METHOD OF MAKING A PORTABLE MIP-BASED ELECTROCHEMICAL SENSOR FOR THE DETECTION OF THE SARS-COV-2 ANTIGEN

Abstract

The current COVID-19 pandemic caused by SARS-CoV-2 coronavirus is expanding around the globe. Hence, accurate and cheap portable sensors are crucially important for the clinical diagnosis of COVID-19. Molecularly imprinted polymers (MIPs) as robust synthetic molecular recognition materials with antibody-like ability to bind and discriminate between molecules are provided here as selective elements in such sensors. Provided are detection assemblies comprising electrochemical sensors having ncovNP-MIP film endowed selectivity against SARS-CoV-2 nucleoprotein (ncovNP) and/or ncovS1-MIP film endowed selectivity against SARS-CoV-2 spike 1 (S1). The ncovNP- or ncovS1-MIP are synthesized electrochemically on portable gold thin-film electrodes system via chronocoulometry or cyclic voltammetry. The sensors show excellent detection capabilities, and high discrimination of interfering proteins.

Claims

1. A detection assembly for detecting at least one SARS-CoV-2 antigen from a sample, the assembly comprising portable electrochemical sensor integrated with a SARS-CoV-2 antigen-molecular imprinted polymer (MIP), wherein the SANS-CoV-2 antigen-MIP is configured to act as a synthetic recognition element selectively detecting and binding the at least one SARS-CoV-2 antigen; and a reading device capable of measuring presence of absence of the antigen.

2. The detection assembly of claim 1, wherein the at least one SARS-CoV-2 antigen is SARS CoV-2 nucleoprotein ncovNP car SARS CoV-2 S1-subunit protein ncovS1.

3. The detection assembly of claim 1, wherein the sample is from an environmental source, such as sewage water, or air to liquid sedimentation sampler; from a human source, such as a swabbing sample from mucous membranes, or blood, urine or saliva sample; or from a laboratory source, such as a recombinant protein expression or purification sample.

4. The detection assembly of claim 1, wherein the detection assembly has a detection limit in a range of 15 to 70 fM, more preferably' 15 to 50 fM, and most preferably 15-30fM.

5. The detection assembly of claim 1, wherein the detection assembly has a quantification limit of 50 to 220 fM, more preferabl 50 to 80 fM and most preferably about 5-51 fM.

6. The detection assembly of claim 1, wherein the assembly has a shelf e more than weeks, preferably more than 7 weeks, and most preferably at least 9 weeks.

7. The detection assembly of claim 1, wherein the detection assembly is configured to discriminate at least E2 (E2 envelope protein of Hepatitis C virus), HCV (Hepatitis C virus antigens), BSA (bovine serum albumin), HSA (human serum albumin), IgG (immunoglobulin (G) and CD48 (Cluster of Differentiation 48) proteins.

8. The detection assembly of claim 1, wherein a polymeric layer coating of the electrochemical sensor is formed from electropolymerizable monomers selected from the group consisting of mPD, 3-aminophenylboronic acid (APBA), dopamine and EDOT; most preferably mPD or APBA, and the polymeric layer coating is deposited by 1-10 mC/cm.sup.2; more preferably 1-7 mC/cm.sup.2, and most preferably by 2-7 mC/cm.sup.2.

9. The detection assembly of claim 1, wherein the detection assembly comprises a potentiostat reader.

10. The detection assembly of claim 9, wherein the potentiostat eader is connectable to a cellular phone.

11. A method to detect presence or absence or quantitative concentration of at least one SARS-CoV-2 antigen in a sample, wherein the method comprises: providing a sensor comprising SARS-Coe-2 antigen molecular imprinted polymer (MIP) integrated with a sensing electrode, such as a thin film electrode (TFE); bringing the sensor in contact with the sample; and detecting the presence or absence of the antigen by differential pulse voltammetry (DPV) or square voltammetry (SWV), wherein presence of the antigen is recorded when limit of detection (LOD) of the sensor is exceeded.

12. The method of claim 11, wherein bringing the sensor in contact with the sample is obtained by incubating the sensor in a buffer, optionally containing a detergent, and comprising the sample, preferably less than 60 minutes, more preferably less than 30 minutes, even more preferably between 20 and 30 minutes, and most preferably 15-20 minutes.

13. The method of claim 11, wherein the antigen is SARS CoV-2 nucleoprotein ncovNP or SARS CoV-2 S1-subunit protein ncovS1.

14. The method of claim 11, wherein the sample is obtained from an environmental source, such as sewage water, or air to liquid sedimentation sampler; from a human source, such as a swabbing sample from mucous membranes, or blood, urine or saliva sample; or from a laboratory source, such as a recombinant protein expression or purification sample.

15. A method for making an electrochemical sensor for detection SARS-CoV-2 antigen, especially ncovNP or ncovS1 from samples, the method comprising the steps of: forming a cleavable linking layer on a metallic surface of sensing electrode surface deposited on an insulating support; immobilizing ncovNP and/or ncovS1 molecules on the cleavable linking layer on the electrode surface; polymerizing a polymer on the antigen -immobilized electrode surface thereby forming a polymeric layer coating on the electrode surface with entrapped antigen molecules; and cleaving off the cleavable linking layer thereby removing the antigen molecules from the polymeric layer and obtaining antigen sensor configured to capture antigens similar to those removed by cleaving off the linking layer.

16. The method of claim 15, wherein the polymer is polymerized from electropolymerizable monomers selected from the group consisting of m-phenylenediamine (mPD), 3-aminophenylboronic acid (APBA), dopamine and 3,4-ethylenedioxythiophene (EDOT), most preferably mPD or APBA.

