Cell line
Cell studies were conducted in two different BSL-3 virology laboratories, the laboratory at Unit of Microbiology, Great Romagna Hub Laboratory, Cesena, Italy and the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna (IZSLER), Brescia, Italy.
Vero E6 cells (ATCC CRL-1586) were used since they are sensitive and permissive to SARS-CoV-2 infection, leading to high titer replication44,45. Vero E6 cells are used for studies of viruses that have an affinity for the ACE2 receptor, this being highly expressed in this cell model and coronaviruses enter cells via this receptor. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin–streptomycin (P/S), and 1% l-glutamine (l-Gln). Culture medium and supplements were all purchased from EuroClone (Milan, Italy). The cell line was cultured following the protocol of growth and maintenance of Vero cell lines46. Cells were seeded at a density of 15,000 cells/well on the OECTs and cultured in 500 µL of DMEM with 2% FBS, 1% penicillin–streptomycin (P/S), and 1% l-glutamine (L-Gln).
Sera samples and virus infection
This study has been performed by using anonymized leftover serum samples. The samples have been collected from healthy blood donors for the detection of the immune response against SARS-CoV-2 by chemiluminescence assay and by PRNT for the evaluation of serum neutralizing activity in vitro.
Our PRNT assay is modified in MNA according to our already published work47. It requires the formation of a virus-antibody (in an equal volume) complex in vitro during incubation for 30 min at 37 °C in thermo shaker and subsequent seeding on cells susceptible to viral infection. After incubation (typically 72 h), the CytoPathic Effect (CPE) is evaluated by fixing and staining the cells with formaldehyde in Crystal violet and, subsequently, reading the absorbance using the spectrophotometer at 560 nm. The highest serum dilution that neutralizes 90% of viral replication and thus its CPE is reported as the neutralizing titer. At the end of the diagnostic workflow, all the samples used in this study were totally anonymized according to the “ANONYMIZING PROCEDURE” of the Great Romagna Hub Laboratory (AVR PPC 09) that has been approved by the local Ethics Committee.
PRNT assays were performed at the BSL-3 laboratory at Unit of Microbiology, Great Romagna Hub Laboratory, Cesena, Italy. The sera were not disrupted at 56 °C for 30 min as no different from the PRNT test was detected in previous tests. The antibody titer against SARS-CoV-2 was detected by SARS-CoV S1/S2 IgG Chemiluminescent ImmunoAssay (LIAISON® SARS-CoV-2 S1/S2 IgG test) and the PRNT of periodic voluntary plasma donors. Neutralizing sera with titer above 80, as determined by standard PRNT evaluation, were used for this work. PRNT tests on the OECT device were carried out using progressive serum dilution of 1:20, 1:40, 1:80, and 1:160.
The SARS-CoV-2 (titred 106.5 TCID50/mL) virus employed for all SN tests was supplied by the Virology Laboratory of Policlinico San Matteo in Pavia (Italy) and is named VR PV10734. In order to mimic, as closely as possible, the conditions of use, a wild-type viral strain belonging to lineage B1 has been employed (GISAID code EPI_ISL_1908157). Viral strain was sequenced using CleanPlex SARS-CoV-2 Flex (Paragon Genomics Inc., Hayward, CA, USA) and Illumina MiSeq (Illumina Inc. San Diego, CA, USA). Viral titers, expressed as TCID50/mL, were calculated according to the Reed and Muench method based on eight replicates for dilution48,49 and Sperman–Karber50. OECT tests employed a dilution of 300 TCID/well, prepared in DMEM/2% FBS.
Cytopathic effect value
After 72 h from the infection, cells were fixed and stained using 300 µL of a 4% formaldehyde (Fisher Chemical, Milan, Italy) solution in Crystal violet (Delcon, Bergamo, Italy) and incubated for 30 min at room temperature. The dye stains only the living cells, fixed to the well, therefore it allows to discriminate the living cells from the dead ones, which are washed away in the next step. After the incubation time, the dye is washed off with tap water and the plate is dried in a hood. Optical micrographs were taken using an inverted optical microscope, with magnification 10× (Hund Wetzlar).
The highest serum dilution capable to neutralize 90% of the CPE is reported as a neutralizing titer. The sera control is taken as a 100% neutralization reference. Hence, it is possible to calculate the optical density which corresponds to the IC90.
OECT device fabrication
Glass substrates (25 × 25 mm2) were cleaned by sonication in distilled water/acetone/isopropanol baths. Afterward, 10 nm of chrome and 50 nm of gold were deposited by thermal evaporation. After that, substrates were treated with air plasma (20 W for 4 min), and then PEDOT:PSS solution was spin-coated (3000 rpm for 10 s) using a Teflon mask. The thin film thickness was 140 ± 10 nm. The solution was made of 94% PEDOT:PSS (Heraeus, Clevios PH1000) with 5% of ethylene glycol (EG) (Sigma-Aldrich), 1% of 3-glycidoxypropyltrimethoxysilane (GOPS), and 0.25% of 4-dodecylbenzenesulfonicacid (DBSA). This suspension was treated in an ultrasonic bath for 10 min and filtered using 1.2 μm cellulose acetate filters (Sartorius) before the deposition. The samples were subsequently baked at 120 °C for 1 h. Planar geometry OECTs were patterned, having two channels with a length (L) of 1 mm and width (W) of 0.75 mm and an inner gate electrode (L = 2 mm, W = 3 mm). The dual-channel configuration doubled up the device monitoring of the same well, thus taking into account possible cell layer dis-homogeneity after seeding, that may conceal the correct outcome. Then, devices were immersed in distilled H2O for 1 h and dried with a nitrogen flux. In the end, a Polydimethylsiloxane (PDMS) transparent, cylindrical well, having an inner diameter and height of 12 and 8 mm, respectively, was bound to the device to realize the culture well.
