磁性微球表面瞬时衍生化学发光新技术检测特定序列DNA的研究

发布时间：2018-08-22 21:32

【摘要】： 现代医学的研究表明：许多疾病如癌症和遗传病的发生都与基因的突变有关，，另外许多传染性疾病是由环境中的病毒、病菌或寄生虫所引起，因此对特定序列DNA的分析以及对DNA链中碱基突变的检测在基因筛选、法医学鉴定、遗传疾病的早期诊断和治疗、病原基因的测定方面具有十分深远的意义。近年来，快速可靠的特定序列DNA检测技术得以迅速发展。大量的研究以酶、同位素、荧光、电化学和化学发光等标记物标记DNA探针，建立高灵敏度、高选择性的特定序列DNA的检测技术。鉴于DNA探针标记物的存在，一定程度上复杂化了传感器的制备和使用，所以无标记型DNA检测技术的研究成为病原基因测定和基因疾病诊断领域新的研究热点之一。化学发光(CL)分析法不需要光源，避免了杂散光的干扰，因此具有极高的灵敏度；且这一方法仪器设备简单、操作简便，因而得到了迅速发展。目前，CL分析成为一个非常活跃的研究领域，已成功地用于药学、生物学、分子生物学、临床医学和环境学中各种物质的检测。2003年，本研究小组首次报道了基于鸟嘌呤特异性化学发光的无标记(瞬时衍生)DNA检测新技术。本论文旨在发展了一系列具有创新意义的磁性微球表面瞬时衍生化学发光DNA检测技术，由以下部分构成：第一章：绪论第一节，介绍了特定序列DNA检测的研究进展及其意义，内容包括：荧光、电化学、表面基质共振和石英微晶天平法等，并列举了近年来它们在该分析领域的典型示例；第二节，介绍了主要的CL体系(如鲁米诺体系、过氧化草酸酯体系、吖啶酯体系和碱性磷酸酯酶体系)的原理、特点以及应用，尤其是在免疫和DNA分析领域的应用。第三节，阐述了本论文的目的和意义，指出了论文的主要研究内容及创新之处。第二章：基于Poly T修饰的磁性微球表面瞬时衍生特定序列DNA化学发光检测新技术 1．端粒特定序列检测新技术端粒是染色体3’末端的重复性序列，由一种核糖核蛋白复合体——端粒酶在一定条件下合成。端粒和端粒酶具有保护细胞不被融合、降解的重要作用，它们量的多少与恶性肿瘤等疾病的发生密切相关，因此这一基因片段的测定对重大疾病的早期诊断和防治具有重要意义。基于磁性微球表面瞬时衍生化学发光，建立了一种新型的端粒特定序列DNA无标记检测技术。整个分析过程由以下三个实验步骤构成：(1)dT_(20)修饰的磁性微球，与连接有dA_(20)的捕获探针杂交；(2)再与端粒特定序列DNA进行第二次杂交；(3)磁性分离洗涤，基于3，4，5-三甲氧基苯甲酰甲醛(TMPG)与端粒中富含的鸟嘌呤(G)反应产生的特异性CL，进行端粒DNA的无标记检测。结果表明：该法具有操作简便、分析快速和灵敏度高等特点。目标序列浓度在5-100 nM浓度范围内具有良好的线性关系，相关系数为0.9918，最低检测浓度为0.5 nM。 2．炭疽杆菌特定序列检测新技术作为一种全球性的生化武器，炭疽杆菌引起了公众以及军事部门的热切关注，建立实时有效的识别、检测技术，对控制其传播具有重要意义。本节以炭疽相关的一段含有30个碱基的序列作为研究对象，构建了无标记CL检测技术，并进一步发展了基于G_(30)序列的放大检测方法。放大检测技术可描述如下：首先通过A_(20)-T_(20)杂交反应将捕获探针固定于磁性微球表面；捕获探针15个核苷酸单位与目标序列相应序列段杂交结合；然后加入报告序列与目标序列的另外15个核苷酸单位进行第三次杂交反应；利用TMPG与目标序列和报告序列中富含的G碱基反应产生的CL来检测。报告序列含有两个功能序列段：一是能够与目标序列的15个核苷酸单位杂交的序列段，另一个是富含G的序列段——(T_2G_(15))_2，结果表明：在6-60 nM的浓度范围内，CL信号呈线性增加(R~2=0.9984)，最低检测浓度为6 nM；采用G_(30)报告序列放大检测技术，目标序列在0.45-6 nM浓度范围内，线性良好(R~2=0.9917)，最低检测浓度为0.45 nM(0.045 pmol)；放大检测技术较不放大技术灵敏度提高了一个数量级，且对A-A单碱基错配识别良好。第三章：基于羧基修饰磁性微球表面瞬时衍生的无标记化学发光检测新技术本章以炭疽杆菌特定序列DNA为研究对象，采用羧基修饰的磁性微球为分离和预富集载体，通过羧基——氨基缩合反应固定捕获探针，杂交目标序列后利用G碱基的特异性CL反应来检测，并在此基础上发展了G_(30)放大检测技术。