WO2020210906A1 - N-heterocylic carbene-based electrochemical biosensors - Google Patents

Abstract

An electrochemical biosensor using ultra-stable N-heterocyclic carbene (NHC)-based self-assembled monolayers (SAMs) is used to detect a target in a sample. A receptor is coupled to the NHC SAM, such as an anti-measles antibody, which allows the electrochemical biosensor to detect whole measles virions. The use of the NHC SAM resulted in greater stability of the biosensor in comparison with known thiol-based SAM biosensors.

Description

N-HETEROCYLIC CARBENE-BASED ELECTROCHEMICAL BIOSENSORS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to United States Patent Application US

62/835,779, filed April 18, 2019, the entire contents of which is hereby incorporate by reference.

FIELD

[0002] The present disclosure relates generally to a biosensor.

BACKGROUND

[0003] Thiol-based self-assembled monolayers (SAMs) are susceptible to oxidation, which results in their eventual desorption from the surface. In turn, thiol desorption from the Au surface results in a short shelf-life for the thiol-based SAMs unless they are stored in inert environments (such as in an argon atmosphere) and under controlled conditions.

[0004] Early studies on the stability of this new class of SAMs showed a surprising resistance to SAM oxidation and chemical attack, far surpassing that of their thiol-based counterparts.¹ However, the commercial availability of NHCs is poor, with no suitable NHCs for biosensor development available for purchase at the time of this work. Described herein is the development of NHC SAMs that can be used in electrochemical biosensors, circumventing the drawbacks of thiol-based SAMs.

SUMMARY

[0005] In one aspect there is described a system for detecting a target in a sample from a subject, the system comprising:

[0006] a substrate having a surface;

[0007] one or more a linkers, each linker having a first end attached to the surface and a second end;

[0008] one or more receptors, wherein each receptor,

[0009] is bound to the second end of one or more of the plurality of linkers; and

[0010] is configured to interact with the target;

[0011] wherein a change in a signal is detectable when said target is bound to said receptor,

[0012] wherein said linker is a N-heterocyclic carbene (NHC), [0013] each said one or more linker may be the same or different,

[0014] each said receptor may be the same or different.

[0015] In one aspect there is described a system for detecting a target in a sample, the system comprising:

[0016] a substrate having a surface;

[0017] one or more linkers, each linker having a first end and a second end;

[0018] for each said linker:

[0019] the first end is attached to said surface of said substrate; and

[0020] the second end either comprises a receptor or does not comprise the receptor;

[0021] wherein said receptor is configured to interact with the target and a change in a signal is detectable when said target is bound to said receptor,

[0022] wherein said linker is a N-heterocyclic carbene (NHC),

[0023] each said one or more linker may be the same or different,

[0024] each said receptor may be the same or different.

[0025] In one example, the second end of two or more linkers comprise said receptor.

[0026] In one example, said target is a polypeptide, a polynucleotide, a lipid, or a small molecule.

[0027] In one example, said polypeptide comprises is derived from a virus virion.

[0028] In one example, said virion comprises a measles virion.

[0029] In one example, said polynucleotide comprises DNA, RNA, PNA, LNA, BNA, or aptamers.

[0030] In one example, the sample comprises a biological sample, an aerosol, or a water sample.

[0031] In one example, the substrate comprises a metal, other conducting non-metal substrate, and/or carbon.

[0032] In one example, the metal is Au, Pt, Cu, Mg, Ag, or other materials.

[0033] In one example, the metal is Au.

[0034] In one example, the NHC is a compound of formula (I):

[0035] In one example, the NHC is a compound of formula (II):

[0036] wherein R₁ and R₂ are independently selected from the group comprising C1

- C24 alkyl group, C1 -C24 substituted alkyl group, C1 -C24 aryl or C1 -C24 substituted aryl group.

[0037] In one example R₁ and R₂ are independently selected from the group comprising methyl, ethyl, iPr, t-Butyl, adamantyl or mesityl, and L is a linking group, preferably an amide, thioether or triazole ring.

[0038] In one example, L is a linking group.

[0039] In one example, said linking group is an amide, thioether or triazole ring.

[0040] In one example, R₁ is iPr, R₂ is iPr, and L is amide.

[0041] In one example, the receptor is a polypeptide.

[0042] In one example, the polypeptide is an antibody.

[0043] In one example, the antibody is an anti-measles H protein antibody.

[0044] In one example, the receptor is a polynucleotide, or a small molecule.

[0045] In one example, the polynucleotide comprises DNA, RNA, PNA, LNA, BNA, or aptamers.

[0046] In one example, further comprising a detectable label.

[0047] In one example, the detectable label is detected electrochemically.

[0048] In one example, the signal is an impedance response. [0049] In one example, the label is ferro/ferricyanide.

[0050] In one example, the label comprises an intercalator.

[0051] In one example, said intercalator comprises methylene blue.

[0052] In one example, the label comprises Ru.

[0053] In one aspect there is described a method of detecting a target in a sample, comprising

[0054] contacting a device of any one of claims 1 to 24 with said sample;

[0055] measuring a change in signal.

[0056] In one example, said change is indicative of a presence or absence or amount of said target in said sample.

[0057] In one example, further comprising:

[0058] determining the quantity of said target in said sample, if present.

BRIEF DESCRIPTION OF THE FIGURES

[0059] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0060] Figure 1 : SAM formation and modification procedure used in the various steps involved in the formation of Fc-SAMs and Ab-SAMs (i.e., the sensors to detect the measles virus) (i, Step 1) 10 mM ester-NHC triflate salt with 20 mM KHCO₃ for 24 hours in methanol, (ii, Step 2) 2,000 eq of KOH in ethanol for 24 hours, (iii) EDC/NHS activation for 1 hour, (iv-a, Step 3a) 1 mg/mL aminoferrocene for 24 hours at 4°C, and (iv-b, Step 3b) 100 mg/mL anti-measles antibody for 24 hours at 4°C.

[0061] Figure 2: (a) Cyclic voltammograms before and after modification of the ester-

NHC SAM to form a Fc-SAM at 50 mV/s. (b) Cyclic voltammograms of the Fc-NHC SAM at multiple scan rates. All scans were performed in 1 M NaCIO₄ with a Pt counter and Ag/AgCI reference.

[0062] Figure 3: Anodic peak current (black circles) and potential (blue squares) as a function of scan rate for a Fc-NHC SAM. All scans were performed in 1 M NaCIO₄ with a Pt counter and Ag/AgCI reference.

[0063] Figure 4: X-ray photoelectron spectra for the (a) Fe 2p and (b) N1 s orbitals of a Fc-modified SAM (black traces, Step 3a in Figure 1) and a deprotected SAM (red trace, Step 2 in Figure 1).

[0064] Figure 5: Schematic showing the effects of the layers of the NHC SAMs constructed in this work on the blocking of the ferro/ferricyanide reaction for the (a) Ester- NHC SAM, (b) COOH-Modified SAM, (c) Ab-modified NHC SAM and (d) the Ab-modified NHC SAM bound to a measles virion. The antibody (ca. 7 x 8 x 10 nm) and measles virus (spherical diameter of ca. 150 nm) are not drawn to scale and are significantly larger than the NHC SAM (ca. 1 nm).

[0065] Figure 6: (a) Nyquist plots and (b) CVs at each stage of NHC-Ab-SAM construction. The black trace corresponds to the electrochemical response of the ester-NHC SAM, while the blue trace represents the response from the deprotected SAM after the removal of the ethyl ester to form a terminal carboxylate functional group. The red trace corresponds to the electrochemical response of the complete Ab-SAM after immobilization of the anti-measles antibody. EIS was performed at the open circuit potential (0.27 V vs.

Ag/AgCI) from 10⁵ to 10^-1 Hz and cyclic voltammetry was performed at 25 mV/s in 1 M 4NaCIO₄ supplemented with 5 mM Fe(CN)₆ ^{3 -/4-}. (c) and (d) represent equivalent circuits used in this Chapter, where CPE is a constant phase element, R is a resistor and W is a Warburg element.

