Methods for assessing the ability of sera to neutralize viruses

Abstract

A method for evaluating the ability of serum to neutralize a virus is disclosed. The method includes the steps of: preparing a solution containing at least one cell, at least one serum, and at least one virus; placing the solution in contact with an organic electrochemical transistor comprising a source electrode, a drain electrode, and a gate electrode, the source electrode and the drain electrode being electrically connected by a conductive channel comprising a conductive or semiconductive polymer; applying a potential difference between the drain electrode and the source electrode, applying a plurality of pulses of potential difference between the gate electrode and the source electrode, and measuring a plurality of values ​​of each of the pulses of current through the channel; calculating an estimate of the response time of the organic electrochemical transistor according to the plurality of values ​​of the measured current; comparing the estimated response time value against a threshold value, and detecting whether the serum has the neutralization ability against at least one virus according to the comparison.

Description

[Technical field]

[0001] The present invention relates generally to the field of in vitro analysis of cell cultures.

[0002] More particularly, the present invention relates to a method for assessing the ability of sera to neutralize a virus. [Background technology]

[0003] The increasing spread of infectious diseases, especially those of the respiratory tract, has driven scientific research to search for new diagnostic and therapeutic strategies. The COVID-19 pandemic caused by the coronavirus (CoV) SARS-CoV-2 has created an unprecedented demand for diagnostic tests and therapeutics against viruses, exacerbating an epidemiological situation in which new potentially pathogenic microorganisms are already abundant.

[0004] Various studies carried out on Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) have demonstrated the presence of specific neutralizing antibodies against those viruses in 80-100% of patients 2 weeks after the onset of symptoms. Although antibodies may suppress the replication of SARS-CoV-2 through virus neutralization, they may also be involved in the pathogenesis and progression of COVID-19 through a process defined as antibody-dependent enhancement.

[0005] For this reason, it is crucial to assess the presence of neutralizing antibodies produced during SARS-CoV-2 infection for prognostic purposes in clinical practice.

[0006] Furthermore, available immunological tests for the quantification of neutralizing antibodies in patients are essential to provide information and data on vaccine efficacy, with the aim of prioritizing different vaccine candidates and supporting vaccine licensure requests. Harmonization of results for tests on neutralizing antibodies (both against SARS-CoV-2 and against pseudoviruses) is particularly important in preclinical testing of vaccines in non-human primates to allow accurate comparison with those obtained in clinical trials.

[0007] Although various methods exist for determining antibody concentrations, the PRNT (plaque reduction neutralization test) is considered the gold standard for measuring the level of neutralizing antibodies. The PRNT was developed by Dulbecco for animal virus inactivation (or neutralization) studies and was modified to measure antibody neutralization titers in serum. The PRNT is already used to test the level of protection of the population of countries affected by other zoonotic coronaviruses, e.g., MERS-CoV. The PRNT requires the formation of virus-antibody complexes in vitro, followed by incubation at 37°C for 30 minutes and subsequent plating on cells susceptible to virus infection. After incubation (typically 72 hours), the cytopathic effect (CPE) is assessed by fixing and staining the cells with formaldehyde and crystal violet, followed by reading the absorbance at 560 nm using a spectrophotometer. The highest serum dilution that neutralizes 90% of the virus replication and therefore its cytopathic effect is reported as the neutralization titer. This technique entails many economic costs, from the materials used to the highly specialized manpower required for the treatment of toxic waste. Moreover, it requires a long time to obtain results (after 72 hours of infection), thus preventing a fast reporting time. Furthermore, formaldehyde and crystal violet are known to have carcinogenic effects and must be handled according to very strict regulations. Due to the high infectivity and pathogenicity of SARS-CoV-2, the virus must be handled in a prescribed biosafety level 3 (BSL-3) facility. PRNT is therefore technically challenging and difficult to automate, which makes it unsuitable for large-scale testing on serum samples, e.g., phase III clinical trials for human vaccines.

[0008] To avoid having to deal with infectious CoV-2, various safety systems based on pseudoviruses at Biosafety Level 2 (BSL2) have been developed. Although they have demonstrated high sensitivity and reliability, the tests are often cumbersome, lengthy and expensive in terms of procedural duration.

[0009] Therefore, there is an increasing need for tests that can evaluate the presence of antibodies in serum, i.e. the neutralizing capacity of serum. In particular, there is a great demand for tests or methods that allow the rapid and limited cost evaluation of the presence of antibodies in serum and the capacity of serum to neutralize the virus. Summary of the Invention [Means for solving the problem]

[0010] The present invention as defined in the appended claim 1 and by the preferred embodiments thereof as defined in the dependent claims 2 to 12 relates to an in vitro method for assessing the ability of a serum to neutralize a virus.

