The Enzyme Mimetic Activity Of Fluorescein And Its Analytical Applications

Nature enzyme,such as HRP,have been exploited for widespread applications such as enzyme-linked immunosorbent assay（ELISA）,biosensors,clinical diagnostics and organic synthesis,owing to their high efficiency and specificity under mild conditions.Considering the intrinsic drawbacks of natural enzymes,such as high cost in large scale production,poor stability of catalytic activities against denaturation,digestion or the variations of environmental conditions,researchers have therefore endeavoured to develop artificial enzymes as viable alternatives.Since Gao and Yan et al.found Fe3O4 magnetic nanoparticles with instinct mimic peroxidase property in 2007,construction of nanozymes has been focus of research over past decade.Compared with natural enzymes,these nanozymes are more stable against denaturation or protease digestion,low in cost,and easy to prepare,store and treat.Because most of them are spontaneous aggregation in aqueous solutions or under high salt conditions to lose the catalytical activity,it is necessary to introduce surface ligands.Nevertheless,it was found that the presence of capping agents may limit their enzyme-like activities.Moreover,the preparation of water-soluble nanozymes and the loading steps of nanomaterials are complicated and time-consuming.The low stability under high salt conditions and unsatisfied batch-to-batch consistence of peroxidase-like activity has unfortunately restricted their further applications in a way.Therefore,it is highly desirable to search for a new mimetic enzyme that possesses highly repeatable activity and highly stable against harsh condition with low cost.For the first time,we found fluorescein possesses mimic peroxidase and visible-light-induced oxidase mimic property.The catalytic mechanism and group effects on enzymatic activity were elucidated in detail.Based on enzymatic activity of fluorescein,colorimetric methods for H₂O₂,carboxylesterse（CaE）and adenosine triphosphate（ATP）detection were successfully construct.The concrete research content is as follow:（1）Fluorescein as an artificial enzyme to mimic peroxidaseWe report for the first time that fluorescein possesses an intrinsic peroxidase-like activity,which can be applied to quantitatively monitor the H₂O₂.Fluorescein can catalytically oxidize 3,3′,5,5′-tetramethylbenzidine（TMB）by H₂O₂ to produce a color reaction and follows the typical Michaelis-Menten kinetics.Results of electron spin resonance（ESR）experiments demonstrated that fluorescein exhibited catalytic ability to decompose H₂O₂ into ·OH radicals.Fluorescein exhibits better durability and stability against stringent conditions comparing with natural enzymes.The robustness of fluorescein makes it suitable for a broad range of applications in the biomedicine,industry and environmental chemistry fields.Our study may open up the possibility of small molecule as enzyme mimetics in biotechnology applications.This will be important for the future development of small molecule based enzyme mimetics.（2）Group effects on the peroxidase-like activity of fluoresceinRecently,an important and intriguing property of fluorescein to function as an artificial peroxidase was found.Although,as a new class of small molecule enzyme mimics,the catalytic activities should be highly associated with the composition and structure,little is known about the mechanism underlying the functional groups mediated enzyme-like activities.Herein,we report for the first time that fluorescein derivatives,i.e.,carboxyfluorescein（CF）and aminofluorescein（AF）possess peroxidase-like activity,and investigate the impact of different functional groups on the catalytic performance.Moreover,by systematically evaluating absorption intensity,electron spin resonance（ESR）signals,enzyme kinetic parameters and activation energies,we obtain evidence that the carboxyl group can enhance catalytic activity and may serve as substrate-binding sites,but the amine groups can even inhibit the catalytic reaction.These observations pave the way for identifying highly effective functional groups that promote the overall performance of fluorescein-based artificial peroxidase.Meanwhile,this work should facilitate the design and construction of tailor-made small molecule peroxidase mimics.（3）Colorimetric determination of carboxylesterse based on the peroxidase-mimicking activity of fluoresceinWe present a new analytical methodology for the carboxylesterse（CaE）detection,by taking advantage of the difference of fluorescein derivatives in peroxidase-mimicking activity reported by our group.Fluorescein diacetate（FDA）has no peroxidase-mimicking activity.CaE causes a rapid release of fluorescein by catalyzing hydrolysis of fluorescein diacetate（FDA）,and in turn the product fluorescein as mimic peroxidase can catalytically oxidize chromogenic substrate 3,3′,5,5′-tetramethylbenzidine（TMB）by H₂O₂ to produce colorimetric signals,providing the quantitative determination of CaE activity.The liner range for CaE sensing is from5 U/L to 5000U/L with a low detection limit of 1.7 U/L,and the proposed sensing method exhibits good selectivity and has been successfully applied to analysis of CaE activity in fetal bovine serum（FBS）.The research put forward a new idea to contruct analytical method using organic small molecule-based enzyme mimics as probe and based on the change of enzyme mimetic activity.Our proposed strategy opens a new horizon for CaE detection.More significantly,to the best of our knowledge,this is the first description of colorimetric strategy using enzyme mimics probe for CaE.