探索新的检测原理和构建新的检测方法是分析化学的重要任务之一。双发射荧光比率 探针可以有效克服单发射荧光探针易受环境因素影响等缺点，以其为基础建立的检测方法 具有稳定性好，重现性高，检测结果可靠性强等优势。本论文制备了不同染料掺杂纳米硅 球，并构建了用于葡萄糖、汞离子以及肝素检测的双发射荧光比率和荧光共振能量转移 （FRET）传感器。主要研究结果如下： 1. 构建了基于FITC掺杂纳米硅球和CdTe QDs的双发射比率探针并应用于葡萄糖的 检测。将荧光素异硫氰酸酯（FITC）包裹于硅球制成发绿色荧光的掺杂纳米硅球（FS）， 为了避免 FITC 的泄露，提高 FITC 的稳定性，在 FS 微球外面再包裹一层硅壳制成双壳纳 米硅球（FSS），然后通过酰胺化反应将红色 CdTe QDs 连接在 FSS 微球表面，制成双发射 比率探针（FSSQ），并将其应用于葡萄糖的定量检测。检测原理为：葡萄糖在葡萄糖氧化 酶（GOD）的作用下，被 O2氧化定量生成葡萄糖酸和过氧化氢（H2O2）；H2O2 在辣根过氧 化酶（HRP）的催化作用下，将多巴胺氧化生成多巴醌。多巴醌是一种缺电子物质，可有 效地淬灭硅球表面红色 CdTe QDs 的荧光，而 FITC 受硅壳的保护荧光强度保持不变。这样， 比率探针 FSSQ 中 FITC 和 CdTe QDs 的荧光比率会随着葡萄糖浓度的变化而变化，由此可 以定量测定葡萄糖的含量。研究结果表明，FITC 和 CdTe QDs 荧光强度比率和葡萄糖在 5~400 μmol•L-1 间呈现良好的线性，葡萄糖的检出限为 1.93 μmol•L-1。该方法具有良好的选 择性和灵敏度，并被用于检测饮料、尿样及血清中葡萄糖的含量，其加标回收率和相对标 准偏差分别为 95.5% ~108.9% 和 1.5% ~4.3%，说明此方法具有较强的实用性和可靠性。 2. 构建了基于RBITC掺杂纳米硅球和核酸染料 SYBR Green I的双发射比率探针并用 于检测 Hg2+。将罗丹明 B 异硫氰酸酯（RBITC）包裹于硅球制成 RBITC 掺杂纳米硅球（RS）， 之后采用酰胺化反应将汞离子适配体（富含 T 碱基的单链 DNA）修饰在 RS 微球表面制成 纳米硅球（RSDNA）。将 RSDNA 纳米硅球和核酸染料 SYBR Green I 相结合形成了可用于 Hg2+检测的双发射比率探针。当体系中存在 Hg2+时，Hg2+会诱导适配体按照 T−Hg2+ −T 形 式结合，使原来的单链 DNA 变成双链 DNA。加入绿色核酸染料 SYBR Green I 后，SYBR Green I 的荧光显著增强，而 RBITC 的荧光受硅壳的保护强度保持不变。这样，SYBR Green I 和 RBITC 的荧光强度比率将随 Hg2+浓度的变化而变化。该方法的线性范围为 5~100 n mol•L-1，线性方程的相关系数 R 为 0.990，最低检出限为 1.4 nmol•L-1。该方法具有良好的 选择性，常见金属离子无干扰。 3. 构建了 SiO2@FITC 掺杂纳米硅球与金纳米颗粒（AuNPs）的 FRET 体系并应用 于肝素的检测。将荧光素异硫氰酸酯（FITC）修饰在纯二氧化硅微球表面制成掺杂纳米硅 球（SiO2@FITC）并将其作为能量供体，金纳米颗粒（AuNPs）作为能量受体构建新型荧 光共振能量转移（FRET）探针用于肝素的检测。检测原理为：当体系中不存在肝素时，鱼 精蛋白会与金纳米颗粒（AuNPs）靠静电作用相互吸引，导致分散的 AuNPs 聚集。聚集后 的 AuNPs 粒径增大，不能与 SiO2@FITC 发生 FRET，SiO2@FITC 荧光强度保持不变；当 体系中存在肝素时，鱼精蛋白会同时结合肝素和 AuNPs，这样会使原先处于聚集态的 Au NPs 恢复分散状态。加入检测探针 SiO2@FITC 后，SiO2@FITC 和 AuNPs 间发生 FRET， 其荧光会被分散的 AuNPs 淬灭，淬灭程度与肝素的量呈一定比例关系，根据此关系可实现 定量检测肝素。实验结果表明，SiO2@FITC 荧光淬灭的程度[(F0-F)/F0]与肝素在 0.02~2.0 g•mL-1 间呈线性关系，检出限为 0.0019 g•mL-1。此外，实验证明肝素浓度的变化会改变 AuNPs 聚集状态，引起检测体系颜色由蓝色逐渐转变成红色。因此，可通过溶液颜色的变 化对肝素进行可视化的定量分析。 综上所述，本论文基于染料掺杂纳米硅球构建的荧光传感器具有特异性好、灵敏度高、 准确度高、方便快捷等优点，适用于复杂基质的快速检测。研究结果具有重要的理论意义 和实际应用价值，为构建新型传感器提供了新的思路和方法。

It is one of the important tasks in analytical chemistry to explore new detection principles and construct new detection methods. Dual emission ratiometric fluorescent probe can effectively overcome the disadvantages of single emission fluorescence probe，which was easily affected by the environmental factors. The ratiometric fluorescent probe has the advantages of good stability, high repeatability, high reliability and others. In this paper, we developed dual emission fluorescence ratiometric sensors and fluorescence resonance energy transfer (FRET) sensor based on different dye−doped silica spheres for detection of glucose, mercury ion and heparin. The main results are as follows: 1. A dual emission ratemetric fluorescent probe based on FITC doped nano silica spheres and CdTe QDs was developed and applied to the detection of glucose. The fluorescein isothiocyanate (FITC) was packaged in silica nanoparticles to prepare doped nano silica spheres with green fluorescence (FS). In order to avoid leakage of FITC and improve the stability of the FITC, FS microspheres was wrapped with another layer of silicon to synthesize double shell silica nanospheres (FSS), and then the red CdTe QDs was connected on the surface of FSS microsphere by amidation reaction to obtain dual emission ratio probe (FSSQ), which was applied to the quantitative detection of glucose. The principle of detection is: after added glucose oxidase (GOD), the glucose was oxidized to generate glucose acid and hydrogen peroxide (H2O2) quantitatively by O2; H2O2 can catalytically oxidize dopamine to dopaquinone by horseradish peroxidase (HRP). Dopaquinone is a kind of deficient–electron molecular, and can effectively quench the fluorescence of the red CdTe QDs on the surface of the silica spheres. However, the fluorescence intensity of FITC protected by silica spheres remained unchanged. In this way, the fluorescence ratio of FITC and CdTe QDs in the ratio probe FSSQ varied with the change of glucose levels, which can be used to determine the content of glucose. The results expressed that the fluorescence ratio of FITC and CdTe QDs showed a good linearity between 5 μmol•L-1 and 400 μmol•L-1 with glucose levels, and the detection limit was 1.93 μmol•L-1 . The method has good selectivity and sensitivity, and can be used to detect the glucose content in beverages, urine and serum, and the recovery rate and relative standard deviation were 95.5%~108.9% and 1.5%~4.3%, respectively, which showed that this method has strong practicality and reliability. 