[0066] Figure 7: Recorded contact angle for four replicates of NHC-modified samples at each stage of the Ab-SAM synthesis, with a representative image shown in the Figure. A 5 m L drop of milliQ water was dropped onto the sample to measure the contact angle. An ANOVA coupled with a Tukey post-hoc test demonstrated a significant difference between the means of all samples compared to each other (p<0.0003).

[0067] Figure 8: (a) Nyquist plot demonstrating the impedance response of the NHC-

Ab-SAM to low (10 ng/mL) or high (1 mg/mL) concentrations of whole measles virus (b) Calculated increase in the resistance of the low frequency arc for duplicate anti-measles biosensors fit to the equivalent circuit shown in Figure 6d. Impedance measurements were performed from 10^-1-10⁵ Hz at the open circuit potential (0.27 V vs. Ag/AgCI) in 1 M NaCIO₄ supplemented with 5 mM Fe(CN)63-/4- with a Pt counter and Ag/AgCI reference.

[0068] Figure 9: Percent resistance increase for thiol (red) and NHC (black) Ab-

SAMs due to increases in measles virus concentration (n=3). (b) is an expanded view of the data in (a). Impedance measurements were performed from 10^-1-10⁵ Hz at the open circuit potential (0.27 V vs. Ag/AgCI) in 1 M NaCI04 supplemented with 5 mM Fe(CN)₆ ^{3 -/4-} with a Pt counter and Ag/AgCI reference.

[0069] Figure 10: Cyclic voltammograms recorded after exposure of (a) a thiol-based

Ab-SAM and (b) a NHC-Ab-SAM to measles virus at concentrations of 0, 10, 25, 50, 100, and 1 ,000 mg/mL. The cyclic voltammetry measurements were performed at 25 mV/s in 1 M NaCI04 supplemented with 5 mM Fe(CN)₆ ^{3 -/4-} with a Pt counter and Ag/AgCI reference. [0070] Figure 1 1 : Impedance response of (a) thiol-based and (b) NHC-based SAMs modified with BSA instead of the anti-measles antibody before and after exposure to 100 mg/mL measles virions. Reported average values are for three replicates. Impedance measurements were performed from 10^-1-10⁵ Hz at the open circuit potential (0.27 V vs. Ag/AgCI) in 1 M NaCI04 supplemented with 5 mM Fe(CN)63-/4- with a Pt counter and Ag/AgCI reference.

[0071] Figure 12: Cyclic voltammograms for (a) thiol-based and (b) NHC-based antimeasles biosensors at 0, 30, 60 and 90 minutes of testing (c) Percent change in the combined resistance of the low and high frequency arcs over 90 minutes for Ab-modified SAMs based on either thiols (red trace) or NHCs (black trace). Sensors were exposed to 1 M NaCIO₄ between tests. Impedance measurements were performed from 10^-1-10⁵ Hz at the open circuit potential (0.27 V vs. Ag/AgCI) in 1 M NaCIO₄ supplemented with 5 mM Fe(CN)₆ ^{3- /4-} with a Pt counter and Ag/AgCI reference.

DETAILED DESCRIPTION

[0072] In one aspect there is described a system for detecting a target in a sample from a subject, the system comprising:

[0073] a substrate having a surface;

[0074] one or more a linkers, each linker having a first end attached to the surface and a second end;

[0075] one or more receptors, wherein each receptor,

[0076] is bound to the second end of one or more of the plurality of linkers; and

[0077] is configured to interact with the target;

[0078] wherein a change in a signal is detectable when said target is bound to said receptor,

[0079] wherein said linker is a N-heterocyclic carbene (NHC),

[0080] each said one or more linker may be the same or different,

[0081] each said receptor may be the same or different.

[0082] In one aspect there is described a system for detecting a target in a sample, the system comprising:

[0083] a substrate having a surface;

[0084] one or more linkers, each linker having a first end and a second end;

[0085] for each said linker:

[0086] the first end is attached to said surface of said substrate; and [0087] the second end either comprises a receptor or does not comprise the receptor;

[0088] wherein said receptor is configured to interact with the target and a change in a signal is detectable when said target is bound to said receptor,

[0089] wherein said linker is a N-heterocyclic carbene (NHC),

[0090] each said one or more linker may be the same or different,

[0091] each said receptor may be the same or different.

[0092] In some examples, the systems described herein may also be referred to as a biosensor, or device.

[0093] It will be appreciated that“system”,“biosensor”, or“device”, may refer to a combination of the reagents necessary for detection and the instrument for deploying them.

[0094] As used herein, the term“support” refers to a material which can bind or attach a substance, such as a linker and/or a plurality of linkers.

[0095] In some examples, a support may be made from any material which has a capability of binding to a biological molecule as used herein via covalent or noncovalent bonds, or which may be induced to have such a capability.

[0096] In some examples, the term "support" means the material to which a linker and/or plurality of linkers is bound or attached.

[0097] The support may be made of a "support material". The support material can be porous or non-porous. Some non-limiting examples of non-porous support materials include plastic substrates, glass substrates, metal substrates and/or silicon substrates. Such substrates can be layered upon each other, and/or layered with porous substrates.

[0098] In some examples, the support material may be treated to provide a chemically reactive group on a surface of the support material. This chemically reactive group can be useful in immobilizing one or more specific binding reagents, such as a linker and/or plurality of linkers, and includes all organic and inorganic groups used in covalent coupling of molecules to solid surfaces and known to persons skilled in the art, such as hydroxyl, carboxyl, amino, sulphonate, thiol, and aldehyde groups, etc.

[0099] In some examples, the support is conducting or conductive. In some examples, the surface is conductive or conductive. In some example both the support and surface is conductive or conductive.

[00100] In some examples, the surface of the support material may be coated or derivatized, e.g., using techniques such as sputtering, vapor deposition and the like, and given a coating of silicon, a metal or other. [00101] In some examples, the support comprises a metal. In some examples, the metal comprises is Au, Pt, Cu, or Mg.

[00102] In some examples, the support comprises conducting oxides.

[00103] The support may have a zone for detection of a target.

[00104] As used herein, the terms "zone," "area," "location" and "site" are used interchangeably to define a region of the device comprising an immobilized specific binding reagent. . The locations may be dots, circles, squares, zones or lines, etc.

[00105] The support may include a sample receiving zone.

[00106] As used herein, the term "sample receiving zone" refers to the portion of the device that is contacted with the sample comprising or suspected of comprising the target.

[00107] As used herein, the term "detection zone" refers to one or more portions of the device that comprises an immobilized specific binding reagent capable of forming a complex with a target in a sample. In some examples, the devices may comprise one or more detection zones. Each detection zone can comprise the same or a different immobilized specific binding reagent.

[00108] As used herein, the term "immobilization" refers to the attachment or entrapment, either chemically or otherwise, of a linker and/or one or more linkers to one or more support materials. A binding reagent can be immobilized on the support material by any suitable methods. For example, binding reagent can be immobilized by absorption, adsorption, or covalent binding to the support material, or by attaching to another substance or particle that is immobilized to the desired location on the support material. In one aspect of the invention, the binding reagent is covalently attached directly or indirectly to the support material.

[00109] As used herein, a "control zone" refers to one or more portions of the device that comprises a control specific binding reagent - a binding reagent that binds specifically to an analyte on one or more control samples.

[00110] A“target”, "target molecule", or“agent” refers any molecule to which a receptor can specifically bind.

[00111] In some examples, a target may, for example, constitute part of a larger molecule.

[00112] In some examples, a target may be at least one of an enzyme substrate, a ligand, an antigen, an antibody, a nucleotide, an amino acid, a peptide, a protein, a nucleic acid, a lipid, a carbohydrate, an organic compound, or an inorganic compound, but is not limited thereto. [00113] A target may, in some examples, be a component of a sample, particularly a sample of a biological material (tissue or body fluid) or a sample derived from a biological material.

[00114] In some examples, a target also includes fragments of any molecule found in a sample.

[00115] In some examples, a target also includes, but is not limited to a metabolite, an amino acid, a herbicide, a pesticide, an environmental pollutant, an analyte, a veterinary drug, a drug, a drug of abuse, and/or a small molecule.

