A new film-forming solution was developed for the efficient immobilization of enzymes on solid substrates. The solution consisted of a biopolymer, chitosan (CHIT), that was chemically modified with a permeability-controlling agent, Acetyl Yellow 9 (AY9), using glutaric dialdehyde (GDI) as a molecular tether. A model enzyme, glucose oxidase (GOx), was mixed with the CHIT-GDI-AY9 solution and cast on the surface of platinum electrodes to form robust CHIT-GDI-AY9-GOx films for glucose biosensing. UV-visible and infrared spectroscopies were used to determine the composition of the films. The optimized films contained on average 1 molecule of AY9/3 glucosamine units of chitosan and 25 free GDI tethers/1 molecule of GOx. The electrochemical assays of the films indicated both a very high efficiency of enzyme immobilization (approximately 99%) and large enzyme activity (60 units cm(-2)). The latter translated into a high sensitivity (42 mA M(-1) cm(-2)) of the Pt/CHIT-GDI-AY9-GOx biosensor toward glucose. The biosensor operated at 0.450 V, had a fast response time (t90% < or = 3 s), and was free of typical interferences, and its dynamic range covered 3 orders of magnitude of glucose concentrations. The lowest actually detectable concentration was 10 microM glucose. In addition, the biosensor displayed a practical shelf life and excellent operational stability, e.g. its response was stable during 24-h testing under continuous polarization and continuous flow of 5.0 mM glucose solution. The proposed approach to enzyme immobilization is simple, efficient, and cost-effective and should be of importance in the development of biosensors based on other enzymes that are more expensive than glucose oxidase.

Endothelial dysfunction and decreased production of nitric oxide (NO) by endothelial NO synthase (eNOS) are implicated in the pathogenesis of hypertension and insulin resistance. Because the potential influence of increased eNOS expression/ activity on these parameters is unclear, the present study examined the effects of eNOS gene therapy on insulin resistance and blood pressure alterations in a fructose-induced hypertension model in rats. As predicted, 2 weeks of fructose consumption in the drinking water resulted in elevated systolic blood pressure and insulin resistance. These and other physiologic alterations were reversed within 2 weeks after a single intravenous injection of a vector containing the human eNOS cDNA (pcDNA3.1-eNOS), whereas injection of an empty vector (pcDNA3.1) was without effect. In support of the beneficial effects of pcDNA3.1-eNOS treatment being because of enhanced eNOS expression and activity, increased eNOS protein levels were documented in aorta, liver, kidney, and heart of fructose-treated rats injected with pcDNA3.1-eNOS, and corresponding elevations in nitrite/nitrate and cGMP concentrations were observed in urine. Furthermore, pcDNA3.1-eNOS treatment prevented fructose-induced decreases in expression levels of insulin receptor substrate-1, the p110 catalytic subunit of phosphatidylinositol 3-kinase, phosphorylated Akt, and phosphorylated AMP-activated protein kinases in liver, aorta, and skeletal muscle. The results of this study cumulatively indicate that gene therapy with human eNOS decreased fructose-induced hypertension and insulin resistance in rats and suggest potential signaling pathways that mediate these effects. These data highlight the potential utility of eNOS gene therapy in the treatment of hypertension and insulin resistance.Nitric oxide (NO), a potent vasodilator constitutively produced by endothelial nitric-oxide synthase (eNOS), is thought to be the endothelium-derived relaxing factor that mediates relaxation in response to acetylcholine, bradykinin, and substance P in vascular beds (Rees and Moncada, 1989). eNOS is the predominant vascular NO synthase isoform and is responsible for the majority of NO production in the vasculature (Moncada and Higgs, 2006). In addition to its effects on the regulation of blood pressure and regional blood flow, NO influences vascular smooth muscle proliferation and inhibits platelet aggregation and leukocyte adhesion (Moncada and Higgs, 2006).A number of lines of evidence implicate eNOS-derived NO as a pivotal regulator of blood pressure, vascular tone, and vascular homeostasis. In vivo inhibition of NO synthase activity by nonhydrolyzable analogs of L-arginine results in a dramatic increase in mean arterial blood pressure (Desjardins and Balligand, 2006), whereas mice genetically deficient in eNOS have impaired endothelium-dependent vasodilator responses to acetylcholine and are hypertensive (Huang et

A direct ionotropic gelation of the polycationic biopolymer chitosan (CHIT) with the polyanionic enzyme lactate oxidase (LOx) was used to form thin biopolymer-enzyme films on the surface of platinum electrodes. The electrochemical assays of such films revealed a well-defined capacity of CHIT for the retention of LOx. The stoichiometry of the CHIT-LOx polyelectrolyte complexes was found to be approximately 1:40, i.e., on average, 1 CHIT chain retained 40 molecules of LOx in the CHIT-LOx films. The enzyme retention was ascribed to strong electrostatic interactions between the LOx and a fraction of the protonated amino groups on the CHIT chains. Although the LOx is inherently unstable outside its natural matrix, it displayed high surface activity of 0.26 units cm(-)(2) in the matrix of CHIT. This correlated with good stability of the biopolymer-enzyme films as demonstrated by a constant response of Pt/CHIT-LOx electrodes to lactate during continuous 24-h testing. When compared to other single-film lactate sensors, the Pt/CHIT-LOx electrodes displayed the best combination of analytical properties in terms of a low detection limit (50 nM), high sensitivity (0.23 A M(-)(1) cm(-)(2)), and fast response time (<1 s). Such a performance validated the CHIT-LOx system as an attractive sensing element for the development of new lactate biosensors.

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