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

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: BIOSENSORS & BIOELECTRONICS; v. 285, p. 9-pg., 2025-10-01.

Resumo

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)

• Processo FAPESP: 21/09706-0 - Desenvolvimento de biossensor eletroquímico baseado na tecnologia CRISPR/Cas para detecção do vírus causador da Leucemia de Células T (HTLV-1) e da bactéria Staphylococcus aureus a partir de dispositivos point-of-care
• Beneficiário: Dagwin Wachholz Júnior
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/22127-2 - Desenvolvimento de novos materiais estratégicos para dispositivos analíticos integrados
• Beneficiário: Lauro Tatsuo Kubota
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 19/00166-2 - Biossensor eletroquímico a base de papel para detecção do Traumatismo Cranioencefálico
• Beneficiário: Patrícia Batista Deroco
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 23/09451-7 - Desenvolvimento de biossensores baseado na tecnologia CRISPR para detecção de genes característico de doenças
• Beneficiário: Bruna Maria Hryniewicz
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 14/50867-3 - INCT 2014: Instituto Nacional de Ciência e Tecnologia de Bioanalítica
• Beneficiário: Marco Aurelio Zezzi Arruda
• Modalidade de apoio: Auxílio à Pesquisa - Temático