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.
Koh
Wei Choon Alvin
WC
Department of Chemistry, Pusan National University, Busan 609-735, Republic of Korea.
Choe
Eun Sang
ES
Lee
Dong Kun
DK
Chang
Seung-Cheol
SC
Shim
Yoon-Bo
YB
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D001921:Brain / Q000201:enzymology*; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D007118:Immunoassay / Q000379:methods*; D008297:Male; D009474:Neurons / Q000201:enzymology*; D052248:Nitric Oxide Synthase Type I / Q000032:analysis / Q000378:metabolism*; D011199:Potentiometry; D051381:Rats; D017207:Rats, Sprague-Dawley
D052248:Nitric Oxide Synthase Type I