Evaluating the Catalytic Efficiency of the Human Membrane-type 1 Matrix Metalloproteinase (MMP-14) Using AuNP–Peptide Conjugates

Abstract

Interactions of plasmonic nanocolloids such as gold nanoparticles and nanorods with proximal dye emitters result in efficient quenching of the dye photoluminescence (PL). This has become a popular strategy for developing analytical biosensors relying on this quenching process for signal transduction. Here, we report on the use of stable PEGylated gold nanoparticles, covalently coupled to dye-labeled peptides, as sensitive optically addressable sensors for determining the catalytic efficiency of the human matrix metalloproteinase-14 (MMP-14), a cancer biomarker. We exploit real-time dye PL recovery triggered by MMP-14 hydrolysis of the AuNP−peptide-dye to extract quantitative analysis of the proteolysis kinetics. Sub-nanomolar limit of detections for MMP-14 has been achieved using our hybrid bioconjugates. In addition, we have used theoretical considerations within a diffusion-collision framework to derive enzyme substrate hydrolysis and inhibition kinetics equations, which allowed us to describe the complexity and irregularity of enzymatic proteolysis of nanosurface-immobilized peptide substrates. Our findings offer a great strategy for the development of highly sensitive and stable biosensors for cancer detection and imaging.

Endoproteolysis of Oligopeptide-Based Coacervates for Enzymatic Modeling

“…Our findings are not unusual because enzyme kinetics on a scaffold (e.g., particle or membrane) have been a topic of debate, with some supporting a slow kinetic model that considers slow diffusion and steric crowdedness, , while others favor a fast kinetic model that emphasizes enzyme hopping and high local concentration . On the one hand, our finding agrees with a recent study by Keating et al, showing that phosphatase exhibits slightly higher activity toward substrates within coacervates compared to free substrates, and kinase activity was not significantly altered by the presence of coacervates .…”

“…S7) and extracting the first-order rate constant, K obs . The K obs was then correlated with the specificity constant by K obs = k cat [ E ]/ K M . This yielded the k cat / K M of 1208 M –1 s –1 , which is about 4-fold less than that of the free substrate (Figure S10).…”

“…The K obs was then correlated with the specificity constant by K obs = k cat [E]/K M . 52 This yielded the k cat /K M of 1208 M −1 s −1 , which is about 4-fold less than that of the free substrate (Figure S10). 41,50 Figure 4d indicates that the K M is 10.6 μM.…”

Supramolecular ‘catch-and-release’ strategy for bioorthogonal fluorogenic imaging across the visible spectrum

“…For this purpose, we explored the use of gold nanoparticles (AuNPs), which are known to be highly efficient cytosolic delivery platforms with the ability to quench proximal dye fluorescence across a broad spectral range. [73][74][75][76] In order to transform the AuNP into a guest quencher, we surfacefunctionalized it with XYL guest conjugated thiolated ligand and promoted the formation of quenched CB7-FL•XYL complex at the AuNP surface (Figure 5). Fluorescence quenching of the CB7-FL dyes by XYL decorated AuNPs (XYL-AuNPs) was investigated by titrating 1 μM solution of CB7-TAMRA (CB7-FL) with an increasing concentration of XYL-AuNP.…”

Plasmonic nanoprobes on single AuNTs for evaluating and monitoring the dynamic release of 2D drug carriers

“…With the development of nanotechnology, various biosensors and drug delivery systems constructed on gold nanoparticle (AuNP) substrates have become practical tools. [13][14][15] For example, Hong and co-workers 16 developed a biosensor that exploits the competition between AuNPs and microRNAs to bind to capture probes immobilized on the AuNP surface, thereby enhancing the sensitivity of detection. Hou and his colleagues 17 reported a phage-based surface plasmon resonance (P-SPR) strategy for detecting carcinoembryonic antigens (CEAs) in undiluted serum samples.…”