CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant

Abstract

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The gold standard method for the diagnosis of SARS-CoV-2 depends on quantitative reverse transcription-polymerase chain reaction till now, which is time-consuming and requires expensive instrumentation, and the confirmation of variants relies on further sequencing techniques. Herein, we first proposed a robust technique-methodology of electrochemical CRISPR sensing with the advantages of rapid, highly sensitivity and specificity for the detection of SARS-CoV-2 variant. To enhance the sensing capability, gold electrodes are uniformly decorated with electro-deposited gold nanoparticles. Using DNA template identical to SARS-CoV-2 Delta spike gene sequence as model, our biosensor exhibits excellent analytical detection limit (50 fM) and high linearity (R2 = 0.987) over six orders of magnitude dynamic range from 100 fM to 10 nM without any nucleic-acid-amplification assays. The detection can be completed within 1 h with high stability and specificity which benefits from the CRISPR-Cas system. Furthermore, based on the wireless micro-electrochemical platform, the proposed biosensor reveals promising application ability in point-of-care testing.

Multifunctional Nanoparticle Platform for Surface Accumulative Nucleic Acid Amplification and Rapid Electrochemical Detection: An Application to Pathogenic Coronavirus

“…cDNA (1 fM) (∼118 copies/μL RNA) (Supporting Information, Table S3), was used to match the average concentration of the clinically extracted SARS-CoV-2 genetic material (∼100 copies/μL RNA) and to compare the cyclic threshold (Ct) values and detection runtime of commercial molecular diagnostic kits and NP-based detection methods with our system using similar amounts of genetic material. [12][13][14][15][16][17][18][19]22,23,35,36 As shown in Figure 4a, the calibration line intersected with the threshold at 2.3 nSLAM cycles, which was ∼1/16th the Ct value of conventional molecular diagnostic tests and significantly shorter than previous NP-based methods. [12][13][14][15][16][17][18][19]22,23 Based on these results, we analyzed E and N2 genes to test whether all target genes of our system shared similar values because the detection of all target genes must be synchronized to reduce the overall runtime.…”

“…The RdRP gene was initially selected as the target for the nSLAM runtime assay. cDNA (1 fM) (∼118 copies/μL RNA) (Supporting Information, Table S3), was used to match the average concentration of the clinically extracted SARS-CoV-2 genetic material (∼100 copies/μL RNA) and to compare the cyclic threshold (Ct) values and detection runtime of commercial molecular diagnostic kits and NP-based detection methods with our system using similar amounts of genetic material. − ,,,, …”

“…NP-based systems have gained much attention and have shown notable results in detecting SARS-CoV-2 genetic material (Table ). − However, the detection performances of these methods possess room for improvement as it is challenging to acquire both high sensitivity and short detection runtime for any single approach. Methods with high sensitivity often involve lengthy procedures, while faster methods generally require greater concentrations of viral genetic material for detection.…”

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

“…The fluorescence and lateral flow assay are so far the most used readout methods in CRISPR/Cas-based detection platforms. Other signal detection methods have also been reported, such as using electrochemical biosensors (E-CRISPR [ 45 , 95 ], PGMs-CRISPR [ 54 ], MOECS [ 120 ]), chemiluminescence enhancement biosensors (CRICED [ 121 ], CLE-CRISPR [ 122 ]), toehold switch-linked colorimetry (NASBACC [ 33 ]), and potentiometry (CRISPR-Chip [ 42 ]). Some CRISPR/Cas-based diagnostic technologies allow for fluorescent signals to be read by the naked eye under blue light (CRISPR-Cas12a-NER [ 48 ], opvCRISPR [ 56 ], CASdetec [ 49 ]) or to be measured with a mobile phone (multiple enhanced CRISPR-Cas13 assay [ 71 ], SHINE [ 69 ]).…”

Evaluation of an automated CRISPR-based diagnostic tool for rapid detection of COVID-19

“…CRISPR-based assays can also detect single-copy targets [ 24 ] and distinguish targets with single base differences [ 25 ] to permit sensitive and accurate mapping of SARS-CoV-2 RNA changes in respiratory and non-respiratory samples. For example, a FnCas9-based CRISPR diagnostic was developed for rapid and accurate detection of major SARS-CoV-2 variants [ 26 ], and a CRISPR-Cas12a-empowered electrochemical biosensor was evaluated for rapid and ultrasensitive detection of the SARS-CoV-2 Delta variant [ 27 ].…”