Quantum Dot-based Ratiometric Fluorescence Assay To Detect Tyrosinase Activity And Screen Its Inhibitors

In recent years,with the advantages of high sensitivity,strong selectivity,simple operation and low experimental cost,fluorescent probe,which can well meet the requirements of new analytical detection technology,has been widely used in chemical and biological sensing,biological imaging,food safety and medical diagnosis.And nowadays,quantum dots(QDs)have become a new generation of fluorescence markers with high expectations in biomedical field due to their good biocompatibility and excellent optical properties,and become a research hotspot of fluorescence analysis methods.Chapter one:IntroductionFirstly,the generation of fluorescence,and the classification and application of fluorescence analysis methods were briefly introduced.Secondly,the concepts,properties,luminescence principles,preparation methods and applications of quantum dots in different fields were described.Then the research status of tyrosinase activity detection was summarized.Finally,the significance and contents of this work were put forward.Chapter two:A ratiometric fluorescence probe based on CdTe quantum dots for tyrosinase activity detectionTyrosinase(TYR)is a typical polyphenol oxidase,found in cells of animals,plants,and fungi.Vitiligo and melanoma are commonly caused by overexpression of TYR.In addition,the TYR imbalance is associated with neurological diseases such as Parkinson's disease.Therefore,monitoring TYR activity is of great significance for early diagnosis of related diseases.The development of a method to detect the activity of TYR has important value for clinical diagnosis.This research synthesized 3-mercapto propionic acid(MPA)stabled CdTe quantum dots(MPA-CdTe QDs).Tyrosinase substrate bisphenol A has been generated to quinones after catalytic.Through the photoelectronic transfer effect(PET),quantum dots' fluorescent will weaken gradually.Introducing a water-soluble ZnCdS/ZnS quantum dot as a reference,which maintained its fluorescence,we built a ratiometric fluorescent probe used in the detection of TYR.The linear range was 0-1 U/mL(R2=0.9928),and the detection limit was 0.1 U/mL(S/N=3).Chapter three:3-Aminophenyl boronic acid-functionalized quantum dot-based ratiometric fluorescence sensor for the highly sensitive detection of tyrosinase activityIn order to further improve the detection sensitivity and enhance the value of the detection method,a dual-emission ratiometric fluorescent probe based on 3-aminophenylboric acid functional quantum dots(APBA-QDs)was established with 6-hydroxycoumarin(6-HC)as the substrate to detect tyrosinase activity.TYR can catalyze 6-hc,which is a mono-hydroxyl compound.After being catalyzed by TYR,it can form a strong fluorescent o-hydroxyl compound,6,7-dihydroxycoumarin.APBA-QDs and 6,7-dihydroxycoumarin form a five-membered ring ester through a special covalent bond between o-hydroxyl and boric acid groups.QDs' fluorescence was weakened due to the introducing of the five-membered trap.With the increase of TYR concentration,the fluorescence at 675 nm from QDs was gradually quenched,while the fluorescence at 465 nm was enhanced by 6,7-dihydroxy coumarin.By measuring the weakened signal at 675 nm and the enhanced signal at 465 nm,the ratio fluorescence method was established to detect the TYR activity.Under the conditions optimized by response surface method,the experimental results show that the linear range of TYR is 0-0.05 U/mL(R2=0.9952).The detection limit was as low as 0.003 U/mL.The application of the probe in real samples such as cell extracts and human serum were also investigated.The sensing strategy can also be used for rapid screening of TYR inhibitors,and satisfactory results were obtained.It was proved that the constructed fluorescent probe not only has high sensitivity and selectivity,but also has certain application potential in the detection of TYR in real samples and the screening of inhibitors.