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Related Experiment Video

Updated: Jun 14, 2026

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
13:22

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Published on: October 23, 2019

New high-throughput screening protease assay based upon supramolecular self-assembly.

Zhijun Zhou1, Yanli Tang, David G Whitten

  • 1Department of Chemical and Nuclear Engineering, The University of New Mexico, Albuquerque, New Mexico 87131, USA.

ACS Applied Materials & Interfaces
|April 2, 2010
PubMed
Summary

This study introduces a novel fluorescent assay for detecting protease activity. A cyanine molecule, linked to a peptide substrate, is released and self-assembles to produce a detectable fluorescent signal upon protease cleavage.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Supramolecular self-assembly of cyanines offers potential for fluorescent enzymatic assays.
  • Previous work demonstrated the utility of cyanine self-assembly in assay development.

Purpose of the Study:

  • To synthesize a covalent adduct of a tetrapeptide (Asp-Glu-Val-Asp, DEVD) and a cyanine (DEVD-cyanine).
  • To investigate the use of DEVD-cyanine as a substrate for Caspase-3 and Caspase-7 proteases.
  • To develop a novel fluorescent assay for protease activity detection based on cyanine self-assembly.

Main Methods:

  • Synthesis of DEVD-cyanine covalent adduct.
  • Enzymatic hydrolysis of DEVD-cyanine by Caspase-3 and Caspase-7 in microwell plates.
  • Detection of liberated cyanine self-assembly on carboxymethylamylose (CMA) and carboxymethylcellulose (CMC) scaffolds.

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Last Updated: Jun 14, 2026

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  • Fluorescence spectroscopy to quantify protease activity.

    • Main Results:

    • DEVD-cyanine was recognized and hydrolyzed by Caspase-3 and Caspase-7.
    • Liberated cyanine self-assembled into J aggregates on CMA/CMC scaffolds, yielding bright fluorescence at 470 nm.
    • Fluorescence intensity correlated with enzyme/substrate concentrations and reaction time, exhibiting saturation kinetics.
    • The Michaelis constant (Km) for DEVD-cyanine was approximately 23 microM.
    • Optimal fluorescence was achieved using a mixture of CMA and CMC scaffolds.

    Conclusions:

    • This work presents the first demonstration of cyanine release from a covalent adduct via protease action, leading to supramolecular self-assembly.
    • The developed method enables the detection of protease activity through enhanced fluorescence.
    • The assay shows potential for sensitive and quantitative protease activity measurements.