Gold Nanoparticle Based Colorimetric Detection Of Thrombin And Sequence-specific DNA

Colorimetric assay of the detection principle is based on the color change of thesolution. This method means simple, fast, and sensitive, so it has been widely appliedin molecular diagnostics, pharmaceutical, environment and other fields. Goldnanoparticle has unique properties, such as optimal, mechanical, thermal andelectrical property, which led to a better label molecular. Because of cheap price,simple protocol, stable character of gold nanoparticles, and high activity of the labeledbiomolecules, it has been applied in many fields. This assay developed thrombin andsequence-specific DNA detection methods based on the colloidal gold as label. Theresearch in my assay consists of three parts:Chapter1: Review-research progress of gold nanoparticlesNanomaterials have been widely used in chemical and biological sensing becauseof their unique optical and mechanical properties. Au NPs are particularly attractive inbioassay by virtue of their facile synthesis, large specific surface area, high chemicalstability, and biocompatibility. This chapter descripted the properties, syntheticmethod and detection method of Au NPs.Chapter2: A simple and sensitive way for direct colorimetric visualization ofthrombin based on gold nanoparticlesA simple, colorimetric turn on‘sensor for ultrasensitive detection of thrombinhas been developed using fbrinogen-modified gold nanoparticles (Fib-Au NPs). Theassay was based on the thrombin-fibrinogen interaction which is part of thephysiological process of blood clotting. The fibrinogen was immobilized on thesurface of96-well plate offering reactive N-oxysuccinimide esters (referred to as NOSgroup) surface. Introducing thrombin and Fib-Au NPs into the fibrinogen-bound96-well plate induced the immobilization of Fib-Au NPs on the surface of96-wellplate through the thrombin mediated converting soluble fibrinogen to insolublecross-linked fibrin. Such process could be detected visually post the HAuCl4-NH2OHredox reaction catalyzed by the Au NPs. The parameters governing the performanceof the assay have been optimized. The detection limit was3.2fM, corresponding to0.16amol thrombin in50μL of sample. Other proteins, such as bovine serumalbumin (BSA), pepsin, trypsin, hemoglobin, lysozyme, and cytochrome c did not show interference with the assay of thrombin. In addition, the work demonstrated thefeasibility of thrombin detection in a complex matrix, showing potential in rapidmedical diagnostics.Chapter3: A simple and sensitive way for direct colorimetric visualization ofsequence-specific DNA based on gold nanoparticlesA simple, rapid, and sensitive method for visual detection of sequence-specificDNA was developed using hairpin DNA as the recognition element andhydroxylamine-enlarged gold nanoparticles (Au-NPs) as the signal producingcomponent. In the assay, we employed a hairpin DNA probe dually labeled withamine and biotin at the5‘-and3‘-end, respectively. The probe was coupled withreactive N-oxysuccinnimide in a DNA-bind96-well plate. Without the target DNA,the immobilized hairpin probe was in a―closed‖state, which kept thestreptavidin-gold off the biotin. Upon approaching the target DNA, the hybridizationbetween the loop sequence and the target broke the short stem duplex. Consequently,biotin was forced away from the96-well plate surface and available for conjugationwith the streptavidin-gold. The hybridization could be detected visually post theHAuCl4-NH2OH redox reaction catalyzed by the Au-NPs. Under the optimizedconditions, the visual DNA sensor could detect as low as100amol of DNA targetswith excellent differentiation ability even single-base mismatch.