SARS-CoV-2 Detection

Abstract

The molecular imprinted polymer comprises at least one recognition site complementary to a template molecule consisting of an amino acid sequence corresponding to a partial sequence of the receptor binding domain of the SARS-CoV-2 spike protein, the amino acid sequence being 50 amino acids or less in length and comprising a sequence selected from (i) NSNNLDSKVGG, (ii) NYNYLYRLFRKS, (iii) YRLFRKSNLKPF, (iv) STEIYQAGSTPC, (v) CNGVEGFNCYF, (vi) GSTPCNGVEGF, (vii) CYFPLQSYGFQP, (viii) GFQPTNGVGYQ, and (ix) LQSYGFQPTNG. A method for preparing the molecular imprinted polymer is also provided. A complex comprising a molecular imprinted polymer and a fluorescent substance is also provided, as are compositions comprising the molecular imprinted polymer and complex of the present invention. The molecular imprinted polymer, complex, and composition of the present invention can be used to detect SARS-CoV-2.

Description

[Technical field]

[0001] The present invention relates to compounds for detecting SARS-CoV-2, methods for their preparation and complexes containing them, as well as compositions for detecting SARS-CoV-2, and more particularly to the use of the aforementioned in detecting SARS-CoV-2. [Background technology]

[0002] The 2019 novel coronavirus, or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), outbreak has led to a pandemic and resulted in both a global public health and economic crisis.

[0003] The international community quickly recognized that both diagnostic and antibody tests are key to preventing further transmission and understanding the morbidity of the virus, respectively, but the demand for reliable, cheap to produce and easy to use test kits remains high.

[0004] Regarding diagnostic testing, reverse transcription polymerase chain reaction (RT-PCR)-based tests are currently the primary means used by health care providers to diagnose the public. RT-PCR tests work by detecting viral ribonucleic acid (RNA) in respiratory samples, often obtained by nasopharyngeal swabs.

[0005] Although RT-PCR testing is widespread, it is problematic. Implementing an RT-PCR test can be labor intensive. The test requires highly skilled staff and can produce false negative results, for example due to sampling error or improper handling of samples. In practice, false positive results can also occur, for example due to cross-contamination of samples. The test is also expensive and has a long turnaround time for results (1-2 days).

[0006] Rapid antigen tests are also used for mass screening, offering a portable and rapid (15 to 30 minutes) assay that can be performed at an individual's convenience. However, their widespread use is limited by their low sensitivity and the fact that they use antibodies as receptors and therefore only function over a narrow temperature and pH range.

[0007] Molecularly imprinted polymers (MIPs) have been proposed as alternative receptors for the detection of SARS-CoV-2.

[0008] In "Proof of Concept for a Quick and Highly Sensitive On-Site Detection of SARS-CoV-2 by Plasmonic Optical Fibers and Molecularly Imprinted Polymers," Cennamo et al. detail the fabrication of a plastic optical fiber (POF)-based surface plasmon resonance (SPR) sensor coupled with an MIP. The MIP was fabricated against the S1 subunit of the spike protein of SARS-CoV-2.

[0009] In “Monoclonal-type plastic antibodies for SARS-CoV-2 based on Molecularly Imprinted Polymers,” Parisi et al. describe the preparation of MIPs using the receptor binding domain (RBD) of SARS-CoV-2 as a template.

[0010] In "Computational analysis of functional monomers used in molecular imprinting for promising COVID-19 detection," Cubuk et al. describe a computational study aimed at identifying templates and suitable monomers for fabricating MIPs against SARS-CoV-2.

[0011] WO 2021 / 195626(A1) discloses the preparation of MIPs against SARS-CoV-2 using the whole virus as a template.

[0012] Methods for making MIPs are also known, see, e.g., U.S. Patent 10,189,934 (B2), Piletsky et al., "Molecular Imprinted Polymers for Cell Recognition," and Malik et al., "Molecular Imprinted Polymers for human viral pathogen detection."

[0013] Whether using the whole virus, the entire S1 subunit, or the entire RBD of SARS-CoV-2 to manufacture MIPs, significant scalability issues arise.

[0014] In contrast to RT-PCR and rapid antigen tests, which are performed to prove whether a person is currently infected with a virus, antibody tests are performed to prove whether a person has ever been infected with the virus. Antibody tests are often performed on a patient's blood sample and work by detecting the presence of a bond between a synthetic antigen and antibodies (if present) in the patient's blood sample. Summary of the Invention [Problem to be solved by the invention]

[0015] Therefore, one object of the present invention is to provide an alternative means for detecting SARS-CoV-2. A further object is to provide such an alternative to currently used polymerase chain reaction-based assays and rapid antigen tests. Objects of specific embodiments of the present invention include providing improved detection means. [Means for solving the problem]

[0016] Thus, the present invention provides a molecular imprinted polymer comprising at least one recognition site complementary to a template molecule consisting of an amino acid sequence corresponding to a subsequence of a receptor binding domain of SARS-CoV-2 spike protein, the amino acid sequence being 50 amino acids or less in length; (i) NSNNLDSKVGG; (ii) NYNYLYRLFRKS, (iii) YRLFRKSNLKPF, (iv) STEIYQAGSTPC, (v) CNGVEGFNCYF, (vi) GSTPCNGVEGF, (vii) CYFPLQSYGFQP, (viii) GFQPTNGVGYQ, and (ix)LQSYGFQPTNG The present invention includes a sequence selected from the following:

[0017] The present invention also provides a method for preparing a molecularly imprinted polymer comprising at least one recognition site for binding to SARS-CoV-2, the method comprising: (a) preparing a carrier material having a template molecule having an amino acid sequence corresponding to a subsequence of a receptor binding domain of the SARS-CoV-2 spike protein, the template molecule being immobilized on the carrier material such that the template molecule is exposed on the surface of the carrier material; (b) contacting a polymerizable composition with a surface; (c) controlling polymerization of the polymerizable composition in contact with the surface to produce a molecularly imprinted polymer; (d) detaching the molecularly imprinted polymer from the surface, the amino acid sequence is 50 amino acids or less in length, (i) NSNNLDSKVGG; (ii) NYNYLYRLFRKS, (iii) YRLFRKSNLKPF, (iv) STEIYQAGSTPC, (v) CNGVEGFNCYF, (vi) GSTPCNGVEGF, (vii) CYFPLQSYGFQP, (viii) GFQPTNGVGYQ, and (ix)LQSYGFQPTNG The present invention includes a sequence selected from the following:

[0018] Further provided is a composite comprising a molecularly imprinted polymer of the present invention and a fluorescent substance.

[0019] Still further, there is provided a composition comprising a molecularly imprinted polymer or composite of the invention.

[0020] The molecularly imprinted polymer of the present invention, the conjugate of the present invention, and the composition of the present invention can all be used for the detection of SARS-CoV-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention provides a molecular imprinted polymer comprising at least one recognition site complementary to a template molecule having an amino acid sequence corresponding to a subsequence of a receptor binding domain of a SARS-CoV-2 spike protein, the amino acid sequence being 50 amino acids or less in length; (i) NSNNLDSKVGG; (ii) NYNYLYRLFRKS, (iii) YRLFRKSNLKPF, (iv) STEIYQAGSTPC, (v) CNGVEGFNCYF, (vi) GSTPCNGVEGF, (vii) CYFPLQSYGFQP, (viii) GFQPTNGVGYQ, and (ix)LQSYGFQPTNG The present invention includes a sequence selected from the following:

[0022] In molecular imprinting, functional monomers (and possibly crosslinking monomers) are polymerized in a suitable solvent in the presence of the compound to be imprinted. The compound to be imprinted is called the template. During the polymerization process, the monomers interact with the template by electrostatic, hydrophobic or other interactions, which results in the formation of binding sites, called recognition sites, in the polymer that are complementary to the template molecule. After the template is removed, the polymer matrix retains the recognition sites that are complementary to the template. Complementary in this sense means that the interacting groups in the polymer are positioned in a way that makes them suitable for interacting with the interacting groups in the template molecule and other molecules that contain the template molecule as a substructure (i.e., in the present invention, SARS-CoV-2).

[0023] The sequence of SARS-CoV-2 is known. SARS-CoV-2 uses the densely glycosylated spike protein to enter host cells. The spike protein is a homotrimeric glycoprotein, with each monomer containing two subunits, S1 and S2, which mediate membrane attachment and membrane fusion, respectively. The receptor binding domain (RBD) of the S1 subunit has been shown to be involved in host cell receptor ligation.

[0024] Although the sequence of the RBD of the SARS-CoV-2 spike protein is known and theoretically any sequence therein can be used as a template, in some downstream applications it is advantageous to select a non-conserved sequence within the target as the template, which ensures that the molecularly imprinted polymer binds to the desired target with high specificity.

[0025] Wrapp et al., 2020 1Supplementary Figure 5 in shows a sequence alignment of the spike protein of SARS-CoV-2 with the spike proteins of two other coronavirus strains, SARS-CoV and RaTG13, with the RBD highlighted, as well as sequences that are conserved and not conserved between the different strains.

[0026] In the present invention, the "at least one recognition site" in the molecular imprinted polymer is complementary to a template molecule consisting of an amino acid sequence corresponding to a partial structure of the receptor binding domain of the SARS-CoV-2 spike protein, the amino acid sequence is 50 amino acids or less in length, and includes a sequence selected from (i) NSNNLDSKVGG, (ii) NYNYLYRLFRKS, (iii) YRLFRKSNLKPF, (iv) STEIYQAGSTPC, (v) CNGVEGFNCYF, (vi) GSTPCNGVEGF, (vii) CYFPLQSYGFQP, (viii) GFQPTNGVGYQ, and (ix) LQSYGFQPTNG.

[0027] The "at least one recognition site" in the molecular imprinted polymer is complementary to a template molecule consisting of an amino acid sequence corresponding to a partial structure of the receptor binding domain of the SARS-CoV-2 spike protein, and preferably the amino acid sequence is 50 amino acids or less in length and includes a sequence selected from (i) NSNNLDSKVGG, (ii) STEIYQAGSTPC, and (iii) CYFPLQSYGFQP.

[0028] In a particularly preferred embodiment, the "at least one recognition site" in the molecularly imprinted polymer is complementary to a template molecule consisting of an amino acid sequence corresponding to a substructure of the receptor binding domain of the SARS-CoV-2 spike protein, the amino acid sequence being 50 amino acids or less in length and comprising the sequence NSNNLDSKVGG.

[0029] In a further particularly preferred embodiment, the "at least one recognition site" in the molecularly imprinted polymer is complementary to a template molecule consisting of an amino acid sequence corresponding to a substructure of the receptor binding domain of the SARS-CoV-2 spike protein, the amino acid sequence being 50 amino acids or less in length and comprising the sequence CYFPLQSYGFQP.

[0030] In the present invention, the size of the template ranges up to 50 amino acids. Preferably, the template is shorter than 50 amino acids, more preferably, the template is shorter than 30 amino acids, and most preferably, the template is shorter than 20 amino acids, for example, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, or 19 amino acids.

[0031] The use of short peptide sequences (as opposed to, for example, the entire spike protein or the entire RBD) significantly reduces reagent costs and also creates high affinity binding sites for specific regions of the target protein, thereby improving the monoclonality of the molecularly imprinted polymer.

[0032] The size of the molecularly imprinted polymers of the present invention will be determined to a large extent by the intended downstream use of the molecularly imprinted polymer, however, preferably the molecularly imprinted polymer is less than 500 nm, more preferably less than 250 nm, even more preferably less than 100 nm, and may be less than 90 nm or even less than 70 nm.

