Method, device and system for detecting a viral agent by means of fluorescence spectroscopy

Abstract

Method for detecting a viral agent, comprising the steps of : providing a sample to be analysed in liquid form; introducing one or more antibodies labelled with one or more fluorophores, said antibodies being configured to selectively bind with one or more proteins of an outer membrane of the viral agent; eliminating unbound labelled antibodies; exposing the s amp 1 e to electromagnetic radiation; detecting a fluorescence response emitted by the labelled antibodies; and detecting the viral agent on the basis of the detected fluorescence response.

Description

[0001] "METHOD , DEVICE AND SYSTEM FOR DETECTING A VIRAL AGENT BY MEANS OF FLUORESCENCE SPECTROSCOPY"

[0002] Cross-Reference to Related Applications

[0003] This Patent Appl ication claims priority from Italian Patent Application No . 102024000027045 filed on November 29 , 2024 , the entire disclosure of which is incorporated herein by reference .

[0004] Technical Field

[0005] The present invention relates to a method, a device and a system for detecting a viral agent .

[0006] Background Art

[0007] The health emergency caused by the global and uncontrolled spread of the SARS-CoV-2 virus has highlighted the fundamental role played by diagnostic techniques as tools which ef fectively contribute to countering the spread of the pandemic . Indeed, thanks to the timely and accurate detection of the presence of a speci fic viral agent in a subj ect , it is possible to implement targeted and timely actions , starting with the isolation of the individual who tests positive for the viral infection and, i f necessary, the administration of an appropriate drug therapy .

[0008] Laboratory tests on biological samples , for example on the nasopharyngeal swab, are the most suitable for timely, ef fectively and to a large extent detecting the presence of viral material and thus providing a practically certain diagnosis of viral infection .

[0009] Currently, the most commonly used laboratory diagnostic tests are those which analyse the biological samples by means of polymerase chain reaction ( PCR) tests , in particular via real-time PCR . Real-time PCR is a molecular biology technique which simultaneously ampli fies fragments of viral nucleic acids ( directly DNA or DNA from an RNA template ) and quanti fies the DNA. However, these diagnostic techniques have important limitations , such as the minimum concentration of biological material to be analysed for the fastest techniques or the analysis time for the most signi ficant quantitative techniques . Furthermore , these techniques based on the detection of the viral nucleic acid must be performed in specialised laboratories by highly quali fied operators and require a long time to prepare the biological sample for analysis . It is therefore evident that in conditions of high diagnostic demand, such as during a pandemic emergency, molecular techniques alone are not suf ficient .

[0010] Given the limitations connected to the PCR-based techniques , alternative methods for detecting viral agents are being developed and some of them are already commercially available . Among those already in clinical use are antigen tests , which detect the presence of viral proteins capable of binding to antibodies , and serological tests , which detect the presence of antibodies corresponding to a given antigen in the blood plasma of the patient to ascertain whether exposure to a given pathogen has occurred .

[0011] Among the techniques under development , the ones which stand out are those which use :

[0012] - mass spectrometry, based on which the presence of viral peptides / proteins is detected;

[0013] - biosensors capable of detecting proteins and viral RNA;

[0014] - microscopy which adopts fluorescent markers and machine learning to identi fy viral particles ;

[0015] - tests carried out on a subj ect ' s breath, analysing biomarkers such as volatile organic compounds ; and

[0016] - skin tests , through which it is possible to detect variations in the amount of lipids circulating in the blood ( dyslipidaemia ) resulting from viral infections , which mani fest themselves at skin level .

[0017] However, the performance of the aforementioned diagnostic techniques under development is still uncertain in terms of both sensitivity and speci ficity and has not yet been validated by regulatory authorities .

[0018] With reference to antigen tests , it is possible to also include the techniques which exploit the potential of fluorescence spectroscopy . Typically, such techniques are based on the detection of the N (nucleocapsid) protein, which is an internal protein of the virus , and work in a time-resolved state , requiring pulsed emitters and pulsed detectors . Therefore , the methods , devices and systems are complex and expensive .

