CRISPR-Cas12a and G4 DNAzyme platform enables label-free detection of RNA, proteins and metal ions
Investigators developed a universal CRISPR/Cas12a-G4 DNAzyme-TMB biosensing platform that converts Cas12a trans-cleavage activity into an enzymatic TMB-H2O2 signal. In proof-of-concept assays, the system detected SARS-CoV-2 RNA with a limit of detection of 100 aM and achieved an AUC of 0.9420 against clinical samples. The platform also quantified prostate-specific antigen across 0 to 100 ng mL-1 with results concordant with clinical chemiluminescence immunoassays, and measured mercury ions in river water with 93% to 107.4% recovery rates comparable to ICP-MS. The study reports that this label-free, rapid biosensor shows analytical promise for multiplexed clinical biomarker screening and environmental monitoring, though large-scale clinical validation remains needed.
The original study
Universal CRISPR/Cas12a-G4 DNAzyme biosensing platform for rapid detection of nucleic acid and non-nucleic acid targets.
- Authors
- Qin H, Liu J, Luo Y, Yu J, Xiao W, Tang Y, et al.
- Journal
- The Analyst
- Type
- Journal Article
- PMID
- 42677878
Original abstract
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system enables sensitive and specific biomolecular detection due to its programmability, high fidelity, and signal amplification. Herein, a novel universal CRISPR/Cas12a-G4 DNAzyme-TMB (Cas-GT) enzymatic biosensing platform was constructed. This platform regulates the catalytic function of G4 DNAzyme through the trans-cleavage activity of Cas12a, achieving an "off/on" response of the TMB-H2O2 enzymatic signal for nucleic acid, protein, and metal ion targets. In proof-of-concept experiments, the detection limit of Cas-GT for in vitro transcribed SARS-CoV-2 RNA reached as low as 100 aM and it distinguished clinical positive from negative patients with good diagnostic performance (AUC = 0.9420). It is also suitable for protein targets, enabling quantitative analysis of prostate-specific antigen (PSA) within the range of 0-100 ng mL-1, with results highly consistent with clinical chemiluminescence immunoassay (CLIA). For small-molecule targets, Cas-GT exhibited good universality, achieving quantitative detection of Hg2+ within the range of 0.06-4 ng mL-1, with recovery rates of 93%-107.4% in spiked river water samples, showing no significant difference from ICP-MS (P = 0.9968). In summary, Cas-GT is a simple, sensitive, rapid, and label-free enzymatic biosensing platform with significant potential for clinical biomarker detection and environmental pollution monitoring.