At the time of first Italian lockdown, in Spring 2020, our scientific group had recently demonstrated the ability of our poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss)-based, planar Organic Electrochemical Transistors (OECTs) to provide the real-time monitoring of in-vitro cell tissue formation, detachment and disruption, induced by external agents. OECTs are three terminal devices, in which the current flowing in the semiconducting channel is modulated by the potential applied to the gate electrode throughout an electrolyte solution. Any impediment positioned between the channel and the gate of the device will hinder the ion flux, slowing down the transistor response to a gate potential switching. Accordingly, OECTs could supply an electrical, real time output reporting cell layer integrity and health.
Thus, we hypothesized that our OECTs were able able to quantify the virus neutralizing antibodies in a patient (either developed after the Sars-Cov-2 infection or due to the vaccination cycles), effectively assessing the actual protection from the virus.
Neutralizing assays are powerful tools used to assess the neutralizing power of antibodies in human serum via monitoring a cell tissue response after exposure to a virus, incubated with progressive dilution of the patient serum. The gold standard technique to for serum neutralization tests is the Plaque Reduction Neutralization Assay (PRNT), which, however, usually requires highly specialized operators, long processing times and toxic waste disposal, thus having huge economic costs. To overcome these issues, we translated our know-how and technology into an electronic platform able to be operated remotely as a multiple neutralizing assay tool. The rendering of a single OECT is reported in Figure 1a, a planar OECT with two separated channels accounting for cell layer inhomogeneity. The use of Pedot:Pss both for the gate and channels grants optical transparency, enabling optical monitoring of the cell line, and full biocompatibility, allowing for the direct seeding and growth of the cell on top of the device active layers. Multiple OECTs are integrated into an electronic platform, equivalent to a multiwell plate, that allows automated, multiplexed measurements, and that can be employed inside a incubator.
In order to assess the cell layer health status, we monitor the transistor time response to voltage pulses on the gate (Figure 1b) : the source-drain current of the OECTs (black dotted line) is reported (with Vds = –0.1 V) while a square wave potential pulse (red line) is introduced on the gate electrode ( Vgs (OFF/ON)= 0.0/3.0 V); the zoomed image shows the channel current normalization (black dotted line) and fitting (green line) with a bi-exponential decay equation. The fitting output are two temporal parameters: the faster, associated to the cell layer polarization, and the slower, reporting the polymeric channel response to the pulse, influenced by the cell layer integrity. The latter is thus used to extract information on the cell health status.
Since we aimed at developing a fast, simple and reliable protocol for quantifying neutralizing antibodies in human serum, we mixed VeroE6 cells, serum and Sars-CoV-2 virus in an Eppendorf tube, before introducing the mix directly onto the OECTs. The cultures were then electrically monitored from remote while stored in the incubator for 44-48 h, obtaining the real-time evaluation of the efficacy/inefficacy of the antibodies present in the patient serum. It is worth reminding that COVID 19-infected cultures with non-neutralizing (inefficient) antibodies would be stressed and disrupted by the virus proliferation, which induces a cytopathic effect, as highlighted in Figure 1c (red). The damage of the cell layer on the OECT would ease the ion flow in and out of the polymeric active areas of the device, thus reducing its time response, extracted with our pulsed measurements. This behaviour is translated in a drop in the normalized time response. On the contrary, a cell culture in which the viral agent is neutralized would report a constantly growing time response (Figure 1c blue).
Figure 1: a) Rendering of the planar OECT, consisting in a glass substrate with Cr/Au contacts and Pedot:Pss channels and gate, enclosed in a PDMS perforated cylinder as culture well. b) Electrical measurements for in-vitro cell health evaluation: on the left, drain current response (black dotted line) to voltage pulses on the gate (red line); on the right, single normalized drain current modulation after a pulse (black dotted line), fitted (green line) with the bi-exponential functional reported in the green rectangular inset. c) Schematicgraphs (left) of the expected OECT responses discriminating a viral-infected culture and a healhty one, with relative relative rendering of OECT cross-section having the cell layer grown on top (right).
Our system proved to be able to discriminate, in real-time, between viral proliferating cultures and viral-neutralized ones, shortening the time of standard neutralization assays that might last over 72h. We report in Figure 2 the complete experimental assay: VeroE6 infected by SARS-CoV-2 (300 TCID50/mL), with and without highly concentrated (dilution 1:10) neutralizing (positive serum = serum P) and non-neutralizing antibodies (negative serum = serum N); control samples, having cells seeded with serum N (dark green line) and P (light green line). OECT time response results are outlined in Figure 2a, while PCR tests, performed at the beginning (0 h) and at the end (48 h) of each experiment to confirm the expected virus proliferation/stop, are reported in Figure 2b. A further validation of our sensor response is obtained by means of optical images (using crystal violet staining) and cytofluorimeter analysis at the end of the experiments, shown in Figure 2c.
Figure 2: a) OECT real-time monitoring of SARS-Cov-2-infected cell lines (red), with neutralizing (blu) and non-neutralizing (magenta) antibodies and sera controls (light and dark green for neutralizing and non-neutralizing antibodies, respectively). The orizontal dashed black line is inserted as the threshold discriminating between viral-proliferating and healthy cell cultures. b) qRT-PCR Allplex Seegene data normalized to the starting value at the beginning of each experiment. ***p < 0.001 c) Cytofluorimeter analysis on cell population and optical micrographs on Crystal Violet stained cells of cell cultures for viral-infected cell lines (red), with neutralizing (blu) and non-neutralizing (magenta) antibodies.White scale bars: 100 μm.
The here proposed technology reduces the procedure time and the risk for the operator, avoiding the use of toxic compounds to fix cells and allowing an automated remote evaluation inside the incubator. The devices are low-cost, re-usable (up to three times at least, after a brief sterilization process), low power-consuming and industrially scalable (as well as the interfaced electronics), thus offering a powerful tool to speed up the viral neutralization screening of Covid-19 on larger scales with higher automated reproducibility, monitoring the population immunity after direct contact with the virus or vaccination. Quantifying the neutralizing antibodies and their temporal decay for single patients could give us a much needed, better understanding of the personal protection and support the sanitary system, guiding vaccination towards the more vulnerable population.
Finally, it is important to highlight that, owing to the aspecific nature of the serum neutralization screening, our OECT devices are versatile and can be employed with different cell lines and viruses: they could be easily transferred to the study of other cytopathic viruses and cell substrates, for extensive, real-time monitoring and operator-safe evaluations over several critical viral infections.