[0011] The basic idea is to estimate the response time value of the organic electrochemical transistor by measuring the growth state of cells cultured directly on the organic electrochemical transistor, and to evaluate the effectiveness of the serum in neutralizing the virus by comparing the variation pattern of the response time value over time against a threshold value expected for healthy cells, which is determined for each cell / virus pair to be analyzed. The ability of the serum to neutralize the virus indicates the presence of antibodies capable of neutralizing the test virus.

[0012] The applicants have realized that the evaluation method according to the invention allows the elimination of the use of formaldehyde, allows objective results to be obtained in real time, allows measurements to be made by non-highly specialized personnel in a significantly shorter time than known techniques, and is suitable for testing laboratories with a low level of expertise.

[0013] Another advantage is that it allows for objective and automated measurements that are mostly performed remotely, considerably reducing the risk of long-term exposure of healthcare workers to the highly infectious virus. Tests are performed in real time inside incubators in Biosafety Level (BSL) 2 / 3 / 4 laboratories without the use of toxic reagents.

[0014] From an economic point of view, an objective measurement can be obtained without the use of extremely expensive, complicated and cumbersome equipment (such as spectrophotometers).

[0015] The manufacture of the device can be carried out on a large scale using low-cost processes, allowing its use in countries with low technological development.

[0016] The present invention is applicable to serum neutralization tests for the identification of antibodies produced against any virus species that causes pathology in animals and humans, even viruses other than SARS-CoV-2.

[0017] Additional features and advantages of the invention will become apparent from the following description of preferred embodiments and variants thereof, given by way of example with reference to the accompanying drawings. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows a block diagram of an electronic device for evaluating the ability of sera to neutralize viruses according to the present invention. [Figure 2A] FIG. 2A shows in greater detail an organic electrochemical transistor used in the electronic device of FIG. [Figure 2B] FIG. 2B shows an equivalent circuit of the organic electrochemical transistor of FIG. 2A. [Diagram 3] FIG. 3 shows a schematic perspective view of an organic electrochemical transistor on the left and a possible physical construction of an organic electrochemical transistor on the right. [Figure 4] FIG. 4 shows a schematic of the time pattern of curves representing time parameters estimating the response time of an organic electrochemical transistor for uninfected cultures and for cultures infected with a virus using non-neutralizing serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition In the context of the present invention, the term "serum neutralization test" means a test capable of evaluating the ability of a serum to neutralize a virus, ie to prevent the infection of cells by a pathogenic agent, preferably a virus.

[0020] In the context of the present invention, the term "neutralizing serum" refers to serum obtained from a human individual or animal that is capable of preventing or reducing infection by a pathogenic agent.

[0021] In the context of the present invention, the term "non-neutralizing serum" means a serum obtained from a human individual or an animal or an artificial serum that is not capable of preventing infection by a pathogenic agent.

[0022] In the context of the present invention, the term "coronavirus" (abbreviated as "CoV") refers to a broad family of respiratory viruses that can cause mild to moderate illnesses ranging from the common cold to respiratory syndromes such as MERS (Middle East Respiratory Syndrome) and SARS (Severe Acute Respiratory Syndrome).

[0023] In the context of the present invention, the term "infection" refers to a process characterized by the invasion and proliferation of unicellular pathogenic microorganisms (infectious agents: bacteria, fungi, protozoa) or viruses into cells. The concept of infection cannot be equated with that of infectious disease or pathology, since there are cases of infection that do not have any pathological signs, i.e. asymptomatic individuals that are carriers of the pathogen.

[0024] In the following description, it should be observed that identical or similar blocks, components or modules are indicated in the figures with the same numerical references even if they are illustrated in different embodiments of the present invention.

[0025] The invention is preferably implemented using a vessel containing a plurality of wells (eg, six), each containing at least one biocompatible organic electrochemical transistor (OECT) that is transparent or semi-transparent to the cell layer.

[0026] Cells are seeded in the wells to create a cell layer, and the organic electrochemical transistor measures the resistance of the cell layer, which depends on its integrity and the health of the cells themselves.

[0027] Serum neutralization tests are based on the fact that the cell cultures or cell lines used in the neutralization assay form a confluent monolayer that represents a physical barrier for the transport of ions. This barrier property can be quantified electrically as the transepithelial electrical resistance (TEER) in impedance spectroscopy experiments.

