GB2615131A - Apparatus and method for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies - Google Patents

Abstract

An electrochemical biosensor for detecting anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies 304 comprising: at least one working electrode 102; a SARS-CoV-2 spike (S) glycoprotein 104 arranged on the surface of the electrode 102; and a redox reporter-tagged secondary antibody 302 located away from the electrode 102; wherein the anti-SARS-CoV-2 spike protein antibodies 304 bind to the spike glycoprotein 104 and are detected using the redox reporters. The spike glycoprotein 104 may be immobilised onto the electrode 102 via passive adsorption or cross-linking. The anti-SARS-CoV-2 antibodies 304 may be detected by measuring a change in electrical potential at the electrode 102. The redox reporter may be ferrocene or anthraquinone. Also claimed is a biosensor detection system for COVID comprising the electrochemical biosensor. Further claimed is a method for detecting anti-SARS-CoV-2 spike protein antibodies 304 comprising: binding a spike glycoprotein 104 onto an electrode 102; depositing reporter-tagged secondary antibodies 302 onto the biosensor, wherein they migrate to the electrode 102; receiving anti-SARS-CoV-2 spike protein antibodies 304 in a sample solution; binding said anti-antibodies 304 to the electrode 102; and detecting said anti-antibodies 304 using a redox reaction at the electrode 102 by redox reporters.

Description

APPARATUS AND METHOD FOR DETECTION OF ANTI-SEVERE ACUTE RESPIRATORY

SYNDROME ASSOCIATED CORONAVIRUS (SARS-COV-2) SPIKE PROTEIN ANTIBODIES

TECHNICAL FIELD

[0001] Various example embodiments relate to an apparatus and a method for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies.

BACKGROUND

[0002] Severe acute respiratory syndrome associated coronavirus (SARS-CoV-2), or severe acute respiratory syndrome coronavirus 2, is a strain of coronavirus that causes coronavirus disease 2019 (COVID-19), the respiratory illness responsible for the COV1D-19 outbreak that was declared a pandemic by The World Health Organization (WHO) on 11 March 2020. A strain of severe acute respiratory syndrome-related coronavirus (SARSr-CoV), SARS-CoV-2 has a simple structure and composition. SARS-CoV-2 is a protein-coated single-stranded positive-stranded RNA virus, which particles are round or elliptical, often pleomorphic, with a diameter of about 60-140nm. The virus is believed to have zoonotic origins, possibly having emerged from a bat SARS-like coronavirus.

[0003] SARS-CoV-2 infects higher animals including humans and is highly infectious and harmful, leading to an increasing number of patients with severe symptoms and deaths. The virus primarily enters human cells by binding to receptor angiotensin converting enzyme 2 (ACE2). Further, epidemiological studies estimate that when no members of the community are immune and no preventive measures are taken, each infection results in 5.7 new infections. Typically, the virus spreads between people through close contact and via respiratory droplets produced from coughs or sneezes. Also, epidemiological studies suggest that aerosols and indirect contact via contaminated surfaces are causes of infection. For most respiratory viruses, patients are typically more contagious when they are most symptomatic. On the other hand, for SARS-CoV-2, there are indications that subclinical infections are a source of many infections.

[0004] However, prevention is better than cure and one resort to prevention is through vaccination. Vaccines have been targeted to spike glycoprotein which is prominent on the SARS-CoV-2 and is critical for recognition with the host cell receptor Angiotensin-Converting Enzyme 2 (ACE2) which then mediate viral cell entry. Antibodies targeted to the spike glycoprotein effectively neutralise the threat of SARS-CoV-2. Further, whether immunity is formed from infection or vaccination, the antibodies for SARS-CoV-2 will largely be targeted to the spike glycoprotein. Currently, vaccination against the SARS-CoV-2 organism is the best recourse to limit the health-damaging effects of infection. Further, the vaccination introduces a safe antigen to stimulate body's immune system to produce specific antibodies. Several vaccines have been approved by the various health agencies across the world, and fortunately, vaccination uptake is high across populations.

100051 With immunity to SARS-CoV-2 becoming an important variable in controlling the pandemic, both the individual level of immunity and the population level of herd immunity requires quantitative measurement. A rapid point-of-care test for the quantitative measure of anti-SARS-CoV-2 antibodies is a vital tool for accurate sero-epidemiological studies. Therefore, there is a need for an improved apparatus and method for the detection of anti-SARS-CoV-2 spike protein antibodies.

SUMMARY

100061 In accordance with an example embodiment, an electrochemical biosensor for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies, may be disclosed. The electrochemical biosensor may comprise at least one working electrode, a recognition element arranged on a surface of the at least one working electrode, wherein the recognition element is SARS-CoV-2 spike glycoprotein, and a signal transduction element, located away from the working electrode, wherein the signal transduction element is a reporter-tagged secondary antibody. Further, the anti-SARS-CoV-2 spike protein antibodies bind to the SARS-CoV-2 spike glycoprotein on the at least one working electrode, detected using electrochemical redox reporters bound to the reporter tagged secondary antibody. In one example embodiment, the SARS-CoV-2 spike glycoprotein is wholly intact spike glycoprotein. Such usage of the electrochemical redox reporters results in detecting anti-SARS-CoV-2 spike protein antibodies.

[0007] Further, the SARS-CoV-2 spike glycoprotein is immobilized onto the at least one working electrode using passive adsorption or via chemical crosslinking. The passive adsorption of the SARS-CoV-2 spike glycoprotein includes hydrophobic interactions between the at least one working electrode and hydrophobic residues of the SARS-CoV-2 spike glycoprotein. The SARS-CoV-2 spike glycoprotein is crosslinked to the at least one working electrode through N-Hydroxysuccinimide ester crosslinking, carbodiimide crosslinking, or hydrazine crosslinking of glycosylated (carbonyl-reactive) residues. Further, the anti-SARS-CoV-2 spike protein antibodies are suspended in a sample solution and migrate to the SARS-CoV-2 spike glycoprotein on the at least one working electrode.

100081 Further, the electrochemical redox reporters are a moiety which are stably redox active within a defined potential window and the electrochemical redox reporters upon oxidation or reduction exchange electrons with an electrode produce a measurable change in electrical properties at the at least one working electrode. Further, the electrochemical redox reporters are attached to the reporter-tagged secondary antibody using at least one of a covalent bond, a hydrogen bonding, a pi-stacking, or hydrophobic interactions. In one example embodiment, the electrochemical redox reporters are at least one of Ferrocene and Anthraquinone. Further, the electrochemical redox reporters are crosslinked to the secondary antibody in an optimized manner by increasing one or more parameters, wherein the one or more parameters include incubation times and concentrations used.

