Abstract
A protease is any enzyme that catalyzes the hydrolysis of proteins into smaller peptide fragments and amino acids, a process known as proteolysis. They are involved in a multitude of normal biological processes as well as in diseases, including cancer, stroke and infections. Here we present a microfluidicbased assay system to detect proteolytic activity using fluorescence resonance energy transfer (FRET) by quantum dot (QD)-peptide conjugates immobilized on microbeads. As an energy donor, QD was immobilized on the microbead surface by the avidin-biotin interaction. As an energy acceptor, the fluorophorelabeled peptide was then associated with QD, thus quenching the photoluminescence (PL) of the QD. The functionalized microbeads were introduced into the microbiochip and captured by a micropillar in the reaction chamber. In the presence of matrix metalloprotease-7 (MMP-7) as a model protease, the PL of QD quenched by fluorophore was recovered due to the proteolytic activity of MMP-7 in the fabricated microbiochip. Moreover, the FRET efficiency induced by MMP-7 was linearly dependent on the logarithmic concentration of MMP-7. This technology is not limited to sensing MMP-7, but could be used to monitor other protease activities (Schematic diagram).
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Shin, H.R. Global activity of cancer registries and cancer control and cancer incidence statistics in Korea. JPMPH 41, 84–91 (2008).
Walther, A. et al. Genetic prognostic and predictive markers in colorectal cancer. Nat. Rev. Cancer 9, 489–499 (2009).
Nagase, H. & Woessner, J.F. Matrix metalloproteinases. J. Biol. Chem. 274, 21491 (1999).
Kong, D.H., Jung, S.H., Lee, S.T. & Ha, K.S. On-chip assay of matrix metalloproteinase-3 activity using fluorescence-conjugated gelatin arrays. BioChip J. 4, 210–216 (2010).
Zucker, S. & Vacirca, J. Role of matrix metalloproteinases (MMPs) in colorectal cancer. Cancer Metastasis Rev. 23, 101–117 (2004).
Förster, T. Fluoreszenz Organischer Verbindungen. 1951. (Vandenhoeck & Ruprecht, Göttingen).
Davis, B.W. et al. FRET detection of proteins using fluorescently doped electrospun nanofibers and pattern recognition. Langmuir (2011).
Kim, J.H. et al. A microfluidic protease activity assay based on the detection of fluorescence polarization. Anal. Chim. Acta 577, 171–177 (2006).
Kim, Y.P., Oh, Y.H., Oh, E. & Kim, H.S. Chip-based protease assay using fluorescence resonance energy transfer between quantum dots and fluorophores. Biochip J. 1, 228–233 (2007).
Furukawa, K., Nakashima, H., Kashimura, Y. & Torimitsu, K. Novel “Lipid-Flow Chip” configuration to determine donor-to-acceptor ratio-dependent fluorescence resonance energy transfer efficiency. Langmuir 24, 921–926 (2008).
Nishioka, T., Frohman, M.A., Matsuda, M. & Kiyokawa, E. Heterogeneity of phosphatidic acid levels and distribution at the plasma membrane in living cells as visualized by a Förster Resonance Energy Transfer (FRET) biosensor. J. Biol. Chem. 285, 35979–35987 (2010).
Carlson, H.J. & Campbell, R.E. Genetically encoded FRET-based biosensors for multiparameter fluorescence imaging. Curr. Opin. Biotechnol. 20, 19–27 (2009).
Lichlyter, D.J., Grant, S.A. & Soykan, O. Development of a novel FRET immunosensor technique. Biosens. Bioelectron. 19, 219–226 (2003).
Popovtzer, R. et al. Novel integrated electrochemical nano-biochip for toxicity detection in water. Nano Lett. 5, 1023–1027 (2005).
Graham, D.L., Ferreira, H.A. & Freitas, P.P. Magnetoresistive-based biosensors and biochips. Trends Biotechnol. 22, 455–462 (2004).
Makohliso, S. et al. Surface characterization of a biochip prototype for cell-based biosensor applications. Langmuir 15, 2940–2946 (1999).
Albers, J., Grunwald, T., Nebling, E., Piechotta, G. & Hintsche, R. Electrical biochip technology-a tool for microarrays and continuous monitoring. Anal. Bioanal. Chem. 377, 521–527 (2003).
Zhao, C. & Wittstock, G. Scanning electrochemical microscopy for detection of biosensor and biochip surfaces with immobilized pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase as enzyme label. Biosens. Bioelectron. 20, 1277–1284 (2005).
Yi, Y.C. et al. Matrix metalloproteinase-7 (MMP-7) polymorphism is a risk factor for endometrial cancer susceptibility. Clin. Chem. Lab. Med. 48, 337–344 (2010).
Crivat, G. et al. Quantum dot FRET-based probes in thin films grown in microfluidic channels. J. Am. Chem. Soc. 132, 1460–1461 (2010).
Buranda, T. et al. Biomimetic molecular assemblies on glass and mesoporous silica microbeads for biotechnology. Langmuir 19, 1654–1663 (2003).
Sukhanova, A. et al. Nanocrystal-encoded fluorescent microbeads for proteomics: antibody profiling and diagnostics of autoimmune diseases. Nano Lett. 7, 2322–2327 (2007).
Bhushan, B. MEMS/NEMS and BioMEMS/BioNEMS: materials, devices, and biomimetics. Nanotribology and Nanomechanics II, 833–945 (2011).
Fernandes, R. et al. Biological nanofactories facilitate spatially selective capture and manipulation of quorum sensing bacteria in a bioMEMS device. Lab. Chip 10, 1128–1134 (2010).
Ko, Y.-J. et al. Real-time immunoassay with a PDMSglass hybrid microfilter electro-immunosensing chip using nanogold particles and silver enhancement. Sens. Actuator B-Chem. 132, 1810–1817 (2008).
Ha, S.-M., Ju, J.-K., Ahn, Y. & Hwang, S.Y. Separation-type multiplex polymerase chain reaction chip for detecting male infertility. Jpn. J. Appl. Phys. 47, 5231–5235 (2008).
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Lee, S.Y., Han, B., Park, C. et al. Development of microbiochip for detection of metalloproteinase 7 using fluorescence resonance energy transfer. BioChip J 7, 164–172 (2013). https://doi.org/10.1007/s13206-013-7210-z
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DOI: https://doi.org/10.1007/s13206-013-7210-z