Preparation Of Fluorescent Metal Nanoclusters And Their Applications In Biomolecular Detection

The content of biomolecules,such as amino acids,dopamine,and various biological enzymes involved in various chemical reactions directly affect the health of biological organisms.Therefore,realizing sensitive detection of biomolecules is significant for the diagnosis and treatment of diseases.To date,several conventional medical techniques for biomolecules analysis have been exploited,such as colorimetry,electrochemistry,mass spectrometry,and chromatography protocols.However,the above-mentioned methods are limited by the complex procedure of sensing or high cost of the apparatus despite their distinct features in terms of favorable sensitivity and reliability.In contrast,fluorescence-related assays are widely utilized in biosensing and bioimaging due to their high sensitivity,simple operation,fast response time,and low cost.Thus,the selection of suitable materials as fluorescent probes and the construction of efficient and sensitive fluorescent sensing platforms are the focus of sensing research topics.Metal nanoclusters（NCs）have broad application prospects in biomedicine and environmental fields owing to their tunable optical properties,low toxicity,and ease of modification.To date,great progress has been made in the study of the physicochemical properties of NCs.However,problems,such as low quantum yield and difficult size control still exist,severely limiting the practical application of NCs.In this thesis,monodisperse 5-methyl-2-thiouracil-stabilized gold nanoclusters were first prepared.Several novel fluorescent NCs were then designed and synthesized by regulating their physical and chemical properties through strategies,such as size adjustment,metal doping,and ligand structure regulation.Combined with a variety of characterization techniques,such as transmission electron microscopy,X-ray photoelectron spectroscopy,Fourier transform infrared spectroscopy,UV-vis absorption spectroscopy,and fluorescence spectroscopy,the morphology,structure,and luminescence mechanism of NCs were systematically explored.By combining the obtained fluorescent NCs with other fluorescent molecules or catalytic materials,a series of fluorescence sensing platforms were constructed for the sensitive detection of biomolecules（α-glucosidase,xanthine,alkaline phosphatase,and sarcosine）.This thesis mainly consisted of six parts:In chapter 1,a brief introduction of the research background of metal nanoclusters was first provided.The synthesis of metal nanoclusters,strategies for regulation of fluorescence properties of metal nanoclusters,and applications in optical sensors of metal nanoclusters were then summarized and discussed.Finally,the research content and significance of this paper were summarized.In chapter 2,gold/silver bimetallic nanoclusters（Au/AgNCs）with tunable fluorescence emission wavelength were synthesized utilizing 5-methyl-2-thiouracil（5-MTU）as a reducing agent and stabilizer.Under the optimal molar ratio of Au/Ag,low concentrations of 5-MTU generated green-emitting Au/Ag（g）NCs,while high concentrations of 5-MTU induced red-emitting Au/Ag（r）NCs.By combining Au/Ag（r）NCs with fluorescent polydopamine（PDA）,a ratiometric fluorescent probe was constructed forα-glucosidase analysis.In this sensing process,the fluorescence emission of Au/Ag（r）NCs at 600 nm were initially quenched by the self-polymerization product of dopamine in the glycine-sodium hydroxide buffer solution through a photoinduced electron transfer（PET）mechanism.The addition ofα-glucosidase to the system led to the hydrolysis of the substrate AAG to form AA.The resultant AA could effectively alleviate the polymerization process of DA,leading to a reduction of the fluorescence intensity of PDA at 470 nm and enhancement in a fluorescent signal of Au/Ag（r）NCs at 600 nm.Quantitative analysis ofα-glucosidase was achieved by monitoring the changes in the ratio of the fluorescence signal