[0033] The present invention also provides a composition comprising the molecularly imprinted polymer of the present invention. Preferably, the molecularly imprinted polymer in such composition has an average size of less than 500 nm, more preferably less than 250 nm, even more preferably less than 100 nm, and may be less than 90 nm or even less than 70 nm.

[0034] Suitable methods for determining the size of molecularly imprinted polymers include dynamic light scattering, such as with a Zetasizer Ultra (Malvern Panalytical), disk centrifugation, nanoparticle tracking analysis, such as with a NanoSight NS300 (Malvern Panalytical), tunable resistive pulse sensing, atomic force microscopy, and electron microscopy. Nanoparticle tracking analysis, tunable resistive pulse sensing, atomic force microscopy, and electron microscopy are particularly suitable for determining the size of single molecularly imprinted polymers. In the examples shown herein, the size of the molecularly imprinted polymers was determined using a NanoSight NS300 instrument.

[0035] Size in relation to molecularly imprinted polymers refers to the diameter of the molecularly imprinted polymer.

[0036] Monomers that can be used to prepare molecularly imprinted polymers include vinyl monomers, allylic monomers, acetylenes, acrylates, methacrylates, acrylamides, methacrylamides, chloroacrylates, itaconates, trifluoromethylacrylates, derivatives of amino acids (e.g., esters or amides), nucleosides, nucleotides, and carbohydrates. Crosslinking monomers that help stabilize the molecularly imprinted polymer may be included. Typical examples of crosslinking monomers suitable for preparing molecularly imprinted polymers include, but are not limited to, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, divinylbenzene, methylenebisacrylamide, ethylenebisacrylamide, N,N'-bisacryloylpiperazine, and N,N'-methylenebis(acrylamide) (BIS). A person skilled in the art would be able to select monomers and crosslinking monomers suitable for a particular template. Alternatively, various combinatorial computational methods could be used to assist in this selection.

[0037] In a preferred embodiment of the present invention, the molecular imprinted polymer comprises at least one monomer selected from the group consisting of (i) N-fluoresceinyl acrylamide, (ii) acrylamide, (iii) tert-butyl acrylate (TBAc), (iv) 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose, (v) 2,2,2-trifluoroethyl methacrylate (CF3), (vi) N-(3-aminopropyl) methacrylamide hydrochloride (APMA), (vii) squaramide monomer 7 (SQ7), and (viii) N,N'-methylenebis(acrylamide) (BIS). More preferably, the molecular imprinted polymer comprises at least N-fluoresceinyl acrylamide and / or 3-O-acryloyl-1,2;5,6-bis-O-isopropylidene-D-glucofuranose. Most preferably, the molecular imprinted polymer comprises all of (i) to (viii). In this context, it is understood that a molecularly imprinted polymer is a polymer and is said to contain a specified monomer if it is produced by polymerizing a mixture of monomers that contain the specified monomer.

[0038] Advantageously, the molecularly imprinted polymers of the present invention are robust and function over a wide range of temperatures and pH, which translates into improved shelf life and storage conditions and means they can function in more challenging environments. They also have excellent biocompatibility and fast binding kinetics.

[0039] The present invention also provides a method for preparing a molecularly imprinted polymer comprising at least one recognition site for binding to SARS-CoV-2, the method comprising: (a) preparing a carrier material having a template molecule having an amino acid sequence corresponding to a subsequence of a receptor binding domain of the SARS-CoV-2 spike protein, the template molecule being immobilized on the carrier material such that the template molecule is exposed on the surface of the carrier material; (b) contacting a polymerizable composition with a surface; (c) controlling polymerization of the polymerizable composition in contact with the surface to produce a molecularly imprinted polymer; (d) detaching the molecularly imprinted polymer from the surface, the amino acid sequence is 50 amino acids or less in length, (i) NSNNLDSKVGG; (ii) NYNYLYRLFRKS, (iii) YRLFRKSNLKPF, (iv) STEIYQAGSTPC, (v) CNGVEGFNCYF, (vi) GSTPCNGVEGF, (vii) CYFPLQSYGFQP, (viii) GFQPTNGVGYQ, and (ix)LQSYGFQPTNG The present invention includes a sequence selected from the following:

[0040] The advantage of this method is that MIPs have a more uniform binding site affinity distribution, providing a means to improve template elution and prevent template leaching downstream.

[0041] Suitable support materials include polymeric resins, polysaccharides, glass, or metal surfaces.

[0042] The support material may be in the form of beads, fibers, membranes, capillaries, etc. In a preferred embodiment of the invention, the support material is glass. More preferably, the support material is in the form of glass beads.

[0043] The polymerizable composition must contain the monomers necessary for polymerization. Suitable monomers are described elsewhere herein.

[0044] Step (c) involves the application of controlled polymerization.Various techniques are known to those skilled in the art for preparing molecularly imprinted polymers by controlled polymerization.

[0045] Examples of controlled polymerization include radical polymerization, such as controlled living radical polymerization (LRP), living anionic polymerization, living cationic polymerization, and controlled condensation polymerization.

[0046] Polymerization can be initiated, for example, by heating, application of electric current (electropolymerization), addition of a redox catalyst(s), persulfate, or peroxide, irradiation including gamma radiation or microwave radiation, or irradiation with ultraviolet or visible light, and typically takes from minutes to hours. In the examples described herein, polymerization was initiated using persulfate.

[0047] In a preferred embodiment of the present invention, the polymerization reaction is terminated at a stage where the size of the synthesized molecularly imprinted polymer is relatively small, the size settings of which are described elsewhere in this specification.

[0048] Once the molecularly imprinted polymer is synthesized, it must be separated and recovered. Separation of the high affinity molecularly imprinted polymer from the immobilized template can be accomplished by heating to disrupt the complexation, by changing the pH of the solution, by changing the ionic strength, or by adding urea, guanidine, or other substances that interact more strongly with the template than the molecularly imprinted polymer.

[0049] In a preferred embodiment of the method, weakly bound materials are removed by washing prior to the separation step.

[0050] Once the molecularly imprinted polymer is recovered, it may be further purified, for example, by chromatography, filtration, and / or electrophoresis.

[0051] For the reasons given above in connection with the molecularly imprinted polymers of the invention, in a preferred embodiment of the method of the invention, the amino acid sequence is 50 amino acids or less in length and comprises a sequence selected from (i) NSNNLDSKVGG, (ii) STEIYQAGSTPC, and (iii) CYFPLQSYGFQP. In a particularly preferred embodiment, the amino acid sequence is 50 amino acids or less in length and comprises the sequence NSNNLDSKVGG. In a further particularly preferred embodiment, the amino acid sequence is 50 amino acids or less in length and comprises the sequence CYFPLQSYGFQP.

[0052] The method of immobilizing template molecule on carrier material is not critical.However, for some methods, it is advantageous for template molecule to have a cysteine ​​residue at one of its termini.Therefore, for those method embodiments in which template molecule does not originally contain a terminal cysteine ​​residue, it is preferred to modify the N-terminus of template molecule to contain an additional cysteine ​​residue.

[0053] In some embodiments of the invention, the additional cysteine ​​residue is added directly to the end of the template molecule, while in other embodiments a spacer is included between the terminal cysteine ​​and the template molecule, hi a preferred embodiment, the spacer comprises a glycine residue.

[0054] Thus, in a particularly preferred embodiment of the invention, the template molecule comprises the amino acid sequence NSNNLDSKVGG and is N-terminally modified to contain a cysteine ​​(C) and a glycine (G) residue such that the molecule used for imprinting is CGNSNNLDSKVGG.

[0055] In some embodiments of the invention, the template molecule is directly attached to the carrier material, while in other embodiments, one or more linkers are included between the carrier material and the template molecule. Suitable linkers for use in the present invention are well known to those skilled in the art. In the examples shown herein, the carrier material used was glass beads, which were silanized with (3-aminopropyl)trimethoxysilane (APTMS) prior to immobilization of the template molecule. APTMS serves as a linker between the surface of the glass beads and the template molecule. In the examples shown herein, APTMS was modified with n-succinimidyl iodoacetate (SIA) prior to immobilization of the template.

[0056] The linkers and spacers function to prevent adverse effects caused by steric hindrance.

[0057] The advantages of the method of the present invention are that it is fast, scalable and uses only short templates.

[0058] The present invention further provides a composite comprising a molecularly imprinted polymer of the present invention and a fluorescent substance.

[0059] Fluorescent materials are molecules that have the ability to absorb light at a particular wavelength, the absorption or excitation wavelength, and subsequently emit light at a higher wavelength, the emission wavelength.

[0060] Attaching fluorescent substances to molecules for visualization purposes is common in the art. Thus, methods for such attachment are also well known. For example, fluorescent substances can be attached via amino groups, thiol groups, or carbohydrate groups. In the examples shown herein, fluorescent substances were attached to the molecular imprinted polymer via amine groups provided by the amino monomers of the molecular imprinted polymer.

[0061] While any fluorescent material can be used in the conjugates of the present invention, for some downstream applications, particularly diagnostic applications, it is advantageous to select a bright and stable fluorescent material.

[0062] Conjugated polymer nanoparticles (CPN)™ are highly fluorescent nanoparticles consisting of a semiconducting light-emitting polymer (LEP) core encapsulated in a biocompatible surfactant. CPNs are particularly suitable for diagnostic applications due to their strong fluorescence, i.e., high sensitivity and photostability. CPNs are around 70-80 nm in size and those currently available from Stream Bio exhibit fluorescence with emission wavelengths between 420-1130 nm. In the examples presented herein, CPN510 and CPN610 (both available from Stream Bio) were conjugated with the molecularly imprinted polymers of the present invention.

[0063] In a preferred embodiment of the present invention, the fluorescent material is CPN.

[0064] The present invention also provides a composition comprising a complex of the present invention.

[0065] The molecularly imprinted polymers, conjugates and compositions of the present invention may be used in a variety of applications.

[0066] It is specifically envisaged that the molecularly imprinted polymers, conjugates and compositions of the present invention are used for detecting SARS-CoV-2. The molecularly imprinted polymers, conjugates and compositions may be used in assays such as diagnostic assays that may be used in medical or clinical settings. Examples of such assays include lateral flow assays (LFA) and enzyme-linked immunosorbent assay (ELISA) type assays that may be used for high throughput screening. The molecularly imprinted polymers, conjugates and compositions of the present invention may be used in sensors.

[0067] In the examples presented herein, nanoMIP-SPR sensors were prepared by covalent immobilization of nanoMIPs onto a 4% mercaptoundecanoic acid (MUDA) chip (prepared using Biacore SIA kit Au surface functionalized with a self-assembled monolayer of 4% MUDA in ethanol). NanoMIP-functionalized screen-printed electrodes (SPEs) were also prepared for use in thermal detection methods.

[0068] The molecularly imprinted polymers, conjugates and compositions of the invention may be used to detect SARS-CoV-2 in a research setting.

[0069] The molecularly imprinted polymers, conjugates and compositions of the present invention may be used as pharmaceuticals.

[0070] The molecularly imprinted polymers of the present invention desirably have a high affinity for SARS-CoV-2. High affinity in the context of this specification means that the affinity with which the molecularly imprinted polymer binds to SARS-CoV-2 is at least 3 times, preferably at least 5 times, more preferably at least 10 times higher than the affinity with which a "blank" molecularly imprinted polymer binds to SARS-CoV-2. Blank in this context means a molecularly imprinted polymer formed in the absence of a template.

[0071] Binding affinity is the strength of the binding interaction between a single molecule and its binding partner, for example in this case between a molecularly imprinted polymer and its target, SARS-CoV-2. Binding affinity is typically measured using the equilibrium dissociation constant (K D ) is measured and reported by K D The lower the value, the higher the binding affinity between the two molecules.

