of a given protein to a bridge involves three major steps.

[0034] Step 1: Synthesize a multifunctional linker molecule with an appropriate reactive handle.

[0035] Step 2: React the probe of interest with a bifunctional linker reagent under selective / non-selective conditions and isolate to give a clickable intermediate.

[0036] Step 3: The clickable intermediate probe is reacted with the corresponding sensor molecule, anchor, or binding molecule, followed by purification to obtain the biosensor probe conjugate.

[0037] Step 1: Synthesis of the bioconjugation reagent: The first step is the synthesis of an appropriate bioconjugation reagent bearing a suitable reactive handle. Below, we refer to example workflows for the preparation of activated phenolic or vinylboronic acid linkers. However, the conjugation chemistry is not limited to specific reagents and can be developed for similar reagent types, such as triazolocarbaldehydes, pyridine-carbaldehydes, ethynylbenzaldehydes, oxazolines, and mixed anhydrides.

[0038] Activated phenols: Multifunctional linker molecules in which the primary reactive handle is a mixed anhydride containing a sulfonamide or sulfonic acid meta to the phenolic oxygen and adjacent to one or more electron-withdrawing groups, including, but not limited to, halogens, polyfluorinated hydrocarbons, nitriles, carboxylic acids and derivatives, and nitro groups. The carbonyl-containing portion of the mixed anhydride contains one or more reactive groups that can undergo selective "click"-type chemistry and can be utilized as a scaffold for further elaboration.

[0039] Example workflow: Under anhydrous conditions, in an inert atmosphere at 0°C, the sulfonyl chloride-phenol is added dropwise with moderate stirring to a solution of the carboxylic acid linker of interest and a catalytic amount of dry tertiary amine in an anhydrous, moderately polar aliphatic solvent. Upon completion of the addition, the reaction is allowed to reach room temperature and stirred until the reaction is complete, as determined via TLC or HPLC. The reaction is then concentrated under reduced pressure, and the residue is redissolved in a minimal amount of organic solvent. This concentrated solution is then purified using appropriate chromatographic methods to yield the final activated phenol linker.

[0040] Vinyl boronate: A multifunctional linker molecule in which the primary reactive handle consists of an E-styrenyl bearing a para-electron donating moiety or an E-vinyl boronate derived from an α-nucleophile / electrophile alkyne, which reacts with a linker bearing one or more reactive groups that can undergo selective "click" type chemistry and serve as a scaffold for further extension.

[0041] Example workflow: Under anhydrous conditions in an inert atmosphere, the alkyne of interest bearing a suitable electrophile or nucleophile is added dropwise to a solution of the linker with the appropriate reaction partner in anhydrous DMF containing a suspension of a large excess of anhydrous potassium carbonate. The solution is heated to 80°C and vigorously stirred overnight. Upon completion of the reaction, as determined by thin-layer chromatography (TLC) or analytical HPLC analysis, the suspension is filtered, diluted with water, and further extracted with ether. The pooled organic extracts are washed thoroughly with brine, then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated crude extract is then purified via an appropriate chromatographic process to obtain the intermediately purified alkyne linker.

[0042] The purified alkyne linker then undergoes catalytic hydroboration under inert, anhydrous conditions by heating overnight at 50°C in toluene with excess pinacolborane and a catalytic amount of carbonylchlorohydridotris(triphenylphosphine)ruthenium(II). The reaction mixture is then cooled and concentrated under reduced pressure. The residue is redissolved in a minimum amount of solvent. This extract is then washed with saturated sodium bicarbonate solution, brine solution, and then dried over anhydrous sodium sulfate. The treated extract is then concentrated and purified via an appropriate chromatographic process to yield the intermediate protected vinyl boronate linker.

[0043] The intermediately protected vinyl boronate linker is then deprotected by dissolving it in methanol and cooling it to 0°C, followed by the dropwise addition of excess aqueous potassium bifluoride. The mixture is stirred for 1 hour, then diluted with water, flash-frozen, and then lyophilized. The residue is then extracted with an organic solvent and filtered to remove solids. The crude extract is then added to an aqueous suspension of silica gel for several hours. The suspension is then filtered, and the filtrate is then extracted with additional organic solvent. The pooled organic extracts are then washed with brine and dried over anhydrous sodium sulfate. The dried organic extract is then concentrated under reduced pressure to obtain the final purified vinyl boronic acid linker.

