【作者】 贾立永；

【导师】 王前；

【作者基本信息】 南方医科大学 ， 临床检验诊断学， 2011， 硕士

【摘要】 研究背景和目的原发性肝癌是发生在肝细胞或肝内胆管细胞的癌,死亡率仅次于胃癌和食管癌,是我国常见的恶性肿瘤之一,且发病率有上升趋势。在我国每年约有11万人死于肝癌,占全球肝癌死亡人数的45%。手术治疗仍是目前治疗原发性肝癌的最好方法,但是手术治疗往往给病人带来巨大痛苦和经济负担。为了降低原发性肝癌的发病率和死亡率、减少手术对病人的身体和精神负担,迫切地需要建立有效的原发性肝癌的筛查方案。肿瘤的筛查最简单实用的方法就是进行肿瘤标志物筛查。肿瘤标志物是癌细胞产生和释放的某些物质,常以抗原、酶、激素、代谢产物的形式存在于肿瘤细胞内或宿主体液中,根据其生化或免疫特性可以识别或诊断肿瘤。理想的肿瘤标记物应具有高特异性,适于人群普查。肿瘤标志物的血清学筛查可以简便的诊断癌症。就肝癌而言,甲胎蛋白(AFP)仍是特异性最强的标志物和诊断肝癌的主要指标。现已经广泛用于肝细胞癌的普查、诊断和判断治疗效果、预测复发。正常成人血清中AFP含量很少,高于20ng/ml就有患肝肿瘤的可能,因此,检测痕量AFP具有重要意义。基于同样的原因,我们对HIV的早期筛查进行了思考。获得性免疫缺陷综合征(Acquired immune deficiency syndrome)简称艾滋病(AIDS),是由人类免疫缺陷病毒(Human immune deficiency virus, HIV)感染所引起的一种严重的传染性免疫缺陷性疾病。据卫生部统计,截至2010年10月底,我国累计报告艾滋病病毒感染者和病人37万多例,其中病人13万多例,死亡6万8千多例。局部地区和特定人群疫情严重,并逐渐由高危人群向一般人群扩散,防治形势非常严峻。由于HIV病毒具有高突变性的特点,目前尚无有效的预防疫苗和根治艾滋病的药物,预防感染成为控制HIV蔓延的主要手段,而HIV感染的实验室诊断是艾滋病预防控制工作的重要组成部分,因此建立敏感实用的检测方法用于监测、诊断或血液筛查,对控制艾滋病的流行显得尤为重要。根据血清学反应不同,HIV可分为HIV-1和HIV-2,其中HIV-1是广泛分布于世界各地,是造成全世界HIV/AIDS流行的主要原因。HIV病毒颗粒呈球形,二十面体立体对称,有gag、pol和env三种结构基因,分别合成核心蛋白(gag)、多聚蛋白(po1)、包膜蛋白(env)三种结构蛋白。HIV变异性很强,各基因的变异程度各不同,其中env基因变异率最高,gag和pol基因相对保守。p24蛋白包被在病毒核酸外面,构成病毒核衣壳的外壳,由gag基因编码前体蛋白,在pol基因编码的蛋白酶的加工下产生,因此p24蛋白氨基酸顺序高度保守,这使其在HIV感染的检测中显示出良好的规律性。p24抗原出现于HIV感染的早期,比抗体出现的时间要早,这是由于急性感染以后病毒发生了爆发性复制,与具有高度传染性的病毒血症相关联。对HIV p24蛋白进行早期检测在HIV早期感染检测、血液筛查、新生儿HIV感染的诊断和监测治疗的效果和病程的进展方面具有重要意义。但是HIV急性感染期后,体内出现特异性抗体,p24抗原与抗体形成免疫复合物,游离抗原浓度很低,常规方法无法检出,因此需要开发新的高敏感性方法,以便在低浓度情况下直接检出p24抗原。由以上分析可以得出,对AFP和HIV p24抗原进行痕量检测具有重要的意义,也成为临床检验诊断学研究的热点。近年来,免疫分析技术由于具有高灵敏度和高特异性,引起了研究者的兴趣。免疫分析技术是实现肿瘤和传染病快速诊断的重要手段,通过获取某种疾病的特异性“诊断蛋白”(diagnostic protein, DP,即抗原／抗体)含量和种类,综合分析可方便了解病情。近年来,免疫分析方法得到了长足的发展,建立了多种灵敏、高效的免疫分析方法,如凝集反应、荧光免疫测定技术(FIA)、放射免疫分析技术(RIA)、酶联免疫吸附测定法(ELISA)、免疫印迹(WB)、化学发光免疫分析技术、电化学发光免疫分析技术、免疫芯片、免疫组织化学等,并且在许多临床疾病诊断中得到了广泛的应用。这些方法极大的促进了临床免疫项目的更新和检测的自动化、智能化以及网络化,但是仍然有许多不足：放射免疫技术是以放射性核素作为示踪元素的一种免疫标记测定技术,伴有不同程度的放射性污染；免疫印迹检测过程需要对蛋白质电泳分离,过程繁琐复杂,耗时较长；免疫组织化学需要借助显微镜的观测且制备繁琐；化学发光和电化学发光需要昂贵的仪器,成本较高；其他如凝集反应、胶体金免疫技术等其特异性和敏感性低,且不能准确定量。基层医疗机构尤其是偏远地区受到条件限制,特别是在急诊手术前需要对病人进行快速筛检,受到常规检测方法灵敏度低、特异性差和需时长、操作复杂的限制常规方法常常不能满足要求；出于隐私和害怕受到歧视的原因,HIV“高危人群”往往不愿意到医院、疾控中心等部门进行检测。综上所述,现有方法不能满足原发性肝癌和HIV感染筛查需求,急需开发一种简便快速、灵敏度高、特异性好、价格便宜、携带方便的快速筛查技术及其相关设备,此类仪器的开发成功对预防和控制艾滋病的传播具有非常重要的意义。1990年Herry等提出了免疫传感器的概念。免疫传感器利用抗体和抗原反应的高亲和力和分子识别能力,结合了传统的免疫测试法和生物传感器技术,使其具有实时输出、高灵敏性、高特异性、操作简便和价格低廉的优点,已经在食品临床诊断、环境监测、食品安全等领域得到广泛应用。围绕以上问题,本论文的工作首先是利用层层自组装技术制备了基于碳纳米管修饰的无酶型新型甲胎蛋白安培免疫传感器,该免疫传感器器对AFP具有很好的灵敏度。其次,通过在玻碳电极表面直接电镀金的方法,通过“三明治”型夹心法制备了新型HIV p24安培免疫传感器,并初步优化了检测条件。研究方法1.基于碳纳米管修饰的无酶型新型甲胎蛋白安培免疫传感器研究首先在玻碳电极(GCE)表面修饰一层羧基化碳纳米管(CNTs),然后利用带负电荷的DNA分子和带正电荷的硫瑾之间的静电作用,层层自组装修饰硫瑾以增强检测信号,然后利用硫瑾的氨基固定纳米金,以便固定抗体,最后利用牛血清白蛋白封闭未结合位点。制备的电极与AFP标准品温育,通过检测传感器电流来对AFP定量。2.基于玻碳电极表面直接电镀金修饰的夹心型HIV p24安培免疫传感器研究将打磨抛光好电极放在在1%的氯金酸溶液中,在-0.2V,用电流时间曲线法施加恒电位20S.然后将电极浸入anti-p24一抗,4℃过夜孵育。充分洗涤,除去非特异性结合抗体,然后用牛血清白蛋白封闭剩余活性位点。这样即制备出基于直接电镀金修饰的p24安培免疫传感器。将传感器与24抗原温育后,形成免疫复合物,洗涤,再将传感器与过氧化物酶标记的二抗(HRP-Ab2)温育,电极表面形成夹心免疫复合物。利用对苯二酚作为信号分子,辣根过氧化物酶催化对苯二酚与过氧化氢的反应,反应前后电流改变值大小与电极表面酶含量多少成正比,而酶的含量与抗原量相关,即传感器电流改变值与抗原量相关,以此作为p24抗原定量依据。结果1成功构建了基于碳纳米管修饰的无酶型新型甲胎蛋白安培免疫传感器,该传感器利用带负电荷的DNA分子与带正电荷的硫瑾直接的静电吸引作用,逐层修饰信号分子硫瑾。当硫瑾修饰五层时,信号达到最大值。在pH7.0,温育时间30min条件下,该传感器在0.5—25ng/ml范围内甲胎蛋白具有良好的检测线性。2成功构建了直接电镀金修饰的电极的夹心型HIV p24安培免疫传感器。该传感器利用电流时间曲线法在玻碳电极表面电镀纳米金后,电极的的导电性和可逆性均提高。同时纳米金可大量固定抗体并保持其生物活性。制备的传感器依次与抗原、二抗反应后,在电极表面形成夹心免疫复合物,二抗上的辣根过氧化物酶催化过氧化氢与对苯二酚的反应,在循环伏案图中表现为氧化电流的减小和还原电流的上升。传感器在0.01—100ng/mL范围内有良好的线性。结论本研究成功采用两种不同的电极修饰方法分别制备了可以高灵敏度检测AFP和p24蛋白的安培免疫传感器,有望为临床上检测原发性肝癌和HIV感染提供新的方法。两种电极的制备表明：1)利用DNA分子可以在电极表面成功修饰多层硫瑾分子,并可以提高传感器的信号强度。2)通过在电极表面直接电镀金的方法可以直接在玻碳电极表面修饰一层纳米金,为抗体的固定提供了一种简单快捷的方法。这两种传感器的制备具有通用性,即只要改变传感器制备过程修饰的抗体,即可制备检测不同蛋白的安培免疫传感器,为其他肿瘤标志物和感染性疾病提供参考。更多还原

