药物及生物大分子的共振瑞利散射和共振非线性散射法研究与应用
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摘要
共振瑞利散射(resonance Rayleigh scattering, RRS)和共振非线性散射(resonance non-linear scattering, RNLS)作为一类新的光散射分析技术兴起并发展于20世纪90年代。RRS分析技术是1993年由Pasternack等在普通的荧光光度计上所建立的,并率先将RRS作为一种分析技术用于核酸的研究和检测；1995年国内刘绍璞教授等在研究离子缔合物RRS的过程中,发现当不同波长的平行单色光通过某些溶液时,在入射光波长的2倍和1/2处也能产生强烈的光散射,首次将它们作为一种光谱分析技术进行研究,并认为这是由于RRS而产生了RNLS,基于此建立了新的痕量分析方法。由于它们灵敏度高,且操作简便快捷而引起了人们的关注,并已成为化学研究中的新的手段,在当前分析化学研究中具有广泛应用前景。
本文首次提出以茜素红及其稀土配合物为光散射探针,研究了其与四环素类药物及生物大分子牛血清白蛋白相互作用的RRS和RNLS光谱,发现稀土离子的加入,使得体系的RRS及RNLS同步增强,据此建立了新的用于痕量药物及生物大分子的分析检测的RRS法和RNLS法,该方法不仅灵敏度高,且分析的线性范围宽；同时以RRS法研究了甲基蓝(MB)与血红蛋白(Hb)的聚集作用,将此法用于Hb的痕量分析具有很高的灵敏度。据此,本论文分以下两部分进行研究：第一部分茜素红-稀土离子配合物为光散射探针的共振瑞利散射和共振非线性散射法测定四环素类药物
(1)在pH 5.75的HAc-NaAc缓冲液中,茜素红(ARS)-La与四环素(TC)形成三元配合物,导致体系的共振瑞利散射(RRS)、二级散射(SOS)和倍频散射(FDS)均增强,光谱最大散射波长分别位于312nm、580nm和290nm,对于RRS在0.004-0.444μg/mL、SOS在0.008-0.444μg/mL和FDS在0.008-0.444μg/mL范围内呈良好的线性关系,TC的检出限分别为2.95 ng/mL(RRS法)、2.47 ng/mL(SOS法)和2.27ng/mL(FDS法),据此建立了灵敏的测定四环素的RRS和RNLS法,检出限达到纳克级。并以SOS法考察了ARS-La-TC体系的反应条件、影响因素等,以标准加入法对生物样品进行分析,结果满意。
(2)在pH 4.4的HAc-NaAc缓冲液中,茜素红(ARS)-Eu与米诺环素(MC)形成三元配合物,导致共振瑞利散射(RRS)增强,首次以染料-稀土配合物为光散射探针,建立了一种测定米诺环素的RRS分析方法。在λ=370nm处,米诺环素浓度在0.073-5.493μg/mL范围内与体系RRS强度呈良好的线性关系,检出限为0.021μg/mL。此方法灵敏度高,简单、快速,具有良好的选择性和重复性,对尿样进行加标回收及用于片剂、胶囊中米诺环素的测定,结果满意。第二部分染料及其稀土离子配合物为光散射探针的共振瑞利散射和共振非线性散射法在生物大分子测定中的研究及应用
(1)在pH 4.5的HAc-NaAc缓冲液中,茜素红(ARS)-La与牛血清白蛋白(BSA)形成三元离子缔合物,导致共振瑞利散射(RRS)、二级散射(SOS)和倍频散射(FDS)同步显著增强,光谱最大散射波长分别位于373 nm、620 nm和310nm,对于RRS在0.034-13.4μg/mL、SOS在0.201-13.40μg/mL和FDS在0.201-10.05μg/mL范围内呈良好的线性关系,对于BSA的检出限分别为0.010μg/mL(RRS法)、0.052μg/mL(SOS法)和0.089μg/mL(FDS法),首次以染料-稀土配合物为光散射探针,建立了灵敏的测定BSA的RRS和RNLS分析法。以RRS法考察了ARS-La与BSA三元离子缔合物的适宜条件、影响因素等,此方法可用于生物样品中蛋白含量的测定,并对样品进行加标回收,结果满意。
(2)以甲基蓝(MB)为光散射探针,应用RRS法研究了与血红蛋白(Hb)相互作用的二元体系的光散射性质。在弱酸性条件下,MB可以通过静电和疏水作用聚集到Hb上并形成离子缔合物,不仅使吸收光谱发生变化,而且使得RRS增强。据此发展了一种新的测定Hb的RRS便捷方法,其线性范围和检出限分别为0.2-20μg/mL和0.012μg/mL。与RRS法相比,分光光度法的线性范围和检出限分别为8-60μg/mL和0.688μg/mL (344 nm),5-60μg/mL和0.398μg/mL (408 nm)。此RRS法可用于尿样中Hb的痕量检测。实验考察了体系的吸收、散射光谱性质和最佳聚合条件,并在最佳条件下研究了分析参数及作用机理。最后首次以RRS法初步讨论了有机小分子对体系的影响。
Resonance Rayleigh scattering (RRS) and Resonance non-linear scattering (RNLS) as a kind of new light scattering technique which is rise and developed in 1990s. The technology of RRS analysis was established in 1993 by Pasternack et al. on a common spectrofluorometer, and firstly it was applied to research and determine DNA. While Prof.Liu Shaopu and his coworkers studying the RRS of process of ionic association, they discovered when monochromatic light of different wavelengths in parallel by some of the solution,2 times the wavelength of the incident light and 1/2 can also have a strong light scattering, respectively. The first time, they used as a spectral analysis technology to study, and thought this is due to resonance Rayleigh scattering from the resonance nonlinear scattering, based on this established a new method of trace analysis. As a new tool in chemical research, RRS and RNLS have received much attention and has a broad application prospects due to their high sensitivity, simplicity and convenient.