该技术操作简单，不放大检测技术可表述为：在一定条件下，羧基磁性微球经过EDC活化后，结合炭疽杆菌特定序列的捕获探针，然后通过杂交反应结合目标序列，洗涤转移后利用特异性反应试剂TMPG直接测定目标序列中的G碱基；G_(30)放大检测技术是一种夹心方法，在上述第一步杂交完成后，加入一段富含G的报告序列，与目标序列另外15个核苷酸单位进行第二次杂交反应，洗涤转移后检测TMPG与目标序列和报告序列中富含的G反应产生的CL信号。结果表明：该方法具有准确可靠、重现性和选择性好的特点。目标序列浓度在2-5 nM范围内，CL信号呈线性增加(R~2=0.9962)，最低可检测浓度为2 nM；G_(30)放大技术，目标序列浓度在50 pM-4 nM范围内，CL信号线性相关性良好(R~2=0.9965)，最低检测浓度为50 pM，较不放大技术灵敏度提高一个数量级。此外，该技术较基于Poly T修饰的磁性微球的不放大及G_(30)放大技术灵敏度提高2.5和10倍，且A-A单碱基错配识别良好。第四章：基于碳纳米管放大的特定序列DNA化学发光检测新技术本章以羧基修饰的磁性微球作为分离以及预富集载体，以碳纳米管(CNTs)这种新型纳米材料作为放大载体，发展了一种高灵敏度CL新技术检测特定序列DNA。该技术利用夹心杂交检测法：(1)将氨基修饰的捕获探针固定于经过EDC活化的羧基修饰的磁性微球表面；(2)通过第一步杂交将目标序列结合于磁性微球表面；(3)表面羧基功能化的碳纳米管，经过EDC活化后结合大量氨基修饰的报告序列形成放大复合物，通过第二步杂交反应将放大复合物结合于磁性微球表面；(4)检测TMPG与目标序列和放大复合物中的G碱基反应产生的CL信号。报告序列由两个功能序列段组成：一是富含G的序列段，二是和目标序列互补的序列段；各类CNTs经过浓硫酸-过氧化氢(9：1)的混合溶液氧化处理后形成表面羧基功能化的CNTs-COOH；通过TEM以及粒度／zeta电位测定仪对其性质进行表征。研究了四种碳纳米管的放大效果，并最终选用两种作为载体进行特定序列DNA放大检测；该方法不需要在测定前将待测组分从分离载体上洗脱下来，也不需要用酶、荧光染料等对核苷酸作进一步的标记，整个实验可以在3-4 h内完成。实验结果表明：该法操作较简便、结果准确、能够高灵敏度检测目标序列；在20 pM-2 nM(2-200 fmol)浓度范围内线性良好(R~2=0.996)，最低检测浓度为10 pM(1 fmol)；较不放大的方法灵敏度提高200倍，并且能较好的区别单碱基错配，为DNA分析提供了一种有效的途径。第五章：基于聚苯乙烯微球作为放大载体的特定序列DNA化学发光检测新技术本章以商品化的链霉亲和素聚苯乙烯微球为放大载体平台，构建了一种省时、省力、高效的放大检测技术。与碳纳米管作为放大载体的技术相比，不需对放大载体进行表面功能化，节省分析时间，且利于放大技术的进一步发展。简单地来说，该技术原理是：结合于磁性微球上的捕获探针以及通过链霉亲和素——生物素反应自组装于聚苯乙烯微球上的报告序列，在目标序列存在条件下三者夹心杂交结合，无目标序列存在，放大复合物不能结合于磁性微球表面。杂交反应结束后，磁性分离去除上清液，利用特异性化学发光反应试剂TMPG对磁性微球上结合的组分进行检测。结果表明：该方法简单方便、稳定可靠、快速灵敏，整个检测过程在2-3 h内完成。在10 pM-1 nM浓度范围内具有良好的线性关系(R~2=0.995)，最低检测浓度为5 pM(0.5 fmol)，较不放大方法提高400倍，较CNTs放大技术提高2倍。错配识别实验表明该方法可以较好的区别目标序列和各种错配序列。综上所述，该法非常适合于高灵敏度检测特定序列DNA。第六章：基于DNAzyme的化学发光新技术检测特定序列DNA 利用羧基修饰的磁性微球作为高效分离载体，基于DNA催化酶(DNAzyme)催化Luminol和H_2O_2产生化学发光的原理，构建了一种新型的磁性微球表面瞬时衍生特定序列DNA检测技术。包括以下分析步骤：(1)羧基磁性微球由EDC活化后，固定氨基修饰的捕获探针；(2)捕获探针与目标序列的15个寡聚核苷酸单位(5'-AAT ATT GAT AAGGAT-3’)杂交；(3)目标序列的另15个寡聚核苷酸单位(5'-GAG GGA TTA TTG TTA-3’)与报告序列的相应序列段进行第二次杂交，报告序列含有一段能够自我折叠形成四聚体的序列——d(TIT GGG TAG GGC GGG TTG GG)，能够特异性结合血红素(Hemin)，形成具有HRP相似催化性能的复合物(DNAzyme)；(4)用Luminol和H_2O_2在碱性条件与洗涤后的磁性微球反应，产生CL进行检测。