基于免疫反应的高灵敏检测方法的建立
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摘要
由于量子点及碳纳米管(CNTs)独特的纳米特性及生物兼容性,它们在免疫分析领域被广泛应用。本论文分别构建了两种基于免疫反应的高灵敏检测方法。基于聚合量子点的超灵敏免疫印迹方法(western blot)用以对复杂蛋白样品进行分析检测,碳纳米管—纸免疫传感器对水中的微囊藻毒素进行检测。
一种免疫方法是：基于量子点的超灵敏蛋白检测免疫印迹方法。碲化镉(CdTe)量子点由于它独特的光学及生物特性,被广泛应用于生物化学标记领域。本论文构建了一种基于量子点的超灵敏蛋白检测免疫印迹方法。由于亲和素功能化的聚合量子点的高亲和性及对检测步骤的简化,使得量化免疫印迹方法成为现实。为了制备聚合量子点,首先需要用生物素化的变性牛血清白蛋白(dBSA)包裹量子点,然后通过生物素与亲和素的相互作用,量子点相互偶联生成聚合量子点。经过一系列的修饰,碲化镉量子点的荧光强度显著增强。把聚合量子点作为免疫印迹的标志物,其追踪微量蛋白比传统染料更灵敏。以蛋白A为例,对基于聚合量子点的免疫印迹方法进行验证,检测线性范围为30pg到1.5ng,检测限达0.84pg。最终在聚偏二氟乙烯膜上的荧光信号至少可以保持40min。进行实际样品检测时,回收率达99.9％到103.0％。
另一种方法是纸—单壁碳纳米管复合物组成的,简单且高效的传感器。碳纳米管传感器的电阻取决于碳纳米管网络之间的间隙宽度,该方法能很好的满足常规毒素的检测要求。本论文对微囊藻毒素进行了检测并建立了标准曲线,线性范围为1.25nmol／L到10nmol／L。检测限达0.6nmol／L(0.6ng／mL),满足了世界卫生组织关于饮用水中微囊藻毒素含量不超过1ng／mL的检测要求。同时,论文还对该方法与ELISA检测方法进行了比较。
Quantum dots and carbon nanotube have been widely used in the field of immunoassay because of their particular speciality and bio-compatibility. In the paper, we researched two detection method based on immunosassy. Ultrasensitive detection of protein by Western blot based on POLY-quantum dot probes were used in micro-protein analyzing, and single walled carbon-nanotubes (SWNTs)-paper sensor were used to detect MC-LR in water.
One of the method is Ultrasensitive detection of protein by Western blot based on POLY-quantum dot probes. With the high affinity of avidin-functionalized POLY-QDs and simplification of the detection process, this enabled the quantitative analysis of protein by Western blotting. To prepare the POLY-QDs, CdTe quantum dots were first coated with biotinylated denatured bovine serum albumin (dBSA), and then via the effect of the biotin-avidin system, the biotinylated dBSA-coated QDs which had strong fluorescence were conjugated. With this series of modifications, the fluorescence intensity of CdTe QDs was significantly increased. Using the POLY-QDs as labels, the signal of Western blotting was more sensitive in tracing the protein than traditional dyeing. In the present study, protein A was applied to POLY-QDs-based Western blotting as a model. The linearity of this method was from 30 pg to 1.5 ng, and the sensitivity was up to 0.84 pictogram. The final fluorescence signal on the PVDF membrane was retained for at least 40 min. The recovery of real samples detection is from 99.9% to 103.0%.
The other is composites obtained impregnation of papers by SWNTs towards very simple and high-performance biosensors. They utilize the strong dependence of electrical conductivity through nanotubes percolation network on the width of nanotubes-nanotube tunneling gap and can potentially satisfy the requirements for the routine toxin monitoring. In this study, we detected MC-LR in water with the SWNTs-paper biosensor. The method has the linear detection range from 1.25 nmol/L to 10 nmol/L. The detection limit was found to be 0.6 nmol/L (0.6 ng/mL), which satisfies the strected World Health Organization standard for MC-LR content in drinking water (1 ng/mL), and is comparable to the detection limit of traditional ELIS A method of MC-LR detection.