[0019] A purpose of the present invention is to provide a method which allows the above problems to be overcome .

[0020] Disclosure of the Invention

[0021] The aforementioned purpose is achieved by a method as claimed in claim 1 .

[0022] The present invention further relates to a device as claimed in claim 11 and to a system as claimed in claim 13 .

[0023] Brief Description of the Drawings

[0024] For a better understanding of the present invention, a preferred embodiment is described hereinafter, by way of non-limiting example and with reference to the accompanying drawings , wherein :

[0025] - Figure 1 is a schematic view of a system according to a first embodiment of the present invention;

[0026] - Figure 2 is a schematic view of a system according to a second embodiment of the present invention;

[0027] - Figures 3 and 4 are respective perspective views of an apparatus of the system;

[0028] - Figure 5 is a perspective view, with transparent parts , of the apparatus of Figures 3 and 4 ; - Figures 6 , 7 and 8 are respective fluorescence spectra as a function of the wavelength, of respective samples ; and

[0029] - Figure 9 is a diagram of fluorescence responses integrated in the spectral band of the instrument , as a function of the time , of the respective samples of Figures 6 , 7 and 8 .

[0030] Detailed Description of the Invention

[0031] With reference to Figures 1 and 2 , there is indicated by 1 a system for detecting a viral agent according to respective embodiments of the present invention .

[0032] The system 1 comprises an emitter 2 of electromagnetic radiation, a detector 3 , and an apparatus 4 operatively interposed between the emitter 2 and the detector 3 .

[0033] Conveniently, the system 1 further comprises a computer 5 communicatively coupled to the detector 3 , for example via an Ethernet connection 6 .

[0034] Preferably, the electromagnetic radiation emitted by the emitter 2 is in the visible spectrum, preferably in the blue range ( from approximately 430 nm to approximately 500 nm) .

[0035] Preferably, the electromagnetic radiation is monochromatic, preferably at 488 nm .

[0036] Conveniently, the emitter 2 comprises a laser source 11 which emits electromagnetic radiation .

[0037] In particular, the laser source 11 comprises a laser diode 12.

[0038] Preferably, the electromagnetic radiation is emitted in continuous-wave mode (i.e. in an uninterrupted manner) , rather than in pulse mode (i.e. in an intermittent manner) .

[0039] In particular, the radiation of the laser diode 12 is used in steady state, i.e. the illumination is substantially constant .

[0040] Preferably, the power of the laser diode 12 is 55 mW.

[0041] Conveniently, the emitter 2 comprises a collimating lens, preferably having focal length f = 4 mm and numerical aperture NA = 0, 6.

[0042] The apparatus 4 is configured to receive an input beam from the emitter 2 and transmit an output beam.

[0043] Conveniently, the input beam is an excitation beam emitted by the emitter 2.

[0044] Conveniently, the output beam is a fluorescence beam transmitted to the detector 3.

[0045] The apparatus 4 (Figures 3, 4 and 5) comprises a device 21 and a container 22 containing the sample to be analysed. The device 21 is configured to house, at least partially, the container 22.

[0046] In particular, the device 21 comprises a substantially box-shaped test tube holder 23, and has an entrance opening

[0047] 31, for the passage of the input beam, and an exit opening

[0048] 32, for the passage of the output beam. Conveniently, the entrance opening 31 and the exit opening 32 are orthogonal with respect to each other . In use , the container 22 is optically interposed between the entrance opening 31 and the exit opening 32 .

[0049] In particular, the device 21 comprises a lower wall 33 , an upper wall 34 , opposite the lower wall 33 , and a side wall 35 defining a first face 41 , a second face 42 adj acent to the first face 41 and orthogonal thereto , a third face 43 adj acent to the second face 42 and orthogonal thereto , and a fourth face 44 adj acent to the third face 43 and orthogonal thereto . The first face 41 has the entrance opening 31 and the second face 42 has the exit opening 32 .

