Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
  • B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
  • B01J20/28004—Sorbent size or size distribution, e.g. particle size
  • B01J20/28007—Sorbent size or size distribution, e.g. particle size with size in the range 1-100 nanometers, e.g. nanosized particles, nanofibers, nanotubes, nanowires or the like
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/3085—Chemical treatments not covered by groups B01J20/3007 - B01J20/3078
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
  • B01J20/28004—Sorbent size or size distribution, e.g. particle size
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

• the present invention relates to compounds for detection of SARS-CoV-2, to methods of producing the same and to conjugates comprising the same, to compositions for detection of SARS-CoV-2 and more particularly to use of the aforementioned in the detection of SARS-CoV-2.
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• RT-PCR reverse transcription polymerase chain reaction
• RT-PCR testing Whilst RT-PCR testing is widespread, there are problems. Conducting RT-PCR tests can be labour intensive: they require highly skilled personnel and false negative results can occur e.g. due to sampling error or improper sample handling. Indeed false positive results may also occur e.g. due to sample cross-contamination. The tests are also expensive and there is a long turn-around time (1-2 days) with respect to results.
• Rapid antigen tests are also used for population screening and offer portable and rapid (15-30 min) analysis that can be performed at the convenience of the individual.
• Molecularly imprinted polymers have been proposed as alternative receptors for use in the detection of SARS-CoV-2.
• Cubuk et al in “Computational analysis of functional monomers used in molecular imprinting for promising COVID-19 detection” describes computational studies aimed at identifying a template and suitable monomers to produce a MIP against SARS-CoV- 2.
• WO 2021/195626 A1 discloses the preparation of MIPs to SARS-CoV-2 wherein the whole virus is used as a template.
• MIPs Methods of producing MIPs are also known - see e.g. US 10 189 934B2, Piletsky et al in “Molecularly Imprinted Polymers for Cell Recognition” and Malik et al in “Molecular Imprinted Polymer for human viral pathogen detection”.
• Antibody tests in contrast to RT-PCR and rapid antigen tests, are carried out in an attempt to establish whether a person may have already had the virus rather than, in the case of RT-PCR and rapid antigen tests, whether the person currently has the virus. Antibody tests are often carried out on blood samples from patients and work by detecting the presence of binding between synthetic antigens and antibodies (if present) in a patient’s blood sample. Accordingly, one aim of the present invention is to provide alternative means for detecting SARS-CoV-2. A further aim is that such means will provide an alternative to the polymerase chain reaction-based assay and rapid antigen tests currently used. An aim of specific embodiments of the invention includes providing improved detection means.
• the invention provides a molecularly imprinted polymer comprising at least one recognition site that is complementary to a template molecule consisting of an amino acid sequence corresponding to a subsequence of the receptor binding domain of SARS-CoV-2 spike protein, wherein the amino acid sequence is no more than 50 amino acids in length and comprises a sequence selected from:
• the invention also provides a method of preparing a molecularly imprinted polymer comprising at least one recognition site which binds SARS-CoV-2 comprising the steps of:
• amino acid sequence is no more than 50 amino acids in length and comprises a sequence selected from:
• conjugate comprising the molecularly imprinted polymer of the invention and a fluorophore.
• compositions comprising the molecularly imprinted polymer or the conjugate of the invention.
• the invention provides a molecularly imprinted polymer comprising at least one recognition site that is complementary to a template molecule consisting of an amino acid sequence corresponding to a subsequence of the receptor binding domain of SARS-CoV-2 spike protein, wherein the amino acid sequence is no more than 50 amino acids in length and comprises a sequence selected from:
• SARS-CoV- 2 The sequence of SARS-CoV-2 is known.
• SARS-CoV-2 makes use of a densely glycosylated spike protein to gain entry into host cells.
• the spike protein is a homotrimeric glycoprotein and each monomer contains 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 engagement.
• 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 subsequence of the receptor binding domain of SARS- CoV-2 spike protein, wherein the amino acid sequence is no more than 50 amino acids in length and comprises a sequence selected from (i) NSNNLDSKVGG, (ii) NYNYLYRLFRKS, (iii) YRLFRKSNLKPF, (iv) STEIYQAGSTPC, (v) CNGVEGFNCYF, (vi) GSTPCNGVEGF, (vii) CYFPLQSYGFQP, (viii) GFQPTNGVGYQ and (ix) LQSYGFQPTNG.
• a template molecule consisting of an amino acid sequence corresponding to a subsequence of the receptor binding domain of SARS- CoV-2 spike protein, wherein the amino acid sequence is no more than 50 amino acids in length and comprises a sequence selected from (i) NS
• 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 subsequence of the receptor binding domain of SARS-CoV-2 spike protein, wherein the amino acid sequence is no more than 50 amino acids in length and comprises a sequence selected from (i) NSNNLDSKVGG, (ii) STEIYQAGSTPC and (iii) CYFPLQSYGFQP.
• 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 subsequence of the receptor binding domain of SARS-CoV-2 spike protein, wherein the amino acid sequence is no more than 50 amino acids in length and comprises the sequence NSNNLDSKVGG.
• 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 subsequence of the receptor binding domain of SARS-CoV-2 spike protein, wherein the amino acid sequence is no more than 50 amino acids in length and comprises the sequence CYFPLQSYGFQP.
• the size of the template is in the range up to 50 amino acids.
• the template will be less than 50 amino acids, more preferably the template will be less than 30 amino acids; most preferred is that the template is less than 20 amino acids, for example 11 , 12, 13, 14, 15, 16, 17, 18 or 19 amino acids.
• the use of short peptide sequences (as opposed to e.g. the whole spike protein or RBD) drastically reduces reagent costs and creates high affinity binding sites for a specific region of the target protein, therefore enhancing the monoclonality of the molecularly imprinted polymer.
