An electrochemical biosensor for the detection of tuberculosis specific DNA with CRISPR-Cas12a and redox-probe modified oligonucleotide.

BACKGROUND: The development of a robust and accurate point-of-care platform for the detection of tuberculosis (TB) biomarkers is important for disease control. In the current study, the detection principle relies on the shredding of PES-modified non-specific ssDNA (Poly T) in the presence of target DNA IS6110, a reliable biomarker for TB diagnosis by the CRISPR-Cas12a mechanism. Cas protein has great potential in the detection of nucleic acids. RESULTS: Herein, we developed a biosensing platform by utilizing the trans cleavage activity of CRISPR-Cas12a into an electrochemical biosensor. Square wave voltammetry technique is used for the analysis of the fabricated biosensing platform. In the presence of target DNA, the trans cleavage activity is observed by a nonspecific ssDNA substrate, PolyT chain. Various concentration of target DNA is tested on the constructed biosensor, the fabricated biosensor successfully detected TB target DNA by trans cleavage of PES-modified poly T. This novel biosensor was able to detect the target DNA, IS6110 with the limit of detection of 14.5 nM within 60 min by trans-cleavage activity of CRISPR-Cas12a and the results revealed the potential of Cas12a-based biosensors as a diagnostic platform. SIGNIFICANCE: This is the first study reporting the CRISPR-Cas12a-based electrochemical sensor for TB. The developed CRISPR-Cas12a endonuclease-based electrochemical biosensor provides a potentially powerful platform for the accurate detection of Mycobacterium tuberculosis.