[0028] During viral infection, the barrier properties are altered due to changes induced during viral replication, which results in an altered TEER.

[0029] Neutralization tests are sensitive and specific, can be applied to all cytopathic viruses, and can measure neutralizing antibody titers after natural exposure, after vaccination, or after passive transfer of maternal antibodies.

[0030] Conventional serum neutralization test methods performed in vitro are based on the inhibition of viral infectivity in cell culture in the presence of neutralizing antibodies in serum.

[0031] The determination of titer may be based on the presence or absence of cytopathic effect.

[0032] Seroneutralization tests are relatively inexpensive when standard laboratory equipment is used, but require more technical capability to perform the tests than other serological methods because the analysis is performed by light microscopy and image evaluation by automated mass screening is difficult to apply. On the other hand, seroneutralization tests are serological methods that can potentially identify neutralizing antibodies in all animal species, including humans.

[0033] Furthermore, for high-risk viral pathogens, visual assessment may entail risk of infection for the operator.

[0034] The present invention envisages the utilization of organic electrochemical transistors for the real-time assessment of the occurrence of cytopathic effects caused by different virus species cultured in cell cultures or cell lines, in serum neutralization assays or to study the dynamics of viral replication in vitro.

[0035] The serum neutralization test involves assaying serial dilutions of human or animal serum to assess the minimum concentration of circulating antibodies capable of neutralizing the virus with a known titer through analysis of the health status of a cell layer by an electronic device according to the present invention.

[0036] Advantageously, the organic electrochemical transistor can be made transparent or semi-transparent, allowing simultaneous optical analysis; moreover, at the end of the monitoring, the cells can be retrieved for possible molecular analysis. This effect paves the way for the realization of real-time electrical neutralization tests based on TEER monitoring.

[0037] The electronic device according to the present invention includes a plurality of dedicated sterilizable wells into which organic electrochemical transistors are inserted.

[0038] The multiple wells are connected to a processing device (eg, on a personal computer) that runs a suitable software program to analyze the collected data.

[0039] Preferably, it is possible to determine in advance (i.e. before testing the sample to be analyzed) a threshold value for distinguishing between healthy cells and infected cells, a threshold value being determined for each type of cell and virus to be tested.

[0040] One embodiment of the present invention relates to an in vitro method for assessing the ability of sera to neutralize a virus.

[0041] The method comprises the steps of: a) preparing a solution comprising at least one cell, at least one serum and at least one virus; b) placing the solution in contact with an organic electrochemical transistor comprising a source electrode, a drain electrode and a gate electrode, the source electrode and the drain electrode being electrically connected by a conductive channel comprising a conductive or semiconductive polymer, and at least one cell being attached to said channel and / or the gate electrode, or at least one cell being attached to a permeable porous support disposed between the conductive channel and the gate electrode; c) applying a potential difference between the drain and source electrodes and applying a plurality of pulses of the potential difference between the gate and source electrodes and measuring a plurality of values ​​of each of the pulses of current through the channel; d) calculating an estimate of the response time of the organic electrochemical transistor as a function of the multiple values ​​of the measured current; e) comparing the value of the estimated response time against a threshold value and detecting whether the serum has a neutralizing capacity against at least one virus depending on said comparison. Includes.

[0042] According to one embodiment, an estimate of the response time is calculated by interpolating multiple values ​​of the measured current into a bi-exponential decay curve and then measuring the value of a time response parameter T1 of the bi-exponential curve.

[0043] In particular, the current I_d through the channel is I_d=a * exp(t / T1)+b * exp(t / T2)+e (In the formula, - t is the time; - exp is the exponential function; - a, b, e are configuration parameters; T1 is a time parameter representing the polymer charging time, which is influenced by the ion-blocking properties of the cell layer; - T2 is a time parameter that depends on the cell layer charging time Calculated using; The current pulse is normalized between a value of 1, which corresponds to the maximum channel current value under conditions of direct polarisation of the organic electrochemical transistor, and a value of 0, which corresponds to the average value of a defined number of points of the channel current when the channel current reaches the stationary regime.

[0044] More specifically, the response time of an organic electrochemical transistor is equal to the value of the time parameter T1 that is then normalized to the value of T1 that it has before cell culture (denoted as T_nc), i.e., according to the following formula: Response time [au] = T1 / T_nc Follow.

