生物标志物传感技术研究
发布时间：2019-02-22 17:37
【摘要】：慢性病是进行性的、不能自然痊愈及很少能够完全治愈的一类疾病,其所包含的疾病主要有糖尿病、高血脂、冠心病、脑卒中、高血压、肿瘤等。慢性病已经成为全世界几乎所有国家成年人的最主要死因,而生物标志物监测是实现慢性病二级预防(早发现、早诊断、早治疗)和对症治疗的基础,控制慢性病快速增长的基本途径。 本论文主要研究多种慢性病相关生物标志物快速检测的方法和实现途径,助力医院临床生物标志物快速测试以及家庭自测,特别是糖尿病、高血脂、痛风及肿瘤患者的自我监测。研究工作主要包括以下几个方面： (1)电化学酶传感技术研究 解决了电化学酶传感器的准确性、一致性和稳定性问题。基于双电极测试系统中电子媒介体作用的新机制,选择适当的检测电压来减少干扰物发生电极反应的概率。选用微反应腔和快速水合的传感层来缩短响应时间,使得电子媒介体的反应比干扰反应优先进行,进一步消除样品中存在的其它电活性物质的干扰。由于电子媒介体、双电极检测技术和短检测时间等因素的协同作用,所制备的尿酸、葡萄糖、胆固醇生物传感器均具有良好的抗干扰能力。正常生理性内源物质基本不干扰测定,外源性药物除极端高值外亦不干扰。采用丝网印刷技术一步完成传感试剂的固定,简化了制备工艺,并且提高了传感器间的一致性。通过优化传感层印浆组成,提高尿酸、葡萄糖、胆固醇生物传感器的储存稳定性。所制备的尿酸传感器的保存期为12个月以上,葡萄糖传感器的保存期为18个月以上,胆固醇传感器的保存期为10个月以上 (2)电化学免疫传感技术研究 以肿瘤标志物甲胎蛋白(AFP)为研究对象,解决了肿瘤标志物电化学免疫传感器研制中的四个共性问题：电化学标记方法、电化学检测方法、生物识别分子的固定化方法和免疫反应模式选择。利用电活性物质甲酸二茂铁标记甲胎蛋白,通过免疫渗滤方式和竞争免疫反应,构建了甲胎蛋白免疫渗滤型电化学传感器。利用胶体金介导辣根过氧化物标记抗体,通过免疫层析方式和夹心免疫反应,构建了甲胎蛋白免疫层析型电化学传感器。采用丝网印刷技术和微量非接触点样技术批量制备了上述两种甲胎蛋白电化学免疫传感器,相比于传统免疫分析方法(如ELISA),两种方法一致性很好。由于不需要频繁的清洗步骤,该方法操作简便,并且节省时间,适于诊所和家庭自检。 (3)条带型半定量胶体金免疫层析传感技术研究 解决了胶体金免疫层析传感技术灵敏度低和不适于定量的问题,实现了三种前列腺癌肿瘤标志物的半定量检测。研究发现：单位面积硝酸纤维素膜上固定化的抗体越多,灵敏度越高；单位数量胶体金上标记的抗体越少,灵敏度越高,但稳定性越差。但是使用牛血清白蛋白来填补胶体金表面的空位和封闭胶体金表面未和抗体结合的位点,可以稳定胶体金溶液和减少非特异性反应。采用多测试线,所制备的快速胶体金免疫层析传感器的检测限和线性范围均达到临床的要求。密封于有干燥剂的塑料袋中常温(25℃)保存,传感器稳定期大于6个月。临床实验研究表明,该方法与ELISA检测结果符合率为97.6%,可以应用于临床,从而满足了应对前列腺癌普查筛选和预后监测等快速检测的需要。
[Abstract]:Chronic diseases are progressive, non-natural and rarely able to cure a class of diseases, including diabetes, hyperlipidemia, coronary heart disease, stroke, hypertension, tumor, etc. Chronic diseases have become the leading cause of death for almost all of the world's adults, and biomarker monitoring is the basis for secondary prevention of chronic diseases (early detection, early diagnosis, early treatment) and symptomatic treatment, and the basic way to control the rapid growth of chronic diseases. This paper mainly studies the method and the way of rapid detection of various chronic diseases related biomarkers, the rapid test of the clinical biomarker of the hospital and the self-supervision of the family self-test, in particular the diabetes, the hyperlipidemia, the gout and the tumor. The study mainly includes the following parties: Surface: (1) Electrochemical enzyme sensing The technical research has solved the accuracy, the consistency and the consistency of the electrochemical enzyme sensor. stability problem. Based on the new mechanism of the action of the electronic mediator in the double-electrode test system, the appropriate detection voltage is selected to reduce the occurrence electrode of the interference object. the reaction time is shortened by selecting a micro-reaction cavity and a rapidly-hydrated sensing layer, so that the reaction of the electronic mediator is preferentially carried out compared with the interference reaction, and the other electric activity existing in the sample is further eliminated. The prepared uric acid, glucose and cholesterol biosensor have good effects due to the synergistic effect of the electronic media, the double-electrode detection technology, the short detection time and the like. Anti-interference ability. The normal physiological endogenous substance does not interfere with the measurement, but the exogenous drug has extreme high value. The method of screen printing is adopted to complete the fixation of the sensing reagent, the preparation process is simplified, and the sensor is improved. the consistency between the uric acid, the glucose and the cholesterol biosensor is improved by optimizing the sensing layer printing paste composition, the storage life of the prepared uric acid sensor is more than 12 months, the storage period of the glucose sensor is more than 18 months, and the storage period of the cholesterol sensor is More than 10 months (2) electrochemical Immunosensor technology was used to study the tumor marker alpha-fetoprotein (AFP). The four common problems in the development of the electrochemical immunosensor for tumor markers are the electrochemical marking method, the electrochemical detection method and the immobilization method of the biological recognition molecule. and the alpha-fetoprotein is constructed by the immunofiltration method and the competitive immune reaction. A percolation type electrochemical sensor is prepared by using colloidal gold to mediate the horseradish peroxidase-labeled antibody, and using the immunochromatographic method and the sandwich immune reaction to construct the alpha-fetoprotein immunity. the electrochemical immunosensor of the two kinds of alpha-fetoprotein is prepared in batches by adopting a screen printing technology and a micro non-contact sample application technology, and compared with a traditional immunoassay method (such as an ELISA), the method has good consistency, and the method is simple and convenient to operate and save time, Suitable for clinic and home self-test. (3) Strip-type semi-quantitative glue The study of the immunochromatographic sensing technology of the body gold solves the problems of low sensitivity and not suitable for quantitative detection of the colloidal gold immunochromatographic sensing technology, and realizes three leading positions. It was found that the more the antibody immobilized on the nitrocellulose membrane in the unit area, the higher the sensitivity, the less the antibody labeled on the unit number of colloidal gold, The higher the sensitivity, the higher the stability. However, bovine serum albumin is used to fill the space on the surface of the colloidal gold and to close the site where the colloidal gold surface is not bound to the antibody, and the glue can be stabilized. bulk gold solution and reduced nonspecific reaction. The test limit of the fast colloidal gold immunochromatographic sensor prepared by using a multi-test line and is sealed in a plastic bag with a drying agent at normal temperature (25 DEG C). The clinical experiment shows that the coincidence rate between the method and the ELISA is 97.6%, which can be applied to the clinic, so as to meet the screening and screening of the prostate cancer.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.0