[0009] Further, redox-reporter tagged secondary antibodies are deposited on the biosensor away from the working electrode. Small volumes of redox-reporter tagged antibodies at predefined concentration are deposited on the bionsensor and dried into position. In the presence of sample solution, redox-reporter tagged secondary antibodies bind the anti-SARSCOV-2 spike protein antibodies allowing for detection of anti-S A RSCOV-2 spike protein antibodies. The pre-defined concentration of reporter-tagged secondary antibody is determined by mixing the anti-SARS-CoV-2 spike protein antibodies with the reporter-tagged secondary antibody and observing electrochemical signal. In one example embodiment, a minimum antibody required is 0.3 micrograms. In one embodiment, a pre-defined amount of glycerol is used in a buffer for the reporter-tagged secondary antibody, to improve resuspension from dried formulation to liquid in the presence of the sample solution. The pre-defined amount of glycerol used in a buffer for the reporter-tagged secondary antibody is 0.5% v/v.

[0010] Further, the SARS-CoV-2 spike glycoprotein is covalently bonded to the at least one working electrode. Further, the electrochemical biosensor is coated with a blocking agent to block non-specific interactions with the at least one working electrode. In one embodiment, the blocking agent blocks unoccupied spaces (e.g. spaces without the SARS-CoV-2 spike glycoprotein) on the working electrode. Further, any vacant space between the spike glycoprotein on the working electrode is blocked using a blocking agent. The blocking agent prevents non-specific interactions with the at least one working electrode. In one example embodiment, the blocking agent is 1% w/v bovine-serum albumin (BSA). Further, 2 microliters of 1% w/v BSA solution is coated for 1 hour at room temperature.

[0011] In accordance with another example embodiment, a biosensor detection system comprising an electrochemical biosensor, may be disclosed. The biosensor detection system may comprise at least one working electrode, a recognition element arranged on a surface of the at least one working electrode, wherein the recognition element is SARS-CoV-2 spike dycoprotein, and a signal transduction element, located away from the working electrode, wherein the signal transduction element is a reporter-tagged secondary antibody. Further, the anti-SARS-CoV-2 spike protein antibodies bind the SARS-CoV-2 spike glycoprotein independently of any other component. The anti-SARS-CoV-2 spike protein antibodies bind to the SARS-CoV2 spike glycoprotein through inherent specificities of anti-SARS-CoV-2 spike protein antibodies to their target antigen. In one example embodiment, the SARS-CoV-2 spike glycoprotein is wholly intact spike glycoprotein.

[0012] Further, the SARS-CoV-2 spike glycoprotein is immobilized onto the at least one working electrode using passive adsorption or via chemical crosslinking. The passive adsorption of the SARS-CoV-2 spike glycoprotein includes hydrophobic interactions between the at least one working electrode and hydrophobic residues of the SARS-CoV-2 spike glycoprotein. The SARS-CoV-2 spike glycoprotein is cross! nked to the at least one working electrode through N-Hydroxysuccinimide ester crosslinking, carbodiimide crosslinking, or hydrazine crosslinking of glycosylated (carbonyl-reactive) residues.

[0013] The anti-SARS-CoV-2 spike protein antibodies are suspended in a sample solution and migrate to the at least one working electrode for the detection of anti-SARS-CoV-2 spike protein antibodies.

[0014] Further, the electrochemical redox reporters are a moiety which are stably redox active within a defined potential window and the electrochemical redox reporters upon oxidation or reduction exchange electrons with an electrode to produce a measurable change in electrical properties at the at least one working electrode. Further, the electrochemical redox reporters are attached to the reporter-tagged secondary antibody using at least one of a covalent bond, a hydrogen bonding, a pi-stacking, or hydrophobic interactions. In one example embodiment, the electrochemical redox reporters are at least one of Ferrocene and Anthraquinone. Further, the electrochemical redox reporters are crosslinked to the secondary antibodies in an optimized manner by increasing one or more parameters, wherein the one or more parameters include incubation times and concentrations used.

[0015] Further, redox-reporter tagged secondary antibodies are deposited on the biosensor away from the working electrode. Small volumes of redox-reporter tagged antibodies at predefined concentrations are deposited on the bionsensor and dried into position. In the presence of sample solution, redox-reporter tagged secondary antibodies bind the anti-SARSCOV-2 spike protein antibodies allowing for detection of antiSARSCOV-2 spike protein antibodies. The pre-defined concentration of reporter-tagged secondary antibody is determined by mixing the anti-SARS-CoV-2 spike protein antibodies with the reporter-tagged secondary antibody and observing electrochemical signals. In one example embodiment, a minimum antibody required is 0.3 micrograms. In one embodiment, a pre-defined amount of glycerol is used in a buffer for the reporter-tagged secondary antibody, to improve resuspension from dried formulation to liquid in the presence of the sample solution. The pre-defined amount of glycerol used in a buffer for the reporter-tagged secondary antibody is 0.5% v/v.

[0016] Further, the SARS-CoV-2 spike glycoprotein is covalently bonded to the at least one working electrode. Further, the electrochemical biosensor is coated with a blocking agent to block non-specific interactions with the at least one working electrode. In one example embodiment, the blocking agent is I % w/v bovine-serum albumin (BSA). Further, 2 microliters of I% w/v BSA solution is coated for 1 hour at room temperature.

[0017] In accordance with yet another example embodiment, a method for detection of anti-SARS-CoV-2 spike protein antibodies, may be disclosed. The method may comprise binding a SARS-CoV-2 spike glycoprotein on at least one working electrode of an electrochemical biosensor, depositing reporter-tagged secondary antibodies on the electrochemical biosensor, wherein upon receiving anti-SARS-CoV-2 spike protein antibodies in a sample solution, the anti-SARS-CoV-2 spike protein antibodies bind to the spike glycoprotein on the at least one working electrode. Further, the reporter-tagged secondary antibodies migrate to the at least one working electrode, where the reporter-tagged secondary antibodies bind to the anti-SARSCoV-2 spike protein antibodies. Further, the method may comprise detecting, using electrochemical redox reporters tagged to the secondary antibodies, the anti-SARS-CoV-2 spike protein antibodies, upon a redox reaction at the at least one working electrode. In one embodiment, a blocking agent blocks unoccupied spaces on the working electrode. Further, any vacant space between the spike glycoprotein on the working electrode is blocked using the blocking agent. The blocking agent prevents non-specific interactions with the at least one working electrode. Such usage of the electrochemical redox reporters results in detecting anti-SARS-CoV-2 spike protein antibodies.