[0045] For further details regarding the calculation of response times, reference can be made to the publication by Francesco Decataldo et al., “Organic Electrochemical Transistors: Smart Devices for Real-Time Monitoring of Cellular Vitality”, Advanced Materials Technologies, 4, 1900207 (2019), including the supplementary “Supporting information”.

[0046] In particular, the plurality of pulses of potential difference (i.e., voltage difference) between the gate electrode and the source electrode are square waves having a difference between high and low values ​​equal to, for example, 0.3 volts; more typically, the high values ​​of the square wave pulses are comprised between 0.1 volts and 0.8 volts and the low values ​​are comprised between -0.3 volts and 0.5 volts.

[0047] The duration of the high value is comprised between 0.005 seconds and 10 seconds, in particular equal to 0.5 seconds; the duration of the low value is comprised between 0.1 seconds and 30 seconds, preferably equal to 1.5 seconds.

[0048] Furthermore, the value of the potential difference (ie, the voltage difference) between the drain electrode and the source electrode is comprised between −0.4 volts and 0.2 volts, and is preferably equal to −0.1 volts.

[0049] The conductive polymer is for example selected from the following list: PEDOT:PSS, PEDOT-S, PEDOT:TOS, PEDOTOH:ClO4, PEDOT-co-PEDOTOH:ClO4, P3HT, PTHS, BBL, p(g2T-TT), PTHS-TMA<+>-co-P3HT, p(gNDI-g2T), p(g0T2-g6T2), polyaniline, polypyrrole, P-90.

[0050] In one embodiment of the present invention, the at least one virus belongs to the Coronaviridae family; it is preferably selected from SARS-CoV, MERS-CoV and SARS-CoV-2. Preferably, the at least one virus is SARS-Cov-2. In other words, the method makes it possible to evaluate the serum neutralization of at least one virus belonging to the Coronaviridae family, preferably SARS-CoV-2.

[0051] Serum is preferably obtained from an individual using techniques known to those skilled in the art. Serum is preferably obtained from an individual who has undergone treatment against at least one virus.

[0052] In a preferred embodiment of the invention, serum is obtained from an individual who has received a vaccine against at least one virus, preferably a vaccine against a coronavirus, more preferably a vaccine against SARS-CoV-2.

[0053] In a further embodiment of the invention, serum is obtained from an individual suffering from an infection caused by at least one virus, preferably caused by a coronavirus, more preferably caused by SARS-CoV-2.

[0054] With reference to FIG. 1, this shows a block diagram of an electronic device 1 for assessing the ability of sera to neutralize viruses, according to the present invention.

[0055] The electronic device 1 comprises an organic electrochemical transistor 4 , a well 5 , electronic driving circuitry 2 and a processing unit 3 .

[0056] The cell layer is attached to the conductive channel and / or gate electrode.

[0057] The electronic device 1 is therefore a sensor that detects in real time the state of the growth of a cell layer attached to the channel and / or gate electrode of the organic electrochemical transistor 4 and has a sufficiently good sensitivity to detect a reduction in the growth of the cell layer caused by the presence of a virus that is not sufficiently neutralized by the serum to be tested.

[0058] The organic electrochemical transistor 4 includes a source electrode 4s, a drain electrode 4d and a gate electrode 4g.

[0059] The source 4s and drain 4d electrodes are electrically connected by a conductive channel comprising a conductive or semiconductive polymer.

[0060] Thus, the current I_d flowing through the conductive channel is modulated by the potential difference applied between the gate and source electrodes through the electrolyte solution.

[0061] The electronic driving circuit 2 is in electrical communication with the organic electrochemical transistor 4 and is capable of generating suitable voltage values ​​to control the operation of the organic electrochemical transistor 4 .

[0062] In particular, the electronic driving circuit 2 includes a first output terminal connected to the gate electrode 4g of the organic electrochemical transistor 4, a second output terminal connected to the source electrode 4s of the organic electrochemical transistor 4, and a third output terminal connected to the drain electrode 4d of the organic electrochemical transistor 4.

[0063] A processing device 3 (e.g. a microprocessor or microcontroller) is electrically connected to the organic electrochemical transistor 4 and is capable of suitably processing measurements of the current I_d through the conductive channel to calculate estimates of the value of the response time of the organic electrochemical transistor 4 as it varies over time, as described in more detail below.

[0064] In particular, the processing device 3 comprises an input terminal adapted to receive the current I_d flowing through the conductive channel of the organic electrochemical transistor 4, and comprises an output terminal adapted to generate a signal S_n indicative of the state of health of at least one cell, the signal S_n being indicative of whether the serum has a neutralizing capacity against the virus under consideration.

