【作者】 朱浩；

【导师】 吴兴才； 朱俊杰；

【作者基本信息】 南京大学 ， 化学， 2015， 硕士

【摘要】 上转换纳米材料(UCNPs)主要是由氧化物、氟化物、卤氧化物等基质通过掺杂三价稀土离子(如Er3+,Eu3+,Yb3+,Tm3+,Ho3+等)得到,经上转换发光过程可将连续吸收的两个或多个泵浦光子转化为比泵浦光波长更短的光,即将低能光转化为高能光。与传统下转换荧光纳米材料相比,UCNPs具有毒性小、化学性质稳定、无光闪烁、不易降解和光致漂白、背景荧光低、光穿透深度深等优点,因此UCNPs在生物传感和成像领域已表现出越来越重要的作用。在本论文工作中,我们基于荧光共振能量转移(FRET)原理利用UCNPs构建了三种简单方便的生物传感用于溶菌酶、胆固醇以及特定DNA序列的检测,其主要内容如下:1.一种简单的基于上转换荧光共振能量转移的生物传感的构建我们利用上转换纳米材料构建了一种FRET传感用于溶菌酶和目标DNA的测定,该传感中上转换纳米粒子和染料分子标记的核酸适配体(aptamer)分别作为能量的供体和受体。我们首先合成了 NaYF4:Yb,Er,并通过阳离子聚合物实现UCNPs和aptamer的连接,以此来猝灭UCNPs的荧光。由于溶菌酶与aptamer之间的亲和作用或互补DNA之间强烈的相互作用,当存在溶菌酶或互补DNA时,上转换材料的荧光得以恢复,且相对荧光强度与溶菌酶或互补DNA的浓度分别在30-210 nM范围以及40-200 nM范围内线性相关,其最低检测限分别为2.5 nM和2.8 nM。我们还利用该方法对成人唾液和血清样中的溶菌酶含量进行了测定,且检测结果与文献报道值相吻合。由于UCNPs是在红外光激发下发光,消除了来自生物样品背景荧光的干扰。该方法避免了 UCNPs复杂的改性步骤,显得简单而方便,对生物分子的测定有一定的参考价值。2.一种基于上转换荧光共振能量转移的成人血清中胆固醇传感的构建上转换纳米材料(UCNPs)由于其具有高信噪比、几乎无光漂白等性质,已经在分析应用中发挥着极其重要的作用。本章我们分别利用β-环糊精(β-CD)衍生物改性的UCNPs和罗丹明B(RB)作为能量的供体和受体构建了一种新型FRET生物传感用于成人血清中胆固醇的检测。该方法对血清中胆固醇检测的选择好,灵敏度高,有望用于临床上胆固醇以及其他生物分子的检测。3.基于上转换纳米材料与金纳米颗粒的荧光共振能量转移对Hepatitis B virus(HBV)DNA的检测本章我们分别利用PEI修饰的上转换纳米材料(NH2-UCNPs)和金纳米粒子(Au NPs)作为能量的供体和受体,构建了 FRET传感用于乙肝病毒(HBV)的一段DNA序列的测定。首先将两条部分互补的短链DNA分别与能量供体(NH2-UCNPs)和受体(Au NPs)相连,在将两种材料混合时,FRET得以发生,NH2-UCNPs的上转换荧光被猝灭。而当加入目标DNA后,由于更稳定双链DNA结构的形成使得Au NPs远离NH2-UCNPs表面,NH2-UCNPs的上转换荧光得以恢复,且相对荧光强度与目标DNA在5.0-50 nM范围内呈线性相关,其最低检测限为1.0 nM。特异性实验结果表明该方法对目标DNA的选择性较好,由于NH2-UCNPs是在红外光激发下发光,消除了来自生物样品背景荧光的干扰,大大提高了方法的检测灵敏度。同时我们对NH2-UCNPS的合成条件进行了讨论和优化,利用水热法合成直接合成了-NH2功能化的UCNPs,方法简单、操作方便。本章提供了一种新的特异性DNA的检测方法,以期为HBV及其他生物分子的检测提供一定的借鉴作用。更多还原

