key: cord-0992362-obnhmfed
authors: Kim, Hyo Eun; Schuck, Ariadna; Lee, See Hi; Lee, Yunjong; Kang, Minhee; Kim, Yong-Sang
title: Sensitive Electrochemical Biosensor Combined with Isothermal Amplification for Point-of-Care COVID-19 Tests
date: 2021-03-18
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2021.113168
sha: 463746fdd3b517d9f5fbe747c622fa8cb15a71ea
doc_id: 992362
cord_uid: obnhmfed

We report an electrochemical biosensor combined with recombinase polymerase amplification (RPA) for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2. The electrochemical biosensor based on a multi-microelectrode array allows the detection of multiple target genes by differential pulse voltammetry. The RPA reaction involves hybridization of the RPA amplicon with thiol-modified primers immobilized on the working electrodes, which leads to a reduction of current density as amplicons accumulate. The assay results in shorter “sample-to-answer” times than conventional PCR without expensive thermo-cycling equipment. The limits of detection are about 0.972 fg/μL (RdRP gene) and 3.925 fg/μL (N gene), which are slightly lower than or comparable to that of RPA assay results obtained by gel electrophoresis without post-amplification purification. The combination of electrochemical biosensors and the RPA assay is a rapid, sensitive, and convenient platform that can be potentially used as a point-of-care test for the diagnosis of COVID-19.

Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease and, based on the pairwise protein 2 sequence analysis, it is part of the species of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3 (Gorbalenya et al., 2020; Kilic et al., 2020; Organization, 2020; Zhou et al., 2020) . The outbreak of can be used for PCR assays, including the ORF1b or ORF8 regions and the nucleocapsid (N), spike (S) protein, sets to detect multiple regions in the virus, fluorescence resonance energy transfer (FRET) quenching is 19 employed to monitor the increase in the number of amplicons indirectly during the reaction in real-time (Liang 20 et al., 2020; Qu et al., 2020) . However, this testing scheme requires complex analytical instruments, expensive 21 reagents, and considerable expertise (Kilic et al., 2020) .

Current serological tests, whether realized using laboratory-based enzyme-linked immunosorbent assay 23 (ELISA) platforms or lateral flow serological assays designed for use as point-of-care tests (POCTs), have 24 rapidly become available but with low accuracy, because antibodies might not be detected in the early stages of 25 infection (Carter et al., 2020; Chan et al., 2020; Espejo et al., 2020; Ji et al., 2020; Tan et al., 2020; Vashist, 26 2020; Xiang et al., 2020) . Currently, only molecular quantitative reverse transcription-polymerase chain reaction 27 (PCR) (RT-qPCR) testing of respiratory tract samples is recommended for the identification and diagnosis of 28 COVID-19 cases (Chan et al., 2020; Corman et al., 2012; Huajun et al., 2020; Joung et al., 2020; Kilic et al., 29 2020; Nagura-Ikeda et al., 2020; Tahamtan and Ardebili, 2020; Tang et al., 2020; Vashist, 2020; Yishan Wang 30 et al., 2020; Yuan et al., 2020; Zhang et al., 2020) . Isothermal amplification techniques, such 31 as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), are 32 alternative methods that feature high sensitivity, specificity, and rapidity under isothermal conditions (Esbin et 33 al., 2020; Joung et al., 2020; Kashir and Yaqinuddin, 2020; Nassir et al., 2020; Won et al., 34 2020; Zhang et al., 2020) . Because these methods significantly simplify the protocol without requiring a 35 thermocycler, isothermal amplification has been combined with various forms of biosensors that provide simple, 36 fast, disposable, and low-cost diagnosis.

In this study, we report an electrochemical biosensor coupled with RPA for the rapid and sensitive detection 38 of SARS-CoV-2. An electrochemical biosensor based on microelectrode array microchips, comprising a 39 reference electrode, counter electrode, and five individual working electrodes, allows for the detection of 40 multiple target genes by differential pulse voltammetry (DPV). The isothermal RPA reaction involves 

The TwistAmp® Basic kit for RPA was purchased from TwistDx Limited (UK). The primers were 53 synthesized and purified by Bionics Inc. (Korea). The plasmids containing a sequence of SARS-CoV-2 N and array. The substrate was cleaned by immersing in 50 mL of acetone and sonicating for 20 min. Acetone residues 66 and remaining foreign substances were cleaned with isopropanol in the same manner as the acetone step, and the 67 substrate was placed in an oven (75 °C) to dry for 10 min. The residue-free bare glass was spin-coated with a 68 positive photoresist (PR) and pre-baked for 10 min at 95 °C. Subsequently, the photomask was aligned over the 69 PR and patterned by a photolithography method using a mask aligner (MA-6, Karl-Suss). Ag was deposited 70 over the substrate (glass/patterned PR layer) using a thermal evaporator system (Evaporation System SHE-6T-71 350D). The fabricated glass/patterned PR/Au thin film layer was soaked in acetone and sonicated until the counter electrode (CE) and working electrode (WE) were fabricated using Au. For the formation of the AgCl 74 layer, simple immersion was performed under FeCl 3 as a bleach solution for 1 min at 30 s intervals, which 75 showed the highest current density of the redox reaction in cyclic voltammetry (CV) measurements.

The N gene and RdRP gene of SARS-CoV-2 were used as target templates (Cheong et al., 2020; Kashir and 79 Yaqinuddin, 2020; Kilic et al., 2020; Won et al., 2020) . The primers were ordered from Bionics (Suwon, Korea) 80 with requested thiol modification at the end of the forward primer. The NTC, including all reagents except 81 template DNA, was used as a negative control. For the RPA reaction, the TwistDx Limited (Maidenhead, UK) 82 protocol was used Kilic et al., 2020; Magro et al., 2017; Yeh et al., 2017) . Rehydration (Akanda et al., 2016; Vogt et al., 2016) . CV measurements were used to investigate the stability and guarantees uniformity, increasing the reproducibility of our device since they were all fabricated under the same least three times, then avoiding an incorrect diagnosis. As a reference electrode, Ag/AgCl was used because it is 105 simple, stable, and has less potential hysteresis against temperature cycles (Kurkina et al., 2011; Punter- 

The relative standard deviation (RSD) of the peak currents of these five electrodes was less than 0.07 μA after The sensitivity of the RPA-coupled electrochemical biosensor was investigated by detecting DPV signals at 164 different template concentrations ranging from 10 9 to 10 3 copies. The assay was carried out for 40 min at human 165 body temperature, and the signals were collected at 5 min intervals. The logistic fittings of the peak current 

In summary, the RPA-coupled electrochemical detection method was proposed for SARS-CoV-2 genome For on-chip RPA, a PDMS slab with a cylindrical hole 25 mm in diameter was prepared for a reaction chamber, 197 and electrodes were coated with thiol-modified primers. During RPA, the amplicon demonstrated a lower DPV 198 signal because of the electrostatic repulsion between the negatively charged amplicon and the electrode. The 199 limit of detection was slightly lower than or comparable to that of the RPA assay results obtained by gel 200 electrophoresis without post-amplification purification. The data suggest that the combination of RPA with an 201 electrochemical biosensor can be utilized as a rapid, sensitive, and convenient DNA detection platform under 202 human body temperature instead of using an external temperature controller.

Coronavirus disease ( COVID-19)

Amperometric and Impedance Monitoring Systems for Biomedical Applications

Fuel Cycle Waste Technol

This work was supported in part by a National Research Foundation of Korea grant funded by the Korean government (No. NRF-2018R1D1A1B05049787).