Department of Biomedical Engineering, University of California, Irvine, California 92617, United States.
Langmuir. 2012 Oct 2;28(39):14055-64. doi: 10.1021/la302708h. Epub 2012 Sep 17.
We report on the functionalization of a micropatterned carbon electrode fabricated using the carbon-MEMS process for its use as a miniature diffusion-free glucose oxidase anode. Carbon-MEMS based electrodes offer precise manufacturing control on both the micro- and nanoscale and possess higher electron conductivity than redox hydrogels. However, the process involves pyrolysis in a reducing environment that renders the electrode surface less reactive and introduction of a high density of functional groups becomes challenging. Our functionalization strategy involves the electrochemical oxidation of amine linkers onto the electrode. This strategy works well with both aliphatic and aryl linkers and uses stable compounds. The anode is designed to operate through mediated electron transfer between 2,5-dihydroxybenzaldehyde (DHB) based redox mediator and glucose oxidase enzyme. The electrode was first functionalized with ethylene diamine (EDA) to serve as a linker for the redox mediator. The redox mediator was then grafted through reductive amination, and attachment was confirmed through cyclic voltammetry. The enzyme immobilization was carried out through either adsorption or attachment, and their efficiency was compared. For enzyme attachment, the DHB attached electrode was functionalized again through electro-oxidation of aminobenzoic acid (ABA) linker. The ABA functionalization resulted in reduction of the DHB redox current, perhaps due to increased steric hindrance on the electrode surface, but the mediator function was preserved. Enzyme attachment was then carried out through a coupling reaction between the free carboxyl group on the ABA linker and the amine side chains on the enzyme. The enzyme incubation for both adsorption and attachment was done either through a dry spotting method or wet spotting method. The dry spotting method calls for the evaporation of enzyme droplet to form a thin film before sealing the electrode environment, to increase the effective concentration of the enzyme on the electrode surface during incubation. The electrodes were finally protected with a gelatin based hydrogel film. The anode half-cell was tested using cyclic voltammetry in deoxygenated phosphate buffer saline solution pH 7.4 to minimize oxygen interference and to simulate the pH environment of the body. The electrodes that yielded the highest anodic current were prepared by enzyme attachment method with dry spotting incubation. A polarization response was generated for this anodic half-cell and exhibits operation close to maximum efficiency that is limited by the mass transport of glucose to the electrode.
我们报告了一种使用碳微机电系统 (MEMS) 工艺制造的微图案化碳电极的功能化，该电极用作微型无扩散葡萄糖氧化酶阳极。基于碳 MEMS 的电极在微观和纳米尺度上具有精确的制造控制，并且具有比氧化还原水凝胶更高的电子导电性。然而，该过程涉及在还原环境中进行热解，这使得电极表面的反应性降低，并且引入高密度的官能团变得具有挑战性。我们的功能化策略涉及通过电化学氧化将胺接头引入电极。该策略适用于脂肪族和芳族接头，并且使用稳定的化合物。该阳极设计通过基于 2,5-二羟基苯甲醛 (DHB) 的氧化还原介体和葡萄糖氧化酶之间的中介电子转移来运行。该电极首先用乙二胺 (EDA) 功能化，作为氧化还原介体的接头。然后通过还原胺化接枝介体，通过循环伏安法确认接枝。通过吸附或附着进行酶固定化，并比较其效率。对于酶附着，通过电氧化氨苯甲酸 (ABA) 接头再次对附着 DHB 的电极进行功能化。ABA 功能化导致 DHB 氧化还原电流减少，这可能是由于电极表面的空间位阻增加，但介体功能得以保留。然后通过 ABA 接头的游离羧基和酶的胺侧链之间的偶联反应进行酶附着。通过干燥点样法或湿点样法进行吸附和附着的酶孵育。干燥点样法要求在密封电极环境之前蒸发酶液滴以形成薄膜，以增加孵育期间电极表面上酶的有效浓度。最后用基于明胶的水凝胶膜保护电极。在无氧磷酸盐缓冲盐水 pH 7.4 中使用循环伏安法测试阳极半电池，以最大程度地减少氧气干扰并模拟体内的 pH 环境。通过干燥点样孵育的酶附着方法制备了产生最高阳极电流的电极。为此阳极半电池生成了极化响应，并且表现出接近最大效率的操作，该效率受葡萄糖向电极的质量传输限制。