Description

SHORT DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1. Illustrates the analysis principle of portable nconNP or nconS1 sensor. Example shows COVID-19 analysis/diagnostics principle by nconNP sensor from clinical samples from nasopharyngeal swab specimens of patients. ncovNP-MIP film is integrated with TFE (thin film electrode). The sensor is incubated 15 minutes in lysis buffer supplemented with sample (here nasopharyngeal swab sample of a patient). The result is read by differential pulse voltammetry from the sensor. Here, a portable differential pulse voltammetry reader is attached to a cell phone and the result is immediately displayed to the user.

[0031] FIG. 2. Shows examples of cyclic voltammograms recorded in 1 M KCl solution containing a 4 mM redox probe K3[Fe(CN)6]/K4[Fe(CN)6] on bare Au-TFE (1), and after the subsequent fabrication steps of ncovNP-MIP: modification by 4-ATP (2) and DTSSP (3), immobilization of ncovNP (4), electrodeposition of PmPD (5) and treatment in 2-ME and acetic acid (6) (see section Sensor fabrication for details). Similar voltammograms were recorded for ncovS1-MIP sensor as well (in ncov-S1-MIP fabrication electrodeposition of PABA was used and the treatment was conducted in DTT and acetic acid, results not shown).

[0032] FIG. 3. Effect of charge densities applied during synthesis of PmPD on the saturated responses (I.sub.n.sat(MIP), I.sub.n.sat(MIP)) of ncovNP-MIP/Au-TFE, NIP/Au-TFE and IF. The I.sub.n.sat(MIP), I.sub.n.sat(MW) were derived from the corresponding adsorption isotherms (FIGS. 12A-C) and the respective IF was calculated according to Eq. 3. The adsorption isotherms were obtained upon the incubation of ncovNP-MIP/Au-TFE and NIP/Au-TFE in LB containing the different concentrations of ncovNP and fitted to Langmuir adsorption model (Eq. 2). Similar effect of charge densities applied during synthesis of PABA on the performance of the resulting ncovS1-MIP was observed (ncovS1-MIP was prepared by potential cycling and the film thickness was controlled by the number of cycles; results not shown).

[0033] FIG. 4. Optimization of time for incubation of ncovNP sensor having PmPD (poly(m-phenylenediamine) synthesized at 2 mC/cm.sup.2. The responses were obtained after incubation of the sensor in LB (lysis buffer) containing 0.1 pM ncovNP. Similar optimization of time for incubation was performed by ncovS1-MIP sensor having PABA (poly 3-aminophenylboronic acid) synthesized by the optimal number of cycles (10) that equalled to about 6 mC/cm.sup.2 (results not shown).

[0034] FIGS. 5A-B. (A) Calibration plot of ncovNP sensor obtained at low concentration range of ncovNP (2-111 fM) in LB. (B) Similar calibration plot in PBS was obtained for nCov S1 (27-194 fM).

[0035] FIG. 6. Selectivity test of the ncovNP sensor showing its responses against the different proteins (S1, E2 HCV, BSA, CD48 and ncovNP) spiked at concentrations (0.04, 0.07, 0.09, and 0.11 pM) in LB. Similar selectivity in PBS was observed by ncovS1 sensor at increasing concentrations 40, 60, 80, 100, 120 fM of different proteins (ncovNP, HSA, E2 HCV, IgG and ncovS1).

[0036] FIGS. 7A-B. (A) The example calibration plots of ncovNP sensors obtained against COVID-19 negative samples in UTM (universal transport medium) of four patients, 20-fold diluted with LB and spiked with 22.2, 44.4, 66.6, 111, 222, 333 fM of ncovNP. (B) The example calibration plots of ncovS1 sensor obtained against COVID-19 negative nasopharyngeal swab samples in SPS solution. Samples were diluted with PBS (1:99) and spiked with 50-400 fM of ncovS1,

[0037] FIGS. 8A-C. (A) Cross-selectivity test of ncovNP sensor showing its responses against ncovNP, S1, and mixture of ncovNP and S1 proteins in COVID-19 negative sample in UTM diluted 20-fold with LB. The concentrations of ncovNP and S1 were selected to simulate their concentration ratio in SARS-CoV-19 virus [32].

[0038] (B) Selectivity of ncovS1 sensor for ncovS1 against ncovNP in COVID-19 negative nasopharyngeal swab samples. The concentration of ncovNP was tenfold higher than the concentration of ncovS1.

[0039] (C) The ncovS1 Sensor responses to SARS-CoV-2 spike protein subunit S1 (ncovS1) and its different strains (S1 UK VOC 202012/01, S1 Brazil P1 and S1 South Africa (SA) VOC 501.V2). The dashed lines represent the LOD determined in FIG. 9B.

[0040] FIGS. 9A-B. (A) The calibration plot of ncovNP sensor obtained by averaging the data in FIG. 7A. The squares represent data points corresponding I.sub.n measured by ncovNP sensor against COVID-19 positive samples in UTM 20-fold diluted with LB.

[0041] (B) The calibration plot (solid line) of ncovS1 sensor obtained by linear regression of the averaged data in FIG. 7B. P4-P8 designated gray squares represent data points corresponding to In measured by ncovS1 sensor against COVID-19 positive samples while P1, P3 black squares are those of negative samples. The error bars represent SDs of three measurements.

[0042] FIG. 10 Stability of ncovNP sensors stored for different time intervals at room temperature. The presented responses were measured against 66.6 fM ncovNP in LB by a pair of sensors right after fabrication or at various time points after 1-9 weeks storage. Similar stability was observed for ncovS1 sensors (results not shown).

[0043] FIG. 11 Shows comparison of electrochemical PmPD growth on Au-TFE and ncovNP-modified Au-TFE. Potentiostatic electrodeposition of PmPD from 10 mM mPD in PBS on bare Au and ncovNP-modified Au working electrode of TFE at 0.6 V vs Ag/AgCl/KCl.

[0044] FIGS. 12A-C shows effect of the sensing layer thickness of rebinding properties of ncovNP sensor. Adsorption isotherms of ncovNP on ncovNP-MIP- and NIP-modified sensors having the sensing layer generated by (a) 1 mC/cm.sup.2, (b) 2 mC/cm.sup.2, (c) 3 mC/cm.sup.2.