[0018] Further, the SARS-CoV-2 spike glycoprotein is immobilized onto the at least one working electrode using passive adsorption or via chemical crosslinking. The passive adsorption of the SARS-CoV-2 spike glycoprotein includes hydrophobic interactions between the at least one working electrode and the hydrophobic residues of the SARS-CoV-2 spike glycoprotein and chemical crosslinking of the SARS-CoV-2 spike glycoprotein includes the use of additional materials to form chemical bonds between the functional groups of the residues being crosslinking. The SARS-CoV-2 spike glycoprotein is crosslinked to the at least one working electrode through an N-Hydroxysuccinimide ester crosslinking, carbodiinaide crosslinking, or hydrazine crosslinking of glycosylated. (carbonyl-reactive) residues.

[0019] Altogether, the apparatus and method according to the exemplary embodiments described herewith allow detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies. Such disclosed technique of detection of anti-SARS-CoV-2 spike protein antibodies with the electrochemical redox reporters results in detecting anti-SARS-CoV-2 spike protein antibodies, provides improved detection and quantification through electrochemical detection methods such as voltammetry, amperometry, and potentiometry. In addition, the disclosed method also allows a redox reaction of the electrochemical redox reporters, which in the proximity of the working electrode, resulting in detection of the anti-SARS-CoV-2 spike protein antibodies.

[0020] To the accomplishment of the foregoing and related ends, one or more aspects comprise the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative aspects and are indicative of but a few of the various ways in which the principles of the aspects may be employed. Other advantages and novel features will become apparent from the following detailed description when considered in conjunction with the drawings and the disclosed aspects are intended to include such aspects and their equivalents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Further embodiments, details, advantages, and modifications of the present example embodiments will become apparent from the following detailed description of the embodiments, which is to be taken in conjunction with the accompanying drawings, wherein: [0022] FIGS. 1A & 18 illustrate an electrochemical biosensor for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies, according to an example embodiment of the subject matter described herein. Figure 1B shows the bare pre-functionalization carbon sensor.

[0023] FIG. 2 illustrates a structure of an antibody. according to an example embodiment of the subject matter described herein.

[0024] FIG. 3 illustrates a diagram showing a primary antibody coupled to a secondary antibody. according to an example embodiment of the subject matter described herein.

[0025] FIG. 4A illustrates a block diagram showing a principle of the working of the electrochemical biosensor, according to an example embodiment of the subject matter described herein.

[0026] FIG. 4B illustrates a block diagram showing a functionalization of a working electrode of the electrochemical biosensor, according to an example embodiment of the subject matter described herein.

[0027] FIG. 5A illustrates a graph illustrating testing results of redox reporters, according to an example embodiment of the subject matter described herein.

[0028] FIG. 58 illustrates a graph illustrating reference standards for Anti-SARS-CoV-2 Immunoglobulins, according to an example embodiment of the subject matter described herein.

[0029] FIG. 6 illustrates a flowchart showing a method for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies, according to an example embodiment of the subject matter described herein.

DETAILED DESCRIPTION

[0030] Some embodiments of this disclosure, illustrating its features, will now be discussed in detail. The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to the listed item or items.

[0031] As used herein and in the appended claims, the singular forms "a,-"an,-and "the-include plural references unless the context clearly dictates otherwise. Although any apparatus and method similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, the apparatus and methods are now described.

[0032] Embodiments of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings in which like numerals represent like elements throughout the several figures, and in which example embodiments are shown. Embodiments of the claims may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The examples set forth herein are non-limiting examples and are merely examples among other possible examples.

[0033] An example embodiment of the present disclosure and its potential advantages are understood by referring to FIGURES I through 6 of the drawings, like numerals being used for like and corresponding parts of the various drawings.

[0034] FIG. IA illustrates an electrochemical biosensor 100 for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies, according to an example embodiment The electrochemical biosensor 100 may comprise at least one working electrode 102, and a recognition element 104 arranged on a surface of the at least one working electrode 102. The at least one working electrode 102 is made at least of platinum, gold, or a carbon allotrope. In one embodiment, the recognition element 104 may be SARS-CoV-2 spike glycoprotein. Further, the recognition element 104 may be interchangeably used as the SARS-CoV-2 spike glycoprotein 104, hereinafter. The SARS-CoV-2 spike glycoprotein 104 is a wholly intact spike glycoprotein. Further, the electrochemical biosensor 100 may comprise a signal transduction element 106, located away from the at least one working electrode 102. The signal transduction element 106 is located at a pre-defined distance from the at least one working electrode 102. In one embodiment, the signal transduction element 106 may be a reporter-tagged secondary antibody. Further, the signal transduction element 106 may be interchangeably used as the reporter-tagged secondary antibody 106 or as redox-reporter conjugated antibodies, hereinafter. Further, reporter-tagged secondary antibodies 106 may be initially located away from the at least one working electrode 102 and only migrate to the at least one working electrode 102, when anti-spike antibodies are bound to the SARS-CoV-2 spike glycoprotein 104 on the at least one working electrode 102. The reporter-tagged secondary antibodies 106 may be the last component to be added to the electrochemical biosensor 100. Further, the electrochemical biosensor 100 may be dried so only resuspension with a sample would allow the reporter-tagged secondary antibodies 106 to be mobile. The electrochemical biosensor 100 may also be referred to as a sensor device. In one embodiment, the electrochemical biosensor 100 may be used to detect the presence of anti-SARS-CoV-2 spike protein antibodies in a sample solution, when then sample solution is passed on the at least one working electrode 102. Further, the SARS-CoV-2 spike glycoprotein 104 may form a first layer of the electrochemical biosensor 100 to which anti-SARS-CoV-2 antibodies recogni7e and bind. FIG. IA is explained in conjunction with FIG. 1B.

[0035] FIG. 1B illustrates a bare carbon sensor pre-functionalization. FIG. 1B describes a base on which the electrochemical biosensor 100 is developed, consisting of the standard three electrode configuration (the working electrode 102, a reference electrode 108, and a counter electrode 110). In the current embodiment, the working electrode 102 may be made of carbon allotrope. Further, the reference electrode 108 and the counter electrode 110 may be made of silver chloride. The working electrode 102 is where the signal is detected. Further, the reference electrode 108 maintains a constant potential and the counter electrode 110 completes the circuit.