In this research, the first proposed method was the alizarin red S (ARS) and its rare earth complexes as light scattering probe. The RRS and RNLS spectra of their interaction between complexes and tetracycline drugs or biological macromolecules of bovine serum albumin were studied, and the added of rare earth ion makes the RRS and RNLS signals of two simultaneous enhanced. Accordingly, new high sensitive RRS method and RNLS method of drugs and biological macromolecules for trace analysis and detection were established with the wide linear range. Simultaneously, the aggregation of Methyl blue (MB) and hemoglobin (Hb) was investigated by RRS method and using for trace analysis of Hb with high sensitivity. Herein, this paper includes two parts:
Part one Determination of tetracycline antibiotics using alizarin red S (ARS)-rare earth ion complexes as a light scattering probe by resonance Rayleigh scattering and resonance non-linear scattering
(1)In buffer solution of HAc-NaAc (pH 5.75), alizarin red S (ARS)-La3+ was combined with tetracycline (TC) and the ternary complexes were formed. This resulted in prominent enhancement of resonance Rayleigh scattering (RRS)、Second-Order scattering (SOS) and Frequency-Double scattering (FDS), The maximum scattering wavelengths were 312 nm for RRS,580 nm for SOS and 290nm for FDS, respectively. The scattering intensity of system was proportional to the concentration of TC in certain ranges. The linear ranges and the detection limits of TC were 0.004-0.444μg/mL and 2.95 ng/mL for RRS method, 0.008-0.444μg/mL and 2.47 ng/mL for SOS method,0.008-0.444μg/mL and 2.27 ng/mL for FDS method, respectively. Based on the enhancement of light scattering, the proposed methods (resonance linear and nonlinear scattering analysis method) can be sensitively applied to the determination of TC, and the detection limit could reach nanogram level. The reacting conditions and influence factors of ARS-La-TC system were also investigated by SOS method. The proposed methods can be applied to the determination of TC content in biological samples by the standard addition method, and the results were satisfied.
(2) In buffer solution of NaAc-HAc (pH 4.4), minocycline (MC) was combined with alizarin red S (ARS) and Eu3+ by intermolecular forces to form ternary complexes, which causing an enhancement of RRS at 370 nm. Based on the enhancement of RRS, a novel method can be applied to the determination of MC. The linear range for the determination of MC was 0.073-5.493μg/mL and the detection limit was 0.021μg/mL. The proposed method was sensitive, simple and fast; it also had well selectivity and repetitiveness. The urine sample was analyzed by the standard addition method; the method could be applied to the determination of MC in tablet and capsule, and the results were satisfied.
