Ultrasensitive dual-mode biosensor for photoelectrochemical and differential pulse voltammetry detection of thrombin based on DNA self-assembly

Abnormal levels of thrombin may be associated with various diseases, such as thrombosis and hemorrhagic diseases, making precise detection of thrombin particularly important. Dual signal detection is a method that enhances detection sensitivity and specificity by simultaneously utilizing two different signals. Its primary advantages include improving detection accuracy and reducing false positive rates, making it particularly suitable for clinical analysis and diagnostics. In this work, we developed a dual signal detection method for thrombin based on DNA self-assembly. This design incorporates an X-DNA structure. The two bottom arms of the X-shaped DNA (X-DNA) are designed to bind to CuInS₂ nanoparticles via dehydration reactions between amine and carboxyl groups. The two top arms of the X-DNA are designed to hybridize with complementary DNA/glucose oxidase (GOx) and DNA/ferrocene (Fc), respectively. Thrombin triggers the hybridization of DNA/GOx and X-DNA, simultaneously causing the dissociation of DNA/Fc from X-DNA. In the Photoelectrochemical mode, GOx can react with O₂ and glucose in the detection solution, resulting in a corresponding decrease in the amount of O₂ acting as an electron acceptor and a decrease in the photoelectric signal. In the Differential Pulse Voltammetry mode, due to the decrease in Fc content, the DPV signal also shows a weakening trend. The detection method exhibits a good linear relationship within the range of 10 fM −10 nM, with a detection limit of 6.89 fM and 5.86 fM. The enhanced analytical sensitivity and specificity of dual signal detection technology offer broad prospects for improving disease diagnosis.