[0072] When the molecularly imprinted polymers, conjugates, and compositions of the present invention are used for the detection of SARS-CoV-2, the molecularly imprinted polymers have a K of less than 500 nM, preferably less than 250 nM, and even more preferably less than 200 nM. DIt is preferred that the ribozyme binds to SARS-CoV-2 via the ribozyme.

[0073] K D Suitable methods for measuring include surface plasmon resonance (SPR), which can be performed, for example, using the Biacore series of instruments (Cytiva) or the MP-SPR Navi instrument (Bionavis).

[0074] The invention will now be further described in specific embodiments with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0075] [Figure 1A] Figure 1 shows two surface plasmon resonance sensorgrams illustrating the binding kinetics of peptide 1 and a nanoMIP imprinted with a recombinant SARS-CoV-2 S protein. [Figure 1B] Figure 1 shows two surface plasmon resonance sensorgrams illustrating the binding kinetics of peptide 1 and a nanoMIP imprinted with a recombinant SARS-CoV-2 S protein. [Diagram 2] FIG. 2 shows the trajectories obtained using a Nanosite NS300 instrument, demonstrating the successful binding of peptide 1 imprinted nanoMIP to CPN510. [Diagram 3] FIG. 3 is a dot plot showing the specificity of binding between peptide 1 and peptide 3 imprinted nanoMIPs and recombinant SARS-CoV-2 S protein. [Figure 4] FIG. 4 is a dot plot showing the specificity of binding between peptide 1 imprinted nanoMIP and SARS-CoV-2. [Diagram 5] FIG. 5 is a dot plot showing the specificity of binding between peptide 1 imprinted nanoMIP and the spike glycoprotein (S1) of SARS-CoV-2. [Figure 6A]Figure 6 shows the raw heat transfer data plotted as Rth versus time for the addition of PBS containing ORF8 of SARS-CoV-2 (1 fg / mL to 10 pg / mL) to screen-printed electrodes (SPEs) functionalized with nanoMIPs for peptide 1 (Figure 6a) and peptide 3 (Figure 6b). [Figure 6B] Figure 6 shows the raw heat transfer data plotted as Rth versus time for the addition of PBS containing ORF8 of SARS-CoV-2 (1 fg / mL to 10 pg / mL) to screen-printed electrodes (SPEs) functionalized with nanoMIPs for peptide 1 (Figure 6a) and peptide 3 (Figure 6b). [Figure 7A] Figure 7 shows the raw data of the heat transfer assay plotted as Rth versus time when PBS containing SARS-CoV-2 S protein RBD (1 fg / mL to 10 pg / mL) was added to a screen-printed electrode (SPE) functionalized with nanoMIP for peptide 1 (Figure 7a) and a dose-response curve plotting the % change in Rth value versus the concentration of SARS-CoV-2 S protein RBD injected into the system (Figure 7b). [Figure 7B] Figure 7 shows the raw data of the heat transfer assay plotted as Rth versus time when PBS containing SARS-CoV-2 S protein RBD (1 fg / mL to 10 pg / mL) was added to a screen-printed electrode (SPE) functionalized with nanoMIP for peptide 1 (Figure 7a) and a dose-response curve plotting the % change in Rth value versus the concentration of SARS-CoV-2 S protein RBD injected into the system (Figure 7b). [Figure 8A] Figure 8 shows the raw data of the heat transfer assay plotted as Rth versus time (Figure 8a) when PBS containing SARS-CoV-2 S protein RBD (1 fg / mL to 10 pg / mL) was added to a screen-printed electrode (SPE) functionalized with nanoMIP for peptide 3, and a dose-response curve (Figure 8b) showing the % change in Rth value plotted against the concentration of SARS-CoV-2 S protein RBD injected into the system. [Figure 8B]Figure 8 shows the raw data of the heat transfer assay plotted as Rth versus time (Figure 8a) when PBS containing SARS-CoV-2 S protein RBD (1 fg / mL to 10 pg / mL) was added to a screen-printed electrode (SPE) functionalized with nanoMIP for peptide 3, and a dose-response curve (Figure 8b) showing the % change in Rth value plotted against the concentration of SARS-CoV-2 S protein RBD injected into the system. [Figure 9A] Figure 9 reports the ability of peptide 1 imprinted nanoMIP to withstand extreme temperatures and pH. Figure 9a shows typical atomic force microscopy (AFM) morphology images (represented by green boxes in the large area images) of a single nanoMIP on a Au surface in air at room temperature, 37°C, and 50°C. Figure 9b shows the corresponding cross-sectional profile plots of the nanoMIP at each temperature. Figure 9c shows box plots comparing the nanoMIP volumes (n=120) from AFM images at various pH levels. Figure 9d compares the thermal response of the nanoMIP-based sensor to a clinical reference fluid (Universal Transport Medium) and to Diet Coca-Cola (pH=3.5). Figure 9e shows a commercial rapid antigen test giving false positive results when Diet Coca-Cola is used as the test fluid. [Figure 9B] Figure 9 reports the ability of peptide 1 imprinted nanoMIP to withstand extreme temperatures and pH. Figure 9a shows typical atomic force microscopy (AFM) morphology images (represented by green boxes in the large area images) of a single nanoMIP on a Au surface in air at room temperature, 37°C, and 50°C. Figure 9b shows the corresponding cross-sectional profile plots of the nanoMIP at each temperature. Figure 9c shows box plots comparing the nanoMIP volumes (n=120) from AFM images at various pH levels. Figure 9d compares the thermal response of the nanoMIP-based sensor to a clinical reference fluid (Universal Transport Medium) and to Diet Coca-Cola (pH=3.5). Figure 9e shows a commercial rapid antigen test giving false positive results when Diet Coca-Cola is used as the test fluid. [Figure 9C]Figure 9 reports the ability of peptide 1 imprinted nanoMIP to withstand extreme temperatures and pH. Figure 9a shows typical atomic force microscopy (AFM) morphology images (represented by green boxes in the large area images) of a single nanoMIP on a Au surface in air at room temperature, 37°C, and 50°C. Figure 9b shows the corresponding cross-sectional profile plots of the nanoMIP at each temperature. Figure 9c shows box plots comparing the nanoMIP volumes (n=120) from AFM images at various pH levels. Figure 9d compares the thermal response of the nanoMIP-based sensor to a clinical reference fluid (Universal Transport Medium) and to Diet Coca-Cola (pH=3.5). Figure 9e shows a commercial rapid antigen test giving false positive results when Diet Coca-Cola is used as the test fluid. [Figure 9D] Figure 9 reports the ability of peptide 1 imprinted nanoMIP to withstand extreme temperatures and pH. Figure 9a shows typical atomic force microscopy (AFM) morphology images (represented by green boxes in the large area images) of a single nanoMIP on a Au surface in air at room temperature, 37°C, and 50°C. Figure 9b shows the corresponding cross-sectional profile plots of the nanoMIP at each temperature. Figure 9c shows box plots comparing the nanoMIP volumes (n=120) from AFM images at various pH levels. Figure 9d compares the thermal response of the nanoMIP-based sensor to a clinical reference fluid (Universal Transport Medium) and to Diet Coca-Cola (pH=3.5). Figure 9e shows a commercial rapid antigen test giving false positive results when Diet Coca-Cola is used as the test fluid. [Figure 9E]Figure 9 reports the ability of peptide 1 imprinted nanoMIP to withstand extreme temperatures and pH. Figure 9a shows typical atomic force microscopy (AFM) morphology images (represented by green boxes in the large area images) of a single nanoMIP on a Au surface in air at room temperature, 37°C, and 50°C. Figure 9b shows the corresponding cross-sectional profile plots of the nanoMIP at each temperature. Figure 9c shows box plots comparing the nanoMIP volumes (n=120) from AFM images at various pH levels. Figure 9d compares the thermal response of the nanoMIP-based sensor to a clinical reference fluid (Universal Transport Medium) and to Diet Coca-Cola (pH=3.5). Figure 9e shows a commercial rapid antigen test giving false positive results when Diet Coca-Cola is used as the test fluid. [Figure 10A] Figure 10 reports the sensitivity of peptide 1-imprinted nanoMIPs to various SARS-CoV-2 antigens and compares the sensitivity of the nanoMIPs to SARS-CoV-2 antibodies. Figure 10a shows typical heat transfer data of nanoMIP-functionalized SPEs upon exposure to PBS containing 1 fg mL-1 to 10 pg mL-1 of SARS-CoV-2 RBD. Figures 10b to 10d show typical dose-response curves (error bars represent SD) showing (i) the thermal response of nanoMIP and antibody sensors to spike protein (nanoMIPs were tested against SARS-CoV-2 spike protein from both alpha and delta variants) (Figure 10b), (ii) the thermal response of nanoMIP and antibody sensors to the RBD of SARS-CoV-2 (Figure 10c), and iii) the thermal response of nanoMIP sensors to each SARS-CoV-2 antigen and negative controls ORF8, IL-6, and HSA. The response of NIP-based sensors to spike protein is also shown (Figure 10d). Figure 10e compares the LoD values ​​of our nanoMIPs to a commercially available rapid antigen test and a number of recently developed antigen tests in the literature. [Figure 10B]Figure 10 reports the sensitivity of peptide 1-imprinted nanoMIPs to various SARS-CoV-2 antigens and compares the sensitivity of the nanoMIPs to SARS-CoV-2 antibodies. Figure 10a shows typical heat transfer data of nanoMIP-functionalized SPEs upon exposure to PBS containing 1 fg mL-1 to 10 pg mL-1 of SARS-CoV-2 RBD. Figures 10b to 10d show typical dose-response curves (error bars represent SD) showing (i) the thermal response of nanoMIP and antibody sensors to spike protein (nanoMIPs were tested against SARS-CoV-2 spike protein from both alpha and delta variants) (Figure 10b), (ii) the thermal response of nanoMIP and antibody sensors to the RBD of SARS-CoV-2 (Figure 10c), and iii) the thermal response of nanoMIP sensors to each SARS-CoV-2 antigen and negative controls ORF8, IL-6, and HSA. The response of NIP-based sensors to spike protein is also shown (Figure 10d). Figure 10e compares the LoD values ​​of our nanoMIPs to a commercially available rapid antigen test and a number of recently developed antigen tests in the literature. [Figure 10C]Figure 10 reports the sensitivity of peptide 1-imprinted nanoMIPs to various SARS-CoV-2 antigens and compares the sensitivity of the nanoMIPs to SARS-CoV-2 antibodies. Figure 10a shows typical heat transfer data of nanoMIP-functionalized SPEs upon exposure to PBS containing 1 fg mL-1 to 10 pg mL-1 of SARS-CoV-2 RBD. Figures 10b to 10d show typical dose-response curves (error bars represent SD) showing (i) the thermal response of nanoMIP and antibody sensors to spike protein (nanoMIPs were tested against SARS-CoV-2 spike protein from both alpha and delta variants) (Figure 10b), (ii) the thermal response of nanoMIP and antibody sensors to the RBD of SARS-CoV-2 (Figure 10c), and iii) the thermal response of nanoMIP sensors to each SARS-CoV-2 antigen and negative controls ORF8, IL-6, and HSA. The response of NIP-based sensors to spike protein is also shown (Figure 10d). Figure 10e compares the LoD values ​​of our nanoMIPs to a commercially available rapid antigen test and a number of recently developed antigen tests in the literature. [Figure 10D]Figure 10 reports the sensitivity of peptide 1-imprinted nanoMIPs to various SARS-CoV-2 antigens and compares the sensitivity of the nanoMIPs to SARS-CoV-2 antibodies. Figure 10a shows typical heat transfer data of nanoMIP-functionalized SPEs upon exposure to PBS containing 1 fg mL-1 to 10 pg mL-1 of SARS-CoV-2 RBD. Figures 10b to 10d show typical dose-response curves (error bars represent SD) showing (i) the thermal response of nanoMIP and antibody sensors to spike protein (nanoMIPs were tested against SARS-CoV-2 spike protein from both alpha and delta variants) (Figure 10b), (ii) the thermal response of nanoMIP and antibody sensors to the RBD of SARS-CoV-2 (Figure 10c), and iii) the thermal response of nanoMIP sensors to each SARS-CoV-2 antigen and negative controls ORF8, IL-6, and HSA. The response of NIP-based sensors to spike protein is also shown (Figure 10d). Figure 10e compares the LoD values ​​of our nanoMIPs to a commercially available rapid antigen test and a number of recently developed antigen tests in the literature. [Figure 10E]Figure 10 reports the sensitivity of peptide 1-imprinted nanoMIPs to various SARS-CoV-2 antigens and compares the sensitivity of the nanoMIPs to SARS-CoV-2 antibodies. Figure 10a shows typical heat transfer data of nanoMIP-functionalized SPEs upon exposure to PBS containing 1 fg mL-1 to 10 pg mL-1 of SARS-CoV-2 RBD. Figures 10b to 10d show typical dose-response curves (error bars represent SD) showing (i) the thermal response of nanoMIP and antibody sensors to spike protein (nanoMIPs were tested against SARS-CoV-2 spike protein from both alpha and delta variants) (Figure 10b), (ii) the thermal response of nanoMIP and antibody sensors to the RBD of SARS-CoV-2 (Figure 10c), and iii) the thermal response of nanoMIP sensors to each SARS-CoV-2 antigen and negative controls ORF8, IL-6, and HSA. The response of NIP-based sensors to spike protein is also shown (Figure 10d). Figure 10e compares the LoD values ​​of our nanoMIPs to a commercially available rapid antigen test and a number of recently developed antigen tests in the literature. [Figure 11A] Figure 11 shows the binding between peptide 1 imprinted nanoMIP and SARS-CoV-2 from clinical samples. Figure 11a is a schematic of a 3D printed loading cell prototype with 1) a thermocouple inlet, 2) a functionalized SPE, 3) an open-bottom reservoir to facilitate manual addition of liquids, and 4) a removable lid to reduce experimental noise. Figure 11b is a photograph of the loading cell shown in Figure 11a. Figure 11c shows a typical dose-response curve obtained using the prototype loading cell for thermal detection of SARS-CoV-2 spike protein (1 fg mL-1 to 10 pg mL-1) in phosphate-buffered saline (PBS). Figure 11d shows the thermal response of the nanoMIP sensor to clinical samples from COVID-positive and COVID-negative patients (n=7). [Figure 11B]Figure 11 shows the binding between peptide 1 imprinted nanoMIP and SARS-CoV-2 from clinical samples. Figure 11a is a schematic of a 3D printed loading cell prototype with 1) a thermocouple inlet, 2) a functionalized SPE, 3) an open-bottom reservoir to facilitate manual addition of liquids, and 4) a removable lid to reduce experimental noise. Figure 11b is a photograph of the loading cell shown in Figure 11a. Figure 11c shows a typical dose-response curve obtained using the prototype loading cell for thermal detection of SARS-CoV-2 spike protein (1 fg mL-1 to 10 pg mL-1) in phosphate-buffered saline (PBS). Figure 11d shows the thermal response of the nanoMIP sensor to clinical samples from COVID-positive and COVID-negative patients (n=7). [Figure 11C] Figure 11 shows the binding between peptide 1 imprinted nanoMIP and SARS-CoV-2 from clinical samples. Figure 11a is a schematic of a 3D printed loading cell prototype with 1) a thermocouple inlet, 2) a functionalized SPE, 3) an open-bottom reservoir to facilitate manual addition of liquids, and 4) a removable lid to reduce experimental noise. Figure 11b is a photograph of the loading cell shown in Figure 11a. Figure 11c shows a typical dose-response curve obtained using the prototype loading cell for thermal detection of SARS-CoV-2 spike protein (1 fg mL-1 to 10 pg mL-1) in phosphate-buffered saline (PBS). Figure 11d shows the thermal response of the nanoMIP sensor to clinical samples from COVID-positive and COVID-negative patients (n=7). [Figure 11D]Figure 11 shows the binding between peptide 1 imprinted nanoMIP and SARS-CoV-2 from clinical samples. Figure 11a is a schematic of a 3D printed loading cell prototype with 1) a thermocouple inlet, 2) a functionalized SPE, 3) an open-bottom reservoir to facilitate manual addition of liquids, and 4) a removable lid to reduce experimental noise. Figure 11b is a photograph of the loading cell shown in Figure 11a. Figure 11c shows a typical dose-response curve obtained using the prototype loading cell for thermal detection of SARS-CoV-2 spike protein (1 fg mL-1 to 10 pg mL-1) in phosphate-buffered saline (PBS). Figure 11d shows the thermal response of the nanoMIP sensor to clinical samples from COVID-positive and COVID-negative patients (n=7). [Figure 12] FIG. 12 shows the size of the nanoMIPs as measured using the NanoSight particle analyzer. [Figure 13A] Figure 13 shows the detection of SARS-CoV-2 spike protein from alpha, beta, and gamma mutants of SARS-CoV-2 in PBS via nanoMIP-LSPR sensor. Figure 13a shows LSPR spectra plotted as wavelength vs. absorbance for the alpha mutant of SARS-CoV-2 at concentrations ranging from 10 aM to 100 nM. Figure 13b shows the change in LSPR wavelength of Ag-MIP complexes with varying concentrations of alpha, beta, and gamma mutants of SARS-CoV-2 from 10 aM to 100 nM. [Figure 13B] Figure 13 shows the detection of SARS-CoV-2 spike protein from alpha, beta, and gamma mutants of SARS-CoV-2 in PBS via nanoMIP-LSPR sensor. Figure 13a shows LSPR spectra plotted as wavelength vs. absorbance for the alpha mutant of SARS-CoV-2 at concentrations ranging from 10 aM to 100 nM. Figure 13b shows the change in LSPR wavelength of Ag-MIP complexes with varying concentrations of alpha, beta, and gamma mutants of SARS-CoV-2 from 10 aM to 100 nM. [Figure 14A] Figure 14 shows the nanoMIP-LSPR mediated detection of SARS-CoV-2 spike protein from alpha, beta and gamma variants of SARS-CoV-2 in serum. Figure 14a shows the LSPR spectra plotted as wavelength vs absorbance for the alpha variant of SARS-CoV-2 at concentrations ranging from 100 fM to 100 nM. Figure 14b shows the shift in LSPR wavelength of Ag-MIP complexes with varying concentrations of alpha, beta and gamma variants of SARS-CoV-2 from 100 fM to 100 nM. [Figure 14B] Figure 14 shows the nanoMIP-LSPR mediated detection of SARS-CoV-2 spike protein from alpha, beta and gamma variants of SARS-CoV-2 in serum. Figure 14a shows the LSPR spectra plotted as wavelength vs absorbance for the alpha variant of SARS-CoV-2 at concentrations ranging from 100 fM to 100 nM. Figure 14b shows the shift in LSPR wavelength of Ag-MIP complexes with varying concentrations of alpha, beta and gamma variants of SARS-CoV-2 from 100 fM to 100 nM. [Figure 15] Figure 15 shows two overlaid surface plasmon resonance sensorgrams showing the binding kinetics of a nanoMIP imprinted with peptide 1 and either recombinant and / or omicron mutant versions of the original SARS-CoV-2 S (spike) protein. EXAMPLES

