Constructing a universal detection platform based on TdT-Cas12a autocycle amplification: From heavy metal ions to pathogenic RNA

We reported a TdT combined with CRISPR/Cas12a driven cascade amplification platform for the sensitive detection of both heavy metal ions and viral targets. • Integrated with reverse fluorescence-enhanced lateral flow strip for visual readout, this platform achieves LODs of 2.5 × 10 −15  M for Pb 2+ and 3.6 × 10 −16  M for DENV-RNA. • The modular design allows flexible replacement of recognition modules, offering a universal solution for on-site rapid monitoring of diverse pollutants in complex environmental matrices. Currently, constructing flexible detection platforms capable of identifying diverse hazardous substances remains a significant challenge. Conventional detection methods are limited by target specificity and lack cross-class universality. Herein, we developed a general platform for sensitive detection of lead ions (Pb 2+ ) and dengue virus (DENV) as two distinct model targets. This platform specifically converted different types of targets into detectable nucleic acid molecules, which would be further recognized and signal amplified by the cascade cycling isothermal amplification system composed of terminal deoxynucleotidyl transferase (TdT) and CRISPR/Cas12a system. TdT has the property of extending the 3′ end of DNA molecule in a template-independent manner, providing long poly-T nucleic acid sequence for binding lots of crRNA, so as to activate the trans-cleavage activity of Cas12a to cleave the hairpin DNA reporters with exposing numerous novel 3′ ends for the same cycle. By combining this strategy with the reverse fluorescence-enhanced lateral flow test strip, the platform achieves a limit of detection (LOD) of 2.5 × 10 −15  M and 3.6 × 10 −16  M for Pb 2+ and DENV-RNA, respectively. Moreover, the detection platform for Pb 2+ in environmental water samples showed excellent agreement with standard methods. It also demonstrated high sensitivity for DENV-RNA detection in complex samples, confirming its reliability and broad applicability potential in practical scenarios. Download: Download high-res image (103KB) Download: Download full-size image