摘要
DNA杂交及多态性检测分析广泛的应用于病毒及遗传疾病的诊断,已经引起分子生物学、药学、生物化学以及分析化学等领域工作者的高度关注。本论文在课题组多年研究的积累及大量文献调研的基础上,合成了荧光高分子聚合物聚[5-甲氧基-2-(3-磺酰化丙氧基)-1,4-苯撑乙烯(MPS-PPV)作为共振光散射光谱探针,及采用三苯甲烷染料溴甲酚绿(BG),表面活性剂十六烷基三甲基溴化铵(CTAB),多环芳烃萘及金属卟啉铜作为探针,结合多种光谱法和电镜分析,探讨其作用机理,建立了5个能够识别完全互补和碱基错配的DNA的新方法,其方法对疾病诊断方面有潜在应用价值;2个测定纳克级核酸、蛋白质的新方法,方法准确度和灵敏度高,简便、快速。
1)合成了水溶性荧光高分子聚合物MPS-PPV,采用1H-NMR,IR对聚合产物的分子结构进行了表征,研究了聚合产物的紫外吸收,荧光性质,并用扫描电镜研究了聚合物的表面形态。将MPS-PPV应用到杂交检测,在pH 7.2的生理Tris-HCl缓冲溶液中,在40℃温度时,杂交反应30 min,在460 nm处产生最大RLS信号。研究了其RLS光谱,荧光光谱性质,反应的电化学性质,探讨了反应机理,机理研究表明,MPS-PPV可以通过CTAB的桥梁作用与带负电荷的双链DNA(P1≈T1)发生静电结合作用,这种作用减弱了Pl≈T1骨架上的负电荷,增强了其骨架的疏水性,最终诱导了MPS-PPV-CTAB和P1≈T1之间相互聚集,导致大的聚集体的形成,这种大的聚集体表现出强的RLS信号放大作用。通过测定放大的RLS信号,完全互补和有碱基错配的DNA序列能很容易地被检测和识别。这种方法不需要对探针DNA和目标DNA序列进行标记,实现了完全互补序列与单碱基错配序列及非互补碱基序列的区分,建立了简单、快速、免标记的DNA杂交检测方法,在疾病的诊断方面有潜在的应用价值。
2)以MPS-PPV为DNA的RLS探针,研究了二者相互作用的机理和反应的RLS光谱,荧光光谱,紫外光谱,原子力显微镜(AFM)特性,建立了纳克级DNA测定的新方法。在pH 5.0的BR缓冲溶液中,MPS-PPV对脱氧核糖核酸与阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)的共振光散射光谱有协同增强作用,在共振光散射波长为342 nm处,发生较大的共振光散射信号。在最佳实验条件下,体系的△IRLS值与鱼精DNA(fsDNA)在一定范围内呈良好的线性关系,其相关系数为0.9996,检测限最低可达3.10 ng/mL。机理研究表明,MPS-PPV、CTAB和DNA之间的结合以静电作用为主,同时还有一定的疏水作用和扦插作用。
3)以MPS-PPV为蛋白质的RLS探针,研究了反应的RLS光谱,荧光光谱,紫外光谱,原子力显微镜(AFM)特性,探讨了二者相互作用的反应机理,建立了纳克级蛋白质测定的新方法。在pH 3.22的BR缓冲溶液中,MPS-PPV与蛋白质通过静电作用和疏水作用,在共振光306 nm处发生较大的共振光散射信号,体系的△IRLS值与牛血清白蛋白(BSA)在一定范围内呈良好的线性关系,其相关系数为0.9991,检测限最低可达3.99 ng/mL。
4)以三苯甲烷类染料溴甲酚绿作为杂交检测探针,探讨了其他不同种三苯甲烷类染料甲基紫,铬天青,亮绿,二甲酚橙和碱性品红与ssDNA,dsDNA的RLS光谱特征,并用Gaussian03计算了染料分子体积对其与DNA作用的影响。研究了溴甲酚绿与dsDNA作用的RLS光谱,荧光光谱性质,紫外光谱,AFM特性,探讨了反应机理,建立了完全互补序列与单碱基错配序列及非互补碱基序列的区分方法。机理研究表明,BG与P1≈T1之间一定存在沟槽作用,这种沟槽作用减弱了P1≈T1骨架上的负电荷,增强了其骨架的疏水性,最终诱导BG-P1≈T1之间相互聚集,从而引起聚集体的形成和强的RLS信号放大作用。
5)以表面活性剂CTAB作为杂交检测探针,探讨了其他不同表面活性剂,阳离子表面活性剂:十六烷基三甲基溴化铵(Cetyltriethylammnonium bromide, CTAB)和溴化十六烷基吡啶(Cetylpyrinium bromide, CPB);阴离子表面活性剂:十二烷基苯磺酸钠(Sodium dodecylbenzene sulphonate, SDBS)和十二烷基磺酸钠Sodium dodecyl sulphonate (SDS),非离子表面活性剂:曲拉通-100(Triton-100, TX-100)和吐温-80(Tween-80, T-80)与sDNA,dsDNA的RLS光谱特征。研究了CTAB与dsDNA作用的RLS光谱,荧光光谱性质,紫外光谱,AFM特性,探讨了反应机理,建立了完全互补序列与单碱基错配序列及非互补碱基序列的区分方法。机理研究表明,CTAB与P1≈T1之间存在静电作用与疏水作用的协同影响,诱导CTAB-P1≈T1聚集,从而引起聚集体的形成和强的RLS信号放大作用。
6)以多环芳烃萘作为杂交检测探针,探讨了其他多环芳烃蒽、荧蒽、芘、菲与sDNA,dsDNA的RLS光谱特征。研究了萘与dsDNA作用的RLS光谱,荧光光谱性质,紫外光谱,AFM特性,探讨了反应机理,建立了完全互补序列与单碱基错配序列及非互补碱基序列的区分方法。萘能和双链DNA(P1≈T1)发生沟槽结合作用,这种结合作用依赖于DNA的G-C碱基序列和萘分子的大小。这种结合减小了P1≈T1骨架的负电荷,增强了其疏水性,从而诱导了萘-P1≈T1之间的疏水结合作用,导致大的聚集体的形成。这种大的聚集体表现出强的RLS信号放大作用,通过测定这种放大的RLS信号,能够准确、简便、快速的检测和识别完全互补和有碱基错配的DNA序列。此方法不需要对探针DNA和目标DNA序列进行标记。在疾病的诊断方面有潜在的应用价值。
7)以卟啉铜作为杂交检测探针,探讨了其他金属卟啉钴,卟啉铬,卟啉镁,卟啉锌,卟啉镍与sDNA,dsDNA的RLS光谱特征。研究了卟啉铜与dsDNA作用的RLS光谱,荧光光谱性质,紫外光谱,AFM特性,探讨了反应机理,实现了完全互补序列与单碱基错配序列及非互补碱基序列的区分。机理研究表明,疏水型金属卟啉Cu(Ⅱ)-TAOPP能和双链DNA(P1≈T1)发生缔合作用,导致大的聚集体的形成。这种大的聚集体表现出强的RLS信号放大作用及荧光猝灭现象。通过测定这种放大的RLS信号,能够准确、简便、快速的检测和识别完全互补和有碱基错配的DNA序列。这种方法不需要对探针DNA和目标DNA序列进行标记,在疾病的诊断方面有潜在的应用价值。