[00116] In a specific example, the target is a component of the measles virus.

[00117] As used herein, the term "measles virus" refers to a virus within the morbilli virus genus of single-stranded, negative -sense, enveloped (non-segmented) RNA virus.

[00118] The term "receptor" refers to a substance which exhibits affinity for and is capable of specifically binding to a target of interest.

[00119] In some examples, specific binding is characterized by a higher binding affinity to a target than to one or several irrelevant molecules which are used as negative controls in a binding assay.

[00120] In some example, a receptor is a biomolecule to which one or more specific kinds of molecules (i.e., targets) may attach. In some examples, a receptor may be at least one of an enzyme substrate, a ligand, an antigen, an antibody, a polynucleotide, an polypeptide, an amino acid, a nucleic acid, a lipid, a carbohydrate, an organic compound, or an inorganic compound, but is not limited thereto.

[00121] The term“small molecule”, as used herein, refers to a chemical agent including, but not limited to a compound, a chemical compound, a composition, a pharmaceutical composition, nucleobases, nucleosides, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds), and salts, esters, carbohydrates, and other pharmaceutically acceptable forms of such compounds.

[00122] The term“polypeptide” as used herein refers to a polymer of amino acids.

The terms“protein” and“polypeptide” are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length.

[00123] Polypeptides typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used. [00124] One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a“polypeptide”. Polypeptides may be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid phase peptide synthesis, etc. The term“polypeptide sequence” or“amino acid sequence” as used herein can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence presented herein is presented in an N-terminal to C-terminal direction unless otherwise indicated.

[00125] The term“derivative” as used herein refers to peptides which have been chemically modified, for example by ubiquitination, labeling, pegylation (derivatization with polyethylene glycol) or addition of other molecules. A molecule is also a“derivative” of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half- life, etc. The moieties can alternatively decrease the toxicity of the molecule, or eliminate or attenuate an undesirable side effect of the molecule, etc.

[00126] The term“polynucleotide” includes, but is not limited to, single- and double- stranded and triple helical molecules. In some examples, polynucleotide generally refers to polynucleotides of between about 3 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an polynucleotides. Polynucleotide may also be referred to as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. In some nonlimiting examples, a polynucleotide includes a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched

polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art. Nucleic acids may be naturally occurring, e.g., DNA or RNA, or may be synthetic analogs, as known in the art. Such analogs may be preferred for use as probes because of superior stability under assay conditions. Modifications in the native structure, including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates;

phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-0'-5'-S-phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH2-5'-0- phosphonate and 3'-NH-5'-0-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage.

[00127] In some examples, a polynucleotide comprises DNA, RNA, PNA, LNA, BNA, or aptamers.

[00128] The term“antibody,” as used herein, refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgG 1 , lgG2, lgG3, lgG4, and the like. In one example, an immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed“alpha,” “delta,”“epsilon,”“gamma” and“mu” respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known. In one example, the antibody is a monoclonal antibody. In another example, the antibodies are humanized, chimeric, human, or otherwise-human-suitable antibodies.“Antibodies” also includes any fragment or derivative of antibodies.

[00129] Antibody fragments include, but are not limited to Fab, F(ab')2, and Fv antibody fragments. The term“epitope” refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules (e.g., amino acid or sugar residues) and usually have specific three dimensional structural characteristics as well as specific charge

characteristics.

[00130] In some examples, the antibodies are“nanobodies”, otherwise known as “VHH antibodies”.

[00131] It will be appreciated that a wide range of samples may be analyzed. [00132] The term "sample" generally refers to material, in non-purified or purified form.

[00133] In some examples, the sample it a In some examples, the term“sample” or “test sample” as used herein refers to a biological sample.

[00134] In some examples, the samples may be water-containing sample, airborne and/or aerosol, etc.

[00135] Samples from biological sources (i.e. biological samples) usually comprise a plurality of analytes, for example targets.

[00136] Biological samples may be obtained from a subject.

[00137] The term“subject”, may refer to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, nonhuman mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In a specific example, the subject is a human.

[00138] Biological samples from a subject include, but are not limited to bodily fluids.

[00139] As used herein the term“bodily fluid” refers to any fluid found in the body of which a sample can be taken for analysis. Non-limiting examples of bodily fluids include blood, plasma, serum, lymph, sudor, saliva, tears, sperm, vaginal fluid, feces, urine or cerebrospinal fluid.

[00140] Biological samples from a subject also include samples derived, e.g., by biopsy, from cells, tissues or organs. This also encompasses samples comprising subcellular compartments or organelles, such as the mitochondria, Golgi network or peroxisomes. Biological samples also encompass gaseous samples, such as volatiles of an organism. Biological samples may be derived from a subject.

[00141] Techniques for obtaining different types of biological samples are known in the art.

[00142] In one example, the NHC is a compound of formula (I):

[00143] In one example, the NHC is a compound of formula (II):

[00144] wherein R₁ and R₂ are independently selected from the group comprising C₁ - C₂₄ alkyl group, C₁-C₂₄ substituted alkyl group, C₁-C₂₄ aryl group or C₁-C₂₄ substituted aryl group. In some examples R₁ and R₂ may be independently selected from the group comprising methyl, ethyl, iPr, t-Butyl, adamantyl or mesityl.

[00145] wherein L is a linking group. In some examples, L may be an amide, thioether or triazole ring. For example, carbodiimide or click chemistry is used to attach a biological marker or redox label.

[00146] In some examples, R₁ is iPr, R₂ is iPr, and L is amide.

[00147] In some example, the linked may be any combination of a carbene contacting the electrode and a functional group that can react with a protein or DNA sequence on the distal end. This could include placing“spacers” between the NHC and the functional group, where spacers could be alkanes, ethylene glycol units (including polyethylene glycol), aromatic groups, or any other number of molecules.

[00148] In some examples, it may be possible to react other functional groups from the protein. The current work has focused on carbodiimide chemistry reacting a carboxyl group from the monolayer with primary amines from the protein. It is possible to do the opposite (amines in the SAM), or other reactions.

[00149] In some examples, the system comprises a detectable label.

[00150] As used herein, a "detectable label" is a substance that is capable of producing a detectable signal.

[00151] In some examples, detection of the label provides an indication of the presence and/or amount of the target in the sample.

[00152] The term "detectable label," as used herein, refers to a label which is observable using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron- spin resonance, ultraviolet/visible absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, electrochemical and electrical methods, including but not limited to impedance measurements.

[00153] Thus, the term“label” includes, but is not limited to, a substance, such as a chemical moiety or protein which is incorporated into a compound and is readily detected. The label can be directly detectable (fluorophore) or indirectly detectable (hapten or enzyme). Such labels include, but are not limited to, radiolabels that can be measured with radiation-counting devices; pigments, dyes or other chromogens that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent labels (fluorophores), where the output signal is generated by the excitation of a suitable molecular adduct and that can be visualized by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems, for example. The label can be a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance; or an enzyme, where there occurs an enzyme- dependent secondary generation of signal, such as the formation of a colored product from a colorless substrate.

[00154] Specific examples of detectable labels include, but are not limited to, a chemiluminescent group, a chromophore, a dye, a fluorophore, a radiolabel, metals, metal nanoparticles, colloidal metal, nano particle colloidal metal, core-shell nanoparticles, such as nanoparticles comprising a dielectric coated with metal. Preferably the metal is selected from gold, silver, platinum and palladium. More preferably the metal is gold.

[00155] In some examples, the detectable label is detected electrochemically.

[00156] In some examples, the detectable label is detected using voltammetry. It will be appreciated that voltammetry refers to any number of other techniques, including but not limited to, cyclic voltammetry, pulsed voltammetry, which would also show the detection.

[00157] In some example, the detectable label is detected using chronoamperometry, or coulometry, which would also show the detection.

[00158] In a specific example, the detectable label is measured using an impedance response.

[00159] In one example the label is ferro/ferricyanide.

[00160] In one example the label comprises an intercalator.

