Development of an Electrochemical Dual H₂S/Ca²⁺ Microsensor and Its In Vivo Application to a Rat Seizure Model

A dual electrochemical microsensor was fabricated for concurrent monitoring of hydrogen sulfide (H₂S) and calcium ions (Ca²⁺), which are closely linked important signaling species involved in various physiological processes. The dual sensor was prepared using a dual recessed electrode consisting of two platinum (Pt) microdisks (50 μm in diameter). Each electrode was individually optimized for the best sensing ability toward a target analyte. One electrode (WE1, amperometric H₂S sensor) was modified with electrodeposition of Au and electropolymerized polyaniline coating. The other electrode (WE2, all-solid-state Ca²⁺-selective electrode) was composed of Ag/AgCl onto the recessed Pt disk formed via electrodeposition/chloridation, followed by silanization and Ca²⁺-selective membrane loading. The current of WE1 and the potential of WE2 in a dual sensor responded linearly to H₂S concentration and logarithm of Ca²⁺ concentration, respectively, without a crosstalk between the sensing signals. Both WE1 and WE2 presented excellent sensitivity, selectivity ($$, i = CO, NO, O₂, NO₂^-, AP, AA, DA, and GABA; and $$, j = Na⁺, K⁺, and Mg²⁺), and fast response time with reasonable stability (during ca. 6 h in vivo experiment). Particularly, WE2 prepared using a mixture of two ionophores (ETH1001 and ETH129) and two plasticizers (2-nitrophenyl octyl ether and bis(2-ethylhexyl) sebacate) showed a very shortened response time (t_R to attain the ΔE/Δt slope of 0.6 mV/min = 3.0 ± 0.2 s, n ≥ 10), a critically required factor for real-time analysis. The developed sensor was utilized for simultaneous real-time monitoring of H₂S and Ca²⁺ changes at the brain cortex surface of a living rat during spontaneous epileptic seizures induced by a cortical 4-aminopyridine injection. The dynamic changes of H₂S and Ca²⁺ were clearly observed in an intimate correlation with the electrophysiological recording of seizures, demonstrating the sensor feasibility of in vivo and real-time simultaneous measurements of H₂S and Ca²⁺.