基于碳点的荧光传感器的制备与应用
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摘要
生物体内的一些微量物质，包括一些阳离子、阴离子和中性分子，在生物活动的调节过程中起着非常重要的作用。近年来，对于这些物质的检测引起了广泛关注。在各种检测技术中，荧光检测法不仅具有操作简单的特点，而且在灵敏度、选择性、响应时间、实时检测（如荧光成像技术）等方面均具有突出的优势，因而被广泛应用与生物检测领域。荧光碳点(Carbon Dots)作为碳纳米材料家族的新型成员，不仅具有优良的光学性能与小尺寸特性，而且还有良好的生物相容性及低细胞毒性，成为替代量子点的最佳选择。因此，本研究通过对荧光碳点的改性，制备了三种基于碳点的荧光检测体系，分别实现了对碘离子、pH、过氧化氢的选择性荧光检测和/或细胞成像。
制备了Cdots-Hg(II)复合物，获得了碘离子的荧光增强型传感器。并采用核磁共振、原子力、红外光谱、紫外-可见光谱、荧光光谱等手段对碳点及基于碳点的检测传感器进行了表征。对于本检测体系，汞离子可以与碳点结合，形成了一种Cdots-Hg(II)复合物，并淬灭了碳点的荧光发射；而碘离子的加入，可以从复合物Cdots-Hg(II)中夺取汞离子形成更加稳定的碘化汞，重新使碳点的荧光恢复，从而实现了对碘离子的荧光检测。该体系可应用于纯水环境和尿液中对碘离子的检测，检测下限为436nM。
将荧光素异硫氰酸酯(FITC)衍生物经共价键接枝到碳点表面，构建了一种碳点为基体的pH荧光比率检测体系。所制备的传感器CD-FITC粒径大小为5nm，其中碳点不仅构成了体系的载体，也是能量转移的供体。当环境的pH发生改变时，FITC基团的结构发生改变，引起其光谱学性质的变化，可接受来自供体的激发态能量而成为能量转移的受体，从而实现了对环境pH值的荧光比率检测。该检测体系在范围为4~8对pH具有较高的灵敏性，且pKa为5.69，可用于细胞内pH的检测。我们发现检测体系具有良好的水分散性、稳定的光谱学特性和很低的细胞毒性，也可以在活细胞内对胞内不同区域的pH进行成像。
设计合成了一种基于碳点的多功能荧光检测体系，构建了基于FRET原理的比率型、具有线粒体靶向功能的过氧化氢荧光检测体系。在构筑荧光传感体系时，我们依次将靶向基团三苯基膦和过氧化氢探针(PF1)接枝到碳点表面(两者含量分别为4.21mg/g和31.47mg/g)。该检测体系具有很好的水溶性、且对过氧化氢的检测具有很好的选择性和抗干扰性，检测下限达到0.75μM。通过细胞毒性和细胞成像实验，发现该检测体系具有很低的细胞毒性和很好的线粒体靶向作用，并可在活细胞内，对外源性(exogenous)和内源性(endogenous)H2O2进行比率型荧光检测。
我们的研究结果表明，作为一种新型的荧光纳米粒子，碳点具有水分散性良好、细胞毒性低、易于表面修饰、光学性质稳定等特点，是一种颇具潜力的生物检测用纳米材料。
Neutral molecules(ROS, RNS), anions (I-, F-), Cations(K+, Ca2+, Fe3+, Zn2+，H+) play avery important role in regulating the balance of living organisms. Currently, the detection ofbiomolecules in living systems has attracted considerable attention.However, due to thecomplexity of physiological system and the diversity of biologicalmolecules, the applicationof many detection methods faces daunting challenges. Amongvarious detection techniques,fluorescence method is considered to be sensitive, simple, rapid, real-time detection; hencethis method has been widely used in biological detection.
Carbon dots (CDs) is a new fluorescence carbon nanomaterial， which possessuniqueoptical properties and smallsize effect. With good biocompatibility and withoutcytotoxicity it may be a nice substitute for QDs. In this dissertation, we prepared three kindsof nanoparticle sensors through the modification of carbon dots. These sensors can sensitivelyand quickly detect iodide, pH and hydrogen peroxide.