[00161] In one example said intercalator comprises methylene blue.

[00162] In one example the label the label comprises a Ru-containing ion or‘contains Ru’. [00163] In one example the label the label comprises ruthenium hexamine.

[00164] Methods of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such a kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[00165] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

EXAMPLES

[00166] As no SAM-based biosensors using NHCs have been reported at the time of this work, a well characterized antibody-based biosensor approach was used in order to reduce the number of variables tested at once. An anti-measles antibody was chosen for this work in order to develop a biosensor capable of detecting the measles virus, due, in part, to the alarming increase in measles cases in recent years.

[00167] While difficulties had been previously experienced by others in synthesizing a carboxyl-terminated NHC that could be analogous to the carboxyl-terminated thiol, they were able to synthesize an ester-terminated NHC (Figure 1 , Step 1) and develop a protocol to “deprotect” the carboxyl group (Figure 1 , Step 2). This allowed the same carbodiimide chemistry to be employed to develop a measles biosensor (Figure 1 , Step 3b). The work herein utilizes an unordered protein immobilization method for the immobilization of the antibody (Figure 1).

[00168] Initial studies aimed at confirming the deprotection and the subsequent modification of the carboxyl group were carried out with aminoferrocene as a trackable proxy in place of the antibody (Figure 1 , Step 3a). The incorporation of the ferrocene group (Fc) allows for cyclic voltammetry to be used in order to verify the success of the modification procedure, as Fc exhibits rapid electron transfer with the underlying Au through a tunneling mechanism. It is shown here that the NHC SAMs could be efficiently deprotected and modified with aminoferrocene, also confirmed through electrochemistry, X-ray photoelectron spectroscopy and contact angle analysis. This led to the development of the first reported electrochemical biosensor, based on NHC SAMs, as well as the first biosensor capable of detecting the intact measles virus within the relevant concentration regimes for human health applications. [00169] 2 Verification of ester-NHC modification with aminoferrocene

[00170] To assess the deprotection and subsequent derivation of the ester-NHC SAM (Figure 1 , Step 1), an electrochemically trackable proxy was used instead of a protein in early experiments. Aminoferrocene was chosen for this purpose, due to the rapid and well understood electron transfer reaction of the ferrocene group (and the reactivity of the primary amine. Figure 2a shows the cyclic voltammograms (CVs) for bare (i.e., unmodified) Au, an ester-NHC SAM (Figure 1 , Step 1), and a ferrocene-modified NHC SAM (Figure 1 , Step 3a). The CV for the ester-NHC SAM (blue trace) demonstrates mostly capacitive behaviour with no redox peaks observed and exhibits significantly smaller double layer charging currents than the bare Au electrode (black trace). Interestingly, the capacitance seems to increase at the anodic end of the scan, which has not been reported previously for NHC SAMs but is similar to what was seen for thiol-based SAMs containing ferrocene. The exact reason for this behaviour is still unclear, but it is possible that there is a reorganization of the monolayer during the potential scan, where the molecules on the Au surface change their orientation such that the thickness of the monolayer is altered (which would result in altered

capacitance). The large cathodic current of the bare Au electrode below 0 V vs. Ag/AgCI, which is likely due to oxygen reduction as the electrolyte was not purged of oxygen, is also heavily suppressed once the ester-NHC SAM is on the Au surface.

[00171] The ferrocene-modified SAM (Figure 1 , Step 3a) exhibits a pair of redox peaks, centered around 0.3 V vs. Ag/AgCI in Figure 2a (red trace), suggesting that the aminoferrocene (hereafter referred to simply as ferrocene, or Fc-NHC) was successfully incorporated into the SAM during the modification procedure. The capacitance of the Fc- NHC SAM is also larger than that of the ester-NHC SAM, which could reflect an effect of the presence of the ferrocene group or, potentially, some loss of the NHC SAM from the Au surface, though the capacitance does not increase to the level of the bare Au electrode (black trace, Figure 2a).

[00172] A scan rate study was also performed on the Fc-NHC SAM, with CVs shown in Figure 2b. The analysis of the anodic peak over the tested scan rates is shown in Figure 3. It is noted that the peak potential increases with a logarithmic trend, as is expected for an irreversible electrochemical reaction (blue squares, Figure 3). Conversely, the peak current increases approximately linearly with scan rate, as is expected for a surface-confined electrochemical reaction (black circles, Figure 3). This provides confidence that the ferrocene is immobilized in the SAM rather than in solution. Interestingly, the cathodic peak broadens significantly at the higher scan rates (Figure 2b). Such behaviour has previously been reported to be due to lateral interactions within a SAM,^8-12 where the oxidation of one molecule impacts the ability of an adjacent molecule to oxidize. One prior report has claimed to observe this behaviour for ferrocene molecules in NHC SAMs, though minimal data was presented in that publication.¹³ There is also a relatively large peak separation, ca. 120 mV, seen in Figure 2a. Such a large peak separation could be indicative of a difference in local environments for the oxidized and reduced states of the ferrocene,¹⁴ where the adjacent molecules to the redox-active ferrocenes can more favourably interact with one oxidation state compared to the other, which can also be influenced by lateral interactions in a monolayer.¹¹·¹². While it is clear from the results of Figures 2 and 3 that the ferrocene moiety is surface-confined and easily addressable via electrochemistry, the peaks in the CVs of Figure 2 are not as sharp as those shown for thiol-based SAMs containing ferrocene. It is plausible that the lateral interactions described above could be the cause of these differences, but effects of the NHC group interacting with the Au electrode cannot be ruled out from these data. Further studies on this behaviour, including computational work, are currently underway to explore this phenomenon.

[00173] The formation of the Fc-NHC SAM (Figure 1 , Step 3a) was further confirmed with X-ray photoelectron spectroscopy (XPS, Figure 4), which demonstrated the presence of Fe after the amide bond was formed between the aminoferrocene and the carboxylate group in the SAM. While the XPS data cannot provide an exact surface coverage, the Fe signal (Figure 4a) can be compared to the signal from other atoms in order to estimate surface coverage. If full conversion of the ester-NHC SAM (step 1 in Figure 1) to the Fc-NHC SAM (Step 3a in Figure 1) had been achieved, the N:Fe ratio is predicted to be 3:1 based on the N and Fe contents of the structure shown in Figure 1 for the Fc-NHC SAM (Step 3a). The Fc-NHC SAM tested in this work has a N:Fe ratio of 3:0.9, which suggests ca. 90% of the monolayer was converted to the Fc-NHC structure shown in Step 3a of Figure 1. This is in stark contrast to the 25% ferrocene coverage observed in the CVs of Figure 2.

[00174] The observed N1 s peak of the Fc-NHC SAM (black trace, Figure 4b) also increases slightly in intensity (by ca. 10%) compared to the deprotected NHC SAM (red trace in Figure 4b) due to the incorporation of the nitrogen from aminoferrocene into the SAM in Step 3a of Figure 1. However, the increase did not match predictions, as a 50% increase was expected based on the N content of the structure outline for the Fc-NHC SAM in Step 3a of Figure 1. This could also indicate some loss of the underlying NHC in the SAM between Steps 2 and 3 of the modification procedure outlined in Figure 1 , though the previously reported stability of NHC SAMs would predict that none of these treatments should compromise the integrity of the NHC SAM.1

[00175] 3 Characterization of the stages of NHC-based measles biosensor construction

[00176] While Section 2 focused on demonstrating the formation of the ester-NHC SAMs (Figure 1 , Step 1) and developing a modification procedure to form the Fc-modified NHC SAM (Figure 1 , Step 3a), Section 3 focuses on capitalizing upon the developed modification procedure to create a biosensor capable of detecting the measles virus. To achieve this, an antibody (Ab) was chosen that would target the H protein on the surface of the measles virion (a single virus particle).^{15 16} The H protein is on the outer surface of the virus, and thus no pre-processing steps should be needed to break open the virus particles, providing a simpler workflow to an end user compared to the common approaches of detecting the internal N protein or the viral genome.^{17 18} This anti-measles antibody was immobilized on the NHC SAM via the carbodiimide crosslinking chemistry to create an Ab- modified SAM (antibody-modified SAM).

