功能化长寿命发光材料在环境重金属和生物分子检测中的应用研究
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摘要
由于荧光分析法具有良好的灵敏性和选择性等优点在环境监测和生命科学中的应用越来越广泛。在早期的荧光分析法中常用的有机荧光染料存在着激发光谱窄、发射光谱宽且不对称、荧光稳定性差等不足以及荧光探针激发发射区域与生物体系内源荧光重叠,背景干扰大,生物荧光探针灵敏性较低、信号易受环境影响等问题影响到有机荧光染料荧光法的应用。因此,通过合成新型长寿命发光材料,利用时间分辨的方法消除样品中荧光背景和固体基质背景光的干扰具有重要的理论与实际意义。本论文主要进行了一系列具有优异发光性能的长寿命发光染料的设计与合成,并将其应用于环境中重金属离子、生物分子和痕量水的检测。本论文提出的检测方法具有高灵敏性和良好的选择性等优点。
主要完成内容如下：
(1)提出了一种基于长寿命发光量子点(QDs)和金纳米颗粒(GNPs)的时间分辨荧光共振能量转移的水体中Hg2+含量的检测方法。该模型包含两条ssDNA探针、长寿命发光的Mn:CdS/ZnS (QDs)以及GNPs。两条富含T碱基的ssDNA探针用以捕获水体中的Hg2+,其中一条ssDNA(探针1)通过5端巯基修饰上QDs,另一条ssDNA (探针2)通过5端巯基修饰上GNPs。QDs用作能量转移供体,GNPs用作能量转移受体。在Hg2+存在的水溶液中,Hg2+促使T碱基对形成T-Hg2+-T结构而使探针1和探针2发生杂交。此时, QDs与GNPs彼此靠近,发生从QDs到GNPs的荧光共振能量转移,使得QDs的荧光显著降低。在浓度范围为1.0×10-8～1.0×10-9Inol/L时,QDs时间分辨荧光强度与Hg2+浓度呈现良好的线性关系,该方法的检测限为0.49nmol/L(缓冲液中)和0.87nmol/L(自来水中)。该方法在实际水样(自来水、江水和湖水)中的检测结果与原子荧光法一致。此外,该方法具有良好的选择性(第2章)。
(2)发展了一种超灵敏的"turn-on"时间分辨荧光传感器用于Hg2+的检测。该方法主要是基于Hg2+引起的富含T碱基的ssDNA的结构变化。水溶性长寿命发光量子点(Mn:CdS/ZnS)作为荧光染料标记上一条富含T碱基的33个碱基的ssDNA(探针2),金纳米颗粒(GNPs)作为量子点荧光淬灭剂标记上一条10个碱基的ssDNA(探针2)。在没有Hg2+存在的样品溶液中,探针1和探针2能够形成杂交结构,使得Mn:CdS/ZnS荧光显著降低。在有Hg2+存在的样品溶液中,Hg2+能够促使探针2形成发夹结构,从而使GNPs标记的探针1从杂交结构中解离出来。此时量子点荧光信号显著升高。该方法的最低检测限为0.18nmol/L.选择性实验表明,该荧光传感器即使是在高浓度其它金属的离子的干扰下仍然对Hg2+具有良好的选择性。利用该传感器成功的对自来水和湖水样品中的Hg2+进行了检测研究(第3章)。
(3)基于T-Hg2+-T结构和量子点(QDs)与氧化石墨烯(GO)之间的荧光共振能量转移,提出了一种对Hg2+进行检测的时间分辨荧光法。作者设计了两段富含T碱基的ssDNA作为Hg2+的捕获探针,其中一条修饰上Mn:CdS/ZnS QDs。在反应体系中Hg2+的加入能促使两段富含T碱基的ssDNA相互杂交形成稳定的T-Hg2+-T结构,使得QDs能够远离GO表面,此时,与没有加入Hg2+的体系相比,体系的荧光信号明显增强。在检测范围为0.2到10nmol/L时,时间分辨荧光强度与Hg2+浓度呈现良好的线性关系,检测限为0.11nmol/L。这个检测限远远低于美国环境保护署对于饮用水中Hg2+含量的限值。该检测方法即使是在有其它金属离子干扰时也能表现出对Hg2+的特异性。该检测方法已成功地应用于实际水样中Hg2+的检测研究(第4章)。
(4)由于金纳米颗粒(GNPs)能够吸附没有与目标物质结合的核酸适配体,而且能够淬灭铽配合物的荧光,因此作者利用非标记的核酸适配体、铽配合物和GNPs提出了一种灵敏性高的蛋白质检测方法。在有凝血酶蛋白存在时,核酸适配体更倾向于与凝血酶结合形成特异性的G-四分体结构而使其不能被吸附于GNPs表面,此时,加入0.5mol/L盐溶液时,GNPs将发生聚集。在低速条件下离心去除聚集的GNPs后,上清液对铽配合物荧光的淬灭性能降低,因此,铽配合物的荧光强度随着凝血酶浓度的增加而增强。由于核酸适配体对凝血酶的特异性识别能力以及GNPs对于铽配合物荧光的强淬灭性,本章中提出的方法对于凝血酶的检测具有良好的选择性和高的灵敏性。在最优化实验条件下,凝血酶浓度在1.0×10-81.0×10-9mol/L时,铽配合物的时间分辨荧光强度与凝血酶浓度呈现良好的线性关系,检测限为0.14nmol/L,该检测限低于很多比色法传感器和一些荧光传感器。该方法成功地应用于复杂生物样品中的凝血酶检测(第5章)。