First, we report on a simple method for the determination of iodide in aqueous solutionby exploiting the fluorescence enhancement that is observed if the complex formed betweencarbon dots and mercury ion is exposed to iodide. The sensor was characterized by XPS,AFM, UV-vis, fluorescence spectrophotometer and FT-IR.Fluorescent carbon dots (C-dots)were treated with Hg(II) ion which causes quenching of the emission of the C-dots. Onaddition of iodide, the Hg(II) ions are removed from the complex due to the strong interactionbetween Hg(II) and iodide. This causes the fluorescence to be restored and the detection limitis~436nM. The test is highly selective for iodide and was used for the determination ofiodide in buffer solution and urine.
Then，we demonstrate a fluorescence resonance energy transfer(FRET)-based ratiometricpH nanosensor with carbon-dot (CD) as the carrier. The sensor was prepared by covalentlylinking a pH-sensitive fluorescent dye (fluorescein isothiocyanate, FITC) onto carbon-dot.The CD-based system exhibits a significant change in fluorescence intensity ratio between pH4and8with a pKa value of5.69. It also displays excellent water dispersibility, good spectralreversibility, satisfactory cell permeability and low cytotoxicity. Following the living celluptake, this nanoplatform with dual-chromatic emissions can facilitate real-time visualization of the pH evolution involved in the endocytic pathway of the nanosensor.
Finally, we demonstrate a multifunctional fluorescent nanoprobe for detectingmitochondrialH2O2. The nanoprobe was prepared by covalently linking amitochondria-targeting ligand (triphenylphosphonium, TPP) and a H2O2recognition element(PFl) onto carbon dots (CDs). The content for the targeting ligand (TPP) and the H2O2recognition element (PFl) were determined to be4.21mg/g and31.47mg/g respectively. Thenanoprobe displays excellent water dispersibility, high sensitivity and selectivity, satisfactorycell permeability, very low cytotoxicity with detection limit of0.75μM. Following the livingcell uptake, this nanoprobe can specifically target and stain the mitochondria; and it can detectthe exogenous H2O2in L929cells, as well as the endogenously produced mitochondrial H2O2in Raw264.7cells upon stimulation by PMA.
This study shows that CDs can serve as promising nano-carriers for fabricating practicalmultifunctional fluorescent nanosensors.
制备了Cdots-Hg(II)复合物，获得了碘离子的荧光增强型传感器。并采用核磁共振、原子力、红外光谱、紫外-可见光谱、荧光光谱等手段对碳点及基于碳点的检测传感器进行了表征。对于本检测体系，汞离子可以与碳点结合，形成了一种Cdots-Hg(II)复合物，并淬灭了碳点的荧光发射；而碘离子的加入，可以从复合物Cdots-Hg(II)中夺取汞离子形成更加稳定的碘化汞，重新使碳点的荧光恢复，从而实现了对碘离子的荧光检测。该体系可应用于纯水环境和尿液中对碘离子的检测，检测下限为436nM。
将荧光素异硫氰酸酯(FITC)衍生物经共价键接枝到碳点表面，构建了一种碳点为基体的pH荧光比率检测体系。所制备的传感器CD-FITC粒径大小为5nm，其中碳点不仅构成了体系的载体，也是能量转移的供体。当环境的pH发生改变时，FITC基团的结构发生改变，引起其光谱学性质的变化，可接受来自供体的激发态能量而成为能量转移的受体，从而实现了对环境pH值的荧光比率检测。该检测体系在范围为4~8对pH具有较高的灵敏性，且pKa为5.69，可用于细胞内pH的检测。我们发现检测体系具有良好的水分散性、稳定的光谱学特性和很低的细胞毒性，也可以在活细胞内对胞内不同区域的pH进行成像。
设计合成了一种基于碳点的多功能荧光检测体系，构建了基于FRET原理的比率型、具有线粒体靶向功能的过氧化氢荧光检测体系。在构筑荧光传感体系时，我们依次将靶向基团三苯基膦和过氧化氢探针(PF1)接枝到碳点表面(两者含量分别为4.21mg/g和31.47mg/g)。该检测体系具有很好的水溶性、且对过氧化氢的检测具有很好的选择性和抗干扰性，检测下限达到0.75μM。通过细胞毒性和细胞成像实验，发现该检测体系具有很低的细胞毒性和很好的线粒体靶向作用，并可在活细胞内，对外源性(exogenous)和内源性(endogenous)H2O2进行比率型荧光检测。
我们的研究结果表明，作为一种新型的荧光纳米粒子，碳点具有水分散性良好、细胞毒性低、易于表面修饰、光学性质稳定等特点，是一种颇具潜力的生物检测用纳米材料。
