| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 97 === We have developed two techniques for the detection of biomolecules of interest. First, 11-MUA-protected gold nanodots (Au NDs) have been prepared and employed for the detection of immunoglobulin G (IgG) in plasma. IgG is the most abundant antibody in plasma, with a normal concentration range over 34.0–105 μM. Plasma IgG at low levels causes poor immune systems, while at high levels can be indicators of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Through the high-affinity binding between protein A and IgG, the fluorescence intensity of protein A-conjugated Au NDs (PA－Au NDs) increases upon increasing IgG concentration. For IgG, this approach provides a limit of detection (LOD) of 10 nM, a linear range over 50 and 250 nM (R2 = 0.998), and high selectivity (117-fold selectivity over transferrin). This approach has been validated by determining the concentrations of IgG in plasma samples from two healthy adults, with results of 45.4 ± 3.2 and 58.0 ± 4.7 μM (three measurements).
Secondly, we have demonstrated the detection of biological thiols through their replacement of Nile Red (NR) from the surfaces of gold nanoparticles (Au NPs). When NR is adsorbed onto Au NPs, FRET and electron transfer occur, leading to fluorescence quenching. Upon addition of the biological thiols to the solutions of NR-Au NPs, NR molecules are released to the bulk solution, leading to increased fluorescence. By monitoring the fluorescence changes, we separately investigated the replacement kinetics of Cys, Hcy and GSH, showing the decreasing order Cys ~ Hcy > GSH. Since Cys in solution at pH 7.00 is oxidized in the air at 95 ℃ more rapidly than Hcy and GSH are, its replacement kinetic is significantly (4-fold) slower than that of the other two. By measuring the differences in fluorescence intensity of Cys solutions with and without being heated for 1 hr, this approach provides an LOD of 10 nM for Cys, with a linear range over 100–1000 nM (R2 = 0.995). This approach is more sensitive than that (LOD 15 nM) of direct replacement without heating. In order to improve the selectivity of this approach toward Cys, polyvinylpyrrolidone (PVP) had been used to modify NR-Au NPs. The replacement of NR from PVP-Au NPs by GSH is slower (20-fold) than those by Cys and Hcy, which allows selective detection of Cys in two prepared solutions composed of Cys (11.74 μM), Hcy (0.301 μM), GSH (3.511 μM), cysteinylglycine (3.584 μM) and γ-glutamylcysteine (0.566 μM), with results of 11.72 μM (recovery 99.8%) and 10.96 μM (recovery 93.4%).
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