OECT integrated system and electrical characterization set-up
All measurements were performed in DMEM as the electrolyte solution. Experiments were performed using an integrated system, the TECH-OECTs, reported in Fig. 1 and in our previous work26. Cells were seeded inside cylindrical PDMS wells, having a diameter of 12 mm. Noteworthy, TECH-OECTs allows to carry out measurements inside the humidified incubator (constant temperature of 37 °C and a CO2 level of 5%), without direct interaction with the virus SARS-Cov-2, thus can be reused. A multiplexer system was used to measure sequentially 12 channels. We measured the source-drain current by means of a Keysight B2912A Source Measure Unit (SMU), while biasing the channel with Vds = –0.1 V and introducing a square wave potential on the gate electrode, from Vgs(OFF) = 0.0 V to Vgs(ON) = 0.3 V, with ton = 0.5 s and toff = 1.5 s. Keysight and the multiplexer were both controlled with customized PC software.
OECT data analysis
Output data were analyzed with a customized Matlab routine: each single channel response to a pulse on the gate was isolated, normalized, and fitted with the bi-exponential curve ({I}_{d}={a},{{exp }}^{-frac{x}{{tau }_{1}}}+{{exp }}^{-frac{x}{{tau }_{2}}}+{e}), as reported in our previous work26. As described elsewhere15, labeling ({tau }_{1}) greater than ({tau }_{2}), ({tau }_{1}) represents the charging time of the PEDOT:PSS influenced by the ion-blocking properties of the cell layer, while ({tau }_{2}) relates to the charging time of the cell layer. Thus, we will focus and examine ({tau }_{1}) as the device time response to a gate potential pulse, averaging its value over five pulses on the same channel and then normalizing it, using the following equation: ({{{{{rm{OECT time response}}}}}}({{{{rm{a.u.}}}}})=tau /{tau }_{{{{{rm{No}}}}}; {{{{rm{Cells}}}}}}), with ({tau }_{{{rm{No}}}} {{{rm{Cells}}}}) is the response time of the device before cell seeding. We then choose 1.2 a.u as the threshold value at the end of the experiment to separate viral-infected cultures (< 1.2 a.u.) from healthy ones (>1.2 a.u.), since it was consistent with the averaged experimental trends carried out in two different laboratories, on three devices, and for the different “incubation mix”.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Viral load is monitored by sampling the cell culture at the end of the experiment and using qRT-PCR Allplex Seegene analysis.
Allplex SARS-CoV-2 Extraction-Free (Seegene Inc., Seoul, Korea) is a real-time qRT-PCR assay that does not require a preparatory RNA-extraction but rather relies on the thermal lysis taking place during the reverse transcription reaction in which the specimen is warmed up to 50 °C for 20 min (reverse transcription) and then to 95 °C for 15 min (polymerase activation). The assay enables the simultaneous detection of three target genes, namely the E gene (common to all Sarbecoviruses), RdRP/S gene, and N gene (both specific for SARS-CoV-2). Sample preparation, reaction setup, and analysis were performed accordingly to the manufacturer’s instructions. In brief, 15 µL of each sample were diluted 1:4 with 45 µL of RNase-free water in a 96-well PCR plate and hence 5 µL were transferred to another plate with 16 µL of PCR master mix, containing 5 µL of MOM (MuDT Oligo Mixture, containing dNTPs, oligos, primers, and Taq-Man 5’ fluorophore / 3’ Black Hole Quencher probes), 5 µL of enzymes, 5 µL of RNase-free water and 1 µL of internal control for every reaction. A positive and negative control were included. The assay was run on a CFX96 real-time thermal cycler (Bio-Rad, Feldkirchen, Germany). The amplification process relies on the first step for cDNA denaturation at 95 °C for 10 s, followed by primers annealing at 60 °C for 15 s and elongation at 72 °C for 10 s (44 cycles). Fluorescent signals were acquired after every amplification cycle for FAM (E gene), Cal Red 610 (RdRP/S gene), Quasar 670 (N gene), and HEX (internal control) fluorophores. In a real-time PCR assay, a positive reaction is detected by the accumulation of a fluorescent signal. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold. Ct levels are inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level the greater the amount of target nucleic acid in the sample). The fold change reported in our graph represents the Ct level at the end of the experiment (T48h), normalized with respect to its initial value at the beginning of the experiment (T0h). Results analysis and target quantification were carried out employing a 2019-nCoV viewer from Seegene Inc.
Cell death evaluation
Cell death was detected by measuring the permeability of the plasma membrane to the normally impermeable fluorescent dye DAPI. At the end of the experiment, cells were washed with PBS and incubated with DAPI for 20 min at room temperature. Cells were then harvested, washed in PBS, and fixed in 4% paraformaldehyde (PFA), and DAPI uptake was quantified by FACS analysis (Cytoflex-S Beckman Coulter, California, USA).
Statistical analysis
Differences between qRT-PCR results were analyzed using a double-sided Student’s t-test. P-values <0.05 were considered statistically significant. Statistical analyses were performed using SPSS version 19.0 (IBM Corp., Armonk, NY).
