Dual electrochemical microsensor for simultaneous measurements of nitric oxide and oxygen: Fabrication and characterization

A planar-type amperometric dual microsensor was developed for the simultaneous measurement of the nitric oxide (NO) and oxygen (O₂) concentrations. The sensor (overall diameter = 500 μm) consisted of a dual working electrode (WE) containing two platinized platinum microdisks (25 μm diameter, WE1, WE2, distance between two disks > 330 μm) and a Ag/AgCl wire reference electrode covered with an expanded poly(tetrafluoroethylene) gas-permeable membrane. The differentiation and concurrent measurements of NO and O₂ were obtained successfully using two sensing WEs with different applied potentials (+0.75 V for WE1 and −0.4 V for WE2). Cross-talk between WE1 and WE2 was eliminated with an optimized internal solution composition. Linear dynamic range, selectivity, sensitivity, detection limit (<5 nM for NO; <500 nM for O₂), and stability (>50 h) were evaluated.     

Bifunctional sensor of nitric oxide and oxygen based on hematite nanotubes embedded in chitosan matrix

A novel hybrid bifunctional sensing platform for simultaneous determination of NO and O₂ has been developed, whereby hematite nanotubes are immobilized into the chitosan matrix onto a gold electrode (labeled as HeNTs-Chi/Au). The HeNTs distributed in porous-structured chitosan matrix not only offer abundant active sites for bifunctional sensing of NO and O₂, but also facilitate oxidation of NO and reduction of O₂ dramatically. Straight calibration curves are achieved in analyte concentration ranges of 5.0 × 10⁻⁸ to 1.25 × 10⁻⁶ mol L⁻¹ for NO and 2.5 × 10⁻⁷ to 6.0 × 10⁻⁶ mol L⁻¹ for O₂. Also, the detection limits are low of 8.0 × 10⁻⁹ mol L⁻¹ for NO and 5.0 × 10⁻⁸ mol L⁻¹ for O₂. Such an efficient bifunctional sensor for NO and O₂ offers great potential in quantitation of NO levels in biological and medical systems, since NO level is highly regulated by various reactive oxygen species.     

Functionalised electrode array for the detection of nitric oxide released by endothelial cells using different NO-sensing chemistries

In a preliminary study aimed at developing strategies for the simultaneous detection of various biologically important molecules, a procedure is described that allows the electrochemical detection of nitric oxide (NO) released by a population of human umbilical vein endothelial cells (HUVEC) by using an array of electrodes comprising three individually addressable electrodes. Each electrode in the array was modified with a different NO-sensitive electrocatalyst, thereby demonstrating the possibility of modifying the individual electrodes in an array with different sensing chemistries. This study opens a doorway to the development of arrays of electrodes for the simultaneous detection of multiple analytes in a complex environment by suitably tailoring the sensitivity and selectivity of each electrode in the array to a specific analyte in the test medium.     

Simultaneous,real-time measurement of nitric oxide and oxygen dynamics during cardiac ischemia-reperfusion of the rat utilizing sol-gel-derived electrochemical microsensors

In this study, we simultaneously measured nitric oxide (NO) and oxygen (O₂) dynamics in the myocardium during myocardial ischemia-reperfusion (IR) utilizing sol-gel modified electrochemical NO and O₂ microsensors. In addition, we attempted to clarify the correlation between NO release in the ischemic period and O₂ restoration in the myocardium after reperfusion, comparing a control heart with a remote ischemic preconditioning (RIPC)-treated heart as an attractive strategy for myocardial protection. Rat hearts were randomly divided into two groups: a control group (n = 5) and an RIPC group (n = 5, with RIPC treatment). Myocardia that underwent RIPC treatment (182 ± 70 nM, p < 0.05) released more NO during the ischemic period than those of the control group (63 ± 41 nM). The restoration value of oxygen tension (pO₂) in the RIPC group significantly increased and was restored to pre-ischemic levels (92.6 ± 36.8%); however, the pO₂ of the control group did not increase throughout the reperfusion period (5.7 ± 7.5%, p = 0.001). Myocardial infarct size measurements revealed a significant decrease in cell death in the myocardium region of the RIPC group (41.44 ± 6.42%, p = 0.001) compared with the control group (60.05 ± 10.91%). As a result, we showed that the cardioprotective effect of RIPC could be attributed to endogenous NO production during the ischemic period, which subsequently promoted reoxygenation in post-ischemic myocardia during early reperfusion. Our results suggest that the promotion of endogenous formation during an ischemic episode might be helpful as a therapeutic strategy for protecting the myocardium from IR injury. Additionally, our NO and O₂ perm-selective microsensors could be utilized to evaluate the effect of drug or treatment.     

