Sequential Flow Controllable Microfluidic Device for G-Quadruplex DNAzyme-Based Electrochemical Detection of SARS-CoV-2 Using a Pyrrolidinyl Peptide Nucleic Acid

초록

The coronavirus disease 2019 (COVID-19) pandemic causedby severeacute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significanthealth issue globally. Point-of-care (POC) testing that can offera rapid and accurate diagnosis of SARS-CoV-2 at the early stage ofinfection is highly desirable to constrain this outbreak, especiallyin resource-limited settings. Herein, we present a G-quadruplex DNAzyme-basedelectrochemical assay that is integrated with a sequential flow controllablemicrofluidic device for the detection of SARS-CoV-2 cDNA. Accordingto the detection principle, a pyrrolidinyl peptide nucleic acid probeis immobilized on a screen-printed graphene electrode for capturingSARS-CoV-2 DNA. The captured DNA subsequently hybridizes with anotherDNA probe that carries a G-quadruplex DNAzyme as the signaling unit.The G-quadruplex DNAzyme catalyzes the H2O2-mediatedoxidation of hydroquinone to benzoquinone that can be detected usingsquare-wave voltammetry to give a signal that corresponds to the targetDNA concentration. The assay exhibited high selectivity for SARS-CoV-2DNA and showed a good experimental detection limit at 30 pM. To enableautomation, the DNAzyme-based assay was combined with a capillary-drivenmicrofluidic device featuring a burst valve technology to allow sequentialsample and reagent delivery as well as the DNA target hybridizationand enzymatic reaction to be operated in a precisely controlled fashion.The developed microfluidic device was successfully applied for thedetection of SARS-CoV-2 from nasopharyngeal swab samples. The resultswere in good agreement with the standard RT-PCR method and could beperformed within 20 min. Thus, this platform offers desirable characteristicsthat make it an alternative POC tool for COVID-19 diagnosis.