[0044] Step 2: Isolation of the clickable intermediate containing the probe of interest: In the first step, the protein probe of interest is buffer-exchanged into a neutral, non-amine buffer containing an optional non-nucleophilic surfactant and, optionally, a stabilizer. A large excess of sodium ascorbate is then added as a freshly prepared aqueous solution, followed by an excess of the linker-vinyl boronate. The resulting mixture is vortexed, centrifuged, and then incubated at 4°C or room temperature for several hours to overnight, depending on the bridge used. Small molecule reactants are removed via centrifugal diafiltration using an appropriately sized membrane filter and, optionally, purified via a suitable chromatography step to yield the capped clickable probe intermediate.

[0045] Alternatively, a capped clickable probe bridge intermediate can be obtained by buffer-exchanging the protein probe of interest into a neutral, non-amine buffer containing optional non-nucleophilic surfactants and stabilizers as needed. The activated linker-phenol is then added in an anhydrous, polar, water-miscible solvent. The resulting mixture is vortexed, centrifuged, and further incubated at 4°C or room temperature for several hours to overnight, depending on the protein used. Small molecule reactants are removed via centrifugal diafiltration using appropriately sized membrane filters and, optionally, purified via a suitable chromatography step.

[0046] Step 3: Purification of the biosensor probe complex In a subsequent step, the crude or purified clickable probe intermediate solution is mixed with a solution of the desired conjugation partner bearing a compatible reactive functional group, typically, but not limited to, a strain-promoted cycloaddition-compatible alkyne. The probe-partner reaction mixture is then incubated at an appropriate temperature, typically 4°C to 50°C. After a sufficient time has passed, the crude reaction mixture is concentrated via diafiltration and purified via size-exclusion HPLC or FPLC using an isocratic mobile phase with PBS and an appropriate buffer, such as a stabilizer or surfactant, as needed. The eluted fraction containing the product of interest is then further concentrated and buffer-exchanged to the desired final buffer composition via diafiltration. The purified product solution is quantified via gel densitometry and UV-Vis spectrophotometry, and purity is assessed by SDS-PAGE and analytical HPLC.

[0047] Attachment of compounds to molecular wires: Conjugates comprising binding probe molecules and multifunctional linkers can be attached to molecular wires described herein that include non-peptide bridges, such as those comprising indium oxide, gold, platinum, ruthenium, graphene, carbon nanotubes, nucleic acids, or polynucleotides.

[0048] Figure 3 demonstrates examples of non-peptide conjugate partners, including nucleic acid-derived conjugate partners, which can be integrated into a multifunctional linker to act as a surface-specific anchor for attaching a probe of interest to an electrode, or integrated as an existing modification of an electrode to enable in situ coupling of the probe of interest on a circuit system. These surface-anchoring conjugate partners consist of a "head" bearing a "click"-reactive moiety compatible with that to be incorporated onto the probe of interest, and an intermediate "spacer" portion, which can be linear or branched, and are generally: peptides, nucleic acids, polyether polyols, aliphatic and functionalized alkyl chains, polyesters, polystyrenes, glycans, polyolefins, and polyamides. The "anchor" portion contains one or more moieties that confer surface / material-specific binding of the probe of interest via either chemical or physical means. The moieties used depend on the surface or material being utilized in the circuit system. These generally include, but are not limited to: protected or free thiols or thiol equivalents, disulfides or other organosulfur moieties that chemically bond to, for example, gold or platinum, polycyclic aromatic hydrocarbons such as pyrene and naphthalene that noncovalently attach to graphene and carbon nanotubes, polycyclic aromatic heterocycles with conformationally constrained heteroatoms that can act as multidentate ligands for chemical bonding to ruthenium surfaces, and silane-based surface modifications that can be used to introduce click-compatible moieties onto a variety of materials.

[0049] In addition, conjugates containing binding probe molecules and polyfunctional linkers can be attached to alpha-helical peptide-bridged molecular wires, as exemplified below. In the first step, bare (unmodified) peptide bridges are suspended in an appropriate aqueous buffer, optionally containing stabilizers or surfactants. At room temperature, a cysteine-selective clickable bioconjugation reagent is added as a solution in a polar, water-miscible solvent. The reaction mixture is then incubated with gentle stirring at temperatures ranging from 4°C to 30°C. After a sufficient time has passed, the crude reaction mixture is filtered to remove solids, and small molecule impurities are removed via diafiltration. The processed reaction mixture is then purified via preparative reverse-phase high-pressure liquid chromatography, typically eluting with a gradient of trifluoroacetic acid containing water and acetonitrile. The eluted fractions containing the product of interest are then diluted with water, frozen, and subsequently lyophilized. The lyophilized product is resuspended in an appropriate buffer to ensure complete neutralization of residual acid. The purified product solution is quantified via gel densitometry and UV-Vis spectrophotometry, and its purity is assessed by SDS-PAGE and analytical HPLC. In a subsequent step, the purified clickable protein intermediate solution is mixed with a solution of the probe of interest, which contains a compatible reactive functional group, typically but not limited to, azide. The probe-bridge reaction mixture is then incubated at an appropriate temperature, typically 4°C to 50°C. After a sufficient time has passed, the crude reaction mixture is concentrated via diafiltration and purified via size-exclusion HPLC using an isocratic mobile phase with appropriate buffers and stabilizers or surfactants, if necessary. The eluted fraction containing the product of interest is then further concentrated and buffer-exchanged to the desired final buffer composition via diafiltration. The purified product solution is quantified via gel densitometry and UV-Vis spectrophotometry, and its purity is assessed by SDS-PAGE and analytical HPLC.

