發(fā)布時間：2018-01-02 23:25

  本文關(guān)鍵詞：毛細管在酶分析和免疫分析方面的研究及應(yīng)用　出處：《東北師范大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 毛細管 酶分析 在線監(jiān)測 免疫傳感器 腫瘤標(biāo)志物

【摘要】：毛細管由于傳質(zhì)距離短、試劑消耗量少、便于攜帶、使用簡便等優(yōu)勢,現(xiàn)已廣泛應(yīng)用于分析化學(xué)中的各個方面。本論文圍繞熔融石英毛細管在酶的在線連續(xù)監(jiān)測及構(gòu)建免疫傳感器方面開展了以下研究工作:1.基于毛細管電泳(CE)技術(shù),成功構(gòu)建了酶反應(yīng)在線分析體系,用于監(jiān)測酶反應(yīng)過程。利用穿過樣品池的相距15μm的兩根同軸毛細管,成功構(gòu)建了毛細管電泳連續(xù)分析的裝置,該裝置設(shè)計簡單,操作方便,通過在樣品池中施加額外的電壓,實現(xiàn)了樣品的在線連續(xù)進樣和毛細管電泳分析。結(jié)合在線快速衍生和毛細管電泳技術(shù),通過在線連續(xù)監(jiān)測脲酶產(chǎn)物(氨)的生成,對脲酶的活性進行了研究。脲酶反應(yīng)過程中,只有產(chǎn)物氨可以與衍生試劑發(fā)生反應(yīng),并在紫外區(qū)(340 nm)有吸收,譜圖斜率即產(chǎn)物生成速率,也是酶反應(yīng)速率。所得米氏常數(shù)Km和最大反應(yīng)速率Vmax分別為2.79±0.15 mM和4.32±0.20 mM/min(n=3),與傳統(tǒng)線外方法所得結(jié)果一致,表明本方法對于在線酶分析測定具有很好的準(zhǔn)確性和可行性�；诖朔椒ㄑ芯苛薈u2+的抑制常數(shù)和機理。根據(jù)Km和Vmax值的變化規(guī)律,得知Cu2+為非競爭型抑制劑,抑制常數(shù)Ki為0.34±0.02μM,該結(jié)果與線外測定結(jié)果相符,證實了該方法在研究酶抑制動力學(xué)方面的準(zhǔn)確性。此研究對于疾病治療中控制脲酶活性、農(nóng)業(yè)上控制土壤中氮肥具有潛在應(yīng)用價值。2.提出了將光門進樣與紫外/可見檢測相結(jié)合的(OG-UV/vis)序列毛細管電泳分析,實現(xiàn)了酶反應(yīng)的全程在線監(jiān)測。光門進樣是一種快速、連續(xù)、可重復(fù)的進樣方式,樣品池中的酶反應(yīng)產(chǎn)物連續(xù)的進入毛細管,在UV/vis檢測之前用相應(yīng)波長的激光束照射一段時間使樣品的紫外吸收減弱,在譜圖中形成負(fù)峰。實驗詳細考察了漂白光強度(激光器功率)、光門進樣時間、運行電壓、衍生試劑濃度對靈敏度及分離的影響。以L-天冬酰胺酶為例,通過同時測定底物的消耗和產(chǎn)物的生成,實現(xiàn)了酶催化反應(yīng)線上全程監(jiān)測,時間分辨率為12s,即每隔12s就可獲得一組底物、產(chǎn)物峰,Km和Vmax值分別為0.25 mM和0.24 mM/min,與傳統(tǒng)線外方法一致,表明了OG-UV/vis的可行性。該方法儀器簡單,分析快速,自動化高,作為連續(xù)在線過程分析的有力工具,可用于在線監(jiān)測各種生物進程。3.制備了多通道微結(jié)構(gòu)毛細管免疫傳感器,并對其性能進行了研究。實驗采用雙抗體夾心法,通過化學(xué)鍵合法將抗體固定于毛細管內(nèi)壁,免疫反應(yīng)完成后,用激光誘導(dǎo)熒光技術(shù)(LIF)進行檢測。與相同截面積的普通毛細管相比,其表面積的增加有利于分子固定和識別,使得靈敏度提高了大約5倍。此外,我們也對封閉劑和包被抗體(Ab1)的濃度進行了優(yōu)化,并在最佳條件下考察了傳感器的批內(nèi)、批間重現(xiàn)性,分別為4.43%和7.14%(n=3),說明該方法具有較好的重現(xiàn)性。4.多通道微結(jié)構(gòu)毛細管熒光免疫傳感器對腫瘤標(biāo)志物的檢測。以檢測原發(fā)性肝癌的特異性標(biāo)志物-甲胎蛋白(AFP)為例,首先繪制了AFP的標(biāo)準(zhǔn)曲線,所得線性范圍、檢出限與傳統(tǒng)ELISA方法進行比較。在最佳條件下考察了傳感器的特異性,實驗結(jié)果表明,所制備的熒光免疫傳感器具有強的抗干擾能力,適用于復(fù)雜樣品的分析。實驗分別測定了單一血清樣品的回收率和多個臨床樣品中的AFP含量,回收率在84.3%-119.0%之間,所得AFP濃度與臨床檢測結(jié)果進行比較,相對誤差小于20.73%,相關(guān)性方程為y=0.888x+0.056。通過更換抗原、抗體體系,所制備的PCF免疫傳感器有望應(yīng)用于對其他腫瘤標(biāo)志物的檢測,在臨床分析中具有潛在的應(yīng)用價值。
[Abstract]:Because the capillary mass transfer distance is short, less reagent consumption, easy to carry, easy to use and other advantages, has been widely used in various aspects of chemical analysis. This paper focuses on aspects of fused silica capillary enzyme in online continuous monitoring and construction of immune sensor to carry out the following research work: 1. based on capillary electrophoresis (CE) technique, was successfully constructed the analysis system for online monitoring of enzyme reaction, enzyme reaction process. Through the use of sample pool is 15 mu m two coaxial capillary, successfully constructed device for continuous analysis of capillary electrophoresis, the device has the advantages of simple design, convenient operation, by applying additional voltage in the sample pool, realizes on-line continuous sampling and sample capillary electrophoresis analysis. Combined with the fast online derivative and capillary electrophoresis technology, products through online continuous monitoring of urease (ammonia) generation of urease activity were studied. The urease reaction process, only the product can react with ammonia and derivatization reagent in the ultraviolet region (340 nm) absorption spectra, the slope of that product formation rate, and enzyme reaction rate. The Michaelis constant Km and the maximum reaction rate of Vmax were 2.79 + 0.15 and 4.32 + 0.20 mM mM /min (n=3). With the traditional line method have the same results show that this method has good feasibility and accuracy of the determination of the analysis for the