DESCRIPTION OF THE INVENTION

Definitions and Abbreviations

[0045] By ncovNP it is meant SARS-CoV-2 nucleocapsid protein. The crystal structure of ncovNP is available at PDB Protein Databank as deposit number 6VYO https://www.rcsb.org/structure/6VYO. Further physical characterization of the protein is provided in the description below:

TABLE-US-00001 Protein Code in Molecular Molecular Isoelectric Name PDB weight, kDa volume, .sup.3 point ncovNP 6VYO 45 85268 10.07

[0046] By ncovS1 it is meant SARS-CoV-2 S1 subunit protein. The crystal structure of ncoS1 is available at PDB Protein Databank as deposit number 6VXX. Further physical characterization of the protein is provided in the description below.

TABLE-US-00002 Protein Code in Molecular Molecular Isoelectric Name PDB weight, kDa volume, .sup.3 point ncovS1 6VXX 75 502733 6.04

[0047] By MIP it is meant molecularly imprinted polymers.

[0048] By TFE it is meant a thin film electrode consisting of a metallic surface e.g. gold as a sensing electrode deposited on an insulating support e.g. glass,

[0049] By antigen it is meant a toxin or other foreign substance which induces an i rnrnune response in the body, especially the production of antibodies.

[0050] By SARS CoV-2 antigens it is meant any biological part of the SARS CoV-2 virus that induces an immune response.

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials similar or equivalent to those described herein can also be used in the practice of the present invention, exemplary materials are described for illustrative purposes.

[0052] As used herein and in the appended claims, the singular form a, and, the include plural referents unless the context clearly dictates otherwise.

[0053] As used herein, the terms about and approximately are used interchangeably and have the meaning a person having ordinary skill in the art would readily understand.

[0054] The terms comprises, comprising, includes, including, having and their conjugates mean including but not limited to. Terms and phrases used in this application, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term including should be read as meaning including, without limitation or the like. The term example is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof. Adjectives such as, e.g.. conventional, n-aditional, known and terms of similar meaning should not be construed as limiting the item described to a given time period, or to an item available as of a given time. But instead, these terms should be read to encompass conventional, traditional, normal, or standard technologies that may he available, known now, or at any time in the future.

[0055] Likewise, a group of items linked with the conjunction and should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as and/or unless expressly stated otherwise. Similarly, a group of items linked with the conjunction or should not be read as requiring mutual exclusivity among that group, but rather should also be read as and/or unless expressly stated otherwise. The presence of broadening words and phrases such as, one or more, at least; but not limited to, or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances, wherein such broadening phrases may be Asent.

[0056] The invention is now described in detail with reference to the Figures.

[0057] Herein, we report for the first time on the development of a MIP-based electrochemical sensor for detection of ncovNP or ncovS1 of SARS-CoV-2 (FIG. 1). The ncovNP molecularly imprinted polymer (ncovNP-MIP) or ncovS1 molecularly imprinted polymer (ncovS1-MIP) were directly generated on a portable sensing electrode, for example gold thin film electrode (TFE) surface through the electrochemical technique. After incubation in a sample solution, the prepared sensors showed remarkable capability to discriminate between the target analyte (ncovNP or ncovS1) and interfering protein such as E2 HCV, BSA, HSA, IgG and CD48 as measured by differential pulse voltammetry or square wave voltammetry in the presence of redox pair. To prove the sensor performance, the clinical diagnostic feasibility of ncovNP-MIP and ncovS1-MIP sensors was studied by analyzing nasopharyngeal swab specimens of patients.

[0058] Preferably, the sensors are prepared by modification of Au-TFE with ncovNP-MIP or ncovS1-MIP film generated from electropolymerizable monomers, selected from the group consisting of mPD, dopamine, APBA, and EDOT. Most preferably the mPD or APBA is used and the resulting polymer is poly-m-phenylenediamine (PmPD) or poly 3-aminophenylboronic acid (PABA).

[0059] The preferable thickness of the sensors determined as the thickness at which the sensor demonstrated the highest IF (imprinting effect) may be selected when the polymer film is deposited by charge density of 1-10 mC/cm.sup.2, more preferably 1-7 mC/cm.sup.2. Most preferably the film is deposited by charge density of 2-7 mC/cm.sup.2.

[0060] The sensors according to this disclosure are highly responsive and specific for the selected antigens. The limit of detection values for the antigens is between 15 and 70 fM, more preferably 15-50 fM and most preferably 15-30fM. The values for limits of quantification of the sensors is between 50-120fM, more preferably 50-80 and most preferably about 50-51 fM. Such values are well suitable to detect antigens from COVED 19-patient samples but also from various environmental samples. Moreover, the sensors according to this disclosure are efficiently discriminating between the antigen to be measured and various interfering proteins, such as E2 HCV, CD48, HAS, IgG and BSA. Moreover, ncovNP-MIP sensor discriminates effectively S1, while ncovS1-MIP sensor discriminates effectively NP. These features ensure that the sensors, and the method provided here is accurate.

[0061] This disclosure provided a portable electrochemical sensor integrated with a molecular imprinted polymer (ncovNP-MIP and ncovS1-MIP) as a synthetic recognition element capable of selective detection of SARS-CoV-2 antigen (ncovNP or ncovS1 respectively). The synthesis parameters of sensors are configured such that the sensor has an ability to selectively rebind the antigen. The sensor has a good linearity and reproducibility in COVID-19 negative clinical samples spiked with ncovNP in the concentration range 0.22-333 fM that resulted in a LOD and LOQ values sufficient to determine the presence of ncovNP in COVID-19 positive samples from nasopharynx swab specimens, which can be expected as well as from other clinical samples or from environmental samples. For ncovS1-MIP sensor linearity response was achieved within a concentration range of 0 to 400 fM. and the LOD and LOQ were determined as 64 fM and 213 fM respectively. The developed sensor that relies on a completely different approach as compared to the currently available SARS-CoV-2 antigen tests, provides a valuable alternative as a portable diagnostic platform for the rapid screening for COVID-19 or quantitative or qualitative analysis of SARS CoV-2 antigens.

