Besic Sabina, Minteer Shelley D
Department of Chemistry, Saint Louis University, St Louis, MO, USA.
Methods Mol Biol. 2011;679:113-31. doi: 10.1007/978-1-60761-895-9_10.
Although enzymes are highly efficient and selective catalysts, there have been problems incorporating them into fuel cells. Early enzyme-based fuel cells contained enzymes in solution rather than immobilized on the electrode surface. One problem utilizing an enzyme in solution is an issue of transport associated with long diffusion lengths between the site of bioelectrocatalysis and the electrode. This issue drastically decreases the theoretical overall power output due to the poor electron conductivity. On the other hand, enzymes immobilized at the electrode surface have eliminated the issue of poor electron conduction due to close proximity of electron transfer between the electrode and the biocatalyst. Another problem is the short-term stability of the catalytic activity of the enzyme that is suspended in free flowing solution. Enzymes in solutions are only stable for hours to days, whereas immobilized enzymes can be stable for weeks to months and now even years. Over the last decade, there has been substantial research on immobilizing enzymes at electrode surfaces for biofuel cell and sensor applications. The most commonly used techniques are sandwich or wired techniques. Sandwich techniques are powerful and successful for enzyme immobilization; however, the enzymes optimal activity is not retained due to the physical distress applied by the polymer limiting its applications as well as the nonuniform distribution of the enzyme and the diffusion of analyte through the polymer is slowed significantly. Wired techniques have shown to extend the lifetime of an enzyme at the electrode surface; however, this technique is very hard to master due to specific covalent bonding of enzyme and polymer, which changes the three-dimensional configuration of enzyme and with that decreases the optimal catalytic activity. This chapter details an entrapment technique where an enzyme will be immobilized within the pores of a hydrophobically modified micellar polymers such as Nafion) and chitosan. This strategy has shown to safely immobilize enzymes at electrode surfaces with shelf and operation lifetimes of more than 2 years.
尽管酶是高效且具有选择性的催化剂，但将它们应用于燃料电池时仍存在一些问题。早期的基于酶的燃料电池中，酶存在于溶液中，而非固定在电极表面。在溶液中使用酶的一个问题是，生物电催化位点与电极之间存在较长的扩散长度，这涉及到传输问题。由于电子传导性差，这个问题极大地降低了理论总功率输出。另一方面，固定在电极表面的酶消除了因电极与生物催化剂之间电子转移距离过近而导致的电子传导不良问题。另一个问题是悬浮在自由流动溶液中的酶催化活性的短期稳定性。溶液中的酶仅能稳定存在数小时至数天，而固定化酶可以稳定存在数周、数月，甚至现在可达数年。在过去十年中，针对将酶固定在电极表面以用于生物燃料电池和传感器应用开展了大量研究。最常用的技术是夹心技术或连线技术。夹心技术在酶固定化方面强大且成功；然而，由于聚合物施加的物理压力，酶的最佳活性无法保留，这限制了其应用，同时酶的分布不均匀，并且分析物通过聚合物的扩散也显著减慢。连线技术已显示可延长酶在电极表面的寿命；然而，由于酶与聚合物的特定共价键合，该技术很难掌握，这会改变酶的三维构型，从而降低最佳催化活性。本章详细介绍了一种包埋技术，即将酶固定在疏水改性的胶束聚合物（如Nafion）和壳聚糖的孔内。这种策略已证明能够将酶安全地固定在电极表面，其储存寿命和操作寿命超过两年。
