WO2021260566A1

WO2021260566A1 - Method for detecting a virus in a liquid medium, molecular sensor for carrying it out and process for the preparation of the molecular sensor

Info

Publication number: WO2021260566A1
Application number: PCT/IB2021/055531
Authority: WO
Inventors: Girolamo D'agostino; Nunzio CENNAMO; Chiara PERRI
Original assignee: Moresense S.R.L.
Priority date: 2020-06-24
Filing date: 2021-06-23
Publication date: 2021-12-30
Also published as: IT202000015145A1; EP4171798A1

Abstract

The present invention relates to a method for detecting a virus in a liquid medium, said virus being characterized by the presence of at least one surface protein, wherein said method comprises: - providing a molecular sensor comprising a receptor element having recognition sites of the surface protein, the receptor element comprising at least one molecularly imprinted polymer obtained using the surface protein of the virus as a templating agent; - putting the liquid medium in contact with the molecular sensor; - detecting the presence of the virus through a variation of a surface property of the receptor element caused by an interaction between the virus and the receptor element. The present invention also relates to a molecular sensor for carrying out the above method. A molecular sensor preparation process and the molecularly imprinted polymer included in the sensor.

Description

METHOD FOR DETECTING A VIRUS IN A LIQUID MEDIUM, MOLECULAR SENSOR FOR CARRYING IT OUT AND PROCESS FOR THE PREPARATION OF THE MOLECULAR SENSOR The present invention relates to a method for detecting a virus in a liquid medium, said virus being characterized by the presence of at least one surface protein. The present invention also relates to the molecular sensor used in the aforementioned method, the process for preparing the aforementioned sensor as well as the molecularly imprinted polymer used in the aforementioned sensor.

The 2019-2020 COVID-19 pandemic is the pandemic of the so-called "new Coronavirus disease" called COVID- 19, which is caused by infection with the SARS-CoV-2 virus. This virus belongs to the Coronaviridae virus family, a family of virus of the order Nidovirales, with genome consisting of positive single-stranded RNA, enveloped by capsid. The structure of Coronaviruses (CoVs) is also characterized by the presence of surface proteins S, the so-called Spike Protein. Spike Proteins are glycoproteins that assemble on the surface of the virion in trimeric units giving the virion the characteristic appearance of a "crown". The external domain of S proteins has a two-domain organization: an N-terminal domain called SI, which is responsible for binding to the receptor, and a C-terminal domain called S2, which is responsible for fusion with the host cell. The CoVs differ from each other in the structure and shape of the surface proteins and consequently in the type of interaction they have with the receptor as well as in the response to environmental stimuli that trigger the fusion of the virus with the membrane of the host cell.

In the specific case of SARS-CoV-2, the surface proteins mediate the attack of the virus on the ACE2 enzyme (Angiotensyn Converting Enzyme) and its fusion by endocytosis with the host cell. The structure of SARS-CoV-2 was first described by Wrapp D. et al. in Science 2020 367(6483):1260-1263. The surface proteins of SARS-CoV-2 have two subunits: the SI subunit, containing the receptor binding domain (RBD) at the C- terminus, binds to the ACE2 receptor on the cell guest; the S2 subunit fuses with the host cell, thanks to an inner membrane fusion peptide (FP), two 7-peptide repeats (HR), a proximal outer membrane region (MPER) and a transmembrane domain (TM).

The extreme speed of spread of this virus, as well as of many other viruses carrying infectious diseases for humans and animals, raises the problem of having techniques and analysis devices that allow diagnosing the occurrence of an infection in a human or animal subject in a precise, effective and rapid way.

Currently, the reference method for the diagnosis of viruses, including SARS-CoV-2 virus, is based on the analysis of samples by the test of reverse transcription polymerase chain reaction (RT-PCR) for the detection of viral RNA.

The tests can be performed on biological samples of respiratory fluid obtained by various methods, including the execution of a nasopharyngeal or oropharyngeal swab, the collection of which is rather annoying and invasive for the patient, or the collection of a sample from the lower respiratory tract (sputum, endotracheal aspirate or broncho-alveolar lavage). The results of the above molecular tests, however, are generally only available 3-6 hours after the execution of the test. Furthermore, despite having a relatively high sensitivity, molecular tests based on the RT-PCR technique can give a false positive or false negative result, especially when carried out on subjects who have recently contracted the infection.

Furthermore, the aforementioned tests have the drawback of using special diagnostic reagents, which are not always easily available, especially in emergencies such as those caused by a pandemic.

In the medical field, methods of analysis based on the use of molecular sensors are also known, i.e. measuring devices that detect molecules of interest in a liquid matrix by means of molecular recognition. Molecular recognition is based on the highly selective interaction of the molecules of interest (target molecule or analyte) with a molecular recognition element (receptor) which may consist of chemical species (chemical sensors) or biological species (biosensors) immobilized on a surface of the transducer of the sensor or close to it. The interaction resulting from molecular recognition alters the surface properties of the receptor. Through the transduction system (for example of the optical or electrical type), the analyte - molecular recognition element interaction is converted into a processable electrical signal.

