Abstract
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.
Highlights
Proteases play an important role in a wide variety of biological processes, including protein digestion, wound healing, apoptosis, fertilization, growth differentiation, and immune system activation [1]
In the fluorescence resonance energy transfer (FRET) assay, the commonly used method for protease activity detection, the peptides labeled with two different fluorophores at two ends are digested by protease in the aqueous phase [4]
The peptide substrate is anchored on a solid surface in the heterogeneous assay, and the enzymatic reaction happens at the solid-liquid interface [5,6]
Summary
Proteases play an important role in a wide variety of biological processes, including protein digestion, wound healing, apoptosis, fertilization, growth differentiation, and immune system activation [1]. The cleavage of low molecular weight peptides leads to a small, undetectable change in the refractive index [16,17,18] It usually requires a signal amplification strategy to detect protease by labeling the peptide substrate with nanomaterials or specific groups [19,20,21,22,23,24]. When the peptide substrate was digested by Cas-3 in the aqueous phase, the biotinylated products (bio-GDEVD and GK-bio) would compete with the substrate to bond NA on the chip surface (Channel 2) This prevents the attachment of bio-GDEVDGK-bio and the follow-up capture of SA-IgG by the avidin-biotin interaction.
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