共振光散射光谱法在生物大分子检测中的应用研究
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摘要
本论文应用共振光散射光谱法、紫外可见光谱法对酶、DNA和肝素等生物大分子的测定进行了较系统的研究，介绍了共振光散射技术与免疫技术相结合在分析中的应用。具体内容如下：
一、第一章对共振光散射分析方法的研究进行了综述，介绍了共振光散射分析方法的原理、应用、现状及发展前景。
二、第二章研究了CdTe纳米粒子在共振光散射分析中的应用。以CdTe纳米粒子为新的共振光散射探针，实现了对菠萝蛋白酶、木瓜蛋白酶和溶菌酶的测定，同时研究了溶菌酶与DNA之间的相互作用,实现了酶和DNA的同时测定。
三、第三章将共振光散射技术与免疫技术相结合，建立了检测羊抗人IgG的方法。改善了共振光散射技术的选择性，提高了分析的灵敏度。
四、第四章将共振光散射技术应用于DNA和肝素含量的测定。重点研究了表面活性剂与绿原酸、厚朴酚、儿茶素、山奈酚相结合在DNA测定中的应用。同时研究了两种阳离子表面活性剂与肝素相互作用的共振光散射光谱。解释了共振光散射强度增强的原因。考查了影响共振光散射强度的各种因素，测定了样品中肝素的含量。
五、第五章研究了DNA对三价稀土离子与槲皮素、山奈酚所形成的络合物的共振光散射淬灭作用。解释了共振光散射强度淬灭的原因。并将选择的共振光散射探针用于DNA的测定。
The resonance light scattering (RLS) technique, not only meets high sensitivity butalso offer the additional benefits of simplicity and versatility. Nowadays it has becomea new spectral analysis technique and has been found wide applications in the analysisof proteins, nucleic acid, drugs, carbohydrates, metallic ions, surfactants, biomolecules,and nanoparticles. Study on the new RLS probe and develop new RLS system todetermination the biomacromolecule bring to more attention and interesting. However,light scattering has suffered from drawbacks such as interference caused by otherparticle in solution and the deficiency of analytic selectivity, which causes somedifficulties to the application of it. In this study, some new system were developed todetermine biomolecules, some content started with overcoming the limitation of lightscattering technique and enhanced the sensitivity of the experiment. We try to combinethe advantages of the high sensitivity and easy operation of RLS technique with thespecificity of immunoreactions, and construct a novel homogeneous immunoassaybased on the measurements of RLS signals with a common spectrofluorometer. Themain contents and some conclusions of the thesis are as follows:
The CdTe nanoparticles which were modified with thioglycolic acid (TGA) or 3-mercaptocarboxylic acid (MPA) can be used as RLS probe to determine bromelain,papain and lysozyme based on the measurement of enhanced resonance light-scattering(RLS) signals. The CdTe nanoparticles which were modified with thioglycolic acid(TGA) or 3-mercaptocarboxylic acid (MPA) combined with enzyme will enhance theRLS intensity, the enhanced RLS intensity is linearly proportional to theconcentration of enzyme. Based on it a novel assay of enzyme is developed. And the interaction of lysozyme and calf thymus DNA (ctDNA) has been studied by resonancelight-scattering (RLS) spectroscopy. Lysozyme as a probe is used to determine nucleicacid, the complex of lysozyme-DNA can cause a strong RLS signal. MeanwhilectDNA can also be used as a probe to determine lysozyme. This system cansynchronously detect lysozyme and DNA. The optimization conditions such as acidity,time, the concentration of probe and coexisting substances of the reaction were alsoexamined and selected. At pH 8.3( bromelain and papain) and pH 7.4 (lysozyme)phosphate buffered saline (PBS), the RLS signals of functionalized nano- CdTe aregreatly enhanced by bromelain, papain and lysozyme, characterized by the peakaround 318 nm, 314 nm and 353 nm,respectively. The liner range is 9.0×10-8-9.0×10-7mol L-1 for bromelain, 4.8×10-8-7.0×10-7 mol L-1 for papain and 0.20-10.7μg mL-1 forlysozyme. At pH 7.2 Britton-Robinson (BR) buffer solution，the RLS signals oflysozyme/ctDNA are greatly enhanced by ctDNA/lysozyme, characterized by thepeak around 306 nm. The linear range is 0.10-25.0μg mL-1 for lysozyme and 0.078-13.0μg mL-1 for ctDNA. The detection limit is 0.041μg mL-1 for lysozyme and 0.024μg mL-1 for ctDNA. The synthetic samples were analyzed with satisfactory results.Pure antigen and antibody has very weak RLS signals under the lowconcentration of antigen and antibody,but the strong RLS signals could be observedobviously after antigen and antibody take the immune precipitation reactions.For agiven concentration of Ab, the enhancement of RLS intensity is in proportion to theconcentration of Ag.The RLS immunoassay based on measuring enhanced RLSsignals using a common spectrofluorometer,was developed.Comparison of the systemof PDDA(Poly diallyldimethylammonium chloride)-Ab-Ag and Ab-Ag is also studied.
When Ab or Ag was mixed with PDDA, the RLS intensity of the solution wasenhanced. When both Ab and Ag were added into the solution containing PDDA, theRLS intensity of the solution was further enhanced. The enhancement of RLSintensity is in proportion to the concentration of Ag, so the immunoassay method ofPDDA-Ab-Ag system was developed. The reasons for the enhancement of RLSintensity is the aggregates of Ab or Ag with PDDA. PDDA is a cationicpolyelectrolyte and not sensitive to the pH change. It has a line-type framework andthe cationic character.The reasons of the enhancement of RLS intensity is theaggregates of Ab or Ag with PDDA by electrostatic interaction and aggregates of Ab with Ag by immunoreaction. Thus, it can be concluded that Ag reacted with Ab andPDDA and produced a new-formed larger compound than Ab-Ag compound whoseRLS intensity was much higher than that of Ag, Ab or PDDA when they existedseparately.Compared to the system only containing Ab-Ag, the sensitivity was furtherenhanced.Compared to traditional assay, this method is much simpler and easier tooperate, as it involves only one step.
The surfactants are widely used in analysis chemistry. In this paper, surfactantswere used to enhance the RLS signal of system in the RLS technique. The interactionbetween four kinds of active components in Chinese herb, chlorogenic acid, magnolol,catechin, kaempferol and calf thymus DNA (ctDNA) has been studied by a resonancelight-scattering (RLS) technique with a common spectrofluorometer. Theinvestigation indicates that surfactants act as a bridge between drugs and ctDNA.The effects of experimental parameters, including surfactant concentration, pH,incubation time and foreign substances, on the enhancement of RLS intensity wereinvestigated. The linear ranges are 0.002-0.1, 0.02-1.0, 0.001-0.08 and 0.1-5.0μg mL-1 for (chlorogenic acid, magnolol, catechin or kaempferol) CTMAB–ctDNA systems,respectively.The assay for ctDNA was successfully applied to determination ofctDNA in synthetic samples.
Using heparin as an analyte, the RLS spectral characteristics of the interaction ofheparin with CTMAB and RBr have been studied.At Britton-Robinson (BR) buffersolution, CTMAB and RBr take positive charge. Heparin takes the negative charge.So the interaction between CTMAB/RBr and heparin may occur. The optimizationconditions of the reaction were also examined and selected. Under the selectedconditions, the enhanced intensities of RLS were proportional to the concentrations ofheparin. They can be determined in the range of 0.01-3.0μg mL-1 and 0.03-3.0μg mL-1 for CTMAB and RBr system, respectively.
Study on the resonance light-scattering (RLS) of Terbium (Ш)（Tb3+)/Europium(Ш) (Eu3+)-quercetin/kaempferol system.The RLS intensity of Tb3+/Eu3+,quercetin/kaempferol and ctDNA are very low. When Tb3+/Eu3+ are mixed withquercetin/ kaempferol, respectively, the RLS intensity of the system is enhanced forthe coordination between Tb3+/Eu3+ and quercetin/ kaempferol. But the RLS intensityof the Tb3+/Eu3+-quercetin/kaempferol system can be quenched by ctDNA. Quercetin/ kaempferol is a kind of flavonoids.If the flavonoids compounds have 3-hydroxylgroup, 4-carbonyl group or 5- hydroxyl group, 4-carbonyl group, they will coordinatewith many metal ions. It can be seen that quercetin/ kaempferol simultaneously hasthe 3-hydroxyl group, 4-carbonyl group and 5- hydroxyl group, 4-carbonyl group, itcan coordinate with Tb3+ or Eu3+. So the RLS intensity can be enhance for theformation of the coordination complex. However, after the addition of ctDNA, theRLS intensity of Tb3+/Eu3+-quercetin/kaempferol complex greatly decreased, whichindicates that the quercetin/ kaempferol in Tb3+/Eu3+-quercetin/kaempferol complex isreleased. The phosphate moieties of nucleic acid chelating with Tb3+ play a key roleduring the quenching process. From these results, we can know that ctDNA andquercetin/ kaempferol existing together will compete to complex with Tb3+ / Eu3+. Atthe same time, because DNA has several binding sites, it will have more advantages.It is considered that there is a displacement of the quercetin/ kaempferol in Tb3+/Eu3+-quercetin/kaempferol complex by ctDNA in Tb3+/Eu3+-quercetin/kaempferol -ctDNAsystem because the binding ability between phosphate groups in ctDNA and Tb3+ /Eu3+ is stronger than that between quercetin/ kaempferol and Tb3+/Eu3+, thus resultingin the decreases of the RLS intensity of the system. The interaction mechanismbetween Tb3+/Eu3+-quercetin/kaempferol and ctDNA is also studied by UV-Vis andfluorescence spectrometry. Based on this, a sensitive method for the determination ofctDNA is proposed.
一、第一章对共振光散射分析方法的研究进行了综述，介绍了共振光散射分析方法的原理、应用、现状及发展前景。
二、第二章研究了CdTe纳米粒子在共振光散射分析中的应用。以CdTe纳米粒子为新的共振光散射探针，实现了对菠萝蛋白酶、木瓜蛋白酶和溶菌酶的测定，同时研究了溶菌酶与DNA之间的相互作用,实现了酶和DNA的同时测定。
三、第三章将共振光散射技术与免疫技术相结合，建立了检测羊抗人IgG的方法。改善了共振光散射技术的选择性，提高了分析的灵敏度。
四、第四章将共振光散射技术应用于DNA和肝素含量的测定。重点研究了表面活性剂与绿原酸、厚朴酚、儿茶素、山奈酚相结合在DNA测定中的应用。同时研究了两种阳离子表面活性剂与肝素相互作用的共振光散射光谱。解释了共振光散射强度增强的原因。考查了影响共振光散射强度的各种因素，测定了样品中肝素的含量。
五、第五章研究了DNA对三价稀土离子与槲皮素、山奈酚所形成的络合物的共振光散射淬灭作用。解释了共振光散射强度淬灭的原因。并将选择的共振光散射探针用于DNA的测定。
The resonance light scattering (RLS) technique, not only meets high sensitivity butalso offer the additional benefits of simplicity and versatility. Nowadays it has becomea new spectral analysis technique and has been found wide applications in the analysisof proteins, nucleic acid, drugs, carbohydrates, metallic ions, surfactants, biomolecules,and nanoparticles. Study on the new RLS probe and develop new RLS system todetermination the biomacromolecule bring to more attention and interesting. However,light scattering has suffered from drawbacks such as interference caused by otherparticle in solution and the deficiency of analytic selectivity, which causes somedifficulties to the application of it. In this study, some new system were developed todetermine biomolecules, some content started with overcoming the limitation of lightscattering technique and enhanced the sensitivity of the experiment. We try to combinethe advantages of the high sensitivity and easy operation of RLS technique with thespecificity of immunoreactions, and construct a novel homogeneous immunoassaybased on the measurements of RLS signals with a common spectrofluorometer. Themain contents and some conclusions of the thesis are as follows:
The CdTe nanoparticles which were modified with thioglycolic acid (TGA) or 3-mercaptocarboxylic acid (MPA) can be used as RLS probe to determine bromelain,papain and lysozyme based on the measurement of enhanced resonance light-scattering(RLS) signals. The CdTe nanoparticles which were modified with thioglycolic acid(TGA) or 3-mercaptocarboxylic acid (MPA) combined with enzyme will enhance theRLS intensity, the enhanced RLS intensity is linearly proportional to theconcentration of enzyme. Based on it a novel assay of enzyme is developed. And the interaction of lysozyme and calf thymus DNA (ctDNA) has been studied by resonancelight-scattering (RLS) spectroscopy. Lysozyme as a probe is used to determine nucleicacid, the complex of lysozyme-DNA can cause a strong RLS signal. MeanwhilectDNA can also be used as a probe to determine lysozyme. This system cansynchronously detect lysozyme and DNA. The optimization conditions such as acidity,time, the concentration of probe and coexisting substances of the reaction were alsoexamined and selected. At pH 8.3( bromelain and papain) and pH 7.4 (lysozyme)phosphate buffered saline (PBS), the RLS signals of functionalized nano- CdTe aregreatly enhanced by bromelain, papain and lysozyme, characterized by the peakaround 318 nm, 314 nm and 353 nm,respectively. The liner range is 9.0×10-8-9.0×10-7mol L-1 for bromelain, 4.8×10-8-7.0×10-7 mol L-1 for papain and 0.20-10.7μg mL-1 forlysozyme. At pH 7.2 Britton-Robinson (BR) buffer solution，the RLS signals oflysozyme/ctDNA are greatly enhanced by ctDNA/lysozyme, characterized by thepeak around 306 nm. The linear range is 0.10-25.0μg mL-1 for lysozyme and 0.078-13.0μg mL-1 for ctDNA. The detection limit is 0.041μg mL-1 for lysozyme and 0.024μg mL-1 for ctDNA. The synthetic samples were analyzed with satisfactory results.Pure antigen and antibody has very weak RLS signals under the lowconcentration of antigen and antibody,but the strong RLS signals could be observedobviously after antigen and antibody take the immune precipitation reactions.For agiven concentration of Ab, the enhancement of RLS intensity is in proportion to theconcentration of Ag.The RLS immunoassay based on measuring enhanced RLSsignals using a common spectrofluorometer,was developed.Comparison of the systemof PDDA(Poly diallyldimethylammonium chloride)-Ab-Ag and Ab-Ag is also studied.
When Ab or Ag was mixed with PDDA, the RLS intensity of the solution wasenhanced. When both Ab and Ag were added into the solution containing PDDA, theRLS intensity of the solution was further enhanced. The enhancement of RLSintensity is in proportion to the concentration of Ag, so the immunoassay method ofPDDA-Ab-Ag system was developed. The reasons for the enhancement of RLSintensity is the aggregates of Ab or Ag with PDDA. PDDA is a cationicpolyelectrolyte and not sensitive to the pH change. It has a line-type framework andthe cationic character.The reasons of the enhancement of RLS intensity is theaggregates of Ab or Ag with PDDA by electrostatic interaction and aggregates of Ab with Ag by immunoreaction. Thus, it can be concluded that Ag reacted with Ab andPDDA and produced a new-formed larger compound than Ab-Ag compound whoseRLS intensity was much higher than that of Ag, Ab or PDDA when they existedseparately.Compared to the system only containing Ab-Ag, the sensitivity was furtherenhanced.Compared to traditional assay, this method is much simpler and easier tooperate, as it involves only one step.
