| The study of protein-small molecule interaction is critical to provide information for new drug designation. In this work, by luminol as luminescence probes, luminol-HAuCl4-lysozyme chemiluminescence (CL) system was established using flow injection analysis (FIA). The interaction of lysozyme with macrolides, sulfonamides and phenolic acids were studied by flow injection chemiluminescence (FI-CL). The binding parameters and thermodynamic parameters were given. The CL system was successfully applied to continuous monitoring of captopril (CAP) and roxithromycin (ROX) in human urine. The content of this thesis included two parts, as follows:Part Ⅰ:ReviewChapter 1 IntroductionThe applications of nanometer materials in the luminescence analysis and FI-CL were described, the common researching methods for the interaction of protein with small moleculars were summarized, and the study on the interaction of lysozyme with small moleculars were given, the significance and contents of this dissertation were described.Part Ⅱ:Research reportsChapter 2 Constructing of luminol-HAuCl4-Iysozyme CL systemIt was found that HAuCl4 could form gold nanoparticles online and enhance the luminol CL intensity. A luminol-HAuCl4-lysozyme CL system was first developed based on the enhancing effect of HAuCl4 on CL intensity from luminol-lysozyme system. The optimum concentrations of luminol, lysozyme, HAuCl4 and NaOH were 2.5 × 10-5,1.0 × 10-7,5.0 × 10-7 and 2.5 × 10-2mol L-1, respectively, flow rate was 2.0 mL min-1 and the mixing tubing length of 10.0 cm. Under the optimum experimental conditions, the stability and repetitiveness of this CL system were tested and exhibited good stability in 5 days with flow system regularly worked over 8 h per day (RSDs<3.5%).Chapter 3 The interaction of lysozyme with macrolides, sulfonamides and phenolic acidsThe interaction behavior of lysozyme with macrolides, sulfonamides and phenolic acids were investigated using FI-CL analysis. It was found that the small moleculars remarkably quench the CL intensity from luminol-HAuCl4-lysozyme system, and the quantitative correlation equation of CL intensity decrements versus the logarithm of the concentration of drugs, △I= ALnC+B was established. By homemade FI-CL model, lg[(Io-I)/I]= nlg[C]+ lgK, the binding constants (K= 104-107 L mol-1 level) and binding sites (n= 1.0) were obtained. The thermodynamic parameters (△H, AS and AG) were given by Van’t Hoff equation and the results showed that the reaction of lysozyme with macrolides, sulfonamides and phenolic acids were spontaneous process. Using AutoDock 4.2, it was found that the small moleculars were located in the hydrophobic pocket of lysozyme, and the values of AG, K were well agreeing with the experimental datas by FI-CL.Chapter 4 Continuous monitoring of CAP in human urineA rapid and sensitive method for determining CAP by FI-CL was proposed. It was found that CAP could remarkably quench the CL intensity of luminol-HAuCl4-lysozyme system, and the decreased CL intensity was linear with the logarithm of CAP concentration over the range from 0.1 to 50.0 ng mL-1 with a detection limit of 0.03 ng mL-1 (3σ). At a flow rate of 2.0 mL min-1, one analysis cycle including sampling and washing could be accomplished within 36 s. By homemade FI—CL model, the binding constant K (7.8 × 105 L mol-1), binding site number n (0.75) and the thermodynamic parameters AH (42.32 KJ mol-1), AS (255.65 J mol-1 K-1), AG (-31.43/-33.60/-36.56 KJ mol-1,288/298/308 K) of CAP to lysozyme were obtained. The results demonstrated that the reaction of CAP binding to lysozyme was a spontaneous process with hydrophobic force. Using molecular docking, it was found that CAP might enter into the hydrophobic pocket of lysozyme and bind to Trp63 of lysozyme with a 1:1 complex formed, leading to the striking inhibition of CL intensity from luminol-HAuCl4-lysozyme system. The proposed FI-CL method was successfully applied to the continuous monitoring of CAP in human urine after oral intake in 7.5 h. The pharmacokinetic parameters of absorption rate constant Ka(2.81 ± 0.05 h-1), elimination rate constant Ke (0.88 ± 0.02 h-1) and elimination half-life time t1/2(0.79 ± 0.01 h) were obtained. This work has been published in Instrumentation Science & Technology (2015,43, 197-213).Chapter 5 Continuous monitoring of ROX in human urineA sensitive and fast method for the determination of ROX by FI-CL analysis is presented, which was based on ROX chould quench the CL intensity of luminol-HAuCl4-lysozyme system. The decrement of CL intensity was linearly proportional to the logarithm of ROX concentration in the range of 0.03-70.0 ng mL-1 with the detection limit of 0.01 ng mL-1 (3σ) and RSDs less than 3.0%. By homemade FI-CL model, the binding constant K (2.1 × 105 L mol-1), binding site number n (0.73) and the thermodynamic parameters △H (21.21 KJ mol-1), △S (173.18 J mol-1 K-1), △G-28.67/-30.36/-32.13 KJ mol-1,288/298/308 K) of ROX to lysozyme were obtained. The results demonstrated that the reaction of ROX binding to lysozyme was a spontaneous process with hydrophobic force. The proposed method was successfully applied to the determination of ROX in human urine after oral intake in 14.0 h and obtained pharmacokinetic parameters (Ka:1.152± 0.05 h-1, Ke:0.092 ± 0.01 h-1,t1/2:7.52 ± 1.01 h). |
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