Part two Determination of biomacromolecules using dye and its rare earth ion complexes as a light scattering probe by resonance Rayleigh scattering and resonance non-linear scattering
(1) In buffer solution of HAc-NaAc (pH 4.5), alizarin red S (ARS)-La3+ was combined with bovine serum albumin (BSA) and form ternary ion-complexes, which resulted in significant enhancement of resonance Rayleigh scattering (RRS)、Second-Order scattering(SOS) and Frequency-Double scattering (FDS). The maximum scattering wavelengths were 373 nm for RRS,620 nm for SOS and 310nm for FDS, respectively. The scattering intensity of system was proportional to the concentration of BSA in certain ranges. The linear ranges and the detection limits of BSA were 0.034-13.40μg/mL and 0.010μg/mL for RRS method,0.201-13.40μg/mL and 0.052μg/mL for SOS method, 0.201-10.05μg/mL and 0.089μg/mL for FDS method, respectively. Based on the enhancing of light scattering, the proposed methods (resonance linear and nonlinear scattering analysis method) can be applied to the determination of BSA, sensitively. The reacting conditions and influence fators of ARS-La-BSA system were investigated by RRS method. The proposed methods can be applied to the determination of protein content in biological samples, and the results were satisfied.
(2)The light scattering characteristics of MB-Hb binary system were investigated by RRS method which using MB as a light scattering probe. In weak acidic condition, methyl blue (MB) could aggregate on hemoglobin (Hb) and form ion-association complexes by electrostatic and hydrophobic interactions. It resulted in not only the changes of the absorption spectrum, but also the enhancement of resonance Rayleigh scattering (RRS). According to these phenomena, a convenient method for the determination of Hb was developed. The linear range and the detection limit for the determination of Hb were 0.2-20μg/mL and 0.012μg/mL, respectively. Compared with RRS method, the linear range and the detection limits for Spectrophotometry (SP) were 8-60μg/mL and 0.688μg/mL (at 344 nm),5-60μg/mL and 0.398μg/mL (at 408 nm), respectively. In this paper, the RRS method has been successfully applied to the determination of trace Hb in urine samples. The spectral characteristics of the absorption and scattering, the optimum conditions of the aggregating were investigated. Besides, the parameters of the analytical chemistry and mechanism of interaction were investigated under the optimum conditions. Finally, the influences of small organic molecules (S.O.M.) were also discussed, firstly.