• the size of the molecularly imprinted polymer of the present invention will be to a large extent dictated by the intended downstream use of the molecular imprinted polymer. That said, suitably the molecular imprinted polymer will be less than 500nm, more suitably less than 250nm and even more suitably less than 100nm, and may be less than 90nm, or even less than 70nm.
• compositions comprising the molecularly imprinted polymer of the invention.
• molecular imprinted polymers in such compositions will have an average size that is less than 500nm, more suitably less than 250nm and even more suitably less than 100nm, and may be less than 90nm, or even less than 70nm.
• Suitable methods for determining the size of molecularly imprinted polymers include dynamic light scattering e.g. using a Zetasizer Ultra (Malvern Panalytical), disc centrifugation, nanoparticle tracking analysis e.g. using 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 provided herein, the size of the molecularly imprinted polymers was determined using a NanoSight NS300 instrument.
• Size in the context of the molecularly imprinted polymer refers to the diameter of the molecularly imprinted polymer.
• Monomers which can be used in the preparation of molecularly imprinted polymers include vinyl monomers, allyl monomers, acetylenes, acrylates, methacrylates, acrylamides, methacrylamides, chloroacrylates, itaconates, trifluoromethylacrylates, derivatives of amino acids (e.g. esters or amides), nucleosides, nucleotides, and carbohydrates.
• Cross-linking monomers which help to stabilise molecularly imprinted polymers, may also be included.
• cross-linking monomers suitable for preparing molecularly imprinted polymers include, but are not limited to, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, divinylbenzene, methylene bisacrylamide, ethylene bisacrylamide, N,N’-bisacryloylpiperazine and N,N'- Methylenebis(acrylamide) (BIS).
• ethylene glycol dimethacrylate trimethylolpropane trimethacrylate
• divinylbenzene divinylbenzene
• methylene bisacrylamide ethylene bisacrylamide
• N,N’-bisacryloylpiperazine and N,N'- Methylenebis(acrylamide) (BIS)
• Those skilled in the art could select monomers and cross-linking monomers suitable for a particular template. Alternatively a variety of combinatorial and computational methods could be used to assist in this selection.
• the molecularly imprinted polymer will comprise at least one monomer 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) Squareamide monomer 7 (SQ7) and (viii) N,N'-Methylenebis(acrylamide) (BIS).
• APMA N-(3-Aminopropyl) methacrylamide hydrochloride
• SQ7 Squareamide monomer 7
• BIOS N,N'-Methylenebis(acrylamide)
• the molecularly imprinted polymer will comprise at least N-Fluoresceinyl acrylamide and/or 3-O-acryloyl-1,2;5,6-bis-O-isopropylidene-D-glucofuranose. Most preferably, the molecularly imprinted polymer will comprise all of (i) to (viii). In this context it is understood that the molecularly imprinted polymer is a polymer and is said to comprise an identified monomer when it has been made by polymerising a mixture of monomers comprising the identified monomer.
• the molecularly imprinted polymers of the present invention are robust and function in wide ranges of temperature and pH which leads to improved shelf-life and storage conditions and means that they can function in more challenging environments. They also have excellent biocompatibility and have fast binding kinetics.
• a method of preparing a molecularly imprinted polymer comprising at least one recognition site which binds SARS-CoV-2 comprising the steps of: (a) providing a carrier substance having a template molecule consisting of an amino acid sequence corresponding to a subsequence of the receptor binding domain of SARS-CoV-2 spike protein immobilised on it so as to be exposed at a surface;
• amino acid sequence is no more than 50 amino acids in length and comprises a sequence selected from:
• An advantage of the method is that the MIPs possess a more homogenous distribution of binding site affinities, and provides means by which template elution is enhanced and downstream template leaching is prevented.
• Suitable carrier substances include polymer resins, polysaccharides, glass or metal surfaces.
• the carrier substance may be in the form of beads, fibres, membranes or capillaries.
• the carrier substance is glass. Further preferred is that the carrier substance is in the form of glass beads.
• Step (c) involves the application of controlled polymerisation.
• Various techniques for producing molecularly imprinted polymers by controlled polymerisation are known to those of skill in the art.
• controlled polymerisation examples include radical polymerisation such as controlled living radical polymerisation (LRP), living anionic polymerisation, living cationic polymerisation, and controlled polycondensation.
• LRP controlled living radical polymerisation
• living anionic polymerisation living anionic polymerisation
• living cationic polymerisation living cationic polymerisation
• controlled polycondensation controlled polycondensation
• Polymerisation can be initiated by e.g. heating, by applying current (electropolymerisation), by the addition of redox catalyst(s), persulfate or peroxides, by irradiation, including gamma radiation or by microwave radiation or by irradiation with UV or visible light and normally takes minutes to hours.
• redox catalyst(s) including gamma radiation or by microwave radiation or by irradiation with UV or visible light and normally takes minutes to hours.
• irradiation including gamma radiation or by microwave radiation or by irradiation with UV or visible light and normally takes minutes to hours.
• polymerisation was initiated using persulfate.
• the polymerisation reaction is terminated at a stage when the size of the synthesized molecular imprinted polymer is relatively small. Preferences with respect to size are discussed elsewhere herein.
• the separation of high affinity molecularly imprinted polymers from the immobilised template can be achieved by heating, which disrupts complex formation, by changing solution pH, changing ionic strength, or through the additional of urea, guanidine or a substance which interacts with the template more strongly than the molecularly imprinted polymer.
• weakly bound material is removed by washing.
• the molecularly imprinted polymers may be purified further e.g. by chromatography, filtration and/or electrophoresis.
• the amino acid sequence is no more than 50 amino acids in length and comprises a sequence selected from: (i) NSNNLDSKVGG, (ii) STEIYQAGSTPC and (iii) CYFPLQSYGFQP.
