Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity
Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity
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DOI：
10.1021/acsami.7b17575
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发表时间：
2018-03-07
影响因子：
9.5
通讯作者：
Bayachou, Mekki
中科院分区：
文献类型：
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作者：
Gunasekera, Bhagya;Abou Diwan, Charbel;Bayachou, Mekki
一氧化氮(NO)的释放抵消了血小板的聚集，防止了血栓在血管内壁的级联。不释放涂层还可以防止与血液接触的医疗器械表面形成血栓。我们以前的工作表明，诱导型一氧化氮合酶(INOS)膜在底物L-精氨酸的酶转化过程中释放NO通量。在这项工作中，我们报道了诱导型一氧化氮合酶加氧酶(INOSoxy)在聚乙烯亚胺(PEI)上的逐层(LBL)薄膜中酶负载的调节。INOSoxy层被静电吸附到PEI层上。INOSoxy溶液的pH值会影响酶的吸附量。溶液中iNOSoxy的总表面负电荷取决于pH，因此决定了蛋白质在带正电的PEI层上的吸附密度。我们使用调节到PHS 8.6和7.0的缓冲iNOSoxy溶液，而使用pH 7.0的生理盐水PEI溶液。原子力显微镜成像显示，在pH 8.6时，iNOSoxy对蛋白质的吸附比在pH 7.0时的iNOSoxy溶液更高。具有PEI/iNOSoxy薄膜的石墨电极对iNOSoxy介导的NO还原具有较高的催化电流。当酶修饰的表面暴露在含有底物和电子源的溶液中时，较高的酶负载量转化为较高的NO通量。分光光度分析表明，在pH为8.6时构建的iNOSoxy/PEI膜比在pH为7.0时构建的膜具有更高的NO通量。对吸附在PEI上的iNOSoxy在pH为8.6和7.0时的傅里叶变换红外光谱分析表明，膜中iNOSoxy的结构不同，这解释了观察到的酶活性的变化。我们的研究结果表明，pH提供了一种策略来优化基于NOS的LBL薄膜中的NOS负载量和酶活性，这使得能够在最少的PEI/NOS层的情况下改善NO的释放。
Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate L-arginine. In this work, we report on the modulation of enzyme loading in layer-by layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.