Research On Novel Biosensing Methods Based On Conjugated Polymer Nanoparticles And Enzyme-Free Amplification

Biosensor technology is a high-tech integrated by biology,chemistry,medicine,physics and other disciplines.Because of its high selectivity,high sensitivity,easy operation,fast response speed and low-cost detection,it has been widely used in biomedical,environmental monitoring,food safety testing,clinical testing and other fields.With the rapid development of Biosensor technology,nanomaterials have become a research object favored by the researchers and have been prepared a variety of nanomaterials,owing to their unique physical and chemical properties.The application of nanomaterials has greatly promoted the development of nanotechnology.At the same time,Functional nanomaterials with its unique photoelectric properties,has been highly concerned about the scientists.It also provided a lot of innovative ideas for the development of new biosensors,and overcomed the shortcomings existing in the traditional methods.The combination of nanotechnology and biosensor tremendously improved the detection performance of biosensors,promoted the development of new biosensors,and driven the rapid development of biosensors.In this thesis,combined with the specificity of conjugated polymer nanomaterials,a serises of nano biosensor strategies have been developed,Meanwhile,major disease markers(such as small molecule,protein etc.)as the test subjects.These methods were developed for the detection of tyrosinase activity and the screening of its inhibitors,small molecule ATP analysis and determination.Compared with the traditional methods,the analytical method established in this paper has the advantages of simple operation,fast response speed,high sensitivity and high selectivity.The details are summarized as follows:Tyrosinase(TYR)is a key enzyme for melanin metabolism,meanwhile,it's the only definite melanin metabolic enzyme at present,which plays an important role in melanogenesis.The pathogenesis of melanoma is related to the level of tyrosinase expression and can be used as a useful biomarker for melanoma activity.Therefore,the detection of TYR activity plays an important role in the study of pathology.In Chapter 2,we established a new sensor based on surface amino-functional fluorescent polymer nanoparticles to detect the activity and inhibitors of tyrosinase.Since its conjugated polymer nanoparticles have large p-conjugated backbone,delocalized electronic structures and large specific surface areas,they become suitable substrates for probe assembly.The method relies on the tyrosine-catalyzed oxidation of melanin-like polymers by tyrosinase(TYR),which is readily assembled on the surface of the conjugated polymer nanoparticles,meanwhile,grown in situ into melanin and quench the fluorescence of the nanoparticles.The method is simple,unlabeled,responsive,highly sensitive,highly specific for detecting tyrosinase activity,and for rapid screening of tyrosinase inhibitors.Therefore,this method provides a new platform for the early diagnosis of tyrosinase-related diseases and the screen of inhibitors.ATP is an important substance of the organism that plays a vital role in maintaining the normal functioning of the organism.It is an important energy currency in living cells,which participates in the physiological and metabolic processes of cells and even organisms.Its content have a great influence on the cell activity,and it has been proved that many diseases are related to abnormal ATP content.Therefore,the development of new methods for the detection of ATP in the clinical diagnosis and metabolic activity in the body is of great significance.In Chapter 3,we established a highly sensitive,highly selective,non-enzymatic amplification method for the detection of ATP,which was based on combination the cleavage of nucleic acid aptamers with the near-mediated hybridization.At the first,the two fragments were cleaved at the appropriate location of the nucleic acid aptamers,followed by the introduction of a short stable sequence at the ends of the two fragments to aid in the binding of the two fragments.The ends of these two nucleic acid aptamers extend a DNA sequence as an initiator for HCR.When the target ATP is present,the distance between the two cleavable nucleic acid aptamers is narrowed,causing the HCR to amplify the reaction,thereby activating the fluorescent signal of the molecular beacon.In addition,we synthesized surface-functionalized fluorescent polymer nanoparticles with positive charge,which can assemble negatively charged DNA probe on its surface by electrostatic adsorption,thereby efficiently transport the ATP probe to intracellular,real-time monitoring of intracellular ATP.The analytical method provides an effective analysis and detection platform,such as the delivery of drugs and genes,protein detection,intracellular tracing,etc.