FIELD OF THE INVENTION

The present invention relates to an in vitro method for the detection of SARS-CoV-2 in an oral biological sample using a colorimetric immunosensor, and the related colorimetric immunosensor.

STATE OF THE ART

In the field of research and diagnostics, colorimetry-based optical biosensors are becoming more prominent on account of their versatility, ease of use and capacity for achieving an extremely low limit of detection (LOD) of the analyte.

Known optical biosensors include colorimetric biosensors based on gold nanoparticles, which utilize a physical property of the gold nanoparticles known as Localized Surface Plasmon Resonance (LSPR) to monitor the change in color when, following the interaction between the analyte to be detected and the gold nanoparticles, clusters of various dimensions are formed.

Optical transducers are of particular interest for the direct (label-free) detection of microorganisms. These sensors are designed to detect the smallest changes in refractive index or thickness that occur when the cells adhere to receptors immobilized on the surface of the transducer, thus correlating the changes in concentration, mass or number of molecules with direct changes in the characteristics of the light. The sensing mechanism is based on converting signals from binding with the target to be detected into physical signals that may be amplified and detected. Nanomaterials, which are characterized by extremely small dimensions and to which appropriate surface modifications may be made, allow highly specific interaction with the biomolecular targets, thus demonstrating enormous potential in the field of biological detections. More generally, gold nanoparticles are used in a wide range of disciplines, including magnetic fluids, catalysis, biotechnology/biomedicine, magnetic resonance imaging and environmental restoration. In the vast majority of these applications, improved functionality of these nanoparticles is observed when their size is smaller than a critical value, which depends on the material but typically is approximately 10-20 nm.

Biosensors that are based on the specificity of interaction between an antigen and the corresponding antibody in order to determine the analyte of interest are generally known as immunosensors. The process of antibody immobilization is a crucial step in the construction of these devices, since the orientation of the antibody molecules on the surface of the electrode significantly affects the performance of a biosensor. In fact, the formation of a layer of antibodies with their binding sites well-oriented and facing the antigen improves the efficiency of the biosensor, making the selection of the immobilization method one of the most important aspects to be considered when designing an immunosensor. Procedures for immobilizing antibodies generally involve physical or chemical adsorption of these molecules.

Among the simplest adsorption processes is mentioned the procedure that utilizes ionic or electrostatic bonds, hydrophobic interactions and Van der Waals bonds between the antibody and the surface (Sharma, Byme, and Kennedy 2016; Um et al. 2011) and does not require the protein to be chemically modified. The main disadvantage of said procedure is that the antibodies are randomly oriented and may therefore not correctly expose the antigen binding sites.

More effective methods for immobilizing antibodies are based on the formation of covalent bonds between the antibody and the gold surface (Alves, Kiziltepe, and Bilgicer 2012; Ho et al. 2010; Vashist et al. 2011; Rahman et al. 2007). For example, biotinylated antibodies may be immobilized on surfaces modified with streptavidin or avidin (Barton et al. 2009; Ouerghi et al. 2002), or the antibodies may be immobilized on surfaces modified with proteins such as protein A or protein G (J. E. Lee et al. 2013; Inkpen et al. 2019; Sharafeldin and Rusling 2019; Fowler, Stuart, and Wong 2007). Lastly, the last decade has seen the development of antibody immobilization methods that involve entrapment in polymeric matrices (Sun et al. 2011; Bereli et al. 2013; Moschallski et al. 2013; Yamazoe 2019).