[00177] As the antibody does not undergo any redox reactions, the impedance and CV response of the SAM was measured in a solution of ferro/ferricyanide ions to assess the degree of blocking of this electron transfer reaction across the monolayer at each stage of sensor fabrication (Fabrication procedure outlined in Figure 1 , blocking shown in Figure 5). The impedance was measured at the open circuit potential, which was stable at ca. 0.27 V vs. Ag/AgCI in this work. If the antibody was successfully immobilized, it was expected that the impedance would increase significantly, as the protein is expected to block the ferro/ferricyanide from reacting with the Au electrode (Figure 5).

[00178] Figure 6a shows that the ester-NHC baseline (blue trace, Step 1 in Figure 1) exhibits ca. 2,500 W of resistance and produces a typical single-arc Nyquist plot with a Warburg diffusion element becoming clear at the low frequency end of the spectrum. This data was fit to the circuit shown in Figure 6c, with the results tabulated in Table 1. The fitting was considered to be good if the c2 value, a measure of how closely the circuit matches the experimental data, was less than 1x10^-3.

[00179] Interestingly, the impedance of the deprotected carboxyl-terminated intermediate exhibits a significantly smaller resistance of ca. 860 W (black trace in Figure 6a, Step 2 in Figure 1).

[00180] This is likely due to the decreased thickness of the monolayer, as shown in Figure 5. This provides a lower barrier to the ferro/ferricyanide electron transfer reaction, with the rate decreasing exponentially with increasing thickness of a monolayer.^{19 20} It is also seen in Table 1 that the measured capacitance during the impedance experiment increased slightly, from 1.3 mF to 1.7 mF, which also supports the presence of a shorter SAM according to the capacitance equations. When fit with constant phase elements, the n value for both the ester-NHC SAM and the deprotected SAM was consistently between 0.97 and 0.99 over 12 measurements and three samples each. This implies that the SAMs are relatively defect- free (i.e., free of areas where the Au is exposed).^{21 22}

[00181] Table 1. Equivalent circuit fitting results for Ab-modified NHC SAM construction*.

* Impedance measurements were performed from 10^-1-10⁵ Hz at the open circuit potential (0.27 V vs. Ag/AsCl) in 1 M NaCIO₄ supplemented with 5 mM Fe(CN)₆ ^{3 -/4-} with a Pt counter and Agt AgCl reference. The NHC SAM and COOH-modified NHC SAM were fit to the equivalent circuit shown in Figure 7 6c. whereas the Ab -modified NHC SAM was fit to the equivalent circuit shown in Figure 7.6d.

[00182] Once the antibody was immobilized on the SAM (Figure 1 , Step 3b), the measured resistance became significantly larger (5,900 W, red trace in Figure 6a), which would be expected from the tethering of a large, non-conductive protein to the outer surface of the monolayer. Interestingly, the impedance response after the Ab was surface- immobilized in Fig 4a demonstrates a stretched arc that fits a two time constant (2 process) equivalent circuit (shown in Figure 6d), with the results shown in Table 1. The high frequency arc (CPE1 and R1) exhibits a similar capacitance to the carboxyl-terminated intermediate stage (1.8 mF), while the low frequency arc (CPE2 and R2) demonstrates a much higher capacitance (14 mF). This low frequency arc also has a different n value from the high frequency arc, ranging between 0.65 and 0.85 over 12 measurements from three different electrodes. As the low frequency arc contributed the majority of the measured resistance, this arc was attributed to the immobilized Ab molecules on the SAM, which was expected to significantly hinder electron transfer across the SAM. This is similar to the blocking seen for TLR-4, though the increase in resistance relative to the observed SAM resistance is larger for the antibody than the TLR-4. The relatively larger increase in resistance could be due to the increased size of the antibody (7 x 8 x 10 nm)²³ compared to the TLR-4 protein (10 x 12 x 2 nm).²⁴

[00183] Similar trends were seen when the SAM was probed with cyclic voltammetry in the 5 mM ferro/ferricyanide solution at each stage during sensor fabrication (Figure 6b and Table 2). When the ester-NHC SAM was tested (blue trace, Step 1 in Figure 1), the ferro/ferricyanide reaction was seen to be kinetically irreversible, with a peak separation of 223 mV. However, when the ester-NHC SAM is deprotected to form the carboxylate- terminated SAM (Step 2 in Figure 1), the ferro/ferricyanide reaction becomes much more reversible. The peak separation decreases to 158 mV and the peak current also decreases substantially. As with the impedance results of Figure 6a, this was to be expected when the thickness of the monolayer on the Au electrode was decreased through the removal of the ethyl group from the ester (Figure 5). This decreased distance between the Au electrode and the ferro/ferricyanide ions allows for increased rates of electron tunneling, which manifests as a more kinetically reversible CV, which is shown in Figure 6b for the COOH-modified SAM.

[00184] Table 2. CV peak characteristics for the voltammograms shown in Figure 6b*.

*Cyclic voltammetry was performed at 25 mV/s in 1 M NaC104 supplemented with 5 inM Fe(CN)₆ ^{3 -/4-}.

[00185] Importantly, the formation of the Ab-modified SAM (Step 3b in Figure 1) results in a large increase in the irreversibility of the ferro/ferricyanide reaction. The peak separation becomes 338 mV and the currents are substantially decreased (Table 2). This mirrors the increased resistance measured by impedance spectroscopy (Figure 6a and Table 1), likely due to the presence of the antibody insulating the electrode from the electrolyte containing the ferro/ferricyanide redox couple.

[00186] Contact angle measurements (Figure 5) also provided evidence that the SAM modifications were successful. The hydrophilicity of the surface was increased after the ester-NHC SAM (Step 3 in Figure 1) was deprotected to from the carboxyl-terminated intermediate (step 2 in Figure 1), and the hydrophilicity increased further after the antibody was immobilized on the SAM (Step 3b in Figure 1). Interestingly, the contact angle was still slightly hydrophilic (i.e., < 90 °) for the ester SAM, even though this SAM terminated in a methyl group. Such terminal groups are typically associated with hydrophobic contact angles (i.e., > 90 °) in SAMs due to their unfavourable interactions with water.^25-27 It was also noted that the contact angle recorded for the Ab-modified SAM was ca. 45 °. This is significantly more hydrophobic than was seen when TLR-4 was immobilized with thiol-based SAMs, typically producing a contact angle of ca. 25 °. It is possible that the antibody itself is not as hydrophilic as the TLR-4, as the chemical groups present on the surface of the proteins, due to the various amino acids that comprise the protein structure, are not identical between TLR-4 and the antibody.

[00187] 4 Confirmation of antibody presence in the antibody-modified SAMs

[00188] A modified enzyme-linked immunosorbent assay (ELISA,) was performed with a peroxidase-modified secondary antibody to confirm that the desired antibody was successfully immobilized on the monolayer surface. Table 3 shows the absorbance at 650 nm, which is the measured output of an ELISA, for a preliminary experiment comparing the different stages of the NHC SAM (as outlined in Figure 1). As expected, minimal responses are observed for the Au, ester-NHC SAM and deprotected NHC SAM samples, as a signal should only be produced in the presence of the antibody. There was a slight increase in signal for the deprotected NHC SAM, which is likely due to trace non-specific adhesion of the secondary antibody used in the ELISA. When the anti-measles antibody-modified SAM was tested, the observed ELISA signal is seen to be significantly larger than the other tested conditions, as would be expected from the successful tethering of the antibody to the SAM (Step 3b in Figure 1).

[00189] Table 3. ELISA analysis of Ab-modified NHC SAM construction*.

*Measured absorbance at 650 nm after a modified ELISA was performed to determine tlie presence of anti-measles antibodies on tlie sample surface.

