A CRISPR/Cas12a-Mediated Dual-Mode Electrochemical Biosensor for Polymerase Chain Reaction-Free Detection of Genetically Modified Soybean.
Haoran GeXiaofu WangJunfeng XuHan LinHuiqian ZhouTingting HaoYangbo WuZhiyong GuoPublished in: Analytical chemistry (2021)
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 Fe3O4@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 1-107 fmol/L for ECL and 10-108 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.
Keyphrases
- crispr cas
- label free
- genome editing
- real time pcr
- loop mediated isothermal amplification
- circulating tumor
- nucleic acid
- gold nanoparticles
- room temperature
- cell free
- single molecule
- sensitive detection
- computed tomography
- ionic liquid
- molecularly imprinted
- high resolution
- genome wide
- risk assessment
- transcription factor
- circulating tumor cells
- human health
- energy transfer