Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China.
Lab Chip. 2010 Mar 7;10(5):639-46. doi: 10.1039/b915762j. Epub 2009 Dec 16.
In this paper, a micro/nanofluidic preconcentration device integrated with an electrochemical detector has been used to study the enrichment of enzymes and homogeneous enzyme reaction kinetics. The enzymes are first concentrated in front of a nanochannel via an exclusion-enrichment effect (EEE) mechanism of the nanochannel integrated in a microfluidics device. If a substrate is electrokinetically transported to the concentrated enzymes, homogeneous enzymatic reaction occurs. The enzymatic reaction product can penetrate through the nanochannel to be detected electrochemically. In this device, the enriched enzymes can be well retained and repeatedly used, thus, the enzymatic reaction occurs in a continuous-flow mode. For demonstration, Glucose oxidase (GOx) was chosen as the model enzyme to study the influence of enzyme concentration on its reaction kinetics. The different concentration of GOx in front of the nanochannel was simply achieved by using different enrichment time. When substrate glucose was introduced electrokinetically, a rapid electrochemical steady-state response could be obtained. It was found that the electrochemical response to a constant glucose concentration increased with the increase of enzyme enrichment time, which is expected for homogeneous enzymatic reactions. Under proper conditions, the electrochemical responds linearly to the glucose concentration ranging from 0 to 15 mM, and the Michaelis constants (K(m)) are relatively low, which indicates a more efficient complex formation between enzyme and substrate. These results suggest that the present micro/nanofluidics device is promising for the study of enzymatic reaction kinetics and other bioassays such as cell assays, drug discovery, and clinical diagnosis.
本文采用微纳流控预浓缩装置与电化学检测器相结合，研究了酶的浓缩和均相酶反应动力学。首先，通过集成在微流控装置中的纳米通道的排除浓缩效应（EEE）机制，将酶浓缩在纳米通道前。如果底物经电泳传输至浓缩酶，就会发生均相酶反应。酶反应产物可穿透纳米通道进行电化学检测。在该装置中，浓缩酶可以很好地保留并重复使用，因此，酶反应以连续流动模式进行。为了验证，选择葡萄糖氧化酶（GOx）作为模型酶，研究酶浓度对其反应动力学的影响。通过改变浓缩时间，简单地实现了纳米通道前不同浓度的 GOx。当电动力学引入底物葡萄糖时，可获得快速的电化学稳态响应。结果发现，对于恒定的葡萄糖浓度，电化学响应随酶浓缩时间的增加而增加，这符合均相酶反应的预期。在适当的条件下，电化学响应与葡萄糖浓度在 0 到 15mM 范围内呈线性关系，米氏常数（K(m)）较低，表明酶与底物之间形成了更有效的复合物。这些结果表明，该微纳流控装置有望用于研究酶反应动力学和其他生物分析，如细胞分析、药物发现和临床诊断。