[0050] Figure 4 shows two representative strategies for incorporating a probe of interest into a circuit system. First, the probe of interest is tagged at the N-terminus with a multifunctional linker containing a "click" reactive moiety. Next, the clickable probe is introduced into a material that has been pre-modified to have a compatible "click" reactive partner, thus linking the probe to the circuit. Alternatively, the N-terminus tagged probe can be inserted into the system via incorporating a material-specific anchor into the multifunctional linker.

[0051] Examples of protein-bridge conjugates and suitable activity / binding assays (Figures 5-12): To demonstrate the versatility of N-terminal conjugation chemistry, we demonstrate a variety of model proteins and enzymes, including polymerases, antigens, antibodies, and the corresponding associated biochemical and binding assays.

[0052] Figures 5-7 depict examples of conjugation to illustrative bridge peptides and activity assays for the corresponding DNA polymerases. Figure 5 shows the selective conjugation of Bst polymerase with a modified short linker A to a molecular wire. Figure 6 shows the conjugation of the same polymerase with an alternative modified long linker B to a molecular wire. The results of a polymerase activity assay highlighting the relative rates of incorporation for the two conjugated materials relative to the starting polymerase are shown in Figure 7. As can be seen, both N-terminal conjugated enzymes retain significant polymerase activity.

[0053] The schematic shown in Figure 9 illustrates protein-protein binding, with the antigen attached to the bridge. The probe on the peptide bridge is the HIV p24 antigen, which has affinity for anti-p24 antibodies. In the presence of the target anti-p24 antibody, the sensor current exhibits a pulse corresponding to the antigen-antibody binding event. The pulse detection rate (and percentage of time in the bound state) increases with increasing concentration of target molecule. Plotting the percentage of time bound versus target concentration provides a typical binding response curve for this antigen-antibody interaction.

[0054] The schematic diagram shown in Figure 11 illustrates antibody-antigen binding, with the antibody attached to a bridge. The probe on the peptide bridge is an anti-interleukin-6 antibody with affinity for the interleukin-6 antigen. In the presence of the interleukin-6 target antigen, the sensor current exhibits a pulse corresponding to the antibody-antigen binding event. Plotting the percentage of time bound versus target concentration provides a typical binding response curve for this antibody-antigen interaction.

[0055] In the schematic shown in Figure 12, the probe on the peptide bridge is Protein A. Protein A has a remarkable ability to bind to the constant (Fc) portion of immunoglobulin molecules from several different species. In the presence of IgG antibodies, the sensor current exhibits a pulse corresponding to an antigen-antibody binding event. Plotting the percentage of time bound versus target concentration yields a typical binding response curve for this antigen-antibody interaction.

[0056] All patents, publications, scientific articles, websites, and other documents and materials referenced or mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it were individually incorporated by reference or set forth in its entirety herein. Applicant reserves the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information, and other referenced materials or documents.

[0057] The specific methods and compositions described herein are representative of preferred embodiments, are illustrative, and are not intended to limit the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon review of this specification and are encompassed within the spirit of the invention as defined by the claims. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention, as illustratively described herein, as applicable, can be practiced in the absence of any element or elements or limitation or limitations not specifically disclosed herein as essential. Thus, for example, in each instance herein, in an embodiment or example of the invention, any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms herein. Additionally, terms such as "comprising," "having," and "containing" are to be interpreted expansively and are not limiting. The methods and processes, as illustratively described herein, as applicable, can be practiced in different order of steps and are not necessarily limited to the order of steps set forth in the specification or claims. Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural unless otherwise specified. Under no circumstances should this patent be construed as limited to the particular examples, embodiments, or methods specifically disclosed herein. Under no circumstances should this patent be construed as limited by any statements made by the examiner or any other officer or employee of the Patent and Trademark Office unless such statements are specifically and expressly adopted without limitation or reservation in the applicant's responsive documents.

