地西泮量热免疫快速检测技术研究

发布时间：2018-07-03 07:20

本文选题：地西泮 + 快速检测　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：食品安全问题是当今全球公共卫生所面临的重要问题,各种人工合成或天然化合物推动着食品工业的迅速发展,而食品安全状况却每况愈下。随着食品污染物持续增多、安全标准更加严格、市场竞争和贸易挑战日益增加,和谐稳定的社会要求人们完善检测技术体系、健全食品药品监管制度、逐步形成食品安全良性循环机制。地西泮属于常见的苯二氮卓类处方药物,可通过化学神经递质作用于大脑及神经从而起到镇静作用,通常口服即可起效,临床当中广泛应用于治疗焦虑、失眠、癫痫、戒断反应以及肌痉挛等。因其代谢产物可在体内富集且仍具有生物活性,已被各国列为畜禽产品中禁止检出化合物。另一方面,由于地西泮具备镇静、催眠作用,犯罪分子常利用这一特点实施犯罪,造成很大的社会危害;并有部分商家在保健品中非法添加类似成分,以片面夸大其产品效果。近来,投毒、误服大量地西泮而中毒的事件引起了政府及社会各界的广泛关注。因此,建立快速、有效的地西泮检测方法是打击此类犯罪活动、监测地西泮非法添加与滥用的一种重要手段。目前已有一系列的分析方法可用于检测相关样品中地西泮的含量,其中包括高效液相色谱(HPLC)、液质联用(LC-MS)、气质联用(GC-MS)以及电喷雾质谱(ESI-MS)等大型仪器分析。此外,还有荧光测定、毛细管电泳及生物传感器检测地西泮的相关报道。尽管这些方法的灵敏度及可靠性已经在分析领域得到确认,然而以上检测方法都存在各自的缺陷,有的需要复杂的样品前处理过程,有的仪器设备昂贵需要专业人员操作,使得这些分析技术往往不适用于现场检测。同时,由于物流、互联网行业高速发展,地西泮及其同类管控药品的获取途径较以往更为便利,这也使地西泮在食品安全及社会事件中出现的频率急剧增加。因此,对于地西泮的检测需求已经从传统的兽药残留向更多方向扩展,亟需一种快速、简便、抗干扰的技术用于实际样品的快速检测。量热生物传感器(enzyme thermistor,ET)是将生物、物理和化学反应中最基本的热量变化转变为检测信号的生物传感装置,具有稳定性强、信号响应不受离子环境或光学特性干扰以及可实现连续监测等优势。其标准设备由内含反应柱的加热恒温装置、注射泵、进样阀、加样环、惠斯通转化放大器、信号采集工作站及连接管路组成。ET作为新型分析仪器已在临床医学、食品安全、环境监测、工业发酵监控等领域得到应用。量热酶联免疫分析法(TELISA)是通过量热传感器来检测酶联免疫吸附试验当中酶催化反应所产生的热信号,相比传统的ELISA具有抗干扰性强、标记酶种类多及免疫吸附剂能够重复使用等优点,还可以实现对待测物的连续监测。本论文以苯二氮卓类药物地西泮为靶标,基于量热传感器及免疫分析技术,建立了可以快速检测地西泮的量热免疫检测技术。该方法抗基质效应性强,通用性良好,无需复杂前处理过程仅需过滤中和稀释,便可直接检测液体样品。并在此基础上进行了多种不同类型实际样品的加标回收实验,通过一系列验证展现出该方法的可靠性,为该快速检测方法在不同领域中广泛应用提供了技术支持;为了进一步提高检测灵敏度,我们引入生物素-亲和素放大系统重新构建了间接竞争免疫检测体系,实现了对目标物更为经济、有效的分析,为量热传感免疫分析技术在食品安全等领域的应用奠定了基础。1、地西泮直接竞争量热免疫检测技术的研究在本研究中,基于流动注射和酶免疫分析技术,利用直接竞争法,设计并构建了快速检测地西泮的量热传感器,并将实样分析结果与传统检测方法进行了比对。其实验原理是利用蛋白G琼脂糖凝胶(PGSFF)作为固相载体,使β-内酰胺酶标记的抗原与样品中目标物竞争结合蛋白G上吸附的地西泮抗体,目标物浓度与酶催化特异性底物氨苄西林产生的量热信号相关。在经过系列条件优化后建立起了检测曲线,最低检出限达到33.71 ng m L-1,线性检测范围为45.37至726.71 ng m L-1。该方法能够实现对地西泮的精确定量,其灵敏度足以检出地西泮对成人的最低起效剂量(2 mg),可适用于地西泮非法添加及投毒的检测。在与五种地西泮结构及功能类似物的交叉反应率对比当中,除结构高度相近的替马西泮外均无交叉反应。对为期3个月的重复性实验结果进行统计学分析,该方法信号响应的变异系数(CV)小于6.17%,且反应柱填料的使用寿命在50次以上。随后选取了包括自来水、橙汁、朗姆酒及口服液等几种具有代表性的饮料及保健品进行了实样检测,加标回收实验的回收率范围在100.15%至116.26%之间,同时采用高效液相色谱法(HPLC)进行方法比对,其结果准确一致,从而进一步证实了本方法在实际应用当中的可靠性。更值得关注的是,我们所建立的检测策略省去了复杂的样品前处理过程,仅用0.22μm的滤器过滤及必要的中和稀释后便可对液体样品直接进行分析,且能够在20分钟之内获得检测结果,使得该方法在快速检测当中具有十分明显的优势。后续研究当中,可进一步优化加样方法,提高整体工作效率,根据反应柱填料特性优化流动相提高其使用寿命。另外,若能充分发挥该传感器抗基质效应的能力,并利用识别元件G蛋白通用性强的特点,实施生物样本中更多类型目标物的检测,有望进一步拓展量热免疫传感技术的应用领域。2、基于生物素-亲和素放大系统的间接竞争量热免疫检测技术研究本研究以量热免疫检测技术为基础,采用ELISA的间接竞争法模型,实现对地西泮检测灵敏度的进一步提高。该方法的检测原理是将地西泮半抗原通过活性酯法直接包被在固相载体标准孔径玻璃珠(CPG)上,使其在结合单克隆抗体时与样品中目标物产生竞争作用,与此同时对β-内酰胺酶进行生物素修饰后,通过生物素化二抗引入生物素-链霉亲和素放大系统,将目标物浓度以酶催化底物氨苄西林的量热响应强度反映出来。实验中对反应体系中的关键条件,包括不同试剂的加入顺序及浓度、比例等进行优化后,建立了地西泮的检测标准曲线,其线性范围为0.1 ng m L-1至1000 ng m L-1;最低检出限为0.12 ng m L-1。选取替马西泮、劳拉西泮、硝西泮、氟西泮及佐匹克隆等地西泮类似物进行特异性研究,线性检测范围内选取的浓度未检测到量热信号响应。随后进行的重复性实验中,99.94-106.25%的回收率也充分验证了检测方法的可靠性。该策略在样品进样30分钟后,即可计算出待测物的浓度。新的检测技术除了具有直接竞争量热免疫传感器自身的优势外,大大提高了检测灵敏度及线性范围,并且增强了传感器中核心元件的寿命及稳定性。未来的研究可针对该策略加样特点,改进量热设备搭建方式,并深入探索流动相体系对间接竞争量热免疫检测技术的影响,建立更为有效、经济的新型量热传感免疫检测策略,为高灵敏检测食品药品非法添加物、农兽药残留提供一个富有应用前景的技术平台。
[Abstract]:The problem of food safety is an important issue facing the global public health. Various synthetic or natural compounds have promoted the rapid development of the food industry, but the food safety situation has gone from bad to worse. With the increasing of the food pollution, the safety standards are more stringent, the market competition and trade challenges are increasing, the harmonious and stable society It will require people to improve the system of testing technology and improve the regulatory system of food and drug, and gradually form a healthy circulation mechanism of food safety. Diazepam belongs to the common benzodiazepine prescription drug, which can act on the brain and nerve by chemical neurotransmitters to play a sedative effect. It can take effect through oral administration, and it is widely used in clinical treatment. Anxiety, insomnia, epilepsy, abstinence reaction and muscle spasm, etc. because their metabolites can be enriched in the body and still have biological activity, which have been listed as banned compounds in livestock and poultry products. On the other hand, because diazepam has a sedative and hypnotic effect, criminals often use this characteristic to commit crimes and cause great social harm. Some businesses have added similar ingredients illegally in health care products to exaggerate the effect of their products unilaterally. Recently, the poisoning of diazepam caused by poisoning and poisoning caused widespread concern in the government and all walks of life. Therefore, the establishment of a rapid and effective method for the detection of diazepam is to attack such crimes and monitor the illegal addition and abuse of diazepam. A series of analytical methods have been used to detect diazepam content in related samples, including high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), GC-MS (GC-MS) and electrospray ionization mass spectrometry (ESI-MS). In addition, fluorescence, capillary electrophoresis and biosensor detection The related reports of diazepam. Although the sensitivity and reliability of these methods have been confirmed in the field of analysis, the above detection methods have their own defects, some need complex sample pre-processing, and some instruments are expensive to be operated by professionals, so that these analytical techniques are often not applicable to field testing. At the same time, because of the rapid development of the Internet industry and the access to diazepam and its similar control drugs, the frequency of diazepam in food safety and social events has increased dramatically. Therefore, the need for diazepam detection has expanded from the traditional veterinary drug residues to more directions. Fast, simple, anti-interference techniques used for rapid detection of actual samples. Enzyme thermistor (ET) is a biological sensing device that transforms the most basic heat change in biological, physical and chemical reactions into a detection signal. It has strong stability, the signal response is not disturbed by the ion environment or optical properties and can be realized. The standard equipment is used as a new analytical instrument in clinical medicine, food safety, environmental monitoring, industrial fermentation monitoring and so on. The standard equipment has been applied in the fields of clinical medicine, food safety, environmental monitoring and industrial fermentation monitoring, such as heating and constant temperature device with internal reaction column, injection pump, injection valve, sample ring, Wheastone conversion amplifier, signal acquisition workstation and connecting pipeline as new analytical instruments. The.ET is used in the fields of clinical medicine, food safety, environmental monitoring, industrial fermentation monitoring and so on. Immunoassay (TELISA) is used to detect the heat signal produced by enzyme catalyzed reaction in the enzyme linked immunosorbent assay (ELISA) by a calorimetric sensor. Compared with the traditional ELISA, it has the advantages of strong anti-interference, many kinds of labelled enzymes and the repeated use of the immune adsorbents, and can also be used to monitor the continuous monitoring of the substances. This paper is based on benzene two azo. Diazepam is a target. Based on the calorimetric sensor and immunoassay technique, a calorimetric immunoassay for the rapid detection of diazepam is established. This method has strong anti matrix effect and good versatility. It can directly detect liquid samples without the need of complex pretreatment process and only need to be filtered and diluted. A series of tests on the same type of actual samples show the reliability of the method through a series of verification. It provides technical support for the wide application of the rapid detection method in different fields. In order to further improve the detection sensitivity, we have introduced the biotin avidin amplification system to reconstruct the indirect competitive immune detection system. In this study, the research of diazepam direct competitive thermal immunoassay (diazepam) in this study is based on the flow injection and enzyme immunoassay technology, and the rapid detection method is designed and constructed by the direct competition method. Diazepam's calorimetric sensor is compared with the traditional method. The experimental principle is that the protein G agarose gel (PGSFF) is used as a solid phase carrier to make the antigen labeled by beta lactamase and the protein G adsorbed on the target object in the sample, and the concentration of the target and the specificity of the enzyme