Exploring a CRISPR/Cas12a-powered impedimetric biosensor for amplification-free detection of a pathogenic bacterial DNA☆

Author(s)

Wachholz Junior, Dagwin ; Pontes, Rafael Goncalves ; Hryniewicz, Bruna M. ; Kubota, Lauro Tatsuo

• Document type: Journal article
• Source: BIOSENSORS & BIOELECTRONICS; v. 285, p. 9-pg., 2025-10-01.

Abstract

Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCRbased methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/ Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a ' s target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria. (AU)

• FAPESP's process: 21/09706-0 - Development of a CRISPR/Cas-based electrochemical biosensor for T-Cell Leukemia virus (HTLV-1) and Staphylococcus Aureus bacteria detection with point-of-care devices
• Grantee: Dagwin Wachholz Júnior
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 13/22127-2 - Development of novel materials strategic for integrated analytical devices
• Grantee: Lauro Tatsuo Kubota
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/00166-2 - Paper-Based Electrochemical Biosensor for Traumatic Brain Injury detection
• Grantee: Patrícia Batista Deroco
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 23/09451-7 - Development of CRISPR-based biosensors for detection of disease-related genes
• Grantee: Bruna Maria Hryniewicz
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/50867-3 - INCT 2014: National Institute of Science and Technology in Bioanalysis
• Grantee: Marco Aurelio Zezzi Arruda
• Support Opportunities: Research Projects - Thematic Grants