[0036] In one embodiment, the electrochemical biosensor 100 may have pre-defined dimensions. In one example embodiment, the dimensions of the electrochemical biosensor 100 may be such that length of the electrochemical biosensor 100 may be 25.4 millimeter (mm), width of the electrochemical biosensor 100 may be 7 mm, and the thickness of the electrochemical biosensor 100 may be 0.625 mm.

[0037] In one embodiment, the electrochemical biosensor 100 may be prepared as described below. Firstly, a solution of 4mg/m1 to 8mg/m1 of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) 11mg/m1 to 22mg/m1 of sulto-N-hydroxysultosuccinimide (sulfo-NHS) may be prepared and added to the at least one working electrode 102 and left to activate at room temperature for 20 minutes. Further, the SARS-CoV-2 spike glycoprotein 104 may be prepared in HBS-EP (0.01 mol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), potential of hydrogen (pH) 7.4, 0.15 mol/L Sodium Chloride (NaC1), 3mmol/L Ethylenediaminetetraacetic acid (EDTA), 0.005% v/v Tween-20 buffer. The HBS-EP may refer to buffer containing all the following components: 0.01mol/L HEPES, 0.15mol/L Sodium Chloride, 3mmol/L EDTA, 0.005% Tween-20. Further, the EDC/sulfo-NHS Carbodiimide crosslinking may be required for the activation of carboxyl groups on the at least one working electrode 102 to bind the primary amines of the SARS-CoV-2 spike glycoprotein 104. When the SARS-CoV-2 spike glycoprotein 104 is bonded on the activated at least one working electrode 102, it then reacts to form a stable carbodiimide bond. In particular, 2n1 of lp g/ml to 100pg/m1 SARS-CoV-2 spike glycoprotein 104 is deposited on the activated at least one working electrode 102 and left to react for I hour at room temperature or overnight in at 4 degrees Celsius. Between 0.1u g/ml to 10Ong/m1 spike glycoprotein may be used. In general, a high concentration is not needed when chemical crosslinking is used, due to a more efficient deposition. But, if spike protein is instead coated by physical adsorption, then a relatively high concentration (e.g. 10pg/m1 or higher) can be used to compensate for the less efficient crosslinking. Surface coverage can be determined by testing the electrochemical biosensor 100 using redox reporter both pre-and post-coating.

[0038] Following the binding of the SARS-CoV-2 spike glycoprotein 104 to the at least one working electrode 102, the unreacted excess EDC and sulfo-NHS are rinsed off. Further, electrochemical biosensor 100 is rinsed with 500p1 I OmmoUL phosphate buffer and then left to dry at room temperature. Once the electrochemical biosensor 100 is dried, it may then be coated with a blocking buffer or a blocking agent. The blocking agent may be an agent, such as a molecule, that can inhibit, block, prevent, or reduce interaction with the at least one working electrode 102 by compounds through non-specific interactions. The electrochemical biosensor 100 may be coated with 5p1 1%w/v Bovine serum albumin (BSA) prepared in lOmmoUL phosphate buffer and left to coat for 30 minutes at room temperature. After the coating period, the electrochemical biosensor 1(X) is rinsed with 500p L 10m moUL phosphate buffer and left to dry. In one embodiment, 2 microliters of 1%w/v BSA solution are coated for 1 hour at room temperature. In one example embodiment, the blocking agent is 1%w/v bovine-serum albumin (BSA).

[0039[ Further, the recognition element 104 or the SARS-CoV-2 spike glycoprotein I 04 may be immobilised onto the at least one working electrode 102 either passively adsorbed or via chemical crosslinking. The SARSCoV-2 spike glycoprotein 104 remains bound to the at least one working electrode 102 at all times during electrochemical biosensor 100 usage. In one embodiment, the SARS-CoV-2 spike glycoprotein 104 may be coated on the at least one working electrode 102 by either passive adsorption or chemical crosslinking. Further, passive adsorption of the SARS-CoV-2 spike glycoprotein 104 may require hydrophobic interactions between the at least one working electrode 102 and hydrophobic residues of the SARS-CoV-2 spike glycoprotein 104.

[0040] In another embodiment, the chemical crosslinking may involve the use of additional materials to form chemical bonds between the functional groups of the residues being crosslinked. Further, the one or more parameters may include, but are not limited to, incubation times and concentrations used.

[0041] The SARS-CoV-2 spike glycoprotein 104 can be crosslinked to the at least one working electrode 102 through at least one of, but not limited to, N-Hydroxysuccinimide ester crossli nking, carbodiimide crosslinking. or hydrazine crosslinking of glycosylated (carbonyl-reactive) residues. In one embodiment, the SARS-CoV-2 spike glycoprotein may be crosslinked to the m least one working electrode 102 through NHydroxysuccinimide ester crosslinking, carbodiimide crosslinking, or hydrazine crosslinking of glycosylated (carbonyl-reactive) residues. Further, crosslinking reagents and protein may be added directly to the at least one working electrode 102, without pre-treatment. In one alternate embodiment, the surface of the at least one working electrode 102 may be primed for crosslinking. Further, the crosslinking may be performed by first treating the surface with acid to generate carboxyl groups. In one example embodiment, the at least one working electrode 102 is coated with 2u12mmol/1 acetic acid. The acetic acid solution is left on the at least one working electrode for 1-2 hours at room-temperature before rinsing the surface with deionised water. The carbodiimide crosslinking materials are prepared in the appropriate buffers such as 8mg/m1 EDC (1-ethy1-3-(3-di methyl am in opropy1)-carbodi im ide hydrochloride) in 0. 1 mol/L compound 2-(N-morphol in o) ethane sulfonic acid (MES) (2-(N-morpholino) ethane sulfonic acid) buffer, and 22mg/m1 in sulfo-NHS (sttlfo-Nhydroxysuccini mide) in 0.1mol/L MES buffer.

[0042] Further, antibodies are proteins that form a critical part of an immune system with key activities in identifying pathogens and neutralising threats. An antibody 200, as shown in FIG. 2, may consist of a fragment antigen-binding (Fab fragment) 202 and fragment crystallizable (Fc) fragment 204. The Fab fragment 202 may have variable regions which provide specificity towards target binding. The Fc fragment 204 may be conserved for each of the 5 classes of the antibodies (lgG, IgM, Ig A, IgE, 1gD). Since the Fc fragment 204 is constant for each class of antibody, it is possible to prepare secondary antibodies 302 which specifically bind other antibodies by immunizing an animal with an antibody from a different species, targeted to the Fc fragment 204. Further, the secondary antibodies 302 are known for the ability to recognise other antibodies, as opposed to primary antibodies 304 which have specificity to the antigen of interest. The electrochemical biosensor 100 uses the secondary antibodies 302, as shown in FIG. 3 specific for human Ig,G and IgM antibodies to detect for the presence of anti-SARS-COV-2 antibodies from human samples of blood, serum, or plasma. The secondary antibody 302 may be coupled to the primary antibody 304, as shown in FIG. 3.

