Transient potentiometry based d-serine sensor using engineered d-amino acid oxidase showing quasi-direct electron...

d-Serine biosensing has been extensively reported based on enzyme sensors using flavin adenine dinucleotide (FAD) -dependent d-amino acid oxidase (DAAOx), based on the monitoring of hydrogen peroxide generated by the enzymatic reaction, which is affected by dissolved oxygen concentration in the measurement environment in in vivo use. Here we report a novel sensing principle for d-serine, transient potentiometry based d-serine sensor using engineered DAAOx showing quasi-direct electron transfer (DET) property. DAAOx Gly52Val mutant, revealed to possess dye-mediated dehydrogenase activity using artificial synthetic electron acceptors, while its oxidase activity was negligible. The enzyme was immobilized on electrode and was modified with amine-reactive phenazine ethosulfate, resulted an enzyme electrode showing quasi-DET type response. Although OCP based monitoring took more than several minutes to obtain steady state OCP value, the time dependent OCP change monitoring, transient potentiometry, provided rapid and sensitive sensor signals. While dOCP/dt based monitoring was suitable for sensing with longer than 5 s time resolution with d-serine concentration range between 0.5 mM and 5 mM, dOCP/d t based monitoring is suitable for d-serine monitoring with much shorter time resolution (less than 1 s) with high sensitivity with wider dynamic range (20 μM-30 mM). The maximum dOCP/d t was -39.2 ± 2.0 mV/s^1/2, the K_m(app) was 1.9 mM, and the lower limit of detection was 20 μM. In addition, d-serine monitoring was also possible in the artificial cerebrospinal fluid. The transient potentiometry based sensing reported in this study will be further utilized to realize miniaturized, continuous, real-time, in vivo sensor for d-serine monitoring.