Preparation and characterization of implantable sensors with nitric oxide release coatings

The widespread use of miniaturized chemical sensors to monitor clinically important analytes such as PO₂, PCO₂, pH, electrolytes, glucose and lactate in a continuous, real-time manner has been seriously hindered by the erratic analytical results often obtained when such devices are implanted in vivo. One major factor that has influenced the analytical performance of indwelling sensors is the biological response they elicit when in contact with blood or tissue (e.g. thrombus formation on the device surface, inflammatory response, encapsulation, etc.). Nitric oxide (NO) has been shown to be a potent inhibitor of platelet adhesion and activation as well as a promoter of wound healing in tissue. Herein, we review recent work aimed at the development of hydrophobic NO-releasing polymers that can be employed to coat catheter-type amperometric oxygen sensors without interfering with the analytical performance of these devices. Such modified sensors are shown to exhibit greatly enhanced hemocompatibility and improved analytical performance when implanted within porcine carotid and femoral arteries for up to 16 h. Further, results from preliminary studies also demonstrate that prototype fluorescent oxygen sensors, catheter-style potentiometric carbon dioxide sensors and subcutaneous needle-type enzyme-based amperometric glucose sensors can also be fabricated with new NO-release outer coatings without compromising the analytical response characteristics of these devices. The NO-release strategy may provide a solution to the lingering biocompatibility problems encountered when miniature chemical sensors are implanted in vivo.     

Direct electrochemistry of hemoglobin and myoglobin at didodecyldimethylammonium bromide-modified powder microelectrode and application for electrochemical detection of nitric oxide

Hemoglobin (Hb) and myoglobin (Mb) were immobilized at the didodecyldimethylammonium bromide (DDAB)-modified powder microelectrode (PME) to fabricate Hb-DDAB-PME and Mb-DDAB-PME. Direct electrochemistry of Hb and Mb were achieved on the DDAB-modified PME. The formal potential was −0.224 V for Hb and −0.212 V for Mb (vs. SCE). The apparent surface concentration of Hb and Mb at the electrode surface was 2.83 × 10⁻⁸ and 9.94 × 10⁻⁸ mol cm⁻². The Hb-DDAB-PME and Mb-DDAB-PME were successfully applied for measurement of NO in vitro. The anodic current peaks for NO oxidation at +0.7 V and the cathodic current peaks for NO reduction at −0.85 V on the CV curves were obtained on the modified electrodes. For detection of NO at +0.7 V, the sensitivity is 3.31 mA μM⁻¹ cm⁻² for Hb-DDAB-PME and 0.6 mA μM⁻¹ cm⁻² for Mb-DDAB-PME. The detection limit is 5 nM for Hb-DDAB-PME and 9 nM for Mb-DDAB-PME. The linear response range is 9-100 and 28-330 nM for Hb- and Mb-modified PME, respectively. For the electrochemical detection of NO at −0.85 V by using Hb-DDAB-PME, the detection sensitivity is 39.56 μA μM⁻¹ cm⁻²; the detection limit is as low as 0.2 μM; and the linear response range is 1.90-28.08 μM.     

Spatio-temporally resolved measurement of quantal exocytosis from single cells using microelectrode array modified with poly L-lysine and poly dopamine

The subsecond, temporal, vesicular exocytosis is ubiquitous, but difficult detecting in communication mechanisms of cells. A microelectrode array(MEA), fabricated by MEMS technology, was applied successfully for real-time monitoring of quantal exocytosis from single pheochromocytoma(PC12) cell.The developed MEA was evaluated by dopamine(DA) using electrochemical methods and the results revealed that the sensitivity of DA was improved to 12659.24 μA L mmol ~(-1)cm~(-2). The modified MEA was used to detect in vitro vesicular exocytosis of DA from single PC12 cells stimulated by concentrated100 mmol L~(-1)K~+cell solution. A total of 592 spikes were measured and analyzed by three parameters and the statistical results revealed the population of each parameter was an approximate Gaussian distribution, and on average, 1.31×10~6 ±9.25×10~4 oxidizable molecules were released in each quantal exocytosis. In addition, results also indicate that a single PC12 cell probably releases the spikes with T ranging from 25.6 ms to 35.4 ms corresponding to I_(max)ranging from 45.6 pA to 65.2 pA. The devices, including a homemade computer interface and the MEA modified with polymer film, provides a new means for further research on the neural, intercellular, communication mechanism.     