In the case of biosensors, molecular recognition is generally based on the selective interaction between a receptor consisting of an antibody immobilized on the sensor surface and an antigen of the biomolecule of interest. In other embodiments, the receptor is a DNA fragment complementary to the DNA of the biomolecule to be determined.

The methods of analysis based on the use of molecular biosensors have the advantage of allowing very rapid analyzes in real time, without requiring special pre-treatment of the samples to be analyzed. Furthermore, they have a high specificity in the recognition of the analyte of interest and very low detection limits of their concentration in the liquid matrix. However, the immobilization of antibodies and DNA fragments on the sensor surface is a complex procedure due to the sensitivity of these receptors to several factors, including temperature, pressure, pH conditions and the use of organic solvents.

The use of molecular sensors in which the receptor is formed by a molecularly imprinted polymer (or MIP) is also known in the state of the art. MIPs are polymeric materials obtained by molecular imprinting polymerization. In general, the molecular imprinting polymerization technique allows preparing polymers having specific recognition sites for a target molecule, carrying out the synthesis of the polymer in the presence of the target molecule of interest, which acts as a templating agent. MIPs are obtained by polymerizing, generally by radical polymerization, at least one functional monomer with a crosslinking agent in the presence of a templating agent molecule, and subsequently removing the templating agent of the formed polymer matrix.

MIPs are mainly used as materials for stationary phases in solid phase extraction or in chromatography. However, applications of MIP as receptors in molecular sensors for the detection of small chemical compounds are also known. An example of application of MIPs for the detection and extraction of perfluoroalkyl compounds from environmental matrices is described in EP3502152 A1.

In consideration of the aforementioned state of the art, the Applicant has set the primary objective of providing an alternative method and analysis device to those known in the art, with which it is possible to detect the presence of a virus having at least one surface protein, in a biological sample and, more generally, in an aqueous liquid medium in a quick, precise and simple way.

The Applicant has found that these and other purposes, which will be better illustrated in the following description, can be achieved through an analysis method, which uses a molecular sensor in which the receptor element comprises a molecularly imprinted polymer obtained using, as a templating agent, a surface protein of the virus to be detected. The MIP thus obtained has a plurality of highly selective recognition sites towards the virus. These sites are capable of interacting with the surface proteins of the virus present in the analyzed sample, binding the latter in a stable manner to the receptor element. The binding interaction between the surface proteins of the virus and the receptor element induces a change in the surface properties of the receptor, which can be converted by means of transduction systems into a processable signal. Advantageously, the interaction between the receptor element and the surface proteins of the virus leads to bind the entire virion to the sensor surface (the size of a virion is approximately 100 nm) with a consequent increase in the response signal of the sensor if the selected detection technique is sensitive to the mass of the analyte, for example, as in the case of optical detection techniques, in particular surface plasmon resonance.

A particular advantage of the sensor is therefore that of being able to confirm the presence of the virus in an active form (i.e. virion). It is noted that, in a liquid medium containing the virus in lysed form, the sensor can detect the presence of surface proteins originally belonging to the virus. However, since in practice the concentration of surface proteins in the matrix of a biological sample of the lysed virus would be insufficient to generate a signal detectable by the sensor, it follows that the sensor also allows precisely excluding the presence of the virus in its active form in the analyzed sample. The sensor therefore allows to overcome the drawbacks of the RT- PCR technique which on the contrary, by determining the presence of the virus indirectly through the identification of traces of its RNA (and not of the virion), does not allow to clearly distinguish the samples coming from potentially contagious subjects from those of non-contagious subjects.

Molecular sensors that use molecularly imprinted polymers for the recognition of the analyte of interest have the advantage of being easily manufactured even on an industrial scale. In particular, these sensors do not require the implementation of difficult procedures for the immobilization of biological recognition molecules (e.g. antibodies and DNA) as conversely is required in molecular biosensors.

According to a first aspect, the present invention therefore concerns a method for detecting a virus in a liquid medium, said virus being characterized by the presence of at least one surface protein, in which said method comprises:

- providing a molecular sensor comprising a receptor element having recognition sites of the surface protein, the receptor element comprising at least one molecularly imprinted polymer obtained using the surface protein of the virus as a templating agent;

- putting the liquid medium in contact with the molecular sensor;

- detecting the presence of the virus through a variation of a surface property of the receptor element caused by an interaction between the virus and the receptor element.