[0065] More particularly, the electronic drive circuit 2 is configured to apply a potential difference V_ds between the drain electrode 4d and the source electrode 4s.

[0066] Furthermore, the electronic drive circuit 2 is configured to apply a number of pulses of a potential difference V_gs between the gate electrode and the source electrode.

[0067] The processing device 3 is configured to receive a plurality of respective values ​​of the pulse of current I_d through the conductive channel of the organic electrochemical transistor 4 .

[0068] Furthermore, the processing device 3 is configured to calculate an estimate of the response time of the organic electrochemical transistor 4 as a function of a plurality of values ​​of the measured current, as will be explained in more detail below.

[0069] Finally, the processing device 3 is configured to compare the estimated response time value against a threshold value TH and generate, depending on said comparison, a health status signal S_n indicating whether the serum has a neutralizing capacity against at least one virus.

[0070] In particular, if the processing device detects that the estimated response time is equal to or greater than the threshold TH, the cell layer is healthy and therefore the virus is neutralized by the test serum; conversely, if the processing device detects that the estimated response time is less than the threshold TH, the cell layer is broken and therefore the virus is active, i.e., the test serum does not have neutralizing capability.

[0071] It can be seen that for simplicity, FIG. 1 shows a single organic electrochemical transistor 4 located in each well 5, but more generally it is possible to have a plurality (e.g. six) of organic electrochemical transistors similar to 4 located in corresponding wells similar to 5: in this case the drive circuit 2 controls the source, drain and gate electrodes of the multiple similar organic electrochemical transistors, and the processing device 3 thus receives and processes measurements of the channel currents generated by the multiple organic electrochemical transistors, thereby generating multiple estimates of the response times of the multiple organic electrochemical transistors.

[0072] Advantageously, the processing device 3 is configured to calculate an estimate of the overall response time as a function of a plurality of estimates of the response time: for example, an average value of the plurality of estimates of the response time is calculated.

[0073] According to a preferred embodiment, an estimate of the response time is calculated by interpolating multiple values ​​of the measured current into a bi-exponential decay curve and then measuring the value of a time response parameter T1 of the bi-exponential curve.

[0074] In particular, the current I_d through the channel has the following biexponential decay curve: I d =a * exp(t / T1)+b * exp(t / T2)+e (In the formula, - t is the time; - exp is the exponential function; - a, b, e are configuration parameters; T1 is a time parameter representing the polymer charging time influenced by the ion-blocking properties of the cell layer; - T2 is a time parameter that depends on the cell layer charging time) It is calculated using:

[0075] The current pulse is normalized between a value of 1, which corresponds to the maximum channel current value under conditions of direct polarization of the organic electrochemical transistor, and a value of 0, which corresponds to the average value of a defined number of points of the channel current when the channel current reaches the steady-state region.

[0076] More specifically, the response time of an organic electrochemical transistor is equal to the value of the time parameter T1 that is then normalized to the value of T1 that it has before cell culture (denoted as T_nc), i.e., according to the following formula: Response time [au] = T1 / T_nc Follow.

[0077] For example, the electronic driving circuit 2 controls the gate 4g and source 4s electrodes with a square wave consisting of five pulses of potential difference (between the gate 4g and source 4s electrodes) having a high value equal to 0.3 volts and a frequency equal to 0.5 Hertz for 12 seconds, the duration of the high value of each pulse being equal to 0.5 seconds and the duration of the low value being equal to 1.5 seconds (i.e. a duty cycle of 25%), maintaining a voltage value of the channel (i.e. the potential difference between the drain 4d and source 4s electrodes) equal to 0.1 volts.

[0078] Subsequently, measurements of the channel current I_d are taken and interpolated to the bi-exponential decay curve, then the average is calculated and the average is normalized.

[0079] For further details on the calculations, see Francesco Decataldo et al., “Organic Electrochemical Transistors: Smart Devices for Real-Time Monitoring of Cellular Vitality”, Advanced Material Technologies, 4, 190207 (2019).

[0080] 2A and 3, these show an organic electrochemical transistor 4 used within the electronic device 1 in more detail.

[0081] The organic electrochemical transistor 4 is implemented in a planar structure including a glass substrate 4-1 on which are disposed two gold metal contacts having a substantially rectangular shape and parallel to each other, each forming a source electrode and a drain electrode.

[0082] The gate electrode is a gold metal contact similarly disposed on the glass substrate 4-1 and disposed between the two gold metal contacts of the source and drain electrodes.