[0076] Example 1: Synthesis of nanoMIPs against SARS-CoV-2 Selection of three peptides derived from SARS-CoV-2 suitable for imprinting Wrapp et al., 2020 1Supplementary Figure 5 in shows a sequence alignment of the spike protein of SARS-CoV-2 with the spike proteins of two other coronavirus strains, SARS-CoV and RaTG13. The RBD is highlighted. We selected three peptide sequences from the RBD of SARS-CoV-2 (peptide 1: NSNNLDSKVGG, peptide 2: STEIYQAGSTPC, and peptide 3: CYFPLQSYGFQP) as potential candidates for imprinting based on their lack of conservation between the respective sequences from SARS-CoV and RaTG13.

[0077] Solid-phase synthesis of nano-MIPs against SARS-CoV-2 Prior to nanoMIP synthesis, peptide 1, peptide 2, and peptide 3 were immobilized on silanized glass beads.

[0078] Based on the requirement of a terminal cysteine ​​for peptide immobilization, a cysteine ​​residue was added to the N-terminus of peptide 1 when ordering peptide 1, peptide 2, and peptide 3 from Ontores, China. In addition, an additional glycine residue was added between the cysteine ​​and the N-terminal asparagine to function as a spacer. Thus, the peptides for imprinting procured from Ontores were as follows: Peptide 1: CGNSNNLDSKVGG Peptide 2: STEIYQAGSTPC Peptide 3: CYFPLQSYGFQP

[0079] Peptide immobilization was performed as follows: Glass beads (approximately 100 μM diameter, obtained from Microbeads AG) were activated by boiling in 4 M NaOH for 10 min. The glass beads were then washed first with deionized water, then with acetone, and then dried at 80° C. for 2 h. The glass beads were then incubated in toluene containing 2% v / v (3-aminopropyl)trimethoxysilane (APTMS) for 3 h, washed with acetone, placed in PBS, pH 7.4, containing 0.2 mg / ml n-succinimidyl iodoacetate (SIA) for 2 h, and washed with acetonitrile. Templates (peptide 1, peptide 2, or peptide 3) were then immobilized on the surface of the glass beads by incubation in a solution of 0.4 mg / ml tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 0.1 mg / ml peptide in PBS, pH 7.4 for a minimum of 4 h. Excess template was removed by washing with water and methanol.

[0080] Imprinted nano-MIPs were synthesized using peptide-coated glass beads as follows: A monomer solution was prepared, sonicated for 10 min, and purged with nitrogen for 5 min. An example of the monomer solution prepared contained 3 mg of N-fluoresceinyl acrylamide, 24.2 mg of acrylamide, 31.7 μL of tert-butyl acrylate (TBAc), 36 mg of 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose, 13.8 μL of 2,2,2-trifluoroethyl methacrylate (CF3), 3.9 mg of N-(3-aminopropyl) methacrylamide hydrochloride (APMA), 1.3 mg of squaramide monomer 7 (SQ7), and 9 mg of N,N'-methylenebis(acrylamide) (BIS). Then, 100 ml of the monomer solution was placed in a 250 ml Duran flask containing 60 g of peptide-coated glass beads. Polymerization was then initiated by adding 0.5 ml of 60 mg / ml ammonium persulfate (APS) containing 60 μL / ml N,N,N',N'-tetramethylethylenediamine (TEMED), and allowed to proceed at room temperature for 1 hour. The contents of the flask were then poured into a solid-phase extraction (SPE) cartridge equipped with a frit with a porosity of 20 μM to separate the glass beads with attached nanoparticles from other components. Next, 10 washing steps using approximately 25 ml of room temperature water were performed to remove low affinity substances. The residual liquid in this washing step was discarded each time. Then, 9 washing steps using 10 ml of ethanol at 60°C were performed to separate the high affinity nanoMIP from the glass beads. The residual liquid in this washing step contained the nanoMIP and was collected each time. The nanoMIP was then concentrated to approximately 10 ml, and the solvent was replaced with distilled water.

[0081] The average diameter of the imprinted nanoMIPs, calculated with a NanoSight NS300, was 59 nm.

[0082] Example 2: Surface Plasmon Resonance (SPR) Analysis of NanoMIPs Against SARS-CoV-2 Peptides In this study, a Biacore 3000 instrument was employed.