2. A dual emission ratemetric probe based on RBITC doped nano silica spheres and nucleic acid dye SYBR Green I was developed and used for the detection of Hg2+ . Rhodamine B isothiocyanate (RBITC) was wrapped into silica nanoparticles to make RBITC doped silica nanospheres (RS), and then the aptamer of mercury ion (single stranded DNA riching in T base) was modified on the surface of RS microspheres to make silica nano spheres (RSDNA) by amidation reaction. RSDNA nano silica spheres were connected with nucleic acid dye SYBR Green I to develop dual emission ratio probe for the detection of Hg2+ . When Hg2+ was existed in the detection system, aptamer was induced mismatch in the form of T−Hg2+ −T, which caused the single stranded aptamer into double stranded DNA. After addition of nucleic acid dye SYBR Green I, the fluorescence of SYBR Green I enhanced significantly, while the fluorescence of RBITC protected by the silica shell kept unchanged. So, the fluorescence ratio of SYBR Green I and RBITC changed with the variation of Hg2+ concentration. The linear range of the method ranged from 5 nmol•L-1 to 100 nmol•L-1 , the linear correlation coefficient R is 0.990, the lowest detection limit is 1.4 nmol•L-1 . The method has good selectivity without common metal ions interference. 3. The FRET system of SiO2@FITC doped nano silica spheres and gold nanoparticles (AuNPs) was developed and applied to the detection of heparin. Doped silica nanospheres (SiO2@FITC) was used as the energy donor which was prepared by modifying fluorescein isothiocyanate (FITC) on the surface of pure silica microspheres, and gold nanoparticles (AuNPs) was used as an energy acceptor to build a novel fluorescence resonance energy transfer (FRET) probe for heparin detection. The principle of detection is: In the absence of heparin, protamine can combine with gold nanoparticles (AuNPs) by electrostatic interactions, resulting in the scattered AuNPs aggregate to larger size. FRET could not occur between aggregated AuNPs and SiO2@FITC, so the fluorescence intensity of SiO2@FITC remains unchanged. On the contrary, in the presence of heparin, protamine can be combined with AuNPs and heparin, which make the aggregated AuNPs revert to dispersed state. After addition of the detection probe SiO2@FITC, FRET was occurred between SiO2@FITC and AuNPs, and the fluorescence of SiO2@FITC was quenched by dispersed AuNPs. Quenching degree and the heparin content showed a certain proportion relations, which can realize quantitative detection of heparin. The experimental results illustrated that the fluorescence quenching degree of SiO2@FITC and heparin showed a linear relationship between 0.02 g•mL-1 and 2.0 g•mL-1 , and the detection limit can reach 0.0019 g•mL-1 . In addition, experiments showed that changes of heparin levels can change the aggregation state of AuNPs, and cause the color of the detection system change from blue to red gradually. Therefore, the method can realize visualized and quantitative analysis of heparin by the change of solution color. In summary, the fluorescence sensors based on dye–doped nano silica spheres have the advantages of good specificity, high sensitivity, high accuracy and convenience, which is suitable for the rapid detection of complex matrix. The results are valuable in theoretical and practical aspects, which provide new ideas and methods for the development of new sensors.