自组装环技术应用于抗菌药检测及药物与蛋白质相互作用机理研究

【作者】 邓凤玉；

【导师】 刘颖；

【作者基本信息】 中央民族大学 ， 环境科学， 2012， 硕士

【摘要】 本文基于抗菌药物分析检测的重要意义和自组装环荧光显微成像技术的特点及其在药物分析中的应用,建立了甲苯磺酸妥舒沙星(TSFX)、左氧氟沙星(LVFX)和洛美沙星(LMX)的分析方法,并应用于鸡血清、鸡肉、肝脏、粪便；兔子血清、兔肉、肝脏和肾脏；药片、胶囊、注射液中药物含量的检测。此外,本文还应用荧光光谱法、紫外-可见吸收光谱法、傅里叶变换红外光谱法和分子模拟的方法研究了甲苯磺酸妥舒沙星TSFX.酒石酸乙酰异戊酰泰乐菌素(ATLL)与牛血清白蛋白(BSA),呋喃西林(NF)与人血清白蛋白(HSA)的相互作用。在pH10.50的NH3-NH4C1缓冲溶液和PVA-124存在下,Mn2+和CTMAB作为敏化剂,甲苯磺酸妥舒沙星(TSFX)在疏水性玻璃表面上形成自组装环。当点样体积为0.2μtL时,线性范围为4.05×10-14～4.28×10-13mol-ring-1(2.02x10-7～2.14×10-6mol·L-1),检出限为4.05×10-15mol·ring-1(2.02×10-8mol·L-1)。实测了甲苯磺酸妥舒沙星片剂中TSFX的含量和兔子灌喂甲苯磺酸妥舒沙星片剂后不同时间血清中TSFX的浓度,平均回收率在90.0～105.0%,相对标准偏差(RSDs)小于3.3%。在pH9.30的NH3-NH4Cl缓冲溶液中,Mn2+和CTMAB作为敏化剂,PVA-124作为辅助成环剂,建立了检测左氧氟沙星(LVFX)的方法,并实测了盐酸左氧氟沙星胶囊、片剂,鸡血清、鸡肉、鸡肝和鸡粪便中LVFX的浓度。当点样体积为0.2μL时,线性范围为5.66×10-14～1.00×10-13mol-ring-1,检出限为5.66x10-15mol-ring-1。该方法应用于鸡血清、鸡肉、鸡肝、鸡粪便和药物制剂(药片、胶囊)中LVFX的测定时回收率为90.0～105.0%,RSDs在0.8～4.0%。在pH9.60的NH3-NH4Cl缓冲溶液和PVA-124存在下,A13+和CTMAB作为敏化剂,洛美沙星(LMX)在二氯二甲基硅烷处理的疏水性固载表面上形成自组装环,据此建立了检测LMX的方法。当点样体积为0.2μL时,线性范围为9.87x10-14～1.47x10-12mol-ring-1检出限为9.87×10-15mol-ring-1(4.93×10-8mol·L-1)。实测了盐酸洛美沙星胶囊、片剂、注射液,兔血液、兔肉、肝脏、肾脏中LMX的浓度,加标回收率为90.6～106.3%,RSDs小于4.2%。模拟生理条件下,应用荧光光谱法、紫外-可见吸收光谱法、傅里叶变换红外光谱法研究了TSFX与BSA的相互作用。