**An ANOVA coupled with a Tukey post-hoc test demonstrated a significant difference between the aliquot deposited antibody and the NHC Ab-modified SAM compared to the other samples (p<0.0001)

[00190] To explore the impact of using a NHC SAM to immobilize the antibody compared to using a thiol-based SAM, 6-mercaptohexanoic acid was used to form a thiol- based SAM in place of the NHC SAM. This thiol was chosen due to the chain length, which matches the length of the deprotected NHC SAM (Step 2 in Figure 1), meaning that the resulting antibody SAMs should be identical in terms of the thickness of the SAMs on the Au surface. As this thiol already has a carboxyl terminal group, there is no need for a

deprotection reaction.

[00191] Table 4 shows the ELISA response for thiol-based and NHC-based SAMs modified to immobilize either bovine serum albumin (BSA) or the anti-measles antibody. The BSA electrodes were used as control samples to ensure that the response from the antibody-modified SAMs was not due to extraneous effects from the SAMs or Au (i.e., adsorption of the ELISA reagents onto the SAMs or non-specific interactions between the reagents and the SAM/Au). The negative control was performed without the presence of any Au, representing the baseline measurement of the experiment. The response from the BSA samples is statistically indistinguishable from the negative control (p<0.0001), as would be expected due to the absence of the anti-measles antibody in those samples. This provides confidence that the results for the antibody samples of the thiol and NHC SAMs is due to the antibody rather than any effects from the underlying structures. [00192] Table 4. ELISA comparison of antibody immobilization with thiol and NHC- based SAMs*

presence of anti-measles antibodies on the sample surface.

**An ANOVA coupled with a Tukey post-hoc test demonstrated a significant difference between the Ab -modified thiol SAM and the NHC-BSA. Tliiol-BSA and negative control samples (p<0.0001).

*** An ANOVA coupled with a Ttikey post-hoc test demonstrated a significant difference between the NHC Ab -modified SAM and the NHC-BSA. Tliiol-BSA and negative control samples (p<0.0001). as well as a significant difference compared to the Ab-modified thiol SAM (p<0.005).

[00193] The ELISA response from the antibody modified SAMs in Table 4 is significantly larger than any of the BSA or negative control samples, indicating the presence of the antibody tethered to the SAMs. Interestingly, the thiol-based SAM shows slightly higher ELISA signals than that of the NHC-based SAMs, indicating that there is more antibody immobilized on the thiol-based SAMs. While small, this difference is consistent over six replicates for each type of SAM. This could be due to the tighter packing of thiol-based SAMs compared to NHC-based SAMs, which would result in denser coverage of the reactive carboxyl groups present in the thiol-based SAM than for the NHC counterpart.^{1 28} This difference would only be compounded by the ca. 25% efficiency of the deprotection reaction (Figure 1 , Step 2), which was calculated from the earlier aminoferrocene results. The fact that the ELISA responses are so similar, even with the expected denser thiol packing and the inefficient NHC deprotection reaction, demonstrates that the relatively large size of the antibody (ca. 7 x 8 x 10 nm) likely overcomes the effects of these differences in the SAMs. [00194] To ensure that the results of Table 3 and 4 are due to the tethering of the antibody to the monolayer, as outlined in Step 3b of Figure 1 , rather than through nonspecific adhesion and physisorption, a series of control experiments was performed. Table 7.5 shows the average ELISA response for the anti-measles antibody tethered to the SAM, drop casted and dried onto bare Au, a baseline measurement for bare Au without the antibody and for a negative control where no Au or antibody was used (i.e., a“background” measurement). Table 5 shows that the signal recorded from the Ab-modified SAM (Step 3b of Figure 1) is significantly larger than all other samples, with over five times the signal of the drop-casted samples. The signals of the bare Au and the negative control samples are statistically indistinguishable based on an analysis of variance, but the drop-casted antibody method produces a statistically different ELISA result. This indicates that drop-casting the antibody did result in some adsorption of the antibody onto the Au surface, though it is significantly less than the SAM modification method. The drop-casting method was electrochemically tested for a response to measles virions in triplicate, but no response was observed and the data were extremely irreproducible. As such, this method was not continued with in future experiments.

[00195] Table 5. ELISA comparison of antibody deposition methods*.

Measured absorbance at 650 nm after a modified ELISA was performed ro determine the presence of anti-measles antibodies on the sample surface.

**An ANOVA coupled with a Tukey post-lioc test demonstrated a significant difference between the drop-casted antibody and the other samples (p<0.0001).

An ANOVA coupled with a Tukey post-hoc test demonstrated a significant difference between the NHC Ab-modified SAM and the other samples (p<0.0001 ).

[00196] 5 Response of the antibody-modified SAMs to the measles virus

[00197] To test the biosensor, measles virions (virus particles) inactivated by gamma radiation, chosen in order to ensure that the structure of the proteins in the virus were preserved, were used. The biosensors were tested electrochemically in a 1 M NaCI04 solution supplemented with 5 mM ferro/ferricyanide, similar to the testing performed in Section 3 to verify the sensor construction. The incubations with the measles virions were performed ex situ in small volumes (300 m L) in order to minimize the generation of biohazardous waste.

[00198] When the anti-measles Ab-modified NHC SAM was exposed to samples containing a range of whole measles viral particle (i.e., virions) concentrations (1 ng/mL - 1 mg/mL), it was observed that the low-frequency arc, previously attributed to the antibody in Section 3, experienced a significant increase in impedance as the measles virus concentration in solution increased (Figure 8). The high-frequency arc experienced little change, as would be expected from the monolayer contributions as the antibody bound its target (as outlined in the equivalent circuit of Figure 6d). If the high frequency arc changed significantly over the course of the experiment, this could indicate that the monolayer was changing during testing. Figure 8a shows representative EIS spectra for a low (10 ng/mL) and a high concentration (1 mg/mL) of the measles virus, clearly showing an increase in the resistance of the low frequency arc and the minimal changes in the resistance of the high frequency arc.

[00199] As no data on the binding affinity of the anti-measles antibody to either purified H-protein or whole measles virions were available at the time of this work, a large range of virus concentrations was tested to determine the binding affinity. Figure 8b depicts the calculated increase in the resistance of the low frequency arc as a function of logarithmic increases in the virus concentration, from 1 ng/mL to 1 mg/mL, for two duplicate biosensors, using the equivalent circuit of Figure 6d. The fitting results for one of the biosensors are shown in Table 6.

Table 7.6. Equivalent circuit fitting for an Ab -modified NHC SAM responding to increases in the concentration of measles virions*.

^*: Impedance measurements were performed from 10^-1-10⁵ Hz at the open circuit potential (0.27 V vs. AgAgCl) in 1 M NaClOr supplemented with 5 mM Fe(CN)₆ ^{3 -/4-} with a Pt counter and AgAgCl reference All data were fit to the equivalent circuit shown in Figure 7.6d.

[00200] The sigmoidal curve shown in Figure 8b is the classical response of an antibody interacting with its target, with no binding (low % increase) observed until ca. 10 mg/mL of measles virions was exposed to the biosensor. The response increased to a plateau at ca. 1 mg/mL of measles virions. The sensors demonstrated exceptional reproducibility at the tested concentrations, with minimal variation until the sensor response was saturated at 1 mg/mL. The EC50, a common measure for the strength of an interaction between an antibody and the target molecule that reflects the concentration needed to achieve 50% of the maximal response, was calculated to be 1 18.1 mg/mL (R2 = 0.990) for the interaction between the surface-immobilized antibody and the measles virions. This represents the concentration of the target molecule (measles virions in this case) needed to achieve half of the maximum response from the biosensor.

[00201] A more detailed study was then conducted at various concentrations of measles virions between 1 mg/mL and 1 mg/mL to generate more data points within the range of measles concentrations where the measles biosensor responded. Figure 9 shows the calculated increase in the resistance of the low frequency arc for triplicate biosensors based on the NHC SAMs and on a comparable thiol-based SAM (6-mercaptohexanoic acid, introduced in Section 3). Similar

[00202] to Figure 8, a linear range in response to 10-100 mg/mL of measles virions was found in this work for the NHC-based sensors (Figure 9, black traces), with a calculated limit of detection of 6 mg/mL (R2 = 0.957). The sensor response began to plateau at higher concentrations, with a minimal increase between 250 mg/mL and 1 mg/mL of the virus, similar to the plateau seen in Figure 8.