• the amino acid sequence is no more than 50 amino acids in length and comprises the sequence NSNNLDSKVGG.
• the amino acid sequence is no more than 50 amino acids in length and comprises the sequence CYFPLQSYGFQP.
• the method by which the template molecule is immobilised on the carrier substance is not critical. However, for some methods, the presence of a cysteine residue at one of the termini of the template molecule is advantageous. As such, for embodiments of the method where the template molecule does not naturally comprise a terminal cysteine residue, it is preferred that the template molecule is modified at the N terminus to comprise an additional cysteine residue.
• the additional cysteine residue is attached directly to the termini of the template molecule whilst in other embodiments a spacer is included between the terminal cysteine and the template molecule.
• the spacer comprises a glycine residue.
• the template molecule comprises the amino acid sequence NSNNLDSKVGG and is modified at the N terminus to comprise a cysteine (C) residue and a glycine (G) residue such that the molecule used for imprinting is CGNSNNLDSKVGG.
• the template molecule is directly attached to the carrier substance whilst in other embodiments one or more linkers are included between the carrier substance and the template molecule.
• Suitable linkers for use in the invention are well known to those of skill in the art.
• the carrier substance used was glass beads and these were silanised using (3- aminopropyl)trimethoxysilane (APTMS) prior to immobilisation of the template molecule.
• the APTMS acts as a linker between the surface of the glass bead and the template molecule.
• the APTMS was modified using n-succinimidyl iodoacetate (SIA) prior to template immobilisation. Linkers and spacers function to prevent adverse effects brought about by steric hindrance.
• SIA n-succinimidyl iodoacetate
• An advantage of the method of the present invention is that it is fast and scalable and uses only a short template.
• conjugate comprising the molecularly imprinted polymer of the invention and a fluorophore.
• Fluorophores are molecules with the ability to absorb light at a particular wavelength, the absorbance or excitation wavelength, and then emit light at a higher wavelength, the emission wavelength.
• fluorophores can be conjugated via amino, thiol or carbohydrate groups.
• the fluorophore was conjugated to the molecularly imprinted polymer via amine groups provided by the amino monomer in the molecularly imprinted polymer.
• any fluorophore can be used in conjugates of the present invention, it is advantageous for some downstream applications, particularly diagnostic applications, to select a fluorophore that is both bright and stable.
• Conjugated Polymer Nanoparticles are highly fluorescent nanoparticles which comprise of a semiconductor light emitting polymer (LEP) core encapsulated within a biocompatible surfactant.
• CPNs are particularly suited for use in diagnostic applications due to their intense fluorescence, which means they have a high level of sensitivity, and their high degree of photostability.
• CPNs are around 70-80nm in size and those currently available from Stream Bio exhibit fluorescence with emission wavelengths from 420nm to 1130nm.
• molecularly imprinted polymers of the invention were conjugated to CPN510 and CPN610, both available from Stream Bio.
• the fluorophore is a CPN.
• compositions comprising the conjugate of the invention.
• the molecularly imprinted polymers, conjugates and compositions of the present invention may be used in a variety of applications. It is specifically envisaged that the molecularly imprinted polymers, conjugates and compositions of the present invention be used in the detection of SARS-CoV-2.
• the molecularly imprinted polymers, conjugates and compositions may be used in assays for example diagnostic assays which may be used in a medical or point of care setting. Examples of such assays include lateral flow assays (LFA) and enzyme-linked immunosorbent assay (ELISA)-type assays which may be used for high throughput screening.
• LFA lateral flow assays
• ELISA enzyme-linked immunosorbent assay
• the molecularly imprinted polymers, conjugates and compositions of the present invention may also be used in sensors.
• nanoMIP-SPR sensors were prepared by covalently immobilising nanoMIPs on 4% mercaptoundecanoic acid (MUDA) chips (prepared using Biacore SIA Kit Au surfaces, functionalised with a self-assembled monolayer of 4% MUDA in ethanol).
• MUDA mercaptoundecanoic acid
• SPEs Screen-Printed Electrodes
• the molecularly imprinted polymers, conjugates and compositions of the present invention may also be used in the detection of SARS-CoV-2 in the research setting.
• the molecularly imprinted polymers, conjugates and compositions of the present invention may also be used as pharmaceuticals. It is desirable that the molecularly imprinted polymers of the present invention have high affinity for SARS-CoV-2.
• High affinity in the context of the present specification means that the affinity with which the molecularly imprinted polymers bind SARS-CoV- 2 exceeds the affinity with which ‘blank’ molecularly imprinted polymers bind SARS- CoV-2 by at least three, preferably at least five, more preferably at least 10 times.
• Blank in this context means molecularly imprinted polymers formed in the absence of template. Binding affinity is the strength of the binding interaction between a single molecule to its binding partner e.g.
• a molecularly imprinted polymer to its target SARS-CoV-2. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD). The smaller the KD value, the greater the binding affinity between the two molecules.
• K D equilibrium dissociation constant
• the molecularly imprinted polymer binds to SARS-CoV-2 with a K D of less than 500nM, preferably less than 250nM and even more preferably less than 200nM.
• Suitable methods for measuring K D include surface plasmon resonance (SPR) which may be conducted using e.g.