【Abstract】 Upconversion nanoparticles(UCNPs)are usually composed of the oxides,fluorides,halogen oxides or other substrates as matrix and doped with trivalent rare earth ions(Er3+,Eu3+,Yb3+,Tm3+,Ho3+ etc.)as energy transfer system.UCNPs can convert low-energy light(NIR or IR)to higher-energy light(UV or visible)through multiple photon absorptions or energy transfers.Compared with the traditional downconversion fluorescent materials,UCNPs attract much more focus due to their unique optical and chemical properties,such as sharp absorption and emission bands,high quantum yields,long lifetimes,low toxicity,superior photostability,low autofluorescence from biosamples and better penetration depth.Therefore,UCNPs can be used as suitable candidates in biosensing and bioimaging.Based on fluorescence resonance energy transfer(FRET)technique,we designed and constructed two new types of sensors for the detection of lysozyme and sequence-specific DNA.The major contents were described as follows.1.A Simple Strategy Based on Upconversion Nanoparticles for a Fluorescent Resonant Energy Transfer BiosensorA novel aptasensor was fabricated for the detection of lysozyme and DNA based on fluorescence resonance energy transfer(FRET)technique between upconversion nanoparticles(UCNPs)and dye labeled aptamer.NaYF4:Yb,Er nanoparticles as UCNPs were synthesized and attached with dye labeled aptamer through a cationic polymer as an electrostatic linker to quench the upconversion fluorescent intensity.The intensity can be restored after the addition of lysozyme or the complementary DNA(target DNA)because of their strong interaction with the aptamer.The sensor provided a linear concentration range from 30 to 210 nM for lysozyme and 40 to 200 nM for the target DNA,the limits of the detection were 2.5 nM and 2.8 nM,respectively.The sensor was also used to monitor lysozyme level in both human saliva and serum samples,and the results were consistent with the reported values.UCNPs can act as excellent emitters due to their low autofluorescence and high penetration depth of biosamples.The method was simple and convenient without the extra procedure of bioconjugations,which was desired to put in use for various targets determination in the future.2.An upconversion nanocomposite for fluorescence resonance energy transfer based cholesterol-sensing in human serumUCNPs are extremely useful for analytical applications,since they display a high signal-to-noise ratio,and their photobleaching can be ignored.Herein,a novel upconversion nanocomposite composed of β-cyclodextrin(β-CD)derivative modified UCNPs and rhodamine B(RB)was prepared for the detection of cholesterol(Cho).The upconversion luminescence emission can serve as a Cho-sensing signal by an effective fluorescence resonance energy transfer(FRET)process,using UCNPs as the donor and RB as the quencher.The sensor for Cho detection in human serum shows excellent sensitivity and selectivity,which has the potential for clinical applications in the analysis of other biological and environmental samples.3.An Upconversion Fluorescent Resonant Energy Transfer Biosensor for Hepatitis B virus(HBV)DNA Hybridization DetectionWe fabricated a novel fluorescent resonant energy transfer(FRET)biosensor for the detection of hepatitis B virus(HBV)DNA using poly(ethylenimine)(PEI)modified upconversion nanoparticles(NH2-UCNPs)as donor and gold nanoparticles(Au NPs)as acceptor,respectively.Two single-stranded DNA,which are partially complementary to each other,were first binded with NH2-UCNPs and Au NPs,respectively.The FRET process occured when NH2-UCNPs and Au NPs were mixed and the upconversion luminescence was quenched.However,upon the addition of the target DNA,Au NPs were far away from NH2-UCNPs and the luminescence could be restored because of the formation of the more stable double-stranded DNA.The sensor provided a linear concentration range from 5.0 to 50 nM for the target DNA and the limit(LOD)of the detection was 1.0 nM.Meanwhile,we optimized and discussed the preparation conditions of NH2-UCNPs,to obtain an optional condition for the preparation.The amino-group functionalized upconversion nanoparticles were directly prepared by the hydrothermal method,which was simple and convenient.The sensor we designed for the target DNA detection shows high sensitivity and good selectivity,which has the potential for clinical applications in the analysis of HBV and other biomolecules.更多还原