老年神經(jīng)退行性疾病和癌癥相關(guān)標(biāo)志物的表面等離子體激元共振與電化學(xué)研究
發(fā)布時(shí)間：2018-11-07 14:24
【摘要】：摘要：阿爾茲海默癥(Alzheimer's disease, AD)由于患病率高、早期癥狀不明顯成為老年神經(jīng)退行性疾病中研究較多的一種。目前普遍認(rèn)為AD發(fā)病的主要原因是淀粉粥樣蛋白(amyloid-beta, Aβ)的產(chǎn)生和清除失衡。因此,Aβ及其相關(guān)蛋白的研究可為AD預(yù)防及治療提供重要信息。癌癥是另外一種威脅人類健康的重大疾病�；颊唧w液(如尿液、血液、唾液、腦脊液)中生物標(biāo)志物的濃度變化能反應(yīng)疾病的發(fā)生、發(fā)展進(jìn)程。因此,疾病標(biāo)志物的檢測(cè)可為疾病的預(yù)警、早期診斷以及后續(xù)治療提供重要依據(jù)。 表面等離子體激元共振(Surface Plasmon Resonance, SPR)是發(fā)生在金屬和電介質(zhì)界面的一種物理現(xiàn)象。通過(guò)檢測(cè)芯片表面折射率或厚度變化研究生物分子之間的相互作用。電化學(xué)技術(shù)通過(guò)檢測(cè)電極表面電活性物質(zhì)的氧化還原而實(shí)現(xiàn)待測(cè)物質(zhì)的定性及定量分析。電化學(xué)測(cè)定時(shí)需要電活性物質(zhì)進(jìn)行標(biāo)記,而SPR檢測(cè)具有實(shí)時(shí)、免標(biāo)記的特點(diǎn),兩種技術(shù)在一定程度上具有互補(bǔ)性。電化學(xué)與SPR都具有靈敏度高、選擇性好、耗樣量少、并適于復(fù)雜體系分析等特點(diǎn),在生命科學(xué)領(lǐng)域中具有廣泛的應(yīng)用前景。 本文采用SPR與電化學(xué)方法對(duì)AD及膠質(zhì)瘤的重要標(biāo)志物進(jìn)行了定量及定性研究。具體工作如下： 1.采用雙通道SPR對(duì)AD的重要標(biāo)志物Aβ(1-42)及其結(jié)合蛋白TTR進(jìn)行了研究。將特異性捕獲腦脊液(CSF)中Aβ(1-42)和TTR的抗體分別固定在SPR兩個(gè)通道上,隨后流動(dòng)注射Aβ(1-42)與TTR標(biāo)準(zhǔn)溶液或腦脊液樣品與復(fù)合物(由親和素和Aβ(1-16)抗體組成)的混合溶液并記錄SPR信號(hào)。該方法對(duì)Aβ(1-42)和TTR的檢測(cè)限分別為4.7pM和1.1nM。且二者在AD病人腦脊液中的濃度均低于健康人。兩種生物標(biāo)志物的同時(shí)檢測(cè)為AD的預(yù)警及早期診斷提供了重要依據(jù)。此外,確定了CSF中Aβ(1-42)與TTR的作用形式。 2.采用可再生的Ni2+-NTA傳感芯片建立了連續(xù)篩選β-分泌酶(BACE1)抑制劑的SPR方法。抑制劑存在下BACE1的活性被抑制,導(dǎo)致BACE1無(wú)法剪切芯片表面的多肽,此時(shí)流動(dòng)注射能識(shí)別多肽片段的抗體會(huì)產(chǎn)生較大的SPR信號(hào)；當(dāng)BACE1的活性保持時(shí),可被抗體識(shí)別的多肽片段被BACE1切割,導(dǎo)致抗體無(wú)法結(jié)合到芯片表面,結(jié)果觀察不到SPR信號(hào)。該方法簡(jiǎn)單、快速、靈敏、檢測(cè)通量高。通過(guò)該方法篩選出兩種潛在的BACE1抑制劑,其半抑制濃度(IC50)與酶聯(lián)免疫法(ELISA)和質(zhì)譜等結(jié)果一致。 3.利用二茂鐵覆蓋的納米金-親和素復(fù)合物進(jìn)行信號(hào)放大建立了電化學(xué)篩選BACE1抑制劑的方法。當(dāng)BACE1的活性被抑制時(shí),同樣觀察不到任何電化學(xué)信號(hào)。該方法可用于BACE1活性的檢測(cè)以及篩選BACE1抑制劑的研究。 4.通過(guò)納米金信號(hào)放大建立了靈敏檢測(cè)癌癥標(biāo)志物miRNA的電化學(xué)方法。生物素標(biāo)記的miRNA(與靶點(diǎn)miRNA具有相同的序列)與靶點(diǎn)miRNA和電極表面固定的DNA探針發(fā)生競(jìng)爭(zhēng)反應(yīng),隨后通過(guò)生物素與親和素的相互作用在電極表面引入二茂鐵覆蓋的納米金-親和素復(fù)合物。通過(guò)測(cè)定二茂鐵的電化學(xué)響應(yīng)從而實(shí)現(xiàn)靶點(diǎn)miRNA的放大檢測(cè)。方法的線性范圍為10fM-2.0pM。采用該方法測(cè)定的膠質(zhì)瘤病人血清中niRNA的濃度是健康人的3.1倍,這與定量聚合酶鏈?zhǔn)椒磻?yīng)(qPCR)結(jié)果一致。該方法靈敏、選擇性好,在實(shí)際樣品檢測(cè)中具有潛在的應(yīng)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,
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：O657.1;R741
[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,
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：O657.1;R741
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