• Figure 1 shows two surface plasmon resonance sensorgrams showing the binding kinetics for nanoMIPs imprinted against peptide 1 and SARS-CoV-2 S recombinant protein
• Figure 2 is a trace obtained using a NanoSight NS300 instrument showing the successful conjugation of a nanoMIP imprinted against peptide 1 to CPN510
• Figure 3 is a dot blot showing specificity of binding between nanoMIPs imprinted against peptides 1 and 3 and recombinant SARS-CoV-2 S protein
• Figure 4 is a dot blot showing specificity of binding between nanoMIPs imprinted against peptide 1 and SARS-CoV-2
• Figure 5 is a dot blot showing specificity of binding between nanoMIPs imprinted against peptide 1 and SARS-CoV-2 Spike Glycoprotein (S1);
• Figure 9a shows typical atomic force microscopy (AFM) height images of an isolated nanoMIP on an Au surface in air (represented by the green box in the larger scale image) at room temperature, 37 °C, and 50 °C.
• Figure 9b shows a corresponding cross-sectional profile plot of the nanoMIP at each temperature.
• Figure 9e shows a commercial rapid antigen test giving a false positive result when Diet Coca-Cola was used as the test liquid;
• Figure 10 reports the sensitivity of nanoMIPs imprinted against peptide 1 to various SARS-CoV-2 antigens and compares the sensitivity of the nanoMIPs to a SARS-CoV-2 antibody.
• Figure 10a shows typical Heat Transfer Method data for a nanoMIP-functionalized SPE upon exposure to PBS containing 1 fg mL -1 – 10 pg mL- 1 of the SARS-CoV-2 RBD.
• Figures 10b-10d show typical dose-response curves (error bars represent the SD) showing the thermal response of (i) nanoMIP and antibody sensors to the spike protein (nanoMIPs were tested against the SARS-CoV-2 spike protein from both the alpha and delta variants) ( Figure 10b) (ii) nanoMIP and antibody sensors to the SARS-CoV-2 RBD ( Figure 10c), and iii) nanoMIP sensors to SARS- CoV-2 antigens and the negative controls ORF8, IL-6, and HSA. The response of NIP- based sensors to the spike protein is also shown (Figure 10d).
• Figure 10e compares the LoD values for nanoMIPs of the present invention with a commercial rapid antigen test and numerous recently developed antigen tests from the literature;
• Figure 11 shows binding between nanoMIPs imprinted against peptide 1 and SARS-CoV-2 from clinical samples.
• Figure 11 a is a schematic illustration of a 3D- printed prototype addition cell: 1 ) thermocouple inlet, 2) functionalized SPE, 3) open- bottomed reservoir to facilitate the manual addition of liquid, and 4) removable lid to reduce experimental noise.
• Figure 11 b is a photograph of the addition cell shown in Figure 11 a.
• Figure 11 c shows a typical dose-response curve obtained when using the prototype addition cell for the thermal detection of the SARS-CoV-2 spike protein (1 fg mL’ 1 - 10 pg mL -1 ) in phosphate-buffered saline (PBS).
• Figure 12 shows the size of nanoMIPs measured using NanoSight particle analyzer
• Figure 13 shows nanoMIP - LSPR sensor mediated detection of SARS-CoV-2 spike proteins from SARS-CoV-2 Alpha, Beta and Gamma variants in PBS.
• Figure 13a shows an LSPR spectrum in wavelength vs absorbance plot for concentrations ranging from 10 aM to 100 nM of the Alpha variant of SARS CoV-2.
• Figure 13b shows the change in LSPR wavelength of the Ag-MIPs complex upon varying the concentration of SARS-CoV-2 Alpha, Beta and Gamma variants from 10 aM to 100 nM;
• Figure 14 shows nanoMIP - LSPR mediated detection of SARS-CoV-2 spike proteins from SARS-CoV-2 Alpha, Beta and Gamma variants in serum.
• Figure 14a shows an LSPR spectrum in wavelength vs absorbance plot for concentrations ranging from 100 fM to 100 nM of the Alpha variant of SARS-CoV-2.
• Figure 14b shows the change in LSPR wavelength of the Ag-MIPs complex upon varying the concentration of SARS-CoV-2 Alpha, Beta and Gamma variants from 100 fM to 100 nM; and
• Figure 15 shows two superimposed surface plasmon resonance sensorgrams showing the binding kinetics for nanoMIPs imprinted against peptide 1 and either the original SARS-CoV-2 S (spike) recombinant protein and or that from the Omicron variant.
• Example 1 Synthesis of NanoMIPs to SARS-CoV-2
• Supplementary Figure 5 of Wrapp et al 2020 1 presents a sequence alignment of the spike protein from SARS-CoV-2 and two other coronavirus strains, SARS-CoV and RaTG13.
• the RBD is highlighted.
• Three peptide sequences (peptide 1 : NSNNLDSKVGG, peptide 2: STEIYQAGSTPC and peptide 3: CYFPLQSYGFQP) from the RBD of SARS-CoV-2 were selected by the present inventors as potential candidates for imprinting based on the lack of conservation with the respective sequences from SARS-CoV and RaTG13.
• Peptides 1 , 2 and 3 were immobilised onto silanised glass beads prior to nanoMIP synthesis.
• Peptide 1 CGNSNNLDSKVGG
• Peptide 2 STEIYQAGSTPC
• Peptide 3 CYFPLQSYGFQP.
• Peptide immobilisation was carried out as follows: glass beads (approximately 100
• ATMS 3-aminopropyl)trimethoxysilane
• the templates (Peptides 1 , 2 or 3) were then immobilised on the surface of the glass beads by incubation in a solution of PBS, pH 7.4 with 0.4 mg/ml tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and 0.1mg/ml of peptide for a minimum of 4 hours. Excess template was removed by washing with water and methanol.
• the peptide coated glass beads were used for the synthesis of imprinted nanoMIPs as follows: monomer solutions were prepared, sonicated for 10 minutes and purged with nitrogen for 5 minutes.