Continuous electrochemical monitoring of nitric oxide production in murine macrophage cell line RAW 264.7

In this study, we realized the continual and long-term electrochemical detection of NO production by stimulated macrophages using modified porphyrinic microsensor. The NO release from RAW 264.7 cells stimulated by lipopolysaccharide started 5 h after the lipopolysaccharide administration. After reaching its maximum at the sixth hour, the stable level of NO production was observed between the seventh and 12th hour of the experiment. This phase was followed by a gradual decline in NO production. A close correlation between the NO signal detected with microelectrode and nitrite accumulation, which had been determined in supernatants removed from stimulated cells, was observed. This finding was utilized for the calibration of the electrochemical experiment. The presence of iNOS enzyme, which constitutes a main requirement for NO production by stimulated macrophages, was confirmed by Western blot analysis of iNOS protein expression at key time points of the corresponding electrochemical experiment. The capability of our microsensor to instantaneously monitor the changes in the NO production by stimulated RAW 264.7 cells was demonstrated by the immediate decrease in the signal due to NO as a response to the addition of iNOS inhibitor into the cell culture medium. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Luminol chemiluminescense reaction: a new method for monitoring nitric oxide in vivo

A new method employing a combination of microdialysis sampling and chemiluminescence (CL) detection was developed to monitor nitric oxide (NO) in vivo. A special probe was designed with an interference-free membrane to achieve very high selectivity for NO. High sensitivity was achieved by optimizing the working system and improving the NO sampling time. This system was used in vivo to monitor blood and brain tissue in rats and rabbits. The system is sensitive enough to detect variations of NO formation under different physiological states. The linear valid range of NO determination is 5 nM to 1 μM, with a 3σ detection limit of 1 nM; real NO concentrations in test animals used in this work were found to be in the range of 1-5 nM or even less. Finally, the effects of body temperature, NO donors, Viagra, NO activators, NO cofactors and NO interference (such as O₂) were investigated carefully in different physiological situations.     

Modified electrode approaches for nitric oxide sensing

Three different methods for the determination of nitric oxide (NO) in solution are described. These are based, respectively, on the use of a horseradish peroxidase (HRP) biosensor or on electrodes modified with films of redox-active transition metal complexes. In the case of the biosensor the enzyme was electrochemically immobilized onto a glassy carbon (GC) electrode. The activity of HRP is inhibited in presence of NO. Thus, the decrease in activity is correlated to the concentration of NO present in solution. The biosensor responds linearly over the range of 2.7×10⁻⁶-1.1×10⁻⁵ M NO with a detection limit (5% inhibition) of 2.0×10⁻⁶ M. In the case of chemically modified electrodes, particular emphasis is placed on materials capable of catalyzing the oxidation of NO. In terms of electrocatalyst, the discussion will centre on electrodeposited films of 6,17-diferrocenyldibenzo[b,i]5,9,14,18-tetraaza[14]annulen]-nickel(II) and indium(III) hexacyanoferrate(III). The resulting sensors exhibited potent and persistent electroacatalytic activity towards the oxidation of NO with low detection limits (1 μM) and good linear relationship between the catalytic current and NO concentrations. In addition, interference due to the presence of nitrate and nitrite have been significantly reduced. According to these results, the described modified electrodes have been used as sensors for the determination of NO generated by decomposition of a typical NO-donor, such as S-nitroso-N-acetyl-d,l-penicillamine (SNAP). A critical comparison of the various methodologies employed is made.     

