Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor.
Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor.
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DOI：
10.1016/j.aca.2014.11.008
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发表时间：
2015-01-07
影响因子：
6.2
通讯作者：
Griebenow K
中科院分区：
文献类型：
--
作者：
Hernández-Cancel G;Suazo-Dávila D;Medina-Guzmán J;Rosado-González M;Díaz-Vázquez LM;Griebenow K
我们通过在玻璃碳（GC）上电沉积金纳米颗粒（AuNP）并随后形成 4-巯基苯甲酸自组装单层（SAM）构建了生物传感器。然后将辣根过氧化物酶 (HRP) 共价固定到 SAM 上。采用两种形式的 HRP：未修饰的和用乳糖化学糖基化的。圆二色性（CD）光谱表明化学糖基化既不改变HRP的三级结构，也不改变血红素环境。使用 HRP-乳糖 1 的生物传感器获得了生物传感器对氢醌的最高灵敏度 (414 nA μM−1 )，而使用未修饰的 HRP 的生物传感器为 378 nA μM−1 。酶的化学糖基化形式比酶的天然形式更快地催化对苯二酚的还原。采用乳糖修饰 HRP 的传感器的检测限 (74 μM) 也低于 HRP 生物传感器 (83 μM)。然而，最重要的是，化学糖基化提高了生物传感器的长期稳定性，在四个月的储存期内保留了 60% 的活性，而 HRP 只能保留 10%。与之前报道的基于酶的生物传感器相比，这些结果凸显了创新稳定方法的改进。
We constructed a biosensor by electrodeposition of gold nano-particles (AuNPs) on glassy carbon (GC) and subsequent formation of a 4-mercaptobenzoic acid self-assembled monolayer (SAM). The enzyme horseradish peroxidase (HRP) was then covalently immobilized onto the SAM. Two forms of HRP were employed: non-modified and chemically glycosylated with lactose. Circular dichroism (CD) spectra showed that chemical glycosylation did neither change the tertiary structure of HRP nor the heme environment. The highest sensitivity of the biosensor to hydroquinone was obtained for the biosensor with HRP-lactose 1 (414 nA μM−1 ) compared to 378 nA μM−1 for the one employing non-modified HRP. The chemically glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly than the native form of the enzyme. The sensor employing lactose-modified HRP also had a lower limit of detection (74 μM) than the HRP biosensor (83 μM). However, most importantly, chemically glycosylation improved the long-term stability of the biosensor, which retained 60% of its activity over a four-month storage period compared to only 10% for HRP. These results highlight improvements by an innovative stabilization method when compared to previously reported enzyme-based biosensors.