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Cancer Nanotechnology pp 63–97Cite as

Multifunctional Concentric FRET-Quantum Dot Probes for Tracking and Imaging of Proteolytic Activity

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1530))

Abstract

Proteolysis has many important roles in physiological regulation. It is involved in numerous cell signaling processes and the pathogenesis of many diseases, including cancers. Methods of visualizing and assaying proteolytic activity are therefore in demand. Förster resonance energy transfer (FRET) probes offer several advantages in this respect. FRET supports end-point or real-time measurements, does not require washing or separation steps, and can be implemented in various assay or imaging formats. In this chapter, we describe methodology for preparing self-assembled concentric FRET (cFRET) probes for multiplexed tracking and imaging of proteolytic activity. The cFRET probe comprises a green-emitting semiconductor quantum dot (QD) conjugated with multiple copies of two different peptide substrates for two target proteases. The peptide substrates are labeled with different fluorescent dyes, Alexa Fluor 555 and Alexa Fluor 647, and FRET occurs between the QD and both dyes, as well as between the two dyes. This design enables a single QD probe to track the activity of two proteases simultaneously. Fundamental cFRET theory is presented, and procedures for using the cFRET probe for quantitative measurement of the activity of two model proteases are given, including calibration, fluorescence plate reader or microscope imaging assays, and data analysis. Sufficient detail is provided for other researchers to adapt this method to their specific requirements and proteolytic systems of interest.

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Acknowledgments

The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), and the University of British Columbia for support of their research. W.R.A. is also grateful for support through a Canada Research Chair (Tier 2) and a Michael Smith Foundation for Health Research Scholar Award.

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Authors and Affiliations

  1. Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, Canada, V6T 1Z1

    Melissa Massey, Jia Jun Li & W. Russ Algar

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  1. Melissa Massey
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  2. Jia Jun Li
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  3. W. Russ Algar
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Correspondence to W. Russ Algar .

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  1. School of Applied Sciences, Mount Ida College, Newton, Massachusetts, USA

    Reema Zeineldin

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Massey, M., Li, J.J., Algar, W.R. (2017). Multifunctional Concentric FRET-Quantum Dot Probes for Tracking and Imaging of Proteolytic Activity. In: Zeineldin, R. (eds) Cancer Nanotechnology. Methods in Molecular Biology, vol 1530. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6646-2_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6646-2_4

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