老年神经退行性疾病和癌症相关标志物的表面等离子体激元共振与电化学研究

发布时间：2018-11-07 14:24

【摘要】：摘要：阿尔兹海默症(Alzheimer's disease, AD)由于患病率高、早期症状不明显成为老年神经退行性疾病中研究较多的一种。目前普遍认为AD发病的主要原因是淀粉粥样蛋白(amyloid-beta, Aβ)的产生和清除失衡。因此,Aβ及其相关蛋白的研究可为AD预防及治疗提供重要信息。癌症是另外一种威胁人类健康的重大疾病。患者体液(如尿液、血液、唾液、脑脊液)中生物标志物的浓度变化能反应疾病的发生、发展进程。因此,疾病标志物的检测可为疾病的预警、早期诊断以及后续治疗提供重要依据。表面等离子体激元共振(Surface Plasmon Resonance, SPR)是发生在金属和电介质界面的一种物理现象。通过检测芯片表面折射率或厚度变化研究生物分子之间的相互作用。电化学技术通过检测电极表面电活性物质的氧化还原而实现待测物质的定性及定量分析。电化学测定时需要电活性物质进行标记,而SPR检测具有实时、免标记的特点,两种技术在一定程度上具有互补性。电化学与SPR都具有灵敏度高、选择性好、耗样量少、并适于复杂体系分析等特点,在生命科学领域中具有广泛的应用前景。本文采用SPR与电化学方法对AD及胶质瘤的重要标志物进行了定量及定性研究。具体工作如下： 1.采用双通道SPR对AD的重要标志物Aβ(1-42)及其结合蛋白TTR进行了研究。将特异性捕获脑脊液(CSF)中Aβ(1-42)和TTR的抗体分别固定在SPR两个通道上,随后流动注射Aβ(1-42)与TTR标准溶液或脑脊液样品与复合物(由亲和素和Aβ(1-16)抗体组成)的混合溶液并记录SPR信号。该方法对Aβ(1-42)和TTR的检测限分别为4.7pM和1.1nM。且二者在AD病人脑脊液中的浓度均低于健康人。两种生物标志物的同时检测为AD的预警及早期诊断提供了重要依据。此外,确定了CSF中Aβ(1-42)与TTR的作用形式。 2.采用可再生的Ni2+-NTA传感芯片建立了连续筛选β-分泌酶(BACE1)抑制剂的SPR方法。抑制剂存在下BACE1的活性被抑制,导致BACE1无法剪切芯片表面的多肽,此时流动注射能识别多肽片段的抗体会产生较大的SPR信号；当BACE1的活性保持时,可被抗体识别的多肽片段被BACE1切割,导致抗体无法结合到芯片表面,结果观察不到SPR信号。该方法简单、快速、灵敏、检测通量高。通过该方法筛选出两种潜在的BACE1抑制剂,其半抑制浓度(IC50)与酶联免疫法(ELISA)和质谱等结果一致。 3.利用二茂铁覆盖的纳米金-亲和素复合物进行信号放大建立了电化学筛选BACE1抑制剂的方法。当BACE1的活性被抑制时,同样观察不到任何电化学信号。该方法可用于BACE1活性的检测以及筛选BACE1抑制剂的研究。 4.通过纳米金信号放大建立了灵敏检测癌症标志物miRNA的电化学方法。生物素标记的miRNA(与靶点miRNA具有相同的序列)与靶点miRNA和电极表面固定的DNA探针发生竞争反应,随后通过生物素与亲和素的相互作用在电极表面引入二茂铁覆盖的纳米金-亲和素复合物。通过测定二茂铁的电化学响应从而实现靶点miRNA的放大检测。方法的线性范围为10fM-2.0pM。采用该方法测定的胶质瘤病人血清中niRNA的浓度是健康人的3.1倍,这与定量聚合酶链式反应(qPCR)结果一致。该方法灵敏、选择性好,在实际样品检测中具有潜在的应用前景。
[Abstract]:Abzheimer's disease (AD), due to its high prevalence and early symptoms, has become more and more effective in the treatment of neurodegenerative diseases in the elderly. It is generally believed that the main cause of the onset of AD is the production and clearance of the amyloid (amyloid-beta, A). Therefore, the study of A-type and its related protein can provide important information for AD prevention and treatment. Cancer is another major disease that threatens human health. The changes in the concentration of biomarkers in the body fluid (e.g., urine, blood, saliva, and cerebrospinal fluid) of the patient can reflect the occurrence and development of the disease. Therefore, the detection of disease markers can provide an important basis for early warning, early diagnosis and follow-up treatment of the disease. Surface Plasmon Resonance (SPR) is a kind of physics that occurs at the interface of metal and dielectric a phenomenon. by detecting a change in the refractive index or thickness of the surface of the wafer, the mutual relation between the biomolecules is investigated. The electrochemical technique achieves the qualitative and quantitative determination of the substance to be tested by detecting the redox of the electroactive species on the surface of the electrode The electrochemical measurement requires the marking of the electroactive species, and the SPR detection has the characteristics of real-time and non-marking, and the two technologies have mutual benefits to a certain extent. Both the electrochemical and the SPR have the