Materials and methods

[0062] 4-aminothiophenol (4-ATP), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), m-phenylenediamine (mPD), bovine serum albumin (BSA), sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate, and sodium dodecyl sulfate, sodium fluoride (NaF), Immunoglobulin G (IgG), and human serum albumin (HSA) were obtained from Sigma-Aldrich. 3-aminophenylboronic acid (APBA) was obtained from Santa Cruz Biotechnology Ethanol 96% was purchased from Estonian Spirit O (Estonia). SARS-Cov-2 nucleoprotein (ncovNP) and S1-subunit of SARS-Cov-2 spike protein and its different strains (S1 UK VOC 202012/01, S1 Brazil P1 and S1 South Africa VOC 501.V2) as well as CD48 protein were provided by Icosagen AS (Estonia). Hepatitis C virus (HCV) surface viral antigen (E2) was obtained from the Institute of Macromolecular Compounds of the Russian Academy of Sciences. 3,3-dithiobis [sulfosuccinimidyl propionate] (DTSSP) and Triton X-100 were purchased from Thermo Fisher Scientific Inc., sulfuric acid, hydrogen peroxide, potassium chloride (KCl), and acetic acid were purchased from Lach-ner, S.R.O. Sodium chloride (NaCl), Tris EDTA buffer concentrate, and ethylenediaminetetraacetic acid (EDTA) were obtained from Fluka analytical. Potassium ferricyanide and ferrocyanide were purchased from Riedel-de Haen. MicruX thin-film electrodes (Au-TFE) were purchased from Micrux Technologies (Spain). All chemicals were of analytical grade and were used as received without any further purification. Ultrapure Milli-Q water (resistivity 18.2 M cm at 25 C., EMD Millipore) was used for the preparation of all aqueous solutions. For producing the sensors and performing the analysis, named compounds could be substituted with similar chemicals from other manufacturers.

Computational Modelling Used for Selection of suitable functional monomer for ncovNP MIP

[0063] The rational selection of a suitable functional monomer for ncovNP-MIP synthesis was based on a computational modeling approach [28]. Briefly the interactions between ncovNP and candidate functional monomers mPD, dopamine (DA), and 3,4-ethylenedioxythiophen (EDOT) were modeled by using molecular docking and quantum chemical calculations. Molecular docking was performed by using Autodock 4.2.6 (from the Scripps Research Institute) on the grid-center with coordinates 13.827; 19.445; 6.256 to find out energetically favorable binding poses of monomers with ncovNP. The crystal structure of ncovNP (6VYO) deposited in Protein Data Bank (PDB) and monomer structures were processed within the AutoDockTools (ADT) software performing structure preparation procedures. The monomer binding poses were generated by molecular docking using Autodock4.2.6 empirical scoring function (GScore) that approximates the monomer-binding free energy and takes into account a number of non-covalent interaction parameters. Quantum chemical calculations were used to estimate the binding energies of hydrogen interactions between sterically accessible proton-accepting amino acids of ncovNP and candidate monomers and performed by using Gaussian 09. The software Rasmol (version 2.7.5.2) was used to find out the sterically accessible proton acceptor group of ncovNP (eg. polar amino acid residues). The strength of H-bond interactions between ncovNP and the monomers was calculated as a sum of association energies between a polar amino acid and the monomer as described before in [28].

[0064] The energies of prepolymerized complex structures of ncovNP with electropolymerizable monomers (mPD, dopamine, and EDOT) in aqueous solutions were determined by the computational approach. The GScore values for the best-scoring binding poses of all three monomers docked to ncovNP were almost similar ranging 25.2 and 29.5 kJ/mol. This indicated that all three monomers were able to form prepolymerization complexes with ncovNP with almost similar stability. At the same time, all the possible H-bond interactions between the respective monomers and the protein (ncovNP) in prepolymerization mixture were estimated by quantum chemical calculations. Table 1 summarizes the association energies of interaction between the polar amino acids of ncovNP and the different monomers (mPD, dopamine, and EDOT).

TABLE-US-00003 TABLE.1 Binding energies of ncovNP with different monomers calculated by Gaussian 09 Monomer Binding energy (kJ/mol) m-PD 3273 EDOT 3122 Dopamine 1124

[0065] As it can be seen mPD forms the strongest network of H-bond interactions around ncovNP molecules as compared to the other 2 monomers. Thus, mPD monomer was selected as the optimal monomer for ncovNP-MIP synthesis.

Sensor Fabrication

[0066] The ncovNP sensor was prepared by modification of Au-TFE with ncovNP-MIP generated from poly-m-phenylenediamine (PmPD). The protocol for synthesis of ncovNP-MIP was developed from [29]: Before modification, Au-TFE was cleaned with a cold piranha solution (H.sub.2SO.sub.4:H.sub.2O.sub.2, 3:1) for 2 minutes, then rinsed with double distilled water and dried under a nitrogen atmosphere. The working electrode of the Au-TFE was modified with 4-ATP by incubating it in 100 mM 4-ATP ethanolic solution for 30 minutes and vortex for 5 minutes in ethanol to remove the non-bound 4-ATP. 4-ATP can be substituted to any compatible amine-terminated thiol or thiocarbonyl, e.g. thiourea. The cleavable linker monolayer was generated by the covalent attachment of DTSSP (3,3-Dithiobis(sulfosuccinimidylpropi onate)) to 4-ATP-Au-TFE via drop casting of 4 L, (10 mM) DTSSP solution in phosphate buffered saline (PBS) for 30 minutes and washed with PBS. For ncovNP immobilization, 4 L of PBS containing 0.55 M of ncovNP was dropped on the cleavable linker modified electrode for 30 minutes and washed with plenty of PBS. The PBS can be substituted to any compatible buffer systems, like citrate buffer or carbonate buffer.

