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

Peroxidase?POD,EC 1.11.1.x?is a kind of natural enzyme with hemoglobin as prostheric group,which widely exists in animals,plants and microbes.Due to strong specificity and high sensitivity,it has been widely applied to many aspects,such as protein,polypeptide,hormone,virus,parasite,biodegradation and nervous system.So far,one of the most widely studied peroxidases is horseradish peroxidase?HRP?,which can catalyze the oxidation of some electron donor substrates,such as o-phenylenediamine?OPD?,terephthalic acid?TA?,luminol etc.,in the presence of H₂O₂ accompanying with colour,fluorescence or chemiluminescence changes.HRP,as an important commercial diagnostic reagent,is often used in biological research.However,the applications of HRP in production practice are still seriously limited owing to high price and easy denaturation under harsh conditions.Ficin?EC 3.4.22.3?,a sulfydryl protease,is isolated from the latex of fig trees.And it has been found that it not only has possesses protease properties but also the property of peroxidase mimetic enzyme,which can also catalyze the oxidation organic substrates by H₂O₂ to produce products with specific color or fluorescence.In addition,except that the optimum conditions for ficin and HRP is similar,ficin can keep higher catalytic efficiency under the conditions of wide ranges of pH,temperature and inhibitors and the price is cheaper.Hence,it is also widely used in the field of environment,biology and medicine for analytical detection.In this work,we have constructed a colorimetric assay for the detection of glucose and two different fluorescence analysis methods for DA detection.?1?We developed a colorimetric biosensing system for glucose detection by coupling the peroxidase-like of ficin and the glucose oxidase?GOx?.GOx can catalyze the oxidation of glucose to produce H₂O₂,then ficin catalyzes the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine?TMB?by H₂O₂ to produce a blue color reaction with a typical absorption peak centered at 652 nm.The present sensing system showed a linear response toward glucose detection over range of 2.0-100?M with a detection limit of 0.5?M and this system was also applied to measuring glucose in human serum.Furthermore,in order to expand the application of ficin in biological sensing,we immobilized ficin onto the SiO₂@Fe₃O₄ NPs,which exhibited the merits of recycling as well as allowing the repeated detection of glucose.Thus it may provide great potential applications in biomedicine,biotechnology and environmental chemistry.?2?Dopamine?DA?,a catecholamine neurotransmitter secreted by the brain,is considered to be an important indicator of the detection of mental diseases in the clinic.As a mimetic peroxidase,ficin can catalyze H₂O₂ decomposition into·OH radicals,which can oxidize non-fluorescent tyramine into fluorescent dityramine.When DA was introduced,·OH radicals can oxidize DA resulting in the oxidation reaction of tyramine was inhibited,and the fluorescence intensity of the system was decreased.Thus,a novel fluorescent probe consisting of the ficin-H₂O₂-tyramine system for determining DA in cerebrospinal fluids?CSF?was developed.Under optimal conditions,the fluorescence intensity decreased linearly with DA concentration in the 0.05-12.0?M?R²=0.995?range with a detection limit of 46 nM.The presented sensing approach suggests its great potential for assay DA in chemical and biological analytical applications.?3?Polydopamine nanoparticles?pDA NPs?,a kind of popular polymer material,have been broadly applied to various areas owing to its excellent physicochemical properties.Herein,we employed ficin as a peroxidase-mimic to catalyze the oxidation DA in the presence of H₂O₂ to its quinone derivative and autopolymerize into synthesize intrinsic fluorescent pDA NPs.Within a certain concentration range,the fluorescence intensity of pDA NPs gradually enhanced with increasing concentration of DA.We developed a fluorescence turn-on method for rapid dopamine?DA?sensing which is based on monitoring the intrinsic fluorescence of pDA NPs.Under the optimum conditions,the strategy showed a good linear relationship from 10 nM to 5.0?M?R²=0.996?and provided an exciting detection limit of 5.5 nM.More importantly,the presented sensing approach has high sensitivity,good selectivity and has been successfully applied to DA sensing in human serum samples.With its good analytical figures of merit,our proposed analytical method is promising for the detection of DA-related diseases.