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Real-time monitoring of enzyme activity in a mesoporous silicon double layer

Nature Nanotechnology volume 4, pages 255–258 (2009)Cite this article

Abstract

The activity of certain proteolytic enzymes is often an indicator of disease states such as cancer1,2, stroke2 and neurodegeneracy3,4, so there is a need for rapid assays that can characterize the kinetics and substrate specificity of enzymatic reactions. Nanostructured membranes can efficiently separate biomolecules5, but coupling a sensitive detection method to such a membrane remains difficult. Here, we demonstrate a single mesoporous nanoreactor 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 (∼100 nm in diameter), traps the protease and acts as the reactor. The lower layer, with smaller pore sizes (∼6 nm), excludes the proteases and other 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 ∼5 nl.

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Figure 1: Scanning electron micrographs (secondary electron image, 5 kV) of the porous silicon double-layer structure used in this study.
Figure 2: a,b, Reflectivity spectrum and corresponding Fourier transform of a porous silicon double-layer film.
Figure 3: Nanoreactor used to process protein and quantify proteolytic activity.
Figure 4: Optical response of the two layers in a pepsin-loaded nanoreactor upon introduction of α-casein.
Figure 5: Kinetics of digestion of α-casein by pepsin in the nanoreactor as a function of casein concentration.

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Acknowledgements

This material is based upon work supported by the National Science Foundation under grant no. DMR-0806859, by the Hitachi Chemical Research Center, and by the UC Discovery Industry-University Cooperative Research Program. M.J.S. is a member of the Moores UCSD Cancer Center and the UCSD NanoTUMOR Center under which this research was conducted, and partially supported by NIH grant U54 CA 119335. M.M.O. thanks the Department of Education, Graduate Assistance in Areas of National Need (GANN) program (P200A030163) for a pre-doctoral fellowship. The authors thank M. Oaks of the Hitachi Chemical Research Center for experimental assistance in obtaining the SEM images.

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  1. Claudia Pacholski

    Present address: Present address: Max-Planck-Institute for Metals Research, New Materials and Biosystems—Department Spatz, Heisenbergstraße 3, 70569 Stuttgart, Germany,

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Department 0358, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093-0358, California, USA

    Manuel M. Orosco, Claudia Pacholski & Michael J. Sailor

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Contributions

M.M.O., C.P. and M.J.S. conceived and designed the experiments. M.M.O. and C.P. performed the experiments. M.M.O., C.P. and M.J.S. analysed the data. M.M.O. and M.J.S. co-wrote the paper.

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Correspondence to Michael J. Sailor.

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Orosco, M., Pacholski, C. & Sailor, M. Real-time monitoring of enzyme activity in a mesoporous silicon double layer. Nature Nanotech 4, 255–258 (2009). https://doi.org/10.1038/nnano.2009.11

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