【Abstract】 Background and objectivesPHC (Primary Hepatocellular Carcinoma) is one of the common malignant tumors occurring in hepatocytes and intrahepatic cholangiocytes, whose mortality rate ranks second only to gastric cancer and esophagus cancer, and its disease incidence shows ascendant tendency. In our country, about 110,000 people die from liver cancer each year, which accounts for 45% of the liver cancer deaths around the world. At the present time, surgical therapy is still the best treatment for PHC, but it also brings the patients great suffering and heavy financial burden. To reduce the disease incidence and mortality rate of PHC and lessen patients’physical and psychological burden of surgery, it is urgently necessary to institute an effective cancer screening program. The most practical and simplest way of tumor screening is to detect the tumor markers.Tumor marker is one substance produced by cancer cells, which usually exists in the tumor cells or the body fluid of hosts in the form of antigen, enzyme, hormone or metabolin, and we can identify and diagnose tumor according to the biochemical and immune characters of them. Ideal tumor marker should be highly specific and suitable for detecting. The serological techniques can easily diagnose cancer. In terms of hepatoma, alpha fetoprotein (AFP), with the highest specificity, is the main index to diagnose liver cancer and is widely applied to cancer screening, cancer diagnosing, evaluating therapeutic effect and predicting recurrence. In general, the content of AFP in human blood serum is very low, it will increase the risk of having liver cancer if the content is greater than 20ng/ml. So, it is very important to evaluate the trace amount of AFP.For the same reason, we took the early HIV screening into consideration. Acquired immune deficiency syndrome (AIDS) is a kind of severe infectious disease caused by infection of human immune deficiency virus (HIV). In China, there are 370,000 patients and infestors in total by the end of October 2010, of which 130,000 are infected and 68,000 have died according to the Ministry of Health’s statistics. The severe epidemic situation of some topical regions and some certain groups and the trend of the disease from high-risk groups to the general population put the prevention and cure in a grim and complex situation. There is no effective vaccine or medicine to prevent and cure the HIV yet because of its high mutability, so preventing infections has become the major method to control its spread. And the laboratory diagnosis of HIV infection is the predominant method for HIV prevention and control, so it is a must to set up a sensitive and practical program for detecting, diagnosing and blood screening.HIV can be categorized as HIV-1 and HIV-2 according to the different reaction of blood serum. What’s more, HIV-1 exists widely all over the world and is the major cause of the wide-spreading of HIV. HIV, which is spherical and a cubically symmetrical icosahedron, has three kinds of structures like gag, pol and env which separately synthesize these three kinds of structural proteins:core protein, multimerin and envelope protein. The variability of HIV is very strong, and the extent of heteromorphosis of different genes is different. The env has the highest heteromorphosis rate, while gag and pol are relatively conservative. The p24 covers on the surface of the viral nucleic acid, which forms the outer covering of the virus nucleocapsid, then gene gag codes precursor protein, gene pol codes protease. So, the amino acid sequence of p24 is highly conservative, which makes it reveals nice regularity in the HIV detection. Antigen p24 occurs in the early stage of HIV infection which is earlier than the occurrence