[0050] According to the illustrated embodiments ( Figures 3 , 4 and 5 ) , the device 21 comprises a first component 51 and a second component 52 substantially complementary to the first component 51 and configured to interlock with it .

[0051] In particular, the first component 51 comprises the lower wall 33 of the device 21 and the second component 52 comprises the upper wall 34 of the device 21 . Optionally, the first component 51 comprises an appendage 53 configured to fix the device 21 to a work surface , for example a portable optical bench . The first component 51 comprises four side plates 51a, 51b, 51c, 51d configured to interlock with respective side plates 52a, 52b, 52c, 52d of the second component 52 . The entrance opening 31 is defined by respective cavities of the respective side plates 51a, 52a defining the first face 41 of the device 21 , and the exit opening 32 is defined by respective cavities of the respective side plates 51b, 52b defining the second face 42 of the device 21 . Preferably, the entrance opening 31 and the exit opening 32 are at the same height and are located in the proximity of , and equidistant from, the edge common to the first face 41 and the second face 42 of the device 21 , so as to minimise the optical path between the entrance opening 31 and the exit opening 32 .

[0052] Conveniently, the device 21 has a through hole 54 configured to be engaged by the container 22 .

[0053] In particular, the hole 54 is located on the upper wall 34 of the device 21 . Preferably, the hole 54 is located in the proximity of the edge common to the first face 41 and the second face 42 of the device 21 .

[0054] Conveniently, the device 21 compri ses a first reflective optics 61 facing the entrance opening 31 .

[0055] In particular, the first reflective optics 61 is associated with the third face 43 , which is opposite the first face 41 , of the device 21 . In use , the container 22 is interposed between the entrance opening 31 and the first reflective optics 61 , which is configured to expand the area of radiation-matter interaction .

[0056] Conveniently, the device 21 comprises a second reflective optics 62 facing the exit opening 32 .

[0057] In particular, the second reflective optics 62 is associated with the fourth face 44 , which is opposite the second face 42 , of the device 21 . In use , the container 22 is interposed between the exit opening 32 and the second reflective optics 62 , which is configured to increase the signal collected by the system .

[0058] According to the illustrated embodiments ( Figures 3 , 4 and 5 ) , the first reflective optics 61 comprises a first mirror 63 and the second reflective optics comprises a second mirror 64 . In particular, the first mirror 63 is carried by the side plate 51c and at least partially housed therein, and the second mirror 64 is carried by the side plate 51d and at least partially housed therein . The first mirror 63 is flat and the second mirror 64 is spherical .

[0059] Alternatively, the first reflective optics 61 comprises confocal or resonant cavities , or multi-pass cells .

[0060] Conveniently, the container 22 is shaped and si zed to contain the volume of the sample to be analysed, for example 100 microlitres , and easily allow the passage of the input beam and of the output beam .

[0061] In particular, the container 22 is a cuvette transparent to , i . e . with a high level o f transmission on, the electromagnetic radiation of interest .

[0062] Preferably, the container 22 is transparent to the visible radiations and is made of plastic material .

[0063] Preferably, the container 22 is substantially prismshaped, for example with a square base , and comprises four side faces 71 , 72 , 73 , 74 facing the respective faces 41 , 42 , 43 , 44 of the device 21 .

[0064] In particular, the first face 71 faces the entrance opening 31 , the second face 72 faces the exit opening 32 , the third face 73 faces the first reflective optics 61 , and the fourth face 74 faces the second reflective optics 62 .

[0065] Conveniently, the device 21 comprises an optical fibre connector 81 coupled to the exit opening 32 .

[0066] According to the first embodiment ( Figure 1 ) , the detector 3 comprises a spectrophotometer 91 .