本文编号：2428446
[Abstract]:Chronic diseases are progressive, non-natural and rarely able to cure a class of diseases, including diabetes, hyperlipidemia, coronary heart disease, stroke, hypertension, tumor, etc. Chronic diseases have become the leading cause of death for almost all of the world's adults, and biomarker monitoring is the basis for secondary prevention of chronic diseases (early detection, early diagnosis, early treatment) and symptomatic treatment, and the basic way to control the rapid growth of chronic diseases. This paper mainly studies the method and the way of rapid detection of various chronic diseases related biomarkers, the rapid test of the clinical biomarker of the hospital and the self-supervision of the family self-test, in particular the diabetes, the hyperlipidemia, the gout and the tumor. The study mainly includes the following parties: Surface: (1) Electrochemical enzyme sensing The technical research has solved the accuracy, the consistency and the consistency of the electrochemical enzyme sensor. stability problem. Based on the new mechanism of the action of the electronic mediator in the double-electrode test system, the appropriate detection voltage is selected to reduce the occurrence electrode of the interference object. the reaction time is shortened by selecting a micro-reaction cavity and a rapidly-hydrated sensing layer, so that the reaction of the electronic mediator is preferentially carried out compared with the interference reaction, and the other electric activity existing in the sample is further eliminated. The prepared uric acid, glucose and cholesterol biosensor have good effects due to the synergistic effect of the electronic media, the double-electrode detection technology, the short detection time and the like. Anti-interference ability. The normal physiological endogenous substance does not interfere with the measurement, but the exogenous drug has extreme high value. The method of screen printing is adopted to complete the fixation of the sensing reagent, the preparation process is simplified, and the sensor is improved. the consistency between the uric acid, the glucose and the cholesterol biosensor is improved by optimizing the sensing layer printing paste composition, the storage life of the prepared uric acid sensor is more than 12 months, the storage period of the glucose sensor is more than 18 months, and the storage period of the cholesterol sensor is More than 10 months (2) electrochemical Immunosensor technology was used to study the tumor marker alpha-fetoprotein (AFP). The four common problems in the development of the electrochemical immunosensor for tumor markers are the electrochemical marking method, the electrochemical detection method and the immobilization method of the biological recognition molecule. and the alpha-fetoprotein is constructed by the immunofiltration method and the competitive immune reaction. A percolation type electrochemical sensor is prepared by using colloidal gold to mediate the horseradish peroxidase-labeled antibody, and using the immunochromatographic method and the sandwich immune reaction to construct the alpha-fetoprotein immunity. the electrochemical immunosensor of the two kinds of alpha-fetoprotein is prepared in batches by adopting a screen printing technology and a micro non-contact sample application technology, and compared with a traditional immunoassay method (such as an ELISA), the method has good consistency, and the method is simple and convenient to operate and save time, Suitable for clinic and home self-test. (3) Strip-type semi-quantitative glue The study of the immunochromatographic sensing technology of the body gold solves the problems of low sensitivity and not suitable for quantitative detection of the colloidal gold immunochromatographic sensing technology, and realizes three leading positions. It was found that the more the antibody immobilized on the nitrocellulose membrane in the unit area, the higher the sensitivity, the less the antibody labeled on the unit number of colloidal gold, The higher the sensitivity, the higher the stability. However, bovine serum albumin is used to fill the space on the surface of the colloidal gold and to close the site where the colloidal gold surface is not bound to the antibody, and the glue can be stabilized. bulk gold solution and reduced nonspecific reaction. The test limit of the fast colloidal gold immunochromatographic sensor prepared by using a multi-test line and is sealed in a plastic bag with a drying agent at normal temperature (25 DEG C). The clinical experiment shows that the coincidence rate between the method and the ELISA is 97.6%, which can be applied to the clinic, so as to meet the screening and screening of the prostate cancer.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.0
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