光散射技术及其在药物生物大分子分析中的应用研究
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摘要
光散射技术自建立以来，已经得到了广泛的应用和发展。本文以共振光散射分析技术为主要研究手段，利用金属纳米粒子的等离子共振光散射性质建立了灵敏测定药物、生物大分子的方法，期望为药物、生物大分子的分析提供一种灵敏的测定方法，为医药、生化领域的研究提供一定的帮助；研究了荧光分析法中的两种干扰因素：拉曼散射和内滤效应，通过荧光光度计将二者结合起来建立了一种新的分析方法，并将其用于金属钴离子的测定，期望进一步开发荧光光度计的功能；共振光散射技术的选择性较差一直是阻碍其发展的不利因素，为了解决这一问题，本文初步建立了高效液相色谱和共振光散射的联用技术，期望通过二者的联用，解决共振光散射技术选择性和重现性差的问题，同时发展一种高选择性高灵敏度的分离分析技术。本文主要研究内容如下：
一．金属纳米材料由于其特殊的物理化学性质已经被广泛的应用于生化药物的分析领域，尤其是金、银纳米粒子的等离子吸收性质，已被用于DNA杂交、免疫分析等，但是它们强的光散射能力的开发却只有少量报道。本文主要将金和银纳米粒子的等离子共振光散射性质应用于药物、生物大分子分析，建立了灵敏的测定药物与生物大分子的方法。
1)．在NaOH和NH_3·H_2O共存的碱性条件下，硝酸银与阿魏酸发生氧化还原反应生成银纳米粒子，产生增强的等离子共振光散射信号，根据产生的共振光散射信号和阿魏酸浓度的关系，建立了等离子共振光散射测定阿魏酸的分析方法。在最佳实验条件下，可检测阿魏酸浓度范围为0.2～2.0μmol L~(-1)，检出限为15.2nmol L~(-1)。研究结果表明，该方法简单、快速、灵敏，本文为其它类似的酚酸类物质的检测提供了一种可靠的方法。此外，本文还提供了一种环境友好的合成银纳米粒子的方法。
2)．在NaOH和NH_3·H_2O共同存在的碱性介质中，硝酸银可以与儿茶酚胺类药物左旋多巴发生氧化还原反应，产生银纳米粒子，进而产生增强的等离子共振光散射信号，根据信号强度和左旋多巴浓度之间的关系，我们建立了等离子共振光散射技术测定左旋多巴的方法，在最佳实验条件下，可检测左旋多巴浓度为0.2～2.0μmolL~(-1)，检出限为20.1nmolL~(-1)。通过理论探讨，我们认为左旋多巴参与反应的活性基团主要是邻二酚，而儿茶酚胺类药物是以具有邻二酚的儿茶酚为母核的一类化合物，因此我们推测其它儿茶酚胺类药物，比如多巴胺、肾上腺素、去甲肾上腺素等，在一定的实验条件下都可以和硝酸银反应生成银纳米粒子而产生增强的等离子共振光散射信号，据此可以建立儿茶酚胺类药物的分析方法。通过以左旋多巴为代表，证明该分析方法简单、快速、灵敏、有效。
3)．凝血酶在血栓形成过程中起重要作用，因此血浆凝血酶浓度的检测对临床疾病的诊断、病程发展、愈后以及疗效的监测和评估等都具有重要的意义。本文以金纳米粒子为探针，将巯基修饰的凝血酶适配子DNA连接于金纳米粒子表面，向该溶液中加入凝血酶后，由于凝血酶和其适配子DNA的反应，通过吸收光谱和透射电子显微镜表征，发现金纳米粒子发生了聚集，从而引起金纳米粒子的等离子共振光散射增强，基于此建立了利用等离子共振光散射技术测定凝血酶的分析方法。实验发现，金纳米粒子的等离子共振光散射信号强度与加入的凝血酶浓度在1.8～9.0nmolL~(-1)范围内呈良好的线性关系，检出限浓度为0.2nmol L~(-1)，可见该方法可以灵敏的检测凝血酶。由于凝血初期产生的凝血酶浓度在纳摩尔水平，因此该方法可用于临床上凝血酶的测定。
二．在荧光分析法中，拉曼散射和内滤效应常常作为干扰因素而存在，通常需要避免或校正。本着“分析科学在于合理的利用信号”的原则，本文通过普通的荧光分光光度计将这两种干扰因素结合起来，以钴(Ⅱ)和钴试剂为研究对象，利用水的拉曼散射信号和钴试剂的内滤效应，通过钴(Ⅱ)加入前后，钴试剂对水的拉曼散射光吸收不同，即发生内滤效应的程度不同，从而引起拉曼散射信号随着钴(Ⅱ)浓度的变化而变化，通过理论推导建立了拉曼散射信号强度与钴(Ⅱ)浓度之间的线性关系，建立了基于水的拉曼散射信号和试剂的内滤效应测定钴(Ⅱ)的分析方法，该方法的检出限为1.2×10~(-7)molL~(-1)，灵敏度与吸收光度法相当。该方法以最常用的溶剂水为探针，利用水的拉曼散射信号，不需要固定的激发波长，可以根据待分析样品选择合适的激发和发射波长，非常灵活、简单，并且提出了一种在荧光光度计上测定非荧光物质的新方法，进一步开发了荧光分光光度计的功能。