[0043] Further, to create electrochemically detectable antibodies, the common redox reporters A nthraqui none and Ferrocene may be conjugated to anti-IgG and anti-IgM antibodies, respectively. The conjugation mechanisms rely on the EDC/NITS carbodiimide reaction described above. Further, derivatives of Anthraquinone and Fenocene containing carboxylic acid groups may be sourced. When reacted with the EDC and the sulfo-NHS the carboxylic acid groups on the redox reporters may be activated to enable binding to primary amine groups which are exposed to lysine residues of antibodies.

[0044] In one example embodiment, the redox reporter molecules Anthraquinone-1-carboxylic acid (AQCOOH) and Ferrocene-carboxylic acid (Fc-COOH) are successively prepared at 0.1mg/mL to 0.5mg/mL, typically 0.5mg/mL, in 0.1M MES buffer and kept at 4°C. Successively, 20mmol/L -800mmol/L, typically 400mmol/L EDC is prepared and added to the AQ-COOH. Then 50mmol/L -800mmol/L. typically 400mmol/L sulfo-NHS is prepared and added to the AQ-COOH. The preparation of the EDC and Sulfo-NHS was repeated and added to the Fc-COOH. Optimum concentrations of EDC and Sulto-NHS were determined on trial and error basis where low concentrations resulted in insufficient crosslinking of redox reporter when final product analyzed on bare carbon sensor, and where high concentrations of EDC/sulfo-NHS resulted in aggregated precipitates in the solution and also poor crosslinking when final product analyzed on bare carbon sensor. The mixture is then left to mix on a vortex at low speed for minimum 15 minutes up to 2 hours, typically 2 hours. Then, the pH value is adjusted to 7 with a non-amine based buffer or alkali, such as 100mmo1/1 carbonate buffer or 50% w/v potassium carbonate, typically 50% w/v potassium carbonate is used. Successively, 0.05mg/mL -lmg/mL antibody solutions can be prepared in HBS-EP buffer (pH7), these are typically prepared at 0.5mg/mL anti-IgM and 0.5mg/mL anti-IgG. The antibody and the reporter mix is then reacted; these can be reacted at varying volumes (200uL -10001.iL) at varying ratios (1:1, 1:2, 1:2.5, 1:3), typically at a 1:2.5 ratio such as by adding 400p L antibody to 1000p L redox reporter/EDC/sulfo-NHS mixture. The concentration and volumes of the antibody solutions reflects the aim to maximize the amount of crosslinked antibody prepared in each batch but also maintain an excess of the redox reporter molecules which have limited solubility in aqueous buffers. E.g. in reaching this embodiment, it was found that at concentrations higher than 0.5mg/ml, AQ-COOH and Fc-COOH have limited solubility in the MES buffer. The tubes are left to mix on the vortex at low speed at either 4 hours at room temperature or overnight at 4°C, to form a concentrated antibody solution, typically left to mix overnight to extend the crosslinking time. The tubes are retrieved the next day from the fridge and the reporter-conjugated antibodies are purified from mixture by ultrafiltration using centrifugal filters or using dialysis tubing. Ultrafiltration allows for rapid purification of crosslinked antibody ultrafiltration using centrifugal filters of using a molecular weight cut-off (MWCO) of either 50 kDa or 100 Is.Da (molecular weight of an antibody is 156 kDa), but ultrafiltration could result in loss of some of the antibody. A more robust but time-consuming purification can alternatively be performed using cellulose dialysis tubing (12 kDa MWCO) which has been pre-wetted in I Ommol/L Phosphate buffer saline, the dialysis tubing containing the crosslinked antibody solution is incubated in lOmmoUL phosphate buffer saline which is exchanged three times every hour to result in purified crosslinked antibody in phosphate buffer saline. With either method the unreacted redox reporter, EDC, and sulfo-NHS, which are supplied in excess to the antibody, are removed from the mixture due to the molecular weight of these components being lower than the cut-off of the membrane of the filter/dialysis tubing. Once purified the reporter-conjugated antibody are transferred to tubes and aliquots are prepared for characterization tests such as protein concentration, degree of conjugation, and homogeneity of mixture. The concentrated reporter-conjugated antibody solutions then stored at -80°C until use. Before coating the reporter-conjugated antibodies on to the biosensor, the concentration antibody solutions are diluted to the desired concentration using lOmmo1/1 Phosphate buffer saline containing 0.05 -0.75% v/v, typically 0.5% v/v glycerol. The glycerol concentration is optimized to counter the improved antibody resuspension against the ability to dry the reporter-conjugated antibody solution onto the biosensor.

[0045] In one example embodiment, Anthraquinone-I -carboxylic acid (AQ-COOH) is prepared at 0.5mg/mL in 0. I mol/L MES buffer and kept in a fridge until used. Further, the EDC may be added directly to the mixture of AQ-COOH to produce a firmd concentration of 400mmol/L. The Sulfo-NHS is also added directed to the mixture of AQ-COOH to produce a final concentration of 400mmol/L. The mixture is then left to mix on a vortex at low speed for 2 hours at room temperature. Meanwhile, anti-IgM antibody is prepared in HBS-EP buffer at 0.5mg/mL. After 2 hours, the pH of the Anthraquinone-1-carboxylic acid/EDC/Sulfo-NHS mixture is adjusted to 7 with 50% w/v potassium carbonate. The mixture is transferred to the anti-IgM at a volume ratio of 1:2.5 by adding 400pE antibody to 100(4tL redox reporter/EDC/sulfo-NHS mixture and gently mixed by inverting the tube up and down. The mixture is left to react for 24 hours on at 4°C in the fridge. After which the antbraquinone-conjugated anti-IgM antibody is purified from the mixture by ultrafiltration using centrifugal filters of using a molecular weight cut-off of 50k Da (molecular weight of antibody is 156k Da). Unreacted anthraquinone, the EDC, and the sulfo-NHS, are removed from the mixture during the centrifugation process. The concentrated antibody solution is transferred to a 1.5mL tube and made up the solution to 5001tL using I Ommol/L Phosphate buffer saline (pH 7.2). Further, 1000_, of the solution is put aside for characterization tests. Finally, the remaining concentrated antibody solution is stored at -80 degree Celsius. The conjugation process is repeated with ferrocene-carboxylic acid and anti-IgG antibody. The reporter tagged antibodies are diluted to the desired concentration using lOrnmol/L Phosphate buffer saline containing 0.5% v/v glycerol.

