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首页> 外文期刊>Electroanalysis >Modification of a capillary for electrophoresis by electrostatic self-assembly of an enzyme for selective determination of the enzyme substrate
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Modification of a capillary for electrophoresis by electrostatic self-assembly of an enzyme for selective determination of the enzyme substrate

机译：电泳毛细管的修饰，通过酶的静电自组装来选择性确定酶底物

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摘要

The integration of a separation capillary for capillary electrophoresis (CE) with an on-column enzyme reaction for selective determination of the enzyme substrate is described. Enzyme immobilization is achieved by electrostatic assembly of poly (diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the GOx produces hydrogen peroxide which migrates the length of the capillary and is detected amperometrically at the capillary outlet. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre- or post-separation chemical modification of the analyte. The enzyme reactor is found to have an optimal response to glucose when a 5:1 mixture of PSS:GOx is used. Under these conditions the limit of detection for glucose is found to be between 5.0 x 10(-4) and 1.3 X 10(-3) M, dependent upon the inner-diameter of the capillary. The apparent Michaelis-Menten constant for the enzyme reaction was determined to be 0.047 (+/- 0.001) M and 0.0037 (+/- 0.0007) M for a 50 and 10 pm inner-diameter capillaries, respectively. These results indicate that the enzyme reaction is efficient, having enzyme kinetics similar to that of a reaction occurring in solution. This enzyme immobilization method was also applied to another enzyme, glutamate oxidase, yielding similar results.
机译：描述了用于毛细管电泳（CE）的分离毛细管与用于选择性确定酶底物的柱上酶反应的集成。酶固定是通过静电组装聚二甲基二甲基氯化铵（PDDA），然后吸附带负电荷的酶葡萄糖氧化酶（GOx）和阴离子聚（苯乙烯磺酸盐）（PSS）的混合物来实现的。葡萄糖与GOx的反应产生过氧化氢，该过氧化氢迁移毛细管的长度，并在毛细管出口处通过安培法进行检测。酶反应发生在毛细管分离过程中，从而可以选择性地确定复杂样品中的底物，而无需对分析物进行分离前或分离后的化学修饰。当使用PSS：GOx的5：1混合物时，发现酶反应器对葡萄糖具有最佳响应。在这些条件下，葡萄糖的检出限在5.0 x 10（-4）至1.3 X 10（-3）M之间，具体取决于毛细管的内径。对于50和10 pm的内径毛细管，酶反应的表观Michaelis-Menten常数分别确定为0.047（+/- 0.001）M和0.0037（+/- 0.0007）M。这些结果表明酶反应是有效的，其酶动力学类似于溶液中发生的反应。这种酶固定方法还应用于另一种酶谷氨酸氧化酶，产生相似的结果。

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