Iowa State University , 2114 Sweeney Hall, 618 Bissell Road , Ames , Iowa 50011 , United States.
Anal Chem. 2018 Apr 17;90(8):5209-5216. doi: 10.1021/acs.analchem.7b05444. Epub 2018 Mar 27.
Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.
水解酶因其工业影响和生物学意义而成为持续研究和改进的课题；然而，测量这些酶活性的能力，特别是在高通量测定中，仅限于已建立的少数几种酶，并且通常涉及测量次要副产物或设计复杂的降解探针。在此，描述了一种基于多功能单壁碳纳米管 (SWNT) 的生物传感器，该传感器易于生产和测量。将水解酶底物制成两亲聚合物，然后用于溶解疏水性纳米管。当目标酶降解包裹物时，由于溶剂可及性和聚集性增加，SWNT 的荧光信号会猝灭，从而可以定量测量水解酶的活性。使用（6,5）手性 SWNT 与多肽和多糖悬浮液，估计了纤维素酶、果胶酶和细菌蛋白酶的周转率。使用经过充分表征的蛋白酶 Proteinase K，记录了低至 5 fM 的浓度的响应。使用建立的胰蛋白酶平板阅读器测定法，将这种纳米管探针测定法与标准技术进行比较。此外，还测量了冻融循环和升高温度对酶活性的影响，表明即使经过 10 次循环冷冻也几乎没有影响，而加热至 60°C 以上则有害。最后，通过生成纤维素酶活性的温度和 pH 响应曲面来快速优化酶的操作条件，在连续扫描 2 小时内确定最佳条件。