[0067] Conveniently, the spectrophotometer 91 is connected to the apparatus 4 via an optical f ibre beam 92 configured to transmit the output beam from the apparatus 4 to the spectrophotometer 91 .

[0068] In particular, the beam 92 is from linear to linear, and is configured to collect , from the exit opening 32 of the device 21 , the output beam and direct it towards a slit of the spectrophotometer 91 .

[0069] Conveniently, the spectrophotometer 91 is configured to detect fluorescence spectra .

[0070] In particular, the spectra are acquired, for example via a piece of software of the computer 5 , and can be analysed in real time , for example via a graphical interface of the computer 5 , or in a deferred manner , with independent algorithms .

[0071] According to the second embodiment ( Figure 2 ) , the detector 3 comprises a photodiode 101 .

[0072] Conveniently, the photodiode 101 is connected to the apparatus 4 via a conditioning assembly 102 conf igured to re-imagine , i . e . collect and refocus , the output beam on the photodiode 101 .

[0073] In particular, the conditioning assembly 102 comprises a doublet of collimating lenses 111 , preferably having focal length f = 60 mm and f / 2 , 5 , a band-pass optical filter 112 operatively downstream of the coll imating lenses 111 , and a camera lens 113 , substantially identical to the collimating lenses 111 , operatively downstream of the optical filter 112 . Conveniently, the optical filter 112 is configured to remove the signal scattered by the laser diode 12 , in particular at 488 nm, and to narrow the sensitivity of the photodiode 101 around the expected fluorescence peak .

[0074] Conveniently, the re-imagined output beam downstream of the lens 113 is detected by the photodiode 101 .

[0075] Conveniently, the photodiode 101 is configured to detect a fluorescence signal integrated in a specific spectral band .

[0076] In particular, the fluorescence emitted by the sample integrated in the wavelength band of the optical filter 112 is acquired .

[0077] Preferably, the detector 3 further comprises a front-end ampli fication stage 114 .

[0078] Preferably, the detector 3 further comprises a processing unit 115 configured to receive a lock-in signal 116 from the ampli fication stage 114 . Conveniently, the processing unit 115 comprises a lock-in system based on FPGA ( Field Programmable Gate Array) controlled by the computer 5 , for example via a graphical interface .

[0079] Preferably, the processing unit 115 is configured to control the emitter 2 via a modulation signal 117 .

[0080] According to the present invention, a method for detecting a viral agent is also provided .

[0081] The method comprises a plurality of steps , described in detail hereinafter .

[0082] The method comprises a step of providing a sample to be analysed in liquid form .

[0083] For example , the sample is a nasopharyngeal swab in which the possible presence of the viral agent , for example the SARS-CoV-2 virus , is to be detected .

[0084] Preferably, the sample is in an isotonic buf fer solution, for example a phosphate buf fered saline ( PBS ) .

[0085] The method comprises a step of introducing one or more antibodies labelled with one or more f luorophores . Such labelled antibodies are configured to selectively bind with one or more proteins of an outer membrane of the viral agent .

[0086] In particular, the viral agent has a plurality of proteins on its outer membrane .

[0087] Each labelled antibody is configured to bind with a protein present on the outer membrane of the viral agent , i f such protein is present . When this occurs , a labelled antibody-viral agent aggregate is formed .

[0088] Preferably, the labelled antibody is configured to bind with the S ( spike ) protein present on the outer membrane of the SARS-CoV-2 virus .

[0089] Di f ferent types of antibodies configured to selectively bind to di f ferent types of proteins can be introduced . Di f ferent types of antibodies can be labelled with di f ferent types of f luorophores .

[0090] Conveniently, the spectroscopic characteristics ( absorption and emission spectra ) of the fluorophores used are known a priori .

[0091] Preferably, the fluorophore is fluorescein, which emits an intense fluorescence , in particular in the range from 520 nm to 530 nm, when excited in the blue range .

