In vitro monitoring of i-NOS concentrations with an immunosensor: the inhibitory effect of endocrine disruptors on i-NOS release.

The amperometric immunosensor has demonstrated the toxicity of endocrine disrupters (EDs) through monitoring the in vitro i-NOS concentration change, where the antibody of inducible nitric oxide synthase (i-NOS) was immobilized on the conducting polymer-gold nanoparticles composite. The performance of the sensor and the experimental parameters affecting the immunoreaction were optimized. Neuronal cells treated by EDs decreased in the in vitro i-NOS concentration. The effect of bisphenol A (BPA) on the i-NOS concentration released in the cells was investigated with different incubation times, and the interfering by nonspecific binding species present in a neuronal cell lysate was also examined. Of all the tested EDs, BPA showed the inhibitoriest effect and the minimum inhibitory concentration of BPA affecting the i-NOS concentration was 0.09 ± 0.005 µM. The result shows that monitoring of i-NOS in the neuronal cells treated by EDs will be a useful method to evaluate the toxic behavior of EDs.

Interactions of Dopamine D1 and N-methyl-D-Aspartate Receptors are Required for Acute Cocaine-Evoked Nitric Oxide Efflux in the Dorsal Striatum.

Alterations in nitric oxide (NO) release in response to psychostimulants in the striatum cause a plastic change contributing to the development and expression of addiction. In this study, regulation of NO efflux evoked by acute cocaine in the dorsal striatum was investigated using real-time detection of NO in vivo. We found that acute systemic injection of cocaine (20 mg/kg) increased NO efflux, which was reduced by the intrastriatal infusion of the dopamine D1 receptor antagonist, SCH23390 (7.5 nmol), and the dopamine D2 receptor agonist, quinpirole (5 nmol). Increased levels of NO efflux by acute cocaine were also reduced by the intrastriatal infusion of the N-methyl-D-aspartate (NMDA) receptor antagonists, MK801 (2 nmol) and AP5 (2 nmol). These findings suggest that interactions of dopamine D1 receptors and NMDA receptors after acute exposure to cocaine participate in the upregulation of NO efflux in the dorsal striatum.

Electropolymerized self-assembled layer on gold nanoparticles: detection of inducible nitric oxide synthase in neuronal cell culture.

Novel nanostructures of gold nanoparticle (AuNP) encapsulated-conductive polymer have been developed to study biosensor probe materials and utilized to detect the concentration of inducible nitric oxide synthase (iNOS). A 2,2':5',5″-terthiophene-3'-benzoic acid (TTBA) monomer was synthesized and self-assembled on gold nanoparticles (AuNPs). The size effects of the AuNPs and TTBA monomer film thickness on the electrode conductivity were examined. Anti-iNOS antibody was covalently bound on an encapsulated-AuNPs polymer layer with self-assembled TTBA. The immunocomplex formation between iNOS and anti-iNOS was directly observed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). This study looked at the applicability of the self-assembled TTBA layer where the results indicated an efficient electrochemical response toward iNOS. The calibration plot of the current response vs. iNOS concentration exhibited a linear relationship in the range of 0.001-0.02 µg/mL. The calibration sensitivity of iNOS was 59.4 ± 0.3 mV/µg mL(-1). The detection limit of iNOS was determined to be 0.20 ± 0.04 ng/mL based on five time measurements (95% confidence level, k = 3, n = 5). Further results show that AuNP-encapsulated conductive polymers are good nanostructured materials as biosensor probes and have a potential application in cell biosensors.

Electrochemical detection of peroxynitrite using a biosensor based on a conducting polymer-manganese ion complex.

A peroxynitrite (ONOO(-)) biosensor has been developed through the preparation of a new manganese-[poly-2,5-di-(2-thienyl)-1H-pyrrole)-1-(p-benzoic acid)] (Mn-pDPB) complex. DPB monomer was first synthesized and polymerized for the purpose of providing a polymer backbone for complex formation with Mn(2+) ion. The Mn-pDPB complex was characterized via Magnetomotive Force (MMF) simulation, X-ray photoelectron spectroscopy (XPS), and cyclic voltammetry. The complex selectively enhanced the reduction process of ONOO(-) which was used as the analytical signal for chronoamperometric detection. A polyethyleneimmine (PEI) layer was coated on the complex surface to increase selectivity and stability. The chronoamperometric calibration plot showed the hydrodynamic range of 2.0 × 10(-8)-5.0 × 10(-7) M. The detection limit was determined to be 1.9 (±0.2) × 10(-9) M based on S/N = 3. The microbiosensor, fabricated on a 100 µm diameter Pt tip, was applied in a real rat plasma sample for the detection of spiked concentrations of ONOO(-). The reliability and long-term stability of the microbiosensor was also examined with YPEN-1 cells in vitro, and the results shown were promising.

