Triple Amperometric Microsensor for Simultaneous Real-Time In Vivo Monitoring of NO, CO, and H ₂ S During Epileptic Seizures

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S) are endogenous gaseous signaling molecules, playing crucial roles in a wide range of biological processes. This study presents the fabrication and evaluation of a triple amperometric microsensor capable of the simultaneous and selective detection of NO, CO, and H₂S for in vivo applications. The sensor integrates three platinum working electrodes into a single device. Each electrode was independently modified with specific metal deposits and selective membranes optimized to enhance specificity toward its target analyte. The sensor's excellent performance in terms of sensitivity, linearity, and selectivity was confirmed. To demonstrate in vivo applicability, the sensor was positioned on the cortical surface of a living rat brain, where it successfully monitored real-time concentration changes of NO, CO, and H₂S during seizure events induced by 4-aminopyridine: NO exhibited the earliest concentration increase, followed sequentially by CO and H₂S, providing the first temporally resolved evidence of in vivo gasotransmitter crosstalk during neural hyperactivity. This work establishes a powerful electrochemical platform for probing multigasotransmitter dynamics in real time.