Construction And Applications Of Fluorescent Metal Nanoclusters Based Sensing Platforms In Biological Enzyme Analysis

Biological enzymes are essential substances for human life activities,which regulate the specific and efficient catalytic reactions to maintain the normal operational function of body.The aberrant expression of important biological enzymes has been confirmed to be closely related to the occurrence of some malignant diseases,and biological enzymes have been regarded as biomarkers for the related clinical diagnosis.Therefore,it is urgent to establish low-cost,simple,rapid and accurate analytical methods of biological enzymes on the purpose of contributing to the diagnosis and accurate treatment of related diseases.Among the numerous detection strategies of biological enzymes,fluorescent methods stand out in the field of biological enzyme sensing as a result of their inherent virtues of simple operation,low cost,fast response,high sensitivity,good selectivity and so on.Specially,selecting fluorescent materials with excellent properties to perform as probes is one of the critical factors for constructing fluorescent methods with high sensitivity and selectivity.Recently,metal nanoclusters(e.g.,Au,Ag and CuNCs)have attracted great attention as a novel type of nanomaterial in the research of nanomaterial.Benefiting from their excellent and unique optical,chemical and electrical properties,such as ultra-small size,inherent magnetism,high catalytic activity and strong luminescent properties,metal nanoclusters have wide application prospects in the fields of biological analysis and imaging,medical diagnosis and treatment,energy,catalysis and electronic equipment.Specially,metal nanoclusters have extremely outstanding fluorescent properties of high optical stability and large Stokes shift.Meanwhile,due to their convenient synthesis strategy,low toxicity and good biocompatibility,metal nanoclusters,performing as a kind of promising fluorescent probes,have been widely used in fluorescent detection,pH and temperature sensing,cell or in vivo bio-imaging and so on.In this paper,on the basis of the excellent fluorescent properties of metal nanoclusters and taking advantages of fluorescent methods,we constructed a series of fluorescence sensing platforms for the efficient,convenient and sensitive analysis of important biological enzymes through the preparation of different metal nanoclusters to act as fluorescent probes,post-modification of metal nanoclusters,integration with other novel materials and so on.Besides,the obtained results also confirmed that our designed sensing strategies exhibited good analytical performance in the complex biological samples.The main contents are as follows:In the first chapter,we studied the research background of metal nanoclusters and systematically discussed and summarized their synthesis,fluorescence properties and fluorescence properties-based applications.Finally,we expounded the design concept and research significance of this paper.In the second chapter,a facile,label-free and sensitive fluorometric strategy for trypsin detection was established based on the fluorescence resonance energy transfer(FRET)between gold nanoclusters(AuNCs)and aggregated gold nanoparticles(AuNPs)via protamine as a bridge.Protamine can trigger the aggregation of AuNPs and link AuNCs with aggregated AuNPs through electrostatic interaction.Compared with monodispersed AuNPs,the UV-vis absorption band of aggregated AuNPs possessed a considerable overlap with the emission spectrum of AuNCs.Thus,the fluorescence of AuNCs was obviously quenched by the aggregated AuNPs through FRET.In the presence of trypsin,protamine was hydrolyzed to lead to the de-aggregation of AuNPs and break the close distance between AuNPs and AuNCs,so the FRET process would be inhibited,and the fluorescence of AuNCs would be recovered.The change in the fluorescence intensity of AuNCs was directly related to the amount of trypsin.Hence trypsin can be determined on the basis of the variation of fluorescence intensity with a linear range of 5–5000 ng m L^–1 and a detection limit1.9 ng m L^–1.This system was used for the detection of trypsin inhibitor.Meanwhile,the sensing method was applied for trypsin detection in human urine and commercial multienzyme tablets samples with satisfactory results.In the third chapter,a fluorescent sensing platform was constructed for adenosine deaminase(ADA)analysis based on the fluorescence resonance energy transfer(FRET)between the split aptamer modified gold nanoclusters(AuNCs)and gold nanoparticles(AuNPs).A single adenosine triphosphate(ATP)aptamer was split into two fragments(referred to as P1 and P2).P1 was covalently attached to the AuNCs at the 5?