Program-Customized Design of MXene-Supported Circuits with Stalker-Regulated DNA Walker for Multiplexed miRNA Detection

DNA walkers can drive nucleic acids to move along designed tracks or processes in DNA circuits, endowing the circuits with highly efficient signal-amplification capability. However, their actuation and movement are difficult to regulate, and integrating them into circuits for high-throughput detection remains challenging. In this work, we propose a novel DNA nanomachine termed "DNA stalker", which can both actuate and regulate the movement of DNA walker while conferring multiplex detection capability to the entire circuit. Herein, DNA stalker can regulate DNA walker via target-responsive catalytic hairpin assembly, and thereafter be integrated with MXene to fabricate the MXene-supported DNA circuits. These DNA circuits operate in an enzyme-free manner, and the inherent limitations of current DNA walkers are overcome solely through rational nucleic acid sequence design. In addition, program-customized design enables the tailored fabrication of the MXene-supported DNA circuits according to the customer-specified target combinations to be detected, without requiring prior knowledge of nucleic acid sequence design when the detection targets are switching. To meet the requirement of clinic detection, a microfluidic device is employed to operate three circuit sets with independent outputs simultaneously, enabling the concurrent detection of six miRNAs in serum samples from healthy individuals and patients with three different types of cancer (breast, cervical and lung cancer). This system achieves highly sensitive and high-throughput detection of miRNAs in serum, providing a powerful tool for the early clinical diagnosis of cancer at the molecular level.