Signal Amplification Strategies For Disease Markers Determination Based On The Photoinduced Oxidase Activity Of Fluorescein

Serum alkaline phosphatase（ALP）is an important biomarker closely related to prostate cancer,bone,liver dysfunction,diabetes and other diseases.It is also a functional enzyme that dephosphorylates the corresponding substrate by hydrolyzing phosphate ester bonds to convert phosphate groups on the substrate molecules into phosphate ions and hydroxyl groups.Similarily,α-fotoprotein（AFP）is a specific serum marker for the diagnosis of primary liver cancer.Because of the significance of these disease markers,it is neccessary to develop rapid and sensitive AFP detection methods.Fluorescein,a small molecule enzyme mimic,can catalyze the oxidation of tetramethylbenzidine（TMB）rapidly under visible light and produce color reaction.In this work,the photocatalytic oxidase activity of fluorecein is regulated by changing its functional groups,synthesizing coordination nanoparticles（CNPs）,and constructing metal-organic framework（MOFs）,thus realizing the detection of ALP and AFP in serum.The method exhibits a wide range of linearity,good specificity,simple instrument and equipment,and provides a prospect for clinical medicine.In addition,this method not only proves that the photocatalytic activity of fluorescein is closely related to its molecular structure,but also that certain metal ions(such as Cu²⁺)inhibit its enzymatic activity.The mechanism of Cu²⁺inhibiting fluorescein activity is revealed.The research content includes the following aspects:（1）Colorimetric determination of ALP and AFP based on photoinduced oxidase activity of fluoresceinFluorescein diphosphate tetrammonium salt（FDP）is selected as the probe of ALP substrate.ALP catalyzes the hydrolysis of FDP with negligible enzyme activity to produce fluorescein with high activity,and catalyzes the oxidation of colorless TMB to blue oxTMB under visible light.Under the optimal reaction conditions,the absorbance of the reacted solution at 652 nm is linearly related to the ALP activity,and it has both good sensitivity and selectivity.Based on this,a colorimetric method for detecting ALP activity in human serum is established.In addition to being a disease marker,ALP can also be used as a marker enzyme in ELISA.In this way,ALP is labeled on AFP antibody,and the colorimetric immunoassay of the disease marker AFP is successfully constructed.There are no significant differences at the 0.05significance level between the methods and hospital detection values（electrochemical detection method）.（2）Reducing background absorbance via a double-lock strategy for highly sensitive detection of ALP and AFPAn ALP-responsive coordination nanomaterial（Ce-FDP CNPs）is synthesized via self-assembly,and background signal induced by escaped fluorescein and FDP are reduced by double locks strategy:1 Phosphorylation of fluorescein;2 Through the strong coordination between cerium ions and phosphate ions,fluorecein is further blocked in the nanomaterials.Under the optimal conditions,ALP catalyzes the rupture of phosphate groups in Ce-FDP CNPs to collapse the material,and releases fluorecein with high activity in a controlled manner.In this experiment,the background absorbance is greatly cutted by the double-lock strategy,and the detection limit was reduced by about 10 times compared with the single FDP.In addition,the immunoassay of AFP was further constructed by labeling the antibody with ALP in enzyme-linked immunosorbent assay.（3）Bifunctional antibody and copper-based organic framework nanocomposites for AFP determinationThis work first reports that Cu²⁺reduces the generation of reactive oxygen radicals in the reaction system,thus effectively inhibiting the photocatalytic activity of fluorescein.Bifunctional AFP antibody and copper-based organic framework nanocomposites（Ab2@Cu-MOFs）are synthesized by simple self-assembly of AFP antibody,Cu²⁺and 4,4’-bipyridine:the binding site of AFP Ab2 exposed on the surface of the nanocomposites specifically recognizes AFP antigen;myriads of Cu²⁺in nanocomposites inhibits the activity of fluorescein.After the formation of the immunecomplex,Cu²⁺in the material are released,and the relationship between absorbance and AFP concentration is established according to its inhibitory effect on fluorescein activity.In this work,Ab2@Cu-MOFs is simple sythesis,uniform size,good dispersion.Also,the center ions in the MOFs works directly,which calls for attention to the function of excavating its own components in materials.