發(fā)布時(shí)間：2018-07-03 07:20

  本文選題：地西泮 + 快速檢測(cè)　； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：食品安全問(wèn)題是當(dāng)今全球公共衛(wèi)生所面臨的重要問(wèn)題,各種人工合成或天然化合物推動(dòng)著食品工業(yè)的迅速發(fā)展,而食品安全狀況卻每況愈下。隨著食品污染物持續(xù)增多、安全標(biāo)準(zhǔn)更加嚴(yán)格、市場(chǎng)競(jìng)爭(zhēng)和貿(mào)易挑戰(zhàn)日益增加,和諧穩(wěn)定的社會(huì)要求人們完善檢測(cè)技術(shù)體系、健全食品藥品監(jiān)管制度、逐步形成食品安全良性循環(huán)機(jī)制。地西泮屬于常見(jiàn)的苯二氮卓類處方藥物,可通過(guò)化學(xué)神經(jīng)遞質(zhì)作用于大腦及神經(jīng)從而起到鎮(zhèn)靜作用,通�？诜�即可起效,臨床當(dāng)中廣泛應(yīng)用于治療焦慮、失眠、癲癇、戒斷反應(yīng)以及肌痙攣等。因其代謝產(chǎn)物可在體內(nèi)富集且仍具有生物活性,已被各國(guó)列為畜禽產(chǎn)品中禁止檢出化合物。另一方面,由于地西泮具備鎮(zhèn)靜、催眠作用,犯罪分子常利用這一特點(diǎn)實(shí)施犯罪,造成很大的社會(huì)危害;并有部分商家在保健品中非法添加類似成分,以片面夸大其產(chǎn)品效果。近來(lái),投毒、誤服大量地西泮而中毒的事件引起了政府及社會(huì)各界的廣泛關(guān)注。因此,建立快速、有效的地西泮檢測(cè)方法是打擊此類犯罪活動(dòng)、監(jiān)測(cè)地西泮非法添加與濫用的一種重要手段。目前已有一系列的分析方法可用于檢測(cè)相關(guān)樣品中地西泮的含量,其中包括高效液相色譜(HPLC)、液質(zhì)聯(lián)用(LC-MS)、氣質(zhì)聯(lián)用(GC-MS)以及電噴霧質(zhì)譜(ESI-MS)等大型儀器分析。此外,還有熒光測(cè)定、毛細(xì)管電泳及生物傳感器檢測(cè)地西泮的相關(guān)報(bào)道。盡管這些方法的靈敏度及可靠性已經(jīng)在分析領(lǐng)域得到確認(rèn),然而以上檢測(cè)方法都存在各自的缺陷,有的需要復(fù)雜的樣品前處理過(guò)程,有的儀器設(shè)備昂貴需要專業(yè)人員操作,使得這些分析技術(shù)往往不適用于現(xiàn)場(chǎng)檢測(cè)。同時(shí),由于物流、互聯(lián)網(wǎng)行業(yè)高速發(fā)展,地西泮及其同類管控藥品的獲取途徑較以往更為便利,這也使地西泮在食品安全及社會(huì)事件中出現(xiàn)的頻率急劇增加。因此,對(duì)于地西泮的檢測(cè)需求已經(jīng)從傳統(tǒng)的獸藥殘留向更多方向擴(kuò)展,亟需一種快速、簡(jiǎn)便、抗干擾的技術(shù)用于實(shí)際樣品的快速檢測(cè)。量熱生物傳感器(enzyme thermistor,ET)是將生物、物理和化學(xué)反應(yīng)中最基本的熱量變化轉(zhuǎn)變?yōu)闄z測(cè)信號(hào)的生物傳感裝置,具有穩(wěn)定性強(qiáng)、信號(hào)響應(yīng)不受離子環(huán)境或光學(xué)特性干擾以及可實(shí)現(xiàn)連續(xù)監(jiān)測(cè)等優(yōu)勢(shì)。其標(biāo)準(zhǔn)設(shè)備由內(nèi)含反應(yīng)柱的加熱恒溫裝置、注射泵、進(jìn)樣閥、加樣環(huán)、惠斯通轉(zhuǎn)化放大器、信號(hào)采集工作站及連接管路組成。ET作為新型分析儀器已在臨床醫(yī)學(xué)、食品安全、環(huán)境監(jiān)測(cè)、工業(yè)發(fā)酵監(jiān)控等領(lǐng)域得到應(yīng)用。量熱酶聯(lián)免疫分析法(TELISA)是通過(guò)量熱傳感器來(lái)檢測(cè)酶聯(lián)免疫吸附試驗(yàn)當(dāng)中酶催化反應(yīng)所產(chǎn)生的熱信號(hào),相比傳統(tǒng)的ELISA具有抗干擾性強(qiáng)、標(biāo)記酶種類多及免疫吸附劑能夠重復(fù)使用等優(yōu)點(diǎn),還可以實(shí)現(xiàn)對(duì)待測(cè)物的連續(xù)監(jiān)測(cè)。本論文以苯二氮卓類藥物地西泮為靶標(biāo),基于量熱傳感器及免疫分析技術(shù),建立了可以快速檢測(cè)地西泮的量熱免疫檢測(cè)技術(shù)。該方法抗基質(zhì)效應(yīng)性強(qiáng),通用性良好,無(wú)需復(fù)雜前處理過(guò)程僅需過(guò)濾中和稀釋,便可直接檢測(cè)液體樣品。并在此基礎(chǔ)上進(jìn)行了多種不同類型實(shí)際樣品的加標(biāo)回收實(shí)驗(yàn),通過(guò)一系列驗(yàn)證展現(xiàn)出該方法的可靠性,為該快速檢測(cè)方法在不同領(lǐng)域中廣泛應(yīng)用提供了技術(shù)支持;為了進(jìn)一步提高檢測(cè)靈敏度,我們引入生物素-親和素放大系統(tǒng)重新構(gòu)建了間接競(jìng)爭(zhēng)免疫檢測(cè)體系,實(shí)現(xiàn)了對(duì)目標(biāo)物更為經(jīng)濟(jì)、有效的分析,為量熱傳感免疫分析技術(shù)在食品安全等領(lǐng)域的應(yīng)用奠定了基礎(chǔ)。1、地西泮直接競(jìng)爭(zhēng)量熱免疫檢測(cè)技術(shù)的研究在本研究中,基于流動(dòng)注射和酶免疫分析技術(shù),利用直接競(jìng)爭(zhēng)法,設(shè)計(jì)并構(gòu)建了快速檢測(cè)地西泮的量熱傳感器,并將實(shí)樣分析結(jié)果與傳統(tǒng)檢測(cè)方法進(jìn)行了比對(duì)。其實(shí)驗(yàn)原理是利用蛋白G瓊脂糖凝膠(PGSFF)作為固相載體,使β-內(nèi)酰胺酶標(biāo)記的抗原與樣品中目標(biāo)物競(jìng)爭(zhēng)結(jié)合蛋白G上吸附的地西泮抗體,目標(biāo)物濃度與酶催化特異性底物氨芐西林產(chǎn)生的量熱信號(hào)相關(guān)。在經(jīng)過(guò)系列條件優(yōu)化后建立起了檢測(cè)曲線,最低檢出限達(dá)到33.71 ng m L-1,線性檢測(cè)范圍為45.37至726.71 ng m L-1。該方法能夠?qū)崿F(xiàn)對(duì)地西泮的精確定量,其靈敏度足以檢出地西泮對(duì)成人的最低起效劑量(2 mg),可適用于地西泮非法添加及投毒的檢測(cè)。在與五種地西泮結(jié)構(gòu)及功能類似物的交叉反應(yīng)率對(duì)比當(dāng)中,除結(jié)構(gòu)高度相近的替馬西泮外均無(wú)交叉反應(yīng)。對(duì)為期3個(gè)月的重復(fù)性實(shí)驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析,該方法信號(hào)響應(yīng)的變異系數(shù)(CV)小于6.17%,且反應(yīng)柱填料的使用壽命在50次以上。隨后選取了包括自來(lái)水、橙汁、朗姆酒及口服液等幾種具有代表性的飲料及保健品進(jìn)行了實(shí)樣檢測(cè),加標(biāo)回收實(shí)驗(yàn)的回收率范圍在100.15%至116.26%之間,同時(shí)采用高效液相色譜法(HPLC)進(jìn)行方法比對(duì),其結(jié)果準(zhǔn)確一致,從而進(jìn)一步證實(shí)了本方法在實(shí)際應(yīng)用當(dāng)中的可靠性。更值得關(guān)注的是,我們所建立的檢測(cè)策略省去了復(fù)雜的樣品前處理過(guò)程,僅用0.22μm的濾器過(guò)濾及必要的中和稀釋后便可對(duì)液體樣品直接進(jìn)行分析,且能夠在20分鐘之內(nèi)獲得檢測(cè)結(jié)果,使得該方法在快速檢測(cè)當(dāng)中具有十分明顯的優(yōu)勢(shì)。后續(xù)研究當(dāng)中,可進(jìn)一步優(yōu)化加樣方法,提高整體工作效率,根據(jù)反應(yīng)柱填料特性優(yōu)化流動(dòng)相提高其使用壽命。另外,若能充分發(fā)揮該傳感器抗基質(zhì)效應(yīng)的能力,并利用識(shí)別元件G蛋白通用性強(qiáng)的特點(diǎn),實(shí)施生物樣本中更多類型目標(biāo)物的檢測(cè),有望進(jìn)一步拓展量熱免疫傳感技術(shù)的應(yīng)用領(lǐng)域。2、基于生物素-親和素放大系統(tǒng)的間接競(jìng)爭(zhēng)量熱免疫檢測(cè)技術(shù)研究本研究以量熱免疫檢測(cè)技術(shù)為基礎(chǔ),采用ELISA的間接競(jìng)爭(zhēng)法模型,實(shí)現(xiàn)對(duì)地西泮檢測(cè)靈敏度的進(jìn)一步提高。該方法的檢測(cè)原理是將地西泮半抗原通過(guò)活性酯法直接包被在固相載體標(biāo)準(zhǔn)孔徑玻璃珠(CPG)上,使其在結(jié)合單克隆抗體時(shí)與樣品中目標(biāo)物產(chǎn)生競(jìng)爭(zhēng)作用,與此同時(shí)對(duì)β-內(nèi)酰胺酶進(jìn)行生物素修飾后,通過(guò)生物素化二抗引入生物素-鏈霉親和素放大系統(tǒng),將目標(biāo)物濃度以酶催化底物氨芐西林的量熱響應(yīng)強(qiáng)度反映出來(lái)。實(shí)驗(yàn)中對(duì)反應(yīng)體系中的關(guān)鍵條件,包括不同試劑的加入順序及濃度、比例等進(jìn)行優(yōu)化后,建立了地西泮的檢測(cè)標(biāo)準(zhǔn)曲線,其線性范圍為0.1 ng m L-1至1000 ng m L-1;最低檢出限為0.12 ng m L-1。選取替馬西泮、勞拉西泮、硝西泮、氟西泮及佐匹克隆等地西泮類似物進(jìn)行特異性研究,線性檢測(cè)范圍內(nèi)選取的濃度未檢測(cè)到量熱信號(hào)響應(yīng)。隨后進(jìn)行的重復(fù)性實(shí)驗(yàn)中,99.94-106.25%的回收率也充分驗(yàn)證了檢測(cè)方法的可靠性。該策略在樣品進(jìn)樣30分鐘后,即可計(jì)算出待測(cè)物的濃度。新的檢測(cè)技術(shù)除了具有直接競(jìng)爭(zhēng)量熱免疫傳感器自身的優(yōu)勢(shì)外,大大提高了檢測(cè)靈敏度及線性范圍,并且增強(qiáng)了傳感器中核心元件的壽命及穩(wěn)定性。未來(lái)的研究可針對(duì)該策略加樣特點(diǎn),改進(jìn)量熱設(shè)備搭建方式,并深入探索流動(dòng)相體系對(duì)間接競(jìng)爭(zhēng)量熱免疫檢測(cè)技術(shù)的影響,建立更為有效、經(jīng)濟(jì)的新型量熱傳感免疫檢測(cè)策略,為高靈敏檢測(cè)食品藥品非法添加物、農(nóng)獸藥殘留提供一個(gè)富有應(yīng)用前景的技術(shù)平臺(tái)。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R155

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