• One example of a monomer solution that was prepared includes 3mg N-Fluoresceinyl acrylamide, 24.2mg Acrylamide, 31.7 ⁇ L Tert-Butyl acrylate (TBAc), 36mg 3-O-Acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose, 13.8 ⁇ L 2,2,2-Trifluoroethyl methacrylate (CF3), 3.9mg N-(3-Aminopropyl) methacrylamide hydrochloride (APMA), 1.3mg Squareamide monomer 7 (SQ7) and 9mg N,N'-Methylenebis(acrylamide) (BIS)).
• TSAc Tert-Butyl acrylate
• CF3 2,2,2-Trifluoroethyl methacrylate
• APMA N-(3-Aminopropyl) methacrylamide hydrochloride
• SQ7 Squareamide monomer 7
• BIOS 9
• NanoMIP-SPR sensors were prepared by covalently immobilising nanoMIPs (from Example 1) on 4% mercaptoundecanoic acid (MUDA) chips (prepared using Biacore SIA Kit Au surfaces, functionalised with a self-assembled monolayer of 4% MUDA in ethanol).
• MUDA mercaptoundecanoic acid
• water was utilized to prime the instrument at a constant flow rate of 5 ⁇ L/minute. Water was utilized as the running buffer throughout the chip preparation at a constant flow rate of 30 ⁇ l/minute. Water was run until a stable baseline was achieved.
• N-hydroxysuccinimide NHS
• EDC 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide
• SARS-CoV-2 S protein-SPR sensors were prepared by covalently immobilising recombinant SARS-CoV-2 S protein (recombinant full-length SARS-CoV-2 spike glycoprotein sourced from The Native Antigen Company) on 4% mercaptoundecanoic acid (MUDA) chips (prepared using Biacore SIA Kit Au surfaces, functionalised with a self-assembled monolayer of 4% MUDA in ethanol).
• MUDA mercaptoundecanoic acid
• Example 4 Conjugation of NanoMIPs to SARS-CoV-2 to CPN NanoMIPs imprinted against peptides 1 and 3 were coupled to Conjugated Polymer Nanoparticles (CPNs), highly fluorescent nanoparticles containing semiconductor Light Emitting Polymer cores encapsulated within a water friendly capping agent, at Stream Bio.
• the CPNs used were CPN510 and CPN610 which have a diameter of 70- 80nm as measured by NanoSight 3000.
• Figure 2 shows the successful conjugation of a nanoMIP imprinted against peptide 1 to CPN510.
• Example 5 Dot-blot analysis of binding between NanoMIPs and SARS-CoV-2 S protein. 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 The Native Antigen Company) were blotted onto nitrocellulose membranes before allowing the membranes to dry.
• the membranes were then blocked by soaking in 5% BSA in PBS- T (0.05% Tween-20 in PBS) for 30-60 minutes at room temperature. Post blocking, the membranes were incubated with (1) NanoMIP-CPN510 (imprinted against peptide 1), (2) NanoMIP-CPN510 (imprinted against peptide 3) or (3) CPN510 only (control) dissolved in 5% BSA in PBS-T for 30-60 minutes at room temperature. The membranes were then washed three times, for 5 minutes each time, with PBS-T. Fluorescence was then observed under UV.
• the detection limit using NanoMIP-CPN510 was 0.3ng, much lower than that achieved using CPN510 only (control) indicating that nanoMIPs imprinted against at least peptide 1 specifically interact with SARS-CoV-2 S protein.
• Example 6 Dot-blot analysis of binding between NanoMIPs and SARS-CoV-2 virus. SARS-CoV-2 (2 x 10 4 pfu) was blotted onto two nitrocellulose membranes. Also blotted on to each of the nitrocellulose membranes was 75ng of recombinant SARS- CoV-2 S protein (sourced from The Native Antigen Company) as a positive control and culture media as a negative control. The membranes were then left to dry.
• the membranes were then blocked by soaking in 5% BSA in PBS-T (0.05% Tween-20 in PBS) for 30-60 minutes at room temperature. Post blocking, the membranes were incubated with NanoMIP-CPN510 (imprinted against peptide 1) or (2) NanoMIP- CPN510 (imprinted against troponin) (negative control) dissolved in 5% BSA in PBS- T for 30-60 minutes at room temperature. The membranes were then washed three times, for 5 minutes each time, with PBS-T. Fluorescence was then observed under UV. As shown in Figure 4, no fluorescence was observed in the area of the nitrocellulose membrane blotted with culture media or when the membrane was incubated with NanoMIP-CPN510 (imprinted against troponin) (negative control).
• NanoMIP-CPN510 imprinted against peptide 1 both with respect to SARS-CoV-2 and also the SARS-CoV-2 S protein positive control. This indicates that nanoMIPs imprinted against at least peptide 1 specifically interact with SARS-CoV-2.
• Example 7 Dot-blot analysis comparing binding between (1) NanoMIPs and four different human coronaviruses (SARS-CoV-2, HCoV-OC43, HCoV-229E and HCoV-HKU1) and (2) antibodies and four different human coronaviruses (SARS- CoV-2, HCoV-OC43, HCoV-229E and HCoV-HKU1).
• SARS-CoV-2 Spike Glycoprotein (The Native Antigen Company), Human Coronavirus OC43 Spike Glycoprotein (S1) (The Native Antigen Company), Human Coronavirus 229E Spike Glycoprotein (S1) (The Native Antigen Company) and Human Coronavirus HKU1 Spike Glycoprotein (S1) (The Native Antigen Company) (0.6 mg/mL) were blotted onto nitrocellulose membranes in triplicate (as shown in the schematic in Figure 5) before allowing the membranes to dry. The membranes were then blocked by soaking in 2 mL BSA (1% in PBS) for 1 hour.
• BSA 1% in PBS
• the membranes were incubated with (1) NanoMIP – CF770 (imprinted against peptide 1) and (2) three different monoclonal antibodies specific to SARS- CoV-2 Spike (RBD), Mab RBD1106-CF770, Mab RBD107-CF770 and Mab RBD5305- CF770 (all prepared in-house and diluted in 0.01% BSA) for 30 minutes.
