A General Biosensing Strategy Based on Cascade Amplification for Enhanced HIV Detection Sensitivity.

Abstract

Objective: Accurate and rapid detection of disease biomarkers is critical for early diagnosis, timely intervention, and effective disease management. Impact Statement: This strategy is exemplified through the development of 2 biosensors for detecting HIV-1 DNA and HIV-1 p24, biomarkers associated with acquired immunodeficiency syndrome (AIDS). Introduction: We propose a general biosensing strategy that leverages a treble signal amplification cascade, demonstrating its versatility and applicability across diverse biomolecular targets. Methods: The method integrates multiple amplification mechanisms to achieve unparalleled sensitivity. Initially, the hybridization of 2 aided probes with the target triggers isothermal amplification facilitated by polymerase and nicking enzyme, providing a robust preliminary signal enhancement. Repeated cycles of primer extension, nicking, and signal primer dissociation then generate multiple signal primers. These primers are further amplified via rolling circle amplification (RCA), resulting in an important secondary signal boost. Finally, the amplified products activate CRISPR-Cas12a-mediated trans-cleavage, achieving a tertiary level of signal enhancement. Results: This cascade amplification approach achieves remarkable sensitivity, with detection limits of 62 aM for nucleic acids and 8.48 pg/ml for proteins, positioning it as a broadly applicable framework. Clinical samples were assayed, which indicates its capability in clinical diagnosis. Conclusion: Beyond HIV detection, the modular design of this strategy allows adaptation for various biomarkers, showcasing its potential as a universal platform for molecular diagnostics in healthcare and research.