A selective nitric oxide nanocomposite biosensor based on direct electron transfer of microperoxidase: removal of interferences by co-immobilized enzymes.

A highly selective nitric oxide (NO) biosensor was developed by immobilizing microperoxidase (MP) onto the MWCNT-poly-5,2':5',2″-terthiophene-3'-carboxylic acid (PTTCA) nanocomposite. Catalase (CAS) and superoxide dismutase (SOD) co-immobilized on the probe successfully protected the interferences of H(2)O(2) and O(2)(-) during NO detection. The nanocomposite layer showed the direct electron transfer processes of the immobilized CAS, SOD, and MP simultaneously at -0.11/+0.04, -0.33/-0.27, and -0.47/-0.40 V vs. Ag/AgCl. Au nanoparticles (AuNPs) were electrodeposited on a glassy carbon surface to enhance the sensitivity of the sensor probe. The layers of CAS/SOD/MP/MWCNT-PTTCA/AuNPs were characterized using SEM, XPS, and QCM. The CAS/SOD/MP/MWCNT-PTTCA/AuNPs probe showed an excellent performance in the electrocatalytic reduction of NO which was attributed to the heme group of MP. Experimental conditions affecting the sensitivity of the biosensor were optimized in terms of pH, temperature, and applied potential. The dynamic range of NO analysis was from 1.0 to 40 µM with the detection limit of 4.3 ± 0.2 nM. The reliability of biosensor was examined for the determination of NO released from the real samples of rat liver, stomach (AGS), and intestinal (HT-29) cancer cells.

Repeated cocaine administration increases nitric oxide efflux in the rat dorsal striatum.

RATIONALE: Repeated injections of cocaine alter extracellular nitric oxide (NO) efflux via interactions between dopamine and glutamate receptor-coupled signaling cascades. OBJECTIVES: Putative cellular mechanisms underlying changes in NO efflux following repeated cocaine administration were investigated. METHODS: Real-time detection of NO efflux using a NO biosensor was mainly performed in the rat dorsal striatum in vivo. RESULTS: Repeated exposure to cocaine (20 mg/kg), once a day for seven consecutive days, increased NO levels. Repeated injections of cocaine also increased the phosphorylation of neuronal nitric oxide synthase (nNOS), and inhibition of nNOS decreased the repeated cocaine-evoked increases in NO levels. Inhibition of protein kinase A, but not protein phosphatases, synergistically increased NO levels elevated by repeated cocaine injections. Blockade of dopamine D1 (D1) receptors or stimulation of dopamine D2 (D2) receptors decreased the repeated cocaine-evoked increases in NO levels. Similarly, blockade of N-methyl-D: -aspartate (NMDA) receptors and group I metabotropic glutamate receptors (mGluRs) or stimulation of group III mGluRs also decreased the repeated cocaine-evoked increases in NO levels. CONCLUSION: Stimulation of D1 receptors or group I mGluRs following repeated cocaine administration upregulates NO efflux via an NMDA receptor-evoked Ca2+ influx, while stimulation of D2 receptors or group III mGluRs downregulates NO efflux. Dephosphorylation of phosphorylated nNOS by protein phosphatases is necessary for upregulating NO efflux in the dorsal striatum after repeated cocaine administration.

Monitoring the activation of neuronal nitric oxide synthase in brain tissue and cells with a potentiometric immunosensor.

An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H(+)-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H(+) ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody-antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4-340.0 microg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was -0.073+/-0.002 mV/microg ml(-1). The detection limit of nNOS was determined to be 0.2 microg/ml based on five-time measurements (95% confidence level, k=3, n=5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis.