An i-Motif Conformational Transition Synergizing with pH-Responsive Reduced Graphene Oxide Field-Effect Transistor for Urease Activity Sensing

Abstract Urease serves as a key diagnostic biomarker, whose abnormal activity is closely associated with oral diseases such as caries and periodontitis. However, detecting trace urease in saliva remains technically challenging. In this work, we report for the first time a C4-rGO-FET biosensor by integrating a conformation-switchable C4-DNA probe with an electrochemically reduced graphene oxide field-effect transistor. The sensing mechanism relies on the pH-induced conformational transition of C4-DNA between a folded i-motif and an unfolded state, which effectively modulates the interfacial charge and thus the carrier distribution in the rGO channel, resulting in a measurable shift in the Dirac point voltage (VCNP). The designed sensor exhibits significantly enhanced pH response (51.57 mV/pH) and reduces the Debye shielding effect. Upon urease-catalyzed hydrolysis of urea, the resulting local pH increase triggers a positive VCNP shift, enabling highly sensitive detection of urease activity. The biosensor demonstrates a wide linear range (1–500 U/L), a low detection limit (0.18 U/L), and good reproducibility. Moreover, it performs reliably in spiked human saliva samples, highlighting its strong potential for point-of-care testing and real-time monitoring in clinical diagnostics.