Reversible release of nitric oxide from an iron(II) nitrosyl complex containing a biomimetic S4N chelate. A facile release of nitric oxide

The release of NO by [Fe(NO)(Et₂NpyS₄)], where (Et₂NpyS₄)^2? = 2,6-bis(2-mercaptophenylthiomethyl)-4-substituted pyridine(2-), has been studied in the absence and presence of a trapping agent. The results show that [Fe(NO)(Et₂NpyS₄)] releases NO spontaneously in solution with a slow rate, k_-NO = 1.7 × 10^?4 s^?1 at 23 °C, in a reversible reaction. NO release becomes faster when the reaction intermediate [Fe(Et₂ NpyS₄)] was trapped by CO, thereby preventing the back reaction. The release of NO was studied as a function of CO concentration and temperature. The reported activation parameters, especially the positive activation entropy values for the release of NO, favor the operation of a dissociative interchange (I_d) mechanism. Thus, [Fe(NO)(Et₂NpyS₄)] can serve as a NO deliverer.     

A novel electrochemical sensor based on nano-structured film electrode for monitoring nitric oxide in living tissues

A sensor exhibited high sensitivity and good selectivity for determination of nitric oxide (NO) was fabricated. The sensor was constructured by coating Nafion/multi-walled carbon nanoubes-chitosan-gold nanoparticles (Nafion/MWNTs-CS-AuNPs) film on glassy carbon electrode (GCE). Several key parameters affecting on the electrochemical response were optimized, such as the film thickness, applied potential and volume of Nafion. The sensor showed good linear relationship with the NO concentration in the range of 1.90 × 10⁻⁸ to 5.40 × 10⁻⁵ M and with the detection limit of 7.60 × 10⁻⁹ M (S/N = 3). Finally, the sensor was successfully applied to the monitoring of NO release from living tissues, including mouse kidney, heart, spleen and liver (a slice). NO release at micro-molar level can be detected while the NO donor l-arginine (l-Arg), nitroglycerin (GTN) and aspirin (ASA) was present. It was interestedly found that the capacities to induce NO generation were in the order of GTN > ASA > l-Arg when these stimulants were converted to the same concentration. In addition, the NO release is associated with the functional groups in these donors.     

Photochemical and pharmacological aspects of nitric oxide release from some nitrosyl ruthenium complexes entrapped in sol–gel and silicone matrices

The entrapped [Ru(terpy)(L)NO](PF₆)₃, where terpy = 2,2′:6′,2″-terpyridine and L = 2,2′-bipyridine (bpy) and 3,4-diiminebenzoic acid (NH · NHq) complexes into sol–gel processed polysiloxane and silicone matrices, shows NO release characteristics when submitted to light irradiation at 355 and 532 nm, as judged by NO measurement using a NO-sensor electrode. The pharmacological properties of doped matrix showed vasodilator characteristics by visible light irradiation, which is of great interest because the target delivery system can avoid the occurrence of side effects possibly by the aquo ruthenium species. All matrices obtained showed to be amorphous materials. The scanning electron micrographs of the matrices showed irregularly shaped particles, with a broad size of 1000 μm for both matrices and homogeneous distribution.     

Synthesis and biological evaluation of 2,4-diaminopteridine derivatives as nitric oxide synthase inhibitor

A series of novel 2,4-diamino-pteridines(9a-1)were synthesized and evaluated as inhibitors of inducible nitric oxide synthase (iNOS)in vitro.It was found that 9a,9d,9e,9h,9i and 91 showed potent inhibitory activities similar to that of methotrexate(MTX),while the activities of 9b,9c,9f,9g,9j and 9k ale stronger than MTX.     

Pyrrole functionalised metalloporphyrins as electrocatalysts for the oxidation of nitric oxide

Pyrrole-functionalised tetracarboxyphenyl porphyrin and trimethoxyphenylcarboxy-phenyl porphyrin containing Ni, Mn and Pd as the central metal ion were used to modify Pt-disk microelectrodes (∅ 50 μm) (by repetitive cyclic voltammetry, dip-dry and pulse-amperometry methods) for the detection of nitric oxide (NO). Electrodes modified with Mn(II) trimethoxyphenylcarboxyphenyl porphyrin using the pulse amperomery approach, were found to be sensitive, stable and fast in response towards the oxidation of NO. Thus, they were used for the detection of NO release from a population of transformed human umbilical vein endothelial cells (T-HUVEC) into a droplet of electrolyte solution following stimulation with vascular endothelial growth factor (VEGF). The electrode surface was covered with an additional layer of Nafion® to prevent interference from anionic molecules such as nitrite.     

Regio- and stereoselective ring opening of aziridines with nitric oxide

Reaction of N-tosyl aziridines with nitric oxide affords the corresponding ring-opened products in regio-, stereoselectivities and excellent yields.     