[00461 Further, redox-reporter conjugated antibodies may be deposited on to the electrochemical biosensor 100 at a specific location away from the at least one working electrode 102. A small volume (0.25p1 -lpl, usually 0.5p1) of conjugated antibodies (at concentrations ranging 10-2000pg/ml, preferably 300pg/m1) is deposited on to a region of the electrochemical biosensor 100 superior to the electrode region. In one embodiment, a pre-defined amount of glycerol may be used in a buffer for the reporter-tagged secondary antibody 106, to improve resuspension from dried formulation to liquid in the presence of the sample solution. In one example embodiment, the pre-defined amount of glycerol used in a buffer for the reporter-tagged secondary antibody 106 is 0.5% v/v. Such deposition may enable the antibodies to be located away from the at least one working electrode 102 until a sample/solution is provided to the electrochemical biosensor 100 to resuspended the antibodies which can then flow over on to the electrode region. The geometric distancing of the components prevents over-loading of the biological materials on the at least one working electrode 102 and prevent the detection of redox-reporter signal in the absence of bound anti-SARS-COV-2 antibodies, and more importantly allows for step-wise binding of anti-SARS-CoV-2 antibodies to the spike glycoprotein first, followed by the reporter-tagged antibodies binding the anti-SARS-CoV-2 antibodies, as shown in FIG. 4A. Further, FIG. 4B illustrates a functionalization of the working electrode 102 of the electrochemical biosensor 100. The working electrode 102, in this example, has deposited thereon both the SARS-CoV-2 spike glycoprotein 104 and the blocking agent 402 or the bovine-serum albumin (BSA), as shown in HG. 48. Further, once the reporter-tagged secondary antibodies 106 are deposited on the electrochemical biosensor 100, it is then dried at room temperature for 10 minutes and then stored at 2-8°C until use or downstream processing (e.g. assembling in microfluidic cartridges). Further, a pre-defined concentration of the reporter-tagged secondary antibody 106 may be determined by mixing the anti-SARS-CoV-2 spike protein antibodies with the reporter-tagged secondary antibody 106. In one example embodiment, a minimum antibody required is 0.3 micrograms.

[0047] The antibodies may have a high degree of specificity to target molecules and lack distinct measurable signal therefore antibodies are often tagged with fluorescent tags or enzymes which allow the presence of the antibody 200 to be measured. Further, the antibody 200 may be tagged with electrochemical reporter molecules or electrochemical redox reporter. The electrochemical redox reporter may be any moiety that has a distinct measurable signal upon being oxidised or reduced. The redox reporter molecules may allow the tagged-antibody to be detected and quantified through electrochemical detection methods such as voltammetry, amperometry, and potentiometry. Further, the electrochemical redox reporters may he any moiety which is stably redox active within a defined potential window. Further, when the electrochemical redox reporters undergo oxidation or reduction the electrons can he exchanged with an electrode to produce a measurable change in electrical properties at the electrode such as current or voltage. Each redox reporter possesses its characteristic reduction potential; the voltage at which oxidation/reduction for the moiety is at optimum. To enable multiplexing of the electrochemical redox reporters it is critical that each redox reporter possess non-overlapping reduction potentials. Further, the electrochemical redox reporters may be attached to the reporter-tagged secondary antibody 106 using at least one of a covalent bond, a hydrogen bonding, a pi-stacking or hydrophobic interactions. In one example embodiment, the electrochemical redox reporters are at least one of Ferrocene and Anthraquinone.

[0048] Ferrocene and Anthraquinone are used as redox reporter molecules which are conjugated to antibodies. Further, a range of redox reporter mediators such a methylene blue, Nile blue, ruthenium hexamine, osmium complexes, cobaltocenium, thionine, and viologen may be used for the antibody labelling. Further, a prerequisite of use the electrochemical redox reporters in a multiplexed system is clear well-distanced peaks. Among the range of redox reporter mediators, ferrocene, methylene blue, ruthenium hexamine, and anthraquinone redox reporters are tested and Anthraquinone and Ferrocene were selected on the bases of well-distanced peaks, as shown in by graph 500 in FIG. 5A. Further, in FIG. 5A, line 502 reflects a peak for Anthraquinone, line 504 reflects a peak for Ferrocene, line 506 reflects a peak for Ruthenium Hexamine, and line 508 reflects a peak for Methylene Blue.

[0049] Further, an operational use of the electrochemical biosensor 100 may require setting the fullyfunctionalised electrochemical biosensor 100 in electrical contact with an analog front-end and contacting the electrochemical biosensor 100 with a sample such as saliva. In one embodiment, the analog front-end may be a hardware used to interface the electrochemical biosensor 100 with the analog signal conditioning circuitry which then manipulates the signal before further processing by an analog-to-digital converter or a microcontroller, resulting in a digital read-out. In one embodiment, the electrochemical biosensor 100 may be inserted into a potentiostat (electrical device providing constant voltage, and processing current measurements). Further, the sample may be added to the electrochemical biosensor 100. The sample may consist of blood, serum, plasma, calibrants, and buffers. Further, a volume of the sample required may range from 10p1 to 40u1. The sample may he introduced to the electrochemical biosensor 100 in multiple ways: directly pipetting the sample on, by flowing the sample across the electrochemical biosensor 100, or using capillary fill by means of a microfluidic cartridge. Further, the anti-SARS-CoV-2 spike protein antibodies may be suspended in a sample solution and migrate to the at least one working electrode 102 for the detection of anti-SARS-CoV-2 spike protein antibodies.

[0050] Further, once the sample is in contact with the electrochemical biosensor 100 an assay time of 5 to 20 minutes (usually 10mins) is required to allow for the antibody binding events to take place. When the assay time is complete the electrochemical signal detection can proceed, alternatively the electrochemical biosensor 100 can be rinsed before signal detection. When rinsed, the electrochemical biosensor 100 is removed from the potentiostat and rinsed using phosphate buffered saline and then re-inserted to the analog front-end and fresh phosphate buffered saline is applied to the electrochemical biosensor 100, and then signal detection proceeds.

