Universal smartphone-assisted label-free CRISPR/Cas12a-DNAzyme chemiluminescence biosensing platform for on-site detection of nucleic acid and non-nucleic acid targets.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) system enables sensitive and specific detection of biomolecules, thanks to its programmability, high fidelity, and powerful signal amplification capabilities. Herein, a universal smartphone-assisted label-free G-quadruplex (G4) DNAzyme-based chemiluminescence CRISPR/Cas12a biosensing platform (G4CLCas) is firstly described that achieves on-site, ultrasensitive visual detection of nucleic acid and non-nucleic acid targets. The G4CLCas-based sensing platform relies on Cas12a trans-cleavage activation that triggers the cleavage of the G4 DNAzyme, resulting in chemiluminescence signals off/on compared to that of the control. Chemiluminescence signals are captured as images that are quantitatively analyzed and visualized using a smartphone-assisted imaging cartridge. Under optimal conditions, G4CLCas achieves a low limit of detection (LOD) of 8.6 aM (∼5.2 copies/μL) for monkeypox virus (MPXV) DNA within the linear concentration range of 10-300 aM and can accurately quantify viral DNA in spiked samples. G4CLCas can also detect non-nucleic acid targets, whereby it achieves a low LOD value of 84.3 nM for adenosine triphosphate (ATP) within the linear concentration range of 2-2000 μM. Here, a label-free, portable, on-site CRISPR/Cas12a chemiluminescence biosensing platform based on the G4 DNAzyme substrates is proposed with potential applications in clinical detection and bioanalytical chemistry research.