Low-cost inkjet-printed nanostructured biosensor based on CRISPR/Cas12a system for pathogen detection

• Published in: Biosensors & bioelectronics 2024-08, Vol.258, p.116340-116340, Article 116340
• Main Authors: Carota, Angela Gilda, Bonini, Andrea, Urban, Massimo, Poma, Noemi, Vivaldi, Federico Maria, Tavanti, Arianna, Rossetti, Marianna, Rosati, Giulio, Merkoçi, Arben, Di Francesco, Fabio
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Biosensing Techniques - instrumentation; CRISPR-Associated Proteins; CRISPR-Cas Systems; CRISPR/Cas12a; DNA, Bacterial - analysis; DNA, Bacterial - genetics; DNA, Single-Stranded - chemistry; Electrochemical biosensing platform; Electrochemical Techniques - methods; Electrodes; Endodeoxyribonucleases; Escherichia coli - genetics; Escherichia coli - isolation & purification; Gold - chemistry; Humans; Inkjet-printed electrodes; Metal Nanoparticles - chemistry; Nanostructures - chemistry; Pathogenic bacteria; Printing; Silver - chemistry; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2024.116340

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. [Display omitted] •Pathogenic bacteria detection by using Cas12a/gRNA and inkjet-printed electrode.•Inkjet-printed nanostructured electrodes outperforms screen-printed ones.•Hairpin-ssDNA-MB with 10 T-loop reporter exhibits better performance.•Cost-effective, easy-to-make inkjet-printed AuNPs electrode.•Cas12a/gRNA and inkjet-printed electrodes: A new potential class of PoC devices.