Determination Methods For Acid Phosphatase And Tyrosinase

Cancer is very dangerous because of its high morbidity and mortality,it is particularly necessary for its early and rapid detection.It has become an effective strategy to construct detection methods based on cancer markers.Among them,biological enzymes are key targets due to their high specificity and catalytic activity.Functional nanomaterials have been widely used to build probes or carriers to improve the specificity and sensitivity of analysis method due to their easy synthesis,flexible modification and excellent photoelectric properties.In this thesis,acid phosphatase(ACP)and tyrosinase(TYR)were selected as representative enzymes to develop a series of highly sensitive,selective and multi-mode detection methods based on various functional nanomaterials.The main contents of this thesis are as follows:(1)A fluorometric method for ACP was established based on the high quantum yield calcein.The coordination between calcein and Eu³⁺quenched the fluorescence of calcein.In the presence of ACP,the L-ascorbic acid-2-phosphate sodium acid(AAP)was hydrolyzed to release phosphate group,which deprived Eu³⁺and restored the fluorescence.The quantitative detection of ACP was realized based on the fluorescence turn-off-on.(2)A europium polymer with red fluorescence was synthesized through the coordination of Eu³⁺,adenine(Ade)and 2,6-pyridinarboxylic acid(2,6-DPA).The substrate phosphopyridoxal(PLP)was bound to europium polymer to bring the aldehyde group close to the polymer,leading to the occurrence of photoinduced electron transfer(PET)and quenched the fluorescence of the polymer.In the presence of ACP,PLP was hydrolyzed to pyridoxal(PL),the phosphate group was exposed and caused the secondary polymerization of the polymer and restored the red fluorescence.The PL underwent intramolecular hemiacetal reaction to produce blue fluorescence.The high selective detection of ACP was realized based on the two signals(blue and red)without false positive error.(3)A non-fluorescent polymer was prepared based on the chelate self-assembly reaction between the neutral red(NR)and the ligand synthesized by ethanediamine and PLP.ACP disrupted the polymer to release NR and generate PL with blue fluorescence.The probe completely avoided the interference of free phosphate groups and realized in vitro fluorescence and UV-vis dual-mode detection,the integrated in-situ and real-time near-infrared fluorescence imaging of endogenous ACP in cells.(4)A novel peroxidase-like nanoparticles were synthesized based on the mechanism of single atom anchoring and protein stabilized metal nanoparticles.TYR was detected through two strategies with tyramine and phenol as substrates,respectively.(?)Tyramine was oxidized to dopamine by TYR,and polydopamine nanoparticles(PDA)were generated in situ under the catalysis of peroxidase-like nanoparticles.The fluorescence resonance energy transfer(FRET)between PDA and fluorescein isothiocyanate(FITC)enhanced the fluorescence of FITC.(?)Phenol reacted with 4-amino-antipyrine(AAP)to produce a red compound(RC)and quenched the fluorescence of FITC due to the internal filtration effect(IFE).Phenol was oxidized by TYR,which inhibited the formation of RC to eliminate the IFE and enhanced the fluorescence of FITC.In this work,the fluorescent and visual detection based on smart phone of TYR was realized,TYR was successfully determined in real serum samples.(5)A highly selective sensor for TYR was constructed based on the self-assembly of metal doped quantum dots,dendritic mesoporous silicon(DMSNs)and TYR-specific polypeptide sequences.The sensor was extended to detect the pesticide herbicide atrazine.This method has been successfully applied to direct detection of TYR in serum samples and indirect assay of atrazine in vegetable samples.