[0058] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as set forth in the claims. Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it will be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0059] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation that removes any subject matter from the genus, regardless of whether the excised material is specifically described herein. Other embodiments are within the scope of the following claims. Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. a current-carrying molecular structure comprising a metal contact on a surface of an electrode, the metal contact being attached to a molecular wire comprising a conjugated moiety; a binding probe molecule conjugated to the molecular wire at the conjugation site, the conjugation of the binding probe molecule to the molecular wire being via a multifunctional linker molecule, the multifunctional linker molecule being covalently attached at one end to the N-terminus of the binding probe molecule and at the other end to the conjugation site on the molecular wire; A biosensor device comprising: A biosensor device in which a circuit is formed that is capable of detecting and / or obtaining detailed information regarding the binding of said binding probe molecules to a target ligand or binding partner.

2. 10. The biosensor device of claim 1, wherein the current-carrying molecular structure comprises: (i) a positive electrode having a metal contact on its surface; and (ii) a negative electrode having a metal contact on its surface.

3. The biosensor device of claim 1 , wherein the molecular wire is an alpha helical peptide or a polynucleotide.

4. The biosensor device of claim 1 , wherein the molecular wire is an alpha helical peptide.

5. 10. The biosensor device of claim 1, wherein the molecular wire is a non-peptide, including graphene, indium oxide thin film ribbon, carbon nanotube, single- or double-stranded polynucleotide, or DNA origami.

6. The biosensor device of claim 4 , wherein the alpha helical peptide has a conjugation site amino acid for attachment to a multifunctional linker molecule.

7. The biosensor device of claim 6 , wherein the conjugation site amino acid is selected from a cysteine ​​(Cys, C), lysine (Lys, K), or tyrosine (Tyr, T) amino acid residue.

8. The biosensor device of claim 1 , wherein the multifunctional linker molecule comprises one or more chemically reactive moieties or surface modification / binding motifs.

9. The biosensor device of claim 1 , wherein the multifunctional linker molecule comprises a vinyl boronate linker.

10. The biosensor device of claim 1 , wherein the multifunctional linker molecule comprises an activated phenolic linker.

11. 10. The biosensor device of claim 8, wherein the one or more reactive moieties comprise a mixed anhydride comprising a sulfonamide or sulfonic acid meta to the phenolic oxygen and adjacent to one or more electron-withdrawing groups including, but not limited to, halogens, polyfluorinated hydrocarbons, nitriles, carboxylic acids and derivatives, and nitro groups.

12. 10. The biosensor device of claim 1, wherein the multifunctional linker molecule comprises a reactive moiety comprising an E-styrenyl with a para-electron donating moiety or a reactive moiety comprising an E-vinyl boronate derived from an α-nucleophile / electrophile alkyne, which reacts with a linker having one or more reactive groups that can undergo selective "click" type chemistry and serve as a scaffold for further extension.

13. The biosensor device of claim 1 , wherein the binding probe molecules comprise proteins, peptides, polypeptides, or protein complexes of biological or synthetic origin.

14. The biosensor device of claim 1 , wherein the bound probe molecules comprise a polymerase.

15. The biosensor device of claim 1 , wherein the bound probe molecules comprise viral antigens.

16. The biosensor device of claim 1 , wherein the binding probe molecules comprise antibodies.

17. 2. The biosensor device of claim 1, wherein the bound probe molecule is selected from a DNA polymerase, an HIV-1 p24 viral antigen, an anti-IL-6 antibody or binding fragment thereof, or protein A.

18. A composite of a molecular wire and a binding probe molecule for use in a biosensor, wherein the molecular wire comprises an alpha-helical peptide having a conjugation site, the binding probe molecule is a protein having an N-terminus, and the conjugation of the binding probe molecule to the alpha-helical peptide is via a multifunctional linker molecule, the multifunctional linker molecule being covalently bonded at one end to the N-terminus of the binding probe molecule and at the other end to the conjugation site in the alpha-helical peptide.

19. 20. The molecular wire and bound probe molecule composite of claim 18, wherein the bound probe molecule comprises a protein selected from a DNA polymerase, an HIV-1 p24 viral antigen, an anti-IL-6 antibody or binding fragment thereof, or protein A.

20. 20. A method for making a conjugate of molecular wire and linked probe molecule according to claim 18, comprising the steps of: (i) synthesizing a multifunctional linker molecule comprising one or more chemically reactive handles; (ii) selecting a linked probe molecule of interest; (iii) reacting the linked probe molecule with a bifunctional linker reagent under selected conditions to generate a clickable intermediate linked probe molecule suitable for carbon-heterobond forming reactions or click chemistry; (iv) reacting the clickable intermediate linked probe molecule with a preselected molecular wire under suitable reaction conditions; and (v) purifying the conjugate of molecular wire and linked probe molecule.

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