考察并优化了各种实验参数，包括磁性微球量、杂交时间和CL检测条件等。在优化的实验条件检测炭疽杆菌特定序列DNA，结果表明：该法具有操作简便和分析快速等特点，目标序列浓度在0.2-20 nM浓度范围内具有良好的线性关系，相关系数为0.9987，并在碱基错配分辨中有一定效果，为DNA检测提供了一种有价值分析手段。第七章：基于Lumino-NaIO_4-环糊精流动注射的表面活性剂分析新技术这章内容是较为早期的工作，旨在发展一种通用型的流动注射CL分析技术，用于检测各种类型的表面活性剂——阳离子型、阴离子型和非离子型表面活性剂。从工业制造、科学研究、家庭用品到环境污染物，表面活性剂是一类随处可见的化合物。由于它们涉及到社会生活的方方面面，对于它们的研究显得尤为重要。其中，发展相应的分析检测方法是对其研究的一个重要方向。本章发展的技术原理是：在碱性溶液中，Luminol-NaIO_4-多羟基化合物(如：环糊精、葡萄糖和甘油等)体系的CL能够被各种类型的表面活性剂不同程度的淬灭，在一定浓度范围内，淬灭程度与表面活性剂的浓度线性相关。我们研究了该现象的反应机制，认为淬灭原因在于多羟基化合物改变了CL反应的微环境。据此发展了适应于三种类型表面活性剂检测的CL技术，阳离子型表面活性剂—CTMAB、阴离子型表面活性剂—SDS和非离子型表面活性剂—Triton X-100。结果发现：三种表面活性剂在4.0μM-0.4 mM浓度范围内线性相关性良好，三种物质的检测灵敏度为2μM，重复测定10μM的表面活性剂所得的相对标准偏差约为3.0％。该技术为多种表面活性剂经过HPLC或CE分离后的柱后衍生分析提供了基础。
[Abstract]:Modern medical research has shown that many diseases, such as cancer and genetic diseases, are related to gene mutations, and many other infectious diseases are caused by viruses, pathogens or parasites in the environment. Therefore, the analysis of specific DNA sequences and the detection of base mutations in DNA strands are involved in gene screening, forensic identification, genetic diseases. In recent years, rapid and reliable DNA detection techniques for specific sequences have been developed rapidly. A large number of studies have been carried out to label DNA probes with enzymes, isotopes, fluorescence, electrochemistry and chemiluminescence markers to establish highly sensitive and selective detection of specific sequence DNA. In view of the existence of DNA probe markers, to a certain extent, the preparation and use of sensors are complicated. Therefore, the study of unlabeled DNA detection technology has become one of the new research hotspots in the field of pathogenic gene detection and gene disease diagnosis.