[0092] The method comprises a step of eliminating unbound labelled antibodies .

[0093] In particular, the labelled antibodies which did not bind to any protein of any viral agent are eliminated, for example because such protein is not present . Preferably, the step of eliminating unbound labelled antibodies comprises the step of washing the sample .

[0094] In particular, the sample is washed, i . e . is exposed to one or more washing steps , to reduce the background fluorescence signal due to the unbound labelled antibodies .

[0095] The method comprises a step of exposing the sample to electromagnetic radiation .

[0096] Conveniently, the step of exposing the sample to electromagnetic radiation is carried out via the emitter 2 .

[0097] Preferably, the electromagnetic radiation is emitted in continuous-wave mode .

[0098] In particular, the sample is exposed to a constant illumination . During the exposure , the sample is illuminated in an uninterrupted manner, rather than in an intermittent manner . Conveniently, the duration of the exposure is chosen so as to avoid the photo-bleaching phenomenon .

[0099] Preferably, the step of exposing the sample to electromagnetic radiation is carried out via the laser source 11 , preferably via the laser diode 12 .

[0100] In particular, the radiation of the laser source 11 is used in steady state , rather than in time-resolved state . The steady state is reached almost immediately when the sample is exposed to the electromagnetic radiation of the laser source 11 .

[0101] Conveniently, the sample is placed in the container 22 which is housed, at least partially, in the device 21 . The container 22 , and thus the sample , is struck several times by electromagnetic radiation, emitted by the emitter 2 , thanks to the first reflective optics 61 and the second reflective optics 62 . In particular , the electromagnetic radiation directly strikes the first face 71 of the container 22 , and indirectly strikes the third face 73 , thanks to the first reflective optics 61 , and the fourth face 74 , thanks to the second ref lective optics 62 . In other words , the first face 71 of the container 22 is struck, preferably in a substantially col limated manner , by the laser diode 12 , and the third face 73 and the fourth face 74 of the container 22 are struck in a backscattered manner .

[0102] The method comprises a step of detecting a fluorescence response emitted by the labelled antibodies .

[0103] In particular, the fluorescence response is detected, by the detector 3 , via spectroscopic techniques , preferably in steady state , i . e . with a constant observation, rather than in a time-resolved state .

[0104] Conveniently, the step of detecting a fluorescence response is carried out via the spectrophotometer 91 , which detects a fluorescence spectrum .

[0105] In particular, the spectrophotometer 91 detects a fluorescence spectrum in a spectral band which comprises at least part of the visible spectrum, preferably from 400 nm to 700 nm, preferably from 500 nm to 600 nm.

[0106] Conveniently, the step of detecting a fluorescence response is carried out via the photodiode 101 , which detects a fluorescence signal integrated in a predetermined spectral band .

[0107] In particular, such spectral band is less broad than that of the spectrophotometer 91 , and is around the range in which the fluorophore emits an intense fluorescence , characteristic of the fluorophore used ( in the case of the fluorescein, from 520 nm to 530 nm) .

[0108] The method comprises a step of detecting the viral agent on the basis of the detected fluorescence response .

[0109] In particular, the fluorescence response detected by the detector 3 is analysed to detect the presence , and possibly the type , of the viral agent .

[0110] Conveniently, i f the step of detecting the fluorescence response is carried out via the spectrophotometer 91 , it is possible to analyse possible fluorescence peaks .

[0111] I f no fluorescence peak is present , it is possible to conclude that no viral agent of interest is present .

[0112] I f a fluorescence peak is present , it is possible to conclude that the viral agent having, on its outer membrane , the protein with which the labelled antibody that emits such fluorescence has bound, is present . For example , i f the antibody is labelled with fluorescein and is configured to bind with the S (spike) protein present on the outer membrane of the SARS-CoV-2 virus, the presence of a fluorescence peak between 520 nm and 530 nm allows to conclude that the SARS- CoV-2 virus is present.