(5)基于凝血酶的两段不同的核酸适配体提出了一种对凝血酶进行检测的时间分辨荧光传感体系。凝血酶的15个碱基的核酸适配体作为捕获探针共价固定在玻璃片表面,29个碱基的核酸适配体作为检测探针与荧光物质结合。在本章中,作者使用铕配合物作为荧光标记物。凝血酶的加入能够使得其两段核酸适配体与其形成三明治结构。本章中铕配合物的时间分辨荧光强度与凝血酶浓度呈现良好的线性关系。本传感体系具有较宽的线性范围和较低的检测限,而且具有良好的选择性(第6章)。
(6)利用自制的铕配合物提出了一种对小分子腺苷进行高灵敏性检测的时间分辨荧光传感器。铕配合物的荧光信号可以用时间分辨的方法检测到,通过时间分辨荧光法可以有效地消除非特异性背景信号的干扰。氨基修饰的腺苷的核酸适配体能够与醛基化的玻片进行共价结合。铕配合物标记的ssDNA能够与腺苷的核酸适配体杂交而结合在玻片表面。当加入腺苷时,腺苷的核酸适配体与腺苷结合从而使得核酸适配体与ssDNA的杂交体系转变为核酸适配体与腺苷的体系,从而解离出铕配合物结合的ssDNA,使得玻片表面的时间分辨荧光信号降低。在最优化条件下,时间分辨荧光强度与腺苷浓度在1.0×10-8-1.0×10-7mol/L之间时呈现良好的线性关系,检测限为5.61mol/L。此传感器还具有优异的选择性,能够作为腺苷检测的潜在方法之一(第7章)。
(7)提出了一种对有机溶剂中水含量进行检测的时间分辨荧光传感器。铕配合物(ETC)由本实验室合成并作为荧光水传感器中的荧光指示剂。为了防止染料泄露,ETC与丙烯酰胺、甲基丙烯酸羟乙酯、交联剂和光敏剂一起光共价聚合在硅烷化的玻片表面。随着有机溶剂中水含量的增加,ETC的时间分辨荧光强度逐渐降低。在水含量在0.0-8.0%(v/v)时,ETC的时间分辨荧光强度与水含量呈现良好的线性关系。在乙醇、四氢呋喃和1,4-二氧六环中的检测限分别为0.056%、0.042%和0.033%。该传感器具有重现性好、可逆性好和响应时间短的优点。传感膜的使用寿命超过一个月(第8章)。
The fluorescence assay is used in environmental monitoring and life science more and more widely due to their excellent sensitivity and good selectivity. In the early days of the fluorescence assay, the organic fluorescent dyes used have the disadvantages of narrow excitation spectra, broad and asymmetry emission spectra, and poor photostability. In addition, the fluorescence spectra of the organic fluorescent dyes probes are usually overlapped with the intrinsic fluorescence of biological systems, resulting high background signal, low sensitivity, and vulnerable to environmental impacts. These weaknesses affect the application of the organic fluorescent dyes based fluorescence assays. Hence, it is important to synthesis novel long life-time fluorescent materials, and eliminate the background signal of samples and solid substrate using time-gated methods. This paper mainly synthesis a series of long life-time dyes with excellent luminescent properties, and then use the synthesized dyes for the detection of heavy metal ions in the environment, biomolecules, and trace water in the organic solvents. The detection methods proposed in the paper have the advantages of high sensitivity and good selectivity.
This paper has completed the following work:
(1) The authors herein described a time-gated fluorescence resonance energy transfer sensing strategy employing water-soluble long lifetime fluorescence quantum dots and gold nanoparticles to detect trace Hg2+ions in aqueous solution. The water-soluble long lifetime fluorescence quantum dots and gold nanoparticles were functionalized by two complementary ssDNA except for four deliberately designed T-T mismatches.The quantum dot was acted as the energy transfer donor, and the gold nanoparticle was acted as the energy transfer acceptor. When Hg2+ions were present in the aqueous solution, DNA hybridization will occur due to the formation of T-Hg2+-T complexes. As a result, the quantum dots and gold nanoparticles are brought into close proximity, which made the energy transfer occurred from quantum dots to gold nanoparticles, leading to the fluorescence intensity of quantum dots decreased obviously. The decrement fluorescence intensity is proportional to the concentration of Hg2+ions. Under the optimum conditions, the sensing system exhibits a same liner range from1×10-9mol/L to1×10-8mol/L for Hg2+ions with the detection limits of0.49nmol/L in buffer and0.87nmol/L in tap water samples. This sensor was also used to detect Hg2+ions from samples of tap water, river water and lake water spiked with Hg2+ions, and the results shown good agreement with the found values determined by Atomic Fluorescence Spectrometer. The sensor also exhibits excellent selectivity (chapter2).
(2) An ultrasensitive "turn-on" fluorescent sensor was presented for determination of Hg2+. This method is mainly based on Hg2+-induced conformational change of a thymine-rich single-stranded DNA. The water-soluble long lifetime fluorescence quantum dot (Mn:CdS/ZnS) acted as the fluorophore, which was labeled on a33-mer thymine-rich single-stranded DNA (probe2). The gold nanoparticles (GNPs) functionalized10-mer single-stranded DNA (probe1) is selected as the quencher to quench the fluorescence of Mn:CdS/ZnS. Without Hg2+in the sample solution, probe1and2could form hybrid-structures, resulting in the fluorescence of Mn:CdS/ZnS decreased sharply. When Hg2+is present in the sample solution, Hg2+-mediated base pairs induced the folding of probe1into a hairpin structure, leading to the release of GNPs-tagged probe2from the hybrid-structures. The fluorescence signal is then increased obviously compared with that without Hg2+Meanwhile, a detection limit of0.18nmol/L is estimated based on3a/slope. Selectivity experiments reveal that the fluorescent sensor is specific for Hg2+even with interference by high concentrations of other metal ions. This sensor is successfully applied to determination of Hg2+in tap water and lake water samples (chapter3).
