This paper reports the fabrication of an insertable amperometric dual microsensor and its application for the simultaneous and fast sensing of NO and CO during acutely induced seizures of living rat brain cortex. NO and CO are important signaling mediators, controlling cerebrovascular tone. The dual NO/CO sensor is prepared based on a dual microelectrode having Au-deposited Pt microdisk (WE1, 76 μm diameter) and Pt black-deposited Pt disk (WE2, 50 μm diameter). The different deposited metals for WE1 and WE2 allow the selective anodic detection of CO at WE1 (+0.2 V vs Ag/AgCl) and that of NO at WE2 (+0.75 V vs Ag/AgCl) with sufficient sensitivity. Fluorinated xerogel coating on this dual electrode provides exclusive selectivity over common biological interferents, along with fast response time. The miniaturized size (end plane diameter < 300 μm) and tapered needle-like sensor geometry make the sensor become insertable into biological tissues. The sensor is applied to simultaneously monitor dynamic changes of NO and CO levels in a living rat brain under acute seizure condition induced by 4-aminopyridine in cortical tissue near the area of seizure induction. In-tissue measurement shows clearly defined patterns of NO/CO changes, directly correlated with observed LFP signal. Current study verifies the feasibility of a newly developed NO/CO dual sensor for real-time fast monitoring of intimately connected NO and CO dynamics.

In this work, we developed a dual amperometric/potentiometric microsensor for sensing nitric oxide (NO) and potassium ion (K(+)). The dual NO/K(+) sensor was prepared based on a dual recessed electrode possessing Pt (diameter, 50 μm) and Ag (diameter, 76.2 μm) microdisks. The Pt disk surface (WE1) was modified with electroplatinization and the following coating with fluorinated xerogel; and the Ag disk surface (WE2) was oxidized to AgCl on which K(+) ion selective membrane was loaded subsequent to the silanization. WE1 and WE2 of a dual microsensor were used for amperometric sensing of NO (106 ± 28 pA μM(-1), n = 10, at +0.85 V applied vs Ag/AgCl) and for potentiometric sensing of K(+) (51.6 ± 1.9 mV pK(-1), n = 10), respectively, with high sensitivity. In addition, the sensor showed good selectivity over common biological interferents, sufficiently fast response time and relevant stability (within 6 h in vivo experiment). The sensor had a small dimension (end plane diameter, 428 ± 97 μm, n = 20) and needle-like sharp geometry which allowed the sensor to be inserted in biological tissues. Taking advantage of this insertability, the sensor was applied for the simultaneous monitoring of NO and K(+) changes in a living rat brain cortex at a depth of 1.19 ± 0.039 mm and near the spontaneous epileptic seizure focus. The seizures were induced with 4-aminopyridine injection onto the rat brain cortex. NO and K(+) levels were dynamically changed in clear correlation with the electrophysiological recording of seizures. This indicates that the dual NO/K(+) sensor's measurements well reflect membrane potential changes of neurons and associated cellular components of neurovascular coupling. The newly developed NO/K(+) dual microsensor showed the feasibility of real-time fast monitoring of dynamic changes of closely linked NO and K(+) in vivo.

This study reports real-time, in vivo functional measurement of nitric oxide (NO) and carbon monoxide (CO), two gaseous mediators in controlling cerebral blood flow. A dual electrochemical NO/CO microsensor enables us to probe the complex relationship between NO and CO in regulating cerebrovascular tone. Utilizing this dual sensor, we monitor in vivo change of NO and CO simultaneously during direct epidural electrical stimulation of a living rat brain cortex. Both NO and CO respond quickly to meet physiological needs. The neural system instantaneously increases the released amounts of NO and CO to compensate the abrupt, yet transient hypoxia that results from epidural electrical stimulation. Intrinsic-signal optical imaging confirms that direct electrical stimulation elicits robust, dynamic changes in cerebral blood flow, which must accompany NO and CO signaling. The addition of l-arginine (a substrate for NO synthase, NOS) results in increased NO generation and decreased CO production compared to control stimulation. On the other hand, application of the NOS inhibitor, l-N(G)-nitroarginine methyl ester (l-NAME), results in decreased NO release but increased CO production of greater magnitude. This observation suggests that the interaction between NO and CO release is likely not linear and yet, they are tightly linked vasodilators.

In this paper, we report the fabrication of a dual microsensor for sensing nitric oxide (NO) and calcium ions (Ca(2+)) and its application for simultaneous NO/Ca(2+) measurements in living rat kidney tissue. NO and Ca(2+) have very important physiological functions and are both intricately involved in many biological processes. The dual NO/Ca(2+) sensor is prepared based on a dual recessed electrode possessing Pt (diameter, 25 μm) and Ag (diameter, 76 μm) microdisks. The Pt disk surface (WE1) is electrodeposited with porous Pt black and then coated with fluorinated xerogel; and used for amperometric sensing of NO. The Ag disk surface (WE2) is chloridated to AgCl, followed by silanization and then Ca(2+) selective membrane loading; and used for potentiometric sensing of Ca(2+). The dual sensor exhibits high sensitivity of WE1 to NO (40.8 ± 6.5 pA μM(-1), n = 10) and reliable Nerntian response of WE2 to Ca(2+) changes (25.7 ± 0.5 mV pCa(-1), n = 10) with excellent selectivity to only NO and Ca(2+) over common interferents and reliable stability (up to ∼4 h tissue experiment). The prepared sensor is employed for real-time monitoring of the dynamic changes of NO and Ca(2+) levels of a rat kidney, which is induced by the administration of 10 mM l-N(G)-nitroarginine methyl ester (l-NAME, a NO synthase inhibitor). Due to the small sensor dimension, location-dependent analyses of NO and Ca(2+) are carried out at two different regions of a kidney (renal medulla and cortex). Higher NO and Ca(2+) levels are observed at the medulla than at the cortex. This study verifies the feasibility for real-time monitoring of intimately connected Ca(2+) and endogenous NO production; and also for localized concentration assessments of both NO and Ca(2+).