[0067] The synthesis of PmPD on ncovNP-modified Au-TFE was performed in the electrochemical cell (ED-AIO-Cell, Micrux Technologies, Spain) connected with the electrochemical workstation (Reference 600TM potentiostat; Gamry Instruments, USA). PmPD film was electrodeposited from PBS containing 10 mM mPD at 0.6 V vs Ag/AgCl/KCl with an optimized charge density. Molecular imprints of ncovNP in the polymer film were generated by treating the polymer film with ethanolic solution of 0.1 M 2-ME to cleave the S-S bond of DTSSP and facilitate the release of ncovNP, followed by washing with the 10% acetic acid solution. A similar procedure was also adopted for the reference film, non-imprinted polymers (NIPs), except by treating with 2-ME to save the covalent attachment of ncovNP in the polymer matrix in NIPs and to avoid formation of the molecular imprints of ncovNP in this film.

[0068] The sensor preparation steps were characterized by cyclic voltammetry (CV) in potential range of 0.2 to 0.2 at a scan rate of 100 mV/s. Cyclic voltammetry was conducted in 1 M KCl solution containing 4 mM redox probe K.sub.3[Fe(CN).sub.6]/K.sub.4[Fe(CN).sub.6].

[0069] Preparation ncovS1 sensor was conducted as follows:

[0070] The ncovS1 sensor was prepared by synthesizing ncovS1-MIP film directly on Au-TFE adapting the surface imprinting strategy previously developed by our group [25,26]. Prior to the modification, the Au-TFME was cleaned in ozone for 15 minutes followed by washing with ethanol, rinsing with MQ and then drying under a nitrogen atmosphere. Modification of the WE of Au-TFE was achieved by incubating it for 30 min in 100 mM 4-ATP ethanolic solution and vortexing for 5 min to remove loosely bound ATP molecules. A monolayer of cleavable linker was obtained via the covalent attachment of DTSSP to the ATP modified Au-TFE by drop casting 10 L (10 mM) DTSSP solution in PBS for 30 min followed by washing with PBS. ncovS1 was immobilized on the DTSSP/ATP-modified Au-TFE by dropping 3 L of PBS containing 0.33 M of ncovS1 for 30 min and washed severally with PBS. Poly(3-aminophenylboronic acid), PAPBA was synthesized on the ncovS1-modified Au-TFE in a set-up consisting of an electrochemical cell (ED-MO-Cell, Micrux Technologies, Spain) connected with the electrochemical workstation (Reference 600TM, Gamry Instruments, USA). PAPBA was electrodeposited from a PBS solution containing 20 mM APBA and 50 mMNaF by cycling the potential between 0.2 to 0.9 V vs Ag/AgCl/KCl. Imprints of ncovS1 were generated in the polymer film by cleaving the SS bond of DTSSP using 50 mM dithiothreitol (DTT) solution for 30 min, followed by washing for another 30 min in 10% acetic acid to remove the ncovS1. The reference, non-imprinted film (NIP), was prepared using a similar protocol but without treatment in DTT to preserve the covalently attached ncovS1 in the polymer thereby avoiding the formation of the target imprint within the matrix.

[0071] Each step of the sensor preparation was characterized by cyclic voltammetry (CV) in the potential range of 0.2 to 0.2 at a scan rate of 100 mV/s and square wave voltammetry (SWV) at a potential range of 0.2 to 0.2 V, pulse amplitude of 12.5 mV, frequency of 10 Hz, and a step potential of 5 mV in 1 M KCl solution containing 4 mM redox probe K.sub.3[Fe(CN).sub.6]/K.sub.4[Fe(CN).sub.6].

Evaluation of the Sensor Performance

[0072] All the samples were prepared in a lysis buffer (LB) (pH=7.2) containing 27.5 mM of Tris-HCl, 12.5 mM of EDTA, 1.5% of Triton X-100 (V/V), and 0.1% SDS diluted with MQ water to the desired volume and stored at 4 C. The lysis buffer can be prepared by using buffering compound replacing Tris-HCl, like phosphate, acetate, citrate, carbonate, Bis-Tris, HEPES, MOPS or other systems known to skilled artisans. The EDTA can be replaced by EGTA or citrate or other chelating agents. The Triton X-100 and SDS can be replaced by other detergents, like nonidet P-40, Tween, IGEPAL or other detergent compounds. The LB did not contain a strong chaotropic agent such as guanidinium chloride in order to minimally affect the native conformations of antigen.

[0073] The rebinding of ncovNP or ncovS1 on the prepared sensors was conducted by means of DPV (differential pulse voltammetry) or SWV (square wave voltammetry). The measurements were performed in the potential range of 0.2 to 0.2 V with the pulse amplitude of 0.025 V, pulse time of 0.025 s, step potential of 0.005 V and scan rate 0.1 V/s in 1 M KCl solution containing 4 mM redox probe K.sub.3[Fe(CN).sub.6]/K.sub.4[Fe(CN).sub.6] at room temperature. The response (I.sub.n) of the sensor was calculated as follows:

I.sub.n=(I.sub.0I)/I.sub.0 (1)

where I.sub.0 and I represent the DPV or SWV anodic peak currents measured after incubation in sample solutions with and without the target analyte (ncovNP or ncovS1), respectively.

[0074] The adsorption isotherm was generated by plotting I.sub.n against the respective concentration of ncovNP or ncovS1 in the measured sample solution. The response at the maximal adsorption, I.sub.n.sat, was derived by fitting the isotherm to the Langmuir adsorption model (eq. 2):

I.sub.n=I.sub.n.satC/(C+K.sub.D) (2)

where, C is the analyte concentration, K.sub.D is the dissociation constant.