(3) a sensitive time-gated fluorescent method for mercury ions (Hg2+) monitoring is developed based on Hg2+-mediated thymine (T)-Hg2+-T structure and the mechanism of fluorescence resonance energy transfer from Mn-doped CdS/ZnS quantum dots to graphene oxide. The authors employ two T-rich single-stranded DNA (ssDNA) as the capture probes for Hg2+, and one of them is modified with Mn-doped CdS/ZnS quantum dots. The addition of Hg2+makes the two T-rich ssDNA hybrids with each other to form stable T-Hg2+-T coordination chemistry, which makes Mn-doped CdS/ZnS quantum dots far away from the surface of graphene oxide. As a result, the fluorescence signal is increased obviously compared with that without Hg2+The time-gated fluorescence intensities are linear with the concentrations of Hg2+in the range from0.2to10nmol/L with a limit of detection of0.11nmol/L. The detection limit is much lower than the U.S. Environmental Protection Agency limit of the concentration of Hg+for drinking water. The time-gated fluorescent method is specific for Hg2+even with interference by other metal ions. Importantly, the proposed method is applied successfully to the determination of Hg2+in natural water samples (chapter4).
(4) Gold nanoparticles (GNPs) can effectively differentiate the unfolded and folded aptamer, and quench the fluorescence of terbium ternary complexes (Tb-complexes), thus the authors herein report a sensitive strategy for protein detection, using label-free aptamer, Tb-complexes and GNPs. In the presence of thrombin, the aptamer is inclined to form G-quartet, and the folded aptamer cannot adsorb on the surface of GNPs, induced the GNPs aggregation in the presence of0.5mol/L salt. After centrifugation at low speed to remove the aggregated GNPs, the quenching capability of the supernatant for Tb-complexes is decreased. The fluorescence intensity of Tb-complexes is increased with the concentration of thrombin increased. Due to the highly specific recognition ability of the aptamer for thrombin and the strong quenching property of GNPs for Tb-complexes, the proposed protocol has good selectivity and high sensitivity for thrombin. Under the optimum conditions, a linear range from1.0×10-9mol/L to1.0×10-8mol/L is obtained with a detection limit of0.14nmol/L, which is much lower than those commonly used colorimetric sensors and some fluorescent sensors. The proposed sensor has been successfully applied in complicated biological samples for thrombin detection (chapter5).
(5) In the present study, the authors report a novel sensitive method for the detection of thrombin using time-resolved fluorescence sensing platform based on two different thrombin aptamers. The thrombin15-mer aptamer as a capture probe was covalently attached to the surface of glass slide, and the thrombin29-mer aptamer was fluorescently labeled as a detection probe. A bifunctional europium complex was used as the fluorescent label. The introduction of thrombin triggers the two different thrombin aptamers and thrombin to form a sandwich structure. The fluorescence intensity is proportional to the thrombin concentration. The present sensing system could provide both a wide linear dynamic range and a low detection limit. The proposed sensing system also presented satisfactory specificity and selectivity (chapter6).
(6) In this protocol, the authors report a time-resolved fluorescence biosensor based on home-made europium complexes for highly sensitive detection of small molecules using adenosine as a model analyte. The fluorophore that used is europium complexes. Its signal can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. The amino modified aptamer probe, which is designed to specifically recognize adenosine, is combined to the aldehyde-group modified glass slide by covalent bond. Europium complex-labeled a short ssDNA, designed to segment hybridize with aptamer probe is immobilized on the glass slide by hybridization reaction. In the presence of adenosine, the aptamer part is more inclined to bounds with adenosine and triggers structure-switching of the aptamer from aptamer/ssDNA duplex to aptamer/target complex. As a result, europium complexes-labeled ssDNA is forced to dissociate from the sensor interface, resulting in time-resolved fluorescence intensity decrease. The decrement intensity is proportional to the amount of adenosine. Under optimized assay conditions, a linear range (1.0×10-8mol/L to1.0×10-7mol/L) is got with low detection limit of5.61nmol/L. The biosensor exhibits excellent selectivity and can provide a promising potential for aptamer-based adenosine detection (chapter7).
(7) A time-gated fluorescence sensor for water content determination in organic solvents was proposed in this paper. A europium ternary complex (ETC) was synthesized and used as the fluorescence indicator in the fabrication of the fluorescence water sensor. To prevent leakage of the fluorophore, ETC was photo-copolymerized with acrylamide,(2-hydroxyethyl)methacrylate,2-hydroxy-2-methyl-1-phenyl-1-propanone, and triethylene glycol dimethacrylate on a glass surface treated with a silanizing agent. The time-gated fluorescence intensity of ETC decreased with increasing of water content in organic solvents. In the range of0.0%～8.0%(v/v), the time-gated fluorescence intensity of ETC changed as a linear function of water content. The detection limits were0.056%,0.042%, and0.033%for ethanol, tetrahydrofuran, and1,4-dioxane, respectively. The sensor exhibited satisfactory reproducibility, reversibility, and a short response time. The sensing membrane was found to have a lifetime of at least one mouth (chapter8).
