Ultrasensitive and Visual Electrochemiluminescence Ratiometry Based on a Constant Resistor-Integrated Bipolar Electrode for MicroRNA Detection

Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.

Currently, portable, low-cost, and easy to operate on-chip analytical units are urgently demanded to meet the requirement for point-of-care testing in resource-limited regions. Herein, a dual-mode lab-on-paper platform is presented, which integrates distance-based visualized readout with ratiometric electrochemiluminescence (ECL) assay in one device. The distance-based measurement is based on a brown visualized strip generated from the oxidation reaction of 3,3'-diaminobenzidine in the presence of H2O2 initiated by horseradish peroxidase (HRP). Notably, visualized semiquantitative results are displayed as the length of a brown bar chart directly on the device-without the need for any data processing or plotting steps, thus avoiding the error caused by the naked eye for distinguishing the color depth. On the contrary, a ratiometric ECL technique was employed for accurate analysis based on the specific biorecognition between Pb2+-dependent DNAzymes and targets. Concretely, upon addition of Pb2+ into the fabricated device, cleaved oligonucleotide fragments connected with HRP functionalized Au nanocubes could permeate through the cellulose on account of their size that is smaller than paper pores, quench the ECL signal of the CdS quantum dots because of resonance energy transfer, and synchronously boost the ECL intensity generated from luminol by catalyzing H2O2. As a consequence, satisfied prediction and accurate monitoring performance was obtained in the range 0.1-2000 nM and 0.01-2000 nM by measuring the length of colored product and the ratio of ECL intensity, respectively. The beneficial advantages of low cost, high efficiency, and the capacity to perform dual-mode assay qualify this innovative device for use with diverse applications.