Intrinsic Peroxidase-like Activity Of Halogen Fluorescein Derivatives And Their Analytical Applications

Horseradish peroxidase?HRP?is one of the most important biological peroxidase in chemical analysis,because of the high activity and substrate specificity under mild conditions,is widely used in biosensor,clinical diagnosis and etc.However,the practical application of HRP is limited by its strict storage requirements,poor stability,and high cost of preparation.Therefore,the search for the use of new substances instead of HRP has become a hot topic.In 2007,Yan's research team discovered the peroxidase mimetic activity of Fe₃O₄ magnetic nanoparticles which was similar to HRP.Since then,the research for preparation,properties and applications of nanoparticles were begun.Compared with natural enzymes,these nanoparticles have the advantages of controllable synthesis,stable,reusable,strong tolerance to high temperature or strong acid and strong base,low cost of preparation and storage.However,the preparation of some nanoparticles was complex,and the catalytic activity has the difference between batch and batch,which leads to the poor reproducibility of the experiment.And the catalytic activity of nanoparticles is closely related to their surface modification groups.Although some of them?such as carboxyl and amino groups?can increase the water solubility of the nanoparticles,they will reduce the catalytic activity of the nanoparticles.Therefore,the shortcomings mentioned above limited the application and development of nanoparticles.In 2016,our team discovered that fluorescein as a small organic molecule,fluorescein also has peroxidase activity similar to HRP.Inspired by this discovery,in this paper we found that 2?,7?-dichlorofluorescein?DCF?and 2',7'-difluorofluorescein?DFF?have the peroxidase-like activity,on the basis on theses findings,the detection of hydrogen peroxide?H₂O₂?,glucose and galactose were established.At the same time,we systematically studied the effect of halogen atoms on the peroxidase-like activity of fluorescein.The specific contents are as follows:?1?We discovered for the first time that DCF as a novel peroxidase mimic can catalyze the oxidation of classical peroxidase substrate TMB in the presence of H₂O₂,producing a colour reaction.Although a variety of peroxidase-likes have been actively developed recently,few study has been made towards the small molecule peroxidase mimic.As a novel peroxidase mimic,DCF was verified to be highly stable,well reproducible and of low cost,exhibiting typical Michealis-Menten kinetics,and showed strong affinity towards H₂O₂ and TMB.On the basis of these findings,we further demonstrated the novel use of DCF for easy and simple detection of H₂O₂ and glucose.This kind of new small molecule peroxidase mimic is expected to be used in medical clinical diagnosis,and this novel finding not only further confirm the peroxidase activity of small molecules of fluorescein derivatives,but also open new opportunities to deepen the knowledge of the new class of small molecule enzyme.?2?The peroxidase-like activity of DFF,was investigated using TMB as a chromogenic substrate in the presence of H₂O₂.DFF could catalyze oxidization of TMB by H₂O₂ to produce a blue coloured oxidation product?ox-TMB?.Effect of various reaction conditions,such as pH,temperature,H₂O₂ concentration and reaction time on the catalytic activity of DFF was studied.The peroxidase-like activity of DFF was found to follow Michaelis–Menten kinetics,and its catalysis accorded with ping-pong mechanism.The calculated kinetic parameters(K_cat)of DFF catalysis showed higher peroxidase-like activity than fluorescein and 2?,7?-dichlorofluorescein?DCF?.According to the radical capture and electron spin resonance?ESR?spectroscopy results,we confirmed that hydroxyl radical?·OH?is the active specie of catalytic process.It is known that the oxidation of galactose by galactose oxidase?GAOx?enzyme leads to the formation of H₂O₂,the H₂O₂ released in this reaction was consequently quantified using DFF as peroxides mimics and TMB as the chromogen.Thus,a combination of above two reactions was exploited to establish a method for galactose detection,moreover,the developed method was applied to detect galactose in urine samples.Our work will facilitate the utilization of DFF intrinsic peroxidase-like activity in medical diagnostics.?3?Fluorescein,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 halogen atoms affected peroxidase-like activity of fluorescein.Herein,we investigated the impact of different halogen atoms on the catalytic performance of fluorescein.By systematically evaluating absorption intensity,enzyme kinetic parameters and activation energies,we obtain evidence that the fluorine atoms position 2 and 7 points can enhance catalytic activity,but the chlorine atoms position 2 and 7 points can even inhibit the catalytic reaction.Correspondingly,the theoretical study further identified these experimental results in molecular-level.This work would not only enhance our understanding of fluorescein-based artificial peroxidase,but also facilitate the design and construction of other types of target-specific artificial enzymes.