Preparation Of Gold Nanoclusters And Their Applications In Chemiluminescent Analysis

Chemiluminescence（CL）is an energy transition process in which chemical reactions take place accompanied by light radiations.Luminol,acridinium ester,peroxyoxalate,and Ru（bpy）₃²⁺CL reaction are four classical CL reaction categories.Luminol-H₂O₂ CL reaction is the most investigated CL system and employed as a promising tool in various fields such as chemistry,biology,medicine,agronomy,etc.Both metal ions and peroxidases were employed to catalyze luminol-H₂O₂ CL reaction early.In recent years,nanomaterials,as a kind of mimetic peroxidase,were reported as catalysts to catalyze the luminol-H₂O₂ CL reaction.These nanomaterials include metal nanoparticles,metal oxide nanoparticles,metal-organic frameworks,carbon dots,graphene oxide,metal nanoclusters,etc.Gold nanoclusters（Au NCs）which are composed of several to dozens of Au atoms have an average size of less than 2 nm.They have exhibited a variety of unique properties such as ultra-small size,good stability,biocompatibility,in particular,CL catalytic activity.In the second chapter,we established a CL detection method for the determination of alkaline phosphatase（ALP）activity by using the luminol-H₂O₂ CL reaction catalyzed by Au NCs.Alkaline phosphatase is an indispensable metalloenzyme present in mammals.Alkaline phosphatase can catalyze the hydrolysis of phosphate monoester bonds in an alkaline environment.Alkaline phosphatase is an important biomarker because the abnormal increase or decrease of its content can predict the occurrence of many diseases,such as liver disease,nephritis,diabetes,anemia,bone disease,and so on.Therefore,alkaline phosphatase activity determination is of great significance.Ascorbic acid is a broad-spectrum free radical scavenger that can effectively quench the luminol-H₂O₂ CL signal.Magnesium ascorbyl phosphate（MAP）is a water-soluble derivative of ascorbic acid.MAP can be hydrolyzed to generate ascorbic acid by alkaline phosphatase,but itself almost has no quenching effect on the Au NCs-catalyzed luminol-H₂O₂ CL reaction.Based on the inhibitory effect of ascorbic acid on the luminol-H₂O₂ CL reaction catalyzed by Au NCs,we established a CL detection method for the determination of alkaline phosphatase activity.We first optimized the experimental conditions including the MAP concentration,the pH of the hydrolysis reaction,the concentration of BSA-Au NCs and H₂O₂,and diluents for luminol working solutions.Under optimal conditions,the CL signal decreased with the increase of alkaline phosphatase concentration.The quenching efficiency（Q）had a linear relationship with the logarithm of the alkaline phosphatase concentration in the range of 0.0027-1.3890 U L^-1.The linear regression equation was Q=0.3439Log C+0.8964,and the regression coefficient was 0.9970.The detection limit was 0.0026 U L^-1.We believed that this was a general strategy for the CL detection of ALP activity because ascorbic acid was an efficient CL quencher for many CL reactions.It has been reported the CL catalytic activity of nanoparticles is related to their surface charge.Our group studied the effect of the surface charge of Au NCs on their CL catalytic activity.We modified ethylenediamine on the surface of BSA-Au NCs to prepare positively charged cationic Au NCs.Compared with negatively charged Au NCs,cationic Au NCs had a higher catalytic activity for the luminol-H₂O₂ reaction.However,the conjugation of Au NCs with antibodies led to a significant decrease of catalytic activity in the luminol CL reaction,because the positive charge of cationic Au NCs would be reduced greatly.To overcome this problem,we would like to prepareβ-carboxamide group-containing charge-reversal Au NCs in chapter three.The charge-reversal Au NCs were stable under neutral or alkaline conditions and hydrolyzed under acidic conditions to release cationic Au NCs with high catalytic activity in the luminol CL reaction.First,we selected maleic anhydride to react with polyethylenimine（PEI）to prepare charge-reversal polymer which containedβ-carboxamide groups.Then,the charge-reversal Au NCs were prepared by using the charge-reversal polymer as a template and glutathione（GSH）as a reducing agent.The CL intensity of the luminol-H₂O₂ CL reaction catalyzed by unhydrolyzed Au NCs was low,while the CL intensity catalyzed by the hydrolyzed Au NCs was enhanced significantly because of the generation of cationic Au NCs after hydrolysis in acidic condition.The experimental results were consistent with our assumptions.The charge-reversal Au NCs had no obvious absorption peak in the range of 200-800 nm.The FL spectrum showed that the maximum emission wavelength of charge-reversal Au NCs was 640 nm.TEM images illustrated that charge-reversal Au NCs had a typical spherical morphology with a particle size of less than 2 nm.The zeta potential of the charge-reversal Au NCs was-23.4 m V,and the zeta potential of the hydrolyzed Au NCs was 24.1 m V.This result demonstrated that the charge-reversal Au NCs were hydrolyzed in the acidic conditions and released cationic Au NCs.In addition,charge-reversal Au NCs showed good stability in 0.1 M Na Cl,0.1 M KCl,0.1 M PBS（pH 7.4）and 0.1 M Tris solution（pH 7-12）within 36 h.In chapter four,we optimized the hydrolysis conditions of charge-reversal Au NCs.The optimal hydrolysis time was 30 min and the HCl concentration was 10 m M.We also established a luminol-H₂O₂ CL reaction catalyzed by the hydrolyzed Au NCs.The optimal CL conditions were 50 m M H₂O₂ and 2 m M luminol prepared in 0.1 M Tris-HCl buffer（pH 8）.Under the optimal conditions,0.02 m M hydrolyzed Au NCs showed a 150-fold enhancement effect on the luminol-H₂O₂ CL reaction.In addition,the reproducibility test showed that the proposed system had good repeatability.We also investigated the effects of some common interfering substances on the hydrolyzed Au NCs-catalyzed luminol-H₂O₂ CL reaction,in which the tolerable concentration of these interferences to the luminol-H₂O₂ CL reaction catalyzed by the hydrolyzed Au NCs was taken as a 10%relative error in the CL signal.For some common ions,the tolerable concentration of Na⁺and K⁺was 5 m M,Mg²⁺and Ca²⁺was 0.05 m M,HPO₄^2-and H₂PO₄^-was 2.5 m M,HCO₃^-was 0.5 m M,and CO₃^2-was0.25 m M.In terms of amino acid,the tolerable concentration of alanine and glycine was 25 m M,L-aspartic acid was 5 m M,L-glutamic acid and L-arginine was 2.5 m M,and L-lysine was 0.025 m M.For some proteins,the tolerable concentration of BSA,calmodulin,streptavidin and Ig G were 25μg/L,5μg/L,2.5μg/L,and 0.5μg/L,respectively.