In accordance with a second aspect, the present invention relates to a molecular sensor for detecting a virus having at least one surface protein in a liquid medium, comprising:

- at least one receptor element having recognition sites of the surface protein of the virus, the receptor element comprising at least one molecularly imprinted polymer obtained using the surface protein as a templating agent;

- a transduction system for generating an optical, electrochemical, acoustic, piezoelectric, electromechanical, optomechanical or electronic signal following bonding between the virus and the receptor element.

In accordance with a third aspect, the present invention relates to a process for preparing a molecular sensor for detecting a virus in a liquid medium, said virus being characterized by the presence of at least one surface protein, comprising: a) preparing a polymerizable mixture comprising:

- at least one functional polymerizable monomer;

- at least one templating agent comprising said surface protein of the virus;

- at least one crosslinking agent;

- at least one polymerization initiator; b) depositing the polymerizable mixture on a surface of a sensor transduction system; c) polymerizing the polymerizable mixture to obtain a crosslinked polymer containing the templating agent; d) removing the templating agent to obtain a molecularly imprinted polymer having recognition sites of the surface protein.

In accordance with a fourth aspect, the present invention relates to a molecularly imprinted polymer having recognition sites of a surface protein of a virus, the polymer being obtained by polymerization of a polymerizable mixture comprising at least one functional polymerizable monomer, in the presence of at least one crosslinking agent and at least one surface protein of the virus as a templating agent.

In accordance with a fifth aspect, the present invention concerns the use of a molecularly imprinted polymer having recognition sites of a surface protein of a virus as a receptor element in a molecular sensor.

For the purposes of the present description and the appended claims, the compositions according to the present invention may "comprise", "consist of" or "essentially consist of" the essential and optional components described in the present description and in the appended claims. For the purposes of this description and the appended claims, the expression "consist essentially of" means that the composition or component may include additional ingredients, but only to the extent that the additional ingredients do not materially alter the essential features of the composition or the component.

The numerical limits and ranges expressed in the present description and in the appended claims also include the mentioned numerical value or the mentioned numerical values. Furthermore, all values and sub ranges of a numerical limit or range are to be understood as specifically included as if they were mentioned explicitly.

Unlike the operational examples, or where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so on used in the specification and claims are to be understood as being modified in all cases by the term "about".

In one embodiment of the invention, the virus belongs to the Coronaviridae family. In another embodiment, the virus is of the SARS-CoV type.

In one embodiment, the surface protein has a mass of 10 - 500 kDa, preferably 50 - 200 kDa.

In a preferred embodiment, the virus is SARS-CoV- 2. In a particularly preferred embodiment, the virus is SARS-CoV-2 and the surface protein is the Si subunit. The structure of SARS-CoV-2 and its surface proteins is described for example in Wrapp D. et al. in Science 2020 367 (6483):1260-1263.

The molecularly imprinted polymer can also be made using two or more different surface proteins, if it is desired, for example, to obtain different recognition sites on the same receptor element of the sensor.

The detection of the virus in the liquid medium can be carried out according to the techniques known in the art. The technique can be selected based on the surface property of the element to be monitored.

Preferably, the detection of the virus is carried out by means of an optical, electrochemical, electrical, acoustic, electro-mechanical, opto mechanical or spectroscopic technique.

In a preferred embodiment, the detection technique is an optical technique, such as for example techniques based on the phenomena of surface plasmon resonance,

In another embodiment, the detection technique is an electronic technique, selected for example from: electro-mechanical technique, opto-mechanical technique or piezoelectric technique. In particular, the detection technique can be implemented through micro- electro-mechanical systems (so-called MEMS).

In a particularly preferred embodiment, the detection technique is based on the phenomenon of surface plasmonic resonance (SPR), which is observed when a polarized electromagnetic radiation strikes a metal film, under conditions of total reflection.

The method according to the invention can be used, for example, to determine the presence of the virus in a wide variety of liquid media, and optionally to determine the present quantity.

In general, the method according to the invention can be applied to determine the presence of a virus in any liquid matrix capable of containing the virions in an active, i.e. non-lysed, form.

In one embodiment, the method according to the invention is applied to a biological sample obtained from any organism, preferably of human or animal origin.

Examples of biological samples are biological fluids, such as blood, saliva, urine, cells, including all the molecular fractions (e.g. proteins, RNA, DNA, etc.) that can be derived from them, originating from healthy or sick subjects.

After collection, the biological sample can be treated for analysis, for example it can be diluted (e.g. with water, saline, buffer solutions, etc.) or stored in viral transport media (e.g. Universal Transport Medium - UTM®).

In particular, the biological sample can be of the type generally used in the known art for the determination of viruses by RT-PCR technique. Examples of biological samples are: samples taken from nasopharyngeal swabs, physiological solutions, sputum, endotracheal aspirate, bronchoalveolar lavage fluid, blood, urine, saliva, sweat, etc.

