Gold nanoparticle-based fluorescence quenching via metal coordination for assaying protease activity

Se Yeon Park,So Min Lee,Gae Baik Kim,Young-Pil Kim

doi:10.1007/s13404-012-0070-9

Abstract

We report a gold nanoparticle (AuNP)-based fluorescence quenching system via metal coordination for the simple assay of protease activity. Carboxy AuNPs (5 nm in core diameter) functioned as both quenchers and metal chelators without requiring further modification with multidentate ligands; therefore, they were strongly associated with the hexahistidine regions of dye-tethered peptides in the presence of Ni(II) ions, leading to notable fluorescence quenching over the varying molar ratios of dye to AuNP. Upon the addition of matrix metalloproteinase-7 (MMP-7), the fluorescent intensity was efficiently recovered in one-pot mixture especially at 10:1–100:1 molar ratios of dye to AuNP. Consequently, the dequenching degree was dependent on the MMP-7 concentration in a hyperbolic manner, ranging from as low as 10 to 1,000 ng mL−1. In this regard, we anticipate that the developed system will give us a general way to construct nanoparticle–dye conjugates and will find applications in the analyses of many other proteases mediating significant biological processes with low background and high sensitivity.

Highlights

The interactions between gold nanoparticles (AuNP) and organic dyes have gained considerable interest in biochemical assay because they provide many advantages regarding quenching efficiency and photostability over the classical dye quencher system [1,2,3,4,5,6]
The ability of AuNPs to induce fluorescence quenching of proximal dyes is reported to be directed by a surface energy transfer process [7,8,9]; the rate of energy transfer from a dye to AuNP depends on the inverse of fourth power of the donor–acceptor separation, which triggers a much longer working distance than that observed in a traditional fluorescence resonance energy transfer system
To construct an efficient quenching system, peptide substrates comprising red dyes (TAMRA) at their N-termini and hexahistidines at their C-termini were mixed with carboxy AuNPs in the presence of Ni(II) ions (Scheme 1a)

Summary

Introduction

The interactions between gold nanoparticles (AuNP) and organic dyes have gained considerable interest in biochemical assay because they provide many advantages regarding quenching efficiency and photostability over the classical dye quencher system [1,2,3,4,5,6]. 1 mM), and MMP-7 protease (10 μL at different stock concentrations) were mixed at a time in 20 mM Tris buffer (pH 7.5) to give a final volume of 100 μL and incubated at 37 °C for 2 h. It was followed by monitoring the emission spectra of the solution using a spectrofluorometer. TAMRA peptide (2.5 μL at 10 μM), MMP-7 (10 μL at different stock concentrations), and 20 mM Tris buffer (77.5 μL) were initially mixed and incubated at 37 °C for 2 h, followed by the addition of AuNPs (2.5 or 5 μL at 1 μM) and NiCl2 (10 μL at 1 mM). Fluorescence intensity was normalized to the background intensity from the control solution without protease

Results and discussion

Histag

Conclusion