Electrochemical determination of nitric oxide and of its derivatives

Nitric oxide (NO) is one of the simplest odd electron species. Furthermore, it is relatively hydrophobic, which is consistent with its role as a diffusible intracellular messenger or as an immune effector. NO is generated in biological systems and plays important roles as a regulatory molecule. The main problem in NO analysis is its extreme reactivity; in aerated water solution it is transformed into nitrite and nitrate, inactive biological forms. Moreover, it may lose an electron forming the NO⁺ ion, involved in the synthesis of nitrosothiols (RSNOs). The main problems encountered in the analytical determination of free NO and of RSNOs in biological systems are the low stability and the very low concentration of these compounds. The determination of nitrates and nitrites may also be difficult when their concentration is in the nmolar range. We describe an electrochemical assay for the determination in the same sample of free NO and of its derivatives in nmolar range. Owing to its high sensitivity, the procedure could also be applied to environmental analyses     

Improved thromboresistance and analytical performance of intravascular amperometric glucose sensors using optimized nitric oxide release coatings

In this work, nitric oxide (NO) release coatings designed for intravenous amperometric glucose sensors are optimized through the use of a polylactic acid (PLA) layer doped with a lipophilic diazeniumdiolated species that releases NO through a proton-driven mechanism. An Elast-Eon E2As polyurethane coating is used to both moderate NO release from the sensor surface and increase the sensor''s linear detection range toward glucose. These sensors were evaluated for thromboresistance and in vivo glucose performance through implantation in rabbit veins. By maintaining NO flux on a similar scale to endogenous endothelial cells, implanted glucose sensors exhibited reduced surface clot formation which enables more accurate quantitative glucose measurements continuously. An in vivo time trace of implanted venous sensors demonstrated glucose values that correlated well with the discrete measurements of blood samples on a benchtop point-of-care sensor-based instrument. The raw measured currents from the implanted glucose sensors over 7 h time periods were converted to glucose concentration through use of both a one-point in vivo calibration and a calibration curve obtained in vitro within a bovine serum solution. Control sensors, assembled without NO release functionality, exhibit distinctive surface clotting over the 7 h in vivo implantation period.     

Phthalocyanines and related complexes as electrocatalysts for the detection of nitric oxide

Metallophthalocyanine (MPc) and metalloporphyrin complexes as well as some metalloproteins including myoglobin, hemoglobin, cyanocobalamin and cytochrome c catalyse the detection of nitric oxide (NO). The catalytic process often involve changes in the oxidation state of the catalyst. These complexes catalyse both the reduction and oxidation of NO. MPc complexes containing electroactive central metals such as CoPc and FePc generally show better catalytic activity towards the detection of NO than complexes containing electroinacive central metals. However, the involvement of ring based redox processes was confirmed for the homogenous NO catalysis using CoPc.     

Ratiometric two-photon excited photoluminescence of quantum dots triggered by near-infrared-light for real-time detection of nitric oxide release in situ

Probe-donor integrated nanocomposites were developed from conjugating silica-coated Mn²⁺:ZnS quantum dots (QDs) with MoS₂ QDs and photosensitive nitric oxide (NO) donors (Fe₄S₃(NO)₇⁻, RBS). Under excitation with near-infrared (NIR) light at 808 nm, the Mn²⁺:ZnS@SiO₂/MoS₂-RBS nanocomposites showed the dual-emissive two-photon excited photoluminescence (TPEPL) that induced RBS photolysis to release NO in situ. NO caused TPEPL quenching of Mn²⁺:ZnS QDs, but it produced almost no impact on the TPEPL of MoS₂ QDs. Hence, the nanocomposites were developed as a novel QDs-based ratiometric TPEPL probe for real-time detection of NO release in situ. The ratiometric TPEPL intensity is nearly linear (R² = 0.9901) with NO concentration in the range of 0.01∼0.8 μM, which corresponds to the range of NO release time (0∼15 min). The detection limit was calculated to be approximately 4 nM of NO. Experimental results confirmed that this novel ratiometric TPEPL probe possessed high selectivity and sensitivity for the detection of NO against potential competitors, and especially showed high detection performance for NIR-light triggered NO release in tumor intracellular microenvironments. These results would promote the development of versatile probe-donor integrated systems, also providing a facile and efficient strategy to real-time detect the highly controllable drug release in situ, especially in physiological microenvironments.