水溶性量子点荧光探针的制备及其分析应用
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摘要
水溶性量子点具有良好的生物应用前景,近年来受到了广泛的关注。与合成油溶性量子点相比,制备水溶性量子点具有试剂价廉、反应条件温和、无需手套箱等特殊反应装置的优点。因此,水相合成量子点已经发展成为量子点合成领域一种重要的方法。为了进一步提高产物质量,简化反应步骤,发展更加便捷的水相量子点合成新方法是目前量子点合成领域的研究热点之一。利用制备的高发光效率水溶性量子点,发展动物重大传染病(猪传染性胸膜肺炎和禽流感)标志物的检测新方法,对控制疾病的传播、减少经济损失、维护我国公共卫生安全具有重要的意思。除此之外,水溶性量子点的化学发光和电化学发光性质研究较少,利用水溶性量子点高灵敏的化学发光和电化学发光性质发展重金属离子和过氧化氢的分析新方法,对控制农业土壤环境污染和水中活性氧的检查具有重要的意义。本论文的主要工作是发展水相量子点的合成新方法；针对猪传染性胸膜肺炎和禽流感两种动物重大传染病,发展更加快速、灵敏的检测新方法；研究水溶性量子点的化学发光和电化学发光性质及其发展重金属离子和过氧化氢的分析新方法。论文主要工作如下：
(1)采用超声辅助法,制备了一种小尺寸、水溶性CdSe量子点；通过紫外-可见吸收光谱、高分辨透射电子显微镜、X射线粉末衍射对合成产物的光学性质、尺寸、形貌和晶体结构进行了表征,探讨了可能的反应机理。这些研究为CdSe量子点的合成提供了一种新的思路,也为其他量子点的合成提供了一种可借鉴的新方法。
(2)以亚碲酸钠为碲源,谷胱甘肽为表面修饰试剂,通过简便的一步合成法在水溶液中制备了高发光效率的、低毒的、生物相容性较好的水溶性CdTe量子点；通过紫外-可见吸收光谱、荧光光谱、X射线粉末衍射、X射线光电子能谱、高分辨透射电子显微镜对合成产物的发光性质、晶体结构、尺寸和形貌就行了表征。在最优化的合成条件下,制备的CdTe量子点最高荧光量子产率达到84%。细胞成像和电化学发光实验表明,产物具有良好的荧光和电化学发光活性。
(3)利用量子点荧光猝灭法,结合纳米金放大效应(溶解纳米金释放大量的金离子),发展了一种高灵敏、高通量检测猪传染性胸膜肺炎的新方法。在最优化的实验条件下,其线性范围为：1：8-1：512标准阳性血清稀释倍数；灵敏度为：1100倍标准阳性血清稀释倍数。与间接血凝实验(128倍标准阳性血清稀释倍数)和酶联免疫吸附法(320倍标准阳性血清稀释倍数)相比,本方法具有更高的灵敏度。对30份临床血清的检测结果表明,本方法与酶联免疫吸附法具有很高的符合率(90.0%)、敏感度(87.5%)和特异性(92.9%)。这为控制该病的传播提供了一种新的、更加灵敏的检测方法。同时,也为其他动物重大疾病标志物的检测提供了一种可以借鉴新思路。
(4)利用免疫层析试纸条作为分离和免疫反应平台,结合纳米金放大效应,构建了一种基于量子点荧光猝灭的高灵敏检测禽流感病毒的新方法。在最优化的条件下,其相对荧光强度与病毒的浓度在2.7×10-11-1.2×10-9 g/mL呈良好的性线关系良好,检出限为9.0×10-11g/mL。与文献报道的方法相比,本方法具有更高的灵敏度。对20份临床血清检测结果表明,本方法和病毒分离法的符合率为90%,敏感性为100%,特异性为88.2%,假阳性为11.8%,假阴性为0,说明该方法具有很强的实际应用价值。
(5)研究了巯基乙酸包裹的CdTe量子点的直接氧化化学发光,发现了尺寸决定的化学发光性质；研究了15种金属离子在量子点-双氧水化学发光体系中的行为及其反应机理；发展了一种基于量子点化学发光分别检测汞、铜、银离子的新方法。其灵敏度分别为3.0×10-8 mol/L(Ag+)、4.0×10-8 mol/L (Cu2+)、6.7×10-8 mol/L (Hg2+)。
(6)研究了巯基包裹的CdTe量子点的直接电化学发光性质,探讨了影响电化学发光强度的各种因素,包括量子点浓度、扫描速率、溶解氧的量,发展了一种基于量子点直接电化学发光检测过氧化氢的新方法。在最优化条件下,其线性范围为：2.0×10-7～1.0×10-5mol/L；灵敏度为：6.0×10-8mol/L。：对浓度为1×10.6 mol/L过氧化氢测定10次,其相对标准偏差是4.82%。与其他伏安生物传感器检测检测过氧化氢的方法相比,本方法具有更高的灵敏度。最后探讨了可能的反应机理。
Due to the potential application in biology, water-soluble quantum dots (QDs) have been attracted much attention in recent years. Compared with the synthesis of TOPO-capped QDs, direct preparation of QDs in aqueous solution has several advantages, such as simple, rapid, efficient and low-cost. Up to date, aqueous-phase synthesis of QDs has been developed an important method. In order to improve the quality of QDs and simplify the reaction process, development of a facile method for the synthesis of QDs is also important in the further. Based on the excellent luminescence properties