In another embodiment, the liquid medium is an aqueous liquid that may have come into contact with a human or animal subject carrier of the virus, for example a sample of civil wastewater, surface and underground drinking water. The molecularly imprinted polymer can be prepared in accordance with the techniques known to the person skilled in the art. It can be obtained, for example, by radical polymerization, of at least one functional monomer in the presence of at least one crosslinking agent and a templating agent comprising the surface protein of the selected virus, and subsequently removing the templating agent from the formed polymeric matrix.

It is also possible to use more than one protein as a templating agent, for example in the case that it is desired to obtain a recognition element having two different types of molecular recognition sites.

The surface proteins of the virus of interest usable as a templating agent can be recombinant proteins, for example of the commercially available type.

Preferably, the functional monomer is a monomer having at least one functional group capable of interacting specifically with the surface protein of the virus and at least one functional polymerizable group. In particular, the functional monomer comprises one or more of the following functional groups: carbonyl group, hydroxyl group and amino group. These functional groups are particularly suitable for interacting by forming hydrogen bonds with the different regions of the surface proteins.

In a preferred embodiment, the functional monomer comprises at least two different groups selected from the aforementioned functional groups, more preferably it comprises all three.

The MIP is preferably obtained starting from a polymerizable mixture comprising two or more different polymerizable monomers.

In consideration of the fact that the surface proteins are bulky chemical species, the MIP preferably has a polymer matrix that is not excessively rigid to easily adapt to the geometric conformation of the protein during polymerization.

Since the protein is easily subject to denaturation, the polymerizable blend is preferably a water-based blend.

In one embodiment, the polymerizable mixture comprises at least one monomer of formula (I)

where:

Ri is H or CH₃;

R₂ is selected from: H or a Ci-Cs alkyl, preferably a, linear or branched, C1-C4 alkyl.

In one embodiment, in the formula (I) R₂ is H.

In another embodiment, in formula (I) R2 is a Ci-Cs alkyl group, preferably a C1-C4, linear or branched alkyl group. The presence of this alkyl group gives the functional monomer a non-polar portion capable of interacting with the non-polar regions of the proteins.

In one embodiment, the polymerizable mixture comprises at least one monomer of formula (II) where:

Ri is H or CH₃; n is an integer in the range 1 - 4, preferably in the range 1 - 2.

In one embodiment, the polymerizable mixture comprises at least one monomer of formula (I) and at least one monomer of formula (II). In this case, the molar ratio between the monomers of formula (I) and those of formula (II), in one embodiment, is in the range from 5:1 to 1:5, preferably from 4:1 to 1:1, more preferably from 3:1 to 1.5:1.

In a preferred embodiment, the polymerizable mixture comprises: acrylamide, N-tert-butylacrylamide and 2-hydroxyethylmethacrylate.

The crosslinking agent is a molecule having at least two functional groups that can be polymerized with the functional monomers of the polymerizable mixture. For this purpose, the crosslinking agents known in the art can be used. Examples of crosslinking agents that can be used are: N,N'-methylene bis- acrylamide, polyethylene glycol dimethacrylate, divinylbenezene, 3- (acryloxy) 2-hydroxypropyl methacrylate.

In a preferred embodiment, the crosslinking agent is N,N'-methylene bis-acrylamide.

The radical polymerization initiator is also of the type known to the person skilled in the art. Preferably, the initiator is selected from the initiators capable of polymerizing the monomers of the polymerizable mixture at a temperature in the range from 0 °C to 40 °C, preferably from 15 °C to 30 °C. At this temperature, indeed, the reaction of polymerization proceeds until the formation of the MIP without alterations of the surface protein used as a templating agent.

In one embodiment, the polymerization initiator comprises persulfate ions and N,N,N',N'- tetramethylethylenediamine (TMED). Alternatively, other pairs of peroxide based Red-Ox initiators can be used.

The polymerizable mixture may further contain water or, more preferably, a buffer solution capable of maintaining the pH at a desired value depending on the protein used as a templating agent, generally in the range of pH = 6-8. In one embodiment, the buffer solution is a phosphate buffer, preferably having a pH of about 7. The relative amounts of the above components in the polymerization mixture may vary over wide ranges.

In one embodiment, the overall concentration of functional monomer is in the range 0.0001 - 0.100 M, preferably in the range 0.001 - 0.050 M.

In one embodiment, the overall concentration of the templating agent in the monomer mixture is in the range of 1*10⁸ M to 5*10³ M, preferably in the range of 4*10⁷ M to 7*10⁴ M.

In one embodiment, the overall concentration of the crosslinking agent in the monomer mixture is in the range of 0.05 M to 1.0 M, preferably in the range of 0.1 M to 0.5 M.

In one embodiment, the overall concentration of polymerization initiator in the monomer mixture is in the range of 0.03% w/v to 0.15% w/v.

