Synthesis Of Fluorescent Metal Nanoclusters And Their Applications In Biosensors

Metal nanoclusters (NCs) consisting of several to tens of atoms are a new class of nanomaterials. The size of the NCs is comparable to the Fermi wavelength of the electrons. The ultra small size of the NCs gives rise to a variety of unique properties different from the large nanoparticles, such as tunable emission, good photostability, high photoluminescence quantum yield, large Stokes shift, low toxicity, and ease of preparation. Owing to these interesting features, metal NCs can be used in the field of metal ions and small biomolecules detection, enzyme activity detection, biological labeling, biological imaging, catalysis, and solar cells. In this thesis, we are focusing on the fluorescent metal NCs and investigate their synthetic methods and applications in the field of biosensors. Four parts of works were carried out as follows:First, enzyme catalyzed substrate hydrolysis isutilized to trigger a "top-down" etching process for the generation of fluorescent NCs, and the changes in emission intensity of the assay solution provide a facile way for the sensing of enzyme activity. Esterase and alkaline phosphatase (ALP) were used as the model enzymes. Proper substrates were designed and synthesized. Analkanethiol ligand6-mercapto-1-hexanol was released because of the substrate hydrolysis. The alkanethiol ligand could etch the AuNPs and fluorescent AuNCs formed in situ. The average size of the as-prepared Au NCs is1.3nm with a uniform size distribution. The Au NCs exhibit intense green emission with a peak maximum at503nm under395nm excitation. The fluorescence quantum yield is5.6%and the average lifetime is552ns. The increase in AuNC emission intensity could be directly related to the amount of enzyme in the assay solution, therefore, esterase and ALP concentration could be determined. The assay allowed the detection of0.1mU/mL esterase and0.01mU/mL ALP, respectively. This is one of the most sensitive nanomaterial-based ALP assays reported to date. In addition, our assay could be used to analyze enzyme activity in complex sample mixtures and evaluate the inhibition effectof the ALP inhibitor Na3VO4.Second, papain was used for the first time as an effective capping and reducing agent for the preparation of Au NCs through a simple green "bottom-up" chemical route. The synthetic route is simple which could be performed only in a basic condition, and the starting materials are commercially available and fairly inexpensive. The as-prepared Au NCs exhibit intense red emission with peak maximum at660nm under the excitation of470nm. Both the solution and the freeze-dried forms of clusters showed strong red emission under UV light. The average diameter of NCs is1.2nm, and they are pretty much uniform in size. The Au NCs are very stable. The fluorescence remained unchanged upon storage at room temperature for3months, and also in buffer solution of pH values in the range of3-12. The fluorescence of Au NCs could be quenched with Cu2+therefore, Cu2+could be sensitively detected using the as-prepred NCs. The detection limit is estimated to be3nM, which is much lower than the maximum safety level of Cu2+(20μM) in drinking water defined by the US Environmental Protection Agency. In addition, the assay method is highly selective for Cu2+detection. Little emission intensity changes were observed in the presence of ten interference metal ions.Third, good biocompatible fluorescent Au NCs stabilized by cysteine were first synthesized through a "top-down" method. The as-prepared Au NCs exhibit intense blue-green fluorescence with peak maximum at495nm under410nm excitation. The fluorescence quantum yield is3.3%. These clusters have good photostability. The average size is1.35nm with good size distribution. The fluorescence of as-prepareAu NCs could be quenched by Cu2+. PPi could recover the Cu2+-quenched Au NCs fluorescence selectively, and thus PPi concentration was determined. ALP could catalyze the hydrolysis of PPi, and Cu2+was released. As a result, the recovered fluorescence was again quenched by the liberated Cu2+, and ALP activity was detected sensitively. Through this "quenching-recovery-quenching" process of NCs, several biologically important molecules were determined using a single sensing system. Our method avoided organic molecules/macromolecules which could simplify the synthesis procedure, and is rather sensitive and convenient. This nanocluster-based label free method provides a new way to for the construction of biosensors.Finally, we prepared fluorescent Ag NCs in situ using Fenton reaction triggered generation of poly(methacrylic acid)(PMAA) as a template, and the changes in emission intensity of the assay solution provide a facile way for the sensing of glucose. The enzymatic oxidation of D-glucose in the presence of O2generated H2O2. Reactive radicals were generated from the Fenton reaction of H2O2and Fe2+, which initiated the polymerization of methacrylic acid (MAA). The product PMAA could be used as template to prepare fluorescent Ag NCs under UV irradiation. The Ag NCs exhibit intense orange emission with peak maximum at585nm under480nm excitation. The average size is1.64with a uniform distribution. The increase in AgNC emission intensity could be directly related to the concentration of glucose in the assay solution, therefore, glucose was detected selectively. Our method avoids the use of organic molecules and did not need to introduce boric acid functionalities to the nanomaterials, thus considerably simplify the sensing procedures. This nanocluster-based label-free fluorometric "turn-on" method provides a new way for selective and cost-effective detection of glucose.In conclusion, combining enzymatic reaction, metal nanoclusters were prepared using "top-down" and "bottom-up" methods. The excellent fluorescent properties make them possible for the sensitive detection of biologically important molecules. Our works provide a new platform for the fluorescent NCs used in the field of biosensors.