三．共振光散射分析技术虽然灵敏度很高，但是其选择性和重现性较差一直是使该技术不尽完美之处；而色谱分离技术的最大优势就在于它们的高分离效率和高选择性，但分析检测的灵敏度不高。本文以高效液相色谱技术为例，以胰岛素为研究对象，初步建立了高效液相色谱和共振光散射的联用技术，该联用技术集两者优势于一体，既提高了共振光散射分析技术的选择性和重现性，同时又提高了高效液相色谱的检测灵敏度，丰富了检测信号的类型。随着研究的深入和联用技术的成熟，共振光散射检测器有望发展成为高效液相色谱的通用检测器。
Since the establishment of the light scattering technique, it has applied widely and developed quickly. In this contribution, based on the resonance light scattering (RLS) technique, we have employed the plasmon resonance light scattering (PRLS) properties of metal nanoparticles and constructed methods for the determination of the Pharmaceuticals and biomacromolecules, which would provide sensitive detection methods for pharmaceuticals and biomacromolecules and may be helpful in the fields of medicine and biochemistry; Raman scattering and Inner Filter Effect (IFE) are the interference sources in spectrofluorometry, we have coupled them together reasonably and proposed a novel approach for the determination of Co(II) by spectrofluorometer, the function of spectrofluorometer can be further exploited; Poor selectivity is always the factor that limits the development of RLS technique, to solve the problem, we have also incorporated the high performance liquid chromatography (HPLC) technique and the RLS technique by employing the insulin as a model system, We hope that the combination of HPLC-RLS technique could improve the selectivity and stability of the RLS technique and then develop a new technique with high slectivity and high sensitivity. The main contents of investigation in the contribution are as follows:
Metal nanoparticles have been found the applications in the fields of pharmaceuticals and biomacromolecules analysis for their unusual physicochemical properties. Of the nanoparticles, especially gold and silver nanoparticles, their plasmon absorption properties have been widely used in the fields, such as DNA hybridization, immunoassay and so on, but the research on their PRLS properties has been little reported. In this contribution, we have focused on the PRLS properties of the nanoparticles and applied them to the pharmaceutical and biomacromolecules analysis.