Among the possible immobilization strategies, the formation of self-assembled monolayers (SAMs) is one of the most widely used methods for immunosensor design. For example, the oriented immobilization of an antibody on the gold surface of an electrode may be achieved by exploiting the formation of SAMs of thiol carboxylic acids (Barreiros dos Santos et al. 2013; Malvano, Pilloton, and Albanese 2018; Wan et al. 2016) or by immobilizing the antibodies on electrochemically deposited layers of cysteamine (Malvano, Pilloton, and Albanese 2018). Moreover, the use of cross-linking agents such as glutaraldehyde has recently been reported, specifically in order to immobilize anti-L. call antibodies on a polyaniline substrate, with interesting results in the detection of this bacterium (Chowdhury et al. 2012). SAMs are therefore widely used as linker agents to immobilize the antibodies in an oriented manner on a gold surface; however, despite the numerous advantages they exhibit in various applications, there are still several aspects that should be taken into consideration in order to understand and control their physical and chemical properties (Vericat et al. 2010; Mandler and Kraus-Ophir 2011; Chaki and Vijayamohanan 2002). A self-assembled monolayer on gold surfaces is often represented as a perfect monolayer in which the molecules are in a perfectly packed configuration. Actually, this idea is far from reality, and quality control of a SAM is a crucial point in many applications. The construction of a well-assembled monolayer depends strongly on the purity of the chemical reagents and solutions used and even the presence of a minimum amount of contaminants, such as for example thiolated molecules that are typical impurities in thiol compounds, may lead to a non-uniform and therefore not ideal layer (C. Y. Lee et al. 2005).

A particularly advantageous technique for immobilizing antibodies on a gold (or more generally metal) surface is that proposed by Della Ventura et al. (“ Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy ” Analyst 144, 6871-6880), according to which antibodies are “activated” by UV radiation and selectively open some disulfide bridges (PIT, Photochemical Immobilization Technique). The sulfur then binds to the surface so that one of the two variable parts is i) exposed to the solution and ii) at a distance of a few nanometers from the surface. The condition ii) results in a reduced steric hindrance of the antibody, which is a particularly important condition when functionalizing nanoparticles which have to cluster; in fact, the reduced steric hindrance leads to a shorter distance between the nanoparticles, which is an important prerequisite for plasmonic interaction to occur between them.

In recent years, various types of immunosensors have been described in studies published in the literature.

Iarossi, M. et al (2018) (“ Colorimetric Immunosensor by Aggregation of Photochemically Functionalized Gold Nanoparticles” ACS Omega 3, 4, 3805-3812) describes a colorimetric immunosensor which uses the phenomenon of surface plasmon resonance of gold nanoparticles as well as the application of this system for the detection of human immunoglobulin IgG.

Liu Y., et al (2015) (“ Colorimetric detection of influenza A virus using antibody - functionalized gold nanoparticles” Analyst 140(12)3989-3995) studied the use of a colorimetric immunosensor based on gold nanoparticles modified with anti-haemagglutinin monoclonal antibodies in order to determine the influenza A virus. However, this document does not demonstrate the effectiveness of the described immunosensor in a clinical setting.

The research described in Della Ventura B. et al (2020) (“ Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs” MedRixv, DOI: https://doi.org/10.1101/2020.08.15.20175489, ACS Sensors 2021, 6, 4269-4271) relates to the use of a colorimetric immunosensor for the detection of SARS-CoV-2 coronavirus in a nasopharyngeal swab.

The ongoing severe pandemic caused by the new Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has posed major challenges for public health in many countries. As a result of the poor specificity of the symptoms of the serious illness caused by the new coronavirus, which is known as COVID-19, confirmation of diagnosis requires laboratory tests to be carried out on respiratory samples and/or on serum samples from patients. The diagnostic tests carried out on a large scale also play a fundamental role in isolating asymptomatic COVID-19 patients, in an attempt to stem the spread of the infection.

Among the methods currently used to diagnose the SARS-CoV-2 coronavims infection, the procedure based on reverse transcription polymerase chain reaction (RT-PCR) plays a primary role. This procedure allows to identify the viral genome in samples from the upper respiratory tract, in particular in samples collected using nasopharyngeal swabs. However, the diagnostic application of PCR has significant limitations due to the complexity of execution, turnaround times, as well as the need for dedicated instruments and trained staff.

The immunological approaches that have been developed for diagnosing COVID-19 also pose significant problems. For example, lateral flow tests, while allowing many samples to be analyzed quickly, are characterized by low sensitivity.

Further problems associated with the methods aimed at diagnosing SARS-CoV-2 infection include the choice of the most suitable samples for detecting viral particles, more specifically intact particles. Among these, the samples of choice are currently samples from the respiratory tract, in particular nasopharyngeal swabs. However, collecting this sample requires a precise operating procedure; i.e., to be valid, the sample has to be collected from the nose by pushing the swab downward, in order to reach the pharynx, and not toward the nasal cavities.

