發(fā)布時(shí)間：2019-03-02 21:33

【摘要】：為了解決傳統(tǒng)的檢測方法存在的檢測費(fèi)事,費(fèi)力,等待時(shí)間長等問題,實(shí)驗(yàn)采用了新的檢測手段——將微流控芯片與表面增強(qiáng)拉曼結(jié)合的方法。即在一塊微流控芯片上完成分析過程的樣品反應(yīng)、分離、檢測等基本操作后,進(jìn)行拉曼檢測與分析,實(shí)驗(yàn)中選用納米銀粒子和納米金球作為拉曼檢測的基底,因金屬納米材料擁有獨(dú)特的光電性質(zhì),特定尺寸的貴金屬納米粒子能夠產(chǎn)生“熱點(diǎn)”效應(yīng),SERS增強(qiáng)因子最高可達(dá)到107~1014倍,從而實(shí)現(xiàn)低濃度待測樣品的高靈敏度的檢測。本文的具體工作內(nèi)容如下:(1)利用微流控滑動(dòng)芯片作為檢測載體,快速檢測環(huán)境中和水產(chǎn)中的羅丹明6G和結(jié)晶紫含量。采用鹽酸氫銨和硝酸銀反應(yīng)制得Ag NPs溶液,并將濃縮后的納米銀粒子和R6G分別加入滑動(dòng)芯片中,選用多通道同時(shí)實(shí)驗(yàn)的方式,依靠微流控芯片的滑動(dòng)將兩者混合后,然后進(jìn)行SERS檢測,可以實(shí)現(xiàn)快速檢測和樣品的低消耗。實(shí)驗(yàn)發(fā)現(xiàn)在滑動(dòng)芯片的檢測區(qū)內(nèi)濃度為10-10mol/L的羅丹明6G依然有明顯的拉曼信號,在拉曼位移1360cm-1處呈現(xiàn)信號的峰值,并且隨著反應(yīng)混合時(shí)間的推移,其信號的強(qiáng)度也會(huì)有所變化,出現(xiàn)梯度最高值,濃度為10-10mol/L的最高信號強(qiáng)度為400,遠(yuǎn)遠(yuǎn)高于空白樣品檢測值。跟蹤結(jié)晶紫(CV)進(jìn)行相同實(shí)驗(yàn),成功驗(yàn)證基于微流控滑動(dòng)芯片的表面增強(qiáng)拉曼的高靈敏性檢測。(2)提出利用回形針來制作三維微流控紙芯片的新方法,能夠適應(yīng)多種不同流體通道的微流控紙芯片。利用簡單的比色檢測來驗(yàn)證自主設(shè)計(jì)的紙芯片的流通性和反應(yīng)靈敏性。在紙芯片上成功進(jìn)行了牛血清白蛋白、二價(jià)鐵離子的快速定量檢測。牛血清白蛋白的線性范圍是5-50μmol/L,檢出限為0.15μmol/L,R2=0.982。二價(jià)鐵離子的線性范圍是0.6μmol/L-12μmol/L,檢出限為0.18μmol/L,R2=0.992。驗(yàn)證了基于回形針的3D微流控紙芯片可用于實(shí)際樣品檢測的可能性。(3)采用SERS技術(shù)完成對紙芯片上腫瘤標(biāo)志物CEA的檢測。首先制備SERS探針標(biāo)記CEA抗體,根據(jù)CEA抗原抗體的特異性結(jié)合作用,與連接在紙芯片上的固定抗體和抗原形成 三明治‖免疫結(jié)構(gòu)。利用SERS檢測待測抗體上的MGITC信號,進(jìn)而間接得到不同濃度抗原對應(yīng)的信號值。CEA濃度分布在1 ng/m L-20ng/m L時(shí),拉曼檢測中標(biāo)記分子的拉曼特征峰強(qiáng)度與檢測物的濃度呈現(xiàn)出良好的線性關(guān)系,實(shí)現(xiàn)了對腫瘤蛋白標(biāo)志物的高靈敏定量檢測,CEA抗原的檢出限為0.3ng/ml。這一檢測方法經(jīng)過拓展后可能適用于更復(fù)雜的多元腫瘤標(biāo)志蛋白聯(lián)合檢測,具有廣泛的應(yīng)用前景。
[Abstract]:In order to solve the problems of detection cost, laborious and long waiting time existing in traditional detection methods, a new detection method, which combines microfluidic chip with surface-enhanced Raman method, is adopted in the experiment. After completing the basic operation of sample reaction, separation, detection and so on a microfluidic chip, Raman detection and analysis are carried out. In the experiment, nano-silver particles and gold nanoparticles are selected as the base of Raman detection. Due to the unique photoelectric properties of metal nanomaterials, the special size of precious metal nanoparticles can produce "hot spot" effect, and the SERS enhancement factor can be up to 107 脳 1014 times, thus realizing the high sensitivity detection of low concentration samples to be tested. The main contents of this paper are as follows: (1) the microfluidic slide chip is used as the detection carrier for rapid detection of Rhodamine 6G and Crystal Violet in environment and aquatic products. The Ag NPs solution was prepared by the reaction of ammonium hydrochlorid and silver nitrate. The concentrated silver nanoparticles and R6G were added to the slide chip respectively. The multi-channel simultaneous experiment