Lead (Pb²⁺) pollution is a serious food safety issue, rapid detection of Pb²⁺ residual in food is vital to guarantee food quality and safety. Here we proposed ratiometric aptamer probes, allowing robust Pb²⁺ supervision in food samples. Pb²⁺ specific aptamer can bolster a transition of G-quadruplex structural response to Pb²⁺; this process can be monitored by N-methyl mesoporphyrin IX (NMM), which is highly specific to G-quadruplex. Particularly, the utilization of G-quadruplex specific dye and terminal-labeled fluorophore allowed to endue ratiometric signal outputs towards Pb²⁺, dramatically increase the robustness for lead detection. The ratiometric G-quadruplex assay allowed a facile and one-pot Pb²⁺ detection at room temperature using a single-stranded DNA aptamer. We demonstrated its feasibility for detecting lead pollution in fresh eggs and tap water samples. The ratiometric G-quadruplex design is expected to be used for on-site Pb²⁺ testing associated with food safety.

Pathogenic infections remain the primary threat for human health, especially the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutations, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we first describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present the diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges such as the development of preamplification-free diagnostic assays and present the emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics.