基于新型固态荧光碳量子点的微生物快速检测技术研究及应用

发布时间：2018-03-02 13:03

  本文选题：碳量子点　切入点：细菌　出处：《中国人民解放军医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的：基于新型纳米荧光材料——固态荧光碳量子点(solid-state fluorescent carbon nanodots,SCDs),结合免疫层析技术和生物成像技术,实现现场条件下快速的微生物特异性检测与总菌监测,为微生物检测／监测提供可供选择的新型技术手段。方法：1.SCDs的合成与光学性能分析：采用微波法一步合成SCDs,对SCDs的宏观和微观结构进行表征,研究其光谱特征、荧光稳定性、耐光漂白性以及溶液pH、有机溶剂、离子等对SCDs荧光量子效率的影响,探讨SCDs作为荧光探针在生物应用中的可行性；2.SCDs在干式快速检测中的应用研究：以SCDs作为荧光标记物,以鼠疫菌的特异性抗体作为生物探针,基于双抗体夹心的免疫层析原理,制备鼠疫菌SCDs免疫层析试纸,并对试纸的检测性能进行系统评价；3.SCDs在液态快速监测中的应用研究：以SCDs作为新型无毒的荧光染料,对以典型细菌为代表的总菌以及HeLa细胞进行快速染色,并对其染色机理进行研究探讨,与商用染料SYT09进行染色性能对比；初步将SCDs染色与流式细胞仪结合,对样品进行总菌计数。结果：1.合成了固态、液态均可以发光的SCDs,平均粒径约2.3nm,分散性好,颗粒表面含有氨基、羟基、羰基等化学基团,量子产率高,在固态和液态分别是37%和28%,λex=360±1nm,λem=458±1nm,发射光谱有激发光依赖性;荧光稳定性强,避光保存30天强度基本不变,耐光漂白；SCDs溶液浓度在6mg/ml时荧光强度最强,pH值3-11,有机试剂10mg/ml PEG20000. 10mg/ml SDS、10mg/ml BSA.1% Triton X-100、1%Tween20以及浓度为0.3molL-1mol/L的Na+、K+、Cl-、PO43-对SCDs的荧光强度均没有影响(P0.05),Zn2+、Cd2+、Mg2+、Ca2+、SO42-、Fe3+、Fe2+、Hg2+、NO3-对SCDs的荧光强度有不同程度的抑制作用(P0.05)。2.在干式快速检测研究中,确定SCDs标记抗体蛋白的条件：SCDs=0.5mg/ml,抗体浓度为50μg/ml；标记缓冲液pH=7.2,0.03mol/L的PB；样品处理液的成分包括50μ1封闭液,3μl 7.5%的干酪素,pH=7.2,37μ1浓度为003mol/L的PB；制备了基于SCDs的鼠疫菌免疫层析试纸,可实现鼠疫菌的特异检测,敏感性可达105CFU/ml。3.在液态快速监测研究中,SCDs可对以典型细菌为代表的总菌1min内进行无差别染色且不受其他生物杂质的干扰；金黄色葡萄球菌、大肠埃希菌、炭疽杆菌芽孢对SCDs的1min渗透率分别是6%、6.7%、5%；核酸对SCDs的荧光强度没有影响(P0.05),SYT09与核酸结合后荧光强度增强(P0.05)。两种染料的染色效果均较好,染色后活菌的荧光强度比死菌的强,染色后浸泡2h、避光保存30天荧光强度均不减弱。结合流式细胞仪,染色后的细菌因为荧光强度高可以与未染色的细菌以及其他杂质相区别,计数结果与未染色的纯菌液呈线性相关(r=0.995,P0.05)。结论：本研究合成的SCDs,具有良好的光学性能,以SCDs作为荧光标记物与荧光染料,经过进一步的优化,新型检测与监测技术可以获得优良的微生物分析性能,从而为新型生物检测技术的发展提供了一种新型的可供选择的荧光纳米探针。
[Abstract]:Objective: the new nano fluorescent material -- solid fluorescent carbon quantum dots (solid-state fluorescent carbon based on nanodots, SCDs), combined with immune chromatography technology and biological imaging technology, realize the field conditions the fast detection of microbial specificity and total bacteria monitoring, provides a new technical means available for microbial detection / monitoring methods: analysis. Synthesis and optical properties of 1.SCDs: using microwave method of one-step synthesis of SCDs, SCDs on the macro and micro structure characterization, study the spectral characteristics of the fluorescence stability, light resistance and pH solution, organic solvent, effect of ions on the fluorescence quantum efficiency of SCDs, SCDs as fluorescent probes in biological application feasibility; application of 2.SCDs in the rapid detection of dry in using SCDs as a fluorescent marker, with specific antibody of Yersinia pestis as biological probes, based on double antibody Sandwich immunochromatographic principle, preparation of Yersinia pestis SCDs immunochromatographic strip, and the detection performance of the test system was evaluated; the research and application of 3.SCDs in rapid monitoring of liquid in using SCDs as a fluorescent dye and nontoxic, fast staining of total bacteria in typical bacteria represented and HeLa cells, and study study on the dyeing mechanism, dyeing performance compared with commercial dye SYT09; initial SCDs staining and flow cytometry with total bacteria counts of samples. Results: 1. the synthesis of solid, liquid can light SCDs, the average particle size of about 2.3nm, good dispersion, the particle surface containing amino and hydroxyl. Carbonyl chemical groups, high quantum yield, in solid and liquid are respectively 37% and 28%, lambda ex=360 + 1nm, em=458 + lambda 1nm, emission spectra of excitation light dependent fluorescence; strong stability, avoid light preservation 30 days strength basically unchanged ,鑰愬厜婕傜櫧锛汼CDs婧舵恫娴撳害鍦，

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