主要完成内容如下：
(1)提出了一种基于长寿命发光量子点(QDs)和金纳米颗粒(GNPs)的时间分辨荧光共振能量转移的水体中Hg2+含量的检测方法。该模型包含两条ssDNA探针、长寿命发光的Mn:CdS/ZnS (QDs)以及GNPs。两条富含T碱基的ssDNA探针用以捕获水体中的Hg2+,其中一条ssDNA(探针1)通过5端巯基修饰上QDs,另一条ssDNA (探针2)通过5端巯基修饰上GNPs。QDs用作能量转移供体,GNPs用作能量转移受体。在Hg2+存在的水溶液中,Hg2+促使T碱基对形成T-Hg2+-T结构而使探针1和探针2发生杂交。此时, QDs与GNPs彼此靠近,发生从QDs到GNPs的荧光共振能量转移,使得QDs的荧光显著降低。在浓度范围为1.0×10-8～1.0×10-9Inol/L时,QDs时间分辨荧光强度与Hg2+浓度呈现良好的线性关系,该方法的检测限为0.49nmol/L(缓冲液中)和0.87nmol/L(自来水中)。该方法在实际水样(自来水、江水和湖水)中的检测结果与原子荧光法一致。此外,该方法具有良好的选择性(第2章)。
(2)发展了一种超灵敏的"turn-on"时间分辨荧光传感器用于Hg2+的检测。该方法主要是基于Hg2+引起的富含T碱基的ssDNA的结构变化。水溶性长寿命发光量子点(Mn:CdS/ZnS)作为荧光染料标记上一条富含T碱基的33个碱基的ssDNA(探针2),金纳米颗粒(GNPs)作为量子点荧光淬灭剂标记上一条10个碱基的ssDNA(探针2)。在没有Hg2+存在的样品溶液中,探针1和探针2能够形成杂交结构,使得Mn:CdS/ZnS荧光显著降低。在有Hg2+存在的样品溶液中,Hg2+能够促使探针2形成发夹结构,从而使GNPs标记的探针1从杂交结构中解离出来。此时量子点荧光信号显著升高。该方法的最低检测限为0.18nmol/L.选择性实验表明,该荧光传感器即使是在高浓度其它金属的离子的干扰下仍然对Hg2+具有良好的选择性。利用该传感器成功的对自来水和湖水样品中的Hg2+进行了检测研究(第3章)。
(3)基于T-Hg2+-T结构和量子点(QDs)与氧化石墨烯(GO)之间的荧光共振能量转移,提出了一种对Hg2+进行检测的时间分辨荧光法。作者设计了两段富含T碱基的ssDNA作为Hg2+的捕获探针,其中一条修饰上Mn:CdS/ZnS QDs。在反应体系中Hg2+的加入能促使两段富含T碱基的ssDNA相互杂交形成稳定的T-Hg2+-T结构,使得QDs能够远离GO表面,此时,与没有加入Hg2+的体系相比,体系的荧光信号明显增强。在检测范围为0.2到10nmol/L时,时间分辨荧光强度与Hg2+浓度呈现良好的线性关系,检测限为0.11nmol/L。这个检测限远远低于美国环境保护署对于饮用水中Hg2+含量的限值。该检测方法即使是在有其它金属离子干扰时也能表现出对Hg2+的特异性。该检测方法已成功地应用于实际水样中Hg2+的检测研究(第4章)。
(4)由于金纳米颗粒(GNPs)能够吸附没有与目标物质结合的核酸适配体,而且能够淬灭铽配合物的荧光,因此作者利用非标记的核酸适配体、铽配合物和GNPs提出了一种灵敏性高的蛋白质检测方法。在有凝血酶蛋白存在时,核酸适配体更倾向于与凝血酶结合形成特异性的G-四分体结构而使其不能被吸附于GNPs表面,此时,加入0.5mol/L盐溶液时,GNPs将发生聚集。在低速条件下离心去除聚集的GNPs后,上清液对铽配合物荧光的淬灭性能降低,因此,铽配合物的荧光强度随着凝血酶浓度的增加而增强。由于核酸适配体对凝血酶的特异性识别能力以及GNPs对于铽配合物荧光的强淬灭性,本章中提出的方法对于凝血酶的检测具有良好的选择性和高的灵敏性。在最优化实验条件下,凝血酶浓度在1.0×10-81.0×10-9mol/L时,铽配合物的时间分辨荧光强度与凝血酶浓度呈现良好的线性关系,检测限为0.14nmol/L,该检测限低于很多比色法传感器和一些荧光传感器。该方法成功地应用于复杂生物样品中的凝血酶检测(第5章)。
(5)基于凝血酶的两段不同的核酸适配体提出了一种对凝血酶进行检测的时间分辨荧光传感体系。凝血酶的15个碱基的核酸适配体作为捕获探针共价固定在玻璃片表面,29个碱基的核酸适配体作为检测探针与荧光物质结合。在本章中,作者使用铕配合物作为荧光标记物。凝血酶的加入能够使得其两段核酸适配体与其形成三明治结构。本章中铕配合物的时间分辨荧光强度与凝血酶浓度呈现良好的线性关系。本传感体系具有较宽的线性范围和较低的检测限,而且具有良好的选择性(第6章)。
