IN VITRO METHOD FOR THE DETECTION OF SARS-COV-2 IN AN ORAL SAMPLE USING A COLORIMETRIC IMMUNOSENSOR AND RELATED COLORIMETRIC IMMUNOSENSOR

Abstract

An in vitro method and a kit for detecting the SARS-CoV-2 virion using a colorimetric immunosensor in an oral biological sample selected from saliva and sputum are provided. The method and kit are based on the use of gold capture nanoparticles carrying on their surface at least one antibody capable of binding a SARS-CoV-2 surface antigen.

Claims

1. An in vitro method for detecting the SARS-CoV-2 virion in an oral biological sample of a subject selected from saliva and sputum, the method comprising: a) contacting said oral 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 SARS-CoV-2 surface antigen being selected from the group consisting of membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof, thereby obtaining a reaction mixture; and (b) determining the formation of a cluster of the gold capture nanoparticles on the surface of the SARS-CoV-2 virion in the reaction mixture, said cluster resulting from an interaction between said at least one antibody and said SARS-CoV-2 surface antigen, the determination being carried out by detecting a change in an optical parameter of the reaction mixture, said change in the optical parameter of the reaction mixture being indicative of the presence of the SARS-CoV-2 virion in the oral biological sample.

2. The in vitro method of claim 1, wherein the change in the optical parameter is a colour change of the reaction mixture detectable by the naked eye.

3. The in vitro method of claim 2, further comprising comparing the detected colour of the reaction mixture with a colorimetric scale.

4. The in vitro method of claim 1, wherein the change in the optical parameter is a reduction in the transmittance value of the reaction mixture measured at a predetermined wavelength in the visible range.

5. The in vitro method of claim 1, wherein the change in the optical parameter is an increase in the absorbance value of the reaction mixture measured at a predetermined wavelength in the visible range.

6. The in vitro method of claim 1, wherein the change in the optical parameter is an increase in the area under the absorption spectrum of the reaction mixture in a wavelength range between 200 nm and 700 nm.

7. The in vitro method of claim 1, wherein the in vitro method is in a competitive format, wherein the colloidal suspension of the gold capture nanoparticles comprises a salt selected from the group consisting of sodium citrate, sodium chloride, potassium phosphate, sodium phosphate, calcium chloride, potassium chloride, and any combination thereof, said salt being present in the colloidal suspension at a concentration ranging from 150 milliMolar (mM) to 250 mM, and wherein the change in the optical parameter is an increase in the absorbance value of the reaction mixture measured at a wavelength in a range from 600 nm to 700 nm.

8. The in vitro method of claim 1, further comprising filtering the reaction mixture obtained in step a) by using a filter element having pores with a diameter ranging from 35 to 65 microns (μm).

9. The in vitro method of claim 1, wherein the change in the optical parameter is detected by a colorimeter or a photometer.

10. A diagnostic kit for detecting the SARS-CoV-2 virion in an oral biological 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 SARS-CoV-2 surface antigen being selected from the group consisting of membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof.

11. The diagnostic kit of claim 10, wherein the colloidal suspension comprises a salt selected from the group consisting of sodium citrate, sodium chloride, potassium phosphate, sodium phosphate, calcium chloride, potassium chloride, and any combination thereof, said salt being present in the colloidal suspension at a concentration ranging from 150 milliMolar (mM) to 250 mM.

12. The diagnostic kit of claim 10, wherein the colloidal suspension is dispensed into a plurality of single disposable test tubes.

13. The diagnostic kit of claim 10, further comprising a support containing a colorimetric scale.

14. The diagnostic kit of claim 10, further comprising a portable colorimeter or a photometer.

15. The diagnostic kit of claim 10, further comprising a filter element having pores with a diameter ranging from 35 to 65 microns (μm).

16. The diagnostic kit of claim 15, wherein the filter element is a hydrophilic polyethylene filter.

17. The in vitro method of claim 4, wherein the change in the optical parameter is a reduction in the transmittance value of the reaction mixture measured at 560 nm.

18. The in vitro method of claim 5, wherein the change in the optical parameter is an increase in the absorbance value of the reaction mixture measured at 560 nm.

