Patent Application
20240426822
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.
Submitted as part of this patent application is a Sequence Listing filed as a XML file named UEMIP1-0002US-SeqList.xml having a file size of 9,000 bytes and generated on Oct. 14, 2022, the content of which is hereby expressly incorporated by reference in its entirety.
The outbreak in 2019 of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has given rise to a pandemic which has in turn led to both a public health and economic crisis on a worldwide scale.
Although the international community quickly recognised that both diagnostic and antibody testing are key to preventing further transmission of the virus and to understanding the prevalence of the virus respectively, testing kits which are reliable, inexpensive to produce and easy to use remain highly sought after.
Regarding diagnostic testing, at present tests based on reverse transcription polymerase chain reaction (RT-PCR) are the predominant means by which healthcare providers are assessing citizens. The RT-PCR tests work by detecting viral ribonucleic acid (RNA) in a respiratory sample often obtained via a nasopharyngeal swab.
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. However, they lack sensitivity and utilize antibodies as receptors, which can only function in narrow temperature and pH ranges, which limits their widespread use.
Molecularly imprinted polymers (MIPs) have been proposed as alternative receptors for use in the detection of SARS-CoV-2.
Cennamo et al 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” details the production of plastic optical fiber (POF) based surface plasmon resonance (SPR) sensors coupled with MIPs. The MIPs were made to the S1 subunit of the SARS-CoV-2 spike protein.
Parisi et al in ““Monoclonal-type” plastic antibodies for SARS-CoV-2 based on Molecularly Imprinted Polymers” describes the preparation of MIPs using the SARS-CoV-2 receptor binding domain (RBD) as a template.
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.
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”.
Using either the whole virus, the whole S1 subunit or the whole RBD of SARS-CoV-2 for the production of MIPs leads to significant issues regarding scalability.
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.
Accordingly, 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:
Further provided is a conjugate comprising the molecularly imprinted polymer of the invention and a fluorophore.
Still further provided are compositions comprising the molecularly imprinted polymer or the conjugate of the invention.
All of the molecularly imprinted polymers of the invention, the conjugates of the invention and the compositions of the invention can be used in the detection of SARS-CoV-2.
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:
In molecular imprinting, functional, and sometimes cross-linking, monomers are polymerised in an appropriate solvent in the presence of the compound to be imprinted. The compound to be imprinted is known as the template. During the polymerisation step, the monomers interact with the template through electrostatic, hydrophobic or other interactions leading to the formation of binding sites in the polymer, known as recognition sites, that are complementary to the template molecule. After removal of the template, 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 placed such that they are in a suitable position to interact with the interacting groups in the template molecule and other molecules that contain the template molecule as a substructure, i.e. in this invention 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.
Although the sequence of the RBD of the SARS-CoV-2 spike protein is known, and theoretically any sequence therein could be used as a template, it is advantageous for some downstream applications to select, as a template, sequences within the target which are non-conserved. This ensures that the molecularly imprinted polymer binds the desired target with high specificity.
Supplementary FIG. 5 of Wrapp et al 20201 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. Also highlighted are sequences which are conserved between the different strains and sequences which are non-conserved.
In the present invention, 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.
Preferred is that 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.
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 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.
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 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.
In the present invention, the size of the template is in the range up to 50 amino acids. Preferably, 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 500 nm, more suitably less than 250 nm and even more suitably less than 100 nm, and may be less than 90 nm, or even less than 70 nm.
Also provided by the invention is a composition comprising the molecularly imprinted polymer of the invention. Suitably, molecular imprinted polymers in such compositions will have an average size that is less than 500 nm, more suitably less than 250 nm and even more suitably less than 100 nm, and may be less than 90 nm, or even less than 70 nm.
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. Typical examples of 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). 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.
In preferred embodiments of the invention, 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). More preferably, 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.
Advantageously, 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.
Also provided by the present invention is a method of preparing a molecularly imprinted polymer comprising at least one recognition site which binds SARS-CoV-2 comprising the steps of:
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.
In preferred embodiments of the invention, the carrier substance is glass. Further preferred is that the carrier substance is in the form of glass beads.
The polymerisable composition must contain the monomers required for the polymerisation. Suitable monomers are discussed elsewhere herein.
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.
Examples of controlled polymerisation include radical polymerisation such as controlled living radical polymerisation (LRP), living anionic polymerisation, living cationic polymerisation, and 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. In the examples described herein, polymerisation was initiated using persulfate.