There is therefore a need to provide diagnostic tests which allow for rapid identification of the SARS-CoV-2 using simple procedures, while maintaining at the same time high specificity and sensitivity parameters.

These and other objects are achieved by means of the in vitro method and the related kit as defined in the attached independent claims, which are suitable for the detection of the SARS- CoV-2 virion in an oral biological sample of a subject selected from saliva and sputum.

Additional features of the invention are identified in the dependent claims, which form an integral part of the description. As will become clear from the following detailed description, the in vitro method according to the invention allows to obtain the diagnostic result in a very short time, in the range of a single minute, and requires minimal quantities of the sample to be analyzed, in the range of approximately one milliliter volume, thereby facilitating sample transport. The particular simplicity of the method, which does not require any sophisticated instruments, also makes it possible to considerably reduce costs.

Therefore, a first object of the present invention is an in vitro method for the detection of the SARS-CoV-2 virion in an oral biological sample of a subject selected from saliva and sputum, comprising the steps of: a) contacting said biological sample with a colloidal suspension of gold capture nanoparticles carrying on their surface at least one antibody capable of binding a SARS-CoV-2 surface antigen, the biological sample being on a swab and being partly released from the swab into the colloidal suspension, thereby obtaining a reaction mixture, wherein the SARS-CoV-2 surface antigen is selected from the group consisting of the membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof; and

(b) determining the formation of a cluster of gold nanoparticles on the surface of the SARS- CoV-2 virion in the reaction mixture, the virion being on the swab and being partly suspended in the reaction mixture, said cluster resulting from the interaction between said antibody and said antigen, the determination being performed by detecting a change in an optical parameter of the reaction mixture, said change in an optical parameter of the reaction mixture being indicative of the presence of the SARS-CoV-2 virion in the oral biological sample, wherein the change in the optical parameter is:

(i) an increase in the transmittance value of the reaction mixture as measured at a wavelength in a range from 520 nm to 540 nm;

(ii) a decrease in the absorbance value of the reaction mixture as measured at a wavelength in a range from 520 nm to 540 nm; and/or

(iii) a decrease in the area under the absorption spectrum of the reaction mixture in a wavelength range between 200 nm and 700 nm. Within the context of the present description, the term “virion” is understood to mean the mature viral particle, including the genome, nucleocapsid, and envelope thereof.

The method according to the invention is based on the physical principle of Localized Surface Plasmon Resonance (LSPR), which consists in the occurrence of coherent and non propagating oscillations of free electrons in metal particles following irradiation with an electromagnetic wave, the frequency of which is in resonance with the surface plasmon. The surface plasmon resonance, which gives the colloidal solution its color, depends on various factors, such as the dimensions of the nanoparticles, and may change significantly when the nanoparticles are in contact with each other, or are in any case at a much smaller distance than their own diameter. In general, the coupling between metal nanoparticles, for example gold nanoparticles, which are present in a colloidal suspension is achieved via the formation of dimers, trimers or larger chains up to the formation of clusters, and involves a change in the plasmon resonance and therefore in the color of the solution, which may also be detected by the naked eye.

According to the invention, the clustering between the gold nanoparticles present in the reaction mixture is brought about by a biological mechanism consisting in the specific interaction between the antibodies immobilized on the surface of said capture nanoparticles and the corresponding antigens present on the surface of the SARS-CoV-2 virus. Thus, clustering occurs only if the SARS-CoV-2 viral particle is present in the sample to be analyzed, thus providing extreme specificity to the method of the invention.

The oral samples suitable for use in the method according to the invention are selected from saliva and sputum.

According to the invention, the saliva or sputum sample contacted with the colloidal suspension of gold capture nanoparticles in step a) is on a swab. In the aforesaid step of the method, a portion of the biological sample is released from the swab into the colloidal suspension. Swabs suitable for collecting an oral biological sample such as saliva or sputum are known and described in the prior art, and therefore their selection and use fall within the ability of the person skilled in the art. Preferably, one of the two ends of the swab or both ends of the swab is/are covered with cotton wool or cotton.