一种免疫方法是：基于量子点的超灵敏蛋白检测免疫印迹方法。碲化镉(CdTe)量子点由于它独特的光学及生物特性,被广泛应用于生物化学标记领域。本论文构建了一种基于量子点的超灵敏蛋白检测免疫印迹方法。由于亲和素功能化的聚合量子点的高亲和性及对检测步骤的简化,使得量化免疫印迹方法成为现实。为了制备聚合量子点,首先需要用生物素化的变性牛血清白蛋白(dBSA)包裹量子点,然后通过生物素与亲和素的相互作用,量子点相互偶联生成聚合量子点。经过一系列的修饰,碲化镉量子点的荧光强度显著增强。把聚合量子点作为免疫印迹的标志物,其追踪微量蛋白比传统染料更灵敏。以蛋白A为例,对基于聚合量子点的免疫印迹方法进行验证,检测线性范围为30pg到1.5ng,检测限达0.84pg。最终在聚偏二氟乙烯膜上的荧光信号至少可以保持40min。进行实际样品检测时,回收率达99.9％到103.0％。
另一种方法是纸—单壁碳纳米管复合物组成的,简单且高效的传感器。碳纳米管传感器的电阻取决于碳纳米管网络之间的间隙宽度,该方法能很好的满足常规毒素的检测要求。本论文对微囊藻毒素进行了检测并建立了标准曲线,线性范围为1.25nmol／L到10nmol／L。检测限达0.6nmol／L(0.6ng／mL),满足了世界卫生组织关于饮用水中微囊藻毒素含量不超过1ng／mL的检测要求。同时,论文还对该方法与ELISA检测方法进行了比较。
Quantum dots and carbon nanotube have been widely used in the field of immunoassay because of their particular speciality and bio-compatibility. In the paper, we researched two detection method based on immunosassy. Ultrasensitive detection of protein by Western blot based on POLY-quantum dot probes were used in micro-protein analyzing, and single walled carbon-nanotubes (SWNTs)-paper sensor were used to detect MC-LR in water.
One of the method is Ultrasensitive detection of protein by Western blot based on POLY-quantum dot probes. With the high affinity of avidin-functionalized POLY-QDs and simplification of the detection process, this enabled the quantitative analysis of protein by Western blotting. To prepare the POLY-QDs, CdTe quantum dots were first coated with biotinylated denatured bovine serum albumin (dBSA), and then via the effect of the biotin-avidin system, the biotinylated dBSA-coated QDs which had strong fluorescence were conjugated. With this series of modifications, the fluorescence intensity of CdTe QDs was significantly increased. Using the POLY-QDs as labels, the signal of Western blotting was more sensitive in tracing the protein than traditional dyeing. In the present study, protein A was applied to POLY-QDs-based Western blotting as a model. The linearity of this method was from 30 pg to 1.5 ng, and the sensitivity was up to 0.84 pictogram. The final fluorescence signal on the PVDF membrane was retained for at least 40 min. The recovery of real samples detection is from 99.9% to 103.0%.
The other is composites obtained impregnation of papers by SWNTs towards very simple and high-performance biosensors. They utilize the strong dependence of electrical conductivity through nanotubes percolation network on the width of nanotubes-nanotube tunneling gap and can potentially satisfy the requirements for the routine toxin monitoring. In this study, we detected MC-LR in water with the SWNTs-paper biosensor. The method has the linear detection range from 1.25 nmol/L to 10 nmol/L. The detection limit was found to be 0.6 nmol/L (0.6 ng/mL), which satisfies the strected World Health Organization standard for MC-LR content in drinking water (1 ng/mL), and is comparable to the detection limit of traditional ELIS A method of MC-LR detection.
引文
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3. Meike L. Schipper, Nozomi Nakayama-Ratchford, Corrine R. Davis. A pilot toxicology study of single-walled carbon nanotubes in a small sample of mice. Nature nanotechnology.2008,3(4):216-221.
4. Jan Becker, Inga Zins, Arpad Jakab. Plasmonic Focusing Reduces Ensemble Linewidth of Silver-Coated Gold Nanorods. Nano letters.2008,8(6): 1719-1723.
5. B. Christoffer Lagerholm, Miaomiao Wang. Multicolor Coding of Cells with Cationic Peptide Coated Quantum Dots. Nano letters.2004,4(10):2019-2022.
6. Weihai Ni, Zhi Yang, Huanjun Chen, Li Li, and Jianfang Wang. Coupling between Molecular and Plasmonic Resonances in Freestanding Dye-Gold Nanorod Hybrid Nanostructures. J. Am. Chem. Soc.2008,130 (21):6692-6693.
7. Jianzhong Shen, Fei Xu, Haiyang Jiang. Characterization and application of quantum dotnanocrystal-monoclonal antibody conjugatesfor the determination of sulfamethazine in milk by fluoroimmunoassay. Anal Bioanal Chem.2007. 389(7-8):2243-2250.
8. Cristina Blasco, Carmen Maria Torres, Yolanda Pico. Progress in analysis of residual antibacterials in food. Analytical Chemistry,2007,26(9):895-913.