The polymerizable blend can be prepared by mixing the selected functional monomer or functional monomers in water or, preferably in a buffer solution, together with the selected protein or proteins, crosslinking agent and polymerization initiator.

In general, to obtain the molecularly imprinted polymer according to the invention, the polymerization reaction can be carried out with one of the polymerization methods known to the person skilled in the art, such as for example: precipitation polymerization, suspension polymerization, emulsion polymerization and dispersion polymerization.

As stated, the molecular sensor comprises the molecularly imprinted polymer as a receptor element and at least one transduction system. The molecular sensor may further comprise a detector for detecting the signal generated by the transduction system.

The molecular sensor can be prepared in several ways.

In one embodiment, the polymerizable mixture is deposited on a surface of the transducer and subsequently polymerized to obtain a support coated with the MIP.

In one embodiment, the surface of the transducer on which the polymer is deposited is a surface of a conductive metal such as gold, silver, copper, platinum or aluminum or alloys containing these metals. In one embodiment, the aforementioned surface is a gold surface. In another embodiment, the surface of the transducer on which the polymer is deposited is a surface of Si0₂ (including glass and quartz).

In the case of a metal surface, in particular gold, to favor the adhesion of the polymer, a binding layer can be deposited on the surface, for example of an alkylentiol compound. Preferably, the binding layer is a self-assembled monolayer (SAM), which can be prepared in accordance with the techniques known to the person skilled in the art.

In one embodiment, the SAM comprises molecules of an alkylenol compound. The molecules of this compound bind to the metal surface by means of the thiol group, leaving the organic portion of the molecule free to interact with the components of the polymerizable mixture.

In one embodiment, the binding monolayer is formed by the compound of formula (III)

CH₂ = CH- (CH₂ ) n-SH ( III) where n is an integer from 1 to 4, preferably from 1 to 2.

In one embodiment, the binding monolayer is formed from the alkylenedisulfide compound of formula (IV)

CH₂=CH- (CH₂ ) _n-S-S- (CH₂ ) _m-CH=CH₂ (IV) where n and m, independently of each other, are an integer from 1 to 4, preferably from 1 to 2.

Examples of compounds of formula (III) are: 2- propene-l-thiol, 3-butene-l-thiol and 4-pentene-l- thiol.

An example of a compound of formula (IV) is diallyl disulfide.

Advantageously, during the polymerization phase of the polymerizable mixture, the external allylic double bond of the compounds of formula (III) and (IV) can be polymerized together with the monomers of the mixture favoring the adhesion of the final polymer to the surface. Furthermore, it has been observed that the binding monolayer allows controlling the growth of the polymer and therefore the thickness of the deposited MIP film.

In one embodiment, the thickness of the binding monolayer is less than 200 nm.

The composition of the binding monolayer can change based on the composition of the receptor surface. In the case of SiO2, for example, it is possible to use allyl alkyl silane compounds (e.g. allyl-trimethylsilane) .

The binding monolayer can be prepared by depositing a solution or emulsion of the thiol or silane compound on the metal surface of the transducer. In one embodiment, the solution or emulsion may have a concentration of thiol or silane compound in the range of 0.005 M to 0.06 M.

The deposition of the polymerizable mixture on the surface of the receptor element, possibly previously coated with a binding monolayer, can be carried out with any of the techniques known to the person skilled in the art, such as drop-coating, spraying, immersion, coating and spin coating. In one embodiment, the polymerization of the deposited mixture is made to take place at a temperature in the range of 15 °C - 30 °C.

The duration of the polymerization reaction depends on several factors, including the amount of deposited polymerizable mixture. In general, the duration of polymerization is in the range of 3-20 minutes.

In one embodiment, the molecularly imprinted polymer is in the form of a thin film, e.g. with a thickness in the range of 20 nm to 200 nm.

The polymerization of the mixture preferably takes place in air.

Once the crosslinked polymer is obtained on the surface of the recognition element, the polymer is treated to remove the templating agent from the polymer matrix and create the recognition sites capable of interacting with the surface proteins of the virus.

Removal of the templating agent can be achieved, for example, by treating the crosslinked polymer containing the templating agent with an aqueous solution containing a proteolytic enzyme (protease), for example trypsin, followed by washing with water.

The washing water can optionally contain at least one surfactant e.g. sodium dodecyl sulfate (SDS).

Advantageously, in one embodiment, at least a second crosslinked polymer layer is grown on the crosslinked and templating agent-free polymer layer, repeating the deposition and polymerization steps described above. It has indeed been observed that the presence of at least a second layer of molecularly imprinted polymer favors the regeneration of the sensor at the end of its use.

In another embodiment, the molecular sensor can be prepared by depositing a previously crosslinked molecularly imprinted polymer on the transducer surface. For example, the polymerizable mixture can be polymerized according to the techniques known in the art so as to obtain a MIP in the form of nanoparticles, which are subsequently deposited on the surface of the transducer.

