Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, 20742.
Biotechnol Bioeng. 2013 Nov;110(11):2994-3002. doi: 10.1002/bit.24971. Epub 2013 Aug 2.
This work aims to develop a repeatable enzyme entrapment method that preserves activity within an amicable environment while resisting activity reduction in the presence of environmental challenges. Advances in such methods have wide potential use in biosensor applications. In this work β-galactosidase (lactase) enzyme was entrapped within hydrogel matrices of acrylamide (ACR) crosslinked with N,N'-methylenebisacrylamide (BIS, non-degradable) or poly(ethylene glycol) diacrylate (PEGDA, degradable) to create "biogels." Diffusivity studies of control, enzyme free, hydrogel constructs showed near-Fickian swelling behavior in PBS regardless of crosslinker type or density. As expected, the swelling rate, Ks , decreased when increasing the crosslink density from 78.6 to 14.7 min⁻¹ over a range of 1-20 mol% PEGDA indicating that diffusivity into the matrix is dependent on crosslink density. Fabricated biogels were evaluated for maintained enzyme activity in the 7 and 8 pH range. PEGDA crosslinked gels consistently showed improved enzymatic activity retention as compared to BIS crosslinked gels. As PEGDA crosslink density increased from 5 to 10 mol%, enzymatic activity retention post-initial entrapment increased. Higher PEGDA crosslink densities between 15% and 40% decreased enzymatic activity due to assumed steric hindrance of the entrapped enzyme and also decreased substrate and product diffusion. Increased enzymatic stability was observed in 40 mol% PEGDA crosslinked gels. The biogels were pH challenged to 8.0 and stability, measured as retention of activity, was observed to be 91%. Free, non-entrapped, solution based enzyme conversion only retained 23% activity under the same pH challenge conditions. No significant loss of active enzyme was determined to elute out of the biogels during storage in PBS or during biogel wash and recycling. This entrapment method illustrates the potential to sterically hinder and diffusively impede enzymes from performing their function. Degradation of the network crosslinks can then potentially enable the reactivation of the enzyme at a site and time dictated by the user.
这项工作旨在开发一种可重复的酶包埋方法，在友好的环境中保持酶的活性，同时抵抗环境挑战下的活性降低。此类方法的进步在生物传感器应用中有广泛的潜在用途。在这项工作中，β-半乳糖苷酶（乳糖酶）被包埋在丙烯酰胺（ACR）的水凝胶基质中，该基质由 N，N'-亚甲基双丙烯酰胺（BIS，不可降解）或聚乙二醇二丙烯酸酯（PEGDA，可降解）交联，形成“生物凝胶”。控制、无酶的水凝胶结构的扩散研究表明，无论交联剂类型或密度如何，在 PBS 中均表现出近菲克扩散行为。如预期的那样，随着交联密度从 78.6 增加到 14.7 min ⁻¹ ，扩散速率 Ks 降低，在 1-20 mol% PEGDA 的范围内表明扩散进入基质依赖于交联密度。制备的生物凝胶在 7 和 8 pH 范围内评估了保持酶活性的能力。与 BIS 交联凝胶相比，PEGDA 交联凝胶始终显示出更好的酶活性保持。随着 PEGDA 交联密度从 5 增加到 10 mol%，初始包埋后酶活性保持增加。较高的 PEGDA 交联密度（15%至 40%）降低了酶活性，这是由于包埋酶的空间位阻所致，并且还降低了底物和产物的扩散。在 40 mol% PEGDA 交联凝胶中观察到增加的酶稳定性。将生物凝胶的 pH 值挑战至 8.0，并测量活性保持率为 91%。在相同的 pH 挑战条件下，非包埋的游离溶液酶转化率仅保留 23%的活性。在 PBS 中储存或在生物凝胶洗涤和回收过程中，没有发现有活性的酶显著损失从生物凝胶中洗脱出来。这种包埋方法说明了通过空间位阻和扩散阻碍酶发挥其功能的潜力。然后，网络交联的降解可能使酶在用户指定的位置和时间重新激活。