[0083] Surface activation and immobilization of nano-MIPs A nanoMIP-SPR sensor was prepared by covalently immobilizing a nanoMIP (from Example 1) onto a 4% mercaptoundecanoic acid (MUDA) chip (prepared using Biacore SIA kit Au surface functionalized with a self-assembled monolayer of 4% MUDA in ethanol).

[0084] More specifically, the device was primed with water at a constant flow rate of 5 μL / min. Water was used as the running buffer throughout the chip preparation at a constant flow rate of 30 μL / min. Water was run until a stable baseline was achieved. A mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (400 uL water containing 2 mg EDC + 3 mg NHS) was injected over the entire sensor surface (8 min at a flow rate of 8 μL / min) to activate the surface (this mixture converts the carboxyl end groups on the chip surface to N-hydroxy-succinimide esters). Then, nanoMIPs (5 nM) were injected into all four flow cells on the chip (8 min to 10 min at a flow rate of 8 μL / min). Finally, unreacted NHS esters were hydrolyzed by injecting carbonate buffer (pH 9.2) (30 min at a flow rate of 5 μL / min to 30 μL / min).

[0085] Detection of interactions between nanoMIPs and SARS-CoV-2 peptides Each of peptide 1, peptide 2, and peptide 3 was conjugated to bovine serum albumin (BSA) prior to testing in SPR.

[0086] After switching the running buffer from water to PBS and obtaining a stable baseline, seven dilutions of BSA or BSA-peptide conjugates (2.33mg / ml, 0.777mg / ml, 259ug / ml, 86ug / ml, 28.8ug / ml, 9.6ug / ml, 3.2ug / ml in PBS) were injected as follows (5 min association phase, 4 min dissociation phase, 28μl / min flow rate): BSA alone was injected on flow cell 1, BSA-peptide 1 on flow cell 2, and BSA-peptide 3 on flow cell 3.

[0087] Good selectivity was observed for the nanoMIP imprinted with peptide 3, and the software showed that K D was calculated to be 160 nM (data not shown).

[0088] Example 3: Surface plasmon resonance analysis of nanoMIPs against the S protein of SARS-CoV-2 In this study, a Biacore 3000 instrument was employed.

[0089] Surface activation and immobilization of SARS-CoV-2 S protein A SARS-CoV-2 S protein-SPR sensor was prepared by covalently immobilizing a recombinant SARS-CoV-2 S protein (full-length SARS-CoV-2 spike glycoprotein recombinant sourced from Native Antigen Company) onto a 4% mercaptoundecanoic acid (MUDA) chip (prepared using Biacore SIA Kit Au surface functionalized with a self-assembled monolayer of 4% MUDA in ethanol).

[0090] More specifically, the device was prepared with water at a constant flow rate of 5 μl / min. Water was also used as the running buffer throughout the chip preparation at a constant flow rate of 30 μl / min. Water was run until a stable baseline was achieved. A mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (400 uL water containing 2 mg EDC + 3 mg NHS) was injected (8 min at a flow rate of 8 μl / min) over the sensor surface to activate the surface (this mixture converts the carboxyl end groups on the chip surface to N-hydroxy-succinimide esters). Then, a control (1% BSA (w / v) and 0.5% Pluronic F127 (w / v)) and two different concentrations of recombinant SARS-CoV-2 S protein (60 nM and 600 nM) were injected into flow cell 1, flow cell 2, and flow cell 3 on the chip, respectively (6 min 15 s at a flow rate of 8 μl / min). Finally, unreacted NHS esters were hydrolyzed by injection of carbonate buffer (pH 9.2) (1 min at a flow rate of 30 μL / min).

[0091] Detection of interaction between SARS-CoV-2 S protein and nanoMIPs After switching the running buffer from water to PBS and obtaining a stable baseline, nanoMIPs imprinted with 30 nM (and its 1:1 dilution series) of peptide 1 were injected (5 min association phase, 3 min dissociation phase, flow rate of 28 μl / min).

[0092] K D was calculated by the software to be 5 nM (FIG. 1a) and 15 nM (FIG. 1b).

[0093] Example 4: Binding of nanoMIPs against SARS-CoV-2 to CPN NanoMIPs imprinted with peptide 1 and peptide 3 were coupled to Stream Bio conjugated polymer nanoparticles (CPNs), highly fluorescent nanoparticles containing a semiconducting light-emitting polymer core encapsulated within a hydrophilic capping agent. The CPNs used were CPN510 and CPN610, with diameters of 70-80 nm as measured on a NanoSight 3000.

[0094] Figure 2 shows that peptide 1-imprinted nanoMIPs were successfully conjugated to CPN510, and it should be noted that while the original unconjugated nanoMIPs were mainly 59 nm, there was a significant change in particle size upon conjugation to CPN510 (see the peak at 119 nm).

[0095] Example 5: Dot blot analysis of binding between nanoMIPs and S protein of SARS-CoV-2 Different concentrations (290ng, 145ng, 73ng, 36ng, 18ng, 9ng, 5ng, 2ng, 1ng, 0.6ng, 0.3ng, and 0.14ng) of recombinant SARS-CoV-2 S protein (sourced from Native Antigen Company) were blotted onto nitrocellulose membranes and then dried. The membranes were then blocked by immersing them in PBS-T (PBS containing 0.05% Tween-20) containing 5% BSA at room temperature for 30 to 60 minutes. After blocking, the membranes were incubated with (1) nanoMIP-CPN510 (peptide 1 imprinted), (2) nanoMIP-CPN510 (peptide 3 imprinted), or (3) CPN510 alone (control) dissolved in PBS-T containing 5% BSA at room temperature for 30 to 60 minutes. The membranes were then washed three times with PBS-T for 5 minutes each. Fluorescence was then observed under UV light. As shown in Figure 3, the detection limit for nanoMIP-CPN510 (peptide 1 imprinted) was 0.3 ng, which was significantly lower than that for CPN510 alone (control), indicating that at least the nanoMIP imprinted with peptide 1 specifically interacts with the S protein of SARS-CoV-2.

[0096] Example 6: Dot blot analysis of binding between nanoMIPs and SARS-CoV-2 virus SARS-CoV-2 (2 × 10 4 pfu) was blotted onto two nitrocellulose membranes. In addition, 75 ng of recombinant SARS-CoV-2 S protein (sourced from Native Antigen Company) was blotted onto each nitrocellulose membrane as a positive control, and medium was blotted as a negative control. The membrane was then left to dry. The membrane was then blocked by immersing it in PBS-T (PBS containing 0.05% Tween-20) containing 5% BSA for 30 to 60 minutes at room temperature. After blocking, the membrane was incubated with nanoMIP-CPN510 (peptide 1 imprinted) or (2) nanoMIP-CPN510 (troponin imprinted) (negative control) dissolved in PBS-T containing 5% BSA at room temperature for 30 to 60 minutes. The membrane was then washed three times with PBS-T for 5 minutes each. Fluorescence was then observed under UV light. As shown in Figure 4, no fluorescence was observed in the areas of the nitrocellulose membrane that were blotted with medium or when the membrane was incubated with nanoMIP-CPN510 (troponin imprinted) (negative control). In contrast, fluorescence was observed when the nitrocellulose membrane was incubated with nanoMIP-CPN510 (peptide 1 imprinted) for both SARS-CoV-2 and the positive control SARS-CoV-2 S protein. This indicates that at least the nanoMIP imprinted with peptide 1 specifically interacts with SARS-CoV-2.

[0097] Example 7: Dot blot analysis comparing (1) the binding between nanoMIPs and four human coronaviruses (SARS-CoV-2, HCoV-OC43, HCoV-229E, and HCoV-HKU1) and (2) the binding between antibodies and four human coronaviruses (SARS-CoV-2, HCoV-OC43, HCoV-229E, and HCoV-HKU1). SARS-CoV-2 spike glycoprotein (S1) (Native Antigen Company), human coronavirus OC43 spike glycoprotein (S1) (Native Antigen Company), human coronavirus 229E spike glycoprotein (S1) (Native Antigen Company), and human coronavirus HKU1 spike glycoprotein (S1) (Native Antigen Company) (0.6 mg / mL) were blotted in triplicate onto nitrocellulose membranes (as shown in the schematic diagram in Figure 5), and then the membranes were dried. The membranes were then blocked by soaking in 2 mL of BSA (1% in PBS) for 1 h. After blocking, the membrane was incubated with (1) nanoMIP-CF770 (peptide 1 imprinted) and (2) three monoclonal antibodies specific for SARS-CoV-2 spike (RBD), namely MabRBD1106-CF770, MabRBD107-CF770, and MabRBD5305-CF770 (all prepared in-house and diluted with 0.01% BSA), for 30 min. The membrane was then washed with 2 mL of PBST for 5 min. Afterwards, the fluorescence was observed under UV. As shown in Figure 5, nanoMIP selectively bound to SARS-CoV-2 spike glycoprotein (S1), but not to human coronavirus OC43 spike glycoprotein (S1), human coronavirus 229E spike glycoprotein (S1), or human coronavirus HKU1 spike glycoprotein (S1). Furthermore, the nanoMIPs were shown to have comparable selectivity and binding to each of the antibodies tested.

[0098] Example 8: Analysis of the binding between nanoMIPs and the RBD of SARS-CoV-2 S protein using a thermal detection method Preparation of nano-MIP-functionalized screen-printed electrodes (SPEs) for use in thermal detection methods An aqueous HCL solution (0.5 M) containing 4-ABA (2 mM) and sodium nitrite (2 mM) was prepared and mixed gently on an orbital shaker for 10 min. A screen-printed electrode (SPE) was immersed in the solution and a voltage of 100 mV s was applied using a Ag / AgCl reference electrode (Alvatek Ltd., Romsey, UK). -1 Cyclic voltammetry was performed from +0.2 V to -0.6 V at 100°C. The resulting electrodes, denoted as SPE / 4-ABA, were thoroughly rinsed with deionized water to remove unbound 4-ABA and dried using nitrogen. The carboxyl groups were then activated by incubation with a PBS buffer solution (pH=5) containing 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (100 mM) and N-hydroxysuccinimide (NHS) (20 mM). 8 μL of the EDC / NHS solution was deposited on the SPE working electrode by drop casting. After 1 h, the electrode was rinsed with deionized water and dried to obtain the SPE / 4-ABA / EDC+NHS electrode. After this, 8 μL of a given nanoMIP solution (peptide 1 imprinted nanoMIP “P1C6” or peptide 3 imprinted nanoMIP “P3C6”) was deposited on the working electrode after gentle stirring. After 3 h, the SPE / 4-ABA / EDC+NHS / nanoMIP electrode was rinsed with deionized water, dried under a gentle stream of nitrogen, and then stored in PBS at 4° C. until use.

[0099] Detection of binding between nanoMIPs and the S protein RBD of SARS-CoV-2 using nanoMIP-functionalized screen-printed electrodes (SPEs) A 3D-printed flow cell was used to facilitate the measurements with the nanoMIP-functionalized SPEs described above. The SPEs were mounted in the cell and the temperature of the liquid was measured by a thermocouple (T2). Freshly prepared nanoMIP-functionalized SPEs were used for each measurement. The flow cell was connected to a heat transfer device as described in van Grinsven et al. The device was operated using LabView software that actively controlled the temperature of the heat sink (copper block, T1), which was set at 37.00 ± 0.02 °C to mimic in vivo conditions. Feedback on the signal was regulated by a proportional-integral-derivative (PID) controller attached to a power resistor (22 Ω) as described in Geerets et al. The PID parameters were fixed at optimal values ​​of P = 1, I = 10, and D = 0.2 in all experiments.