• the membranes were then washed with 2mL PBST for 5 minutes. Fluorescence was then observed under UV.
• the nanoMIPs selectively bound the SARS- CoV-2 Spike Glycoprotein (S1) and did not bind to any of 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.
• Example 8 Analysis of binding between NanoMIPs and SARS-CoV-2 S protein RBD using thermal detection methods. Preparation of nanoMIP-functionalized Screen-Printed Electrodes (SPEs) for use in thermal detection methods.
• SPEs Screen-Printed Electrodes
• a solution of 4-ABA (2 mM) and sodium nitrite (2 mM) in aqueous HCl (0.5 M) was prepared and gently mixed on an orbital shaker for 10 minutes.
• Screen-Printed Electrodes (SPEs) were submerged into the solution and cyclic voltammetry was performed from +0.2 V to -0.6 V at 100 mV s -1 using an Ag/AgCl reference electrode (Alvatek Ltd., Romsey, UK).
• the obtained electrode, denoted SPE/4-ABA was thoroughly rinsed with deionised water to remove any unbound 4-ABA and dried with nitrogen.
• EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
• NHS N- hydroxysuccinimide
• NanoMIP imprinted against peptide 1 ‘P1C6’ or NanoMIP imprinted against peptide 3 ‘P3C6’ was deposited onto the working electrodes following a gentle vortex.
• the SPE/4-ABA/EDC+NHS/nanoMIP electrodes were rinsed in deionised water and dried with a gentle stream of nitrogen before being stored in PBS at 4°C until use.
• SPEs nanoMIP-functionalised Screen-Printed Electrodes
• a SPE was mounted into the cell and a thermocouple measured the temperature in the liquid (T 2 ).
• T 2 a freshly prepared nanoMIP-functionalised SPE was used.
• the flow cell was connected to a heat-transfer device as described by van Grinsven et al.
• the device was steered with LabView software that actively controls the temperature of the heat sink (copper block, T 1 ), which was set to 37.00 ⁇ 0.02 °C to mimic in-vivo conditions.
• a proportional- integral-derivative (PID) controller attached to a power resistor (22 ⁇ ) regulated the feedback on the signal as described in Geerets et al.
• the flow cell was filled with PBS and left for 30 minutes to ensure stabilisation of the baseline temperature signal before a first injection of PBS was added to act as the blank measurement.
• Solutions (3 mL) of increasing concentrations of each target biomarker (SARS-CoV-2 S protein RBD or SARS-CoV-2 ORF8 (as a negative control)) (0-10 pg mL -1 ) were prepared in PBS prior to experiments and stored at 4°C until required.
• Each biomarker injection was performed at 250 ⁇ L min -1 for 12 minutes using a LSP02-1B dual channel syringe (Longer Precision Pump Co., Hebei, China) pump. Following each biomarker injection, the system was allowed to stabilise for 30 minutes prior to the next injection.
• the thermal resistance (Rth) was determined throughout the experiments by dividing the temperature gradient (T1-T2) over the power required to keep the heat sink at 37.00°C.
• the R th and standard deviation (SD) were calculated using the average of 600 data points from the baseline signal of each concentration and the initial PBS injection, respectively ( Figures 6, 7a, and 8a). This data was used to construct dose-response curves, from which the limit of detection (LoD) was calculated using the three-sigma method in the linear range of the sensor ( Figures 7b and 8b).
• the error bars in the graphs relate to the SD values.
• FIG. 6 shows that the thermal response to SARS-CoV-2 ORF8 is very limited whereas Figures 7 and 8 show that a good response was observed with SARS-CoV-2 S protein RBD for nanoMIPs imprinted against both peptides 1 and 3.
• Example 9 Analysis of nanoMIP robustness The capability of MIPs imprinted against peptide 1 to withstand extremes of temperature and pH was investigated. Temperature Atomic force microscopy (AFM) was utilized to examine how increasing temperature impacted the morphology of adsorbed nanoMIPs by imaging the same nanoMIPs at room temperature, 37 0 C, and 50 0 C. AFM measurements were performed on a JPK Nanowizard 4 XP Bioscience (Bruker, Nano GmbH, Berlin, Germany).
• the chips 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 at 75 °C. The chips were then rinsed in milli- Q water and dried with nitrogen. NanoMIP solutions were diluted in milli-Q water to ⁇ 2.54 ⁇ g mL -1 , drop-cast (20 ⁇ L) onto the Au-coated surfaces, and allowed to dry in ambient conditions for a minimum of 4 h in a Petri dish. A high temperature heating stage (HTHS, JPK BioAFM – resolution of 0.1 °C) was used as a temperature controller to facilitate imaging at 37°C and 50°C.
• HTHS high temperature heating stage
• FIGa and 9b which show typical AFM images and a corresponding cross-sectional profile plot respectively show that the nanoMIP morphology was unaffected by increasing temperature.
• NanoMIP volume was calculated using Gwyddion. Tracking numerous droplets revealed minimal changes in mean nanoMIP volume ( ⁇ 6% decrease) from room temperature to 50 °C. Thus, nanoMIP morphology remains consistent across relatively large temperature ranges.
• SPR binding analysis using a Biacore 3000 instrument, was also performed against the SARS-CoV-2 spike protein using nanoMIPs that had experienced autoclaving for 15 minutes with a maximum temperature of 121 0 C.
• nanoMIP-SPR sensors were prepared by covalently immobilising nanoMIPs (from Example 1), either as directly prepared or after autoclave at 121 0 C for approximately 15 minutes, on 4% mercaptoundecanoic acid (MUDA) chips (prepared using Biacore SIA Kit Au surfaces, functionalised with a self-assembled monolayer of 4% MUDA in ethanol) in line with the process directly below.
