A CRISPR/Cas12a-Mediated Dual-Mode Electrochemical Biosensor for Polymerase Chain Reaction-Free Detection of Genetically Modified Soybean

Abstract

A clustered regularly interspaced short palindromic repeats (CRISPR)/ Cas12a-mediated dual-mode electrochemical biosensor without polymerase chain reaction (PCR) amplification was designed for sensitive and reliable detection of genetically modified soybean SHZD32-1. A functionalized composite bionanomaterial Fe 3 O 4 @AuNPs/DNA-Fc&Ru was synthesized as the signal unit, while a characteristic gene fragment of SHZD32-1 was chosen as the target DNA (tDNA). When Cas12a, crRNA, and tDNA were present simultaneously, a ternary complex Cas12a-crRNA-tDNA was formed, and the nonspecific cleavage ability of the CRISPR/Cas12a system toward single-stranded DNA was activated. Thus, the single-stranded DNA-Fc in the signal unit was cleaved, resulting in the decrease in the fast scan voltammetric (FSV) signal from ferrocene (Fc) and the increase in the electrochemiluminescence (ECL) signal from ruthenium complex (Ru) inhibited by Fc. The linear range was 1−10 7 fmol/L for ECL and 10−10 8 fmol/L for FSV, and the limit of detection (LOD) was 0.3 fmol/L for ECL and 3 fmol/L for FSV. Accuracy, precision, stability, selectivity, and reliability were all satisfied. In addition, PCR-free detection could be completed in an hour at room temperature without requiring complicated operation and sample processing, showing great potential in the field detection of genetically modified crops.

Advances in Electrochemical Techniques for the Detection and Analysis of Genetically Modified Organisms: An Analysis Based on Bibliometrics

“…Deng et al [61] used a label-free sensing technology to directly reflect the differences in DNA hybridization with changes in electrode performance. Ge et al [62] recently reported a CRISPR/Cas12a-mediated dual-mode PCR, genosensor, and sequence became burst keywords over the next few years, but they did not last very long. In 2018, amplification and reduced graphene oxide became the new burst keywords until 2021.…”

“…Deng et al [61] used a label-free sensing technology to directly reflect the differences in DNA hybridization with changes in electrode performance. Ge et al [62] recently reported a CRISPR/Cas12a-mediated dual-mode did not last very long. In 2018, amplification and reduced graphene oxide became the new burst keywords until 2021.…”

“…Deng et al [61] used a label-free sensing technology to directly reflect the differences in DNA hybridization with changes in electrode performance. Ge et al [62] recently reported a CRISPR/Cas12a-mediated dual-mode Cluster analysis of keywords can exhibit the research focus formed by different keywords in this topic. Figure 8 shows the clustering results of keywords, with 11 clusters formed based on similarity of content.…”

“…Deng et al [61] used a label-free sensing technology to directly reflect the differences in DNA hybridization with changes in electrode performance. Ge et al [62] recently reported a CRISPR/Cas12a-mediated dual-mode for electrochemical detection of GM soybean. In addition to soybeans, GM rice [63] and E. coli [64] tests are also included in this cluster.…”

Nucleic acid isothermal amplification-based soft nanoarchitectonics as an emerging electrochemical biosensing platform

“…This is advantageous when compared to the above-mentioned ratiometric strategies that also showed two readouts, however, the two readouts are from the same detecting method. For example, electrochemical/ECL dual- mode, 138,139 electrochemical/photoelectrochemical (PET) dualmode, 140,141 and ECL/PET dual-mode 142 sensing strategies have been reported for improved cross-validating detection. Zhou et al combined the electrochemistry method and surface-enhanced Raman scattering (SERS) technology to realize a dual-mode assay for miRNA.…”