[0075] The optimal thickness of the sensing layer (ncovNP-MIP or ncovS1-MIP) in the sensor was elucidated after calculation of the molecular imprinting effect or the imprinting factor (IF) for every pair of ncovNP-MIP or ncovS1-MIP- and NIP-modified sensors characterized by the same thicknesses:

IF=I.sub.n.sat(MIP/I.sub.n.sat(NIP) (3)

where, I.sub.n.sat(MIP) and I.sub.n.sat(NIP) depict I.sub.n.sat responses of ncovNP-MIP or ncovS1-MIP- and NIP-modified sensors at saturation. Further, the sensors with the optimal thickness were used to determine their analytical performance in prepared and real samples solution.

[0076] The selectivity of the sensor was assessed by comparing the responses against the target (i.e. ncovNP or ncovS1) and interfering analytes (i.e., BSA, HSA, E2 HCV, IgG, and CD48) at equivalent concentrations.

[0077] Limit of Detection (LOD), and Limit of Quantitation (LOQ) of the sensors were derived from linear regression of their calibration plots obtained in LB or PBS and with for example COVID-19-negative clinical samples (see below) containing the known concentration of ncovNP or ncovS1:

LOD=3.Math.SD/b (4)

LOD=10.Math.SD/b (5)

where SD and b represents the standard deviation and the slope of the regression line, respectively. LOD and LOQ values can be similarly obtained for any sample of interest, including environmental samples (wastewater) or laboratory samples. As an example of clinical samples solutions of nasopharyngeal specimens in UTM (universal transport medium) from four COVID-19 negative and four COVID-19 positive patients were obtained from the SYNLAB Eesti medical laboratory (Estonia). Similarly, the clinical samples could be saliva, blood, urine, or stool samples collected by standard procedures and prepared into lysis buffer as follows. The presence or absence of the viral infection in the clinical samples was confirmed with RT-PCR method. Before the analysis, the samples were 20-fold diluted with LB by vortexing for 30 min to facilitate the viral protein release as well as to decrease the concentration of interfering species present in UTM. The samples from four COVID-19-negative patients were spiked with ncovNP at a concentration range of 22.2 -333 fM and used to construct the calibration plot and derive LOD and LOQ values.

[0078] As another example, the clinical samples obtained from SYNLAB Eesti medical laboratory (Estonia) consisted of nasopharyngeal specimens of three negative and five positive COVID-19 patients in sample preservation solution (SPS), (Jiangsu Mole Bioscience Co., Ltd). Their COVID-19 status was previously confirmed with the RT-PCR method. The samples were 100 fold diluted in PBS i.e., 1:99 (SPS:PBS). Calibration plot was plotted using the diluted negative samples spiked with an increasing concentration (0-400 fM) of ncovS1 and the LOD and LOQ values were derived. Reading DPV or SWV signals from ncovNP sensors was performed by electrochemical workstation (Reference 600TM, Gamry Instruments, USA) or a portable potentiostat (EmStat3 Blue and Sensit Smart, PalmSens BV, The Netherlands).

Preparation and Optimization of the Sensor

[0079] The ncovNP-MIP synthesis strategy is based on the electrochemical surface imprinting [25,29,31]. All the fabrication steps were monitored by CV (cyclic voltammogram) measurements (FIG. 2). It can be seen that while the modification of Au-TFE by 4-ATP monolayer almost did not affect anodic/cathodic current peaks of the redox couple indicating that this short thiol was too thin to effectively block electron transfer taking place on the electrode surface, on the other hand, after the subsequent attachment ncovNP via DTSSP, the redox current peaks were significantly reduced. Surprisingly, it was still possible to initiate PmPD growth at a rather low potential (0.6 V vs Ag/AgCl/KCl) comparable with that at which the similar reaction happens at a bare Au-electrode (FIG. 11). The redox peaks completely disappeared after PmPD electrodeposition indicating non-conducting polymer film formation. However, a treatment of the modified electrode in mercaptoethanol and acetic acid caused a prominent increase in the peak current indicating supposedly, elution of ncovNP from the PmPD matrix and formation of molecular cavities, which tunneled the charge transfer across the ultra-thin non-conducting PmPD layer to the electrode. Similar effects were observed during the preparation of ncovS1-MIP sensor.

[0080] Since the present imprinting approach allows the generation of macromolecular imprints situated at/or close to the polymer film surface, the deposition of polymer with an appropriate film thickness is of crucial importance in order to avoid irreversible entrapment of a protein and infeasibility of its removal during the subsequent washing out procedure. Another reason to optimize the thickness of PmPD or PABA is to provide sufficient change transfer through the imprints to the electrode for following responsive DPV sensing of protein rebinding on the resulting ncovNP-MIPs or ncov S1-MIPs.

[0081] In order to determine the thickness effect of the sensing layer on the sensor, the charge densities of 1, 2, and 3 mC/cm.sup.2 were imposed to generate PmPD for building ncovNP-MIP. The resulting ncovNP sensors having the different thicknesses of ncovNP-MIP were assessed in terms of their capability to rebind ncovNP from LB solutions determining IFs (Eq. 3) from analysis of the respective adsorption isotherms (FIG. 12). The appropriate thickness of ncovNP-MIP for building ncovNP sensor was determined as the thickness at which the sensor demonstrated the highest IF. As it can be seen the most pronounced imprinting effect (IF=5.8) was achieved in the case of PmPD film deposited by 2 mC/cm.sup.2 (FIG. 3). Apparently, with this charge, the polymer generated at the surface of electrode confines the anchored ncovNPs, but does not hinder their successful extraction during ncovNP washing procedure. Thus, the resulting polymer is endowed with the ncovNP-selective molecular cavities capable to transduce ncovNP binding events showing the responsive inhibition of charge transfer to the modified electrode. In case of ncovS1-MIP cyclic voltammetry was used to synthesize PAPBA, charge of 6.1 mC/cm.sup.2 gave an optimal polymer.