In preferred embodiments of the invention, 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.
Once the molecularly imprinted polymers are synthesized they must be separated and collected. 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.
In preferred embodiments of the method, prior to the separation step, weakly bound material is removed by washing.
Once collected, the molecularly imprinted polymers may be purified further e.g. by chromatography, filtration and/or electrophoresis.
For the reasons recited above in relation to the molecularly imprinted polymer of the invention, in preferred embodiments of the method of the invention, 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.
In a particularly preferred embodiment, the amino acid sequence is no more than 50 amino acids in length and comprises the sequence NSNNLDSKVGG. In a further particularly preferred embodiment, 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.
In some embodiments of the invention, 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. In preferred embodiments the spacer comprises a glycine residue.
Thus in a particular preferred embodiment of the present invention, 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.
In some embodiments of the invention, 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. In the examples provided herein, 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. In the examples provided herein, 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.
An advantage of the method of the present invention is that it is fast and scalable and uses only a short template.
Further provided by the invention is a 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.
Conjugation of fluorophores to molecules for visualization purposes is common in the art. As such, methods for such conjugation are also well known. By way of example, fluorophores can be conjugated via amino, thiol or carbohydrate groups. In the examples provided herein, the fluorophore was conjugated to the molecularly imprinted polymer via amine groups provided by the amino monomer in the molecularly imprinted polymer.
Although 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 (CPNs)™ 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 70-80 nm in size and those currently available from Stream Bio exhibit fluorescence with emission wavelengths from 420 nm to 1130 nm. In the examples provided herein, molecularly imprinted polymers of the invention were conjugated to CPN510 and CPN610, both available from Stream Bio.
In preferred embodiments of the present invention, the fluorophore is a CPN.
Also provided by the present invention are 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. The molecularly imprinted polymers, conjugates and compositions of the present invention may also be used in sensors.
In the examples provided herein, 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). NanoMIP-functionalized Screen-Printed Electrodes (SPEs) were also prepared for use in thermal detection methods.
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. in the present case 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.
When the molecularly imprinted polymers, conjugates and compositions of the present invention are used in the detection of SARS-CoV-2, it is preferred that the molecularly imprinted polymer binds to SARS-CoV-2 with a KD of less than 500 nM, preferably less than 250 nM and even more preferably less than 200 nM.
Suitable methods for measuring KD include surface plasmon resonance (SPR) which may be conducted using e.g. the Biacore series of instruments (Cytiva) or the MP-SPR Navi instruments (BioNavis).
The invention is now further described in specific examples with reference to the accompanying drawings in which:
Selection of Three Peptides from SARS-CoV-2 Suitable for Imprinting
Supplementary FIG. 5 of Wrapp et al 20201 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.
On the basis that a terminal cysteine was required for peptide immobilisation, when peptides 1, 2 and 3 were ordered from Ontores, China, a cysteine residue was added to the N terminus of peptide 1. A further glycine residue was also added between the cysteine and the N terminal asparagine, functioning as a spacer. Thus, the peptides sourced from Ontores for use in imprinting were:
Peptide immobilisation was carried out as follows: glass beads (approximately 100 μM in diameter, sourced from Microbeads AG) were activated by boiling in 4M NaOH for 10 minutes. The glass beads were then washed with firstly deionised water and then acetone and then dried at 80° C. for 2 hours. The glass beads were then incubated in toluene with 2% v/v (3-aminopropyl)trimethoxysilane (APTMS) for 3 hours, washed with acetone and placed in PBS, pH 7.4 with 0.2 mg/ml n-succinimidyl iodoacetate (SIA) for two hours before being washed with acetonitrile. 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 0.1 mg/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 3 mg N-Fluoresceinyl acrylamide, 24.2 mg Acrylamide, 31.7 μL Tert-Butyl acrylate (TBAc), 36 mg 3-O-Acryloyl-1,2:5,6-bis-O-isopropylidene-D-glucofuranose, 13.8 μL 2,2,2-Trifluoroethyl methacrylate (CF3), 3.9 mg N-(3-Aminopropyl) methacrylamide hydrochloride (APMA), 1.3 mg Squareamide monomer 7 (SQ7) and 9 mg N,N′-Methylenebis(acrylamide) (BIS)). 100 ml of the monomer solution was then put into a 250 ml Duran flask containing 60 g of the peptide coated glass beads. Polymerisation was then initiated by adding 0.5 ml of 60 mg/ml ammonium persulphate (APS) with 60 μL/ml of N,N,N′,N′-tetramethylethylenediamine (TEMED) and carried out at room temperature for 1 hour. The contents of the flasks were then poured into Solid Phase Extraction (SPE) cartridges fitted with 20 μM porosity frits in order to separate the glass beads with attached nanoparticles from the other components. 10 washing steps, each with approximately 25 ml of room temperature water, were then performed to remove low affinity material. The residue from each of these washing steps was discarded. High affinity nanoMIPs were then detached from the glass beads by performing 9 washing steps, each with 10 ml of 60° C. ethanol. The residue from each of these washes, containing the nanoMIPs, was collected. The nanoMIPs were then concentrated down to approximately 10 ml and solvent exchanged to distilled water.