of QDs, development of QDs-based fluorescence immunoassay for the detection of disease of animals is extremely suitable for detecting, preventing, or controlling animal-borne disease outbreaks.In addition, the chemiluminescence (CL) and electrogenerated chemiluminescence (ECL) properties of QDs were seldom investigated. Therefore, development of QDs-based CL and ECL methods for the analysis of heavy metal ions and hydrogen peroxide with high sensitivity are very important in soil environment protection field and active oxygen detection. The main results were as follows:
(1)A novel method for the preparation of water-soluble and small-particle size CdSe QDs has been proposed under high-intensity ultrasonic irradiation. The as-prepared products have been characterized by Ultraviolet-visible (UV-vis) absorption spectra, X-ray powder diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM).A possible reaction mechanism was also investigated.
(2) A simple, rapid, cost-efficient and convenient method has been developed for synthesis of water-soluble CdTe QDs in ambient atmospheric conditions.Using this method, the preparation of Te precursor and growth of CdTe QDs were achieved with one-step synthesis route. Under the optimal conditions, the as-prepared CdTe QDs possessed a high photoluminescence quantum yield (84%), a narrow size distribution (full width at half maximum=30 ran), small particle size (2.6nm) and low cytotoxicity. The photoluminescence and ECL behaviors of as-prepared CdTe QDs show their potential application in cell imaging and ECL biosensing with high sensitivity.
(3)We detected the antibody of Actinobacillus Pleuropneumoniae (APP) using a QDs-based indirect fluroimmunoassay. Under the optimal conditions, the linear range for the dilution coefficient of the ApxIV antibody (standard positive serum) was in the dilution range of 1:8-1:512.The limits of detection (LOD)for the assay was 1100 times dilution of standard positive serum. Compared with the LOD of ApxⅣ-ELISA (320 times dilution) and IHA(128 times dilution), the proposed method had a higher sensitivity for the determination of ApxⅣantibody of APP.This is the first investigation for measuring the ApxIV antibody via the quenching effect of AuCl4- to QDs, and it showed great potential for numerous applications in immunoassays. We believe that the proposed method is extremely suitable for detecting, preventing, or controlling animal-borne disease outbreaks.