[0113] If multiple fluorescence peaks emitted by different types of labelled antibodies configured to bind with different types of proteins present on the outer membrane of different types of viral agents are present, it is possible to conclude that such viral agents are present. In other words, it is possible to conclude that different types of viral agents are present.

[0114] Conveniently, if the step of detecting the fluorescence response is carried out via the photodiode 101, it is possible to analyse a value of the fluorescence signal integrated in the predetermined spectral band associated with the photodiode 101.

[0115] If such value is below a threshold value, it is possible to conclude that the viral agent having, on its outer membrane, the protein with which the labelled antibody that emits fluorescence in the predetermined spectral band associated with the photodiode 101 is configured to bind, is not present.

[0116] If such value is above a threshold value, it is possible to conclude that the viral agent having, on its outer membrane, the protein with which the labelled antibody that emits fluorescence in the predetermined spectral band associated with the photodiode 101 is configured to bind, is present . For example , i f the antibody is labelled with fluorescein and is configured to bind with the S ( spike ) protein present on the outer membrane of the SARS-CoV-2 virus and the predetermined spectral band associated with the photodiode 101 is from 520 nm to 530 nm, the exceeding of a threshold value of the fluorescence signal integrated in the predetermined spectral band associated with the photodiode 101 allows to conclude that the SARS-CoV-2 virus is present .

[0117] Preferably, the threshold values are established via experiments performed previously with VLPs (Virus-Like Particles ) , used to reproduce virus models , and / or with the isotonic buf fer solution alone , for example the phosphate buf fered saline ( PBS ) .

[0118] Optionally, the step of detecting the fluorescence response comprises the step of detecting a fluorescence spectrum in a first spectral band, obtaining a second spectral band on the basis of the fluorescence spectrum detected in the first spectral band, and detecting a fluorescence signal integrated in the second spectral band .

[0119] In particular, the first spectral band is that of the spectrophotometer 91 and the second spectral band is that of the photodiode 101 . The first spectral band is broader than the second spectral band, which is contained in the first spectral band .

[0120] In other words , the step of detecting the fluorescence response is carried out via both the spectrophotometer 91 and the photodiode 101 . Preferably, the spectrophotometer 91 and the photodiode 101 are used sequentially . In particular, first the spectrophotometer 91 is used and then the photodiode 101 .

[0121] In particular, the spectrophotometer 91 is used in the first spectral band to detect a fluorescence spectrum . I f a fluorescence peak is present in a second spectral band, contained in the first spectral band, the photodiode 101 is used to detect a fluorescence signal integrated in the second spectral band . In other words , the predetermined spectral band of the photodiode 101 is the second spectral band, which is obtained on the basis of the fluorescence spectrum detected in the first spectral band .

[0122] Considering the system 1 , it is possible to combine the first embodiment ( Figure 1 ) and the second embodiment ( Figure 2 ) . For such purpose , it is suf ficient to act on the detector 3 , since the emitter 2 and the apparatus 4 can be the same for the two illustrated embodiments .

[0123] In particular, it is possible to first use the detector 3 comprising the spectrophotometer 91 ( Figure 1 ) and then the detector 3 comprising the photodiode 101 ( Figure 2 ) .

[0124] Examples of experimental results obtained with the system 1 are illustrated in Figures 6 to 9 .

[0125] In particular, such figures relate to the step of detecting a fluorescence response emitted by the labelled antibodies . Such step is carried out via the spectrophotometer 91 in Figures 6 to 8 , and via the photodiode 101 in Figure 9 .