[0082] Rebinding time was another parameter that was taken into consideration. The sensor was incubated in the range of 5 and 60 minutes with LB solution containing 0.1 pM ncovNP. As it can be seen the sensor response became saturated and reached equilibrium after 15 minutes (FIG. 4). Therefore, 15 minutes was selected as the optimal time to rebind ncovNP at the surface of ncovNP sensor. Optimization of rebinding time of ncovS1 on ncovS1 was carried out by recording the responses after incubation in PBS containing 0.05 ng/mL (0.67 pM) ncovS1.

Performance of the Sensor

[0083] The calibration plot of ncovNP sensor against ncovNP in LB depicts the pseudo-linear increase of the sensor response with ncovNP concentration up to 111 fM (FIG. 5A). The calculated LOD and LOQ values were 15 and 50 fM (0.7-2.2 g/mL), respectively that fell well within the range of ncovNP level present in real samples of COVID-19 patients [9,32,33]. Similar calibration plot in PBS was obtained for ncov S1 (27-194 fM) (FIG. 5B).

[0084] The selectivity of the ncovNP sensor against ncovNP was explored by evaluating its ability to discriminate between target and interfering proteins such as S1 (75 kDa, pI 6.0), E2 HCV (47 kDa, pI 8.2), CD48 (22 kDa, pI 9.3), and BSA (66 kDa, pI 4.7). Selectivity of ncovS1 sensor was studied against increasing concentrations (40, 60, 80, 100, and 120 fM) of different proteins (ncovNP, HSA, E2, IgG and S1) in PBS The selection of these proteins as interfering proteins was based on their size, isoelectric point, molecular weight, and possible presence in real samples. Thus, CD48 was selected due to its close isoelectric point and smaller molecular mass, E2 HCVas a protein having molecular weight close to ncovNP, S1, being a subunit of SARS-Cov-2 spike protein, can be present in the real clinical samples along with ncovNP. As it can be seen, the response of ncovNP sensor was considerably higher against ncovNP as compared to the responses against the interfering proteins demonstrating thus the appreciable selectivity of the fabricated device towards ncovNP (FIG. 6) and promising uncompromised performance in clinical samples.

[0085] Table 2 below shows physical characteristic of the interfering proteins that were used to study the selectivity of the device.

TABLE-US-00004 Protein Code in Molecular weight, Molecular volume, Isoelectric Name PDB kDa .sup.3 point CD48 2PTV 22 16853 9.3 BSA 3V03 66 48160 4.7 E2 4MWF 47 99263 8.24 HSA 1E78 67 47902 4.7 IgG 1HZH 152 223482 8.8 ncovNP 6VYO 45 85268 10.07 ncovS1 6VXX 75 502733 6.04

Sensor Performance and Stability in Clinical Samples

[0086] Performance of ncovNP sensor in clinical samples was assessed by samples prepared from nasopharyngeal swab specimens of patients as example. Samples can be originating from human source, obtained by standard clinical swab sampling techniques from mucous membranes, or from blood, urine or saliva, obtained by standard clinical sampling methods, or the samples can be obtained from environmental source, like sewage water or air to liquid sedimentation sampler, or the samples can be obtained from laboratory source, like recombinant protein expression or purification samples. For measuring the samples ncovNP or ncovS1 sensor can be used to detect NP or S1 antigens respectively.

[0087] In the example, first, the sensor was calibrated in COVID-19 negative samples spiked with known concentrations of ncovNP (FIG. 7A). The sensor showed a pseudo-linear response versus ncovNP concentration in the range of 0.22-333 fM. LOD (27 fM) and LOQ (90 fM), were obtained by linearly regressing the averaged data of four COVID-19 negative patients. Thus, one can suppose that the patient' samples producing I.sub.n exceeding the values of 0.22 a.u. (corresponds to LOD) can be considered as COVID-19 positive. Moreover, the sensor demonstrated appreciable selectivity to ncovNP, since its response was almost insensitive to addition of S1 in the COVID-19 negative sample, but raised immediately after ncovNP was spiked to the sample (FIG. 8A). These results promise that capability of the sensor to respond towards ncovNP will not be much disturbed in COVID-19 positive samples, where the other viral proteins can be presented. Second, the sensor was calibrated in COVID-19 negative samples spiked with known concentrations of ncovS1 in the range 50-400 fM (FIG. 7B). LOD and LOQ were determined as 64 fM and 213 fM respectively. The selectivity of the sensor was further studied in COVID-19 negative samples by spiking the sample with varying concentrations of either ncovS1, ncovNP or their mixture. The concentrations of both proteins were selected to simulate their concentration ratio, 1:10 (ncovSl:ncovNP) in SARS-CoV-2 virus [32]. As observed in FIG. 8B, the responses induced on the sensor by the increasing concentration of ncovNP are below the LOD (64 fM) indicating no recognition for ncovNP. Whereas, the sensor demonstrates remarkably increasing responses, above the LOD, to ncovS1 concentrations at tenfold lower values compared to that of ncovNP. Moreover, the responses induced by the mixture of both proteins, ncovS1:ncovNP (1:10) are comparable to that from ncovS1 thus indicating that the presence of ncovNP in the sample would not interfere, to any significant extent, with the sensor specific recognition of ncovS1 thereby enabling its accurate analysis.

