To immobilize enzymes at the surface of a nanoparticle-based electrochemical sensor is a common method to construct biosensors for non-electroactive analytes. Studying the interactions between the enzymes and nanoparticle support is of great importance in optimizing the conditions for biosensor design. This can be achieved by using a combination of analytical methods to carefully characterize the enzyme nanoparticle coating at the sensor surface while studying the optimal conditions for enzyme immobilization. From this analytical approach, it was found that controlling the enzyme coverage to a monolayer was a key factor to significantly improve the temporal resolution of biosensors. However, these characterization methods involve both tedious methodologies and working with toxic cyanide solutions. Here we introduce a new analytical method that allows direct quantification of the number of immobilized enzymes (glucose oxidase) at the surface of a gold nanoparticle coated glassy carbon electrode. This was achieved by exploiting an electrochemical stripping method for the direct quantification of the density and size of gold nanoparticles coating the electrode surface and combining this information with quantification of fluorophore-labeled enzymes bound to the sensor surface after stripping off their nanoparticle support. This method is both significantly much faster compared to previously reported methods and with the advantage that this method presented is non-toxic.

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将酶固定在基于纳米颗粒的电化学传感器的表面是构建用于非电活性分析物的生物传感器的常用方法。研究酶与纳米颗粒支持物之间的相互作用对于优化生物传感器设计条件至关重要。这可以通过使用分析方法的组合来仔细表征传感器表面的酶纳米颗粒涂层，同时研究酶固定的最佳条件来实现。通过这种分析方法，发现控制酶到单层的覆盖是显着提高生物传感器的时间分辨率的关键因素。但是，这些表征方法既涉及繁琐的方法，又涉及有毒氰化物溶液。在这里，我们介绍了一种新的分析方法，该方法可以直接量化涂覆有金纳米颗粒的玻璃碳电极表面上固定化酶（葡萄糖氧化酶）的数量。这是通过利用电化学剥离方法直接定量测定覆盖电极表面的金纳米颗粒的密度和大小，并将此信息与剥离传感器纳米颗粒支持物后结合到传感器表面的荧光团标记酶的定量结合而实现的。与先前报道的方法相比，该方法不仅速度快得多，而且具有所呈现的这种方法无毒的优点。这是通过利用电化学剥离方法直接定量测定覆盖电极表面的金纳米颗粒的密度和大小，并将此信息与剥离传感器纳米颗粒支持物后结合到传感器表面的荧光团标记酶的定量结合而实现的。与先前报道的方法相比，该方法不仅速度快得多，而且具有所呈现的这种方法无毒的优点。这是通过利用电化学剥离方法直接定量测定覆盖电极表面的金纳米颗粒的密度和大小，并将此信息与剥离传感器纳米颗粒支持物后结合到传感器表面的荧光团标记的酶的定量相结合而实现的。与先前报道的方法相比，该方法不仅速度快得多，而且具有所呈现的这种方法无毒的优点。

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