IN VITRO METHOD FOR THE DETECTION OF SARS-COV-2 IN A SAMPLE FROM THE UPPER RESPIRATORY TRACT 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 a biological sample from the upper respiratory tract of a subject is provided. The biological sample is a nasopharyngeal swab sample. The in vitro 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 a biological sample from the upper respiratory tract of a subject, the method comprising: a) resuspending the biological sample in a buffer solution, thereby obtaining a sample solution; b) taking a portion from said sample solution and contacting said portion 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 c) 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 biological sample from the upper respiratory tract.

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 biological sample from the upper respiratory tract is selected from the group consisting of nasal swab sample, nasopharyngeal swab sample, pharyngeal swab sample and oropharyngeal swab sample.

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

9. A diagnostic kit for detecting the SARS-CoV-2 virion in a biological sample from the upper respiratory tract of a subject, the kit comprising: (i) a buffer solution suitable for resuspending the biological sample; and (ii) 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.

10. The diagnostic kit of claim 9, wherein the colloidal suspension is dispensed into a plurality of single disposable test tubes.

11. The diagnostic kit of claim 9, further comprising a support containing a colorimetric scale.

12. The diagnostic kit according to of claim 9 or 10, further comprising a portable colorimeter or a photometer.

13. 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.

14. 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

[0064] The following experimental examples are provided for illustrative purposes only. Therein, reference is made to the accompanying drawings, wherein:

[0065] 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.

[0066] 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)

[0067] 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.

[0068] 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

[0069] The surface of the gold nanoparticles was functionalized by using the mechanism known as Photochemical Immobilization Technique (PIT), as described in FIG. 1.

[0070] 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).

[0071] 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).

[0072] 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.

[0073] Purification of the obtained samples was carried out by centrifugation at 6 G for 10 minutes.

Example 3: Sample Preparation

[0074] The nasopharyngeal swab, after collection using an appropriate stick, was resuspended in Universal Transport Medium (UTM) (Copan Diagnostics, Inc.), an isotonic solution commonly used to transport samples in which the presence of viruses is to be detected. Among various ingredients, the UTM buffer contains: Hank's balanced salt solution, bovine serum albumin, L-cysteine, gelatin, sucrose, L-glutamic acid, HEPES buffer, phenol red, vancomycin, amphotericin B, and colistin.

Example 4: Detection

[0075] Gold capture nanoparticles prepared and functionalized as described above were used for the SARS-CoV-2 virus detection experiments.

[0076] Briefly, after performing the nasopharyngeal swab, a volume of 100 μL of the buffer solution containing the virus was added and mixed with the colloidal suspension of capture gold nanoparticles for about 1 minute. 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.

[0077] For transmittance or absorbance analysis, a defined volume of the nasopharyngeal swab, after being resuspended, was deposited by a sterile disposable Pasteur pipette in a test tube (Wheaton type) already containing the gold capture nanoparticle suspension.

[0078] Alternatively, defined volumes of the test sample and the gold capture nanoparticle suspension were dispensed together into a supplied cuvette.

[0079] 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.

[0080] Subsequently, 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.

[0081] 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.

[0082] 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 nasopharyngeal swab sample was dispensed and assayed directly in the disposable reaction tube pre-packaged with the colloidal suspension of gold capture nanoparticles.

[0083] 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.

[0084] 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.