• MUDA mercaptoundecanoic acid
• Water was utilized to prime the instrument at a constant flow rate of 5 ⁇ L/minute.
• Water was utilized as the running buffer throughout the chip preparation at a constant flow rate of 30 ⁇ l/minute. Water was run until a stable baseline was achieved.
• N-hydroxysuccinimide NHS
• EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
• Example 10 Comparison of binding between NanoMIPs and SARS-CoV-2 antigens and SARS-CoV-2 antibodies and SARS-CoV-2 antigens using thermal detection methods.
• NanoMIP-functionalized Screen-Printed Electrodes (SPEs) using nanoMIPs imprinted against peptide 1) were made in line with the procedure in Example 8.
• SARS-CoV-2 antibody-functionalized SPEs were also made in line with the procedure in Example 8.
• SARS-CoV-2 antigens either recombinant full-length SARS-CoV-2 spike glycoprotein (alpha variant) sourced from The Native Antigen Company, recombinant SARS-CoV- 2 spike receptor binding domain (RBD) (delta variant) sourced from Abbexa, Cambridge, UK or SARS-CoV-2 RBD from the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit (Dundee, UK)
• RBD recombinant SARS-CoV- 2 spike receptor binding domain
• thermocouples measured the heat sink (T 1 ) and liquid reservoir (T 2 ) temperatures every second, and the thermal resistance (R th ) was obtained by dividing the temperature gradient (T 1 -T 2 ) over the power required to maintain the heat sink at 37.00 ⁇ 0.02 °C.
• the thermal measurement device was controlled using LabView software and a proportional-integral-derivative (PID) controller attached to a power resistor (22 ⁇ ) regulated the feedback on the signal.
• PID proportional-integral-derivative
• the liquid reservoir was filled with PBS and left for 30 min to ensure stabilization of the baseline Rth signal.
• five spiked PBS solutions (3 mL) with increasing concentrations of the SARS-CoV-2 antigen (1 fg mL -1 - 10 pg mL -1 ) were injected into the flow cell at a rate of 250 ⁇ L min -1 for 12 min using an automated syringe pump (LSP02-1 B, Longer Precision Pump Co., Hebei, China).
• LSP02-1 B Longer Precision Pump Co., Hebei, China
• the system was allowed to stabilize for 30 min prior to each subsequent injection.
• the experiments produced raw thermal data plots which displayed a step-wise increase in Rth where each stabilized plateau represents the injection of an increasingly concentrated spiked solution (see Figure 10a).
• Dose-response curves were composed from the thermal plots by taking the mean and standard deviation (SD) of the stabilized plateau from each concentration injection. Limit of detection (LoD) values were calculated from the dose-response curves using the three-sigma method (3 x reference SD) in the linear range.
• SARS-CoV-2 antibody-functionalized SPEs were used to facilitate a direct comparison between the sensing performance of the nanoMIPs and those of antibody receptors.
• the versatility of nanoMIP receptors was also examined by measuring their thermal response against the SARS-CoV-2 RBD (see Figure 10c). Antibody receptors were also tested for the purpose of direct comparison.
• the LoD value for the SARS-CoV-2 RBD was ⁇ 20 times smaller for the nanoMIP sensor (3.9 ⁇ 1 .0 fg mL -1 ) compared to the antibody sensor (85.5 ⁇ 15.0 fg mL -1 ). Consequently, this demonstrates that the nanoMIPs possessed greater versatility than antibodies as they had a comparable LoD for the full-length spike protein but a significantly lower LoD for the RBD.
• the selectivity of the nanoMIP sensor was also comprehensively examined using three negative controls which are common interferents in clinical samples: open reading frame 8 (ORF8), interleukin-6 (IL-6), and human serum albumin (HSA).
• ORF8 open reading frame 8
• IL-6 interleukin-6
• HSA human serum albumin
• NIPs were prepared using the same synthesis protocol as nanoMIPs except they were not exposed to a target epitope during polymerization, and therefore had no specific cavities to facilitate target binding.
• the thermal response of a nanoMIP-functionalized SPE to the spike protein was ⁇ 6 times larger compared to the NIP-functionalized SPE (0.04 °C W -1 ) showing that specific binding occurred between the spike protein and nanoMIP cavities.
• the LoD values of the nanoMIP sensor, a commercial rapid antigen test, and numerous recently developed antigen tests from the literature 4-15 are presented in Figure 10e. Table 1 below provides further details of the experimental setup.
• the LoD value for the nanoMIP sensor is ⁇ 6000 times lower than the commercial rapid antigen test (20 pg mL -1 ).
• Example 11 Use of NanoMIPs to detect SARS-CoV-2 in clinical samples
• a prototype 3D-printed resin addition cell ( Figure 11a and 11b) was developed for clinical analysis with disposable components.
• the thermal measurement device was controlled using LabView software and a proportional- integral-derivative (PID) controller attached to a power resistor (22 ⁇ ) regulated the feedback on the signal.
• PID proportional- integral-derivative
• thermal detection experiments were performed using the SARS-CoV-2 spike protein in PBS.
• the addition cell sensor showed a good thermal response to the spike protein (Figure 11c) as the LoD value (7.0 ⁇ 4.0 fg mL- 1 ) was very similar to the results obtained when using the flow cell design (9.9 ⁇ 2.5 fg mL -1 (Example 10)).
• UTM and VPM Virtual preservation medium
• 100 ⁇ L of the reference liquid (UTM/VPM) was pipetted into the reservoir and the R th signal was allowed to stabilize for 10 min.
• NanoMIP synthesis Peptide 1 was immobilised onto silanised glass beads prior to nanoMIP synthesis in line with the procedure in Example 1.
