Lipidic Nanoclusters-Assisted Spatial Confinement Signal Amplification for Ratiometric MicroRNA Sensing

MicroRNAs (miRNAs) have served as crucial tumor markers in the identification of cancer. miRNA biosensors tend to be reliable, sensitive, specific, and efficient, especially in complex biological samples and clinical samples. Catalytic hairpin assembly (CHA) has successfully offered the prospect of efficiently amplifying the signal based on recycling of the target sequences. However, the traditional CHA amplifier includes dissociative DNA probes based on free diffusion and random collisions, which leads to delayed reaction kinetics, inadequate amplification, and false-positive signals. Herein, we developed an improved CHA amplifier by installing the CHA probes on a ratiometric fluorescence-based 3D lipidic nanocluster (RF-LNC), enabling sensitive detection of miRNA-21 in a complex environment with accelerated reaction kinetics. The RF-LNC is a lipidic micelle that is decorated with hairpin probes f-HP2 (modified with Cy5, f-HP2-Cy5) and f-HP1 (modified with Cy3, f-HP1-Cy3). Once exposed to the target miR-21, the two-hairpin probe-based CHA can be activated. Then, self-powered strand displacement precession can light up the lipidic nanoclusters (LNCs), generating amplified fluorescence signals finally. We demonstrate that the RF-LNC system is endowed with some outstanding features such as enhanced detection sensitivity and reaction kinetics and shows excellent detection accuracy by minimizing hybridization events with the disappearance of the target. In addition, the simple yet robust system exhibits an improved nuclease resistance owing to all termini of the DNA sequences being hidden from degrading enzyme (nuclease) attack, making the system suitable for operating under a complex environment.