Description

[0070] The following experimental examples are provided for illustrative purposes only. Therein, reference is made to the accompanying drawings, wherein:

[0071] FIG. 1 is a schematic representation of the method for functionalizing the surface of gold nanoparticles with type G immunoglobulins (Photochemical Immobilization Technique, PIT). IgG antibodies are irradiated with UV rays using a lamp of appropriate power, causing the reduction of disulfide bridges at specific positions in the light chain 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 portions of the antibody free.

[0072] FIG. 2 is a schematic representation of the method of the invention. The colloidal suspension of gold nanoparticles functionalized with anti-SARS-CoV-2 antibodies is contacted with the sample containing the virus, thus forming a reaction mixture. Following the clustering of the functionalized gold nanoparticles around the viral particle, the reaction mixture changes colour. As the concentration of viral particles increases, the shift towards blue increases.

EXPERIMENTAL SECTION

Example 1: Preparation of the Colloidal Gold Solution (Synthesis of Nanoparticles)

[0073] 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, tetrachloroauric acid is first solubilized in water and the addition of sodium citrate causes reduction of the gold, resulting in the production of a gold seed and subsequently the growth of gold around it. The synthesis reaction consisted in mixing 1 mL of HAuCl4 (10 mg/mL) and 2 mL of sodium citrate dihydrate (25 mg/mL) in 100 mL of milliQ (ultrapure) water. The operating temperature was maintained at 90° C., with gentle stirring. The formation of gold nanoparticles was identified by a drastic change in the colour of the solution from yellow to orange.

[0074] At the end of the synthesis, the solution was centrifuged at 6 G for 30 minutes, thereby obtaining gold nanoparticles ready to be functionalized.

Example 2: Functionalization

[0075] The surface of the gold nanoparticles was functionalized by using the mechanism known as Photochemical Immobilization Technique (PIT), as described in FIG. 1.

[0076] Briefly, IgG antibodies directed against the membrane protein (Membrane, M), the envelope protein (Envelope, E), and the spike protein (S) of the SARS-CoV-2 virus were used (0.1 mg/mL).

[0077] A quartz cuvette containing the antibody solution at a concentration of 1 μg/mL was inserted into a specially designed UV lamp and irradiated with UV rays for 30 seconds in order to obtain the reduction of some disulfide bridges in specific positions of the antibody. Subsequently, gold nanoparticles having a diameter of 20 nm in size were functionalized, obtaining a concentration of nanoparticles with the anti-envelope antibody of 10.sup.10 nanoparticles (np)/mL, a concentration of nanoparticles with the anti-spike antibody of 10.sup.10 np/mL, and a concentration of nanoparticles with the anti-membrane antibody of 10.sup.10 np/mL. Any empty spaces left on the gold nanoparticles were then blocked by using a solution containing BSA (50 μg/mL).

[0078] Finally, the colloidal suspensions containing the three different antibodies were mixed together in 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.

[0079] Purification of the obtained samples was carried out by centrifugation at 6 G for 10 minutes.

Example 3: Sample Preparation

[0080] The saliva sample was collected using a swab and immediately transferred into a volume of 0.5 ml of the colloidal suspension of functionalized gold nanoparticles. Any virion on the swab was released by vigorously rotating the swab on its own axis for about 10 seconds in the suspension. The present inventors found 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 the sampling. 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.

[0081] Optionally, in order to remove the mucosal component present in the samples, the reaction mixture obtained by mixing the saliva or sputum samples with the colloidal suspension of the gold capture nanoparticles was allowed to drip through a hydrophilic polyethylene filter with pores of 50 microns in diameter and collected in an underlying cuvette. The time taken for this passage varied over a 3-10-minute interval according to the viscosity of the saliva. After the filtration, the reaction mixture was read.

Example 4: Detection

[0082] Gold capture nanoparticles prepared and functionalized as described above were used for the SARS-CoV-2 virus detection experiments.

[0083] Briefly, after mixing the saliva or sputum sample with the colloidal suspension of the gold capture nanoparticles, a colour change occurred in the reaction mixture that went from orange to blue passing through purple. In particular, the shift towards blue increased with increasing concentration of virus. The simple buffer solution in which no colour change was seen was used as a negative control.

[0084] For transmittance or absorbance analysis, a defined volume of the saliva or sputum sample, after being resuspended, was deposited by a sterile disposable Pasteur pipette in a test tube (Wheaton type) already containing the gold capture nanoparticle suspension. Alternatively, defined volumes of the test sample and the gold capture nanoparticle suspension were dispensed together into a supplied cuvette.

