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.
We developed an axis-mode donor-DNA-acceptor electrochemical system to distinguish whether electron transfer in DNA occurs by tunneling or hopping. In the axismode, rigid stem-loop DNA was designed with the redox probe Ag + embedded at the axis of the strand through a C−Ag + −C mismatch, which was immobilized onto the electrode surface in a saturated manner. Thus, the rotation, swing, and bending of the DNA strand were restricted and then the number of Ag + , the distance L between Ag + and the electrode, and the chemical environment could be precisely controlled. In addition, fast scan cyclic voltammetry was applied to realize the in situ redox reaction of Ag + , without diffusion away from the electrode and the ensuing deconstruction of the stem-loop DNA. In this case, as a direct indicator of rate, the peak Faradaic current i p was extracted and used to fit the tunneling mechanism i ∝ exp (−βL) and the hopping mechanism i ∝ L −η . The value of β was determined to be 0.100 Å −1 , which is consistent with the range of 0.1∼1.5 Å −1 reported previously, while η was determined to be 0.677, which is completely beyond the correct range of 1 ≤ η ≤ 2, demonstrating that electron transfer in DNA occurs by tunneling instead of hopping or that tunneling dominates. Additionally, current additivity and the irrelevance of the base sequence illustrate this point again. Thus, the possibility of independent parallel tunneling currents in DNA strands is revealed, which is helpful for recognizing the feasibility of DNA-based wires and devices.
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