Enzyme-based E-RNA sensor array with a hairpin probe: Specific detection of gene mutation

Abstract

An enzyme-based E-RNA sensor for the sequence-specific detection of nucleic acids was reported. The RNA sensor employed a “stem-loop” DNA probe which was immobilized on a 16-unit gold array surface via pre-labeled with thiol group (–SH), and pre-labeled biotin serves as an affinity tag for the enzyme (avidin-horseradish peroxidase) binding. The stem-loop structure of the sensor probes kept the biotin from being approached by a bulky avidin-horseradish peroxidase (avidin-HRP) by means of the steric hindrance. However, the probes would undergo a significant conformational change after DNA/RNA hybridization by forcing biotin away from the electrode, resulting the avidin-HRP access to the labeled biotin. Thus, target DNA/RNA hybridization event could be sensitively transduced via the enzymatically amplified electrochemical voltage and current signals, which were then measured by means of cyclic voltammetry and amperometric detection. The results showed that a single-base mismatch in target RNA could be selectively detected by the E-RNA sensor array, indicating that this E-RNA sensing strategy based on enzyme amplification is versatile with great potential in the construction of stem-loop based biosensors for the detection of nucleic acid mutations.