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. 2022 Aug 15:1221:340120.
doi: 10.1016/j.aca.2022.340120. Epub 2022 Jul 2.

Ultrasensitive SARS-CoV-2 diagnosis by CRISPR-based screen-printed carbon electrode

Lina Wu  1 , Xinjie Wang  2 , Chengyuan Wu  3 , Xizhong Cao  4 , Taishan Tang  4 , He Huang  5 , Xingxu Huang  6
Affiliations

Ultrasensitive SARS-CoV-2 diagnosis by CRISPR-based screen-printed carbon electrode

Lina Wu et al. Anal Chim Acta. .

Abstract

Early and accurate diagnosis of SARS-CoV-2 was crucial for COVID-19 control and urgently required ultra-sensitive and rapid detection methods. CRISPR-based detection systems have great potential for rapid SARS-CoV-2 detection, but detecting ultra-low viral loads remains technically challenging. Here, we report an ultrasensitive CRISPR/Cas12a-based electrochemical detection system with an electrochemical biosensor, dubbed CRISPR-SPCE, in which the CRISPR ssDNA reporter was immobilized onto a screen-printed carbon electrode. Electrochemical signals are detected due to CRISPR cleavage, giving enhanced detection sensitivity. CRISPR-SPCE enables ultrasensitive SARS-CoV-2 detection, reaching as few as 0.27 copies μL-1. Moreover, CRISPR-SPCE is also highly specific and inexpensive, providing a fast and simple SARS-CoV-2 assay.

Keywords: CRISPR/Cas12a; CeO(2) nanorods; Detection; SARS-CoV-2; Screen-printed carbon electrode.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Scheme 1
Scheme 1
Schematic illustration of the functional principle of CRISPR-SPCE.
Fig. 1
Fig. 1
(A) TEM image of PAH dispersed CeO2 nanorods; (B) XPS survey spectrum of the CeO2 nanorods.
Fig. 2
Fig. 2
(A) Cyclic voltammetry of PAH/Fc-labeled ssDNA modified SPCE biosensor (a) and CeO2/PAH/Fc-labeled ssDNA modified SPCE (b); (B) DPV of modified SPCE before (a) and after incubation without target DNA (b), as well as after incubation with target DNA (c); (C) PAGE analysis of the ssDNA trans-cleavage ability of Cas12a. Lane 1: target DNA + Fc-labeled ssDNA; lane 2: Cas12a + target DNA + Fc-labeled ssDNA; lane 3: crRNA + target DNA + Fc-labeled ssDNA; lane 4: Cas12a + crRNA + Fc-labeled ssDNA; lane 5: Cas12a + crRNA + target DNA + Fc-labeled ssDNA.
Fig. 3
Fig. 3
Design of specific crRNAs targeting the E gene for SARS-CoV-2 detection.
Fig. 4
Fig. 4
Optimization of the detection conditions. Evaluation of the effects of (A) CeO2 concentration; (B) PAH concentration; (C) Fc-labeled ssDNA concentration; (D) Incubation time and (E) Mg2+ concentration; (F) Comparison of signal change with Fc-labeled ssDNA of different lengths.
Fig. 5
Fig. 5
(A) DPV with different concentrations of target DNA; (B) The linear calibration curve in the range of 2.0 × 10−8 to 5.0 × 10−5 ng μL−1.
Fig. 6
Fig. 6
The selectivity of the proposed system in the detection of SARS-CoV-2.
See this image and copyright information in PMC

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