本文首次提出以茜素红及其稀土配合物为光散射探针,研究了其与四环素类药物及生物大分子牛血清白蛋白相互作用的RRS和RNLS光谱,发现稀土离子的加入,使得体系的RRS及RNLS同步增强,据此建立了新的用于痕量药物及生物大分子的分析检测的RRS法和RNLS法,该方法不仅灵敏度高,且分析的线性范围宽；同时以RRS法研究了甲基蓝(MB)与血红蛋白(Hb)的聚集作用,将此法用于Hb的痕量分析具有很高的灵敏度。据此,本论文分以下两部分进行研究：第一部分茜素红-稀土离子配合物为光散射探针的共振瑞利散射和共振非线性散射法测定四环素类药物
(1)在pH 5.75的HAc-NaAc缓冲液中,茜素红(ARS)-La与四环素(TC)形成三元配合物,导致体系的共振瑞利散射(RRS)、二级散射(SOS)和倍频散射(FDS)均增强,光谱最大散射波长分别位于312nm、580nm和290nm,对于RRS在0.004-0.444μg/mL、SOS在0.008-0.444μg/mL和FDS在0.008-0.444μg/mL范围内呈良好的线性关系,TC的检出限分别为2.95 ng/mL(RRS法)、2.47 ng/mL(SOS法)和2.27ng/mL(FDS法),据此建立了灵敏的测定四环素的RRS和RNLS法,检出限达到纳克级。并以SOS法考察了ARS-La-TC体系的反应条件、影响因素等,以标准加入法对生物样品进行分析,结果满意。
(2)在pH 4.4的HAc-NaAc缓冲液中,茜素红(ARS)-Eu与米诺环素(MC)形成三元配合物,导致共振瑞利散射(RRS)增强,首次以染料-稀土配合物为光散射探针,建立了一种测定米诺环素的RRS分析方法。在λ=370nm处,米诺环素浓度在0.073-5.493μg/mL范围内与体系RRS强度呈良好的线性关系,检出限为0.021μg/mL。此方法灵敏度高,简单、快速,具有良好的选择性和重复性,对尿样进行加标回收及用于片剂、胶囊中米诺环素的测定,结果满意。第二部分染料及其稀土离子配合物为光散射探针的共振瑞利散射和共振非线性散射法在生物大分子测定中的研究及应用
(1)在pH 4.5的HAc-NaAc缓冲液中,茜素红(ARS)-La与牛血清白蛋白(BSA)形成三元离子缔合物,导致共振瑞利散射(RRS)、二级散射(SOS)和倍频散射(FDS)同步显著增强,光谱最大散射波长分别位于373 nm、620 nm和310nm,对于RRS在0.034-13.4μg/mL、SOS在0.201-13.40μg/mL和FDS在0.201-10.05μg/mL范围内呈良好的线性关系,对于BSA的检出限分别为0.010μg/mL(RRS法)、0.052μg/mL(SOS法)和0.089μg/mL(FDS法),首次以染料-稀土配合物为光散射探针,建立了灵敏的测定BSA的RRS和RNLS分析法。以RRS法考察了ARS-La与BSA三元离子缔合物的适宜条件、影响因素等,此方法可用于生物样品中蛋白含量的测定,并对样品进行加标回收,结果满意。
(2)以甲基蓝(MB)为光散射探针,应用RRS法研究了与血红蛋白(Hb)相互作用的二元体系的光散射性质。在弱酸性条件下,MB可以通过静电和疏水作用聚集到Hb上并形成离子缔合物,不仅使吸收光谱发生变化,而且使得RRS增强。据此发展了一种新的测定Hb的RRS便捷方法,其线性范围和检出限分别为0.2-20μg/mL和0.012μg/mL。与RRS法相比,分光光度法的线性范围和检出限分别为8-60μg/mL和0.688μg/mL (344 nm),5-60μg/mL和0.398μg/mL (408 nm)。此RRS法可用于尿样中Hb的痕量检测。实验考察了体系的吸收、散射光谱性质和最佳聚合条件,并在最佳条件下研究了分析参数及作用机理。最后首次以RRS法初步讨论了有机小分子对体系的影响。
Resonance Rayleigh scattering (RRS) and Resonance non-linear scattering (RNLS) as a kind of new light scattering technique which is rise and developed in 1990s. The technology of RRS analysis was established in 1993 by Pasternack et al. on a common spectrofluorometer, and firstly it was applied to research and determine DNA. While Prof.Liu Shaopu and his coworkers studying the RRS of process of ionic association, they discovered when monochromatic light of different wavelengths in parallel by some of the solution,2 times the wavelength of the incident light and 1/2 can also have a strong light scattering, respectively. The first time, they used as a spectral analysis technology to study, and thought this is due to resonance Rayleigh scattering from the resonance nonlinear scattering, based on this established a new method of trace analysis. As a new tool in chemical research, RRS and RNLS have received much attention and has a broad application prospects due to their high sensitivity, simplicity and convenient.