online enzyme. Based on this method to study the inhibition constants and the mechanism of Cu2+. According to the variation of Km and Vmax value, that Cu2+ as a non competitive inhibitor, the inhibition constant Ki was 0.34 + 0.02 M the results, and line determination results, confirmed the accuracy of this method in the study of enzyme inhibition kinetics. The research for the control of urease activity in the treatment of diseases, control of soil nitrogen in agriculture has the potential application value of.2. is proposed to be light The door of sampling and ultraviolet / visible detection combination (OG-UV/vis) sequence analysis of capillary electrophoresis, realized the on-line monitoring of the whole enzyme reaction. Optical gate injection is a rapid, continuous, sample into repeat, into the capillary enzyme reaction products in the sample cell continuous, UV to sample the corresponding wavelength of the laser beam irradiation time in UV/vis detection before the decrease of absorption, formation of negative peaks in the spectrum. The experiment investigated bleaching light intensity (laser power), light door operation voltage, injection time, derivatization reagent concentration separation effect on sensitivity and to L- asparaginase as an example, by generating simultaneous determination of substrate consumption and product, the enzyme catalytic reaction online monitoring, time resolution of 12s, is that every 12s can get a set of substrate, product peak, Km and Vmax were 0.25 mM and 0.24 mM/ min, and the traditional line method Consistent, show that OG-UV/vis is feasible. The method is simple, rapid analysis, high automation, as a powerful tool for continuous online analysis process, can be used for online monitoring of various biological processes for preparation of.3. multi-channel micro structure capillary immunosensor, and its performance was studied. The experiment using double antibody sandwich method by chemical bonding will be fixed on the inner wall of the capillary, antibody, immune response after using laser induced fluorescence technique (LIF) were detected. Compared with the same cross-sectional area of the common capillary, its surface area increased with the increasing of molecular recognition and fixed, the sensitivity increased by about 5 times. In addition, we also on sealant and coating antibody (Ab1) concentration were optimized, and the optimum conditions were investigated in batch batch sensor, reproducibility, respectively 4.43% and 7.14% (n=3), which shows that the new method has a good reproducibility of.4. Multi channel micro capillary fluorescence immunosensor for the detection of tumor markers. The specific marker detection of primary liver cancer alpha fetoprotein (AFP) as an example, first draw the standard curve of AFP, the linear range, detection limit and traditional ELISA method were compared. The optimum conditions were investigated specific the sensor, the experimental results show that the fluorescence immunosensor prepared has strong anti-interference ability, suitable for the analysis of complex samples. The experiment tested the content of AFP recovery single serum samples and multiple clinical samples, the recovery rate was 84.3%-119.0%, compared with AFP concentration and clinical detection results, the relative error is less than 20.73%, the correlation equation was y=0.888x+0.056. by replacing the antigen antibody system for PCF immune sensor preparation is expected to be applied to other tumor markers in clinical detection. The analysis has potential application value.

【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：O658.9;TP212.3

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