实验结果表明,TSFX与BSA的作用属于静态猝灭过程,结合常数Kα为2.58×104L·mol-1(298K),结合位点数n≈1,作用力类型主要为静电作用力。根据Foster能量转移理论求得TSFX与BSA第212位色氨酸残基间的距离r=3.42nm。同步荧光光谱和三维荧光光谱数据显示TSFX能够改变BSA的构象,色氨酸残基所处微环境疏水性降低。采用FT-IR对BSA与TSFX作用前后BSA二级结构的变化进行了定量分析,在298K当TSFX:BSA从0∶1变化到10∶1时,α-螺旋从48.5%降低到38.6%,β-折叠从23.3%降低到18.3%,而β-转角从15.3%增加到24.1%,无规卷曲从12.9%增加到19.0%,TSFX与BSA的作用使得BSA的二级结构变得松散。应用荧光光谱法、傅里叶变换红外光谱法和紫外-可见吸收光谱法研究了ATLL与BSA相互作用及Zn2+、Cu2+对ATLL与BSA相互作用的影响。实验表明有无Zn2+、Cu2+存在时ATLL与BSA的作用都是静态猝灭机制。Zn2+使结合作用的有效猝灭常数降低,ATLL的药效增加,而Cu2+增大了有效猝灭常数,使ATLL在血液中的储备时间延长。热力学参数表明氢键和疏水作用力在反应中起主要作用,Zn2+、Cu2+对作用力类型没有影响。根据Foster能量转移理论求出了BSA第212位色氨酸残基与ATLL司的平均距离。应用同步荧光和三维荧光对ATLL对BSA构象的影响进行了研究,表明ATLL改变了色氨酸和酪氨酸残基微环境的极性。红外光谱结果显示ATLL引起了BSA二级结构由α-螺旋和β-折叠结构向β-转角和无规卷曲转变,BSA分子结构的松散程度增加。紫外光谱表明Zn2+对ATLL与BSA相互作用的影响可能是通过Zn2+与ATLL竞争结BSA,而Cu2+可能是形成Cu2+-ATLL复合物,通过离子架桥作用影响BSA与ATLL的作用。应用荧光光谱法、紫外-可见吸收光谱法、傅里叶变换红外光谱法和分子模拟的方法研究了NF与HSA的相互作用。实验结果表明NF对HSA荧光猝灭是一个静态猝灭过程,氢键和范德华力在维持复合物稳定中起主要作用。根据Foster非辐射能量转移理论求出了能量给体(Trp-214)和能量受体(NF)之间的距离r,表明在NF与HSA的相互作用中存在非辐射能量转移。取代实验表明NF在HSA上有一个结合位点位于site I。分子模拟进一步确定了NF在HSA上的具体结合信息,如NF主要是通过NF的N11与Lue283；NF的N14与Lue283、Ser287；NF的O7与Ser287的氢键起作用等。三维荧光光谱显示NF与HSA作用后HSA构象发生改变,色氨酸残基微环境的极性降低。红外光谱结果表明NF与HSA的作用引起HSA二级结构由α-螺旋和β-折叠向β-转角和无规卷曲结构的转变,α-螺旋从54.2%降低到45.8%,β-折叠从18.5%降低到15.7%,β-转角从21.7%增加到23.8%,无规卷曲结构从5.6%增加到14.7%。更多还原

