Low-cost inkjet-printed nanostructured biosensor based on CRISPR/Cas12a system for pathogen detection
Carota, Angela Gilda (University of Pisa. Department of Chemistry and Industrial Chemistry)
Bonini, A (University of Pisa. Department of Biology)
Urban, Massimo (Institut Català de Nanociència i Nanotecnologia)
Poma, Noemi (University of Pisa. Department of Biology)
Vivaldi, Federico Maria (University of Pisa. Department of Chemistry and Industrial Chemistry)
Tavanti, Arianna (University of Pisa. Department of Biology)
Rossetti, Marianna (Institut Català de Nanociència i Nanotecnologia)
Rosati, Giulio (Institut Català de Nanociència i Nanotecnologia)
Merkoçi, Arben (Institut Català de Nanociència i Nanotecnologia)
Di Francesco, Fabio (University of Pisa. Department of Chemistry and Industrial Chemistry)

Data:	2024
Resum:	The escalating global incidence of infectious diseases caused by pathogenic bacteria, especially in developing countries, emphasises the urgent need for rapid and portable pathogen detection devices. This study introduces a sensitive and specific electrochemical biosensing platform utilising cost-effective electrodes fabricated by inkjet-printing gold and silver nanoparticles on a plastic substrate. The biosensor exploits the CRISPR/Cas12a system for detecting a specific DNA sequence selected from the genome of the target pathogen. Upon detection, the trans-activity of Cas12a/gRNA is triggered, leading to the cleavage of rationally designed single-strand DNA reporters (linear and hairpin) labelled with methylene blue (ssDNA-MB) and bound to the electrode surface. In principle, this sensing mechanism can be adapted to any bacterium by choosing a proper guide RNA to target a specific sequence of its DNA. The biosensor's performance was assessed for two representative pathogens (a Gram-negative, Escherichia coli, and a Gram-positive, Staphylococcus aureus), and results obtained with inkjet-printed gold electrodes were compared with those obtained by commercial screen-printed gold electrodes. Our results show that the use of inkjet-printed nanostructured gold electrodes, which provide a large surface area, in combination with the use of hairpin reporters containing a poly-T loop can increase the sensitivity of the assay corresponding to a signal variation of 86%. DNA targets amplified from various clinically isolated bacteria, have been tested and demonstrate the potential of the proposed platform for point-of-need applications.
Ajuts:	European Commission 101008701 European Commission 825694 Agencia Estatal de Investigación CEX 2021-001214-S European Commission 101029884 Agència de Gestió d'Ajuts Universitaris i de Recerca 2021/SGR-01464
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, i la comunicació pública de l'obra, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original. No es permet la creació d'obres derivades.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	CRISPR/Cas12a ; Electrochemical biosensing platform ; Inkjet-printed electrodes ; Pathogenic bacteria
Publicat a:	Biosensors & bioelectronics, Vol. 258 (April 2024) , art. 116340, ISSN 1873-4235

DOI: 10.1016/j.bios.2024.116340

11 p, 4.7 MB

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