Real-time monitoring of enzyme activity in a mesoporous silicon double layer.
Real-time monitoring of enzyme activity in a mesoporous silicon double layer.
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DOI：
10.1038/nnano.2009.11
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发表时间：
2009-04
影响因子：
38.3
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具有不同孔径的双层介孔硅用作纳米反应器，可以通过光学反射率实时分离，过滤和量化酶反应的动力学。这种微小的反应器可用于以无标记的方式快速表征各种分离的酶。某些蛋白酶的活性通常是疾病状态如癌症、中风和神经退行性变的指标，因此，需要能够表征酶促反应的动力学和底物特异性的快速测定。纳米结构膜可以有效地分离生物分子，但结合灵敏的检测方法仍然很困难。在这里，我们报告了一个单一的介孔纳米反应器，可以隔离和实时定量的蛋白酶的反应产物。该反应器由两层由晶体硅电化学制备的多孔膜组成。具有大孔径的上层捕获蛋白酶并充当反应器，而具有较小孔径的下层排除大蛋白并捕获反应产物。消化片段渗透到下层会产生可测量的光学反射率变化，这使得可以在约5纳升的体积内实时无标记定量酶动力学。
A double layer mesoporous silicon with different pore sizes functions as a nano-reactor that can isolate, filter and quantify the kinetics of enzyme reactions in real-time by optical reflectivity. This tiny reactor may be used to rapidly characterize a variety of isolated enzymes in a label-free manner. Activity of certain protease enzymes is often an indicator of disease states such as cancer, stroke, and neurodegeneracy, and thus, there is a need for rapid assays that can characterize the kinetics and substrate specificity of enzymatic reactions. Nanostructured membranes can efficiently separate biomolecules but coupling a sensitive detection method remains difficult. Here we report a single mesoporous nano-reactor that can isolate and quantify in real-time the reaction products of proteases. The reactor consists of two layers of porous films electrochemically prepared from crystalline silicon. The upper layer with large pore sizes traps the protease enzymes and acts as the reactor while the lower layer with smaller pore sizes excludes the large proteins and captures the reaction products. Infiltration of the digested fragments into the lower layer produces a measurable change in optical reflectivity and this allows label-free quantification of enzyme kinetics in real-time within a volume of approximately 5 nanoliters.