As illustrated in greater detail in the following experimental part, the present inventors have surprisingly found that the method of the invention allows immunological detection of the SARS-CoV-2 virus in the saliva or sputum of a subject. Despite the undoubted advantage of the ease of collecting this type of sample, using these samples in a method as described above has so far proved particularly difficult, if not impossible, not only due to the high salt concentration in these samples, which causes non-specific clustering of the gold nanoparticles, making the biosensor unusable, but also due to the presence of mucus. The latter, although not affecting the kinetics of clustering, contains opaque substances that alter the absorption spectrum. Unfortunately, the variability of the mucus concentration (not only among people, but also over time for the same person) makes it impossible to standardize the contribution of the mucus to the optical reading. A further challenge with the use of unprocessed saliva in immunological tests is the high concentration of proteins present in this sample, which may interfere with the antigen-antibody interaction.

All these considerations highlight the peculiarity of the salivary matrix compared to other solutions containing the virion, such as, for example, that consisting of a Virus Transport Medium (VTM). In order to demonstrate the importance of the peculiarity of each matrix, which makes it almost impossible to directly apply the analysis technique from one matrix to the other, it is sufficient to note that the “gold standard” technique provided by RT-PCR is ineffective for, if not inapplicable to, the salivary matrix as such.

As indicated above, the metal nanoparticle used in the method according to the invention is a gold nanoparticle.

Preferably, the gold nanoparticle has a diameter ranging between 1 nm and 100 nm, more preferably between 2 nm and 40 nm. Most preferred is a gold nanoparticle having a diameter of 20 nm. In the method according to the invention, the at least one antibody on the surface of the gold capture nanoparticle is capable of binding a SARS-CoV-2 surface antigen selected from the group consisting of the membrane protein (M), the envelope protein (E), the spike protein (S), and any combination thereof.

In a preferred embodiment, the gold capture nanoparticles carry on their surface at least one antibody capable of binding the SARS-CoV-2 surface antigen membrane protein (M), at least one antibody capable of binding the SARS-CoV-2 surface antigen envelope protein (E), and at least one antibody capable of binding the SARS-CoV-2 surface antigen spike protein (S).

As is known in the art, the aforementioned viral proteins contribute together to the formation of the external viral envelope. Of these proteins, the spike protein is responsible for the binding of SARS-CoV-2 to the host cell by promoting the fusion of the viral envelope with the cell membrane.

The antibody molecules suitable for use in the method according to the invention include, by way of non-limiting example, monoclonal or polyclonal antibodies, monomeric (Fab) or dimeric (F(ab’)2) antibody fragments, single-chain antibody fragments (scFv), or any binding protein derived from an antibody scaffold.

According to the invention, it is contemplated that anti-SARS-CoV-2 antibodies as defined above may be present on the gold capture nanoparticle, in any possible combination.

Procedures suitable for achieving the immobilization of one or more antibody molecules on the surface of a gold nanoparticle are known in the art. For this purpose, for example, the photochemical PIT technique described above may be used, in which the immobilization of the antibodies on the gold surface in the correct orientation is achieved by irradiating these molecules with UV light. Although the PIT technique has advantages over other processes, the selection of the most appropriate antibody immobilization method for use within the scope of the present invention falls well within the skills of the person skilled in the art.

Preferably, the amount of gold capture nanoparticles as defined above in the colloidal suspension is in the range from 1 to 10 ²⁰ nanoparticles (np)/mL based on the total volume of the suspension, more preferably from 10 to 10 ¹⁵ np/mL. In an even more preferred embodiment, the amount of gold capture nanoparticles is 10 ¹⁰ np/mL on the total volume of the suspension.

Advantageously, the method according to the invention enables the SARS-CoV-2 viral particle to be detected in its entirety in the test sample due to the ability of the gold nanoparticles carrying antibodies specifically directed against the viral surface proteins to cluster on the surface of the virion by forming a layer around said virion. Therefore, as a result of the specific determination of active viral particles, the method according to the invention is particularly suitable for identifying cases in which the SARS-CoV-2 infection is still ongoing.

