Synthesis Of Gold Naoclusters Under Acidic And Alkaline Conditions And Their Applications On Protease Detection

Recent advances in nanotechnology have given rise to a new class of fluorescentlabels, i.e. fluorescent metal nanoclusters. These nanoclusters are of significantinterest because they can provide the missing link between atomic and nanoparticlebehavior in metals. Composed of a few to a hundred atoms, their sizes are comparableto the Fermi wavelength of electrons, resulting in molecule-like properties includingdiscrete electronic states and size-dependent fluorescence. Ultrasmall size, goodbiocompatibility and photostability make them ideal fluorescent lables andbiodetection probes. This thesis focused on the preparation and application of goldnanoclusters based on the coating, stability and reduction by lysozyme. The mainconclusions in this thesis are as follows.(1) Firstly, we investigated the effects of pH, reaction time on the size andfluorescence properties of gold nanoclusters, and also analyzed the influence of theseconditions on the regulation of nanoclusters during their formation. We studied thechanges in lysozyme stabilized AuNCs followed by pH adjustment. The result showsthat the bule-emitting AuNCs will gradually turn to red-emitting AuNCs when pHgoes up. When pH goes down, the fluorescence intensity of red-emitting AuNCsdrops quickly. The results of this research not only helps understanding of thesynthesis mechanism of gold nanoclusters, but also helps to control the size ofAuNCs.(2) Pepsin is an active digestive enzyme present in the acidic environment ofanimal stomachs, and has been widely used to prepare bioactive peptides in the foodindustry. In this work, a simple fluorescence sensor for scissor-based detection ofpepsin activity was developed by using lysozyme-stabilized gold nanoclusters(AuNCs@Lyz) in aqueous media. Under acidic conditions (pH3.0), enzymaticdigestion of AuNCs@Lyz with pepsin results in a signifcant decrease of fuorescenceintensity. Notably, its acidic environment not only helps maintain the maximumfluorescence of gold nanoclusters, but also ensures the highest pepsin enzymaticactivity. In addition to offering high selectivity because of the unique proteolyticaction of pepsin under acidic conditions, this facile method provides high sensitivity. With the sensing system, the linear range for pepsin detection is found to be1μg/mLto100μg/mL, with a detection limit of0.6ng/mL at a signal-to-noise ratio of3.(3) Alkaline is a kind of enzyme widely used in various fields includingbiochemical engineering, food production, drug and washing industry. In this paper, aquick and sensitive assay for alkaline protease has been established based on thehydrolysis activity of protease to the lysozyme which is used to coat and stabilize thegold nanoclusters (Au NCs). Through the experiments, we obtained the optimumreaction condition. The optimum volume ratio of Au NCs and alkaline proteasesolution is1:9, while the optimum reaction temperature and reaction time are40oCand3h, respectively. Under these conditions, the linear range of this method is from2μg/mL to2000μg/mL (4×10-5~0.04unit/mL) with a limit of detection of0.1μg/mL(2×10-6unit/mL) and good selectivity. Therefore, this fluorescence detection shows itspotential in the detection of real samples.