[0088] To further elucidate the sensor selective recognition for the target it was tested in the samples prepared by spiking the desired amount of S1 protein of different mutated strains of the SARS-CoV-2 virus such as S1 UK VOC 202012/01, S1 Brazil P1, S1 South Africa VOC 501.V2 to COVID-19 negative samples. As seen in FIG. 8C, the sensor demonstrated the highest response for the imprinted target (ncovS1) at both concentrations. Although all strains induced a concentration-dependent sensor response, their responses, especially at the lower concentration, are analytically not significant since they fall below or are comparable to the estimated sensor's detection limit, thus highlighting the sensor's preference for ncovS1 against other strains

[0089] To explore the feasibility to use the sensors for COVID-19 diagnostics, they were tested against the RT-PCR confirmed COVID-19 samples. As it can be seen, four COVID-19 positive samples, obtained from the laboratory, and tested by ncovNP sensor caused the responses, In, higher than those against of the COVID-19 negative samples containing no ncovNP (y-intercept, 0.172 a.u.) or at least containing ncovNP at LOD (0.22 a.u.) (Table 3, FIGS. 9A-B).

TABLE-US-00005 TABLE 3 Comparison of measurements made by ncovNP-MIP sensor and RT-PCR for COVID-19 positive nasopharynx swab specimens. Patient ncovNP sensor response ncovNP conc. number (ct*) (I.sub.n)(a.u) (fM) Patient 5 (27) 0.254 45 11 Patient 6 (27) 0.331 90 11 Patient 7 (25) 0.466 168 11 Patient 8 (22) 0.544 214 12 *cycle threshold value as determined by RT-PCR

[0090] It should be noted, samples of patient 6, patient 7 and patient 8, produced I. (0.331, 0.466 and 0.544 a.u.) corresponding to that caused ncovNP at concentrations higher or close to LOQ (0.33 a.u.), while I.sub.n against the sample of patient 5 was higher than at LOD (0.22 a.u.). Despite the cycle threshold (Ct) values from RT-PCR cannot be directly interpreted as viral load still we observed a correlation between them and I.sub.n of the sensor: the lower Ct the higher I.sub.n. It is clear that the sensor is responding to the presence of viral ncovNP in COVID-19 positive samples and demonstrated thus its potential in development express tests for COVID-19.

[0091] Five COVID-19 positive samples which had been previously validated by RT-PCR were tested with the ncovS1 sensor. FIG. 9A clearly shows that all five samples effected responses that are higher (In =0.4, 0.36, 0.34, 0.37, 0.38) than LOD (In=0.32), Table 4. While samples from patients 5 and 7 produced responses close to the LOQ value, samples of patients 4 and 8 show higher responses than LOQ. The sample from patient 6 caused the lowest response, which still established a significant distinction from the LOD. Moreover, it is worthy to note that the negative samples are below the LOD. This affirms that the increased responses received from the positive patients are due to the presence of ncovS1 in the samples.

TABLE-US-00006 TABLE 4 ncovS1 sensor responses (I.sub.n) to COVID-19 positive samples and the associated ncovS1 concentration. Patient ncovS1 sensor response ncovS1 conc. number (ct*) (I.sub.n)(a.u) (fM) Patient 4 (16) 0.40 0.02 280 20 Patient 5 (26) 0.36 0.01 170 10 Patient 6 (19) 0.34 0.01 120 10 Patient 7 (12) 0.37 0.02 200 20 Patient 8 (10) 0.38 0.01 220 10 *cycle threshold value as determined by RT-PCR

EXAMPLE: THE SENSORS HAVE AT LEAST 9 WEEKS SHELF LIFE

[0092] Finally, to examine the stability of the sensors, twelve (12) sensors were prepared and periodically, once a week, tested by the LB diluted COVID-19 negative samples spiked with ncovNP at concentration of 66.6 fM. The finding confirms that the response (I.sub.n) of the as-prepared sensors remained the same after up to 9 weeks of storage, which reveals that ncovNP sensors have excellent long term stability (FIG. 10) i.e. it has a long shelf life.

CONCLUSION

[0093] In this invention, we have developed for the first time a portable electrochemical sensor integrated with a molecular imprinted polymer (ncovNP-MIP and ncovS1-MIP) as a synthetic recognition element capable of selective detection of SARS-CoV-2 antigen (ncovNP or S1 respectively). The synthesis parameters of sensors were optimized and the ability of the prepared sensor to selectively rebind the antigen was demonstrated. As an example, the sensor demonstrated a good linearity and reproducibility in COVID-19 negative clinical samples spiked with ncovNP in the concentration range 0.22-333 fM that resulted in a LOD and LOQ values sufficient to determine the presence of ncovNP in COVID-19 positive samples of nasopharynx swab specimens. In addition, the sensor demonstrated a good linearity and reproducibility in COVID-19 negative clinical samples spiked with ncovS1 in the concentration range 50-400 fM that resulted in a LOD and LOQ values sufficient to determine the presence of ncovNP in COVID-19 positive samples of nasopharynx swab specimens. It is foreseen that the sensor could be used in detecting COVID-19 positive samples of other complex biological specimens.

[0094] The developed sensor that relies on a completely different approach as compared to the currently available SARS-CoV-2 antigen tests, could represent a valuable alternative as a portable diagnostic platform for the rapid screening for COVID-19.

[0095] It will be readily understood by one of ordinary skill in the relevant art that the present invention has broad utility and application. Although the present invention has been described and illustrated herein with referred to certain embodiments, it will be apparent to those of ordinary skill in the art that other embodiments may perform similar functions and/or achieve like results, and that the described embodiments are for illustrative purposes only. Thus, it should be understood that various features and aspects of the disclosed embodiments can be combined with, or substituted for one another in order to form varying modes of the disclosed invention. Many different embodiments such as, variations, adaptations, modifications, and equivalent arrangements are implicitly and explicitly disclosed by the embodiments described herein, and thus fall within the scope and spirit of the present invention.

[0096] Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, product or use of the invention, and vice versa.

[0097] Further, the discussed prior art is not an admission by Applicant and should not be construed that the current invention does not antecede and is not patentable over the discussed prior art, but has merely been presented to better define the knowledge in the field to a skilled artisan and to the reader in general.

ACKNOWLEDGEMENT

[0098] This research was supported by Estonian Research Council grants: COVSG34 and PRG307.

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