of antibody, which results from that the viruses copy quickly after being infected, and they combine with the highly contagious viruses. Early detection of p24 is of great significance in early HIV detection, blood screening, and diagnosis of HIV infection of new infants, treatment effect and process. But after an acute infection period of HIV, the specific antibody occurs in the body, and the p24 antigen and antibody compose immune complex which makes the concentration of free antigen lower, then common methods can’t be used to detect the p24 antigen. So we need develop new methods with high sensitivity to detect directly the p24 antigen at low antigen concentration.As a conclusion of the work mentioned above, to evaluate and detect the AFP and HIV-p24 antigen is of great significance, it is also a hot topic of clinical examinations. In recent years, immunoassay has attracted the interests of many researchers because of its high sensitivity.Immunoassay is a major method to fulfill the rapid diagnostic of tumors and infectious diseases. It can easily recognize the patients’ condition through obtaining the content and sorts of the diagnostic protein (DP, antigen/antibody) of certain disease and making analysis. In recent years, immune analysis method has got considerable development, a lot of immune analysis methods have been developed, such as agglutination reaction, Fluorescence immune assays (FLA), radio immune assays technique (RIA), enzyme-linked immune sorbent assay (ELISA, western blot (WB), chemiluminescence immune assay, electrochemiluminescence immune assay, immunochip, immunohistochemistry and so on. These methods are widely used in clinical diagnosis and greatly promote the update, automation, intelligentization and networking of clinical immune projects. But there are still quite a few deficiencies. For example, RIA is always accompanied with more or less radioactive pollutions; WB needs ionophortic separation, which is time-consuming and complex; immunohistochemistry needs microscope to observe and its preparation is complex; chemiluminescence and electrochemiluminescence needs expensive instruments; the other methods all have low sensitivity and specificity. Basic medical institutions especially the remote areas are limited by varieties of conditions. The common methods can not meet the needs of quick screening for the patients due to their low sensitivity, bad specificity and complex process. For fearing of being pried about privacy and discriminated, HIV high-risk groups are not willing to go to hospital or the centers for disease control. In conclusion, the now available methods can not satisfy the needs of quick screening of liver cancer and HIV. So it is urgent to develop a new technique and associated instruments which are highly sensitive, specific, cheap, quick, convenient and portable. It will be of great significance in preventing and controlling the spread of HIV.In 1990, Herry put forward the conception of immunosensor. It makes use of the high affinity and molecular recognition ability of antigen and antibody and combines traditional immune test technique with biosensor technique, which makes it be quick, highly sensitive and specific, convenient and cheap. It has been widely applied to food clinical diagnosis and food security, environmental monitoring and many other fields. This thesis aims to prepare a new self-assembled amperometric immunosensor which is sensitive to AFP, then, prepare a new HIV-p24 amperometric immunosensor through plating gold on the surface of the carbon electrode and the sandwich method. Methods1. Research on the new enzymefree amperometric immunosensor