Fluoroimmunoassay Based On Quantum Dot For The Detection Of Several Pesticide And Veterinary Drugs Residum

Quantum dots (QDs) are somewhat spherical nanocrystals in the size range of 1-10 nm diameter. These QDs are a brand new class of luminescent inorganic fluorophores, which have several important advantages over conventional fluorescent dyes. In recent years, QDs have gained increasing attention and played important roles in the fields of biochemistry, fluorescence immunoassay, genomics, proteome, cell biology and molecular biology, etc. Aiming at this important research direction, the development of QDs was summarized, and then the following several works have mainly performed by taking QDs preparation, QDs characterization and QDs applications in pesticide and veterinary drugs residue analysis as the line of this dissertation. 1. A rapid and sensitive indirect competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of chlorpyrifos in drinking water has been developed.Based on QDs as the fluorescence label coupled with secondary antibody (Ab2), the cFLISA allowed for chlorpyrifos determination in a liner working range of 15.2-205.5ng-mL-1. The 50% inhibition value (IC50) and the limit of detection (LOD) of the cFLISA were 50.2ng mL-1 and 8.4ng mL-1 respectively. In water sample analysis, the results of cFLISA were similar to those obtained from cELISA and HPLC, while the detection time by cFLISA was reduced 0.5h compared with ELISA. It showed that cFLISA could be used as a new screening method for the detection of pesticide residue in food and envirement.2. Water-soluble CdTe QDs were synthesized directly and coupled with streptavidin (SA) as the fluorescent probe. Based on QDs-SA conjugate and ELISA, a novel fluoroimmunoassay (QDs-SA-cFLISA) has been developed for the detection of chlorpyrifos in drinking water.Water-soluble CdTe QDs were prepared in aqueous solution by using 3-mercaptopropyl acid as the stabilizer and then characterized by fluorescence spectroscopy and TEM, respectively. The results show that these QDs have good optical properties, such as narrow spectral line widths and continuous absorption profiles, which provide a powerful foundation for further applications. A rapid and sensitive competitive fluorescence-linked immunosorbent assay (QDs-SA-cFLISA) based on QDs-SA conjugate has been developed for the detection of chlorpyrifos in drinking water. The sensitivity of QDs-SA-cFLISA increased by 5.5 folds and the detection time reduced 1h compared to conventional ELISA. In real sample analysis, the results of QDs-SA-cFLISA were similar to those obtained from a ELISA and a HPLC. This study showed that QDs-SA-cFLISA was more rapid and sensitive than conventional ELISA. Therefore, it can be used as a novel screening method for the detection of pesticide residue.3. Based on direct coating of small molecular hapten (Hap) on the surface of microtiter plates and QDs-SA fluorescent probe, a new fluoroimmunoassay (QDs-SA-direct Hap coated cFLISA) has been developed for the detection of chloramphenicol (CAP).In this Hap directly coated assay, the surface of microtiter plates was treated with a glutaraldehyde (GA) polymer network to introduce aldehyde groups for crosslinking of CAP with amino groups. Compared with the plates coated with Hap-carrier protein conjugate, the modified plates presented significantly high antibody and antigen (Ab-Ag) affinity and showed excellent stability. And then the biotinylated monoclonal antibody (mAb) and QDs-SA conjugate were employed in this assay for amplification of signals. The sensitivity of QDs-SA-direct Hap coated cFLISA increased by approximate 5 folds and the stability improved greatly. In sample analysis, the results of CAP detected by this assay were in accordance with which obtained by conventional ELISA and HPLC. Therefore, it is an attractive alternative compared to conventional immunoassays in routine supervision for residue detection in food and environment.