[00203] Interestingly, while the thiol-based anti-measles biosensor also responded over a similar concentration range, it produced a much lower response to changes in the concentration of the measles virions (Figure 9, red traces). The maximum change of the thiol-based SAM is ca. 60% increase at 1 mg/mL, significantly smaller than the ca. 650% increase observed for the same concentration of measles on the NHC-based SAM. This relative insensitivity to changes int he concentration of the measles virions could be due to the tighter packing of the thiol-based SAMs, which results in a larger baseline resistance and might not provide significant gaps between thiols that could be blocked when the antibody binds to the virus particles.

[00204] The infectious dose for the measles virus, based on primate studies, is approximately 10^L6-10^L8 virus particles.^29-31 Based on electron microscopy data, the measles virus exhibits a spherical shape with a diameter of ca. 150 nm,15 and a density of 1.23 g/mL, based on density gradient separation results.³² This converts to a minimum infectious dose for primates of between 1-100 mg/mL, which corresponds extremely well with the linear range and limit of detection of the NHC-based measles biosensor shown in Figure 9.

[00205] Cyclic voltammograms collected in a solution containing equimolar ferrocyanide and ferricyanide (Figure 7.10) confirmed the impedance data, demonstrating the increasing resistance of the thiol-based (Figure 10a) and NHC-based (Figure 10b) biosensors with increasing concentrations of measles virions. While both sensors exhibit similar peak potentials and currents initially, the NHC-based sensor exhibits a marked increase in peak separation and decrease in peak current as the concentration of measles virions is increased. This is a hallmark trait of increasing irreversibility of a diffusion- controlled redox reaction, such as the ferro/ferricyanide reaction, and is confirmation of the increased resistance seen in the low frequency arc of the impedance spectroscopy data shown in Figure 8-9 and Table 6. [00206] Interestingly, the thiol-based sensor (Figure 10a) exhibits much smaller changes in the CV over the same range of concentrations of measles virions, confirming the relative insensitivity (i.e., the lower response when compared to the NHC-based sensor) seen with impedance spectroscopy in Figure 9. The observed voltammograms for the thiol- based sensors did pass significantly more current than the TLR-4 biosensors when no ferrocene was present, likely due to the decreased length of the thiol alkyl chain (6 carbons) compared to the previously tested thiols (1 1 carbons).

[00207] When the cyclic voltammograms of Figure 10 are further analyzed, the trend of increasing kinetic irreversibility becomes more pronounced. Table 7 shows the characteristics of the cathodic peaks shown in Figure 10. As the measles concentration was increased the anodic peak of the NHC-based sensor shifted outside of the potential scan window, only the cathodic peak was analyzed. Table 7 shows that the thiol-based sensor exhibits a decrease in peak current of ca. 35% between the baseline (0 mg/mL) and 1 ,000 mg/mL of measles virions. Comparatively, the NHC-based sensor exhibits a ca. 80% decrease in peak current over the same concentration ranges. While these differences are quite pronounced, they are not as large as the differences recorded in Figure 9 for the change in the resistance of the low frequency arc as measured by impedance spectroscopy (ca. 60 and 650% changes for the thiol and NHC-based sensors, respectively).

[00208] Table 7. Analysis of the Cathodic peaks in the CVs of Figure 10*.

* Cyclic voltammetry measurements were performed at 25 mV/s in 1 M XaC lOi supplemented with 5 niM Fe(CN)₆ ^{3 -/4-} with a Pt counter and Ag AgCl reference. [00209] Unexpectedly, the peak potentials recorded in Table 7 show that the thiol- based sensor exhibited greater changes than the NHC-based sensor. The cathodic peak for the thiol-based sensor shifted from 137 to 101 mV vs. Ag/AgCI, whereas the NHC-based sensor moved from 1 19 to 96.5 mV vs. Ag/AgCI. This could be an artifact of measuring the peak characteristics with a regression baseline, as the cathodic peaks of the NHC-based sensor in Figure 10b do appear to shift significantly over the tested range of measles concentrations.

[00210] Together, Figures 8-10 and Tables 7 and 8 represent the first reported demonstration of an electrochemical biosensor response towards measles virions on any SAM, as previous reports have only explored detecting the human immune response towards the measles virus rather than the causative agent itself. This work is also the first reported biosensor based on NHC monolayers, demonstrating that the approaches refined over the previous decades for thiol-based SAMs could be applied on this new type of self- assembled monolayer.

[00211] 6 Control experiments confirming antibody-based biosensor mechanism

[00212] To confirm that the strong responses to the measles virus shown in Section 5 were not due to physical adsorption of the measles virus onto the SAM surface, a control experiment (Figure 1 1) was performed, where the anti-measles antibody was replaced with bovine serum albumin (BSA) on both thiol (Figure 1 1 a) and NHC-based SAMs (Figure 11 b). The results from fitting the data to the equivalent circuit of Figure 6d is shown in Table 8.

[00213] When exposed to 100 mg/mL of the measles virus, there is a slight decrease seen in the measured resistance of the control electrodes. The thiol-based SAMs modified with BSA exhibits a 32% decrease over three replicates, while the NHC-based SAMs exhibits only a 18% decrease over three replicates. This is in stark contrast to the ca. 300% increase over the baseline that was observed for the Ab-modified NHC SAMs for the same virus concentration (Figures 8 and 9).

[00214] Table 8. Equivalent circuit fitting for thiol and NHC-based sensors responding to 100 mg/mL of the measles virus*

*Impedance measurements were performed from 10^-1- 10⁵ Hz at the open circuit potential (0.27 V vs. Ag/AgCl) in 1 M NaClOt supplemented with 5 nM Fe(CN)₆ ^{3 -/4-} with a Pt counter and AgAgCl reference. All data were lit to the equivalent circuit shown in Figure 7 6d.

[00215] These observed decreases in the resistance could be due to some weakly adsorbed BSA or SAM molecules being lost to the electrolyte solution during testing, which would explain why both the low (i.e., antibody/BSA) and high (i.e., monolayer) frequency arcs experience some changes (Table 8). As BSA has no known affinity towards the measles virus and is routinely used as an inactive blocking layer, this provides strong evidence that the responses shown in Figures 8-10 are due to the interaction of the immobilized anti-measles antibody and the measles virus.

[00216] Interestingly, the resistance of the thiol-based BSA electrodes in Figure 10 decreased more than did the NHC-based BSA electrodes after exposure to 100 mg/mL of the measles virus, which could indicate issues with the underlying SAMs desorbing during the exposure of the sensor to the measles virus. To further explore this, the“drift” of both the thiol and NHC-based Ab-modified sensors was explored by immersion in 1 M NaCIO₄ between measurements instead of exposure to the measles virus. Voltammograms collected every 30 minutes from 0-90 minutes show a clear trend in the thiol-based sensors (Figure 12a), with the currents decreasing and the overall voltammogram appearing more irreversible as time progressed. Conversely, the NHC-based sensor (Figure 12b) experienced a slight increase in the observed currents between 0 and 30 minutes, with the voltammograms exhibiting little change between 30 and 90 minutes.

[00217] While the increase observed for the NHC-based sensor could be due to removal of weakly adsorbed SAM or antibody molecules, the decrease in current seen over time for the thiol-based sensor could represent a reorganization of the SAM structure. It has been reported that thiol-based SAMs undergo a reorganization when switching between solvents,³³ which could be occurring over the 90-minute time period of the experiment shown in Figure 1 1. Such reorganizations could include lateral diffusion of the SAM molecules within the monolayer or alterations in the angle of the SAM molecules with respect to the surface normal, with the goal of increasing lateral interactions between SAM molecules and decreasing the overall surface energy of the monolayer to the most stable configuration.

[00218] The resulting more organized SAM should result in a larger resistance between the underlying Au and the electrolyte.^{20 3435} This was assessed by impedance spectroscopy over a 29 similar 90-minute time course, with the impedance response measured every 15 minutes (Figure 12c). Minimal changes were observed for the NHC- based sensor over a 90-minute period (Figure 12c, black trace), while the thiol-based sensor experienced >200% change in the total combined resistance of the low and high frequency arcs (Figure 12c, red trace). This is in line with previous reports, highlighting the superior stability of NHC-based SAMs compared to their thiol counterparts ^{1 36}.