Neutral molecules(ROS, RNS), anions (I-, F-), Cations(K+, Ca2+, Fe3+, Zn2+，H+) play avery important role in regulating the balance of living organisms. Currently, the detection ofbiomolecules in living systems has attracted considerable attention.However, due to thecomplexity of physiological system and the diversity of biologicalmolecules, the applicationof many detection methods faces daunting challenges. Amongvarious detection techniques,fluorescence method is considered to be sensitive, simple, rapid, real-time detection; hencethis method has been widely used in biological detection.
Carbon dots (CDs) is a new fluorescence carbon nanomaterial， which possessuniqueoptical properties and smallsize effect. With good biocompatibility and withoutcytotoxicity it may be a nice substitute for QDs. In this dissertation, we prepared three kindsof nanoparticle sensors through the modification of carbon dots. These sensors can sensitivelyand quickly detect iodide, pH and hydrogen peroxide.
First, we report on a simple method for the determination of iodide in aqueous solutionby exploiting the fluorescence enhancement that is observed if the complex formed betweencarbon dots and mercury ion is exposed to iodide. The sensor was characterized by XPS,AFM, UV-vis, fluorescence spectrophotometer and FT-IR.Fluorescent carbon dots (C-dots)were treated with Hg(II) ion which causes quenching of the emission of the C-dots. Onaddition of iodide, the Hg(II) ions are removed from the complex due to the strong interactionbetween Hg(II) and iodide. This causes the fluorescence to be restored and the detection limitis~436nM. The test is highly selective for iodide and was used for the determination ofiodide in buffer solution and urine.
Then，we demonstrate a fluorescence resonance energy transfer(FRET)-based ratiometricpH nanosensor with carbon-dot (CD) as the carrier. The sensor was prepared by covalentlylinking a pH-sensitive fluorescent dye (fluorescein isothiocyanate, FITC) onto carbon-dot.The CD-based system exhibits a significant change in fluorescence intensity ratio between pH4and8with a pKa value of5.69. It also displays excellent water dispersibility, good spectralreversibility, satisfactory cell permeability and low cytotoxicity. Following the living celluptake, this nanoplatform with dual-chromatic emissions can facilitate real-time visualization of the pH evolution involved in the endocytic pathway of the nanosensor.
Finally, we demonstrate a multifunctional fluorescent nanoprobe for detectingmitochondrialH2O2. The nanoprobe was prepared by covalently linking amitochondria-targeting ligand (triphenylphosphonium, TPP) and a H2O2recognition element(PFl) onto carbon dots (CDs). The content for the targeting ligand (TPP) and the H2O2recognition element (PFl) were determined to be4.21mg/g and31.47mg/g respectively. Thenanoprobe displays excellent water dispersibility, high sensitivity and selectivity, satisfactorycell permeability, very low cytotoxicity with detection limit of0.75μM. Following the livingcell uptake, this nanoprobe can specifically target and stain the mitochondria; and it can detectthe exogenous H2O2in L929cells, as well as the endogenously produced mitochondrial H2O2in Raw264.7cells upon stimulation by PMA.
This study shows that CDs can serve as promising nano-carriers for fabricating practicalmultifunctional fluorescent nanosensors.
引文
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