In this research, the first proposed method was the alizarin red S (ARS) and its rare earth complexes as light scattering probe. The RRS and RNLS spectra of their interaction between complexes and tetracycline drugs or biological macromolecules of bovine serum albumin were studied, and the added of rare earth ion makes the RRS and RNLS signals of two simultaneous enhanced. Accordingly, new high sensitive RRS method and RNLS method of drugs and biological macromolecules for trace analysis and detection were established with the wide linear range. Simultaneously, the aggregation of Methyl blue (MB) and hemoglobin (Hb) was investigated by RRS method and using for trace analysis of Hb with high sensitivity. Herein, this paper includes two parts:
Part one Determination of tetracycline antibiotics using alizarin red S (ARS)-rare earth ion complexes as a light scattering probe by resonance Rayleigh scattering and resonance non-linear scattering
(1)In buffer solution of HAc-NaAc (pH 5.75), alizarin red S (ARS)-La3+ was combined with tetracycline (TC) and the ternary complexes were formed. This resulted in prominent enhancement of resonance Rayleigh scattering (RRS)、Second-Order scattering (SOS) and Frequency-Double scattering (FDS), The maximum scattering wavelengths were 312 nm for RRS,580 nm for SOS and 290nm for FDS, respectively. The scattering intensity of system was proportional to the concentration of TC in certain ranges. The linear ranges and the detection limits of TC were 0.004-0.444μg/mL and 2.95 ng/mL for RRS method, 0.008-0.444μg/mL and 2.47 ng/mL for SOS method,0.008-0.444μg/mL and 2.27 ng/mL for FDS method, respectively. Based on the enhancement of light scattering, the proposed methods (resonance linear and nonlinear scattering analysis method) can be sensitively applied to the determination of TC, and the detection limit could reach nanogram level. The reacting conditions and influence factors of ARS-La-TC system were also investigated by SOS method. The proposed methods can be applied to the determination of TC content in biological samples by the standard addition method, and the results were satisfied.
(2) In buffer solution of NaAc-HAc (pH 4.4), minocycline (MC) was combined with alizarin red S (ARS) and Eu3+ by intermolecular forces to form ternary complexes, which causing an enhancement of RRS at 370 nm. Based on the enhancement of RRS, a novel method can be applied to the determination of MC. The linear range for the determination of MC was 0.073-5.493μg/mL and the detection limit was 0.021μg/mL. The proposed method was sensitive, simple and fast; it also had well selectivity and repetitiveness. The urine sample was analyzed by the standard addition method; the method could be applied to the determination of MC in tablet and capsule, and the results were satisfied.
Part two Determination of biomacromolecules using dye and its rare earth ion complexes as a light scattering probe by resonance Rayleigh scattering and resonance non-linear scattering
(1) In buffer solution of HAc-NaAc (pH 4.5), alizarin red S (ARS)-La3+ was combined with bovine serum albumin (BSA) and form ternary ion-complexes, which resulted in significant enhancement of resonance Rayleigh scattering (RRS)、Second-Order scattering(SOS) and Frequency-Double scattering (FDS). The maximum scattering wavelengths were 373 nm for RRS,620 nm for SOS and 310nm for FDS, respectively. The scattering intensity of system was proportional to the concentration of BSA in certain ranges. The linear ranges and the detection limits of BSA were 0.034-13.40μg/mL and 0.010μg/mL for RRS method,0.201-13.40μg/mL and 0.052μg/mL for SOS method, 0.201-10.05μg/mL and 0.089μg/mL for FDS method, respectively. Based on the enhancing of light scattering, the proposed methods (resonance linear and nonlinear scattering analysis method) can be applied to the determination of BSA, sensitively. The reacting conditions and influence fators of ARS-La-BSA system were investigated by RRS method. The proposed methods can be applied to the determination of protein content in biological samples, and the results were satisfied.
(2)The light scattering characteristics of MB-Hb binary system were investigated by RRS method which using MB as a light scattering probe. In weak acidic condition, methyl blue (MB) could aggregate on hemoglobin (Hb) and form ion-association complexes by electrostatic and hydrophobic interactions. It resulted in not only the changes of the absorption spectrum, but also the enhancement of resonance Rayleigh scattering (RRS). According to these phenomena, a convenient method for the determination of Hb was developed. The linear range and the detection limit for the determination of Hb were 0.2-20μg/mL and 0.012μg/mL, respectively. Compared with RRS method, the linear range and the detection limits for Spectrophotometry (SP) were 8-60μg/mL and 0.688μg/mL (at 344 nm),5-60μg/mL and 0.398μg/mL (at 408 nm), respectively. In this paper, the RRS method has been successfully applied to the determination of trace Hb in urine samples. The spectral characteristics of the absorption and scattering, the optimum conditions of the aggregating were investigated. Besides, the parameters of the analytical chemistry and mechanism of interaction were investigated under the optimum conditions. Finally, the influences of small organic molecules (S.O.M.) were also discussed, firstly.
引文
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