Materials and reagents: Monkey African green kidney (Vero) cell cultures (ATCC CCL-81) were originally provided from the LGC Promochem (Teddington, UK). Dulbecco’s Modified Eagle Medium (DMEM), Fetal bovine serum (FBS), antibiotics (streptomycin–penicillin), l-glutamine & l-alanine, and trypsin/EDTA were purchased from Sigma-Aldrich (Taufkirchen, Germany). Monoclonal antibodies against SARS-CoV-2 Spike protein S1 subunit (No. ABIN6952616) were purchased from antibodies-online.com.

Cell culture and sensor fabrication: Cell culture was performed according to Apostolou et al. 2020. Briefly, Vero cells were cultured in Dulbecco medium with 10 % fetal bovine serum (FBS), 10 % antibiotics (streptomycin-penicillin) and 10 % l-glutamate, l- alanine (nutrient). The cells were removed from the culture vessel by adding 0.05 % trypsin / EDTA (1X) for 10 min at 37 °C and the cells were harvested by centrifugation (2 min / 1200 rpm) at a final density of 2.5 × 106 mL−1.

Membrane-constructed cells were generated by electro-insertion of monoclonal antibodies against the S1 SARS-CoV-2 Spike protein subunit in the Vero cell membrane. Briefly, cells were detached and harvested after centrifugation (6 min / 1000 rpm / 25 °C). The cell pellet was resuspended in 400 μL PBS (pH 7.4) containing 5 μg mL-1 antibody and incubated for 20 min at 4 °C. After incubation, the cell-antibody mixture was transferred to electroporator (Eppendorf Eporator, Eppendorf AG, Germany) cuvettes (4 mm) and electroinserted by applying two square electrical pulses at 1800 V / cm. The mixture was then transferred to a Petri dish (60 × 15 mm2) containing 3 mL of medium and incubated at 37 °C and 5% CO2 for 24 h. The medium was then discarded from the Petri dish and the Vero / anti-S1 cells were mechanically removed and collected with the medium in Eppendorf tubes.

Definition of the Lower Limit of Detection (LOD): In order to define the LOD of the system we used a positive sample with a viral concentration of 4 × 1011genome copies (gc)/mL. Subsequently, sequential 10−1 dilutions (10 dilutions in total) of the sample were prepared to a final concentration of 40gc/mL.

Samples were first added to the top of each carbon electrode (20 μL). Then, biosensors with Vero membrane cells made with monoclonal antibodies (20 μL ≈ 5 × 104 cells) were added. Cell response was recorded as a time series of potentiometric measurements (in Volts). Each measurement lasted 180 s and 360 values ​​per sample were recorded at a sampling rate of 2 Hz. After each measurement, the cell responses were downloaded to a cloud server and calculations were performed, based on a newly developed algorithm that yielded results on the presence of SARS-CoV-2 that appeared on the smartphone screen. Each sample was tested eight times using a set of eight individual sensors and each experiment was performed in duplicate.

The sensitivity and specificity of the BERA were determined using 110 samples with a range of Ct values, and 136 negative samples all confirmed with RT-PCR. All samples were analyzed at the Laboratory of Hygiene and Epidemiology of the University of Thessaly, a Biosafety Level 2 laboratory, from July 2020-Sempember 2020. All samples were sent for consented molecular laboratory investigation from symptomatic patients, as part of the routine diagnostic procedure. After the test, the samples were stored at -80⁰C. The number of the samples used as well as their Ct Values are shown in detail in Table 1 .

Specificity and sensitivity of the method according to Ct values and different prevalence rates of the disease.

● Sensitivity, that is the probability that a test result will be positive when the disease is present (true positive rate) was calculated for each group according to the following type: a / (a + b).

● Specificity, that is the probability that a test result will be negative when the disease is not present (true negative rate) was calculated according to the following type: d / (c + d).

● Positive predictive value, that is the probability that the disease is present when the test is positive was calculated for each group according to the following type:

PPV = Se x Prevalence / Se x Prevalence + (1-Sp) x (1-Prevalence).

● Negative predictive value, that is the probability that the disease is not present when the test is negative was calculated for each group according to the following type:

NPV = Sp x (1-Prevalence) / (1-Se) x Prevalence + Sp x (1-Prevalence).

Nucleic acids were extracted from specimens using the iPrepTM PureLink® Virus Kit and iPrepTM Purification Instrument (Invitrogen) according to the manufacturer’s instructions. Four hundred μl from a commercially available viral transport medium (VTM) for the safe transfer of virus were transferred to the strip and viral RNA was eluted to a final volume of 100 μl.

Specific SARS-CoV-2 RNA (E gene) was detected with the RIDA®GENE SARS-CoV-2 kit (R-Biopharm, Germany, CE, IVD) in an ABI STEP ONE (ThermoFisher Scientific) real time validated system (Being validated, provide reference citation). The PCR conditions were set according to the manufacturer’s indications.

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