期刊
BIOSENSORS-BASEL
卷 11, 期 10, 页码 -出版社
MDPI
DOI: 10.3390/bios11100362
关键词
surface plasmon resonance; protease; caspase; avidin-biotin interaction
资金
- Program for Innovative Research Team of Science and Technology in the University of Henan Province [21IRTSTHN005]
- National Natural Science Foundation of China [21705166]
This study presented a heterogeneous surface plasmon resonance (SPR) method for protease detection by integrating homogeneous reaction, with enhanced sensitivity through signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG).
The heterogeneous assays of proteases usually require the immobilization of peptide substrates on the solid surface for enzymatic hydrolysis reactions. However, immobilization of peptides on the solid surface may cause a steric hindrance to prevent the interaction between the substrate and the active center of protease, thus limiting the enzymatic cleavage of the peptide. In this work, we reported a heterogeneous surface plasmon resonance (SPR) method for protease detection by integration of homogeneous reaction. The sensitivity was enhanced by the signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG). Caspase-3 (Cas-3) was determined as the model. A peptide labeled with two biotin tags at the N- and C-terminals (bio-GDEVDGK-bio) was used as the substrate. In the absence of Cas-3, the substrate peptide was captured by neutravidin (NA)-covered SPR chip to facilitate the attachment of SA-IgG by the avidin-biotin interaction. However, once the peptide substrate was digested by Cas-3 in the aqueous phase, the products of bio-GDEVD and GK-bio would compete with the substrate to bond NA on the chip surface, thus limiting the attachment of SA-IgG. The method integrated the advantages of both heterogeneous and homogeneous assays and has been used to determine Cas-3 inhibitor and evaluate cell apoptosis with satisfactory results.
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