10051] Further, the electrochemical measurement of the redox reporter may be conducted via a number of methods including amperometry, voltammetry, coulometry, open-circuit potential, and impedance spectroscopy, without departing from the scope of the disclosure. In one example embodiment, the technique used in the electrochemical biosensor 100 is cyclic voltammetry. Voltammetry is where the voltage is varied in a series of discrete steps across a potential range, with cyclic voltammetry the potential ranges between two values. As a result of the varying voltage the at least one working electrode 102 may be in a state of excess voltage causing oxidation and reduction to the at least one working electrode 102. This produces a characteristic current response which is recorded as a function of the applied voltage. The output signal is recorded by a data acquisition system. The output signal is displayed as a peak or series of peaks at the reduction potential (where species are undergoing redox reaction) of the species. The data may then be analysed using the peak features such as peak positions, peak heights, peak widths, and peak areas. The data may then be interpreted against a standard curve for concentration against peak feature or using more advanced machine learning tools. The electrochemical biosensor 100 may be tested using WHO reference standards for Anti-SARS-CoV-2 Immunoglobulins, as shown by graph 510, in FIG. 5B. In one example embodiment, the cyclic voltammetry parameters are as follows: Scan range -1.2V to IV, voltage step 0.005V, scan rate 50mV/s. Further, in FIG. 5B, line 512 reflects a peak for 20/150 Anti-sarcov-2 High, line 514 reflects a peak for 20/148 Anti-sarcov-2 Mid, and line 516 reflects a peak for 20/144 Anti-sarcov-2 Low.

[0052] FIG. 6 illustrates a flowchart 600 showing a method for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies. FIG. 6 is described in conjunction with FIGS. 1-5. At first, the electrochemical biosensor 100 may bind a SARS-CoV-2 spike glycoprotein 104 on at least one working electrode 102, at step 602. Firstly, a solution of 4mg/m1 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) 11mg/m1 sulfo-N-hydroxysulfosuccinimide (NHS) may be prepared and added to the at least one working electrode 102 and left to activate at room temperature for 20 minutes. Further, the SARS-CoV-2 spike glycoprotein 104 may be prepared in HBS-EP (0.01 mol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 0.15 mol/L NaC1, 3mmol/L EDTA, 0.005% v/v Tween-20) buffer. Further, the EDC/sulfoNHS Carbodiimide crosslinking may be required for the activation of the carboxyl groups on the at least one working electrode 102 to bind the primary amines of the SARS-CoV-2 spike glycoprotein 104. When the SARS-CoV-2 spike glycoprotein 104 is deposited on the activated at least one working electrode 102, it then reacts for a stable carbodiimide bond. In particular, 2p1 of 1pg/m1 SARS-CoV-2 spike glycoprotein 104 is deposited on the activated at least one working electrode 102 and left to react for 1 hour at room-temperature or overnight in at 4 degrees Celsius.

[0053] Further, the method may include depositing reported-tagged secondary antibodies 106 on the electrochemical biosensor 100, at step 604. Further, redox-reporter conjugated antibodies may be deposited on to the electrochemical biosensor 100 at a specific location away from the at least one working electrode 102. A small volume (0.25p1 -lul, usually 0.5p1) of conjugated antibodies (at concentrations ranging 10-2000pg/ml, usually 300pg/m1) is deposited on to a region of the electrochemical biosensor I 00 superior to the electrode region. Such deposition may enable the antibodies to be located away from the at least one working electrode 102 until a sample/solution is provided to the sensor to resuspend the antibodies which can then flow over on to the electrode region.

[0054] Further, the method may include receiving anti-SARS-CoV-2 spike protein antibodies in a sample solution to react with the reporter-tagged secondary antibodies 106, at step 606. Further, the anti-SARS-CoV2 spike protein antibodies may be suspended in a sample solution and migrate to the at least one working electrode 102 for the detection of anti-SARS-CoV-2 spike protein antibodies. Further, the method may include binding the received anti SARS-CoV-2 spike protein antibodies to the at least one working electrode 102, at step 608. Further, the method may include detecting the anti-SARS-CoV-2 spike protein antibodies using electrochemical redox reporters, at step 610. Further, the electrochemical biosensor 100 may facilitate detecting, using electrochemical redox reporters. the anti-SARS-CoV-2 spike protein antibodies, upon a redox reaction at the at least one working electrode. Such usage of the electrochemical redox reporters results in detecting anti-SARS-CoV-2 spike protein antibodies. Further, antibodies may be tagged with electrochemical reporter molecules or electrochemical redox reporter. The electrochemical redox reporter may be any moiety that has a distinct measurable signal upon being oxidised or reduced. The redox reporter molecules may allow the tagged-antibody to be detected and quantified through electrochemical detection methods such as voltammetry, amperometry, and potentiometry. Further, the electrochemical redox reporters may be any moiety that is stably redox active within a defined potential window. Further, when the electrochemical redox reporters undergo oxidation or reduction the electrons can be exchanged with an electrode to produce a measurable change in electrical properties at the electrode such as current or voltage. Each redox reporter possesses its characteristic reduction potential; the voltage at which oxidation/reduction for the moiety is at optimum. To enable multiplexing of the electrochemical redox reporters it is critical that each redox reporter possess non-overlapping reduction potentials. Further, the electrochemical redox reporters may be attached to the reporter-tagged secondary antibody 106 using at least one of a covalent bond, a hydrogen bonding, a pi-stacking, or hydrophobic interactions. In one example embodiment, the electrochemical redox reporters are at least one of Ferrocene and Antluaquinone.

[0055] It will be apparent to one skilled in the art that the above-mentioned components of the electrochemical biosensor 100 have been provided only for illustration purposes. In one example embodiment, the electrochemical biosensor 100 may include an input device, output device etc. as well, without departing from the scope of the disclosure.

[0056] The detailed description section of the application should state that orders of method steps are not critical. Such recitations would later support arguments that the step order in a method claim is not critical or fixed. Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.

[0057] While the above embodiments have been illustrated and described, as noted above, many changes can be made without departing from the scope of the example embodiments. For example, aspects of the subject matter disclosed herein may be adopted on alternative operating systems. Accordingly, the scope of the example embodiments is not limited by the disclosure of the embodiment. Instead, the example embodiments should be determined entirely by reference to the claims that Rfflow.

