• CRISPR/Cas
• electrochemical biosensor
• inkjet-printing
• pathogens
• point-of-care

Infectious diseases pose a global threat, and biosensors offer a low-cost alternative to traditional diagnostics. The use of CRISPR/Cas defined new strategies for the detection of nucleic acids. Precise endonuclease activity of this programmable and highly specific system is triggered upon the binding with the complementary target oligonucleotide. This thesis presents a CRISPR/Cas12a-based electrochemical biosensor for bacteria detection developed utilizing low-cost inkjet-printing gold electrodes. The activation of Cas12a collateral activity triggered the non-specific cleavage of a methylene blue (MB)-labelled ssDNA reporter bound to the electrode, disrupting electron transfer and reducing the square wave voltammetry (SWV) signal. A comparative analysis revealed that biosensors fabricated using inkjet-printed gold electrodes outperformed commercial screen-printed ones. The biosensor’s specificity was demonstrated for Escherichia coli and Staphylococcus aureus, and its effectiveness was validated using clinical isolates from real samples. The biosensor was integrated with a microfluidic circuit, a printed heater, and an NFC antenna for smartphone readout. Isothermal amplification was implemented to lower the detection limit, while the Cas12a/gRNA complex was freeze-dried to simplify operations and storage. Overall, this thesis highlights the potential of CRISPR/Cas12a-based electrochemical biosensors for low-cost, portable pathogen detection for decentralized diagnostics.