(4) We have developed QDs-AuCl4--based immunochromatographic strip biosensor for the simple, rapid and sensitive detection of avian influenza (AI) virus.Under the optimal conditions, the linear range for the concentration of AI virus was in the range of 2.7×10-9-3.2×10-8 g/mL. The LOD for the assay was 9.0×10-10g/mL. This sensor has the benefits of conventional GITS and high sensitivity of QDs probe, and shows potential application in animal-borne disease. Compared with previously reported methods, the proposed method had high sensitivity for the detection of AI virus. In combination with multiplex GITS,this sensor will allow multiplexed detection of viruses and will make a portable device possible for point-of-care applications.
(5) The behaviors of 15 kinds of metal ions in the thiol-capped CdTe QDS-H2O2 CL reaction were investigated in detail. The results showed that Ag+, Cu2+ and Hg2+ could inhibit CdTe QDs and H2O2 CL reaction. A novel CL method for the selective determination of Ag+, Cu2+ and Hg2+ was developed, based on their inhibition of the reaction of CdTe QDs and H2O2. Under the optimal conditions, good linear relationships were realized between the CL intensity and the.logarithm of concentrations of Ag+,Cu2+ and Hg2+.The linear ranges were from 2.0×10 to 5.0×10-8 mol/L for Ag+, from 5.0×10-6 to 7.0×10-8 mol/L for Cu2+ and from 2.0×10-5 to 1.0×10-7 mol/L for Hg2+, respectively. The LOD (S/N=3) were 3.0×10-8,4.0×10-8 and 6.7×10-8 mol/L for Ag+, Cu2+ and Hg2+, respectively. A possible mechanism for the inhibition of CdTe QDs and H2O2 CL reaction was also discussed.
(6) The ECL from thiol-capped CdTe QDs was investigated.The ECL emission was occurred at-1.1 V and reached a maximum value at-2.4 V when the potential was cycled between 0.0 and-2.5V. The reduced species of CdTe QDs could react with the coreactants to produce the ECL emission. The CdTe QD concentration(6.64×10-7 mol/L)of ECL is lower than that (1.0×10-3 mol/L)of CL. Based on the enhancement of light emission from thiol-capped CdTe QDs by H2O2 in the negative electrode potential, a novel method for the determination of H2O2 was developed. The light intensity was linearly proportional to the concentration of H2O2 between 2.0×10-7 and 1.0×10-5 mol/L with a LOD of 6.0×10-8 mol/L.Compared with most of previous reports, the proposed method has higher sensitivity for the determination of H2O2. In addition, the ECL spectrum of thiol-capped CdTe QDs exhibited a peak at around 620 nm, which was substantially red shifted from the FL spectrum, suggesting the surface states play an important role in this ECL process.
(1)采用超声辅助法,制备了一种小尺寸、水溶性CdSe量子点；通过紫外-可见吸收光谱、高分辨透射电子显微镜、X射线粉末衍射对合成产物的光学性质、尺寸、形貌和晶体结构进行了表征,探讨了可能的反应机理。这些研究为CdSe量子点的合成提供了一种新的思路,也为其他量子点的合成提供了一种可借鉴的新方法。