[00219] 7 Response of antibody-modified SAMs after storage

[00220] One of the main motivations for exploring the use of NHC SAMs in a biosensor design was related to their reported stability compared to that of thiol-based SAMs. An important aspect of stability, especially for any commercial application of these sensors, is to consider their shelf-life (i.e., their stability with time of storage). Table 9 shows the response of thiol and NHC-based sensors to 100 mg/mL of the measles virus either directly after sensor construction (“no storage”) or after being stored for two weeks in 1 M NaCIO₄ at 4 °C after sensor construction (“2 weeks @ 4 °C”). This temperature was chosen as it is the supplier’s recommended temperature for storage of the antibody to maintain functionality.

[00221] Table 9. Response of Ab-modified SAMs to 100 mg/mL measles virus over time*.

Table 7,9. Response of Ab-modified SAMs to 100 mg/mL measles virus over time*

* Change in the res or Ab-modified SAMs based on eit (n=3) Sensors were either tested of storage in 1 M NaClO₄ at 4°C d from 10^-1- 10⁵ Hz at the open circ ted with 5 mM Fe(CN)₆ ^{3 -/4-} with a [00222] As already demonstrated in Figure 9, the thiol-based sensor exhibits a lower response when a freshly prepared sensor was tested and compared to a NHC-based SAM. Interestingly, the response after 2 weeks of storage for both the thiol and NHC-based sensors was much weaker (i.e., smaller changes in resistance for the same concentration of the measles virus) than the response of sensors that were tested immediately, indicating that there is some degradation to both sensor system. It is important to highlight that, in this study, it cannot be concluded if these changes in sensor resistance are due to the degradation of the underlying SAM or of the anti-measles antibody tethered to the outer surface of the SAM.

[00223] It was noted that the NHC-based sensors still responded positively towards the virus after 2 weeks of storage in 1 M NaCIO₄ at 4 °C, albeit to a lesser degree than before (Table 9). Interestingly, the variability of the NHC-based sensors also increased after 2 weeks of storage, with a standard deviation in the measured resistance of 8% without storage increasing to 30% after storage. Conversely, the thiol-based sensor did not respond to the measles virus after 2 weeks of storage. Instead, a decrease in the resistance was observed, although the variability before and after storage was comparable for the thiol- based sensors (Table 9). This decrease could be due to weakly adsorbed alkanethiol molecules on the SAM being lost to solution during exposure to the measles virus. It is thus clear from these results that the stability of the NHC-based anti-measles sensors is far superior to that of its thiol-based counterparts.

[00224] 8 Summary

[00225] As described herein there is provided the first biosensor based on NHC monolayers, as well as the first electrochemical biosensor capable of detecting whole measles virions. It was shown that the ester-NHC SAMs could be efficiently deprotected to form carboxyl-terminated SAMs capable of being reacted with either aminoferrocene or antibodies through EDC/NHS chemistry. The measles biosensor fabricated in this work was shown to have a linear response to the intact measles virus between 10-100 mg/mL, with a limit of detection of 6 mg/mL. This encompasses the range of virus concentrations relevant to human health applications, as well as requiring no pre-treatment steps to break open the virus before detection.

[00226] Importantly, the NHC-based sensor showed minimal drift, with less than 10% change in the measured baseline after 90 minutes of repeated testing. This was not the case for the thiol-based counterpart, which experienced a ca. 200% change in the same conditions. One of the main purposes for the use of NHC SAMs in the biosensor design was to increase the shelf-life of sensors, as thiol-based systems have significant requirements that make commercialization efforts harder. When stored for two weeks at 4 °C, the NHC- based sensors were still able to respond to the presence of the measles virus, albeit at a decreased level. This was a significant improvement over the comparable thiol-based system, which was unable to detect high concentrations of the measles virus after storage in similar conditions.

[00227] The embodiments described herein are intended to be examples only.

Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[00228] All publications, patents and patent applications mentioned in this

Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00229] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A system for detecting a target in a sample from a subject, the system comprising:

a substrate having a surface;

one or more a linkers, each linker having a first end attached to the surface and a second end;

one or more receptors, wherein each receptor,

is bound to the second end of one or more of the plurality of linkers; and is configured to interact with the target;

wherein a change in a signal is detectable when said target is bound to said receptor,

wherein said linker is a N-heterocyclic carbene (NHC),

each said one or more linker may be the same or different,

each said receptor may be the same or different.

2. A system for detecting a target in a sample, the system comprising:

a substrate having a surface;

one or more linkers, each linker having a first end and a second end;

for each said linker:

the first end is attached to said surface of said substrate; and the second end either comprises a receptor or does not comprise the receptor;

wherein said receptor is configured to interact with the target and a change in a signal is detectable when said target is bound to said receptor,

wherein said linker is a N-heterocyclic carbene (NHC),

each said one or more linker may be the same or different,

each said receptor may be the same or different.

3. The system of claim 1 , wherein the second end of two or more linkers comprise said receptor.

4. The system of any one of claims 1 to 3, wherein said target is a polypeptide, a polynucleotide, a lipid, or a small molecule.

5. The system of claim 4, wherein said polypeptide comprises is derived from a virus virion.

6. The system of claim 5, wherein said virion comprises a measles virion.

7. The system of claim 4, wherein said polynucleotide comprises DNA, RNA, PNA, LNA, BNA, or aptamers.

8. The system of any one of claims 1 to 7, wherein the sample comprises a biological sample, an aerosol, or a water sample.

9. The system of any one of claims 1 to 8, wherein the substrate comprises a metal, other conducting non-metal substrate, and/or carbon.

10. The system of claim 9, wherein the metal is Au, Pt, Cu, Mg, Ag, or other materials.

11. The system of claim 10, wherein the metal is Au.

12. The system of any one of claims 1 to 1 1 , wherein the NHC is a compound of formula (I):

13. The system of any one of claims 1 to 1 1 , wherein the NHC is a compound of formula (II):

wherein R₁ and R₂ are independently selected from the group comprising C1 - C24 alkyl group, C1-C24 substituted alkyl group, C1-C24 aryl or C1-C24 substituted aryl group.

14. The system of claim 13, wherein R₁ and R₂ are independently selected from the group comprising methyl, ethyl, iPr, t-Butyl, adamantyl or mesityl, and L is a linking group, preferably an amide, thioether or triazole ring.

15. The system of claim 13 or 14, wherein L is a linking group.

16. The system of claim 15, wherein said linking group is an amide, thioether or triazole ring.

17. The system of claim 13, wherein R₁ is iPr, R₂ is iPr, and L is amide.

18 The system of any one of claims 1 to 17, wherein the receptor is a polypeptide.

19. The system of claim 18, wherein the polypeptide is an antibody.

20. The system of claim 19, wherein the antibody is an anti-measles H protein antibody.

21. The system of any one of claims 1 to 17, wherein the receptor is a polynucleotide, or a small molecule.

22. The system of claim 21 , wherein the polynucleotide comprises DNA, RNA, PNA, LNA, BNA, or aptamers.

23. The system of any one of claims 1 to 22, further comprising a detectable label.

24. The system of claim 23, wherein the detectable label is detected electrochemically.

25. The system of claim 24, where the signal is an impedance response.

26. The system of any one of claims 23 to 25, wherein the label is ferro/ferricyanide.

27. The system of any one of claims 23 to 25, wherein the label comprises an intercalator.

28. The system of claim 27, wherein said intercalator comprises methylene blue.

29. The system of claim 23 or 24, wherein the label comprises Ru.

30. A method of detecting a target in a sample, comprising: contacting a device of any one of claims 1 to 29 with said sample; measuring a change in signal.

31. The method of claim 30, wherein said change is indicative of a presence or absence or amount of said target in said sample.

32. The method of claim 31 , further comprising:

determining the quantity of said target in said sample, if present.