(6)利用自制的铕配合物提出了一种对小分子腺苷进行高灵敏性检测的时间分辨荧光传感器。铕配合物的荧光信号可以用时间分辨的方法检测到,通过时间分辨荧光法可以有效地消除非特异性背景信号的干扰。氨基修饰的腺苷的核酸适配体能够与醛基化的玻片进行共价结合。铕配合物标记的ssDNA能够与腺苷的核酸适配体杂交而结合在玻片表面。当加入腺苷时,腺苷的核酸适配体与腺苷结合从而使得核酸适配体与ssDNA的杂交体系转变为核酸适配体与腺苷的体系,从而解离出铕配合物结合的ssDNA,使得玻片表面的时间分辨荧光信号降低。在最优化条件下,时间分辨荧光强度与腺苷浓度在1.0×10-8-1.0×10-7mol/L之间时呈现良好的线性关系,检测限为5.61mol/L。此传感器还具有优异的选择性,能够作为腺苷检测的潜在方法之一(第7章)。
(7)提出了一种对有机溶剂中水含量进行检测的时间分辨荧光传感器。铕配合物(ETC)由本实验室合成并作为荧光水传感器中的荧光指示剂。为了防止染料泄露,ETC与丙烯酰胺、甲基丙烯酸羟乙酯、交联剂和光敏剂一起光共价聚合在硅烷化的玻片表面。随着有机溶剂中水含量的增加,ETC的时间分辨荧光强度逐渐降低。在水含量在0.0-8.0%(v/v)时,ETC的时间分辨荧光强度与水含量呈现良好的线性关系。在乙醇、四氢呋喃和1,4-二氧六环中的检测限分别为0.056%、0.042%和0.033%。该传感器具有重现性好、可逆性好和响应时间短的优点。传感膜的使用寿命超过一个月(第8章)。
The fluorescence assay is used in environmental monitoring and life science more and more widely due to their excellent sensitivity and good selectivity. In the early days of the fluorescence assay, the organic fluorescent dyes used have the disadvantages of narrow excitation spectra, broad and asymmetry emission spectra, and poor photostability. In addition, the fluorescence spectra of the organic fluorescent dyes probes are usually overlapped with the intrinsic fluorescence of biological systems, resulting high background signal, low sensitivity, and vulnerable to environmental impacts. These weaknesses affect the application of the organic fluorescent dyes based fluorescence assays. Hence, it is important to synthesis novel long life-time fluorescent materials, and eliminate the background signal of samples and solid substrate using time-gated methods. This paper mainly synthesis a series of long life-time dyes with excellent luminescent properties, and then use the synthesized dyes for the detection of heavy metal ions in the environment, biomolecules, and trace water in the organic solvents. The detection methods proposed in the paper have the advantages of high sensitivity and good selectivity.