was used to mix the two solutions by the sliding of the microfluidic chip. Then SERS detection can achieve rapid detection and low consumption of samples. It is found that Rhodamine 6G with the concentration of 10-10mol/L in the detection region of the sliding chip still has obvious Raman signal, and it shows the peak value of the signal at the Raman shift 1360cm-1, and with the passage of the reaction mixing time, the raman signal appears at the peak of the signal at the Raman shift 1360cm-1. The intensity of the signal also changes, and the maximum gradient value appears. The maximum signal intensity of 10-10mol/L is 400, which is much higher than that of blank sample. The high sensitivity detection of surface enhanced Raman based on microfluidic slide chip was successfully verified by the same experiment of tracking crystal violet (CV). (2) A new method of fabricating 3D microfluidic paper chip by using paper clip was proposed. Can adapt to a variety of different fluid channels microfluidic paper chip. A simple colorimetric test was used to verify the flowability and sensitivity of the self-designed paper chip. The rapid quantitative detection of bovine serum albumin (BSA) and divalent iron ion on paper chip was successfully carried out. The linear range of bovine serum albumin was 5-50 渭 mol / L, the detection limit was 0.15 渭 mol / L, and R2 was 0.982 2. The linear range of divalent iron ion is 0.6 渭 mol / L-12 渭 mol / L, the detection limit is 0.18 渭 mol / L, and R2 is 0.992.The detection limit is 0.18 渭 mol / L and the detection limit is 0.18 渭 mol / L. The possibility that 3D microfluidic paper chip based on paper clip can be used for real sample detection is verified. (3) the detection of tumor marker CEA on paper chip is completed by using SERS technology. First, the SERS probe labeled CEA antibody was prepared. According to the specific binding effect of CEA antigen antibody, the immobilization antibody and antigen formed the sandwich immune structure with the immobilized antibody and antigen attached to the paper chip. SERS was used to detect the MGITC signal on the antibody to be tested, and then the corresponding signal values of different concentrations of antigen were obtained indirectly. When the concentration of CEA was 1 ng/m L-20ng/m / L, There is a good linear relationship between the intensity of Raman characteristic peak of the labeled molecule and the concentration of the detection substance in Raman detection. The detection limit of CEA antigen is 0.3 ng / ml for the detection of tumor protein markers. This method may be applied to the combined detection of more complex multiple tumor marker proteins, and has a wide application prospect.
【學(xué)位授予單位】：煙臺(tái)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O657.37

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