[0126] Figures 6 to 8 illustrate fluorescence responses , as a function of the wavelength, of respective samples with respective concentrations of viral agent , in particular of virus-like particles (VLPs ) . Each sample has a volume of 105 microlitres , contains virus-like particles and labelled antibodies with a fluorescent protein, for example EGFP (Enhanced Green Fluorescent Protein) , and is illuminated by the laser diode 12 which emits a monochromatic electromagnetic radiation at 488 nm and is configured to have a probed volume of approximately 1 millilitre . For each sample , the spectrophotometer 91 detects the fluorescence spectrum emitted by the fluorescein-labelled antibodies , bound to the virus-like particles . Conveniently, the peak around 488 nm, due to the illumination by the laser diode 12 , must be neglected . The data processing is based on the average of di f ferent spectra acquired with a fixed exposure time , chosen so as to avoid the photo-bleaching phenomenon, and on the subtraction of the background spectra due to the electrical noise of the spectrophotometer 91 and the buf fer . In particular, Figure 6 relates to a first sample S I with a first concentration of virus-like particles , Figure 7 relates to a second sample S2 with a second concentration of virus-like particles , lower than the first concentration, and Figure 8 relates to a third sample S3 with a third concentration of virus-like particles , lower than the second concentration .

[0127] Conveniently, the spectrum profile is similar in Figures 6 to 8 . In particular , the fluorescein, which is excited in the blue range , emits an intense fluorescence around 520 nm . The intensity decreases as the concentration decreases , thus it is relatively decreasing from the first sample S I ( Figure 6 ) to the third sample S3 ( Figure 8 ) . Conveniently, accurate statistical algorithms can be applied to distinguish the fluorescence from the noise .

[0128] Figure 9 illustrates fluorescence responses , as a function of the time , of the first sample S I , of the second sample S2 and of the third sample S3 . For each sample , the photodiode 101 detects a fluorescence signal emitted by the fluorescein-labelled antibodies , bound to the virus-like particles , integrated in the wavelength band of the optical filter 112 , preferably from 520 nm to 530 nm . Conveniently, also the signal received when the container 22 only contains the phosphate buf fered saline ( PBS ) is detected .

[0129] Conveniently, each signal is substantially constant . The intensity decreases as the concentration decreases , thus it is relatively decreasing from the first sample S I to the third sample S3 and is even lower for the PBS , the value of which can be used as the aforementioned threshold value .

[0130] For each sample , the number of virus-like particles present in the 105 microlitres and the number of virus-like particles detected in the probed volume of approximately 1 millilitre are reported in the following table .

[0131] Upon examination of the characteristics of the method, of the device 21 and of the system 1 , the advantages of the present invention are evident .

[0132] In particular, the method is simple , quick and cost- ef fective .

[0133] Providing the sample to be analysed in liquid form allows an operational simpli fication in terms of instrumentation . Indeed, it is suf ficient to place the sample in liquid form in the container 22 which can be a commercial disposable micro-cuvette , easily available , instead of depositing the sample on a membrane which is typically necessary, for example , in the antigen tests . The fluorophores used can be commercial f luorophores , easily available , with spectroscopic characteristics ( absorption and emission spectra ) well known a priori .

[0134] The labelled antibodies are configured to selectively bind with one or more proteins of an outer membrane of the viral agent , and allow to use live viral particles , unlike the methods which analyse broken virions such as , for example , the techniques based on the detection of the N (nucleocapsid) protein, which is an internal protein of the virus . In other words , intact viruses can be detected, without the need to break them down . This allows to reduce the false positives in the ampli fication of the RNA and the false negatives in the molecular and antigen tests , where the degradation of the viral products is always possible .

[0135] Eliminating the unlabelled antibodies , in particular by exposing the sample to one or more washing steps , allows to reduce the background fluorescence signal caused by unbound labelled antibodies and to increase the reliability of the test .

[0136] The use of the spectrophotometer 91 allows to detect the fluorescence response over a broad spectrum and to simultaneously analyse fluorescence peaks due to di f ferent types of fluorophores , potentially associated with di f ferent types of viral agents .

[0137] The use of the photodiode 101 allows to detect viral agents even in low concentrations and to increase the sensitivity of the detection .