This paper has completed the following work:
(1) The authors herein described a time-gated fluorescence resonance energy transfer sensing strategy employing water-soluble long lifetime fluorescence quantum dots and gold nanoparticles to detect trace Hg2+ions in aqueous solution. The water-soluble long lifetime fluorescence quantum dots and gold nanoparticles were functionalized by two complementary ssDNA except for four deliberately designed T-T mismatches.The quantum dot was acted as the energy transfer donor, and the gold nanoparticle was acted as the energy transfer acceptor. When Hg2+ions were present in the aqueous solution, DNA hybridization will occur due to the formation of T-Hg2+-T complexes. As a result, the quantum dots and gold nanoparticles are brought into close proximity, which made the energy transfer occurred from quantum dots to gold nanoparticles, leading to the fluorescence intensity of quantum dots decreased obviously. The decrement fluorescence intensity is proportional to the concentration of Hg2+ions. Under the optimum conditions, the sensing system exhibits a same liner range from1×10-9mol/L to1×10-8mol/L for Hg2+ions with the detection limits of0.49nmol/L in buffer and0.87nmol/L in tap water samples. This sensor was also used to detect Hg2+ions from samples of tap water, river water and lake water spiked with Hg2+ions, and the results shown good agreement with the found values determined by Atomic Fluorescence Spectrometer. The sensor also exhibits excellent selectivity (chapter2).
(2) An ultrasensitive "turn-on" fluorescent sensor was presented for determination of Hg2+. This method is mainly based on Hg2+-induced conformational change of a thymine-rich single-stranded DNA. The water-soluble long lifetime fluorescence quantum dot (Mn:CdS/ZnS) acted as the fluorophore, which was labeled on a33-mer thymine-rich single-stranded DNA (probe2). The gold nanoparticles (GNPs) functionalized10-mer single-stranded DNA (probe1) is selected as the quencher to quench the fluorescence of Mn:CdS/ZnS. Without Hg2+in the sample solution, probe1and2could form hybrid-structures, resulting in the fluorescence of Mn:CdS/ZnS decreased sharply. When Hg2+is present in the sample solution, Hg2+-mediated base pairs induced the folding of probe1into a hairpin structure, leading to the release of GNPs-tagged probe2from the hybrid-structures. The fluorescence signal is then increased obviously compared with that without Hg2+Meanwhile, a detection limit of0.18nmol/L is estimated based on3a/slope. Selectivity experiments reveal that the fluorescent sensor is specific for Hg2+even with interference by high concentrations of other metal ions. This sensor is successfully applied to determination of Hg2+in tap water and lake water samples (chapter3).
(3) a sensitive time-gated fluorescent method for mercury ions (Hg2+) monitoring is developed based on Hg2+-mediated thymine (T)-Hg2+-T structure and the mechanism of fluorescence resonance energy transfer from Mn-doped CdS/ZnS quantum dots to graphene oxide. The authors employ two T-rich single-stranded DNA (ssDNA) as the capture probes for Hg2+, and one of them is modified with Mn-doped CdS/ZnS quantum dots. The addition of Hg2+makes the two T-rich ssDNA hybrids with each other to form stable T-Hg2+-T coordination chemistry, which makes Mn-doped CdS/ZnS quantum dots far away from the surface of graphene oxide. As a result, the fluorescence signal is increased obviously compared with that without Hg2+The time-gated fluorescence intensities are linear with the concentrations of Hg2+in the range from0.2to10nmol/L with a limit of detection of0.11nmol/L. The detection limit is much lower than the U.S. Environmental Protection Agency limit of the concentration of Hg+for drinking water. The time-gated fluorescent method is specific for Hg2+even with interference by other metal ions. Importantly, the proposed method is applied successfully to the determination of Hg2+in natural water samples (chapter4).