Detection of DNA hybridization and polymorphism widely used in the diagnosis of viral and genetic diseases, has caused the attention of molecular biology, pharmacology, biochemistry and analytical chemistry and other workers. On the basis of the accumulation of years of research and the investigation and research a large number of references, we synthesis the fluorescent polymer poly [5- methoxy -2-(3-sulfonated propoxy) -1,4- phenylene vinylene (MPS-PPV) as a resonance light scattering probe, and use of triphenylmethane dyes bromocresol green (BG), surfactant cetyltrimethylammonium bromide (CTAB), polycyclic aromatic hydrocarbons naphthalene (NAP) and metal Cu (Ⅱ)meso-(4-alkoxyphenyl) porphyrin (Cu(Ⅱ)-TAOPP) as probes, and used various of spectra means and electron microscopic technology to study the mechanism of the reactions. established 5 new methods of accurate, simple and quickly identify fully complementary and mismatched DNA base pairs and 2 new analytical methods of nucleic acids and protein.
1) Synthesized a water soluble fluorescent polymer poly [5-methoxy-2-(3-sulfonyl isopropoxide)-1, 4-PPV (MPS-PPV), used 1H-NMR, IR to characterize the molecular structure, researched the UV absorption, fluorescence properties of polymer and utilized scanning electron microscope to study on surface morphology of the polymer. The results showed that the maximum UV-vis spectra of polymer was at 450nm with a wide absorption peak, fluorescence peak located at 550 nm and its surface has a microporous honeycomb structure with an irregular appearance which closed to a ball shape.We applied MPS-PPV into the determination of DNA hybridization,inspected the best conditions of the reaction, studied the properties of RLS spectra, fluorescence spectra and electrochemical reaction, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of polymer/ssDNA and polymer/dsDNA, and established a new non-labeled methods for the determination of DNA hybridization. In pH7.2 Tris-HCl buffer, at 40 oC, hybridization reaction for half an hour, RLS of polymer-DNA system with the maximum scattering peak located at 460 nm. Under the optimum conditions,the enhanced RLS intensity was proportional to the concentration of target DNA over the range of 0.3×10-7~1.0×10-7mol/L. Mechanism research indicated that MPS-PPV can interact with negatively charged double-stranded DNA (dsDNA, P1≈T1) by electrostatic binding in the presence of CTAB. The binding effect weakened the negative charge of P1≈T1 skeleton, enhanced its skeleton’s hydrophobicity, and led the aggregation of MPS-PPV-CTAB and P1≈T1, resulting in the amplification of RLS signal. By measuring the amplification of RLS signal, complete complementary and bases mismatched DNA sequences can easily be detected and identified. This method does not need to lable the target DNA and probe DNA sequence. The novel method is simple and fast, and has potential application value in disease diagnosis.