-end(P1-AuNCs)through the amide bond,and P2 was labeled with AuNPs at the 3?-end(P2-AuNPs)through the Au–S bond.In the presence of ATP,ATP specifically bound with its split aptamer(P1 and P2)to link P1-AuNCs and P2-AuNPs together,thus the fluorescence of P1-AuNCs was quenched via FRET from P1-AuNCs to P2-AuNPs.With the addition of ADA,ATP was transformed into inosine triphosphate,and then P1 and P2 were released to cause the fluorescence recovery of the system.So a split aptamer based fluorescent platform for ADA detection can be established via the fluorescence intensity change of the system.This platform showed a good linear relationship between the fluorescence intensity change of P1-AuNCs in the system and ADA concentration in the range of 2–120 U L^–1,and the limit of detection was 0.72 U L^–1.Moreover,the detection of ADA in human serum sample demonstrated the accuracy and applicability of the method for ADA detection in real sample.In the fourth chapter,fluorescent silver nanoclusters(AgNCs)were synthesized by one facile,low-cost and green method with histidine acting as templating agent and ascorbic acid performing as reducing agent.In addition,we designed a new strategy for fabricating bioresource derived N-containing porous carbon supported iron(Fe/NPC).Through the integration of Fe/NPC and AgNCs,a novel dual-channel biosensing system was strategically fabricated for sensitively determining acetylcholinesterase(ACh E).Fe/NPC with the oxidase-like activity can catalyze the oxidation of colorless 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)to its oxidation product(ox ABTS).The ox ABTS possessed the apparent absorbance and ability to effectively quench the fluorescence of AgNCs through inner filter effect.However,ACh E can catalyze the hydrolysis of acetylthiocholine to thiocholine,which can inhibit the Fe/NPC-regulated oxidation of ABTS to cause the absorbance fading and fluorescence recovery of AgNCs.The limits of detection of ACh E can achieve as low as 0.0073 and 0.0032 U L^–1 by the colorimetric and fluorometric modes,respectively.This dual-signal system was applied to analyze human erythrocyte ACh E and its inhibitor with robust analytical performance.In the fifth chapter,a ratio fluorescence method was developed for T4polynucleotide kinase(PNK)analysis based on the in situ formation of dual-emitting graphene quantum dots-copper nanoclusters(GQDs-CuNCs)nanohybrid.Amino capped single-stranded DNA(ss DNA)was firstly used to modify GQDs(GQDs-ss DNA)and then hybridize with its complementary DNA strand to form double-stranded DNA(ds DNA)functionalized GQDs(GQDs-ds DNA).The ds DNA on GQDs-ds DNA surface can act as the effective template for preparation CuNCs with fluorescence emission at 594 nm.When ds DNA of GQDs-ds DNA was phosphorylated through T4 PNK and subsequently degraded via?exonuclease to produce mononucleotides and GQDs-ss DNA,the formation of fluorescence CuNCs in GQDs-CuNCs was blocked due to the lack of effective ds DNA substrate,during which the fluorescence of GQDs at 446 nm in nanohybrid was nearly not influenced.Thus,with the CuNCs serving as the reporter and GQDs acting as the reference signal,T4 PNK activity can be monitored through the fluorescence intensity ratio(F₅₉₄/F₄₄₆)change of the system in the range of 0.01–10 U m L^–1 with a detection limit of 0.0037U m L^–1.Furthermore,the practicality of this T4 PNK activity analysis strategy in complex sample was performed in cell lysates.In the sixth chapter,a ratio fluorometric sensing platform was constructed for ultra-sensitive detection of alkaline phosphatase(ALP)by integrating silver ions(Ag⁺)modified gold nanoclusters(Ag-AuNCs)and o-phenylenediamine.The AuNCs fluorescence was greatly enhanced via simply modifying with Ag⁺.With the addition of ALP,substrate ascorbic acid 2-phosphate was transformed to ascorbic acid(AA).Then,a redox reaction between KIO₃ and AA was employed to produce I₂/I^–and dehydro-ascorbic acid(DHA).The I₂/I^–caused the notable fluorescence quenching of Ag-AuNCs at 600 nm through I^–combining with Ag⁺and I₂ triggered oxidative etching and aggregation of Ag-AuNCs.Meanwhile,DHA reacted with o-phenylenediamine to form a fluorescent quinoxaline derivative with apparent emission at 438 nm.Therefore,by measuring fluorescence intensity ratio(F₄₃₈/F₆₀₀)of this fluorometric platform,ALP activity was sensitively monitored with a low detection limit of 0.0035 U L^–1.Moreover,the practical application was demonstrated by ALP analysis in human serum with high accuracy and reliability.In the seventh chapter,we systematically summarized the contents of this thesis,and highlighted the future perspectives.