9. Reinhard Zeleny, Franz Ulberth, Petra Gowik, Joachim, Polzer. Developing new reference materials for effective veterinary drug-residue testing in food-producing animals. Analytical Chemistry,2006,25(9):927-936.
10. A.A.M. Stolker, U.A.Th. Brinkman. Analytical strategies for residue analysis of
veterinary drugs and growth-promoting agents in food-producing animals—a review. Journal of Chromatography A,2005,1067:15-53.
11. Richard L Ornberg, Theresa F Harper, Hongjian Liu. Western blot analysis with quantum dot fluorescence technology:a sensitive and quantitative method for multiplexed proteomics. Nature methods.2004,2(1):79-81.
12. Rumiana Bakalova, Zhivko Zhelev, Hideki Ohba. Quantum Dot-Based Western Blot Technology for Ultrasensitive Detection of Tracer Proteins. J. Am. Chem. Soc.2005,127(26):9328-9329.
13.张淑华,李巧,潘熙萍,徐敬波.微囊藻毒素-LR的研究进展.现代预防医学.2009.17,3236-3239.
14.杨希存,张洪霞.微囊藻毒素毒作用机制研究进展.河北医学.2009.31,2126-2128
15.邓鹏,薛文通,胡鹏,王兆华,杜方岭.微囊藻毒素致毒机理及防治方法研究进展.中国食物与营养.2008.08,52-53
16. Puerto Maria, Pichardo Silvia, Jos Angeles, Camean Ana Maria. Comparison of the toxicity induced by microcystin-RR and microcystin-YR in differentiated and undifferentiated Caco-2 cells. Toxicon.2009.54,161-169.
17. Zegura B, Zajc I, Lah TT, et al. Patterns of microcystin-LR induced alteration of the expression of genes involved in response to DNA damage and apoptosis. Toxicon,2008,51(4):615-623.
18. Teneva I, Mladenov R, Popov N, et al. Cytotoxicity and apoptotic effects of microcystin-LR and anatoxin-a in Mouse lymphocytes. Folia Biologica,2005, 51(3):62-67
19. Zhou L, Yu H, Chen K. Relationship between microcystin in drinking water and colorectal cancer. Biomed Environ Sci,2002,15(2):166-171.
20. Hao, X.L.; Kuang, H.; Li, Y.L.; Yuan Y.;et al. Development of an Enzyme-Linked Immunosorbent Assay for the Alfa-Cyano Pyrethroids Multiresidue in Tai Lake Water. Journal of Agricultural and Food Chemistry, 2009,57(8):3033-3039.
21. Peng C F, Chen Y W, Chen W, Xu C L, Kim J M, Jin Z Y Development of a
sensitive heterologous ELISA method for analysis of acetylgestagen residues in animal fat. Food Chemistry,2008,109(3):647-653.
22. Wei Ma, Wei Chen, Ruirui Qiao. Rapid and sensitive detection of microcystin by immunosensor based on nuclear magnetic resonance. Biosensors and Bioelectronics.2009,25(1):240-243.
23. Bong Sup Shim, Wei Chen, Chris Doty. Smart Electronic Yarns and Wearable Fabrics for Human Biomonitoring made by Carbon Nanotube Coating with Polyelectrolytes. Nano letters.2008.8(12):4151-4157.
24. Haibing Li, and Fengge Qu. Synthesis of CdTe Quantum Dots in Sol-Gel-Derived Composite Silica Spheres Coated with Calixarene as Luminescent Probes for Pesticides. Chem. Mater.2007,19(17):4148-4154.
25. Mingyuan Gao, Stefan Kirstein, and Helmuth Molhwald. Strongly Photoluminescent CdTe Nanocrystals by Proper Surface Modification. J. Phys. Chem. B.1998,102(43):8360-8363.
26. Bruchez M J, Moronne M, Gin P, et al. Semiconductor Nanocrystals as Fluorescent biological Labels. Science,1998,281(5385):2013-2015.
27. Chan W C, Nie S. Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection. Science,1998,281(5385):2016-2018.
28. Irwin Chen, Yoon-Aa Choi, and Alice Y. Ting. Phage Display Evolution of a Peptide Substrate for Yeast Biotin Ligase and Application to Two-Color Quantum Dot Labeling of Cell Surface Proteins. J. Am. Chem. Soc,2007, 129(20):6619-6625.
29. Brian M. Lingerfelt, Hedi Mattoussi, Ellen R. Goldman. Preparation of Quantum Dot-Biotin Conjugates and Their Use in Immunochromatography Assays. Anal. Chem.,2003,75(16):4043-4049.
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