CRISPR-Cas13a-powered electrochemical biosensor for the detection of the L452R mutation in clinical samples of SARS-CoV-2 variants

Abstract

Since the end of 2019, a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has deprived numerous lives worldwide, called COVID-19. Up to date, omicron is the latest variant of concern, and BA.5 is replacing the BA.2 variant to become the main subtype rampaging worldwide. These subtypes harbor an L452R mutation, which increases their transmissibility among vaccinated people. Current methods for identifying SARS-CoV-2 variants are mainly based on polymerase chain reaction (PCR) followed by gene sequencing, making time-consuming processes and expensive instrumentation indispensable. In this study, we developed a rapid and ultrasensitive electrochemical biosensor to achieve the goals of high sensitivity, the ability of distinguishing the variants, and the direct detection of RNAs from viruses simultaneously. We used electrodes made of MXene-AuNP (gold nanoparticle) composites for improved sensitivity and the CRISPR/Cas13a system for high specificity in detecting the single-base L452R mutation in RNAs and clinical samples. Our biosensor will be an excellent supplement to the RT-qPCR method enabling the early diagnosis and quick distinguishment of SARS-CoV-2 Omicron BA.5 and BA.2 variants and more potential variants that might arise in the future.

Recent advances using MXenes in biomedical applications

“…195 Electrochemical sensors for microorganisms using MXene-based nanomaterials are summarized in Table 4. Exosomes, 196–199 bacteria, 200–205 viruses, 149,206–209 and even cancer cells 114,171 can be measured through appropriate surface modification of electrodes. The lowest sensing range is about 10 particles per μL.…”

“…194 Chen et al used electrodes made of MXene–AuNP composites for improved sensitivity and the CRISPR/Cas13a system for high specificity in detecting the single-base L452R mutation (in the SARS-Cov-2 virus) in RNAs and clinical samples. 208 Kim et al developed a field-ready cyanobacterial pretreatment device and an electrochemical clustered regularly interspaced short palindromic repeats (EC-CRISPR) biosensor. 200 The biosensor was composed of CRISPR/Cpf1 protein conjugated with MXene on an Au microgap electrode (AuMGE) integrated into a printed circuit board (PCB).…”

Ultra‐Fast Single‐Nucleotide‐Variation Detection Enabled by Argonaute‐Mediated Transistor Platform

“…Single-nucleotide variations (SNVs), as the genetic basis of many diseases related to susceptibility and individual response to therapy, are significant biomarkers in not only biological research but also pharmacology, disease diagnosis, and population genetics in the postgenomic era. [1][2][3][4][5][6] Genome-wide association studies have identified SNVs associated with diverse human illnesses, such as cancer, neurodegenerative disease, cardiovascular disease, reaction (PCR, 1-2 h), reverse transcriptase droplet digital PCR (RT-ddPCR, ≈24 h), isothermal amplification (0.5-1 h), CRISPR-Cas (0.5-2 h), colorimetric assay [15] (≈0.5 h), electrochemistry [27] (≈45 min) and so on. However, to realize sensitive SNV detection, these methods normally require nucleic acid amplification, which increases operational complexity and takes tens of minutes to obtain the result.…”

AND-Logic Cascade Rolling Circle Amplification for Optomagnetic Detection of Dual Target SARS-CoV-2 Sequences

“…One of the key mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spike L452R (corresponding to single-nucleotide variant T22917G), is synthesized as our target sequence (i.e., target input, TAR), which was reported to be associated with an increased viral infectivity 30,31 and was used to track the pandemic. 32 A highly conserved SARS-CoV-2 sequence (belonging to ORF1ab coding region, NSP6 gene) is synthesized as our reference input (REF). 33 As one of the inputs for the AND-logic gate, REF can be regarded as an internal reference and positive control for TAR detection, which can potentially increase the sensing specificity and lower the risk of false-positive results.…”

“…Herein, we demonstrate a homogeneous AND-logic cascade RCA strategy for single-nucleotide variant discrimination and the real-time analysis of dual target nucleic acid sequences. One of the key mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spike L452R (corresponding to single-nucleotide variant T22917G), is synthesized as our target sequence (i.e., target input, TAR), which was reported to be associated with an increased viral infectivity , and was used to track the pandemic . A highly conserved SARS-CoV-2 sequence (belonging to ORF1ab coding region, NSP6 gene) is synthesized as our reference input (REF) .…”