CRISPR/Cas12a-powered nanoconfined biosensing platform with hybrid chain reaction cascading guanine nanowire amplification for ultrasensitive dual-mode detection of lipopolysaccharide†

Abstract

Traditional endotoxin detection methods face challenges in sensitivity, interference resistance, and reliability. This study develops a CRISPR/Cas12a-powered nanoconfined biosensing system that integrates mesoporous nanoengineering with a hybrid chain reaction (HCR) cascading guanine nanowire (G-wire) dual amplification strategy for ultrasensitive dual-mode detection of lipopolysaccharide (LPS). By leveraging a vertically ordered mesoporous silica membrane (VMSM) as a molecular sieve and CRISPR trans-cleavage activity modulator, the system achieves precise regulation of Ru(bpy)₃²⁺ adsorption via LPS-suppressed HCR assembly. This architecture enables physical confinement-mediated electrochemiluminescence (ECL) and fluorescence (FL) signal transduction, with dual-mode outputs providing mutual validation for enhanced reliability. The biosensor exhibits superior sensitivity with detection limits of 3.4 pg mL⁻¹ for ECL and 1.4 pg mL⁻¹ for FL, while also offering a broad dynamic range (0.005–100 ng mL⁻¹), significantly outperforming conventional LPS assays. The CRISPR-triggered HCR cascading G-wire dual amplification synergizes with nanoconfinement of VMSM to ensure robust anti-interference performance in complex matrices, validated by recovery rates of 97.8–102.5% in real samples. By integrating CRISPR programmability with nanoengineered signal amplification, this work establishes a transformative paradigm for portable, high-precision endotoxin detection in clinical diagnostics, industrial monitoring, and environmental safety applications.