For virus determination, the liquid medium that contains or is believed to contain the virus is placed in contact with the molecular sensor for an incubation time sufficient to allow the surface proteins of the virus to interact with the molecular recognition sites of the sensor. Generally, the incubation time is in the range from 30 seconds to 15 minutes.

In one embodiment, the incubation is carried out at a temperature in the range of 15 °C - 37 °C, preferably at room temperature (25 °C).

Before measurement, the sensor surface can be washed with water, preferably deionized water (e.g. Milli-Q® water).

At the end of the measurement, the sensor can be regenerated by washing with aqueous solutions of a proteolytic enzyme (e.g. trypsin) and possibly a surfactant (e.g. SDS), operating in the same way as illustrated for the removal of the templating agent.

In one embodiment, the measurement system used is an optical measurement system, in particular an SPR measurement system.

In one embodiment, the measurement system comprises: - at least one molecular sensor for detecting a virus in a liquid medium according to the present description;

- at least one signal source for sending an input signal to said molecular sensor;

- at least one detector for detecting an output signal transmitted by said molecular sensor.

This measurement system is illustrated in more detail in the examples.

The following embodiments are provided merely for illustrative purposes of the present invention and must not be construed as limiting the scope of protection defined by the appended claims.

EXAMPLES

In the following examples, reference will be made to the attached figures, which illustrate:

- Figure 1, a schematic representation of an optical fiber SPR sensor containing a recognition element comprising a MIP according to the present invention;

- Figure 2, a schematic representation of a measurement system comprising the SPR sensor of figure i;

- Figure 3, transmission resonance spectra of the plasmon excitation of an SPR sensor before and after the deposition of the molecularly imprinted polymer;

- Figure 4, transmission resonance spectra of the plasmon excitation of the SPR sensor in the presence of a physiological solution containing the protein used as a templating agent for the MIP;

- Figure 5, transmission resonance spectra of the plasmon excitation of the SPR sensor in the presence of a biological sample preserved in physiological solution;

- Figure 6, transmission resonance spectra of the plasmon excitation of an SPR sensor in the presence of a biological sample stored in universal transport medium (UTM®).

In the aforementioned schematic figures, the dimensions of the various elements are not proportional to the actual dimensions.

1, Preparation of a MIP according to the present invention

The polymerization mixture was prepared by mixing the following functional monomers and crosslinking agent in a phosphate buffer (pH = 7.4, 15 mM concentration) :

- 0.01876 M acrylamide

- N-tert-butylacrylamide 0.008736 M

- 2-hydroxyethyl methacrylate 0.0111 M

- N, N'-methylene bisacrylamide 0.1874 M.

The mixture (10 mL) was prepared at room temperature with the aid of an ultrasonic bath and degassed with nitrogen gas.

The surface protein of interest was then added in an amount equal to 1*10⁶ M to the mixture.

The protein used for the preparation is the SI subunit of the SARS-Cov-2 virus marketed by Sino

Biological (SARS-CoV-2(2019-nCov) Spike Protein (SI Subunit, His tag)).

To start the polymerization reaction at room temperature, N,N,N ',N'-tetramethylethylenediamine

(0.06% v/v) and ammonium persulfate (0.08% w/v) are then added to the mixture. The mixture was immediately used for deposition on the sensor.

2. Preparation of the optical sensor

With reference to Figures 1 and 2, an optical sensor 1 was prepared as described in the publication Cennamo et al., Sensor and Actuators B, 188 (2013), 221-226, starting from an optical fiber having a core 2 in polymethylmethacrylate (PMMA) and an external coating 3 (cladding) in a fluorinated polymer (cladding thickness equal to 20 micrometers; core thickness equal to 980 micrometers). For ease of manipulation of the sensor, the optical fiber has been housed in a recess obtained inside a resin platform 5. A part of the outer covering 3 of the optical fiber has been removed until a portion of the core 2 of the optical fiber is left exposed. The cladding 3 was removed for a length of about 10 mm. The removal of the cladding involved approximately 50% of the circumference of the optical fiber.

To make an analysis region 9, the uncovered portion of the core was abraded with an abrasive paper so as to obtain a substantially planar surface 4. On the exposed surface 4 a layer of photoresist material 6 (Microposit S1813), 1.5 micrometers thick, was deposited by spin coating and then a conductive layer formed by a gold film 7 (60 nm thick) by sputtering. The photoresist state, which is optional, improves the coupling of the incident radiation with the plasmon of the gold layer.

In Figures 1 and 2, elements 2, 3, 6 and 7 form the transduction system (or transducer). A binding monolayer 10 of 2-propene-l-thiol compound was deposited over the conductive film 7 of the transducer. For this purpose, a solution in deionized water containing 2.15 mL/L of the above compound (deposited amount equal to approximately 40 microliters/cm²) was deposited on the gold surface at room temperature. The surface was left exposed to air for about 12 hours, in the absence of light and then washed with deionized water at room temperature.