[0100] For all measurements, the flow cell was filled with PBS and left for 30 min to ensure stabilization of the baseline temperature signal, after which PBS was injected first as a blank measurement. -1 to 10 pg mL -1 ) A PBS solution (3 mL) of each target biomarker (SARS-CoV-2 S protein RBD or SARS-CoV-2 ORF8 (as a negative control)) was prepared prior to the experiment and stored at 4 °C until required. For the injection of each biomarker, a LSP02-1B dual channel syringe (Longer Precision Pump Co., Hebei, China) pump was used to inject 250 μL min -1 The injection was performed over a 12 min period. After each biomarker injection, the system was allowed to stabilize for 30 min before the next injection. Throughout the experiment, the thermal resistance (R th ) was obtained. R thThe mean and standard deviation (SD) were calculated using the average of 600 data points from the baseline signal at each concentration and the first PBS injection, respectively (Figure 6, Figure 7a, and Figure 8a). This data was used to generate dose-response curves and the limit of detection (LoD) was calculated using the 3 sigma method in the linear range of the sensor (Figure 7b and Figure 8b). The error bars in the graphs relate to the SD values. Figure 6 shows that the thermal response to the ORF8 of SARS-CoV-2 is very limited, whereas Figures 7 and 8 show that for both peptide 1 and peptide 3 imprinted nanoMIPs, a good response was observed with the SARS-CoV-2 S protein RBD.

[0101] Example 9: Analysis of nanoMIP robustness The ability of peptide 1-imprinted MIPs to withstand extremes of temperature and pH was investigated.

[0102] temperature Using atomic force microscopy (AFM), the effect of increasing temperature on the morphology of the adsorbed nanoMIP was investigated by imaging the same nanoMIP at room temperature, 37°C, and 50°C.

[0103] AFM measurements were performed on a JPK Nanowizard 4 XP Bioscience (Bruker Nano GmbH, Berlin, Germany). The cantilever length was approximately 225 μm and the spring constant was approximately 48 N m. -1 Measurements were performed in air using a PPP-NCL-W probe (Nanosensors, Neuchâtel, Switzerland) in tapping mode. The cantilever length was approximately 140 μm and the spring constant was approximately 0.1 N m -1Measurements were performed in liquid in quantitative imaging (QI) mode using a MLCT-E probe (Bruker, CA, USA). Au-coated Si chips were used as substrates (Si-Mat, Kaufering, Germany). Prior to drop casting, the chips were cleaned by immersion for 5 min in a 5:1:1 mixture of Milli-Q water, ammonia, and hydrogen peroxide heated to 75 °C. The chips were then rinsed with Milli-Q water and dried using nitrogen. The nanoMIP solution was diluted to approximately 2.54 μg mL -1 The solution was diluted to 0.05 mL, drop-cast (20 μL) onto the Au-coated surface, and dried in a Petri dish for a minimum of 4 h at ambient conditions. A high temperature heating stage (HTHS, JPK Bio AFM - 0.1 °C resolution) was used as the temperature controller to facilitate imaging at 37 °C and 50 °C.

[0104] Figures 9a and 9b, which show typical AFM images and corresponding cross-sectional profile plots, respectively, demonstrate that the morphology of the nanoMIP was not affected by increasing temperature.

[0105] The volume of the nanoMIPs was calculated using Gwyddion. Tracking multiple droplets revealed that the average volume of the nanoMIPs changed minimally (a decrease of about 6%) from room temperature to 50 °C.

[0106] Thus, the morphology of the nanoMIP remains unchanged over a relatively wide temperature range.

[0107] The nanoMIPs autoclaved at a maximum temperature of 121 °C for 15 min were also used to perform SPR binding analysis on the SARS-CoV-2 spike protein using a Biacore 3000 instrument.

[0108] More specifically, two nanoMIP-SPR sensors were prepared by covalently immobilizing the prepared nanoMIP (from Example 1) onto a 4% mercaptoundecanoic acid (MUDA) chip (prepared using Biacore SIA kit Au surface functionalized with a self-assembled monolayer of 4% MUDA in ethanol) either as is or after autoclaving at 121° C. for approximately 15 min, according to the process described immediately below.

[0109] The device was primed with water at a constant flow rate of 5 μl / min. Water was used as the running buffer at a constant flow rate of 30 μl / min throughout the chip preparation. Water was run until a stable baseline was achieved. A mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (400 uL water containing 2 mg EDC + 3 mg NHS) was injected over the entire sensor surface (8 min at a flow rate of 8 μL / min) to activate the surface (this mixture converts the carboxyl end groups on the chip surface to N-hydroxy-succinimide esters). NanoMIPs (5 nM) were then injected into all four flow cells on the chip (8 min to 10 min at a flow rate of 8 μL / min). Finally, unreacted NHS esters were hydrolyzed by injecting carbonate buffer (pH 9.2) (30 min at a flow rate of 5 μL / min to 30 μL / min).

[0110] After switching the running buffer from water to PBS and obtaining a stable baseline, five dilutions of SARS-CoV-2 S protein (recombinant full-length SARS-CoV-2 spike glycoprotein sourced from Native Antigen Company) (30 mM, 15 mM, 7.5 mM, 3.75 mM, and 1.88 mM in PBS) were injected (approximately 5 min binding phase, at least 2 min dissociation phase, flow rate of 28 μl / min) along with appropriate controls in the control flow path. The results from the two chips were then overlaid and the KD values ​​of the nanoMIPs calculated by the software were very similar before and after autoclaving (7 nM and 3 nM, respectively), demonstrating that the binding affinity was not affected after exposure to high temperatures. Autoclaving (sterilization) is a great advantage in the shelf life of nanoMIPs, as it facilitates long-term storage of nanoMIPs in water without bacterial degradation. In contrast, antibodies experience a significant decrease in affinity at temperatures above 37°C.

[0111] pH The ability of nanoMIPs to withstand extreme pH was also assessed by measuring the volume of adsorbed nanoMIPs (n=120) in liquids with various pH levels using AFM. More specifically, QI measurements were performed at room temperature (23±1°C) in liquids with different pH levels (5.5 to 8.5) within the AFM operating conditions. The first measurements were performed in pure Milli-Q water (pH 5.5), and then the pH was increased by adding ammonia.

[0112] The volume of the nanoMIPs was calculated using Gwyddion as described above and revealed that the average nanoMIP volume changed very little from pH 5.5 to pH 8.5 (a decrease of about 3%), highlighting that the morphology of the adsorbed nanoMIPs remained unchanged over a wide pH range (see Figure 9c).

[0113] Furthermore, the thermal response of the developed nano-MIP test (see Example 10) to a clinical reference fluid (Universal Transport Medium, UTM) was compared to that to Diet Coca-Cola (pH=3.5). The results demonstrated that there was no statistically significant difference between the thermal responses of the two fluids (see FIG. 9d). In contrast, a commercially available antibody-based rapid antigen test is highly affected by changes in pH, resulting in false positive results when an acidic soft drink (Diet Coca-Cola) is used as the test fluid (see FIG. 9e).

[0114] Example 10: Comparison of binding between nanoMIPs and SARS-CoV-2 antigens with binding between SARS-CoV-2 antibodies and SARS-CoV-2 antigens using thermal detection methods A nanoMIP-functionalized screen-printed electrode (SPE) (with nanoMIP imprinted with peptide 1) was fabricated according to the procedure in Example 8.

[0115] SARS-CoV-2 antibody-functionalized SPEs were also prepared according to the procedure in Example 8.

[0116] Detection of binding between nanoMIPs or SARS-CoV-2 antibodies and SARS-CoV-2 antigens using nanoMIP-functionalized screen-printed electrodes (SPEs) or SARS-CoV-2 antibody-functionalized screen-printed electrodes (SPEs) The nanoMIP-functionalized SPE was implemented in a 3D-printed resin flow cell to create an interface between a heat sink and a reservoir for thermal detection of SARS-CoV-2 antigens (either recombinant full-length SARS-CoV-2 spike glycoprotein (alpha mutant) from Native Antigen Company, recombinant SARS-CoV-2 spike receptor binding domain (RBD) (delta mutant) from Abbexa (Cambridge, UK), or SARS-CoV-2 RBD from the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit (Dundee, UK). The temperature of the heat sink (T1) and the temperature of the reservoir (T2) were measured every second by two thermocouples, and the thermal resistance (R th ) was obtained. When the target was attached to the nanoMIP, the heat transfer at the solid-liquid interface decreased (the temperature gradient increased), and R th This has led to a not inconsiderable rise in

[0117] For all experiments, the thermal measurement device was controlled using LabView software and regulated the feedback on the signal by a proportional-integral-derivative (PID) controller attached to a power resistor (22 Ω). The PID parameters were optimized to reduce noise and were set to P = 1, I = 13, and D = 0.2.

[0118] The reservoir was filled with PBS and left for 30 minutes. th The baseline signal was then stabilized by gradually increasing concentrations of SARS-CoV-2 antigen (1 fg mL -1 to 10 pg mL -1 ) Five types of PBS solutions (3 mL) were dispensed at 250 μL min using an automated syringe pump (LSP02-1B, Longer Precision Pump Co., Ltd., Hebei, China). -1The flow cell was injected at 1000 rpm for 12 min. The system was allowed to stabilize for 30 min before the next injection. th Raw thermal data plots were obtained showing a stepwise increase in where stable plateaus represented the injection of solutions of increasing concentrations (see Figure 10a). Dose-response curves (Figures 10b to 10d) were constructed from the thermal plots by taking the mean and standard deviation (SD) of the stable plateaus at each concentration injection. Limits of detection (LoD) values ​​were calculated from the dose-response curves using the 3 sigma method (3 × baseline SD) in the linear range.

[0119] A SARS-CoV-2 antibody-functionalized SPE was used to facilitate a direct comparison of the sensing performance of the nanoMIP with that of the antibody receptor. Thermal detection results (see Figure 10b) showed that the response to the SARS-CoV-2 spike protein (alpha variant) was significantly higher than that of the nanoMIP-based sensor (LoD = 9.9 ± 2.5 fg mL). -1 ) and antibody-based sensors (LoD = 8.9 ± 4.1 fg mL -1 ) were found to be very similar to those of the spike protein, highlighting that the specificity of the nanoMIP for the spike protein is comparable to the antibodies used in commercial tests.

[0120] The ability of the nanoMIP receptor to detect viral mutations was also investigated by measuring its specificity for the SARS-CoV-2 spike protein (delta mutant). The obtained LoD (6.1 ± 2.9 fg mL -1 The results (Figure 10b) show that the nanoMIP sensor was effective in detecting the delta mutant, as the α-mutant α-reactive protein (A) was very similar to the value obtained for the alpha mutant. This demonstrates that the nanoMIP sensor is capable of equally detecting low amounts of both the alpha and delta mutant spike proteins, making it very promising for potential commercial applications where there are ongoing concerns about reduced validity of the test due to the presence of novel variants.

[0121] The versatility of the nanoMIP receptor was also investigated by measuring the thermal response to the RBD of SARS-CoV-2 (see Figure 10c). For direct comparison, an antibody receptor was also tested. The LoD value for the RBD of SARS-CoV-2 was 10.1 ± 15.0 fg mL−1 (85.5 ± 15.0 fg mL−1). -1 ) compared with approximately 20 times lower for the nanoMIP sensor (3.9 ± 1.0 fg mL -1 ). As a result, this demonstrates that nanoMIPs are more versatile than antibodies, since the LoD for the full-length spike protein was comparable to antibodies, but the LoD for the RBD was significantly lower.