（4）Fluorescein as artificial peroxidase for antibody-free and label-free colorimetric sensing of ATPHerein,an unprecedented colorimetric sensing probe for ATP was rationally designed on the basis of ATP remarkable inhibition on the peroxidase-like activity of fluorescein.ATP is covalently conjugated on fluorescein via the reaction between the triphosphate moiety on ATP and the carboxyl group on fluorescein,resulting in suppress of the catalytic performance of fluorescein to catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine（TMB）by H₂O₂ to produce a color reaction.Importantly,our analytical results exhibited an excellent sensitive response to ATP with a low detection limit of 3.2 n M and a wide linear range of 7.5 n M-500 n M.This proposed sensing strategy was successfully applied to ATP detection in fetal bovine serum（FBS）.Significant features of this new sensing probe are its simplicity,sensitivity and cost-effectiveness because of antibody-free and label-free.（5）Fluorescein as visible-light-induced oxidase mimicA novel visible-light-induced mimic oxidase activity of fluorescein was unprecedentedly found.Fluorescein can catalytically oxidize typitical chromogenic substrates e.g.3,3′,5,5′-tetramethylbenzidine（TMB）,2,2’-azino-bis（3-ethylbenzthiazoline-6-sulfonic acid）diammonium salt（ABTS）or o-phenylenediamine（OPD）to produce a color reaction under visible light irradiation.The catalytic reaction follows the typical Michaelis-Menten kinetics and exhibits higher affinity to TMB than that of HRP.In addition,we elucidated the photocatalytic mechanism in detail and found h+ and O₂^·-were the main reactive species account for TMB oxidation.Notably,fluorescein uses oxygen as a mild and green electron acceptor（not relies on unstable and destructive H₂O₂）,and the high catalytic activity can be attain fast and simply only through 10 min visible light irradiation.Significantly,its activity is easily controlled by visible light as a simple,clean,and rapid mean.（6）Group effects on visible-light-stimulated mimic oxidase activity of xanthene dyesFluorescein as one of the most commonly used fluorescent xanthene dye has wide application.The visible-light-stimulated mimic oxidase activity activities of eleven common and typical xanthene dyes with different structural characteristics,and the impact of different functional groups on the catalytic performance were further investigated.The eleven kinds of xanthene dyes including fluorescein（F）,eight types of fluorescein derivatives,（i.e.carbolxyfluorescein（CF）,aminofluorescein（AF）,calcein（CC）,2’,7’-dichlorofluorescein（DCF）,fluorescein chloride（FC）,fluorescein diacetate（FDA）,eosin B（EB）and erythrosine Y（EY））and two types of cationic dyes（i.e.,rhodamine B（Rh B）and rhodamine 6G（Rh6G））,with varied functional groups were used to explore effects of groups on the enzymatic activities.Except for FC and FDA,the other 8 kinds of xanthene dyes（CF,AF,CC,DCF,EY,EB,Rh B and Rh6G）possess visible-light-stimulated mimic oxidase activity Furthermore,by systematically evaluating absorption intensity and enzyme kinetic parameters,the important role of carboxyl groups and electrostatic affinity between 3,3′,5,5′-tetramethylbenzidine（TMB）substrate and functional groups of xanthene dyes were emphasized.The amine groups can even inhibit the catalytic reaction.The carboxyl groups and amine groups carboxylic electron-withdrawing groups can improve the the affinity to TMB substrate.However,the ester groups and amine groups are not helpful for the affinity.This word should facilitate the design of new class of visible light induced enzyme mimics with excellent performance.（7）Fluorescein as visible-light-induced oxidase mimetic for colorimetric detection of carboxylesterseCarboxylesterase（CaE）play the vital roles in drug metabolism,toxin detoxication and anticancer prodrug activation.It was discovered that human plasma carboxylesterase could be a novel serologic biomarker candidate for hepatocellular carcinoma,so ultrasensitive and selective detection of CaE in serum is very help for applying therapeutic prodrugs in the clinical practice.A new colorimetric method for CaE detection was constructed using fluorescein diacetate（FDA）hasing no photocatalytic activity as probe,on basis of the visible-light-induced oxidase-mimicking acivtity of fluorescein The CaE sensing platform was elaborated on the basis of fluorescein,in situ generated by CaE catalyzing FDA hydrolysis,possessing excellent photocatalytic capability to transform colorless 3,3′,5,5′-tetramethylbenzidine（TMB）into blue oxidized TMB（ox TMB）.Our proposed sensing platform displayed excellent analytical performance for the detection of CaE in a wide linear range from 0.04 to 20 U/L and a low detection limit of 0.013 U/L,which superior to the existing optical analytical methods.Moreover,the proposed sensing strategy has been successfully applied to CaE detection in fetal bovine serum（FBS）.