[0083] A conductive or semiconductive polymer layer 4-4 overlies the terminal portion of the gold contact of the gate electrode.

[0084] Furthermore, a similar conductive or semiconductive polymer layer 4-5 overlaps each of the gold contacts of the source and drain electrodes in the central portion, thus forming the conductive channel of the organic electrochemical transistor 4.

[0085] It is possible to observe that the cell layers 4-7 and 4-6 to be examined grow directly on the polymer layer 4-5 of the channel and on the polymer layer 4-4 at the gate terminal, respectively.

[0086] The polymer used is for example Pedot:Pss (poly(3,4-ethylenedioxythiophene) polystyrenesulfonate), which is transparent or semi-transparent and thus allows the acquisition of optical images of the cell layers 4-6 and 4-7.

[0087] Under suitable polarization conditions of the gate electrode, source electrode and drain electrode, a flow of positive ions (cations) and negative ions (anions) occurs through the cell layer 4-7 into and out of the polymer material 4-5 at the source 4s and drain 4d electrodes; further, a flow of positive ions (cations) and negative ions (anions) occurs through the cell layer 4-6 into and out of the polymer material 4-4 at the gate electrode 4g.

[0088] Thus, the organic electrochemical transistor 4 monitors the flow of ions through the cell layers 4-6, 4-7 cultured directly on the organic electrochemical transistor 4, and a time parameter (response time) indicative of the state of health of the cell layers 4-6, 4-7 is extracted (by the processing device).

[0089] Indeed, the health and integrity of the cell layers 4-6, 4-7 (cultured in direct contact with the organic electrochemical transistor 4) modifies the rate at which ions flow into the active polymer material (Pedot:Pss): the response time of the organic electrochemical transistor 4 is a time parameter estimated by a suitable algorithm created using a software program executed on the processing device 3.

[0090] Alternatively, it is possible to fabricate the organic electrochemical transistor 4 using a vertical geometry, i.e. the gate electrode is immersed in the solution at a defined distance (for example 2 mm) from the conductive channel: in this case the cell layer is grown on a permeable porous support (transwell), such as a 24 mm Costar Transwell support from Corning or a 0.4 μm Costar Transwell support from Corning, which is placed between the conductive channel and the vertical gate electrode. Thus, the cells are grown on a permeable porous support, which limits the flow of ions through the cell layer and consequently affects the value of the response time of the organic electrochemical transistor 4.

[0091] The present invention allows the performance of a serum neutralization test using an organic electrochemical transistor 4 as a substrate for cell culture: the organic electrochemical transistor 4 is inserted into an electronic device 1 with different culture wells for the analysis of different serum dilutions; cells are then suspended in different dilutions of neutralizing serum together with a virus (e.g., SARS-CoV-2) and then seeded and cultured directly on the organic electrochemical transistor 4.

[0092] The electronic device 2 monitors the progression of the tissue in real time and distinguishes between healthy cells (where the virus has been neutralised) and infected cells (ie, with active virus which reduces cell proliferation).

[0093] Known serum neutralization techniques allow the evaluation of the neutralization effect only at the end of the experiment (60 or even 72 hours). The advantage of using the electronic device 2 is that it has data in real time, thus increasing the information obtained during the experiment and possibly reducing the response time. Indeed, it is possible to quantify a given serum once a defined threshold value for the test cells and viruses has been reached.

[0094] Furthermore, the use of an organic electrochemical transistor 4 has the advantage of amplifying the output signal generated, thus improving the sensitivity of the electronic device 1 functioning as a sensor.

[0095] In particular, serum neutralization experiments performed using the organic electrochemical transistor 4 allow the last neutralizing dilution of serum to be known after 44 hours (versus a 72 hour wait in known tests).

[0096] Furthermore, known methods require staining using a toxic reagent (formaldehyde) and final titration is performed by subjective optical analysis or else requires spectrophotometric equipment.

[0097] In contrast, the use of organic electrochemical transistors 4 makes it possible to avoid the use of toxic reagents and maintain objective measurements without the use of spectrophotometers (which are expensive and cumbersome and therefore not always present in laboratories with biological containment levels such as BSL2 / BSL3 / BSL4).

[0098] In fact, the apparatus associated with the organic electrochemical transistor 4 can be made small and portable, optionally with disposable devices suitable for this type of laboratory.

[0099] Finally, because the neutralization test is a test that can be performed on all animal species (does not require the use of species-specific antibodies / antibody pairs), the use of the OLED4 can be extended to zoonotic and epidemiological studies, along with the evaluation of vaccine efficacy and duration of immune coverage.

