本文選題：碳量子點　切入點：細(xì)菌　出處：《中國人民解放軍醫(yī)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：基于新型納米熒光材料——固態(tài)熒光碳量子點(solid-state fluorescent carbon nanodots,SCDs),結(jié)合免疫層析技術(shù)和生物成像技術(shù),實現(xiàn)現(xiàn)場條件下快速的微生物特異性檢測與總菌監(jiān)測,為微生物檢測／監(jiān)測提供可供選擇的新型技術(shù)手段。方法：1.SCDs的合成與光學(xué)性能分析：采用微波法一步合成SCDs,對SCDs的宏觀和微觀結(jié)構(gòu)進(jìn)行表征,研究其光譜特征、熒光穩(wěn)定性、耐光漂白性以及溶液pH、有機(jī)溶劑、離子等對SCDs熒光量子效率的影響,探討SCDs作為熒光探針在生物應(yīng)用中的可行性；2.SCDs在干式快速檢測中的應(yīng)用研究：以SCDs作為熒光標(biāo)記物,以鼠疫菌的特異性抗體作為生物探針,基于雙抗體夾心的免疫層析原理,制備鼠疫菌SCDs免疫層析試紙,并對試紙的檢測性能進(jìn)行系統(tǒng)評價；3.SCDs在液態(tài)快速監(jiān)測中的應(yīng)用研究：以SCDs作為新型無毒的熒光染料,對以典型細(xì)菌為代表的總菌以及HeLa細(xì)胞進(jìn)行快速染色,并對其染色機(jī)理進(jìn)行研究探討,與商用染料SYT09進(jìn)行染色性能對比；初步將SCDs染色與流式細(xì)胞儀結(jié)合,對樣品進(jìn)行總菌計數(shù)。結(jié)果：1.合成了固態(tài)、液態(tài)均可以發(fā)光的SCDs,平均粒徑約2.3nm,分散性好,顆粒表面含有氨基、羥基、羰基等化學(xué)基團(tuán),量子產(chǎn)率高,在固態(tài)和液態(tài)分別是37%和28%,λex=360±1nm,λem=458±1nm,發(fā)射光譜有激發(fā)光依賴性;熒光穩(wěn)定性強(qiáng),避光保存30天強(qiáng)度基本不變,耐光漂白；SCDs溶液濃度在6mg/ml時熒光強(qiáng)度最強(qiáng),pH值3-11,有機(jī)試劑10mg/ml PEG20000. 10mg/ml SDS、10mg/ml BSA.1% Triton X-100、1%Tween20以及濃度為0.3molL-1mol/L的Na+、K+、Cl-、PO43-對SCDs的熒光強(qiáng)度均沒有影響(P0.05),Zn2+、Cd2+、Mg2+、Ca2+、SO42-、Fe3+、Fe2+、Hg2+、NO3-對SCDs的熒光強(qiáng)度有不同程度的抑制作用(P0.05)。2.在干式快速檢測研究中,確定SCDs標(biāo)記抗體蛋白的條件：SCDs=0.5mg/ml,抗體濃度為50μg/ml；標(biāo)記緩沖液pH=7.2,0.03mol/L的PB；樣品處理液的成分包括50μ1封閉液,3μl 7.5%的干酪素,pH=7.2,37μ1濃度為003mol/L的PB；制備了基于SCDs的鼠疫菌免疫層析試紙,可實現(xiàn)鼠疫菌的特異檢測,敏感性可達(dá)105CFU/ml。3.在液態(tài)快速監(jiān)測研究中,SCDs可對以典型細(xì)菌為代表的總菌1min內(nèi)進(jìn)行無差別染色且不受其他生物雜質(zhì)的干擾；金黃色葡萄球菌、大腸埃希菌、炭疽桿菌芽孢對SCDs的1min滲透率分別是6%、6.7%、5%；核酸對SCDs的熒光強(qiáng)度沒有影響(P0.05),SYT09與核酸結(jié)合后熒光強(qiáng)度增強(qiáng)(P0.05)。兩種染料的染色效果均較好,染色后活菌的熒光強(qiáng)度比死菌的強(qiáng),染色后浸泡2h、避光保存30天熒光強(qiáng)度均不減弱。結(jié)合流式細(xì)胞儀,染色后的細(xì)菌因為熒光強(qiáng)度高可以與未染色的細(xì)菌以及其他雜質(zhì)相區(qū)別,計數(shù)結(jié)果與未染色的純菌液呈線性相關(guān)(r=0.995,P0.05)。結(jié)論：本研究合成的SCDs,具有良好的光學(xué)性能,以SCDs作為熒光標(biāo)記物與熒光染料,經(jīng)過進(jìn)一步的優(yōu)化,新型檢測與監(jiān)測技術(shù)可以獲得優(yōu)良的微生物分析性能,從而為新型生物檢測技術(shù)的發(fā)展提供了一種新型的可供選擇的熒光納米探針。
[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 ,鑰愬厜婕傜櫧錛汼CDs婧舵恫嫻撳害鍦，

本文編號：1556633