• the peptide coated glass beads were used for the synthesis of imprinted nanoMIPs as follows: a monomer solution (see Example 1) was degassed under vacuum and sonicated for 5 min, and then purged with N 2 for 20 min before being added to 60 g of peptide 1-coated glass beads.
• Polymerization was initiated by adding an ammonium persulfate aqueous solution (800 ⁇ L, 60 mg/mL) and N,N,N',N'- tetramethylethylenediamine (24 ⁇ L), both from Sigma Aldrich. The headspace was flushed with N 2 and the bottle sealed with a screw cap. Polymerization was carried- out at room temperature for 1 h. Subsequently, the content of the polymerization vessel was poured into a solid-phase extraction (SPE) cartridge (60 mL) equipped with a frit (20 ⁇ m porosity). A total of 9 washes with 20 mL of distilled water at 20 oC were carried out to remove low affinity nanoMIPs, polymer and unreacted monomer.
• SPE solid-phase extraction
• the SPE cartridge containing the solid-phase was placed in a water bath at 70 °C for 15 min.
• An aliquot of 20 mL of distilled water pre-warmed at 65 °C was poured into the SPE to collect the high-affinity nanoMIPs. This action was repeated 5 times, until about 100 mL of a solution of high-affinity nanoMIPs in water were collected.
• the collected solution was concentrated down and dialysed with a SnakeSkin membrane (10 kDa molecular weight cut-off).
• Imprinted nanoMIPs had an average diameter of 69.3nm, as calculated by NanoSight NS300 ( Figure 12).
• nanoMIPs imprinted against peptide 1 were integrated in a silver nanoparticle (AgNP) based localized surface plasmon resonance (LSPR) sensor.
• AgNP silver nanoparticle
• LSPR localized surface plasmon resonance
• a nanoMIP stock solution (5 pg/ml in DI water) was dispensed on the Ag-LSPR chips. Afterwards, the Ag chips were placed inside a humidified chamber for 3h to ensure that the nanoMIPs were immobilized on the surface of the Ag nanoparticles. Since the nanoMIPs bear amine groups, electrostatic interactions between the negative LSPR Ag surface and the positively charged nanoMIPs (-Ag with NH3 +) resulted in binding of the nanoMIPs to the surface. After this the Ag substrates were thoroughly rinsed with DI water to remove any loosely-bound polymers from the electrode surface. The chips were then stored at 4°C.
• 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 (all purchased from antibodies-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) in PBS.
• the LSPR performance of the nanoMIP-LSPR sensor against spike proteins of human coronavirus strains HCoVOC43, HKU1 and HCoV-229E was tested as was the LSPR performance of a NIP-based LSPR sensor.
• the sensor demonstrated successful detection of all SARS- CoV-2 variants.
• Figure 13a shows a typical LSPR sensor response upon binding of various concentrations of the alpha variant together with the corresponding absorbances.
• Figure 13b shows the change in LSPR wavelength of the Ag-MIPs complex upon varying the concentration of Alpha, Beta and Gamma from 10 aM to 100 nM.
• the limit of detection (LOD) using wavelength data was found at 466.37 nm, 467.13 nm and 467.71 nm which corresponds to 9.71 fM, 7.32 fM and 8.81 pM respectively for Alpha, Beta and Gamma.
• the LOD was computed using empirical formula involving the use of limit of blank (LOB) and standard deviations of the measurements, where blank refers to the effect of PBS on the MIPs (without any proteins) – see below.
• LOD calculation To obtain the LOD of the LSPR sensor, the wavelength change was calculated using equation 1 ad 2 and then converted to the concentration of spike proteins of Alpha, Beta and Gamma variants.
• Limit of blank mean blank + 1.645 (SD blank ) (Eq.1)
• Limit of detection (LOD) LOB + 1.645 (SD lowest concentration ) (Eq.2)
• Figure 14a shows a typical LSPR sensor response to blood serum spiked with various concentrations of the Alpha spike protein.
• Figure 14b shows the change in LSPR wavelength of the Ag-MIPs complex upon varying the concentration of Alpha, Beta and Gamma spike proteins from 10 aM to 100 nM.
• the LOD computed for Alpha and Beta using wavelength data was found to be at 457.54 nm and 460.49 nm. These LOD values correspond to concentrations of 14 fM for Alpha and 94 fM for Beta. The calculated LOD was 130 fM (at 457.37 nm) for wavelength changes observed for Gamma binding. The method for determining LOD values is shown above. Note that for LOB calculation, necessary for computing LOD, measurements in serum without protein were considered as blank samples in the present experiments.
• the LSPR signal was acquired using an in-house setup which consists of components purchased from Ocean Optics: spectrometer FLAME-T-XR1 -ES, reflection probe QR400-7-SR-BX, UV-Vis patch connectors, DH-2000 Deuterium-Tungsten Halogen lamp (DH 2000-S-DUV-TTL), RTL-T stage, and Ocean View software. Prior to the acquisition of the LSPR spectrum, dark and reference signals for background noise cancellation were measured using a glass slide as a reference. This glass slide was the same substrate on which Ag were deposited.
• This reference substrate was generated by complete removal of Ag nanoparticles from one of the substrates by sonicating the substrate in acetone for 1 hr and then using isopropanol wipes to clean the surfaces. All generated data were analysed and plotted using the in-built functionality of the GraphPad Prism 9 software.
• Example 13 Surface Plasmon Resonance (SPR) Analysis of NanoMIPs to SARS- CoV-2 spike proteins from two different SARS-CoV-2 variant strains.
• NanoMIP-SPR sensors were prepared by covalently immobilising nanoMIPs (from Example 1 ) to CM5 chips from Cytiva in line with manufacturers guidelines.
• 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. Sequence Summary

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