Analytical Application In Biological Detection Based On The Enhanced Peroxidase-like Activity Of Ficin

Enzymes,a class of biocatalysts composed of proteins and RNA,possess high catalytic efficiency and specificity,and play a leading role in biological processes such as metabolism,nutrition and energy conversion.Peroxidases,such as horseradish peroxidase(HRP),are a class of oxidoreductases produced by microorganisms or plants that can catalyze the oxidation of peroxidase substrates by hydrogen peroxide as an electron acceptor.Natural peroxidase has good specificity and high catalytic efficiency,which has been widely used in many biochemical analyses.However,due to its high production cost,difficulty in preservation,and poor tolerance to acid base and high temperature,its practical application is largely restricted.In recent years,artificial peroxidase mimics have attracted considerable attentions due to their long-term storage,lower cost and stronger stability against harsh conditions,which are considered as the alternatives for natural enzymes.Regrettably,some of these non-bioperoxidase mimics typically require complex preparation and modification procedures to avoid aggregation,with batch-to-batch differences in activity.Therefore,it is highly desirable to develop artificial peroxidase mimetics based on natural enzymes.Ficin is a natural thiol protease with peroxidase-mimicking activity.However,the peroxidase activity of ficin is relatively low,which limits its application in some aspects.In this paper,in order to enhance the peroxidase activity of ficin,ficin was encapsulated in a mesoporous metal-organic framework to form ficin@PCN-333(Fe),or N-ethylmaleimide(NEM)and tris(2-carboxyethyl)phosphine hydrochloride(TCEP)were used to reasonably block the thiol group of ficin to synthesize ficin-NEM(ficin-N)and ficin-TCEP-NEM(ficin-TN).Besides,in order to explore the excellent peroxidase catalytic performance of the three ficin derivatives,TMB-H2O2 chromogenic system was used to detect L-Cysand glucose in human serum successfully.(1)A mesoporous metal-organic framework,PCN-333(Fe),was prepared using H3 TATB,anhydrous ferric chloride and trifluoroacetic acid as raw materials,and used to encapsulate ficin to form ficin@PCN-3333(Fe).The results showed that the peroxidase activity of ficin@PCN-3333(Fe)increased about 3 times than that of ficin,and the kinetic parameters showed that ficin@PCN-3333(Fe)had higher affinity and catalytic constant for TMB and H2O2.Taking advantages of the high peroxidase-like activity,ficin@PCN-3333(Fe)was used for colorimetric determination of glucose with a low detection limit of 97 nM,and was successfully used to detect glucose in human serum.(2)Two procedures,i.e.,direct blocking with N-ethylmaleimide(NEM),or using tris(2-carboxyethyl)phosphine hydrochloride(TCEP)to interrupt disulfide bonds then blocking thiol groups with NEM,were applied to block thiol groups of ficin,ficin-NEM(ficin-N)and ficin-TCEM-NEM(ficin-TN)were produced,respectively.Compared with ficin,the peroxidase activity of ficin-N and ficin-TN increased about 2.5 times and5 times,respectively.A colorimetric method for the determination of biothiol was established under optimal conditions based on ficin-TN enhanced peroxidase activity,since biothiol inhibited the catalytic activity of ficin.Furthermore,this method has high sensitivity and can be used to detect L-cysteine in human serum.(3)Because thiol is very important the protease activity of ficin,ficin-TN obtained after reasonable blocking of the thiol group of ficin possesses the reduced protease activity and the enhanced peroxidase activity.Based on this,a colorimetric method with high sensitivity and selectivity was developed for the one-step detection of glucose.Compared to ficin,ficin-TN has a wider detection range and lower detection limit(88nM),and our one-step method is simpler and save-time compared with other two-step methods.In addition,the method can be used to detect glucose in human serum with broad application prospects.