Imprinted nanoMIPs had an average diameter of 59 nm, as calculated by NanoSight NS300.
A Biacore 3000 instrument was employed in this study.
Surface Activation and nanoMIP Immobilisation.
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).
In more detail, water was utilized to prime the instrument at a constant flow rate of 5 μL/minute. Water was utilized as the running buffer throughout the chip preparation at a constant flow rate of 30 μl/minute. Water was run until a stable baseline was achieved. A mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (2 mg EDC+3 mg NHS in 400 μL of water) was injected (8 minutes at a flow rate of 8 μL/minute) across the sensor surface to activate the surface (the mixture converts the carboxylic terminal groups on the chip surface into N-Hydroxy-succinimide esters). NanoMIPs (5 nM) were then injected (8-10 minutes at a flow rate of 8 μL/minute) into all four flow cells on the chip. Finally, unreacted NHS esters were hydrolysed by injecting carbonate buffer (pH 9.2) (30 minutes at a flow rate of between 5 and 30 μL/minute).
Detection of Interaction Between nanoMIPs and SARS-CoV-2 Peptides
Prior to testing in SPR, each of peptides 1, 2 and 3 were conjugated to Bovine Serum Albumin (BSA).
Once a stable baseline was obtained having switched running buffer from water to PBS, 7 dilutions of BSA or BSA-peptide conjugates (2.33 mg/ml, 0.777 mg/ml, 259 ug/ml, 86 ug/ml, 28.8 ug/ml, 9.6 ug/ml, 3.2 ug/ml in PBS) were injected (5 minutes association phase, 4 minutes dissociation phase, 28 μl/minute flow rate) as follows: BSA alone was injected into flow cell 1, BSA-peptide 1 into flow cell 2 and BSA-peptide 3 into flow cell 3.
Good selectivity was observed for nanoMIPs imprinted against peptide 3, for which the software calculated a KD of 160 nM (data not shown).
A Biacore 3000 instrument was employed in this study.
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).
In more detail, water was utilized to prime the instrument at a constant flow rate of 5 μl/minute. Water was also utilized as the running buffer throughout the chip preparation at a constant flow rate of 30 μl/minute. Water was run until a stable baseline was achieved. A mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (2 mg EDC+3 mg NHS in 400 μL of water) was injected (8 minutes at a flow rate of 8 l/minute) across the sensor surface to activate the surface (the mixture converts the carboxylic terminal groups on the chip surface into N-Hydroxy-succinimide esters). 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 then injected (6 minutes 15 seconds at a flow rate of 8 l/minute) into flow cells 1, 2 and 3 respectively on the chip. Finally, unreacted NHS esters were hydrolysed by injecting carbonate buffer (pH 9.2) (1 minute at a flow rate of 30 μl/minute).
Detection of Interaction Between SARS-CoV-2 S Protein and nanoMIPs
Once a stable baseline was obtained having switched running buffer from water to PBS, 30 nM (and 1:1 serial dilutions thereof) nanoMIPs imprinted against peptide 1 were then injected (5 minutes association phase, 3 minutes dissociation phase, 28 μl/minute flow rate).
The software calculated a KD of 5 nM (
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 70-80 nm as measured by NanoSight 3000.