Chemiluminescence (CL) analysis method does not need light source and avoids the interference of stray light, so it has very high sensitivity; moreover, this method has been developed rapidly because of its simple equipment and easy operation. At present, CL analysis has become a very active research field and has been successfully used in pharmacy, biology, molecular biology, clinical medicine and so on. Detection of various substances in environmental sciences. In 2003, our team reported for the first time a new technique for the detection of unmarked (transient derivative) DNA based on guanine specific chemiluminescence.
This paper aims to develop a series of innovative techniques for the detection of transient derivatization chemiluminescent DNA on magnetic microspheres.
Chapter 1: Introduction
The first section introduces the research progress and significance of DNA detection for specific sequences, including fluorescence, electrochemistry, surface matrix resonance and quartz microcrystalline balance, and lists their typical examples in this field in recent years; the second section introduces the main CL systems (such as luminol system, oxalate peroxide system, acridine ester system). The principles, characteristics and applications of the system and alkaline phosphatase system, especially in the fields of immunity and DNA analysis. Section 3, the purpose and significance of this paper are described, and the main research contents and innovations are pointed out.
Chapter 2: A New Chemiluminescence Detection Technique for Instantaneous Derivation of Specific DNA Sequences on the Surface of Magnetic Microspheres Modified by Poly T
1. new technology for telomere specific sequence detection
Telomere is a repetitive sequence at the 3'end of chromosome. Telomere is synthesized by a ribonucleoprotein complex, telomerase, under certain conditions. Telomere and telomerase play an important role in protecting cells from fusion and degradation. Their amount is closely related to the occurrence of malignant tumors and other diseases. Therefore, the determination of this gene fragment is of great significance. Based on the surface transient derivatization chemiluminescence of magnetic microspheres, a novel labeless detection technique for telomere-specific DNA sequence was developed. The whole analysis process consists of three experimental steps: (1) dT_ (20) modified magnetic microspheres hybridized with a capture probe connected with dA_ (20); (2) the detection of DNA sequence was carried out using a magnetic microsphere. The results showed that the method was simple, rapid and sensitive. The correlation coefficient was 0.9918 and the minimum detection concentration was 0.5 nM.
2. new technology for detection of anthrax specific sequences
As a global biochemical weapon, Bacillus anthracis has attracted great attention from the public as well as military departments. Establishing real-time and effective identification and detection technology is of great significance to control its spread. The amplification detection method based on G_ (30) sequence is developed. The amplification detection technique can be described as follows: Firstly, the capture probe is fixed on the surface of the magnetic microspheres by A_ (20) - T_ (20) hybridization reaction; 15 nucleotide units of the capture probe are hybridized with the corresponding sequence segments of the target sequence; then the report sequence is added to the other 15 nuclei of the target sequence. The report sequence contains two functional segments: one is a sequence segment capable of crossing 15 nucleotide units of the target sequence, the other is a sequence segment rich in G - (T_2G_ (15)) _2, the result table Ming: In the concentration range of 6-60 nM, the CL signal increases linearly (R~2=0.9984) and the minimum detection concentration is 6 nM; the target sequence is linearly good (R~2=0.9917) and the minimum detection concentration is 0.45 nM (0.045 pmol) in the concentration range of 0.45-6 nM using G_ (30) report sequence amplification detection technology; amplification detection technology is more sensitive than non-amplification technology. It is one order of magnitude higher and has good recognition for A-A single base mismatch.
The third chapter: a new technology of unlabeled chemiluminescence detection based on carboxyl modified magnetic microspheres surface.
In this chapter, the specific sequence DNA of Bacillus anthracis was studied. Carboxyl modified magnetic microspheres were used as separation and preconcentration carriers, fixed capture probes were used by carboxyl-amino condensation reaction, hybridized target sequences were detected by G-base specific CL reaction. On this basis, G_ (30) amplification detection technology was developed. Simple, non-amplified detection technique can be described as follows: under certain conditions, carboxyl magnetic microspheres activated by EDC, combined with the capture probe of specific sequence of Bacillus anthracis, then combined with the target sequence by hybridization reaction, washed and transferred using specific reaction reagent TMPG to directly determine the G base in the target sequence; G_ (30) amplified detection technique It is a sandwich method. After the first step of hybridization, a G-rich report sequence is added to the target sequence for the second hybridization. After washing and transferring, CL signals generated by the G-rich reaction between TMPG and target sequence and report sequence are detected. In the range of 2-5 nM, the CL signal increases linearly (R~2=0.9962) and the lowest detectable concentration is 2 nM. In G_ (30) amplification technique, the CL signal has good linear correlation (R~2=0.9965) and the lowest detectable concentration is 50 pM, which improves the sensitivity of non-amplification technique. In addition, the sensitivity of this technique is 2.5 and 10 times higher than that of non-amplification and G_ (30) amplification based on poly T modified magnetic microspheres, and the A-A single base mismatch recognition is good.