(4) Gold nanoparticles (GNPs) can effectively differentiate the unfolded and folded aptamer, and quench the fluorescence of terbium ternary complexes (Tb-complexes), thus the authors herein report a sensitive strategy for protein detection, using label-free aptamer, Tb-complexes and GNPs. In the presence of thrombin, the aptamer is inclined to form G-quartet, and the folded aptamer cannot adsorb on the surface of GNPs, induced the GNPs aggregation in the presence of0.5mol/L salt. After centrifugation at low speed to remove the aggregated GNPs, the quenching capability of the supernatant for Tb-complexes is decreased. The fluorescence intensity of Tb-complexes is increased with the concentration of thrombin increased. Due to the highly specific recognition ability of the aptamer for thrombin and the strong quenching property of GNPs for Tb-complexes, the proposed protocol has good selectivity and high sensitivity for thrombin. Under the optimum conditions, a linear range from1.0×10-9mol/L to1.0×10-8mol/L is obtained with a detection limit of0.14nmol/L, which is much lower than those commonly used colorimetric sensors and some fluorescent sensors. The proposed sensor has been successfully applied in complicated biological samples for thrombin detection (chapter5).
(5) In the present study, the authors report a novel sensitive method for the detection of thrombin using time-resolved fluorescence sensing platform based on two different thrombin aptamers. The thrombin15-mer aptamer as a capture probe was covalently attached to the surface of glass slide, and the thrombin29-mer aptamer was fluorescently labeled as a detection probe. A bifunctional europium complex was used as the fluorescent label. The introduction of thrombin triggers the two different thrombin aptamers and thrombin to form a sandwich structure. The fluorescence intensity is proportional to the thrombin concentration. The present sensing system could provide both a wide linear dynamic range and a low detection limit. The proposed sensing system also presented satisfactory specificity and selectivity (chapter6).
(6) In this protocol, the authors report a time-resolved fluorescence biosensor based on home-made europium complexes for highly sensitive detection of small molecules using adenosine as a model analyte. The fluorophore that used is europium complexes. Its signal can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. The amino modified aptamer probe, which is designed to specifically recognize adenosine, is combined to the aldehyde-group modified glass slide by covalent bond. Europium complex-labeled a short ssDNA, designed to segment hybridize with aptamer probe is immobilized on the glass slide by hybridization reaction. In the presence of adenosine, the aptamer part is more inclined to bounds with adenosine and triggers structure-switching of the aptamer from aptamer/ssDNA duplex to aptamer/target complex. As a result, europium complexes-labeled ssDNA is forced to dissociate from the sensor interface, resulting in time-resolved fluorescence intensity decrease. The decrement intensity is proportional to the amount of adenosine. Under optimized assay conditions, a linear range (1.0×10-8mol/L to1.0×10-7mol/L) is got with low detection limit of5.61nmol/L. The biosensor exhibits excellent selectivity and can provide a promising potential for aptamer-based adenosine detection (chapter7).
(7) A time-gated fluorescence sensor for water content determination in organic solvents was proposed in this paper. A europium ternary complex (ETC) was synthesized and used as the fluorescence indicator in the fabrication of the fluorescence water sensor. To prevent leakage of the fluorophore, ETC was photo-copolymerized with acrylamide,(2-hydroxyethyl)methacrylate,2-hydroxy-2-methyl-1-phenyl-1-propanone, and triethylene glycol dimethacrylate on a glass surface treated with a silanizing agent. The time-gated fluorescence intensity of ETC decreased with increasing of water content in organic solvents. In the range of0.0%～8.0%(v/v), the time-gated fluorescence intensity of ETC changed as a linear function of water content. The detection limits were0.056%,0.042%, and0.033%for ethanol, tetrahydrofuran, and1,4-dioxane, respectively. The sensor exhibited satisfactory reproducibility, reversibility, and a short response time. The sensing membrane was found to have a lifetime of at least one mouth (chapter8).
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