• bacteria
• biosensor
• Cas12a
• CRISPR
• detection
• EIS
• impedance
• sepsis

In this work, CRISPR/Cas system capability to recognize and cleave specific DNA sequences is exploited to detect the presence of bacterial DNA in a sample. Two common pathogens involved in health care associated infections and sepsis (Gram negative Escherichia coli and Gram positive Staphylococcus aureus) were chosen as a proof of concept, but the system could be easily expanded to include several human pathogens, including viruses. For each microorganism, a species-specific gene was selected and amplified by PCR. We decided to use Cas12a as the biological part of the sensor, since it is a class 2 type V effector of CRISPR/Cas system, which has a collateral aspecific single strand DNase activity triggered by double strand DNA target recognition mediated by a specific guide RNA. This activity was first tested in polyacrylamide gel electrophoresis (PAGE) using a 50 nucleotide linear reporter, confirming the ability of the Cas12a/crRNA complex to selectively recognize the desired target only. Meanwhile, gold electrodes were functionalized with a mixed self-assembled monolayer of 6-mercaptohexanol and HS-(CH2)6-linker modified 20 nucleotides linear ssDNA. Electrochemical impedance spectroscopy (EIS) signal was recorded before and after the reporter cleavage performed by the collateral activity of a Cas12a/sgRNA/activator complex, revealing a significant difference in the charge transfer resistance parameter. Moreover, validation of the system’s ability to correctly detect a panel of clinical E. coli and S. aureus isolates of different origin was performed to provide information on the reproducibility of the developed system.