[0122] The selectivity of the nanoMIP sensor was also comprehensively tested using three negative controls, open reading frame 8 (ORF8), interleukin-6 (IL-6), and human serum albumin (HSA), which are common interfering substances in clinical samples. A high degree of binding occurred between the SARS-CoV-2 antigen and the nanoMIP, which resulted in the highest concentration (10 pg mL -1 ) spike protein (0.23°C W -1 ) and RBD(0.35°C W -1 ) for ΔR th In contrast, the negative control produced minimal binding, which resulted in ORF8 (0.00°C W -1 ), IL-6 (0.06°C·W -1 ), and HSA(0.05°C W -1 ) for ΔR thThe values ​​were significantly lower. The results demonstrated that the thermal response of the nanoMIP sensor was significantly higher for the SARS-CoV-2 antigen compared to the negative control, highlighting the superior selectivity of the nanoMIP sensor. An additional control experiment was also performed using a non-imprinted polymer (NIP) immobilized on an SPE (NIP-functionalized SPE prepared according to the procedure in Example 8). The NIP was prepared using a similar synthetic protocol as the nanoMIP, except that it did not expose the target epitope during polymerization, and therefore did not have a specific cavity to facilitate target binding. The thermal response of the nanoMIP-functionalized SPE to the spike protein was significantly higher than that of the NIP-functionalized SPE (0.04 °C·W -1 ), indicating that specific binding occurs between the spike protein and the cavity of the nanoMIP.

[0123] Figure 10e shows the results of a comparison of the nanoMIP sensor, a commercially available rapid antigen test, and the literature 4~15 The LoD values ​​of a number of recently developed antigen tests are shown in Table 1 below. The LoD values ​​for the nanoMIP sensor are comparable to those of a commercially available rapid antigen test (20 pg mL -1 ), which is approximately 6000 times lower than that of the conventional nanoMIP receptor. This remarkably low LoD highlights that the nanoMIP receptor could be a valuable tool in producing rapid antigen tests that are sensitive enough to perform effective population screening. Furthermore, the LoD of the nanoMIP sensor is one of the lowest of the LoDs of recently developed antigen tests in the literature. This demonstrates that thermal detection using nanoMIPs can compete with the best antigen tests in the recent literature, with the added advantage of being able to withstand extreme temperatures and pH.

[0124] [Table 1-1]

[0125] [Table 1-2]

[0126] Example 11: Use of nanoMIPs to detect SARS-CoV-2 in clinical samples A 3D printed resin additive cell prototype (FIGS. 11a and 11b) was developed for clinical analysis using disposable parts. As in Example 10, the thermal measurement device was controlled using LabView software and the feedback on the signal was regulated by a proportional-integral-derivative (PID) controller attached to a power resistor (22 Ω). The PID parameters were optimized to reduce noise and were set to P=1, I=14, and D=0.

[0127] To validate the design of the spike cell, we performed a thermal detection experiment using PBS containing the SARS-CoV-2 spike protein. The spike cell sensor showed a good thermal response to the spike protein (Figure 11c), with its LoD value (7.0 ± 4.0 fg mL -1 ) was significantly lower than the results obtained using the flow cell design (9.9 ± 2.5 fg mL -1 It was very similar to (Example 10).

[0128] After initial validation, clinical measurements were performed using COVID-positive (positive after <20 PCR cycles) and COVID-negative patient samples (n=7).

[0129] UTM and VPM (virus storage medium) were used as reference solutions for negative and positive samples, respectively. During the measurement, 100 μL of reference solution (UTM / VPM) was pipetted into the reservoir and R thThe signal was allowed to stabilize for 10 minutes. Afterwards, the reference solution was removed using a pipette and 100 μL of sample was added. It should be noted that the use of a pipette reduces adverse effects on the system (e.g., flow and the addition of air bubbles) compared to the use of a syringe pump. In this case, the sample volume is equivalent to that obtained with a throat swab and a nasal swab (100 μL), the measurement time is shortened to about 15 minutes, and the operation of the device is simple, which is very advantageous for clinical analysis.

[0130] The thermal detection results (Figure 11d) show that specific binding to the nanoMIP cavity occurs during the measurement of positive samples, resulting in an average ΔR th The value increased (0.27°C·W -1 In contrast, when measuring negative samples, only non-specific binding occurs to the nanoMIP, with a mean ΔR th is 0.00℃·W -1 It became. ΔR th The full range of values ​​is for the negative sample (0.10 °C W -1 ) compared to the positive sample (0.41°C W -1 ), which is due to the variability of viral load in different COVID-positive patients. Importantly, the maximum ΔR in negative samples th is the minimum ΔR in positive samples th The measurement time was approximately one-quarter of that of the control, so there was no overlap between positive and negative measurements. This highlights that the nanoMIP sensor has excellent sensitivity and specificity for the detection of SARS-CoV-2 in clinical samples. Moreover, the measurement time of the nanoMIP sensor (approximately 15 min) is comparable to commercially available rapid antigen tests, which is of great importance for potential mass screening applications.

[0131] Example 12: Analysis of binding between nanoMIPs and alpha, beta, and gamma variants of SARS-CoV-2 Synthesis of nano-MIPs Following the procedure of Example 1, peptide 1 was immobilized on silanized glass beads prior to nanoMIP synthesis.

[0132] Imprinted nanoMIPs were synthesized using peptide-coated glass beads as follows: The monomer solution (see Example 1) was degassed under vacuum, sonicated for 5 min, purged with N2 for 20 min, and added to 60 g of peptide 1-coated glass beads. Polymerization was initiated by adding aqueous ammonium persulfate (800 μL, 60 mg / mL) and N,N,N',N'-tetramethylethylenediamine (24 μL) (both from Sigma Aldrich). The headspace was flushed with N2 and the bottle was sealed with a screw cap. Polymerization was carried out at room temperature for 1 h. The contents of the polymerization vessel were then poured into a solid phase extraction (SPE) cartridge (60 mL) equipped with a frit (porosity 20 μm). A total of nine washes with 20 mL of distilled water at 20° C. were performed to remove the low affinity nanoMIP, polymer, and unreacted monomer. The SPE cartridge containing the solid phase was then placed in a water bath at 70° C. for 15 min. An aliquot of 20 mL of distilled water pre-warmed to 65 °C was poured into the SPE to recover the high affinity nanoMIPs. This was repeated five times to recover approximately 100 mL of high affinity nanoMIPs in water. To ensure complete removal of potential unreacted monomers from the majority of nanoparticles, the recovered solution was concentrated and dialyzed using a SnakeSkin membrane (molecular weight cutoff 10 kDa).

[0133] The average diameter of the imprinted nanoMIP, calculated with NanoSight NS300, was 69.3 nm (Figure 12).

[0134] Assembling the Sensor Once the peptide 1-imprinted nanoMIP was fabricated, it was integrated into a silver nanoparticle (AgNP)-based localized surface plasmon resonance (LSPR) sensor.

[0135] More specifically, 50 μl of nanoMIP stock solution (5 μg / ml in DI water) was dispensed onto the Ag-LSPR chip. The Ag chip was then placed in a humidified chamber for 3 h to ensure that the nanoMIPs were immobilized on the surface of the Ag nanoparticles. Since the nanoMIPs have amine groups, the nanoMIPs were bound to the surface by electrostatic interactions (-Ag and NH3+) between the negative LSPR Ag surface and the positively charged nanoMIPs. After this, the Ag substrate was thoroughly rinsed with DI water to remove any loosely bound polymer from the electrode surface. The chip was then stored at 4 °C.

[0136] Detection performance of nanoMIP-LSPR sensor The LSPR performance of the nanoMIP-LSPR sensor was then evaluated by detecting spike proteins of alpha, beta, and gamma variants of the SARS-CoV-2 virus in PBS (all purchased from Antibody Online: alpha, SARS-CoV-2 spike protein lineage B.1.1.7, product number: ABIN6963738; beta, SARS-CoV-2 spike protein lineage B.1.351, product number: ABIN6963739; gamma, SARS-CoV-2 spike protein lineage P.1, product number: ABIN6964442). As a control, the LSPR performance of the nanoMIP-LSPR sensor was tested against the spike proteins of human coronavirus strains HCoVOC43, HKU1, and HCoV-229E, and the LSPR performance of the NIP-based LSPR sensor was also tested.

[0137] As shown in Figure 13, the sensor successfully detected all SARS-CoV-2 mutants. Figure 13a shows the typical LSPR sensor response upon binding to various concentrations of the alpha mutant along with the corresponding absorbance. Figure 13b shows the change in LSPR wavelength of the Ag-MIP complex upon varying the concentrations of alpha, beta, and gamma from 10 aM to 100 nM.

[0138] The limits of detection (LODs) using wavelength data were found to be 466.37 nm, 467.13 nm, and 467.71 nm for alpha, beta, and gamma, corresponding to 9.71 fM, 7.32 fM, and 8.81 pM, respectively. The LODs were calculated using empirical formulas, which involved using the limit of blank (LOB) and standard deviation of the measurements, where blank refers to the effect of PBS (no protein) on the MIP (see below).

[0139] Calculating LOD To obtain the LOD of the LSPR sensor, the wavelength shift was calculated using Equation 1 and Equation 2 and then converted to the concentration of alpha, beta, and gamma mutant spike proteins.

[0140] Blank Limit (LOB) = Average ブランク +1.645(SD ブランク )(Formula 1) Limit of detection (LOD) = LOB + 1.645 (SD 最低濃度 )(Formula 2)

[0141] After successful detection of virus samples in PBS, similar experiments were performed with human serum, a clinically relevant fluid. In particular, alpha, beta, and gamma spike proteins at concentrations of 100 fM, 10 pM, 1 nM, and 100 nM were added to the serum and the changes in wavelength and absorbance were measured. As in the above experiments with PBS, control experiments were also performed with spike proteins from human coronavirus strains HCoVOC43, HKU1, and HCoV-229E to test the LSPR performance of the NIP-based LSPR sensor. Figure 14a shows a typical LSPR sensor response to serum spiked with various concentrations of alpha spike protein. Figure 14b shows the change in LSPR wavelength of the Ag-MIP complex upon varying the concentrations of alpha, beta, and gamma spike proteins from 10 aM to 100 nM.

[0142] The calculated LODs for alpha and beta using the wavelength data were found to be 457.54 nm and 460.49 nm. These LOD values ​​correspond to concentrations of 14 fM for alpha and 94 fM for beta. For the observed wavelength shift for gamma binding, the calculated LOD was 130 fM (457.37 nm). The method for determining the LOD values ​​is described above. Note that in the calculation of the LOB required to calculate the LOD, the measurements in protein-free serum were considered as the blank sample for this experiment.

[0143] For each of the above experiments in PBS and serum, all spike proteins, including the human coronavirus spike protein used as a control, were prepared or diluted in different concentrations (10 aM to 100 nM) with PBS / serum. The Ag-MIP-functionalized substrate was exposed to each protein at different concentrations. This was done by drop-casting 50 μl of sample onto the Ag-MIP. After exposing the sensor surface to a given concentration, a 20 min incubation was performed to allow the protein to interact with the MIP. The sensor surface was then washed with PBS (both PBS and serum samples) and the LSPR signal was measured. To acquire the signal, multiple spectra were acquired by ocean view software (see below) for 30 seconds and an average of 10 spectra was displayed with a boxcar width of 5. The LSPR spectra were then saved and the wavelength / absorbance changes were recorded, analyzed, and plotted using Graphpad Prism software.

[0144] LSPR signals were acquired using an in-house setup consisting of components purchased from Ocean Optics: FLAME-T-XR1-ES spectrometer, QR400-7-SR-BX reflectance probe, UV-VIS patch connector, DH-2000 deuterium-tungsten halogen lamp (DH2000-S-DUV-TTL), RTL-T stage, and Ocean View software. Prior to acquiring the LSPR spectrum, a dark reference signal was measured using a glass slide as a reference to cancel background noise. This glass slide was the same as the substrate on which Ag was deposited. This reference substrate was created by completely removing Ag nanoparticles from one of the substrates by sonicating it in acetone for 1 hour and then wiping the surface with isopropanol. All data generated were analyzed and plotted using the built-in features of GraphPad Prism 9 software.

