Amino Acid Incorporated Nanoflowers With Mimetic Peroxidase Activity For The Detection Of Glutathione

Natural enzymes are traditional biocatalysts with highly specific and efficient catalytic activity and are widely used in food processing,environmental monitoring,medical testing,etc.However,the inherent limitations of natural enzymes(easy denaturation,high cost,laborious preparation and difficult recovery)have limited their wider application.Since the 1950s,researchers have constructed highly stable,low-cost artificial enzymes designed to mimic natural enzymes using alternative materials.Nanoflower,a new type of artificial enzyme,has attracted much attention in the past two decades due to its obvious advantages(high catalytic activity,low cost,and easy mass production)compared with natural and conventional artificial enzymes,and has been applied to replace natural enzymes in biosensing,chemical analysis,and food industry.Amino acid hybrid nanoflower is a representative nanoenzyme with high potential application in bioassay field due to its large specific surface area,less agglomeration,low cost and high recycling rate.Glutathione is a tripeptide widely found in living organisms and has antioxidant properties.The level of glutathione in human serum is often associated with the pathogenesis of many diseases,such as Alzheimer's disease,malignant tumors,chronic renal failure,cataracts,etc.Therefore,the detection of glutathione levels in serum plays an important role in the early diagnosis and pathological analysis of diseases.Among the many methods to detect glutathione,the colorimetric assay has the advantages of simplicity,rapidity and sensitivity.Therefore,in this paper,we want to construct a colorimetric assay platform for the detection of glutathione with D-amino acid hybrid nanoflowers.This work was carried out as follows:(1)Nineteen D-amino acid hybrid nanoflowers were synthesized and compared for their catalytic activity in simulated peroxidases,and the D-isoleucine hybrid nanoflowers with the highest specific activity were selected for characterization using scanning electron microscopy,Raman spectroscopy,UV–vis diffuse reflectance spectroscopy,and X-ray electron spectroscopy.The results show that D-amino acids are key to the formation of nanoflowers and can influence the morphology,structure,and elemental components of nanoflowers.(2)The enzymatic properties of D-amino acid hybrid nanoflowers were investigated using D-isoleucine hybrid nanoflowers as a representative.It was found that D-isoleucine hybrid nanoflowers have the same enzymatic kinetics as natural peroxidase,they are peroxidase nanozyme.The hybrid nanoflowers present the maximum mimetic activity at p H 4.5 and 45°C.The Michaelis–Menten parameters of this nanoflower were calculated,and the Km value of H₂O₂ was 1.02 m M and Vmax value was 5.77×10^-8 M s^-1,the Km value of TMB was 2.71 m M and Vmax value was3.02×10^-8 M s^-1.The nanoflower enzyme has a higher affinity for the substrate H₂O₂than the natural peroxidase.(3)A colorimetric assay system was constructed with D-isoleucine hybrid nanoflower as the main enzyme,and the concentration of glutathione in solution was detected rapidly,sensitively and conveniently.The linear range of the assay system was1-30?M,and the detection limit was as low as 95.52 n M.With good specificity,stability and reusability,the assay system has broad development potential and market prospect in clinical diagnosis and treatment.