Proximity hybridization induced molecular machine for signal-on electrochemical detection of α-synuclein oligomers.

α-synuclein oligomer is a marker of Parkinson's disease. The traditional enzyme-linked immunosorbent assay for α-synuclein oligomer detection is not conducive to large-scale application due to its time-consuming, high cost and poor stability. Recently, DNA-based biosensors have been increasingly used in the detection of disease markers due to their high sensitivity, simplicity and low cost. In this study, based on the DNAzyme-driven DNA bipedal walking method, we developed a signal-on electrochemical sensor for the detection of α-syn oligomers. Bipedal DNA walkers have a larger walking area and faster walking kinetics, providing higher amplification efficiency compared to conventional DNA walkers. The DNA walker is driven via an Mg2+-dependent DNAzyme, and the binding-induced DNA walker will continuously clamp the MB, resulting in the proliferation of Fc confined near the GE surface. The linear range and limit of detection were 1 fg/mL to 10 pg/mL and 0.57 fg/mL, respectively. The proposed signal-on electrochemical sensing strategy is more selective. It will play a significant role in the sensitive and precise electrochemical analysis of other proteins.

Electrochemical biosensing for E.coli detection based on triple helix DNA inhibition of CRISPR/Cas12a cleavage activity.

BACKGROUND: Escherichia coli (E.coli) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract, posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors. Therefore, the development of a simple and sensitive method for analyzing E.coli would make significant contributions to human health and food safety. RESULTS: We constructed an electrochemical biosensor based on triple-helical DNA and entropy-driven amplification reaction (EDC) to inhibit the cleavage activity of Cas12a, enabling high-specificity detection of E.coli. Replacing antibodies with nucleic acid aptamers (Apt) as recognition elements, we utilized the triple-helical DNA generated by the binding of DNA2 and DNA5/DNA6 double-helical DNA through the entropy-driven amplification reaction to inhibit the collateral cleavage activity of clustered regularly interspaced short palindromic repeats gene editing system (CRISPR) and its associated proteins (Cas). By converting E.coli into electrical signals and recording signal changes in the form of square wave voltammetry (SWV), rapid detection of E.coli was achieved. Optimization of experimental conditions and data detection under the optimal conditions provided high sensitivity, low detection limits, and high specificity. SIGNIFICANCE: With a minimal detection limit of 5.02 CFU/mL and a linear range of 1 × 102 - 1 × 107 CFU/mL, the suggested approach was successfully verified to analyze E.coli at various concentrations. Additionally, after examining E.coli samples from pure water and pure milk, the recoveries ranged between 95.76 and 101.20%, demonstrating the method's applicability. Additionally, it provides a feasible research direction for the detection of pathogenic bacteria causing other diseases using the CRISPR/Cas gene editing system.