On top of the binding monolayer 10, an aliquot of the polymerizable mixture prepared as described in point 1 (about 50 microliters/cm²) was deposited. The platform was left at room temperature for 10 minutes to allow the polymer to cross-link and then washed with deionized water. The deposition, polymerization and washing steps were repeated a second time to obtain a receptor 8.

The removal of the templating agent was performed by treating the surface of the receptor 8 with a 5% aqueous sodium dodecyl sulfate (SDS) solution (50 microliters for 5 minutes). The sensor was then treated with trypsin (concentration 4xl0⁸ M) in phosphate buffer (pH = 7.4, 15 mM) to fragment the protein and subsequently washed with the aqueous solution of SDS and rinsed with water.

3. Measurement system

With reference to Figure 2, a measurement system 21 configured for spectrum interrogation has been set up. To this end, a white light source 22 (halogen lamp with emission band 360-1700 nm) was connected to one end of the optical fiber to send, inside the core 2, an incident radiation 23 to the analysis region 9. A detector 24 (spectrometer) with a detection range of 200-850 nm was connected to the other end of the optical fiber 2.

The spectrometer 24 has the function of recording the resonance spectrum of the plasmon excitation, which occurs at the interface between the gold film 7 and the receptor 8, when the incident radiation 25 (input signal) hits the analysis region 9. Following the interaction, in fact, the analysis region 9 transmits an output signal 26, which is detected by the spectrometer 24. When the receptor 8 is in contact with a liquid sample 20 to be analyzed, it detects the possible presence of the virus by binding it selectively to the receptor 8 through its spike proteins and causing a change in the refractive index of the receptor 8. The variation between the input signal to the spectrometer 24 (plasmon resonance peak in the presence of a virus-free liquid matrix) and the output signal of (plasmon resonance peak in the presence of a liquid matrix containing the virus) with consequent displacement of the plasmon resonance peak recorded by the spectrometer 24. The magnitude of the peak shift is a function of the analyte concentration.

Alternatively, the input signal can be generated from a monochromatic source, at a predetermined wavelength, by recording the intensity of the radiation transmitted to the detector as an output signal. In this case, the presence of the virus is determined on the basis of the variation in the intensity of the detected output signal, due to the interaction of the compound with the virus.

4. Measurement tests

The efficacy of the molecular sensor in detecting a virus was tested on two biological samples taken from the same subject and stored in two different liquid media, namely physiological solution (sample 1) and UTM® viral transport medium (sample 2). Both samples tested positive for SARS-CoV-2 virus with the real time RT-PCR molecular investigation.

The two samples were tested on two different sensors (sensor 1 and sensor 2), both prepared as described in point 2.

Each of the two samples was analyzed in undiluted form and after one or more dilutions with physiological solution.

For analysis, a sufficient amount of a sample was deposited on the surface of the receptor 10 to completely cover the receptor (approximately 25-50 microliters). The sample was left to incubate for 10 minutes and then washed with deionized water to proceed with the recording of the spectrum.

At the end of each measurement, the polymer receptor of the sensor was treated with 5% aqueous sodium dodecyl sulfate (SDS) solution (50 microliters for 5 minutes) to dissolve the pericapsid envelope of the virus and inactivate it. The sensor was then regenerated by treating the receptor 8 according to the procedure used for the removal of the templating agent described in point 2 above.

Figure 3 shows the SPR transmission spectra for sensor 1:

- before deposition of receptor 10 (spectrum a),

- after deposition of receptor 10 (spectrum b), and

- after the removal of the templating agent and the formation of the recognition sites from the templating agent (spectrum c).

The shift of the resonance peak towards higher wavelength values (red shift) confirms the presence of the receptor 10 on the sensor. After trypsin treatment, a partial blue shift (at lower wavelengths) is observed due to the removal of the templating agent from the polymer. The extent of the shift between spectra a and b (about 10-20 nm) is compatible with a receptor thickness lower than 100 nm.

Figure 4 shows the SPR spectra for sensor 1 placed in contact with a physiological solution containing the same protein (concentration 6.5 * 10⁶ M) used for the preparation of the molecularly imprinted polymer (spectrum a) compared with the spectrum of the sensor in the absence of the solution (spectrum b). Comparison of spectra a and b highlights the response of the sensor to the protein.

Figure 5 shows the SPR transmission spectra recorded on the sensor la in contact with the sample 1, in comparison with the spectrum of the physiological solution alone. In the presence of the virus bound to the receptor, a peak shift of about 3 nm is observed on the undiluted sample. The shift is also instrumentally measurable on the diluted sample up to a dilution ratio 1:500.

