Surface Plasmon Resonance for Protease Detection by Integration of Homogeneous Reaction

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.

Switch-on Fluorescence Analysis of Protease Activity with the Assistance of a Nickel Ion-Nitrilotriacetic Acid-Conjugated Magnetic Nanoparticle

“…The nanomaterials usually exhibit superior quenching ability to molecular quenchers due to the long-range energy transfer. However, immobilization of peptide substrates on the solid surface may cause the steric hindrance effect to affect the configurational freedom of peptides, thus limiting their interaction with proteases and decreasing the cleavage efficiency [38,39]. In addition, peptide immobilization requires laborious and time-consuming procedures.…”

A General, Label-Free and Homogeneous Electrochemical Strategy for Probing of Protease Activity and Screening of Inhibitor

“…Traditional methods, such as enzyme-linked immunosorbent assay (ELISA), gel electrophoresis and high-performance liquid chromatography (HPLC), always involve tedious separation/washing steps, complicated equipment and/or low sensitivity for protease detection [ 2 , 3 ]. To overcome these shortcomings, various novel methods have been developed recently, including mass spectrometry, fluorescence, colorimetric assays, quartz crystal microbalance, surface plasma resonance (SPR), surface-enhanced Raman scattering (SERS), and electrochemical, photoelectrochemical or electrochemiluminescent assays [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. As a typical homogeneous assay, fluorescence methods have been broadly applied because of their distinguish merits of high sensitivity and fast response.…”

“…Despite the enhanced sensitivity, the electrode modified with redox-labeled peptide may suffer from obvious signal loss after long-term preservation [ 12 ]. Moreover, in heterogeneous assays, the access of substrate peptide immobilized on the electrode surface to the catalytic center of protease may be relatively blocked due to the steric effect [ 4 , 7 , 13 ]. Thus, immobilization-free homogeneous electrochemical biosensors have aroused wide interests for protease detection.…”