1). In the coexistence of NaOH and NH_3-H_2O medium, it was found that, Ferulic Acid (FA) acts as a reducing agent and can be oxidized by AgNO_3, resulting in the formation of silver nanoparticles (NPs) which give the enhanced PRLS signals, the detection method of FA was constructed according to the linear relationship between the PRLS intensities and the concentrations of FA. Under the optimal conditions, the FA concentrations of 0.2~2.0umol L~(-1) could be detected and the detection limit was 15.2nmol L~(-1). The results have shown that the method is simple, rapid and sensitive, it also provides a reliable detection method for other phenolic compounds. What's more, the present reaction maybe provides the basis of an environmentally friendly approach for the synthesization of silver nanoparticles.
2). In the alkaline medium with the coexistence of NaOH and NH_3H_2O, one of the Catecholamines, L-DOPA can be oxidized by AgNO_3, resulting in the formation of silver NPs and the enhanced PRLS signals, the method for L-DOPA detection was constructed according to the linear relationship between the PRLS intensities and the concentrations of L-DOPA. Under the optimal conditions, the L-DOPA concentrations of 0.2～2.0μmolL~(-1) could be detected and the detection limit was 20.1nmol L~(-1). By the mechanism investigations, we conclude that the groups oxidized in L-DOPA are the two adjacent phenol groups, while all the catecholamine compounds possess the two adjacent phenol groups, so all of other catecholamine compounds, such as dopamine, adrenaline, noradrenaline, could be anticipated to be oxidized by AgNO_3 under the corresponding conditions, resulting in the formation of silver NPs and the enhanced PRLS signal, so the detection methods for the catecholamine compounds could be constructed as L-DOPA. The results of detection L-DOPA have shown that the method is simple, rapid, sensitive and reliable.
3).Thrombin plays an important role in the course of thrombus formation, so the detection of thrombin has important significances for the diseases diagnosis, evolution, evaluation of curative effect. In this contribution, gold nanoparticles (NPs) stabilized with citrate were functionalized by the thiolated thrombin binding aptamer (TBA), the gold NPs are served as a probe, when thrombin was added into the solution, the gold NPs would be aggregated for thrombin reacted with TBA, which was testified by the absorption spectra and the TEM images of gold NPs, and the then the PRLS signals were enhanced, so we set up a method for the detection of thrombin based on the PRLS signals of gold NPs. The results showed that the PRLS intensities were in proportion to the thrombin concentrations over the range of 1.8～9.0nmol L~(-1), the detection limit of thrombin was 0.2 nmol L~(-1), so it is a sensitive method for thrombin detection. During the initial phase of coagulation thrombin is produced in nanomolar concentrations, so the present method can be used for the clinical detection of thrombin.
Both Raman scattering and IFE are the interference sources and usually should be avoided or corrected in spectrofluorometry. We suppose analytical sciences lie on the reasonable application of signals only if the intensity of the signals has simple functional relationship with the content of analytes. In this contribution, we propose a quantitative method based on the IFE of reagents on the Raman scattering signals of solvent by taking the complexation of Co(II) with 4-[(5-chloro-2-pyridyl)azo]-l,3-diaminobenzene (5-C1-PADAB) as a model system on a common spectrofluorometer. In a solution of 5-C1-PADAB, the Raman scattering signals of water are decreased owing to the IFE of 5-C1-PADAB, after the addition of Co(II) which can form the complex with 5-C1-PADAB and consumes the reagent, then the Raman scattering signals were found recovered, based on the linear relationship between the enhanced Raman scattering intensities and the concentrations of Co(II), Co(II) was detected, the detection limit is 1.2×10~(-7) mol L~(-1) which is comparable to the generally used UV-Vis spectrophotometry. In the present method, the commonly used solvent, water, serves as a probe, and the Raman scattering signals of water are used, the obtain of Raman scattering does not require flexed excitaion wavelength, so we can freely select the excitation wavelength according to the analyte, which is very flexible and simple. What's more, a novel method for detection of some non-fluorescent compounds was proposed in a common spectrofluorometer, the function of spectrofluorometer was further exploited.