【Abstract】 Based on the significance of determination of antimicrobial, the characteristic and application of self-ordered ring (SOR) fluorescent microscopic imaging technique, in this paper we developed the methods for the determination of tosufloxacin tosylate (TSFX), levofloxacin (LVFX) and lomefloxacin (LMX), and employed the developed methods to detect the content of above drugs in tablet, capsule and injecta, the content of TSFX in rabbit serum, LVFX in chiken serum, muscle, liver, manure and LMX in rabbit serum, muscle, liver and kidney after dosing, respectively. Besides, The interactions of TSFX and BSA, ATLL and BSA, NF and HSA were studied by fluorescence spectra, UV-vis absorption spectra and FT-IR spectra and molecular modeling methods.In the pH10.50NH3-NH4C1buffer solution and poly(vinyl alcohol)-124(PVA-124), with Mn2+and cetyltrimethyl ammonium bromide (CTMAB) as sensitizer, a highly sensitive and simple method is developed for the determination of TSFX by the SOR fluorescent microscopic imaging technique. When the droplet volume is0.2μL, TSFX in the range of4.05x10-14-4.28x10-13mol/ring (2.02x10-7-2.14x10-6mol/L) can be detected, and the limit of detection (LOD) can reach4.05x10-15mol/ring (2.02x10" mol/L). This developed methods was applied to determine the content of TSFX in the tablet and in the rabbit serum of different time after dosing. The recovery is90.0-105.0%and relative standard deviations (RSDs) are less than3.3%.Based on the SOR fluorescent microscopic imaging technique on a hydrophobic glass slide with Mn2+and CTMAB as sensitizer, PVA-124and NH3-NH4Cl buffer (pH9.30) as the medium, a method has been developed for determining LVFX residues in chicken muscle, chicken liver, chicken manure, the concentrations in chicken serum and the content of LVFX in tablet and capsule. When the droplet volume is0.2μL, LVFX in the range of5.66xl0-14-1.00x10-13mol/ring can be detected, and the limit of detection (LOD) can reach5.66x10-15mol/ring (2.83x10-8mol/L). It is a viable method for the determination of LVFX in these samples with the recoveries of90.0-105.0%and RSDs0.8-4.0%. The results indicate that the method applied to chicken tissue, manure and serum is reliable and applicable.With Al3+and CTMAB as sensitizer, PVA-124and NH3-NH4Cl (pH9.60) as the medium, a simple and sensitive SOR technique was successfully developed to determination LMX. When the droplet volume is0.2μL, LMX in the range of9.87x10-14-1.47x10-12mol/ring can be detected, and the detection limit can reach9.87x10-15mol/ring (4.93x10-8mol/L). This developed methods was applied to determination LMX content in pharmaceutic preparation(tablet, capsule and injecta), concentrations in rabbit serum and residues in rabbit tissue, urine. It was proved to be a viable method for analysis of LMX in those samples with the recoveries90.6-106.3%and RSDs less than4.2%.The interaction of tosufloxacin tosylate (TSFX) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy, UV-vis spectroscopy and FT-IR spectroscopy. The results indicated that the intrinsic fluorescence of BSA was quenched by TSFX through a static quenching mechanism, and the effective binding constants (Kα) were obtained to be2.58x104L/mol (298K) by means of the modified Stern-Volmer equation. Thermodynamic parameters showed that electrostatic interaction was mostly responsible for the binding of TSFX to BSA. The binding distance (r) between TSFX and Trp-212was determined to be3.90nm according to Foster non-radiative energy transfer theory. BSA had a single class of binding site at Sudlow’sites I in subdomain IIA for TSFX. The effects of TSFX on the conformation of BSA were analyzed by synchronous fluorescence spectra and three-dimensional fluorescence spectra, and the results exhibited that the hydrophobicity of tryptophan microenvironment was decreased. In FT-IR spectra, Fourier self-deconvolution, secondary derivative and the curve-fitting process were carried out to obtain the components of BSA secondary structure. At298K when the molar ratio of TSFX to BSA changed from0:1to10:1, the a-helix decreased from48.5%to38.6%, β-sheet changed from23.3%to18.3%, while β-turn had an increase from15.3%to24.1%, random structure increased from12.9%to 19.0%. This indicated that TSFX induced unfolding of the polypeptides of BSA.The interaction of acetylisovaleryltylosin tartrate (ATLL) and bovine serum albumin (BSA) without or with Zn2+and Cu2+has been studied by fluorescence spectroscopy, FT-IR spectroscopy and UV-vis spectroscopy. The fluorescence of BSA was quenched by ATLL through a static quenching mechanism. The effective quenching constant (Ka) of ATLL to BSA decreased with Zn2+and increased with Cu2+. Thermodynamic parameters revealed that hydrogen bonds and hydrophobic forces played significant roles in the binding of ATLL to BSA. The polarity of tryptophan and tyrosine residues changed on addition of ATLL regardless with or without Zn2+and Cu2+. FT-IR spectra showed that ATLL changed a-helix and β-sheet of BSA into β-turn and random structure. And the adding of Zn2+and Cu2+further loosen the polypeptides of BSA. The UV-vis spectra indicated that the effects of Zn2+on ATLL binding to BSA may through a competition binding, and Cu2+possibly formed Cu2+-ATLL complex via metal ion bridge.The interaction of nitrofurazone (NF) and human serum albumin (HSA) has been studied by fluorescence spectroscopy, UV-vis spectroscopy, FT-IR spectroscopy and molecular modeling methods. The results showed that the fluorescence of HSA was quenched by NF in a static quenching mechanism. Thermodynamic parameters revealed that hydrogen bonds and van der Waals force played the major role during the interaction. The calculated binding distance (r) indicated that the non-radioactive energy transfer coming into being in the interaction between NF and Trp-214of HSA. HSA had a single class of binding site at Sudlow’sites I in subdomain IIA for NF, which was verified by the displacement experiment. The molecular modeling study further confirmed the specific binding sites of NF on HSA, such as the interaction between N11and Lue283; N14and Lue283, Ser287;07and Ser287; predominately through hydrogen bonds. Three-dimensional fluorescence spectra indicated that the polarity around the tryptophan residues decreased and the conformation of HSA changed after adding NF. FT-IR spectra showed that NF could induce the polypeptides of HSA unfolding because it changed α-helix and β-sheet into β-turn and random structure of HAS with the content of α-helix reducing froom54.2％to45.8%, β-sheet reducing froml8.5%to15.7%,β-turn increasing from21.7%to23.8%and random structure from5.6%to14.7%.更多还原