Preparation Of Metal Nanocluster Enzymes And Their Applications In Bioanalysis

Atomically precise metal nanoclusters（MNCs）are a class of potential biocatalysts due to their abundant active sites and good biocompatibility.Since MNCs have precise atomic configurations,and the size of MNCs is between discrete atoms and nanoparticles,which provides an opportunity to study the structure-activity relationship between atomic composition and its enzyme-like activity.This paper takes MNCs as the research object,explores the improvement of the activity of cluster enzymes by single atom substitution,further explores their catalytic mechanism,and successfully applies them to the simple,sensitive and visual detection of a variety of biomolecules,the main research contents are as follows:1.Au₂₅ NCs as peroxidase-like enzyme for ALP activity detection:We designed and synthesized Au₂₅（MPA）₁₈ NCs with atomic-level precise structure,and explored their peroxidase-like activity and catalytic mechanism.By logical adjustment of Au₂₅ enzyme activity,a simple PPi detection sensor is designed.Based on the principle that alkaline phosphatase（ALP）can hydrolyze PPi and thus restore the peroxidase activity of Au₂₅,we used the color reaction of Au₂₅ catalyze H₂O₂ oxidation TMB as a signal amplifier and built an ALP activity comparison sensor platform with PPi as a substrate,and calculated that ALP detection limit is 0.49 U/L.This analysis method can be extended to other types of substrates-H₂O₂system and be used to determine other types of phosphatase.This work will help design other MNCs with simulated enzyme activity to directly detect some important biomolecules.2.Enhancement of Au₂₅ enzyme-like activity by single Ag atom substitution and the biological applications:Based on the precise atomic structure of Au₂₅ NCs,we wante to study the structure-activity relationship of cluster enzymes by substituting individual atoms.Au and Ag have similar electronic structures,we replaced the Au atom in Au₂₅ with an Ag atom and found that its enzyme-like activity was improved.We adopted the Au₂₄Ag₁ NCs replaced by a single Ag atom using the water phase synthesis method,and studied its catalytic mechanism and steady-stricken dynamics.By calculating the kinetic constants and specific activity（SA）of Au₂₄Ag₁ and Au₂₅,it is found that Au₂₄Ag₁ has smaller K_m,larger V_max and higher SA than Au₂₅,proving that the substitution of a single Ag atom can increase the activity of Au₂₅ NCs.Next,based on the inhibitory effect of phosphonic compounds on clusterzyme activity and the hydrolysis of ALP on phosphate compounds,we designed colorimetric sensors to detect PPi,ATP,ADP and ALP,and the detection limit of ALP was as low as 0.04 U/L.On this basis,according to the logic adjustment of enzyme activity,simple optical logic gates were constructed,and the optical signal was converted into digital signal,so that expanded the application of cluster enzymes.3.Enhancement of Au₂₅ enzyme-like activity by single Pt atom substitution and its biological applications:Platinum（Pt）-based catalyst has always been one of the most efficient catalysts.among which Pt single atoms have high enzyme-like activity.We successfully prepared Au₂₄Pt₁ NCs by single-atom substitution,not only exploring their enzyme-like activity and catalytic mechanism,but also performing steady-state kinetic analysis.The kinetic constants obtained were significantly better than that of Au₂₅.Through colorimetric analysis,the absorbance of Au₂₄Pt₁ was 2.3 times higher that of Au₂₅,indicating that the substitution of Pt atoms greatly improved the activity of clusterzymes.Next,we constructed PPi and ALP biosensors with high sensitivity and selectivity,where the detection limit of ALP was as low as0.014 U/L,and the expected results were also obtained in human serum samples.In addition,Au₂₄Pt₁ has also been successfully applied to the sensing platform for the determination of TAC in commercial beverages and the detection of small biological molecules such as glutathione,cysteine and dopamine,which has broadened the biological application of clusterzymes.