[0145] Example 13: Surface Plasmon Resonance (SPR) Analysis of NanoMIPs Against SARS-CoV-2 Spike Proteins from Two SARS-CoV-2 Mutants In this study, a Biacore T200 instrument was employed.

[0146] Surface activation and immobilization of nano-MIPs NanoMIP-SPR sensors were prepared by covalently immobilizing nanoMIP (from Example 1) onto a Cytiva CM5 chip according to the manufacturer's guidelines.

[0147] More specifically, the device was prepared with water at a constant flow rate of 5 μL / min. Water was used as the running buffer throughout the chip preparation at a constant flow rate of 5 μL / min. Water was run until a stable baseline was achieved. A mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (400 uL water containing 2 mg EDC + 3 mg NHS) was injected over the entire sensor surface (8 min at a flow rate of 5 μL / min) to activate the surface (the mixture converts the carboxyl end groups on the chip surface to N-hydroxy-succinimide esters). NanoMIPs (5 nM) were then injected into several flow cells on the chip (7 min at a flow rate of 5 μL / min). Finally, unreacted NHS esters were deactivated by injecting ethanolamine into water.

[0148] Detection of interactions between nanoMIPs and SARS-CoV-2 spike proteins from two SARS-CoV-2 variants Once a stable baseline was obtained by switching the running buffer from water to PBS, five dilutions (1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM, and 100 nM in PBS) of recombinant SARS-CoV-2 S protein (recombinant full-length SARS-CoV-2 spike glycoprotein sourced from Native Antigen Company, Inc., either Wuhan Hu1 original or Omicron strain) were injected sequentially with minimal dissociation time occurring only due to transition from one solution to the next (309 s at 5 μl / min). The results of the two spike protein variants were then overlaid so that the relative magnitude of the binding response could be compared. As shown in Figure 15, the nanoMIP continued to function despite the mutation in the Omicron strain spike protein, with only a slight degradation in performance. D It was not considered necessary to derive a value.

[0149] (References) 1.Wrapp et al., Science 367, 1260-1263 (2020) 2.van Grinsven, B.; Eersels, K.; Peeters, M.; Losada-Perez, P.; Vandenryt, T.; Cleij, T. J.; Wagner, P. The Heat-Transfer Method: A Versatile Low-Cost, Label-Free, Fast, and User-Friendly Readout Platform for Biosensor Applications. ACS Appl. Mater. Interfaces 2014, 6, 13309-13318. 3.Geerets, B.; Peeters, M.; van Grinsven, B.; Bers, K.; de Ceuninck, W.; Wagner, P. Optimizing the Thermal Read-out Technique for MIP-Based Biomimetic Sensors: Towards Nanomolar Detection Limits. Sensors 2013, 13, 9148-9159. 4.Diagnostics Wantai SARS-CoV-2. WANTAI SARS-CoV-2 Ag Rapid Test (Colloidal Gold); 2020. 5.Grant, B. D.; Anderson, C. E.; Williford, J. R.; Alonzo, L. F.; Glukhova, V. A.; Boyle, D. S.; Weigl, B. H.; Nichols, K. P. SARS-CoV-2 Coronavirus Nucleocapsid Antigen Detecting Half-Strip Lateral Flow Assay toward the Development of Point of Care Tests Using Commercially Available Reagents. Anal. Chem. 2020, 92, 11305-11309. 6.Zhang, C.; Zhou, L.; Du, K.; Zhang, Y.; Wang, J.; Chen, L.; Lyu, Y.; Li, J.; Liu, H.; Huo, J.; Li, F.; Wang, J.; Sang, P.; Lin, S.; Xiao, Y.; Zhang, K.; He, K. Foundation and Clinical Evaluation of a New Method for Detecting SARS-CoV-2 Antigen by Fluorescent Microsphere Immunochromatography. Front. Cell. Infect. Microbiol. 2020, 10, 10.3389 / fcimb.2020.553837. 7.Liu, D.; Ju, C.; Han, C.; Shi, R.; Chen, X.; Duan, D.; Yan, J.; Yan, X. Nanozyme Chemiluminescence Paper Test for Rapid and Sensitive Detection of SARS-CoV-2 Antigen. Biosens. Bioelectron. 2021, 173, 10.1016 / j.bios.2020.112817. 8.Rahmati, Z.; Roushani, M.; Hosseini, H.; Choobin, H. Electrochemical Immunosensor with Cu2O Nanocube Coating for Detection of SARS-CoV-2 Spike Protein. Microchim. Acta 2021, 188, 10.1007 / s00604-021-04762-04769. 9.Raziq, A.; Kidakova, A.; Boroznjak, R.; Reut, J.; Opik, A.; Syritski, V. Development of a Portable MIP-Based Electrochemical Sensor for Detection of SARS-CoV-2 Antigen. Biosens. Bioelectron. 2021, 178, 10.1016 / j.bios.2021.113029. 10.Seo, G.; Lee, G.; Kim, M. J.; Baek, S. H.; Choi, M.; Ku, K. B.; Lee, C. S.; Jun, S.; Park, D.; Kim, H. G.; Kim, S. J.; Lee, J. O.; Kim, B. T.; Park, E. C.; Kim, S. Il. Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor. ACS Nano 2020, 14, 5135-5142. 11.Moitra, P.; Alafeef, M.; Alafeef, M.; Alafeef, M.; Dighe, K.; Frieman, M. B.; Pan, D.; Pan, D.; Pan, D. Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene 11 Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles. ACS Nano 2020, 14, 7617-7627. 12.Mavrikou, S.; Moschopoulou, G.; Tsekouras, V.; Kintzios, S. Development of a Portable, Ultra-Rapid and Ultra-Sensitive Cell-Based Biosensor for the Direct Detection of the SARS-CoV-2 S1 Spike Protein Antigen. Sensors 2020, 20, 10.3390 / s20113121. 13.Zhang, M.; Li, X.; Pan, J.; Zhang, Y.; Zhang, L.; Wang, C. Ultrasensitive Detection of SARS-CoV-2 Spike Protein in Untreated Saliva Using SERS-Based Biosensor. Biosens. Bioelectron. 2021, 190, 10.1016 / j.bios.2021.113421. 14.Yakoh, A.; Pimpitak, U.; Rengpipat, S.; Hirankarn, N.; Chailapakul, O.; Chaiyo, S. PaperBased Electrochemical Biosensor for Diagnosing COVID-19: Detection of SARS-CoV-2 Antibodies and Antigen. Biosens. Bioelectron. 2021, 176, 10.1016 / j.bios.2020.112912. 15. Fabiani, L.; Saroglia, M.; Galata, G.; De Santis, R.; Fillo, S.; Luca, V.; Faggioni, G.; D’Amore, N.; Regalbuto, E.; Salvatori, P.; Terova, G.; Moscone, D.; Lista, F.; Arduini, F. Magnetic Beads Combined with Carbon Black-Based Screen-Printed Electrodes for COVID-19: A Reliable and Miniaturized Electrochemical Immunosensor for SARS-CoV2 Detection in Saliva. Biosens. Bioelectron. 2021, 171, 10.1016 / j.bios.2020.112686.

[0150] (Overview of the array)

[0151] [Table 2]

Claims

1. A molecularly imprinted polymer comprising at least one recognition site complementary to a template molecule consisting of an amino acid sequence corresponding to a partial sequence of the receptor binding domain of SARS-CoV-2 spike protein, the amino acid sequence is 50 amino acids or less in length and comprises the sequence NSNNLDSKVGG; The polymer (i) N-fluoresceinyl acrylamide, (ii) acrylamide, (iii) tert-butyl acrylate (TBAc); (iv) 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose, (v) 2,2,2-trifluoroethyl methacrylate (CF3), (vi) N-(3-aminopropyl)methacrylamide hydrochloride (APMA), (vii) squaramide monomer 7 (SQ7), and (viii) N,N'-methylenebis(acrylamide) (BIS) 1. A molecularly imprinted polymer comprising at least one monomer selected from the group consisting of:

2. The molecularly imprinted polymer of claim 1, wherein the molecularly imprinted polymer comprises monomers of N-fluoresceinyl acrylamide and / or 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose.

3. The molecularly imprinted polymer of claim 2, wherein the molecularly imprinted polymer comprises monomers of N-fluoresceinyl acrylamide and 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose.

4. The molecularly imprinted polymer of claim 1, wherein the molecularly imprinted polymer comprises monomers of N-fluoresceinyl acrylamide, acrylamide, tert-butyl acrylate (TBAc), 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose, 2,2,2-trifluoroethyl methacrylate (CF3), N-(3-aminopropyl)methacrylamide hydrochloride (APMA), squaramide monomer 7 (SQ7), and N,N'-methylenebis(acrylamide) (BIS).

5. 5. The molecularly imprinted polymer of claim 1, wherein the size of the polymer is less than 500 nm.

6. 5. The molecularly imprinted polymer of claim 1, wherein the size of the polymer is less than 250 nm.

7. 5. The molecularly imprinted polymer of claim 1, wherein the size of the polymer is less than 100 nm.

8. 1. A method for preparing a molecularly imprinted polymer comprising at least one recognition site that binds to SARS-CoV-2, comprising: (a) providing a carrier material having a template molecule comprising an amino acid sequence corresponding to a partial sequence of the receptor-binding domain of the SARS-CoV-2 spike protein, wherein the template molecule is immobilized on the carrier material so as to be exposed on the surface; (b) providing a polymerizable composition in contact with the surface; (c) controlling polymerization of the polymerizable composition in contact with the surface to form a molecularly imprinted polymer; (d) separating the molecularly imprinted polymer from the surface and the immobilized template molecules; the amino acid sequence is 50 amino acids or less in length and comprises the sequence NSNNLDSKVGG; The polymer is (i) N-fluoresceinyl acrylamide, (ii) acrylamide, (iii) tert-butyl acrylate (TBAc); (iv) 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose, (v) 2,2,2-trifluoroethyl methacrylate (CF3), (vi) N-(3-aminopropyl)methacrylamide hydrochloride (APMA), (vii) squaramide monomer 7 (SQ7), and (viii) N,N'-methylenebis(acrylamide) (BIS) at least one monomer selected from the group consisting of:

9. The method of claim 8, wherein the polymerizable composition comprises monomers of N-fluoresceinyl acrylamide and / or 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose.

10. The method of claim 9, wherein the polymerizable composition comprises monomers of N-fluoresceinyl acrylamide and 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose.

11. The method of claim 8, wherein the polymerizable composition comprises monomers of N-fluoresceinyl acrylamide, acrylamide, tert-butyl acrylate (TBAc), 3-O-acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose, 2,2,2-trifluoroethyl methacrylate (CF3), N-(3-aminopropyl)methacrylamide hydrochloride (APMA), squaramide monomer 7 (SQ7), and N,N'-methylenebis(acrylamide) (BIS).

12. 12. The method of any one of claims 8 to 11, wherein the N-terminus of the template molecule is modified to include an additional cysteine ​​residue.

13. 13. The method of claim 12, wherein the template molecule is modified to include a glycine residue between the cysteine ​​residue and the last residue of the template.

14. (i) a molecularly imprinted polymer according to any one of claims 1 to 7; (ii) a fluorescent material; and A complex comprising:

15. Use of (1) the molecularly imprinted polymer of any one of claims 1 to 7, or (2) the conjugate of claim 14 in detecting SARS-CoV-2.

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