Integrating an entropy-driven DNA circuit with a tetrahedral scaffold as a generic in situ electrochemical biosensor for amplified detection of microRNAs

Abstract

Detection of carcinogenesis-related miRNAs presents significant challenges due to their low abundance and high specificity, necessitating highly sensitive and reliable analytical methods. Herein, we propose a generic in situ electrochemical biosensor for the sensitive and effective detection of miRNAs by rationally integrating an entropy-driven DNA circuit (EDC) with a tetrahedral scaffold. The key advancement of this work is the implementation of tetrahedral DNA nanostructures (TDNs) as both a scaffold and substrate for the EDC directly on the electrode surface. TDNs, which are readily decorated with ordered orientation and well-controlled spacing, enhance hybridization efficiency and facilitate essential structural interactions within the EDC, achieving a performance comparable to that of homogeneous liquid-phase reactions. Identifying a target miRNA is achieved with complementary probes that trigger a cascade of structural rearrangements leading to the immobilization of numerous biotin-labeled signal strands on the electrode surface. This accumulation of biotinylated strands ensures that the initial interfacial hybridization event is subsequently amplified and translated into electrochemical signals via cascaded signal amplification. The resulting electrochemical signals are directly proportional to the concentration of the target miRNA, offering a highly sensitive detection platform with a detection limit as low as 74 aM and a dynamic range spanning from 100 aM to 100 pM. The biosensor's performance is validated using biological samples derived from B[a]PDE-exposed cells, where significantly elevated miR-96 levels are detected, consistent with qRT-PCR results. This demonstrates the potential of the proposed biosensor for early cancer diagnosis and monitoring of cancer-related miRNA biomarkers.