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Probing the kinetics of quantum dot-based proteolytic sensors

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Abstract

As an enzyme superfamily, proteases are rivaled only by kinases in terms of their abundance within the human genome. Two ratiometric quantum dot (QD) Förster resonance energy transfer-based sensors designed to monitor the activity of the proteolytic enzymes collagenase and elastase are investigated here. Given the unique material constraints of these sensing constructs, assays are realized utilizing excess enzyme and fixed substrate in progress curve format to yield enzyme specificity or k cat/K m ratios. The range of k cat/Km values derived is 0.5–1.1 mM−1 s−1 for the collagenase sensor and 3.7–4.2 mM−1 s−1 for the elastase sensor. Of greater interest is the observation that the elastase sensor can be well represented by the Michaelis-Menten model while the collagenase sensor cannot. The latter demonstrates increased specificity at higher peptide substrate/QD loading values and an apparent QD-caused reversible inhibition as the reaction progresses. Understanding the detailed kinetic mechanisms that underpin these types of sensors will be important especially for their further quantitative utilization.

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Acknowledgements

SAD acknowledges an ASEE post-doctoral fellowship through NRL. The authors acknowledge the NRL Nanosciences Institute and DTRA JSTO MIPR # B112582M.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC, 20375, USA

    Sebastián A. Díaz, Anthony P. Malonoski & Igor L. Medintz

  2. Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory, Washington, DC, 20375, USA

    Kimihiro Susumu & Eunkeu Oh

  3. Sotera Defense Solutions, Columbia, MD, 21046, USA

    Kimihiro Susumu & Eunkeu Oh

  4. Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany

    Romina V. Hofele

Authors
  1. Sebastián A. Díaz
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  2. Anthony P. Malonoski
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  3. Kimihiro Susumu
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  4. Romina V. Hofele
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  5. Eunkeu Oh
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  6. Igor L. Medintz
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Correspondence to Igor L. Medintz.

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Díaz, S.A., Malonoski, A.P., Susumu, K. et al. Probing the kinetics of quantum dot-based proteolytic sensors. Anal Bioanal Chem 407, 7307–7318 (2015). https://doi.org/10.1007/s00216-015-8892-y

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  • DOI: https://doi.org/10.1007/s00216-015-8892-y

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