Dimeric DNA Tetrahedron-Incorporated Nanopipette: Split Aptamer-Mediated Signal Amplification for Ultrasensitive...

Solid-state nanopore electroanalysis enables single-molecule resolution while maintaining multiple advantages of ultrahigh sensitivity, label-free operation, and high-throughput applications. However, current solid-state nanopore configurations fail to capture detectable signals during the translocation of subnanoscale small molecules (SMs) due to the dimensional incompatibility, rapid transit dynamics, and inherent signal-to-noise constraint. Herein, a novel sensing strategy utilizing the DNA tetrahedron (DTN) as an amplifier was proposed for nanopipette-based small-molecule detection. A pair of split aptamers (SApts) highly specific to the target SM was separately integrated on one vertex of DTN, yielding the conjugate DTN-SApts. Upon binding to SM targets, the SApts reassembled to trigger the formation of dimeric DTN (D-DTN). Compared to monomeric DTN-SApts, these target-bound D-DTN generated substantially enhanced resistive pulse sensing (RPS) signals during translocation through an ∼50 nm nanopipette. By leveraging this size-amplified signal transduction mechanism, the DTN-incorporated nanopipette emerges as a powerful platform for highly sensitive detection of SMs. Specifically, for ATP analysis, this approach exhibits exceptional analytical performances, featuring an ultralow limit of detection (0.24 nM), high selectivity, and a superior signal-to-noise ratio (SNR). Furthermore, we employed this platform to investigate the interaction kinetics between DTN-SApts and ATP and to validate its practical applicability by quantifying ATP in serum samples. These promising results motivate us to leverage the synergistic DTN-nanopipette system for detecting various small molecules or metal ions, including vancomycin and Hg²⁺. Such a DTN-incorporated nanopipette amplifier holds great promise for developing high-precision diagnostic platforms targeting clinically molecules.