The Off-on Fluorescent Probe Based On Quantum Dots And Its Application In Protein Detection

Quantum dots have been extensively applied in fluorescent detection due to its unique optical and chemical properties.Compared with the traditional fluorometric reagents,quantum dots possess the advantages of,narrow emission spectra,wide excitation spectra,tunable emission wavelength,and easy modification,Abnormal protein biomarker level is associated with the metabolic disorder which is mainly caused by the pathological change.The concentration of protein in body fluids is considered as an important indicator for disease diagnosis and treatment,such as the enzyme,antigen,antibody and so on.In recent years,the detection of protein based on quantum dot-linked immunosorbent assay has attracted much attention due to its high sensitivity and selectivity.However,there are still some problems with this mode,such as complicated sample preparation,separation of free labelling,and high cost.Therefore,it is of great significance to develop accurate and simple methods for protein detection.In order to reduce background signals and improve the detection specificity and sensitivity,we proposed the off-on fluorescence and homogeneous assay relying on the enzymatic hydrolysis,the antigen-antibody binding and aptamer-target combination.The main research contents are described as following:(1)Refer to the method we created,the quantum dots with different emission wavelengths were synthesized by hydrothermal process.The quenching effects of cytochrome c and protamine on quantum dots were investigated,and it was found that these two proteins quenched the fluorescence of quantum dots strongly,especially on those with small particle size.In addition,we investigated the probe comprised of quantum dots and cytochrome c or protamine,respectively.The experimental results show that the hydrolysis reaction of cytochrome c is time-consuming and its hydrolysate is unstable.Therefore,protamine-quantum dot complex dyads are more suitable for trypsin detection.In this work,protamine is employed both as quencher to quantum dots and protease hydrolysis substrates of trypsin.In the presence of trypsin,the protamine can be hydrolyzed to small peptides and the fluorescence of quantum dots will be recovered.Therefore,a label-free and sensitive method for trypsin detection can be realized by means of the recovery of quantum dot fluorescence.(2)An off-on fluorescence and homogeneous immunoassay for alpha fetoprotein(AFP)has been developed by using the copper complex-labelled antibody(Cu-Ab)as the novel QTL(quencher-tether-ligand)probe.The Cu-Ab acted both as the quencher of quantum dots and the capture of AFP.Upon the addition of the AFP,the strong antigen-antibody combination can break up the low fluorescent ionic ensemble.Thus,Cu-Ab can be moved away from quantum dots leading to the enhancement of quantum dots fluorescence.Compared with the antibody labelled with commercial ruthenium complex(Ru-Ab),this proposed Cu-Ab possesses the advantages of low price and high quenching efficiency.Such studies will expand the specific sensing assay based on the quenching effect of metal complex to quantum dot fluorescence.(3)Based on the research in chapter 2,we introduce aptamer as a ligand that recognize the target analyte.A stable QTL(quencher-tether-ligand)complex was synthesized by the association of the copper complex-labelled streptavidin(Cu-SA)with the biotinylated aptamer(Biotin-APT).The QTL complex can be employed as a quencher to quantum dot fluorescence and the recognition of the thrombin.In the absence of the thrombin,the QTL complex can absorb on the surface of quantum dots and quench its fluorescence.However,the strong binding between aptamer and thrombin can reduce the quenching efficiency of the complex.The rapid and specific detection of thrombin can be realized based on the good linear relationship between fluorescence intensity recovered and thrombin concentration.