According to one embodiment, the method of the invention further comprises the step of filtering the reaction mixture obtained in step a) by means of a filter element having pores with a diameter ranging from 0.5 to 5.0 microns (pm), preferably from 1.0 to 4.0 pm, for example a diameter of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 pm.

As illustrated above, in the method according to the invention, the formation of a cluster of gold nanoparticles on the surface of the SARS-CoV-2 virion is determined by detecting a change in an optical parameter of the reaction mixture, using three different approaches.

In a first detection embodiment, the detected change in the optical parameter is an increase in the transmittance value of the reaction mixture as measured at a wavelength ranging from 520 nm to 540 nm, preferably from 525 nm to 535 nm, for example at a wavelength of 525, 526, 527, 528, 529, 530, 531, 532, 533, 534 or 535 nm. According to the above embodiment, the measurement of the transmittance value of the reaction mixture may be carried out using a photometer or colorimeter instrument, preferably calibrated with a standard solution having a transmittance value of 100%.

In a second detection embodiment, in the method of the invention the detected change in the optical parameter is a decrease in the absorbance value of the reaction mixture as measured at a wavelength ranging from 520 nm to 540 nm, preferably from 525 nm to 535 nm, for example at a wavelength of 525, 526, 527, 528, 529, 530, 531, 532, 533, 534 or 535 nm. A suitable instrument for performing said absorbance measurement is, for example, a spectrophotometer that may be portable or benchtop.

According to a third detection embodiment, the detected change in the optical parameter is a change in the absorption spectrum of the reaction mixture in a wavelength range between 200 nm and 700 nm. In this embodiment, the change in the optical parameter may be, for example, the decrease in the area under the absorption spectrum of the reaction mixture in a wavelength range between 200 nm and 700 nm.

Within the context of the present description, the expression “wavelength in the visible range” is understood to refer to a wavelength between approximately 390 nm and approximately 760 nm.

In the detection embodiments described above, the method according to the invention also allows to carry out a quantitative measurement that is indicative of the SARS-CoV-2 viral load present in the test sample. Indeed, the use of a standard curve in addition makes it possible to obtain an “absolute” measurement of the viral load.

It is envisaged that, according to the invention, the various embodiments for detecting the change in the optical parameter as detailed above may be used in the method of the invention in combination or alternatively to one another depending on need, and the selection of the detection mode is well within the ability of the person skilled in the art. Without wishing to be bound by any theory, the present inventors believe that the changes in the optical parameters in the reaction mixture as defined above are determined by the clustering of the nanoparticles induced by the presence of the virion (or parts thereof). The proximity of the nanoparticles leads to their plasmonic interaction, resulting in a change in the resonance wavelength and thus in the spectrum (color).

In one embodiment of the method of the invention, the detected change in the optical parameter consists of a color change of the reaction mixture that may be detected by the naked eye.

In this embodiment, a further optional step consists in comparing the detected color of the reaction mixture with a colorimetric scale. This step increases the interpretative quality of the result.

It is also important to note that, in the embodiment illustrated above, advantageously there is no need to use any instruments.

As previously stated, a kit including means suitable for carrying out the method according to the invention is also included within the scope of the present invention.

Therefore, a second aspect of the present invention is a diagnostic kit for the detection of the SARS-CoV-2 virion in an oral sample of a subject selected from saliva and sputum, comprising a colloidal suspension of gold capture nanoparticles carrying on their surface at least one antibody capable of binding a SARS-CoV-2 surface antigen, the antigen being selected from the group consisting of the membrane protein (M), the envelope protein (E), the spike protein (S), and any combination thereof, the kit being characterized by further comprising one or more swabs suitable for collecting an oral biological sample.

According to the invention, the one or more swabs contained in the kit are as defined above in relation to the method. In one embodiment, the diagnostic kit of the invention also comprises a support containing a colorimetric scale, for example a colorimetric strip.

According to another embodiment, the diagnostic kit of the invention also comprises a portable colorimeter or photometer.