[0100] FIG. 4 shows a schematic diagram of the time patterns of two curves representing two real-time estimates of the response time of the organic electrochemical transistor 4, one curve 50-1 relating to a culture in the presence of neutralizing serum, i.e. not infected with a virus, and the other curve 50-2 relating to a culture infected with a virus in the presence of a non-neutralizing serum, with respective optical images showing the morphology of the cell culture on the organic electrochemical transistor 4.

[0101] The values ​​of curves 50-1, 50-2 are normalized to the initial value at time 0, when cell seeding begins and no cells have attached to the organic electrochemical transistor or formed a cell layer; therefore the response time values ​​have no unit of measurement, i.e., they are expressed in arbitrary units, abbreviated as au.

[0102] It can be seen that the response time curve for the cell culture in the presence of neutralizing serum, i.e. not infected by the virus, has a substantially rising pattern due to the regular growth of the cell layer on the organic electrochemical transistor 4, and therefore the estimated response time of the organic electrochemical transistor 4 has a stable value above the threshold value TH defined based on the cell layer under consideration and the virus under test. The maximum value achieved for the estimated response time depends on the cell culture under consideration.

[0103] For example, for the virus SARS-CoV-2 cultured in Vero E6 cells, the threshold TH is equal to 1.2 a.u. and the response time estimate reaches a value of about 2 a.u. after about 44 hours, while in other types of healthy cells, the response time estimate can reach values ​​of 16-20 a.u.

[0104] The response time curve 50-2 for the cell culture infected with virus in the presence of non-neutralizing serum initially shows a slower substantially increasing pattern, and then after a time of about t1=24 hours, it shows a transition towards a substantially decreasing pattern, with values ​​below the threshold TH due to the reduced growth of the cell layer caused by the presence of virus that is not sufficiently neutralized by serum: the estimated response time of the organic electrochemical transistor 4 therefore increases over time and has a value that decreases below the threshold TH.

[0105] In other words, - a low value of the response time indicates a decrease in the cell layer, i.e. the viral activity has impaired the growth of the cell layer, which therefore did not proceed sufficiently; - A high value of the response time indicates a healthy cell layer, i.e. the viral activity was neutralized by the serum and therefore the growth of the cell layer continued regularly.

[0106] In particular, the response time estimate at time t1=24 hours falls below the threshold TH=1.2, which is predictive for healthy growth of the cell layer under consideration.

[0107] The threshold is set based on the cells and viruses under study, making the technique adaptable to a wide range of effects.

[0108] Thus, the electronic device 1 has the ability to identify the ability of serum to neutralize viruses and indirectly assess the health status of the cell layers in the multiple wells.

[0109] With reference to FIG. 2B, this shows an equivalent circuit of the organic electrochemical transistor 4 .

[0110] The equivalent circuit is obtained from a theoretical model reported by Salleo's group and based on the work of Roisin's group.

[0111] Rsol represents the resistance of the solution containing cells, serum and virus.

[0112] Rp and Cp represent the resistance and capacitance of the channel of the organic electrochemical transistor 4.

[0113] Rcell1 and Ccell1 represent the resistance and capacitance of the cell layer 4-7 in the channel between the source and drain electrodes.

[0114] Rcell2 and Ccell2 represent the resistance and capacitance of cell layer 4-6 at the gate electrode.

[0115] With reference to FIG. 3, on the right hand side, a possible physical construction of an organic electrochemical transistor 4 is shown.

[0116] It can be observed that the organic electrochemical transistor 4 has a substantially rectangular shape with equal sides of 25 mm and 26 mm.

[0117] The width of the gold contacts of the source and drain electrodes is equal to about 1.5 mm.

[0118] The width of the gold contacts of the gate electrode is equal to about 5 mm and the conductive polymer layer overlying the gold contacts of the gate electrode has a substantially rectangular shape with equal sides of about 5 mm and about 2 mm.

[0119] The conductive polymer layer centrally overlying each of the source and drain electrode gold contacts (ie, the channel) has a substantially rectangular shape with equal sides of about 2 mm and about 1.5 mm.

[0120] Preferably, the ratio of the area of ​​the gate terminal to the area of ​​the channel is comprised between 3-20.