The surfactants are widely used in analysis chemistry. In this paper, surfactantswere used to enhance the RLS signal of system in the RLS technique. The interactionbetween four kinds of active components in Chinese herb, chlorogenic acid, magnolol,catechin, kaempferol and calf thymus DNA (ctDNA) has been studied by a resonancelight-scattering (RLS) technique with a common spectrofluorometer. Theinvestigation indicates that surfactants act as a bridge between drugs and ctDNA.The effects of experimental parameters, including surfactant concentration, pH,incubation time and foreign substances, on the enhancement of RLS intensity wereinvestigated. The linear ranges are 0.002-0.1, 0.02-1.0, 0.001-0.08 and 0.1-5.0μg mL-1 for (chlorogenic acid, magnolol, catechin or kaempferol) CTMAB–ctDNA systems,respectively.The assay for ctDNA was successfully applied to determination ofctDNA in synthetic samples.
Using heparin as an analyte, the RLS spectral characteristics of the interaction ofheparin with CTMAB and RBr have been studied.At Britton-Robinson (BR) buffersolution, CTMAB and RBr take positive charge. Heparin takes the negative charge.So the interaction between CTMAB/RBr and heparin may occur. The optimizationconditions of the reaction were also examined and selected. Under the selectedconditions, the enhanced intensities of RLS were proportional to the concentrations ofheparin. They can be determined in the range of 0.01-3.0μg mL-1 and 0.03-3.0μg mL-1 for CTMAB and RBr system, respectively.
Study on the resonance light-scattering (RLS) of Terbium (Ш)（Tb3+)/Europium(Ш) (Eu3+)-quercetin/kaempferol system.The RLS intensity of Tb3+/Eu3+,quercetin/kaempferol and ctDNA are very low. When Tb3+/Eu3+ are mixed withquercetin/ kaempferol, respectively, the RLS intensity of the system is enhanced forthe coordination between Tb3+/Eu3+ and quercetin/ kaempferol. But the RLS intensityof the Tb3+/Eu3+-quercetin/kaempferol system can be quenched by ctDNA. Quercetin/ kaempferol is a kind of flavonoids.If the flavonoids compounds have 3-hydroxylgroup, 4-carbonyl group or 5- hydroxyl group, 4-carbonyl group, they will coordinatewith many metal ions. It can be seen that quercetin/ kaempferol simultaneously hasthe 3-hydroxyl group, 4-carbonyl group and 5- hydroxyl group, 4-carbonyl group, itcan coordinate with Tb3+ or Eu3+. So the RLS intensity can be enhance for theformation of the coordination complex. However, after the addition of ctDNA, theRLS intensity of Tb3+/Eu3+-quercetin/kaempferol complex greatly decreased, whichindicates that the quercetin/ kaempferol in Tb3+/Eu3+-quercetin/kaempferol complex isreleased. The phosphate moieties of nucleic acid chelating with Tb3+ play a key roleduring the quenching process. From these results, we can know that ctDNA andquercetin/ kaempferol existing together will compete to complex with Tb3+ / Eu3+. Atthe same time, because DNA has several binding sites, it will have more advantages.It is considered that there is a displacement of the quercetin/ kaempferol in Tb3+/Eu3+-quercetin/kaempferol complex by ctDNA in Tb3+/Eu3+-quercetin/kaempferol -ctDNAsystem because the binding ability between phosphate groups in ctDNA and Tb3+ /Eu3+ is stronger than that between quercetin/ kaempferol and Tb3+/Eu3+, thus resultingin the decreases of the RLS intensity of the system. The interaction mechanismbetween Tb3+/Eu3+-quercetin/kaempferol and ctDNA is also studied by UV-Vis andfluorescence spectrometry. Based on this, a sensitive method for the determination ofctDNA is proposed.
引文
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