Preferably, the portable photometer is provided with a tungsten lamp and a monochromator capable of isolating a wavelength ranging from 520 nm to 540 nm, preferably from 525 nm to 535 nm, for example a wavelength of 525, 526, 527, 528, 529, 530, 531, 532, 533, 534 or 535 nm.

Preferably, the portable colorimeter is provided with a diode capable of emission at 525 nm or 530 nm.

Among the portable instruments suitable for use in the kit of the invention, the portable photometer model Genotrix Cube 1 from the company Genotrix is cited by way of example.

In the diagnostic kit of the invention, the colloidal suspension comprising the gold capture nanoparticles may be dispensed into a plurality of single disposable tubes.

Alternatively, said colloidal suspension may be provided in a single package, for example in a dedicated dropper device.

In a further embodiment, the diagnostic kit of the invention also comprises a filter element as previously defined in connection with the method of the invention.

According to this embodiment, the filter element may be housed inside a disposable test tube, preferably in proximity to one end of the body of the test tube opposite the end where the colloidal suspension is introduced.

Within the scope of the invention, the filter element in the kit is preferably a polypropylene filter. The following experimental examples are provided purely by way of illustration. Reference is made therein to the appended drawings, wherein:

Fig. 1 is a schematic representation of the method for functionalizing the surface of gold nanoparticles with type G immunoglobulin (“Photochemical Immobilization Technique,” RGG). The IgG antibodies are irradiated with UV rays using the “Trylight” lamp, leading to a reduction in disulfide bridges at specific positions in the light chain, i.e. the constant part, of the antibody. The production of thiols allows the formation of a covalent bond between the antibody and the surface of the gold nanoparticle, leaving one of the two antigen- recognition antibody portions free;

Fig. 2 is a schematic representation of the method of the invention. The swab used to collect the oral biological sample is introduced into the colloidal suspension of functionalized gold nanoparticles, which suspension has an OD value preferably equal to 1.5. Virions possibly present on the swab are partly released into the suspension. Both the released virions and the virions that remain trapped in the cotton of the swab contribute to “removing” free functionalized gold nanoparticles from the suspension by binding them, and this results in a change in the absorption spectrum. More specifically, a reduction in the area of the spectrum of the free nanoparticles and the formation of a spectrum caused by the clustering of the nanoparticles around the suspended virions are observed. This leads to a reduction in absorbance at the absorption peak of the “free” functionalized gold nanoparticles, which is observed at 530 nm when the diameter of the nanoparticles is 20 nm;

Fig. 3 is a graph which shows the correlation between positive and negative results for SARS-CoV-2 infection in ten subjects, as measured by means of the method of the invention on saliva samples, and expressed as absorbance value at the wavelength of 525 nm and 530 nm, and by means of the PCR method on nasopharyngeal samples, and expressed as threshold cycle (Ct) values. The dotted line indicates a threshold discriminating positive and negative cases of viral infection with high specificity and sensitivity. At the bottom right of the graph, a circle highlights a subject with proven SARS-CoV-2 infection who tested positive using the method of the invention, but who was not detected using real-time PCR.

Experimental section Example 1: Preparation of the colloidal gold solution (synthesis of the nanoparticles)

For their experiments, the present inventors obtained the synthesis of gold nanoparticles having a diameter of approximately 20 nm using a variant of a protocol known in the art (the Turkevich method). According to this protocol, tetrachloro auric acid is first dissolved in water and, with the addition of sodium citrate, a reduction of the gold occurs together with the production of a gold seed, and subsequently the growth of the gold around said seed is promoted. The synthesis reaction consisted in mixing 1 mL of HAuCU (10 mg/mL) and 2 mL of sodium citrate dihydrate (10 mg/mL) in 100 mL of MilliQ (ultrapure) water. The operating temperature was kept at 90°C, with slow stirring. The formation of the gold nanoparticles was identified by a drastic change in the color of the solution from yellow to orange.

At the end of the synthesis, the solution was subjected to centrifugation at 6 G for 30 minutes, thus obtaining the gold nanoparticles ready for functionalization.

Example 2: Functionalization

In order to functionalize the surface of the gold nanoparticles, the mechanism known as “photochemical immobilization technique” (PIT) was used, as shown in Fig. 1.

