S9.6 Antibody-Mediated Bivalent Capture Biosensor Using Vacuum-Assembled HCR Amplifier for One-Pot, Record-Fast and Ultrasensitive Detection of Nucleic Acid.

Electrochemical biosensors utilizing hybridization chain reaction (E-HCR) have witnessed substantial advancement over the past two decades, yet achieving simultaneous rapid and ultrasensitive detection remains a challenge with current strategies. Herein, we report a portable, wireless E-HCR biosensor that leverages S9.6 antibody-mediated bivalent capture for ultrasensitive nucleic acid detection, achieving a record-fast assay completion time. The detection mechanism involves target nucleic acid-triggered opening of a hairpin probe, followed by hybridization with a preassembled HCR/HRP amplifier. The resulting target/hairpin/HCR/HRP complex contains two segments of DNA/RNA heteroduplex, enabling efficient capture by an S9.6 antibody-modified screen-printed carbon electrode (SPCE) through bivalent S9.6 antibody-heteroduplex interactions. The bivalent capture strategy demonstrates a 1.6-fold enhancement over single-site S9.6 antibody-heteroduplex binding and a 3.1-fold improvement in capture efficiency compared to monovalent hybridization. This one-pot strategy offers three unique advantages. First, the integration of bivalent capture, homogeneous hybridization, and preformed HCR/HRP amplifiers enables the heterogeneous E-HCR assay to be completed within 34 min, significantly faster than conventional methods. Second, optimization of HCR amplifier and background signal suppression achieves a high signal-to-noise ratio, facilitating ultrasensitive nucleic acid detection. Third, the biosensor features wireless signal output and utilizes low-cost SPCE, making it suitable for point-of-care applications. Collectively, these unique merits enable the bivalent capture biosensor to achieve portable, one-pot, rapid, and ultrasensitive nucleic acid detection, addressing limitations in current E-HCR biosensing platforms.