catalyze. The measurement curve was set up after a series of conditions, the minimum detection limit reached 33.71 ng m L-1, and the linear detection range was 45.37 to 726.71 ng m L-1.. The method was able to achieve accurate quantitative determination of diazepam, and the sensitivity was sufficient to detect the minimum starting dose of diazepam (2 mg). It can be applied to the detection of diazepam's illicit addition and poisoning. In the cross reaction rate compared with the structure and functional analogues of diazepam in five kinds of diazepam, there is no cross reaction except the structure height similar to diazepam. The results of repeated experiments for 3 months are statistically analyzed, the variation coefficient of the signal response (CV) of this method is less than 6.1 7%, and the service life of the reacting column is more than 50 times. Then several representative drinks and health care products, including tap water, orange juice, rum and oral liquid, are tested. The recovery rate of the reclaim experiment is between 100.15% and 116.26%, and the method is compared with the method of high performance liquid chromatography (HPLC) at the same time. The results are accurate and consistent, which further confirms the reliability of this method in practical applications. It is more worthy of concern that the detection strategy we have established saves the complex sample pretreatment process and can analyze the liquid sample directly with only 0.22 M filter filter and the necessary neutralization dilution, and can be in 20 minutes. In the follow-up study, the method of adding sample can be further optimized to improve the overall working efficiency and improve the service life of the flow phase according to the characteristics of the column packing. The characteristics of the G protein of other components, the implementation of the detection of more types of objects in biological samples, it is expected to further expand the application field of thermal immunosensing technology,.2, based on the biotin avidin amplification system of indirect competitive thermal immunoassay technology research based on the calorimetric immunoassay technology as the basis of the use of ELISA The test principle of diazepam detection is to further improve the sensitivity of diazepam detection. The detection principle of this method is that the diazepam semi antigen is directly coated with the standard pore glass bead (CPG) by the active ester method, so that it can compete with the target in the sample with the monoclonal antibody and at the same time to beta lactamase. After biotin modification, the biotin two resistance was introduced into the biotin streptomycin amplification system, and the target concentration was reflected by the calorimetric response intensity of the enzyme catalyzed ampicillin. The key conditions in the reaction system, including the CIS order, concentration and proportion of different reagents, were optimized. The standard curve for the detection of diazepam was 0.1 ng m L-1 to 1000 ng m L-1; the minimum detection limit was 0.12 ng m L-1. to select diazepam, Laura Si, nitazepam, fluoxazepam, and zoepone, and other diazepam analogues were specifically studied. The concentration of the selected concentration in the linear detection range was not detected by the calorimetric response. The following was carried out. In the repeatability experiment, the recovery rate of 99.94-106.25% also fully verified the reliability of the detection method. The strategy can calculate the concentration of the object to be measured after 30 minutes of sample sampling. The new detection technique greatly improves the sensitivity and linear range of detection, and increases the detection sensitivity and the linear range. The life and stability of the core components in the sensor can be studied in the future. In the future, we can improve the construction mode of the calorimetric equipment and explore the influence of the mobile phase system on the indirect competitive thermal immunoassay. Illicit additives and Veterinary Veterinary Drug Residues provide a promising technology platform.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R155

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