decorated with carbon nanotubesFirst, coat a layer of carboxylic carbon nanotubes (CNTs) on the surface of the glassy carbon electrode (GCE); then decorate the thionine with self-assembled monolayer to strengthen the detecting signals by making use of the electrostatic reaction between the DNA molecule with negative charge and the thionine with positive charge; and then use the amino of thionine to fix the nano-Au so as to fix the antigen; finally, use the BSA (Bovine Serum Albumin) to seal up the points which are not linked. Incubate the prepared electrode and the AFP standard, and then quantify the AFP through detecting the sensor’s current.2. Research on the new sandwich HIV-p24 amperometric immunosensor decorated with glassy carbon electrode plated with goldPlace the polished electrode into the 1% chloroaurate solution, and exert constant potential to it for 30s by chronoamperometric method at -0.2V. Then dip the electrode into the anti-p24 Ab1, and incubate them at 4℃for one night. After that, wash it and eliminate the unspecific antibody, use the BSA to seal up the rest points which are not linked. Then the p-24 sensor is prepared. Incubate the sensor and p24 antigen which formates immune complex, wash it, and then incubate the sensor and the HRP-Ab2 marked with peroxidase, and formates sandwicn immune complex on the surface of the electrode. Take the hydroquinone as the signal molecule, and use HRP (horse radish peroxidase) to catalyze the hydroquinone and peroxide. The changing values of the current before and after the reaction is proportional to the content of enzyme on the surface of the electrode, while the changing value is connected with the amount of antigen, so we quantify the p24 according to the changing of the current.Results1. We successfully construct a new enzymefree amperometric immunosensor decorated with carbon nanotubes, which decorates the thionine by layer through making use of the electrostatic reaction between the DNA molecule with negative charge and the thionine with positive charge. When the thionine is decorated with five layers, the signal reaches the highest. At pH7.0, after being incubated for 30min, the sensor has nice detecting linear between 0.5ng to 25ng.2. We successfully construct a new sandwich HIV-p24 amperometric immunosensor decorated with glassy carbon electrode plated with gold. After plating gold on the surface of the electrode, the electrical conductivity and reversibility of electrode improve greatly, and nano-Au can nicely fix numbers of antibody so as to keep its bioactivity. The sensor reacts successively with antigen and Ab2, sandwich immune complex forms on the surface of the electrode. The reaction catalyzed by HRP between peroxide and hydroquinone shows that the oxydic current decreases and the reduced current increases. This sensor has nice detecting linear between 0.01ng/ml to 100ng/ml.ConclusionsThis research prepared the highly sensitive amperometric immunosensor used for detecting AFP and p24 in two different electrode-decorated methods. This sensor is expected to provide a new method to detect liver cancer and HIV. These two methods show:1) the DNA molecule can decorate the thionine multilayerly, and thus improving the signal intensity of the sensor.2) Plating gold on the surface of the electrode can form a layer of nano-Au on te glassy carbon electrode, which can conveniently fix the antibody. These two methods use the same principle that we can construct different amperometric immunosensors which detect differnent proteins by changing the decorating antibodies. It can provide reference for the other cancer markers and infectious diseases.更多还原