Research On The Fluorescent Detection Methods For Biological Enzyme Activity Using Carbon Quantum Dots As Probe And Dopamine As Substrate

Carbon dots(CDs)is a new type of zero-dimensional carbon nanomaterial with particle size below 10 nm.CDs have been extensively applied in biosensing,bioimaging and drug delivery due to the excellent optical properties,good biocompatibility and simple synthetic routes.Especially in the field of biomedicine,the formed interaction systems of fluorescent probe CDs and special materials or molecule or ions through the inner filter effect(IFE)or F?rster resonance energy transfer(FRET)or photoinduced electron transfer(PET)have been used for the analysis of specific targets.The bioactive molecules such as dopamine(DA)not only play physiological roles on human body,but also serve as substrates for enzymatic catalysis which can be oxidized to o-dopaquinone.Furthermore,DA can be oxidized and self-polymerize into polydopamine(pDA)under alkaline(pH>7.5)conditions with oxygen.Enzyme is a class of highly catalytic protein.It plays an important role in the fields of disease diagnosis,health assessment,and food safety.In this paper,we used carbon dots as probe and dopamine as substrate.Based on the special effects of carbon dots and substances such as o-dopaquinone and polydopamine,two new fluorescence sensing strategies have been established and high sensitivity detection of tyrosinase and acid phosphatase have been achieved.The main research content of this thesis was divided into the following two chapters: Chapter 1: Determination of tyrosinase activity by fluorescence sensor based on N-CDs and research on its mechanismIn this chapter,amine riched CDs(N-CDs)were prepared from citric acid,glutathione and polyethylene polyamine.Dopamine was oxidized to o-dopaquinone with the catalyzation of tyrosinase(TYR)and quenched the fluorescence of N-CDs effectively.Based on this principle,employing N-CDs as probe,a facile and effective homogeneous fluorescence strategy for monitoring TYR activity was developed.The experimental results showed that the linear range of TYR detection was 0.05-6.0 U/mL,and the detection limit was 0.039 U/mL.And this method was successfully applied to the detection of TYR activity in human serum samples,indicating the feasibility for clinical practice.In addition,we synthesized two other CDs with different nitrogen doping degrees,and compared the response results of CDs with different nitrogen doping degrees to the TYR catalyzed DA.By investigating the mechanism of CDs and o-dopaquinone,it could be found that the improved nitrogen doping degree and amino content of CDs were favorable for obtaining more effective and sensitive performance for the determination of TYR activity.Chapter 2: Fluorescence sensor for acid phosphatase detection based on enzyme catalytic oxidation and polymerization of dopamineIn this chapter,DA was devised for the first time as a new substrate for the enzymatic catalysis of acid phosphatase(ACP).ACP could induce DA to form pDA under acidic and neutral conditions.The formed pDA could quench the fluorescence emission behavior of CDs by internal filtration effect.While,alkaline phosphatase(ALP)failed to serve such function for DA under similar conditions.Based on this principle,we developed a novel fluorescence sensing strategy for monitoring ACP activity using fluorescent CDs as signal indicator.The experimental results showed that the method for detecting ACP illustrated a good analytical performance with wide linear range of 1-60 U/L and detection limit of 0.45 U/L.In particular,this method showed high selectivity for ACP detection under neutral conditions which could distinguish ACP from common interferents including ALP.In addition,the spiked recovery of ACP in human serum samples was determined.The results of the recovery experiment showed that this method has a good application prospect in the clinical and biomedical fields.