For comparison, undiluted sample 1 was subjected to molecular testing with RT-PCR, where it was positive after 36 PCR cycles (test method cut-off equal to 40 cycles).

The method according to the invention therefore has a significantly lower detection limit than the RT- PCR test.

Figure 6 shows the SPR transmission spectra of sample 2 stored in UTM recorded on sensor 2, compared with the spectrum of UTM liquid medium only. Even in this case, in the presence of the virus bound to the receptor, a peak shift of about 3 nm is observed on the undiluted sample. The shift is instrumentally measurable on the diluted sample up to a dilution ratio 1:10.

For comparison, undiluted sample 2 was also subjected to molecular testing with RT-PCR, where it was positive after 36 PCR cycles.

The virus detection tests on samples 1 and 2 carried out using two different sensors prepared with the same procedure described above show completely comparable results, thus confirming the high reproducibility of the method and the reliability of the sensor according to the invention.

The examples also confirm that the sensor and the method according to the present invention allow a virus to be detected quickly and effectively, both in qualitative and quantitative terms, in a liquid medium such as a biological sample with a very low detection limit.

Claims

1. A method for detecting a virus in a liquid medium, said virus being characterized by the presence of at least one surface protein, wherein said method comprises:

- putting the liquid medium in contact with the molecular sensor;

2. The method according to claim 1, wherein the virus belongs to the Coronaviridae family.

3. The method according to any one of claims 1 to 2, wherein the virus is a SARS-CoV virus, preferably SARS-CoV-2.

4. The method according to claim 3, wherein the virus is SARS-CoV-2 and the surface protein is the SI subunit of said virus.

5. The method according to any one of claims 1 to

4, wherein the detection is carried out by means of an optical, electrochemical, electrical, acoustic or spectroscopic technique, preferably by surface plasmon resonance.

6. The method according to any one of claims 1 to

5, wherein the molecularly imprinted polymer is obtained by copolymerization of: at least one monomer of formula (I)

where:

Ri is H or CH₃;

R2 is selected from: H or a C1-C8 alkyl, preferably a, linear or branched, C1-C4 alkyl;

- at least one monomer of formula (I)

where:

Ri is H or CH₃; n is an integer in the range 1 - 4, preferably in the range 1 - 2, in the presence of at least one crosslinking agent and said surface protein of the virus as a templating agent.

7. The method according to claim 6, wherein the polymerizable mixture comprises: acrylamide, N-tert- butylacrylamide and 2-hydroxyethylmethacrylate.

8. The method according to any one of claims 1 to 7, wherein the liquid medium is a biological fluid obtained from a human or animal subject.

9. A molecular sensor for detecting a virus having at least one surface protein in a liquid medium, comprising:

10. The molecular sensor according to claim 9, wherein the receptor element comprises a metal or SiCt substrate having a surface coated with the molecularly imprinted polymer.

11. A process for preparing a molecular sensor for detecting a virus in a liquid medium, said virus being characterized by the presence of at least one surface protein, comprising: a) preparing a polymerizable mixture comprising:

- at least one functional polymerizable monomer;

- at least one templating agent comprising said surface protein of the virus;

- at least one cross-linking agent;

12. The process according to claim 11, wherein said polymerizable mixture comprises:

- at least one monomer of formula (I)

where:

Ri is H or CH₃;

R2 is selected from: H or a C1-C8, alkyl preferably a linear or branched C1-C4 alkyl;

- at least one monomer of formula (I)

where:

13. The process according to claim 11 or 12, wherein the polymerizable mixture comprises: acrylamide, N-tert-butylacrylamide and 2- hydroxyethylmethacrylate .

14. The process according to any one of claims 11 to 13, wherein the surface on which the polymerizable mixture is deposited is a gold or Si0₂ surface.

15. The process according to claim 14, wherein the polymerizable mixture is deposited on a gold surface on which it has been previously deposited a layer of:

- a compound of formula (III)

where n is an integer from 1 to 4, preferably from 1 to 2, or

- a compound of formula (IV)

where n and m, independently of each other, are an integer from 1 to 4, preferably from 1 to 2.

16. A molecularly imprinted polymer having recognition sites of a surface protein of a virus, the polymer being obtained by polymerization of a polymerizable mixture comprising at least one functional polymerizable monomer, in the presence of at least one crosslinking agent and at least one surface protein of the virus as a templating agent.

17. A molecularly imprinted polymer according to claim 16, wherein the polymerizable mixture comprises:

- at least one monomer of formula (I)

where:

Ri is H or CH₃;

R₂ is selected from: H or a C1-C8, alkyl, preferably a linear or branched C_1-C₄ alkyl;

- at least one monomer of formula (I)

where:

18. Use of a molecularly imprinted polymer according to claim 16 or 17 as a receptor element in a molecular sensor.