Claims (25)

1. CLAIMSWhat is claimed is: 1. An electrochemical biosensor (100) for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV-2) spike protein antibodies, wherein the electrochemical biosensor comprises: at least one working electrode (102); a recognition element (104) arranged on a surface of the at least one working electrode (102), wherein the recognition element (104) is SARS-CoV-2 spike glycoprotein; and a signal transduction element (106), located away from the at least one working electrode (102), wherein the signal transduction element (106) is a reporter-tagged secondary antibody; wherein the anti-SARS-CoV-2 spike protein antibodies bind to the SARS-CoV-2 spike glycoprotein on the at least one working electrode (102), using electrochemical redox reporters bound to the reporter tagged secondary antibody.
2. 2. The electrochemical biosensor (100) according to claim 1, wherein the SARS-CoV-2 spike glycoprotein is wholly intact spike glycoprotein.
3. 3. The electrochemical biosensor (100) according to claim 2, wherein the SARS-CoV-2 spike glycoprotein is immobilized onto the at least one working electrode (102) using passive adsorption or via chemical crosslinking.
4. 4. The electrochemical biosensor (100) according to any preceding claim, wherein the anti-SARS-CoV2 spike protein antibodies are suspended in a sample solution and miwate to the SARS-CoV-2 spike glycoprotein on the at least one working electrode (102).
5. 5. The electrochemical biosensor (100) according to any preceding claim, wherein the passive adsorption of the SARS-CoV-2 spike glycoprotein includes hydrophobic interactions between the at least one working electrode (102) and hydrophobic residues of the SARS-CoV-2 spike glycoprotein.
6. 6. The electrochemical biosensor (100) according to any preceding claim, wherein the at least one working electrode (102) is made at least of platinum, gold, or a carbon allotrope.
7. 7. The electrochemical biosensor (IOU) according to any preceding claim, wherein the electrochemical redox reporters are crosslinked to secondary anti-IgG and anti-IgM antibodies in an optimized manner by increasing one or more parameters, wherein the one or more parameters include incubation times and concentrations used.
8. 8. The electrochemical biosensor (100) according to any preceding claim, wherein the SARS-CoV-2 spike glycoprotein is crosslinked to the at least one working electrode (102) through NHydroxysuccinimide ester crosslinking, carbodiimide crosslinking, or hydrazine crosslinking of glycosylated (carbonyl-reactive) residues.
9. 9. The electrochemical hiosensor (1 00-) according to any preceding claim, wherein a pre-defined concentration of the reporter-tagged secondary antibody is determined by mixing the anti-SARS-CoV2 spike protein antibodies with the reporter-tagged secondary antibody.
10. 10. The electrochemical biosensor (100) according to any preceding claim, wherein the reporter-tagged secondary antibody (106) is deposited on the electrochemical biosensor (100) at a pre-defined concentration.
11. 11. The electrochemical biosensor (100) according to any preceding claim, wherein the electrochemical redox reporters are a moiety which are stably redox active within a defined potential window and the electrochemical redox reporters upon oxidation or reduction exchange electrons with an electrode produce a measurable change in electrical properties at the at least one working electrode.
12. 12. The electrochemical biosensor (100) according to any preceding claim, wherein the electrochemical redox reporters are attached to the reporter-tagged secondary antibody using at least one of a covalent bond, a hydrogen bonding, a pi-stacking or hydrophobic interactions.
13. 13. The electrochemical biosensor (100) according to any preceding claim, wherein the electrochemical redox reporters are at least one of Ferrocene and Anthraquinone.
14. 14. The electrochemical biosensor (100) according to any preceding claim, wherein the SARS-CoV-2 spike glycoprotein is covalently bonded to the at least one working electrode.
15. 15. The electrochemical biosensor (100) according to any preceding claim, wherein a pre-defined amount of glycerol is used in a buffer for the reporter-tagged secondary antibody, to improve resuspension from dried formulation to liquid in the presence of the sample solution.
16. 16. The electrochemical biosensor (100) according to any preceding claim, wherein the pre-defined amount of glycerol used in a buffer for the reporter-tagged secondary antibody is 0.5% v/v.
17. 17. The electrochemical biosensor (100) according to any preceding claim, wherein the electrochemical biosensor (100) is coated with a blocking agent (402) to block interaction with the at least one working electrode.
18. 18. The electrochemical biosensor (100) according to any preceding claim, wherein the blocking of the interaction is optimized by reducing the volume and incubation time with the blocking agent (402).
19. 19. The electrochemical biosensor (100) according to any preceding claim, wherein the blocking agent (402) is 1% w/v bovine-serum albumin (BSA).
20. 20. The electrochemical biosensor (100) according to any preceding claim, wherein 2 microliters of 1% w/v BSA solution is coated for 1 hour at room temperature.
21. 21. A biosensor detection system comprising an electrochemical biosensor (100), wherein the electrochemical biosensor is implemented according to any preceding claim
22. 22. A method (60() for detection of anti-severe acute respiratory syndrome associated coronavirus (SARS00V-2) spike protein antibodies, the method comprising: binding (602) a SARS-CoV-2 spike glycoprotein on at least one working electrode (102) of an electrochemical biosensor (100); depositing (604) reporter-tagged secondary antibodies on the electrochemical biosensor (100), wherein the reporter-tagged secondary antibodies migrate to the at least one working electrode (102); receiving (606) anti-SARS-CoV-2 spike protein antibodies in a sample solution, to react with the reporter-tagged secondary antibodies; binding (608) the received anti-SARS-CoV-2 spike protein antibodies to the at least one working electrode (102); and detecting (610), using electrochemical redox reporters, the anti-SARS-CoV-2 spike protein antibodies, upon a redox reaction at the at least one working electrode (102).
23. 23. The method of claim 22, wherein the SARS-CoV-2 spike glycoprotein is immobilized onto the at least one working electrode (102) using passive adsorption or via chemical crosslinking.
24. 24. The method of claim 22 or 23, wherein the passive adsorption of the SARS-CoV-2 spike glycoprotein includes hydrophobic interactions between the at least one working electrode and the hydrophobic residues of the SARS-CoV-2 spike glycoprotein and chemical crosslinking of the SARS-CoV-2 spike glycoprotein includes the use additional materials to form chemical bonds between the functional groups of the residues being crosslinking.
25. 25. The method of claim 22 or 23, wherein the SARS-CoV-2 spike glycoprotein is crosslinked to the at least one working electrode through an N-Hydroxysuccinimide ester crosslinking, carbodiimide crosslinking, or hydrazine crosslinking of glycosylated (carbonyl-reactive) residues.