intensity(F₆₀₀/F₄₇₀).In addition,the constructed ratiometric fluorescent probe based on Au/Ag（r）NCs/PDA can be used for the screening ofα-glucosidase inhibitor acarbose.In chapter 3,lysozyme-functionalized 5-methyl-2-thiouracil-stabilized gold nanoclusters（5-MTU-LZ@GNCs）were designed and synthesized based on the robust interaction between 5-methyl-2-thiouracil and lysozyme.By combining yellow-emitting 5-MTU-LZ@GNCs with iron-doped nanosheets（Fe/CNS）with peroxidase-like activity,a fluorescence detection platform with high sensitivity and strong anti-interference ability was fabricated.In the presence of O₂,xanthine was catalyzed by xanthine oxidase（XOD）to generate uric acid and hydrogen peroxide（H₂O₂）.The resultant H₂O₂ was catalyzed by Fe/CNS to generate reactive oxygen species(ROS,O₂^·-/¹O₂),followed by oxidation of p-phenylenediamine（PPD）to PPDox.Then,the resultant PPDox quenched the fluorescence emission of5-MTU-LZ@GNCs at 550 nm through fluorescence resonance energy transfer（FRET）.Sensitive analysis of xanthine was achieved by monitoring the changes in the fluorescence intensity of the sensing system.The constructed fluorescent sensing platform based on 5-MTU-LZ@GNCs and Fe/CNS not only provided novel inspirations for xanthine-related assay,but also broadened the application of nanoclusters/nanozymes in the field of bioanalysis.In chapter 4,lysozyme-functionalized 5-methyl-2-thiouracil-stabilized gold/silver bimetallic nanoclusters（5-MTU/Lys Au/AgNCs）were synthesized based the synergistic effect between Au and Ag and the interaction between 5-MTU and lysozyme.An"off-on-off"fluorescent sensing platform was established for sensitive determination of alkaline phosphatase（ALP）by the integration of 5-MTU/Lys Au/AgNCs and the nanozyme with a graphene structure（Fe-G Nanozymes）.The fluorescence of 5-MTU/Lys Au/AgNCs at 600 nm was first quenched by indigo carmine（IC）through the inner filter effect（IFE）.After the introduction of H₂O₂ and Fe-G nanozyme into the system,the generated reactive oxygen species(ROS,¹O₂/O₂^·–)promoted the oxidative degradation of IC,simultaneously restoring the fluorescence emission of 5-MTU/Lys Au/AgNCs.Moreover,ALP effectively catalyzed the hydrolysis of L-ascorbic acid-2-phosphate（AAP）to generate ascorbic acid（AA）,which inhibited the oxidative degradation of IC,thereby reducing the fluorescence intensity of 5-MTU/Lys Au/AgNCs.By the integration of ALP/AAP with Fe-G Nanozyme/IC,a fluorescent sensing platform based on enzyme/nanozyme cascade reaction was constructed for the specific analysis of ALP.In addition,this sensing method obtained satisfactory results for the analysis of ALP in serum samples.In chapter 5,yellow-emitting gold nanoclusters（AuNCs）were prepared with5-methyl-2-thiouracil acting as the stabilizer.In addition,nitrogen-doped copper nanosheet（CuT@N NS）was designed and synthesized by pyrolysis of the tryptophan‐based Cu‐containing nanosheet（CuT NS）and graphitic carbon nitride（g-C₃N₄）.A fluorescent sensing platform for sarcosine was constructed by utilizing AuNCs as a fluorescent signal indicator and CuT@N NS as a peroxidase substitute.In this sensing process,sarcosine was first catalyzed by sarcosine oxidase（SOx）to generate hydrogen peroxide（H₂O₂）.The as-obtained CuT@N NS decomposed H₂O₂into superoxide radicals(O₂^·-)and further catalyzed the oxidative coupling of4-aminoantipyrine（AAP）with phenol to pink-red quinone-imine dye（p-QID）.Then,p-QID quenched the fluorescence emission of AuNCs at 560 nm through inner filter effect（IFE）.By integrating sarcosine/SOx with CuT@N NS/IC,a fluorescence sensing platform based on enzyme/nanozyme cascade reaction was constructed for the specific detection of sarcosine.Additionally,the favorable accuracy and practicality of this fluorescence sensing platform was demonstrated by analyzing the sarcosine content in urine samples.In chapter 6,we systematically summarized the research works in this thesis and provided future outlooks for the development of metal nanoclusters.