Application Of The Photoinduced Oxidase Activity Of Fluorescein In Analytical Chemistry And Photocatalytic Production Of Hydrogen Peroxide

Natural enzymes,with high catalytic efficiency and strong substrate specificity under mild reaction conditions,not only play an especially important role in life activities,but also widely used in various fields such as biotechnology,production and life,medicine and chemical industry.Unfortunately,natural enzymes are difficult to preserve and have low tolerance under extremely harsh conditions.These inherent defects seriously restrict the practical application of natural enzymes.Researchers have been working to find and develop new materials with mimic enzyme activities（i.e.,mimic enzymes or called like enzyme）to replace natural enzymes for solving the problem.Nano-enzymes could maintain high catalytic activity in harsh environment and have a good application prospect.However,the synthesis or modification of nanomaterials may be affected by the differences between batches,which would be a problem hindering the development of nano-enzymes.Recently,the discovery of small molecule mimic enzymes had provided important ideas for finding new mimic enzyme materials.Fluorescein is a small organic dye molecule with the peroxidase-like activity and the visible light induced oxidase-like enzyme activity.At present,the mimic enzyme activity of fluorescein had been applied in biological detection and immunoassay.It’s worth emphasizing that fluorescein,as a visible light induced oxidase-like enzyme,didn’t need to use unstable H₂O₂ in the reaction.And the catalytic reaction could be regulated by controlling the light on/off during the experiment.The whole process has the advantages of moderation,green cleaning and controllability.Based on the above facts,we first determined the critical micelle concentration（CMC）of cationic surfactants by regulating the visible light induced oxidase-like activity of fluorescein.Secondly,the immunoassay method for the determination of alkaline phosphatase（ALP）and alpha-fetoprotein（AFP）was established through the reduction of oxidation state TMB（ox-TMB）by the strong reducing substance ascorbic acid（AA）based on the chronotropic reaction of chronotropic substrates 3,3’,5,5’-tetramethylbenzidine（TMB）catalyzed by fluorescein.Finally,other important functions of fluorescein have also been found in this work.As a metal-free photocatalyst,fluorescein could catalyze the production of hydrogen peroxide（H₂O₂）from H₂O and O₂ under visible light.We carried out a series of experiments to explore the performance and fluorescein mechanism of the photocatalytic process.The specific research content of this paper is as follows:（1）Determining the CMC of cationic surfactants based on the inhibition of the visible light induced oxidase-like activity of fluorescein.Fluorescein could catalyze oxidation of chromogenic substrate TMB to produce blue ox-TMB with the maximum absorption peak at 652 nm under the visible light irradiation.The positively charged cationic surfactant in the solution could combine with negatively charged fluorescein due to electrostatic attraction,which weakens the photoinduced oxidase-like activity of fluorescein,accompanied by the color change of the product ox-TMB.Moreover,the inhibition degree of enzyme activity increased with the increase of the surfactant concentration.The mimic enzyme activity was almost completely inhibited when the CMC value was reached.Three kinds of cationic surfactants cetyltrimethylammonium bromide（CTAB）,hexadecyltrimethylammonium chloride（CTAC）and myristyltrimethylammonium bromide（TTAB）were selected in the experiment.The CMC of cationic surfactants was determined according to the relationship between their concentration and the absorbance of ox-TMB at 652 nm.This method provides a new idea for determining the CMC value.（2）Immunoassay determination of ALP and AFP based on the visible light induced oxidase-like activity of fluorescein.Sodium L-ascorbyl-2-phosphate（AAP）could be hydrolyzed to produce AA under the catalysis of ALP.AA is a strong reducing substance that can reduce ox-TMB from blue to colorless.A colorimetric method for detecting ALP enzyme activity was established based on the chromogenic system of fluorescein-TMB.There was a linear relationship between 0.20 m U/m L and 100 m U/m L with the limit of detection was 0.20m U/m L.At the same time,this method can also be used to determine the tumor marker AFP due to ALP was often used as a labeling reagent in immunoassay.The linear range was 0.20-60 ng/m L and the limit of detection was 0.17 ng/m L.It also could be used in actual sample analysis.This method was simple and easy to operate.It is expected to be used in the detection of other biomolecules.（3）Fluorescein as a metal-free photocatalyst could catalyzes the production of H₂O₂from H₂O under the visible light.We found that fluorescein could catalyze H₂O and O₂ to produce H₂O₂ under the visible light irradiation.It was confirmed that the photocatalytic process produced H₂O₂through the oxidation of H₂O and the reduction of O₂.Firstly,fluorescein generates hole（h⁺）and e^-under the visible light,h⁺oxidizes H₂O to produce H⁺and O₂ while e^-reduces O₂ to produce superoxide anion radical(O₂^·-),which continued to react with e^-and H⁺to produce H₂O₂.The yield of this method reaches 783μmol·g^-1·h^-1.As a metal-free photocatalyst,fluorescein has the advantages of cheap,low energy consumption and no need for complex synthesis steps.