Research Of The Visual Biochip Technology Based On Nanoparticles

Objective Fluorescence detection has been a conventional detection method of DNAmicroarray.The method relies on an expensive laser-based confocal scanner,and the costof the quipment restricts its use in the research field and makes it unsuitable for thereadig of microarrays in a wide clinical.Therefore, visual detection method is greatpotential for it overcome the drawbacks. A sensitive and visual detection technologywas established, and which would make DNA microarray more applied in the field ofmolecular assay.Methods A series of HRP substrate was synthesized. Nanogold modified withtryamine was synthesized and prepared.A new nanogold complex substrate wasdeveloped by bioconjugate technology,and nanogold particles deposited on the vicinityof enzyme site directly under catalysis of HRP. The products was tested by visual DNAmicroarrays. The new nanogold particle was used in visual etection of DNA microarrayand protien,and the sensitivity was assessed. Detection sensitivity amongTSA-GLSS(TSA-Immuno gold silver staining)GLSS and T-Au were compared.A novel visual detection method on the basis of the QDs coupled with silverenhancement was developed. The method was applied in the DNA microarraytechnique. A novel visual detection method based on TSA (Tryamine signalamplification) coupled with QDS (quantum dot coupled with silver enhancement) wassuccessfully established.TSA-QDS (Tryamine signal amplification-quantum dotcoupled with silver enhancement):First,each zone of the slide was load with the dilutionof,Streptavidin-HRP,Biotin-Tyramine,Streptavidin-QDs in turn after hybridization oftargets DNA,and incubation was necessary after the slide was loaded with each dilution.Second,the result was obtained after silver staining. The images were scanned by avisible light scanner. Finally, Detection were systematically optimized. Brucella wasselected as target in this study. Detection sensitivity among TSA-QDS(TSA-QuantumDots-Silver enhancemen) and TSA-Cy3were compared. Results A new T-Au substrate was prepared according follwing methods:Nanogold-NHS reacted with1.1fold Tyramine at50℃in dark for1.5hours. Theproducts of T-Au detection as follow: each zone of the slide was load with the dilutionof streptavidin-HRP and T-Au turn in after hybridization of targets DNA,and incubationwas necessary after the slide was loaded with each dilution.then silver strin solutionswere blended and added to slide.Finally,the visual signals were scanned andquantificated. The optimized conditions were: T-Au1:50, the glass slides wereincubated at37℃for25min and stain time of4-5min. The sensitivity of T-Au detectionand TSA-GLSS was identical, but the former was more convenient for one step wasomitted. Detection limit of T-Au and TSA-GLSS both reached103CFU mL.The procedure was set and detection paraneters included dilution ratio of theregents,incubation temperature,and silver stain time.The optimized conditions were:Biotin-Tyramine1:4000, streptavidin-QDs1:50, the glass slides were incubated at37℃for25-30min and stain time of6-7min. Detection limit of the method reached103CFU/mL. The sensitivity of TSA-QDS detection and TSA-Cy3was identical.Detection limit of TSA-QDS and TSA-Cy3both reached103CFU mL.Conclusion In this study, new Nanogold substrate was developed and used indetection of DNA Microarrays. This novel method showed the characteristics of thesimple operation and the visual results．High sensistive and visual detection could beimplemented using the method, and TSA-GLSS could be replaced by it. In chapter2anovel visual detection method based on the quantum dot coupled with silverenhancement was successfully established．In a word, this novel visual detectionmethod based on QDs is a good method that requires very less instruments, and theresults of the method can be easily visualized. Furthermore, it also provide a valuableresearch view of the application of QDs in other areas.