As we know, the RLS technique is very sensitive for the detection, but its poor selectivity and stability are the limitations for being as a perfect technique. While high separation resolution and selectivity are the advantages of chromatography all the time, the disadvantage lies in the poor detection sensitivity. Here HPLC is chosen as a model, combining the advantages of the both techniques, the incorporation of HPLC-RLS was carried out in the contribution, the incorporated technique not only improves the selectivity and stability of the RLS technique, but also enhances the detection sensitivity of HPLC and enriches the detection types of HPLC. With the deep research and developed incorporated technique, the RLS detector is hopeful for HPLC.
一．金属纳米材料由于其特殊的物理化学性质已经被广泛的应用于生化药物的分析领域，尤其是金、银纳米粒子的等离子吸收性质，已被用于DNA杂交、免疫分析等，但是它们强的光散射能力的开发却只有少量报道。本文主要将金和银纳米粒子的等离子共振光散射性质应用于药物、生物大分子分析，建立了灵敏的测定药物与生物大分子的方法。
1)．在NaOH和NH_3·H_2O共存的碱性条件下，硝酸银与阿魏酸发生氧化还原反应生成银纳米粒子，产生增强的等离子共振光散射信号，根据产生的共振光散射信号和阿魏酸浓度的关系，建立了等离子共振光散射测定阿魏酸的分析方法。在最佳实验条件下，可检测阿魏酸浓度范围为0.2～2.0μmol L~(-1)，检出限为15.2nmol L~(-1)。研究结果表明，该方法简单、快速、灵敏，本文为其它类似的酚酸类物质的检测提供了一种可靠的方法。此外，本文还提供了一种环境友好的合成银纳米粒子的方法。
2)．在NaOH和NH_3·H_2O共同存在的碱性介质中，硝酸银可以与儿茶酚胺类药物左旋多巴发生氧化还原反应，产生银纳米粒子，进而产生增强的等离子共振光散射信号，根据信号强度和左旋多巴浓度之间的关系，我们建立了等离子共振光散射技术测定左旋多巴的方法，在最佳实验条件下，可检测左旋多巴浓度为0.2～2.0μmolL~(-1)，检出限为20.1nmolL~(-1)。通过理论探讨，我们认为左旋多巴参与反应的活性基团主要是邻二酚，而儿茶酚胺类药物是以具有邻二酚的儿茶酚为母核的一类化合物，因此我们推测其它儿茶酚胺类药物，比如多巴胺、肾上腺素、去甲肾上腺素等，在一定的实验条件下都可以和硝酸银反应生成银纳米粒子而产生增强的等离子共振光散射信号，据此可以建立儿茶酚胺类药物的分析方法。通过以左旋多巴为代表，证明该分析方法简单、快速、灵敏、有效。
3)．凝血酶在血栓形成过程中起重要作用，因此血浆凝血酶浓度的检测对临床疾病的诊断、病程发展、愈后以及疗效的监测和评估等都具有重要的意义。本文以金纳米粒子为探针，将巯基修饰的凝血酶适配子DNA连接于金纳米粒子表面，向该溶液中加入凝血酶后，由于凝血酶和其适配子DNA的反应，通过吸收光谱和透射电子显微镜表征，发现金纳米粒子发生了聚集，从而引起金纳米粒子的等离子共振光散射增强，基于此建立了利用等离子共振光散射技术测定凝血酶的分析方法。实验发现，金纳米粒子的等离子共振光散射信号强度与加入的凝血酶浓度在1.8～9.0nmolL~(-1)范围内呈良好的线性关系，检出限浓度为0.2nmol L~(-1)，可见该方法可以灵敏的检测凝血酶。由于凝血初期产生的凝血酶浓度在纳摩尔水平，因此该方法可用于临床上凝血酶的测定。
二．在荧光分析法中，拉曼散射和内滤效应常常作为干扰因素而存在，通常需要避免或校正。本着“分析科学在于合理的利用信号”的原则，本文通过普通的荧光分光光度计将这两种干扰因素结合起来，以钴(Ⅱ)和钴试剂为研究对象，利用水的拉曼散射信号和钴试剂的内滤效应，通过钴(Ⅱ)加入前后，钴试剂对水的拉曼散射光吸收不同，即发生内滤效应的程度不同，从而引起拉曼散射信号随着钴(Ⅱ)浓度的变化而变化，通过理论推导建立了拉曼散射信号强度与钴(Ⅱ)浓度之间的线性关系，建立了基于水的拉曼散射信号和试剂的内滤效应测定钴(Ⅱ)的分析方法，该方法的检出限为1.2×10~(-7)molL~(-1)，灵敏度与吸收光度法相当。该方法以最常用的溶剂水为探针，利用水的拉曼散射信号，不需要固定的激发波长，可以根据待分析样品选择合适的激发和发射波长，非常灵活、简单，并且提出了一种在荧光光度计上测定非荧光物质的新方法，进一步开发了荧光分光光度计的功能。
三．共振光散射分析技术虽然灵敏度很高，但是其选择性和重现性较差一直是使该技术不尽完美之处；而色谱分离技术的最大优势就在于它们的高分离效率和高选择性，但分析检测的灵敏度不高。本文以高效液相色谱技术为例，以胰岛素为研究对象，初步建立了高效液相色谱和共振光散射的联用技术，该联用技术集两者优势于一体，既提高了共振光散射分析技术的选择性和重现性，同时又提高了高效液相色谱的检测灵敏度，丰富了检测信号的类型。随着研究的深入和联用技术的成熟，共振光散射检测器有望发展成为高效液相色谱的通用检测器。