(2)以亚碲酸钠为碲源,谷胱甘肽为表面修饰试剂,通过简便的一步合成法在水溶液中制备了高发光效率的、低毒的、生物相容性较好的水溶性CdTe量子点；通过紫外-可见吸收光谱、荧光光谱、X射线粉末衍射、X射线光电子能谱、高分辨透射电子显微镜对合成产物的发光性质、晶体结构、尺寸和形貌就行了表征。在最优化的合成条件下,制备的CdTe量子点最高荧光量子产率达到84%。细胞成像和电化学发光实验表明,产物具有良好的荧光和电化学发光活性。
(3)利用量子点荧光猝灭法,结合纳米金放大效应(溶解纳米金释放大量的金离子),发展了一种高灵敏、高通量检测猪传染性胸膜肺炎的新方法。在最优化的实验条件下,其线性范围为：1：8-1：512标准阳性血清稀释倍数；灵敏度为：1100倍标准阳性血清稀释倍数。与间接血凝实验(128倍标准阳性血清稀释倍数)和酶联免疫吸附法(320倍标准阳性血清稀释倍数)相比,本方法具有更高的灵敏度。对30份临床血清的检测结果表明,本方法与酶联免疫吸附法具有很高的符合率(90.0%)、敏感度(87.5%)和特异性(92.9%)。这为控制该病的传播提供了一种新的、更加灵敏的检测方法。同时,也为其他动物重大疾病标志物的检测提供了一种可以借鉴新思路。
(4)利用免疫层析试纸条作为分离和免疫反应平台,结合纳米金放大效应,构建了一种基于量子点荧光猝灭的高灵敏检测禽流感病毒的新方法。在最优化的条件下,其相对荧光强度与病毒的浓度在2.7×10-11-1.2×10-9 g/mL呈良好的性线关系良好,检出限为9.0×10-11g/mL。与文献报道的方法相比,本方法具有更高的灵敏度。对20份临床血清检测结果表明,本方法和病毒分离法的符合率为90%,敏感性为100%,特异性为88.2%,假阳性为11.8%,假阴性为0,说明该方法具有很强的实际应用价值。
(5)研究了巯基乙酸包裹的CdTe量子点的直接氧化化学发光,发现了尺寸决定的化学发光性质；研究了15种金属离子在量子点-双氧水化学发光体系中的行为及其反应机理；发展了一种基于量子点化学发光分别检测汞、铜、银离子的新方法。其灵敏度分别为3.0×10-8 mol/L(Ag+)、4.0×10-8 mol/L (Cu2+)、6.7×10-8 mol/L (Hg2+)。
(6)研究了巯基包裹的CdTe量子点的直接电化学发光性质,探讨了影响电化学发光强度的各种因素,包括量子点浓度、扫描速率、溶解氧的量,发展了一种基于量子点直接电化学发光检测过氧化氢的新方法。在最优化条件下,其线性范围为：2.0×10-7～1.0×10-5mol/L；灵敏度为：6.0×10-8mol/L。：对浓度为1×10.6 mol/L过氧化氢测定10次,其相对标准偏差是4.82%。与其他伏安生物传感器检测检测过氧化氢的方法相比,本方法具有更高的灵敏度。最后探讨了可能的反应机理。
Due to the potential application in biology, water-soluble quantum dots (QDs) have been attracted much attention in recent years. Compared with the synthesis of TOPO-capped QDs, direct preparation of QDs in aqueous solution has several advantages, such as simple, rapid, efficient and low-cost. Up to date, aqueous-phase synthesis of QDs has been developed an important method. In order to improve the quality of QDs and simplify the reaction process, development of a facile method for the synthesis of QDs is also important in the further. Based on the excellent luminescence properties of QDs, development of QDs-based fluorescence immunoassay for the detection of disease of animals is extremely suitable for detecting, preventing, or controlling animal-borne disease outbreaks.In addition, the chemiluminescence (CL) and electrogenerated chemiluminescence (ECL) properties of QDs were seldom investigated. Therefore, development of QDs-based CL and ECL methods for the analysis of heavy metal ions and hydrogen peroxide with high sensitivity are very important in soil environment protection field and active oxygen detection. The main results were as follows:
(1)A novel method for the preparation of water-soluble and small-particle size CdSe QDs has been proposed under high-intensity ultrasonic irradiation. The as-prepared products have been characterized by Ultraviolet-visible (UV-vis) absorption spectra, X-ray powder diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM).A possible reaction mechanism was also investigated.