Different concentrations (290 ng, 145 ng, 73 ng, 36 ng, 18 ng, 9 ng, 5 ng, 2 ng, 1 ng, 0.6 ng, 0.3 ng and 0.14 ng) 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. As shown in
SARS-CoV-2 (2×104 pfu) was blotted onto two nitrocellulose membranes. Also blotted on to each of the nitrocellulose membranes was 75 ng 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
0.5 μL of SARS-CoV-2 Spike Glycoprotein (S1) (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
Preparation of nanoMIP-Functionalized Screen-Printed Electrodes (SPEs) for Use in Thermal Detection Methods.
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. The carboxyl group was then activated through incubation with a solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (100 mM) and N-hydroxysuccinimide (NHS) (20 mM) in PBS buffer (pH=5). Dropcasting was utilised to deposit 8 μL of the EDC/NHS solution onto the working electrode of the SPEs. After 1 hour, the electrodes were rinsed with deionised water and dried to obtain the SPE/4-ABA/EDC+NHS electrodes. Following this, 8 μL of a given nanoMIP solution (NanoMIP imprinted against peptide 1 ‘P1C6’ or NanoMIP imprinted against peptide 3 ‘P3C6’) was deposited onto the working electrodes following a gentle vortex. After 3 hours, 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.
Detection of Binding Between NanoMIPs and SARS-CoV-2 S Protein RBD Using nanoMIP-Functionalised Screen-Printed Electrodes (SPEs).
3D-printed flow cells were used to facilitate measurements using the nanoMIP-functionalised SPEs described above. A SPE was mounted into the cell and a thermocouple measured the temperature in the liquid (T2). For each measurement, 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, T1), 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 (22Ω) regulated the feedback on the signal as described in Geerets et al. The PID parameters were fixed for all experiments at optimised values of P=1, I=10, and D=0.2.
In all measurements, 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 μL 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 Rth 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 (
The capability of MIPs imprinted against peptide 1 to withstand extremes of temperature and pH was investigated.
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° C., and 50° C.
AFM measurements were performed on a JPK Nanowizard 4 XP Bioscience (Bruker, Nano GmbH, Berlin, Germany). Measurements in air were carried out in tapping mode using PPP-NCL-W probes (Nanosensors, Neuchatel, Switzerland) with a cantilever length of ˜225 μm and spring constant of ˜48 N m−1. Measurements in liquid were performed in Quantitative Imaging (QI) mode using MLCT-E probes (Bruker, Ca, USA) with a cantilever length of ˜140 μm and spring constant of ˜0.1 N m−1. 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 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 μg mL−1, drop-cast (20 μL) 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.
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° C.
In more detail, two nanoMIP-SPR sensors were prepared by covalently immobilising nanoMIPs (from Example 1), either as directly prepared or after autoclave at 121° 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.
Water was utilized to prime the instrument at a constant flow rate of 5 μL/minute. Water was utilized as the running buffer throughout the chip preparation at a constant flow rate of 30 μl/minute. Water was run until a stable baseline was achieved. A mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (2 mg EDC+3 mg NHS in 400 μL of water) was injected (8 minutes at a flow rate of 8 μL/minute) across the sensor surface to activate the surface (the mixture converts the carboxylic terminal groups on the chip surface into N-Hydroxy-succinimide esters). NanoMIPs (5 nM) were then injected (8-10 minutes at a flow rate of 8 μL/minute) into all four flow cells on the chip. Finally, unreacted NHS esters were hydrolysed by injecting carbonate buffer (pH 9.2) (30 minutes at a flow rate of between 5 and 30 μL/minute).
Once a stable baseline was obtained having switched running buffer from water to PBS, 5 dilutions (30, 15, 7.5, 3.75 and 1.88 mM in PBS) of SARS-CoV-2 S protein (recombinant full-length SARS-CoV-2 spike glycoprotein sourced from The Native Antigen Company) were injected (circa 5 minutes association phase, at least 2 minutes dissociation phase, 28 μl/minute flow rate) with suitable controls in control channels. The results from the two chips were then superimposed, and the software calculated very similar KD values for the nanoMIPs before and after autoclaving (7 and 3 nM, respectively), demonstrating that there is no impact on binding affinity after exposure to high temperatures. This is highly advantageous for nanoMIP shelf-life as autoclaving (sterilization) facilitates their long-term storage in water without bacterial degradation. In contrast, antibodies experience significant deterioration in affinity at temperatures above 37° C.
pH
AFM was also used to assess the ability of nanoMIPs to withstand extremes of pH by measuring the volume of adsorbed nanoMIPs (n=120) in liquid at various pH levels. More specifically, QI measurements were performed at room temperature (23±1° C.) in liquid at different pH levels (5.5-8.5) within the operating conditions of the AFM. Initial measurements were carried out in pure milli-Q water (pH 5.5) and the pH was subsequently increased with the addition of ammonia.