[0138] The combined use of the spectrophotometer 91 and the photodiode 101 has a synergi stic ef fect and allows for a double checking of the measurements .

[0139] Preferably, the method works in a steady state . In particular, the emission of electromagnetic radiation, by the emitter 2 , i s continuous , and the observation of the fluorescence response , by the detector 3 , is constant . This allows for a decrease in complexity and costs with respect to the techniques which work in a time-resolved state and require pulsed emitters and pulsed detectors .

[0140] The use of the first reflective optics 61 and / or the second reflective optics 62 makes the device 21 more ef ficient .

[0141] In particular, the combined use of the first reflective optics 61 and the second reflective optics 62 allows to increase the performance of the system 1 by 50% .

[0142] The first component 51 and the second component 52 of the device 21 can be easily manufactured, for example via three-dimensional printing .

[0143] Finally, it is clear that modi fications and variations can be made to the device 21 and the system 1 without departing from the scope of protection defined by the claims . For example , the first reflective optics 61 and the second reflective optics 62 of the device 21 can be reversed ,

[0144] The emitter 2 of the system 1 can comprise an LED .

Claims

CLAIMS1 . Method for detecting a viral agent , comprising the steps of :- providing a sample to be analysed in liquid form;- introducing one or more antibodies labelled with one or more f luorophores , said antibodies being configured to selectively bind with one or more proteins of an outer membrane of the viral agent ;- eliminating unbound labelled antibodies ;- exposing the sample to electromagnetic radiation;- detecting a fluorescence response emitted by the labelled antibodies ; and- detecting the viral agent on the basis of the detected fluorescence response .2 . Method as claimed in claim 1 , wherein the sample is in an isotonic buf fer solution .3 . Method as claimed in claim 1 or 2 , wherein the fluorophore is fluorescein .4 . Method as claimed in any of the preceding claims , wherein the step of eliminating the antibodies comprises the step of washing the sample .5 . Method as claimed in any of the preceding claims , wherein the step of exposing the sample to electromagnetic radiation is carried out via a laser source ( 11 ) .6 . Method as claimed in any of the preceding claims ,wherein the electromagnetic radiation is emitted in continuous-wave mode .7 . Method as claimed in any of the preceding claims , wherein the electromagnetic radiation is in the visible spectrum .8 . Method as claimed in any of the preceding claims , wherein the step of detecting the fluorescence response comprises the step of detecting a fluorescence spectrum via a spectrophotometer ( 91 ) .9 . Method as claimed in any of the preceding claims , wherein the step of detecting the fluorescence response comprises the step of detecting a fluorescence signal integrated in a predetermined spectral band via a photodiode ( 101 ) .10 . Method as claimed in any of the preceding claims , wherein the step of detecting the fluorescence response comprises the step of detecting a fluorescence spectrum in a first spectral band, obtaining a second spectral band on the basis of the fluorescence spectrum detected in the first spectral band, the first spectral band being broader than the second spectral band, and detecting a fluorescence signal integrated in the second spectral band .11 . Device for detecting a viral agent as claimed in the method of any of the preceding claims , the device ( 21 ) being configured to house , at least partially, a container(22) containing the sample to be analysed, the device (21) comprising an entrance opening (31) and an exit opening (32) for the passage of respective beams of electromagnetic radiation, and a first reflective optics (61) facing one of the entrance opening (31) and the exit opening (32) .

12. Device as claimed in claim 11, comprising a second reflective optics (62) facing the other of the entrance opening (31) and the exit opening (32) .

13. System for detecting a viral agent as claimed in the method of any of claims 1 to 10, comprising an emitter(2) of electromagnetic radiation, a detector (3) and an apparatus (4) operatively interposed between the emitter (2) and the detector (3) , wherein the apparatus (4) comprises the device (21) as claimed in claim 11 or 12 and a container (22) containing the sample to be analysed, the container(22) being housable, at least partially, in the device (21) .

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