(2) A simple, rapid, cost-efficient and convenient method has been developed for synthesis of water-soluble CdTe QDs in ambient atmospheric conditions.Using this method, the preparation of Te precursor and growth of CdTe QDs were achieved with one-step synthesis route. Under the optimal conditions, the as-prepared CdTe QDs possessed a high photoluminescence quantum yield (84%), a narrow size distribution (full width at half maximum=30 ran), small particle size (2.6nm) and low cytotoxicity. The photoluminescence and ECL behaviors of as-prepared CdTe QDs show their potential application in cell imaging and ECL biosensing with high sensitivity.
(3)We detected the antibody of Actinobacillus Pleuropneumoniae (APP) using a QDs-based indirect fluroimmunoassay. Under the optimal conditions, the linear range for the dilution coefficient of the ApxIV antibody (standard positive serum) was in the dilution range of 1:8-1:512.The limits of detection (LOD)for the assay was 1100 times dilution of standard positive serum. Compared with the LOD of ApxⅣ-ELISA (320 times dilution) and IHA(128 times dilution), the proposed method had a higher sensitivity for the determination of ApxⅣantibody of APP.This is the first investigation for measuring the ApxIV antibody via the quenching effect of AuCl4- to QDs, and it showed great potential for numerous applications in immunoassays. We believe that the proposed method is extremely suitable for detecting, preventing, or controlling animal-borne disease outbreaks.
(4) We have developed QDs-AuCl4--based immunochromatographic strip biosensor for the simple, rapid and sensitive detection of avian influenza (AI) virus.Under the optimal conditions, the linear range for the concentration of AI virus was in the range of 2.7×10-9-3.2×10-8 g/mL. The LOD for the assay was 9.0×10-10g/mL. This sensor has the benefits of conventional GITS and high sensitivity of QDs probe, and shows potential application in animal-borne disease. Compared with previously reported methods, the proposed method had high sensitivity for the detection of AI virus. In combination with multiplex GITS,this sensor will allow multiplexed detection of viruses and will make a portable device possible for point-of-care applications.
(5) The behaviors of 15 kinds of metal ions in the thiol-capped CdTe QDS-H2O2 CL reaction were investigated in detail. The results showed that Ag+, Cu2+ and Hg2+ could inhibit CdTe QDs and H2O2 CL reaction. A novel CL method for the selective determination of Ag+, Cu2+ and Hg2+ was developed, based on their inhibition of the reaction of CdTe QDs and H2O2. Under the optimal conditions, good linear relationships were realized between the CL intensity and the.logarithm of concentrations of Ag+,Cu2+ and Hg2+.The linear ranges were from 2.0×10 to 5.0×10-8 mol/L for Ag+, from 5.0×10-6 to 7.0×10-8 mol/L for Cu2+ and from 2.0×10-5 to 1.0×10-7 mol/L for Hg2+, respectively. The LOD (S/N=3) were 3.0×10-8,4.0×10-8 and 6.7×10-8 mol/L for Ag+, Cu2+ and Hg2+, respectively. A possible mechanism for the inhibition of CdTe QDs and H2O2 CL reaction was also discussed.
(6) The ECL from thiol-capped CdTe QDs was investigated.The ECL emission was occurred at-1.1 V and reached a maximum value at-2.4 V when the potential was cycled between 0.0 and-2.5V. The reduced species of CdTe QDs could react with the coreactants to produce the ECL emission. The CdTe QD concentration(6.64×10-7 mol/L)of ECL is lower than that (1.0×10-3 mol/L)of CL. Based on the enhancement of light emission from thiol-capped CdTe QDs by H2O2 in the negative electrode potential, a novel method for the determination of H2O2 was developed. The light intensity was linearly proportional to the concentration of H2O2 between 2.0×10-7 and 1.0×10-5 mol/L with a LOD of 6.0×10-8 mol/L.Compared with most of previous reports, the proposed method has higher sensitivity for the determination of H2O2. In addition, the ECL spectrum of thiol-capped CdTe QDs exhibited a peak at around 620 nm, which was substantially red shifted from the FL spectrum, suggesting the surface states play an important role in this ECL process.
引文
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