2) We applied MPS-PPV into the determination of DNA, studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM, discussed the reaction mechanism, and proposed a simple and speedy assay for DNA determination. In pH5.0 BR buffer, MPS-PPV has synergistic effect in the RLS signal of CTAB-DNA with the maximum scattering peak located at 342 nm. Under the optimum conditions , the enhanced RLS intensity was proportional to the concentration of fsDNA, the related coefficient is 0.9996, and the limit of detection is 3.10 ng/mL. Mechanism research indicated that electrostatic force is the main force among MPS-PPV, CTAB and DNA, together with hydrophobic effect and intercalation.
3) We applied MPS-PPV into the determination of protein, studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM, discussed the reaction mechanism, and proposed a simple and speedy assay for protein determination. In pH 3.22 BR buffer, MPS-PPV interacted with BSA (Bovine Serum Albumin) by electrostatic force and hydrophobic effect with the maximum scattering peak located at 342 nm. The enhanced RLS intensity was proportional to the concentration of BSA, the related coefficient is 0.9991, and the limit of detection is 3.99 ng/mL.
4) We applied triphenylmethane dye Bromocresol Green (BG) into the determination of DNA hybridization, discussed the properties of RLS spectra of other different kinds of triphenylmethane dyes methyl violet, chromazurine, bright green, xylenol orange and alkaline magenta with double strand DNA (dsDNA) and single strand DNA (ssDNA) and utilized Gaussian03 to calculate the molecular volume of those dyes and the effects on the interaction with DNA. We studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM of BG-dsDNA, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of BG/ssDNA and BG/dsDNA , and established a new non-labeled methods for the determination of DNA hybridization. Mechanism research indicated that BG can interact with negatively charged double-stranded DNA (dsDNA, P1≈T1) by groove binding. The binding effect weakened the negative charge of P1≈T1 skeleton, enhanced its skeleton’s hydrophobicity, and led the aggregation of BG and P1≈T1, resulting in the amplification of RLS signal.
5) We applied cationic surfactant CTAB into the determination of DNA hybridization, discussed the properties of RLS spectra of other different kinds of surfactants CPB, SDBS, SDS, TX-100 and T-80 with dsDNA and ssDNA. We studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM of CTAB-dsDNA, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of CTAB/ssDNA and CTAB/dsDNA,and established a new non-labeled methods for the determination of DNA hybridization. Mechanism research indicated that CTAB can interact with dsDNA (P1≈T1) by electrostatic force and hydrophobic effect leading the aggregation of CTAB and P1≈T1,resulting in the amplification of RLS signal.
6) We applied PAH (polycyclic aromatic hydrocarbon) naphthalene into the determination of DNA hybridization, discussed the properties of RLS spectra of other different kinds of PAHs anthracene, fluoranthene, pyrene, phenanthrene with dsDNA and ssDNA. We studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM of naphthalene-dsDNA, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of naphthalene/ssDNA and naphthalene/dsDNA, and established a new non-labeled method for the determination of DNA hybridization. Mechanism research indicated that naphthalene can interact with dsDNA (P1≈T1) by groove binding which depends on G-C sequenees of dsDNA and the volume of naphthalene. The binding effect weakened the negative charge of P1≈T1 skeleton, enhanced its skeleton’s hydrophobicity, and led the aggregation of naphthalene and P1≈T1, resulting in the amplification of RLS signal. By measuring the amplification of the RLS signal, entirely complementary and bases mismatched DNA sequences can easily be detected and recognized. The method has potential application value in disease diagnosis.
7) We applied Cu (Ⅱ)meso-(4- alkoxyphenyl) porphyrin(Cu(Ⅱ)-TAOPP) into the determination of DNA hybridization, discussed the properties of RLS spectra of other different kinds of metalloporphyrin Co(Ⅱ)-TAOPP, Cr(Ⅱ)-TAOPP, Mg(Ⅱ)-TAOPP, Zn(Ⅱ)-TAOPP and Ni(Ⅱ)-TAOPP with dsDNA and ssDNA. We studied the properties of RLS spectra, fluorescence spectra, UV absorption spectra and AFM of Cu(Ⅱ)-TAOPP-dsDNA, discussed the reaction mechanism, proposed a simple and speedy assay for specific oligonucleotide sequences and single-base mismatch based on the different RLS signals of Cu(Ⅱ)-TAOPP/ssDNA and Cu(Ⅱ)-TAOPP/dsDNA, and established a new non-labeled methods for the determination of DNA hybridization. Mechanism research indicated that hydrophobic Cu(Ⅱ)-TAOPP can interact with dsDNA(P1≈T1) leading the aggregation of Cu(Ⅱ)-TAOPP and P1≈T1. The large aggregates showed strong RLS signal amplification and fluorescence quenching. By measuring the amplification of the RLS signal, entirely complementary and bases mismatched DNA sequences can easily be detected and recognized. The method has potential application value in disease diagnosis.
引文
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