Gold Nanoparticles-Based Colorimetric Assay for Cathepsin B Activity and the Efficiency of Its Inhibitors

Abstract

Cathepsin B has been suggested to be a prognostic marker of melanoma, glioma, and a variety of cancers such as brain, breast, colon, esophageal, gastric, lung, ovarian, and thyroid cancers. Cathepsin B inhibitors have also been considered as anticancer drug candidates; hence, there has been a growing need for a probe which enables the selective and simple detection of cathepsin B and its inhibitors. For the purpose of selective assay, a cathepsin B-specific substrate, N,N'-diBoc-dityrosine-glycine-phenylalanine-3-(methylthio)propylamine (DBDY-Gly-Phe-MTPA) was synthesized in this study. Phe-MTPA, which was produced via cathepsin B-catalyzed hydrolysis of DBDY-Gly-Phe-MTPA, allowed aggregation of gold nanoparticles (AuNPs) leading to a color change from red to blue. When tested for cathepsins B, L, and S, this assay method exhibited AuNPs color change only in reaction to cathepsin B. The limits of detection for cathepsin B was 10 and 5 nM in the 1 and 2 h hydrolysis reactions, respectively. The efficiency of cathepsin B inhibitors such as leupeptin, antipain, and chymostatin was easily compared by the degree of color change. Moreover, IC50 values of leupeptin, antipain, and chymostatin were found to be 0.11, 0.48, and 1.78 μM, respectively, which were similar to the results of previous studies. Therefore the colorimetric assay of cathepsin B and cathepsin B inhibitors using DBDY-Gly-Phe-MTPA and AuNPs allowed not only the selective but also the simple assay of cathepsin B and its inhibitors, which was possible with the naked eye.

Sensitive colorimetric assays for α-glucosidase activity and inhibitor screening based on unmodified gold nanoparticles

“…However, with the addition of AGIs, the α-glucosidase activity is inhibited and cysteine induced aggregation of AuNPs would be recovered. The employing of unmodified AuNPs provides a simple assay avoiding the involved complicated and time-consuming functional manufacture of AuNPs [36][37][38]. This colorimetric assay of α-glucosidase activity and inhibitor screening is simple and cost effective, which may have great potential application in the drug screening and clinical diagnose in the future.…”

Solution-based nanosensors for in-field detection with the naked eye

“…For example it has been shown that the protease cathepsin B can be detected with high sensitivity and specificity when the enzyme hydrolyses the substrate N,N -diBoc-dityrosine-glycinephenylalanine-3-(methylthio)-propylamine (DBDY-Gly-Phe-MTPA) in the presence of gold nanoparticles. 101 The resulting product of the enzymatic reaction (Phe-MTPA) is positively charged and can induce the aggregation of the gold nanoparticles, which results in the characteristic change in the colour of the solution from red-to blue-coloured (Fig. 7B).…”

Gold Nanoparticle-Based Colorimetric and Electrochemical Methods for Dipeptidyl Peptidase-IV Activity Assay and Inhibitor Screening

“…For example, because of their high extinction coefficients and the unique size-dependent optical properties, AuNP-based colorimetric assays have been reported for detection of many types of analytes, including monitoring the enzyme activity [21,22,23,24,25,26] and measuring the concentration levels of nucleic acids [27,28,29], proteins [30,31], metal ions [32,33,34] and other small molecules [35,36,37,38,39]. As to the assay of protease activity with AuNPs as the probes, two strategies could be commonly used.…”

“…In the first method, peptide substrate containing two binding tags can crosslink AuNPs before the enzymatic reaction, thus inducing their aggregation and color change [24]. The second method is based on the non-crosslinking AuNPs aggregation in which the peptide substrate either before or after the cleavage reaction could cause the loss of surface charges of AuNPs and destroy the stability of the negatively charged AuNPs [25,26]. Since DPP-IV is a peptidase responsible for the cleavage of X-Pro-peptide, where X represents an amino residue, the previously reported AuNP-based colorimetric protease assays pave us a new way for the detection of DPP-IV by rationally designing the sequence of DPP-IV substrate.…”