The fourth chapter: a new DNA chemiluminescence detection technology based on the specific amplification of carbon nanotubes.
In this chapter, carboxyl-modified magnetic microspheres were used as separation and preconcentration carriers, and carbon nanotubes (CNTs) were used as amplification carriers to develop a novel CL technique with high sensitivity for the detection of specific DNA sequences. (3) Carboxylated carbon nanotubes (CNTs) were activated by EDC and combined with a large number of amino-modified report sequences to form amplified complexes. The amplified complexes were bonded to the surface of magnetic microspheres by the second-step hybridization reaction; (4) Check the surface of the magnetic microspheres. CL signals produced by the reaction of TMPG with the target sequence and the G base in the amplified complex were measured. The report sequence consists of two functional segments: one is a G-rich sequence segment, the other is a complementary sequence segment with the target sequence, and all kinds of CNTs were oxidized by concentrated sulfuric acid-hydrogen peroxide (9:1) mixed solution to form surface carboxyl functionalized CNTs. - COOH; characterized by TEM and particle size/zeta potentiometer. The amplification effect of four kinds of carbon nanotubes was studied. Finally, two kinds of carriers were selected as carriers for amplification detection of specific DNA sequences. This method does not need to elute the components from the separation carrier before determination, nor does it need enzymes, fluorescent dyes and so on. The experimental results show that the method is simple, accurate and can detect target sequences with high sensitivity; the linearity is good in the concentration range of 20 pM-2 nM (2-200 fmol) (R~2=0.996), and the minimum detection concentration is 10 pM (1 fmol); the sensitivity is improved compared with the method without amplification. It is 200 times higher and can better distinguish single base mismatch, thus providing an effective way for DNA analysis.
The fifth chapter: a new DNA chemiluminescence detection technology based on polystyrene microspheres as amplification vectors.
In this chapter, a time-saving, labor-saving and efficient amplification detection technique based on commercial streptavidin polystyrene microspheres is proposed. Compared with carbon nanotubes as amplification carriers, there is no need for surface functionalization of amplification carriers, which saves analysis time and is conducive to further development of amplification technology. The principle of this technique is that the capture probe on magnetic microspheres and the report sequence self-assembled on polystyrene microspheres by streptavidin-biotin reaction are combined by sandwich hybridization in the presence of the target sequence. No target sequence exists and the amplified complex can not be combined on the surface of magnetic microspheres. The results showed that the method was simple, reliable, rapid and sensitive. The whole detection process was completed within 2-3 hours. There was a good linear relationship between the concentration of 10 pM-1 nM (R~2=0.995) and the minimum detection. The detection concentration is 5 pM (0.5 fmol), 400 times higher than that of the non-amplification method and 2 times higher than that of the CNTs amplification technique. Mismatch recognition experiments show that the method can distinguish the target sequence from the mismatch sequence.
The sixth chapter: a new chemiluminescence technology based on DNAzyme to detect specific sequence DNA.
Based on the principle of chemiluminescence of Luminol and H_2O_2 catalyzed by DNA zyme, a novel technique for the detection of specific sequence DNA on the surface of magnetic microspheres was developed by using carboxyl-modified magnetic microspheres as efficient separation carriers. (3) Another 15 oligonucleotide units of the target sequence (5'-GAG GGA TTA TTA-3') were hybridized with the corresponding sequence segments of the report sequence for the second time, and the report sequence contained a segment that could self-fold to form tetramers. The sequence D (TIT GGG TAG GGC GGG TTG GG) specifically binds hemin to form a complex (DNA zyme) with HRP-like catalytic properties; (4) CL is produced by the reaction of Luminol and H_2O_2 with washed magnetic microspheres in alkaline conditions. Various experimental parameters, including the amount of magnetic microspheres and impurity, are investigated and optimized. The results showed that the method was simple and rapid. The linear relationship between the concentration of target sequence and the concentration of target sequence was good in the range of 0.2-20 nM. The correlation coefficient was 0.9987. The method was effective in the discrimination of base mismatch and was suitable for DNA detection. A kind of measurement is provided.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

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