characteristics of high sensitivity, good selectivity, less sample consumption, and being suitable for complex system analysis, and has wide application in the field of life science. In this paper, the important markers of AD and glioma were quantified by SPR and electrochemical method. and qualitative research. The work of the body is as follows: 1. The important marker A-(1-42) and the binding protein of the two-channel SPR pair AD are adopted TTR was studied. The antibodies that specifically capture the A-(1-42) and TTR in the cerebrospinal fluid (CSF) were fixed to the two channels of the SPR, respectively, followed by a mixed solution of a TTR standard solution or a cerebrospinal fluid sample and a complex (consisting of a pro-and A-(1-16) antibody) with a TTR standard solution or a cerebrospinal fluid sample. and the detection limit of the A-type (1-42) and the TTR is respectively 4-7. pM and 1.1nM, both of which were in the cerebrospinal fluid of the AD patient The concentration of the two biomarkers is lower than that of a healthy person, and the early warning and the early diagnosis of the AD are simultaneously detected by the two biological markers An important basis for the break is provided. In addition, the concentrations of A-(1-42) in the CSF are determined. The role of TTR. 2. A continuous screening of the yeast-secreting enzyme (BACE) was established using a regenerable Ni2 +-NTA sensing chip 1) The SPR method of the inhibitor. The activity of the BACE1 in the presence of the inhibitor is inhibited, resulting in the BACE1 being unable to cut the polypeptide on the surface of the chip, at this time, the flow injection can recognize that the antibody of the polypeptide fragment will generate a large SPR signal; when the activity of the BACE1 is maintained, it can be recognized by the antibody the peptide fragment was cut by the BACE1, causing the antibody to be unable to bind to the surface of the wafer, the method is simple, Two potential BACE1 inhibitors, the semi-inhibitory concentration (IC50) and the enzyme-linked immunosorbent assay (ELI) were screened by this method. (SA) and mass spectrometry. 3. The signal amplification was established by using the nano-gold-affinity complex covered with ferrocene. The method of screening a BACE1 inhibitor. When the activity of BACE1 is inhibited, The method can be used for detecting the activity of BACE1, and screening of the BACE1 inhibitor. The electrochemical method for measuring the miRNA of the cancer marker. The biotin-labeled miRNA (with the same sequence as the target miRNA) is competitive with the target miRNA and the DNA probe immobilized on the surface of the electrode, Ferrocene-covered nano-gold-affinity complex. The electrochemical response of ferrocene was determined. The amplification and detection of target miRNAs should be achieved. The linear range of the method is 10fM-2.0pM. the method is sensitive and has good selectivity,
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：O657.1;R741

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