Briefly, IgG antibodies directed against the membrane protein {Membrane, M), envelope protein {Envelope, E) and spike protein (S) of the SARS-CoV-2 virus were used (0.1 mg/mL).

A quartz cuvette containing the antibody solution at a concentration of 1 pg/mL was inserted into the Trylight lamp and irradiated with UV rays for 30 seconds so as to obtain the reduction of some disulfide bridges in specific positions of the antibody. Subsequently, gold nanoparticles having a diameter of 20 nm were functionalized, so as to obtain a concentration of nanoparticles with the anti-envelope antibodies of 10 ¹⁰ nanoparticles (np)/mL, a concentration of nanoparticles with the anti-spike antibodies of 10 ¹⁰ np/mL, and a concentration of nanoparticles with the anti-membrane antibodies of 10 ¹⁰ np/mL. Any empty spaces left on the gold nanoparticles were then blocked using a solution containing BSA (50 pg/mL).

Finally, the colloidal suspensions containing the three different antibodies were mixed together at a ratio of 1:1:1 so as to obtain a single suspension of gold nanoparticles carrying the three anti-SARS-CoV-2 antibodies, thus significantly increasing the specificity of the system.

Purification of the obtained samples was carried out by centrifugation at 7000 g for 10 minutes.

Example 3: Preparation of the sample

The saliva sample was collected in the oral cavity, in the region between the gums and the cheek, by means of a cotton swab, and immediately transferred into a volume of 0.5 mL of the colloidal suspension of functionalized gold nanoparticles. If present, the virions on the cotton swab are released partly into the suspension, by vigorously rotating the swab around its axis for approximately 10 seconds, and partly remain immobilized in the cotton of the swab. The viral particles released into the colloidal suspension are recognized by the functionalized gold nanoparticles that cluster around the virions, while other gold nanoparticles recognize the virions that have remained trapped in the cotton, and cluster around them. Both methods of nanoparticle binding to the virions, i.e. in suspension and on the swab, result in a significant reduction in the nanoparticles in solution. Therefore, the reading of the absorbance value of the reaction mixture at a wavelength between 520 nm and 540 nm, more specifically the decrease in the absorbance value at one of said wavelengths, for example at 525 nm or 530 nm, is the “sensing” parameter. It follows that if a person tests positive, the absorbance value at one of the wavelengths indicated above tends toward zero; in the opposite case, the absorbance value of the reaction mixture does not change with respect to the absorbance value of the starting nanoparticles.

The present inventors observed that, unlike the prior art assays, the method according to the invention surprisingly does not require the saliva sample to be resuspended in a buffer solution after collection. The present inventors also found that other stirring methods, for example stirring the test tube up and down in an uncoordinated manner, do not achieve the same result in the same times, also showing lack of repeatability and reproducibility.

Optionally, the reaction mixture obtained by mixing the saliva or sputum samples with the colloidal suspension of the gold capture nanoparticles was left to drip through a polypropylene filter having a thickness of 2 mm and pores of 1.0 pm in diameter, and collected in an underlying cuvette. The time taken for this step varied within a range of 2-5 minutes, depending on the viscosity of the saliva. After filtration, the reaction mixture was read.

Example 4: Detection

For SARS-CoV-2 virus detection experiments, gold capture nanoparticles prepared and functionalized as described above were used.

Briefly, after having mixed the saliva or sputum sample with the colloidal suspension of the gold capture nanoparticles, a color change occurred in the reaction mixture that went from orange to blue passing through purple. In particular, the shift toward blue was greater as the virus concentration increased. A simple colloidal solution of the gold capture nanoparticles in which no reduction in the absorbance at a wavelength between 520 nm and 540 nm was observed was used as a negative control.

For transmittance or absorbance analysis, the entire volume of the colloidal solution that has come into contact with the swab that carries the oral biological sample was injected into a microcuvette in order to facilitate the reading in the spectrophotometer, as well as improve the reading with the naked eye.