As above, nanoMIP volume was calculated using Gwyddion which revealed negligible changes to the mean nanoMIP volume from pH 5.5 to 8.5 (˜3% decrease), highlighting that adsorbed nanoMIP morphology was consistent across a broad pH range (see
Additionally, the thermal response of the developed nanoMIP tests (see Example 10) to a clinical reference liquid (universal transport medium, UTM) and Diet Coca-Cola (pH=3.5) were compared. The results demonstrated that there was no statistically significant difference between the thermal response of the two liquids (see
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.
Detection of Binding Between NanoMIPs or SARS-CoV-2 Antibodies and SARS-CoV-2 Antigens Using nanoMIP- or SARS-CoV-2 Antibody-Functionalised Screen-Printed Electrodes (SPEs).
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)) were thermally detected by mounting nanoMIP-functionalized SPEs into 3D-printed resin flow cells to create an interface between the heat sink and liquid reservoir. Two thermocouples measured the heat sink (T1) and liquid reservoir (T2) temperatures every second, and the thermal resistance (Rth) was obtained by dividing the temperature gradient (T1-T2) over the power required to maintain the heat sink at 37.00±0.02° C. As the target attached to the nanoMIPs, heat transfer at the solid-liquid interface was reduced (larger temperature gradient), which led to a measurable increase in the Rth.
For all experiments, the thermal measurement device was controlled using LabView software and a proportional-integral-derivative (PID) controller attached to a power resistor (22Ω) regulated the feedback on the signal. The PID parameters were optimized to reduce noise and were set at P=1, I=13, and D=0.2.
The liquid reservoir was filled with PBS and left for 30 min to ensure stabilization of the baseline Rth signal. Subsequently, 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 μL min−1 for 12 min using an automated syringe pump (LSPO2-1B, 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
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 thermal detection results (see
The ability of the nanoMIP receptors to detect virus mutations was also examined by measuring their specificity against the SARS-CoV-2 spike protein (delta variant). Results show (
The versatility of nanoMIP receptors was also examined by measuring their thermal response against the SARS-CoV-2 RBD (see
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). A high degree of binding occurred between the SARS-CoV-2 antigens and nanoMIPs, which led to large ΔRth values at the highest concentration (10 pg mL−1) for the spike protein (0.23° C. W−1) and RBD (0.35° C. W−1). In contrast, minimal binding occurred with the negative controls, which led to significantly lower ΔRth values for ORF8 (0.00° C. W−1), IL-6 (0.06° C. W−1), and HSA (0.05° C. W−1). The results demonstrated that the thermal response of the nanoMIP sensor was considerably greater for SARS-CoV-2 antigens compared to the negative controls, which highlighted the excellent selectivity of the nanoMIP sensor. An additional control experiment was also performed using non-imprinted polymers (NIPs) immobilized to SPEs (NIP functionalised SPEs made in line with the procedure in Example 8). 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 literature4-15 are presented in
and spike
Rapid Test
with double antibody
and
combined with a
mL−1
modified with
CV and
used for
chip
mL−1
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mL−1
properties measured
-based
immobilized onto Si
mL−1
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with spike antibodies
used
for detection.
-
and
black-
. DPV used for
indicates data missing or illegible when filed
A prototype 31D-printed resin addition cell (
To validate the addition cell design, 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 (
After initial validation, clinical measurements were performed using COVID-positive (positive after <20 PCR cycles) and negative patient samples (n=7).
UTM and VPM (Viral preservation medium) were used as reference liquids for the negative and positive samples, respectively. During the measurements, 100 μL of the reference liquid (UTMNPM) was pipetted into the reservoir and the Rth signal was allowed to stabilize for 10 min. Following this, the reference liquid was pipetted out and 100 μL of the sample was added. Note that use of a pipette resulted in less disturbance to the system (e.g., flow, addition of air bubbles) compared to using a syringe pump. This is highly advantageous for clinical analysis since the sample volume was similar to that collected by a throat and nasal swab (100 μL), measurement time was reduced to −15 min, and device operation was straightforward.