[0085] The reaction mixture obtained with the preparation procedures described above was mixed by stirring the test tube/cuvette, or by pipetting, thereby allowing the formation of the clusters of functionalized gold nanoparticles on the surface of the SARS-CoV-2 virion.

[0086] Subsequently, and optionally after filtering, the test tube or cuvette was housed in the supplied portable reader, which provided absorbance or transmittance values indicative of the positivity/negativity of the sample, i.e., the presence or absence of SARS-CoV-2 viral particles.

[0087] In the experiments carried out by the present inventors, a portable photometer was used, which was equipped with a tungsten lamp and a monochromator capable of isolating the wavelength at 560 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.

[0088] In their experiments, the present inventors alternatively used a colorimeter device equipped with a diode capable of emitting at 560 nm and, similarly to the above-mentioned device, of returning a transmittance value. In this procedural mode, after being resuspended, 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.

[0089] The transmittance values measured in the experiments described above are indicative of the amount of SARS-CoV-2 viral particles present in the test sample.

[0090] In order to measure the absorbance of the reaction mixture, the inventors also used a benchtop spectrophotometer instrument which is capable of emitting in a wide spectrum of wavelengths ranging, for example, from 200 to 700 nm. The absorbance measurements taken at the different wavelengths allowed the area under the absorption spectrum to be calculated, thus providing a “relative” quantitative determination of the viral load. The viral load measurement can become “absolute” by means of a calibration of the described technique.

[0091] Table 1 below shows the results obtained in 6 patients (3 positive and 3 negative) using the PCR method and the method according to the invention in parallel. For the PCR analysis, the data is 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 is expressed as absorbance values (Abs). The term “x” represents a negative sample.

TABLE-US-00001 TABLE 1 PCR threshold (Ct) Abs@560 nm 18 0.325 22 0.294 35 0.224 x 0.171 x 0.195 x 0.176

[0092] Table 1 shows that negative samples give an absorbance value of 0.18±0.01. Even considering the 3 SD criterion, the sample with the lowest viral load (Ct=35) is clearly distinguished from the negative samples. In fact, values higher than 35 for the PCR threshold are considered negative. These results demonstrate that the method according to the invention has a detection limit comparable to the PCR.

Example 5: Method of the Invention with a Competitive Configuration

[0093] For the experiments carried out using the method of the invention with a competitive configuration, a particularly unstable colloidal suspension of capture nanoparticles was produced. To this end, the following two steps were carried out: 1) a 160 mM concentration of sodium citrate was used during the step of production in water; 2) the excess reagents were removed by centrifuging at 4.5 G for 30 minutes, and the precipitate was resuspended in an aqueous solution containing 160 mM sodium citrate. Subsequently, the addition of saliva containing SARS-CoV-2 viral particles to the colloidal suspension of capture nanoparticles obtained as described above caused a shift of the resonance peak in the absorption spectrum of the reaction mixture different from that caused by the reaction mixture to which uninfected saliva was added and in which, therefore, the clustering of the nanoparticles was solely induced by salts. In fact, the present inventors observed a very bright colour change of the reaction mixture in the absence of the virion, whereas they witnessed a slight colour change in the presence of the virus.

[0094] In the method of the invention with the direct configuration, as described in Example 4, an absorbance peak of the reaction mixture is observed at a wavelength of approximately 560 nm, whereas in the method of the invention with the competitive configuration, the peak of the reaction mixture, which is much larger, is observed at a wavelength in the visible range of 600-700 nm.

[0095] Table 2 below shows the results obtained in 6 patients (3 positive and 3 negative) using the PCR method and the method according to the invention in parallel. The data is expressed as indicated above with reference to Table 1. The term “x” represents a negative sample.

TABLE-US-00002 TABLE 2 PCR threshold (Ct) Abs@560 nm Abs@600 nm 15 0.274 0.157 19 0.252 0.133 22 0.214 0.122 x 0.199 0.046 x 0.187 0.052 x 0.192 0.064

[0096] The results shown in Table 2 demonstrate the presence of measurable differences between samples containing SARS-CoV-2 (positive cases) and virus-free samples (negative cases).