Since the establishment of the light scattering technique, it has applied widely and developed quickly. In this contribution, based on the resonance light scattering (RLS) technique, we have employed the plasmon resonance light scattering (PRLS) properties of metal nanoparticles and constructed methods for the determination of the Pharmaceuticals and biomacromolecules, which would provide sensitive detection methods for pharmaceuticals and biomacromolecules and may be helpful in the fields of medicine and biochemistry; Raman scattering and Inner Filter Effect (IFE) are the interference sources in spectrofluorometry, we have coupled them together reasonably and proposed a novel approach for the determination of Co(II) by spectrofluorometer, the function of spectrofluorometer can be further exploited; Poor selectivity is always the factor that limits the development of RLS technique, to solve the problem, we have also incorporated the high performance liquid chromatography (HPLC) technique and the RLS technique by employing the insulin as a model system, We hope that the combination of HPLC-RLS technique could improve the selectivity and stability of the RLS technique and then develop a new technique with high slectivity and high sensitivity. The main contents of investigation in the contribution are as follows:
Metal nanoparticles have been found the applications in the fields of pharmaceuticals and biomacromolecules analysis for their unusual physicochemical properties. Of the nanoparticles, especially gold and silver nanoparticles, their plasmon absorption properties have been widely used in the fields, such as DNA hybridization, immunoassay and so on, but the research on their PRLS properties has been little reported. In this contribution, we have focused on the PRLS properties of the nanoparticles and applied them to the pharmaceutical and biomacromolecules analysis.
1). In the coexistence of NaOH and NH_3-H_2O medium, it was found that, Ferulic Acid (FA) acts as a reducing agent and can be oxidized by AgNO_3, resulting in the formation of silver nanoparticles (NPs) which give the enhanced PRLS signals, the detection method of FA was constructed according to the linear relationship between the PRLS intensities and the concentrations of FA. Under the optimal conditions, the FA concentrations of 0.2~2.0umol L~(-1) could be detected and the detection limit was 15.2nmol L~(-1). The results have shown that the method is simple, rapid and sensitive, it also provides a reliable detection method for other phenolic compounds. What's more, the present reaction maybe provides the basis of an environmentally friendly approach for the synthesization of silver nanoparticles.