The tube or cuvette was then placed into the supplied portable reader, which provided the absorbance or transmittance values indicative of the positivity/negativity of the sample, i.e. the presence or absence of SARS-CoV-2 viral particles. Absorbance readers suitable for measuring absorbance (OD) at 525 nm and 530 nm were used in the experiments carried out by the present inventors.

In particular, a portable photometer was used which was equipped with a tungsten lamp and a monochromator capable of isolating the wavelength at 525 nm and 530 nm, after suitable calibration with a standard. A simple “blank”, i.e. a sample assigned a 100% transmittance or 0% absorbance, was used as the standard . Disposable cuvettes containing the reaction mixture were used for the photometric analysis.

In their experiments, the inventors alternatively used a colorimeter device equipped with a diode capable of emitting at 525 nm or 530 nm and returning an absorbance value, analogously to the device described above. In this procedure mode, after resuspension, the saliva or sputum sample was dispensed and assayed directly in the disposable reaction tube pre-packaged with the colloidal suspension of gold capture nanoparticles.

The absorbance values measured in the experiments described above are indicative of the amount of SARS-CoV-2 viral particles present in the test sample.

In order to measure the absorbance of the reaction mixture, the inventors also used a benchtop spectrophotometer instrument capable of emitting in a wide spectrum of wavelength ranging, for example, from 200 to 700 nm. The absorbance measurements detected at the various wavelengths allowed to calculate the area under the absorption spectrum, thus providing a “relative” quantitative determination of the viral load. The measurement of the viral load can become “absolute” by means of a calibration of the described technique.

Table 1 below shows the results obtained on six patients (3 positive and 3 negative for SARS- CoV-2 infection), using the PCR method on nasopharyngeal swabs and the method according to the invention on unprocessed saliva samples in parallel. For the PCR analysis, the data are expressed as the cycle threshold (Ct), which corresponds to the PCR reaction cycle in which the emitted fluorescence exceeds the threshold. For the analysis according to the method of the invention, the data are expressed in absorbance values (Abs). The term “x” indicates a negative sample.

Table 1

Table 1 shows that negative samples give an absorbance value equal to 1.5+0.2. Even taking into consideration the 3SD criterion, the sample with the lowest viral load (Ct = 30) may be clearly distinguished from the negative samples. In fact, values higher than 30 for the PCR threshold are considered negative.

The graph in Fig. 3 shows the results of a further validation study carried out by the present inventors on a larger cohort of subjects. Briefly, during this study the correlation between the positive or negative results for SARS-CoV-2 infection was examined as detected in the examined subjects by the PCR method on nasopharyngeal samples (expressed as cycle threshold values (Ct)) and, in parallel, by the method of the invention on unprocessed saliva samples (expressed as absorbance values (OD)).

As shown in the graph of Fig. 3, the response is significantly different for negative subjects, who have an absorbance similar to the starting solution (colloidal suspension of the capture nanoparticles), which has an OD equal to 1.5 in the wavelength range between 525 nm and 530 nm, and for positive subjects who have a lower absorbance. In the graph, a threshold discriminating positive and negative cases with high specificity is also shown as a dotted line. As is well known, values higher than Ct=35 are often considered to be negative or, in any case, indicative of an absence of contagiousness. Therefore, the method of the invention has a limit of detection comparable with PCR.

Of note, moreover, the method of the invention enabled a subject with proven SARS-CoV- 2 infection who had been classified as a negative case according to the PCR analysis (sample enclosed by a circle in the graph in Fig. 3) to be detected as a positive case. Without wishing to be bound by any theory, the inventors believe that the superior sensitivity of the method of the invention as compared with the real-time PCR method is based on the following considerations: high specificity of the immunosensors, for which recognition with antibodies guarantees a specificity that is known to be very high, and higher concentration of virus in saliva, which allows intercepting infected (and infecting) persons with high viral load especially in the oral cavity, whereas the region of choice for the analysis by real-time PCR is the nasopharyngeal cavity. In conclusion, the results obtained by the present inventors in the validation studies described above demonstrate a clear correlation in the samples examined between the decrease in OD and the Ct value, and also reveal a significant sensitivity of the method of the invention, making it suitable for quantitative viral load measurements