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Chemiluminescent determination of the activity of caspase-3 using a specific peptide substrate and magnetic beads

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Abstract

We report on a new scheme for the determination of the activity of caspase-3 using a specific peptide labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as a chemiluminescent (CL) probe and on the development of magnetic separation technology. Firstly, the ABEI-labeled and biotinylated peptide was prepared and conjugated to streptavidin-coated magnetic beads (MBs) to form f-MBs (functionalized magnetic beads). The f-MBs contain a site (DEVD, Asp-Glu-Val-Asp) that is cleaved by caspase-3. Upon cleavage, the terminal residue attached to ABEI can dissociate from the f-MBs and can be used for CL detection. CL intensity is linearly related to the concentration of caspase-3 in the range 1.0 to 600 ng mL−1, with a detection limit of 0.3 ng mL−1. The relative standard deviation of the assay is 3.6 % at a level of 50 ng mL−1 of caspase-3 (for n = 11). The CL assay has been applied to the determination of caspase-3 in Jurkat cell extract with recoveries between 96.6 % and 106.1 % (n = 5).

A chemiluminescence assay for the detection of caspase-3 activity using N-(4-aminobutyl)-N-ethylisoluminol labeled specific peptide as CL probe coupling the magnetic separation technology was developed. The developed method has been applied to determination of caspase-3 in Jurkat cells extract with a satisfactory.

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  1. Department of Chemistry and Chemical Engineering, Ankang University, Ankang, 725000, China

    Yaping Li

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  1. Yaping Li
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Correspondence to Yaping Li.

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Li, Y. Chemiluminescent determination of the activity of caspase-3 using a specific peptide substrate and magnetic beads. Microchim Acta 177, 443–447 (2012). https://doi.org/10.1007/s00604-012-0798-1

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  • DOI: https://doi.org/10.1007/s00604-012-0798-1

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