2). In the alkaline medium with the coexistence of NaOH and NH_3H_2O, one of the Catecholamines, L-DOPA can be oxidized by AgNO_3, resulting in the formation of silver NPs and the enhanced PRLS signals, the method for L-DOPA detection was constructed according to the linear relationship between the PRLS intensities and the concentrations of L-DOPA. Under the optimal conditions, the L-DOPA concentrations of 0.2～2.0μmolL~(-1) could be detected and the detection limit was 20.1nmol L~(-1). By the mechanism investigations, we conclude that the groups oxidized in L-DOPA are the two adjacent phenol groups, while all the catecholamine compounds possess the two adjacent phenol groups, so all of other catecholamine compounds, such as dopamine, adrenaline, noradrenaline, could be anticipated to be oxidized by AgNO_3 under the corresponding conditions, resulting in the formation of silver NPs and the enhanced PRLS signal, so the detection methods for the catecholamine compounds could be constructed as L-DOPA. The results of detection L-DOPA have shown that the method is simple, rapid, sensitive and reliable.
3).Thrombin plays an important role in the course of thrombus formation, so the detection of thrombin has important significances for the diseases diagnosis, evolution, evaluation of curative effect. In this contribution, gold nanoparticles (NPs) stabilized with citrate were functionalized by the thiolated thrombin binding aptamer (TBA), the gold NPs are served as a probe, when thrombin was added into the solution, the gold NPs would be aggregated for thrombin reacted with TBA, which was testified by the absorption spectra and the TEM images of gold NPs, and the then the PRLS signals were enhanced, so we set up a method for the detection of thrombin based on the PRLS signals of gold NPs. The results showed that the PRLS intensities were in proportion to the thrombin concentrations over the range of 1.8～9.0nmol L~(-1), the detection limit of thrombin was 0.2 nmol L~(-1), so it is a sensitive method for thrombin detection. During the initial phase of coagulation thrombin is produced in nanomolar concentrations, so the present method can be used for the clinical detection of thrombin.
Both Raman scattering and IFE are the interference sources and usually should be avoided or corrected in spectrofluorometry. We suppose analytical sciences lie on the reasonable application of signals only if the intensity of the signals has simple functional relationship with the content of analytes. In this contribution, we propose a quantitative method based on the IFE of reagents on the Raman scattering signals of solvent by taking the complexation of Co(II) with 4-[(5-chloro-2-pyridyl)azo]-l,3-diaminobenzene (5-C1-PADAB) as a model system on a common spectrofluorometer. In a solution of 5-C1-PADAB, the Raman scattering signals of water are decreased owing to the IFE of 5-C1-PADAB, after the addition of Co(II) which can form the complex with 5-C1-PADAB and consumes the reagent, then the Raman scattering signals were found recovered, based on the linear relationship between the enhanced Raman scattering intensities and the concentrations of Co(II), Co(II) was detected, the detection limit is 1.2×10~(-7) mol L~(-1) which is comparable to the generally used UV-Vis spectrophotometry. In the present method, the commonly used solvent, water, serves as a probe, and the Raman scattering signals of water are used, the obtain of Raman scattering does not require flexed excitaion wavelength, so we can freely select the excitation wavelength according to the analyte, which is very flexible and simple. What's more, a novel method for detection of some non-fluorescent compounds was proposed in a common spectrofluorometer, the function of spectrofluorometer was further exploited.
As we know, the RLS technique is very sensitive for the detection, but its poor selectivity and stability are the limitations for being as a perfect technique. While high separation resolution and selectivity are the advantages of chromatography all the time, the disadvantage lies in the poor detection sensitivity. Here HPLC is chosen as a model, combining the advantages of the both techniques, the incorporation of HPLC-RLS was carried out in the contribution, the incorporated technique not only improves the selectivity and stability of the RLS technique, but also enhances the detection sensitivity of HPLC and enriches the detection types of HPLC. With the deep research and developed incorporated technique, the RLS detector is hopeful for HPLC.
引文
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