CRISPR/Cas12a-triggered ordered concatemeric DNA probes signal-on/off multifunctional analytical sensing system for ultrasensitive detection of thalassemia

Abstract

Based on CRISPR/Cas12a triggered ordered concatemeric DNA probes, a “on/off” self-powered biosensor is developed to achieve highly sensitive detection of thalassemia gene CD142 through open-circuit potential-assisted visual signal output. The ingeniously constructed glucose oxidase (GOD)-functionalized ordered concatemeric DNA probe structure can significantly amplify signal output, while the coupled CRISPR/Cas12a system is served as a “signal switch” with excellent signal-transducing capabilities. When the ordered concatemeric DNA probe structure is anchored on electrode, the response signal of the sensing system is in the “signal on” mode. While, the presence of the target activates the non-specific cleavage activity of the CRISPR/Cas12a system, causing the sensing system to switch to the “signal off” mode. In the detection system, GOD catalyzes the oxidation of glucose to produce hydrogen peroxide, which further catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to form a color product, enabling visual signal of the target through naked-eye color contrast. By employing a multifunctional analytical mode combining electrochemical and visual signal outputs, accurate determination of the target is achieved, with linear ranges of 0.0001–100 pM, and detection limits of 48.1 aM (S/N = 3). This work provides a reference method for sensitive detection of thalassemia genes and holds great diagnostic potential in biomedical applications.