The thermal detection results (
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 N2 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 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 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 μm porosity). A total of 9 washes with 20 mL of distilled water at 20° C. were carried out to remove low affinity nanoMIPs, polymer and unreacted monomer. Afterwards, 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. To ensure complete removal of any potential unreacted monomer from the bulk of the nanoparticles, 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 69.3 nm, as calculated by NanoSight NS300 (
Once produced the nanoMIPs imprinted against peptide 1 were integrated in a silver nanoparticle (AgNP) based localized surface plasmon resonance (LSPR) sensor.
In more detail, 50 μl of a nanoMIP stock solution (5 μg/ml in DI water) was dispensed on the Ag-LSPR chips. Afterwards, the Ag chips were placed inside a humidified chamber for 3 h 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.
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 (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. For controls, 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.
As shown in
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.
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.
Following the successful detection of virus samples in PBS, similar experiments were conducted using the clinically relevant fluid human serum. In particular, serum was spiked with 100 fM, 10 pM, 1 nM and 100 nM concentrations of Alpha, Beta and Gamma spike protein and wavelength and absorbance shifts were measured. As for the above experiments using PBS, control experiments were also conducted using spike proteins of human coronavirus strains HCoVOC43, HKU1 and HCoV-229E and the LSPR performance of a NIP-based LSPR sensor was also tested.
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.
For each of the above experiments in PBS and serum, all spike proteins, including those of the human coronavirus used as controls, were prepared or diluted with PBS/serum at different concentrations (10 aM to 100 nM). The Ag-MIP functionalised substrate was exposed to different concentrations of the respective protein. This was done by drop-casting 50 μl of the sample onto the Ag-MIPs. After exposing the surface of the sensor to a given concentration, a 20 min incubation period was included to allow the protein to interact with the MIPs. Thereafter, the surface of the sensor was washed with PBS (for both PBS and serum samples) and the LSPR signal was measured. To acquire the signal, a 30 s period was included during which the ocean view software (see below) acquired multiple spectrum and displayed an average of 10 spectrums with a box car width of 5. The LSPR spectra were later saved and wavelength/absorbance shifts were recorded, analyzed and plotted using Graphpad Prism software.
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.
A Biacore T200 instrument was employed in this study.
Surface Activation and nanoMIP Immobilisation.
NanoMIP-SPR sensors were prepared by covalently immobilising nanoMIPs (from Example 1) to CM5 chips from Cytiva in line with manufacturers guidelines.
In more detail, water was utilized to prime the instrument at a constant flow rate of 5 μL/minute. Water was utilized as the running buffer throughout the chip preparation at a constant flow rate of 5 μl/minute. Water was run until a stable baseline was achieved. A mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (2 mg EDC+3 mg NHS in 400 μL of water) was injected (8 minutes at a flow rate of 5 μL/minute) across the sensor surface to activate the surface (the mixture converts the carboxylic terminal groups on the chip surface into N-Hydroxy-succinimide esters). NanoMIPs (5 nM) were then injected (7 minutes at a flow rate of 5 μL/minute) into multiple flow cells on the chip. Finally, unreacted NHS esters deactivated by injecting ethanolamine in water.
Detection of Interaction Between nanoMIPs and SARS-CoV-2 Spike Proteins from Two Different SARS-CoV-2 Variants
Once a stable baseline was obtained having switched running buffer from water to PBS, 5 dilutions (1.23, 3.7, 11.11, 33.33 and 100 nM in PBS) of recombinant SARS-CoV-2 S protein (recombinant full-length SARS-CoV-2 spike glycoprotein sourced from The Native Antigen Company, either designated as Wuhan Hu 1 original strain or Omicron strain) were injected (309 seconds at 5 μl/minute) sequentially with minimal dissociation time only due to moving from one solution to the next. The results from the two spike protein variants were then superimposed such that the relative magnitude of binding response could be compared. As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 21197636.0 | Sep 2021 | EP | regional |
This application is a 35 U.S.C. § 371 U.S. national stage patent application which claims the benefit of priority and is entitled to the filing date of International Patent Application PCT/EP2022/076119, filed Sep. 20, 2022, an international patent application which claims the benefit of priority and is entitled to the filing date pursuant to 35 U.S.C. § 119(a) of EP Patent Application 21197636.0, filed Sep. 20, 2021, the content of each of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/076119 | 9/20/2022 | WO |
