Study Of Fluorescence Resonance Energy Transfer Systems And Their Application In Homogeneouse Immunoassays

Fluorescence resonance energy transfer (FRET) is a analytical method of molecular emission spectroscopy, which is based on non-radiative energy transfer between fluorescent materials. In recent years, research work on FRET developed rapidly and is applied widely in the structure and function determination of protein and nucleic acid, measuration of interaction between ligands and receptors, etc. FRET studies mainly focus on the investigation of structure and interaction of protein and nucleic acid. However, FRET studies on biological analysis are relatively less, especially on immunological analysis. In the area of immunoassays based on FRET, the linkers between energy donors and acceptors, labeling degree of donor and acceptor conjugates and match of donor and acceptor have not been researched thoroughly. In this thesis, a number of FRET systems and application in homogeneous competitive immunoassays were studied. The main results are as follows:1) In the preliminary stage, three FRET systems of F(ab')2-QDs/mouse IgG-RBITC, mouse IgG-FITC/rabbit anti mouse IgG-TRITC and mouse IgG-FITC/rabbit anti mouse IgG-Dylight 549 were studied for the feasibility of homogeneous competitive immunoassay based on FRET. The results showed that QDs and RBITC, FITC and TRITC were good matches for FRET donor and acceptor. Although in theory FITC and Dylight 549 can become a good donor-acceptor pair, the result of FRET was not obvious. This may be due to the low F/P value of acceptor conjugate. In the choice of linkers between donor and acceptor, F(ab')2-mouse IgG and rabbit anti mouse IgG-mouse IgG were both good linkers that meet the conditions for occurrence of FRET and FRET phenomena were quite obvious. In the first two systems, the relationship between FRET signals and concentration of competititors were quite obvious, confirming homogeneous competitive immunoassay based on FRET is possible.2) In order to shorten the distance between donor and acceptor to improve the efficiency of FRET, SA-biotin was used as a linker between donor and acceptor. FRET system of SA-FITC/biotin-Dylight 549 applied for homogeneous competitive immunoassay was studied. In this study, biotin was found to enhance the fluorescence intensity of SA-FITC conjugates, which would improve the sensitivity of analysis methods combined with FRET. The results showed that, FITC and Dylight 549 is ideal for FRET energy donor and acceptor pair. In the FRET process, not only the fluorescence intensity of donor could be observed but also the fluorescence changes of acceptor could be observed. The FRET process was more apparent when BAS was used as the link, which would shorten the distance between donor and acceptor. In addition, a homogeneous competitive analysis method for the determination of biotin was established based on FRET with fluorescence enhancement. This method is simple, short time-consuming (no more than 15 minutes), highly sensitive, wide linear range (0.8-9.8nM), and can be applied for the detection of biotin in actual sample (such as milk).3) The influence of molar ratio of dyes to protein (F/P value) on FRET efficiency was studied. The earth chelates BHHCT-Eu3+was used as the energy donor and Dylight 649 as the energy acceptor. Goat anti-mouse IgG (antibody)-mouse IgG (antigen) immunocomplex was used as the linker (BHHCT-Eu3+labeled to goat anti-mouse IgG, Dylight 649 labeled to mouse IgG). The results showed that FRET efficiency was high only when F/P value was appropriate (about 10). When F/P value was too low, the average distance between the donor and acceptor fluorophores was large to cause low FRET efficiency. High F/P value (over 20) would influence the binding activity between antibody and antigen, which also resulted in low FRET efficiency.4) A homogeneous immunoassay based on FRET for the detection of AFP was established. Rare earth chelates BHHCT-Eu3+was used as energy donor labeling to AFP antigen, Dylight 649 AFP as energy acceptor conjugating to monoclonal antibody. The method is simple, short time-consuming (no more than 30 minutes) and wider linear range (0.05-2μg/mL), and can be used for AFP antigen screening.A number of FRET systems applied in homogeneous competitive immunoassay were studied in this thesis, including different energy donor-acceptor pairs (traditional organic dyes, quantum dots, lanthanides) and different linkers between donor and acceptor (antigen-antibody system, biotin-streptavidin system). The guide line of choosing FRET donor-acceptor pairs and links was discussed. The influence of molar ratio of dyes to protein on FRET efficiency was studied, which would provide new ideas were for homogeneous immunoassay based on FRET.