Organic Electrochemical Transistors as Versatile Tool for Real-Time and Automatized Viral Cytopathic Effect Evaluation

Abstract

In-vitro viral studies are still fundamental for biomedical research since studying the virus kinetics on cells is crucial for the determination of the biological properties of viruses and for screening the inhibitors of infections. Moreover, testing potential viral contaminants is often mandatory for safety evaluation. Nowadays, viral cytopathic effects are mainly evaluated through end-point assays requiring dye-staining combined with optical evaluation. Recently, optical-based automatized equipment has been marketed, aimed at the real-time screening of cell-layer status and obtaining further insights, which are unavailable with end-point assays. However, these technologies present two huge limitations, namely, high costs and the possibility to study only cytopathic viruses, whose effects lead to plaque formation and layer disruption. Here, we employed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss) organic electrochemical transistors (OECTs) for the real-time, electrical monitoring of the infection of cytolytic viruses, i.e., encephalomyocarditis virus (EMCV), and non-cytolytic viruses, i.e., bovine coronavirus (B-CoV), on cells. OECT data on EMCV were validated using a commercially-available optical-based technology, which, however, failed in the B-CoV titration analysis, as expected. The OECTs proved to be reliable, fast, and versatile devices for viral infection monitoring, which could be scaled up at low cost, reducing the operator workload and speeding up in-vitro assays in the biomedical research field.

Keywords: BCoV; ECMV; bovine coronavirus; cytolytic virus; encephalomyocarditis virus; non-cytolytic virus; organic electrochemical transistor; virus replication.

Figure 1

(a) An OECT render schematic. (b) The TECH-OECT system used for the measurements inside the incubator. The schematic non-cytolytic (c) and cytolytic (d) viral action (top) and micrograph (bottom) are on the cell lines.

Figure 2

The OECT real-time monitoring of HRT-18 cell growth infected with B-CoV in suspension (a) and adhesion (b) for 48 h. Each OECT time response point is the average of five consecutive measurements, having the standard deviation reported as the curve shadow. The normalized Incucyte^® data of HRT-18 cell growth, infected with B-CoV in suspension (c) and adhesion ((d), the data were taken and normalized after viral incubation). The experiments were stopped 72 h post virus infection, except for the Incucyte screening in adhesion, which was stopped after 48 h since a clear cytopathic effect had already been monitored by the OECTs.

Figure 3

OECT (a) and Incucyte^® (b) real-time monitoring of HRT-18 cells, infected with progressively diluted B-CoV concentrations in suspension. Each OECT time response point is the average of five consecutive measurements, having the standard deviation reported as a curve shadow. (c) The cytofluorimeter analysis of the HRT-18 dead population after 72 h of B-CoV incubation, at increasing viral dilutions (from left to right).

Figure 4

The OECT (a) and Incucyte^® (b) real-time monitoring of VERO E6 cells, infected with progressively diluted EMCV inocula, compared to a healthy cell growth, as the control. Each OECT time response point is the average of five consecutive measurements, having the standard deviation reported as the curve shadow. (c) The optical micrographs on the active area of the devices after 48 h of incubation, with different dilutions of EMCV and the control over a standard healthy growth, using a red staining dye for dead cells. White scale bars = 150 µm. * p < 0.05 denotes a significant difference compared to the control.

Figure 5

The OECT (a) and Incucyte^® (b) real-time monitoring of VERO E6 cells, infected with progressively diluted EMCV, compared to healthy cell growth, as the control. Each OECT time response point is the average of five consecutive measurements, having the standard deviation reported as the curve shadow. (c) The optical micrographs on the active area of the devices after 48 h of EMCV incubation at different dilutions and the control over a standard healthy growth, using a red staining dye for dead cells. * p < 0.05 denotes a significant difference compared to the control.