[0121] For example, 800 x 800 μm 2 Gate electrode and 300×400μm 2 It is possible to obtain a channel area of

Claims

1. 1. An in vitro method for assessing the ability of serum to neutralize a virus, comprising: a) preparing a solution comprising at least one cell, at least one serum, and at least one virus; b) placing the solution in contact with an organic electrochemical transistor comprising a source electrode, a drain electrode and a gate electrode, wherein the source electrode and the drain electrode are electrically connected by a conductive channel comprising a conductive or semiconductive polymer, and wherein the at least one cell is attached to the channel and / or the gate electrode, or the at least one cell is attached to a permeable porous support disposed between the conductive channel and the gate electrode; c) applying a potential difference between the drain electrode and the source electrode, applying a plurality of pulses of the potential difference between the gate electrode and the source electrode, and measuring a plurality of values ​​of each of the pulses of current through the channel; d) calculating an estimate of a response time of the organic electrochemical transistor as a function of the plurality of values ​​of the measured current; e) comparing said value of said estimated response time against a threshold value and detecting whether said serum has neutralizing ability against said at least one virus depending on said comparison. A method comprising:

2. 2. The method of claim 1, wherein the value of the estimated response time is calculated by interpolating the plurality of values ​​of the measured current to a bi-exponential decay curve and then measuring values ​​of a time response parameter of the bi-exponential curve.

3. The current I_d through the channel is given by the following equation: I_d=a ＊ exp(t / T1)+b ＊ exp(t / T2)+e (In the formula, - t is the time; - exp is the exponential function; a, b, e are setting parameters; T1 is a time parameter representing the polymer charging time, which is influenced by the ion-blocking properties of the cell layer; - T2 is a time parameter that depends on the cell layer charging time) is calculated using the current pulse is normalized between a value of 1, which corresponds to the maximum channel current value under conditions of direct polarization of the organic electrochemical transistor, and a value of 0, which corresponds to the average value of the channel current over a predetermined number of points when the channel current reaches a steady state region; 3. The method of claim 2, wherein the response time of the organic electrochemical transistor is equal to the value of the time parameter T1 over time normalized to the value of T1 it had before preparing the solution.

4. the plurality of pulses of the potential difference between the gate electrode and the source electrode are square waves with a defined difference between a high value and a low value, the high value of the square wave being comprised between 0.1 volts and 0.8 volts and the low value of the square wave being comprised between −0.3 volts and 0.5 volts, in particular the potential difference between the high value and the low value of the square wave being equal to 0.3 volts; the duration of said high value of each pulse is comprised between 0.005 seconds and 10 seconds, and in particular is equal to 0.5 seconds; 3. The method according to claim 1, wherein the duration of the low value is comprised between 0.1 seconds and 30 seconds, in particular equal to 1.5 seconds, and the value of the potential difference between the drain electrode and the source electrode is comprised between -0.4 Volts and 0.2 Volts, in particular equal to 0.1 Volts.

5. Before step a), - preparing a further solution comprising said at least one cell and said at least one serum; - placing said further solution in contact with said organic electrochemical transistor; applying the potential difference between the drain electrode and the source electrode and applying the plurality of pulses of potential difference between the gate electrode and the source electrode, and measuring a further plurality of values ​​of current through the channel and through the at least one cell, respectively; - calculating said threshold value as a function of said further plurality of values ​​of said measured current; 3. The method of claim 1 or 2, comprising:

6. 3. The method of claim 1 or 2, wherein the conductive polymer is selected from PEDOT:PSS, PEDOT-S, PEDOT:TOS, PEDOTOH:ClO4, PEDOT-co-PEDOTOH:ClO4, P3HT, PTHS, BBL, p(g2T-TT), PTHS-TMA<+>-co-P3HT, p(gNDI-g2T), p(g0T2-g6T2), polyaniline, polypyrrole, and P-90.

7. 3. The method of claim 1 or 2, wherein the at least one virus belongs to the Coronaviridae family.

8. 8. The method of claim 7, wherein the at least one virus is selected from SARS-CoV, MERS-CoV, and SARS-CoV-2.

9. 8. The method of claim 7, wherein the at least one virus is SARS-Cov-2.

10. 3. The method of claim 1 or 2, wherein the serum is obtained from an individual who has received treatment against the at least one virus.

11. 11. The method of claim 10, wherein said at least one treatment is a vaccine against said at least one virus, preferably a vaccine against a coronavirus, more preferably a vaccine against SARS-CoV-2.

12. 3. The method of claim 1 or 2, wherein the serum is obtained from an individual who has developed an infection caused by the at least one virus, preferably caused by a coronavirus, more preferably caused by SARS-CoV-2.

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