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

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant health issue globally. Point-of-care (POC) testing that can offer a rapid and accurate diagnosis of SARS-CoV-2 at the early stage of infection is highly desirable to constrain this outbreak, especially in resource-limited settings. Herein, we present a G-quadruplex DNAzyme-based electrochemical assay that is integrated with a sequential flow controllable microfluidic device for the detection of SARS-CoV-2 cDNA. According to the detection principle, a pyrrolidinyl peptide nucleic acid probe is immobilized on a screen-printed graphene electrode for capturing SARS-CoV-2 DNA. The captured DNA subsequently hybridizes with another DNA probe that carries a G-quadruplex DNAzyme as the signaling unit. The G-quadruplex DNAzyme catalyzes the H2O2-mediated oxidation of hydroquinone to benzoquinone that can be detected using square-wave voltammetry to give a signal that corresponds to the target DNA concentration. The assay exhibited high selectivity for SARS-CoV-2 DNA and showed a good experimental detection limit at 30 pM. To enable automation, the DNAzyme-based assay was combined with a capillary-driven microfluidic device featuring a burst valve technology to allow sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion. The developed microfluidic device was successfully applied for the detection of SARS-CoV-2 from nasopharyngeal swab samples. The results were in good agreement with the standard RT-PCR method and could be performed within 20 min. Thus, this platform offers desirable characteristics that make it an alternative POC tool for COVID-19 diagnosis.

Opinion in Recent Nanomaterial-Based Electrochemical Platforms for Infectious Disease

“…Recent nanomaterial-based platforms integrated with various device designs to enable simultaneous and rapid detection have been demonstrated to diagnose tuberculosis (TB) [12], leptospirosis [13,14], sepsis [15], Human Immunodeficiency Virus (HIV) [16], Hepatitis C Virus (HCV) [16], Hepatitis B Virus (HBV) [17,18], and COVID-19 [19]. Pornprom et al [12] described a paperbased electrochemical biosensor using gold/carboxyl graphene nanocomposite (AuPs/GCOOH)-modified electrode for detecting a specific biomarker indicating the onset of TB infection.…”

“…In addition to these detection schemes, integrated electrochemical sensor microfluidic devices have been introduced to detect both single and multiple analytes simultaneously [16,20]. For example, Naorungroj et al [19] presented a G-quadruplex DNAzyme-based electrochemical sensor integrated with a sequential flow controllable microfluidic device for the detection of COVID-19. This combined platform allows sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion.…”

Construction of Fluorescent G-Quadruplex Nanowires for Label-Free and Accurate Monitoring of Circular RNAs in Breast Cancer Cells and Tissues with Low Background

“…34,35 G-quadruplex is a highorder structure formed by the folding of DNA/RNA containing tandemly repeated guanines, and it has been widely applied in bioimaging, drug transportation, and catalytic reactions. 36,37 Herein, we demonstrate the construction of fluorescent G-quadruplex nanowires for label-free and accurate monitoring of circRNAs with low background by the integration of a proximity ligation-RCA cascade with light-up G-quadruplex. In comparison with the reported proximity ligation-RCA strategies, 38−40 this assay involves only a linear primer and a padlock probe, which greatly simplifies the probe design and avoids the risk of nonspecific amplification caused by multiple primers.…”

“…Rolling circle amplification (RCA) is a classical and well-developed nucleic acid amplification method, and it can achieve exponential amplification of target within a short reaction time. , Proximity ligation techniques endow ligation-based methods with excellent specificity. In proximity ligation, two ends of the DNA probe are splinted to adjacent sites by a complementary sequence and then ligated by isothermal/thermostable ligases. , G-quadruplex is a high-order structure formed by the folding of DNA/RNA containing tandemly repeated guanines, and it has been widely applied in bioimaging, drug transportation, and catalytic reactions. , Herein, we demonstrate the construction of fluorescent G-quadruplex nanowires for label-free and accurate monitoring of circRNAs with low background by the integration of a proximity ligation-RCA cascade with light-up G-quadruplex. In comparison with the reported proximity ligation-RCA strategies, − this assay involves only a linear primer and a padlock probe, which greatly simplifies the probe design and avoids the risk of nonspecific amplification caused by multiple primers.…”