| 中文摘要 |
半导体聚合物量子点(semiconducting polymer dots，Pdots)是一类由荧光共轭聚合物为原料合成的水溶性纳米粒子，兼具荧光共轭聚合物和纳米材料的特性。Pdots作为一类性能优良的纳米荧光团，在生物传感、成像和光动力治疗等领域得到了广泛的应用。然而，目前Pdots探针的识别基团比较单一，具有近红外激发或靶向定位功能的比率型Pdots荧光探针鲜有报道。 基于此，本论文旨在利用Pdots的高亮度、放大的能量转移作用、易于功能化、以及生物相容性好等特性，通过不同策略进行表面化学调控，以实现Pdots的功能化修饰，进而发展具有特定功能的Pdots荧光探针，探索其在比率荧光传感与成像中的应用。主要工作如下： 1)采用β-环糊精(β-cyclodextrin，β-CD)修饰聚(9,9-二辛基芴-交替-[2,1',3]苯并噻二唑)(poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)]，PFBT) Pdots，构筑了一种比率型胆固醇(cholesterol，Cho)传感器。将功能化Pdots(CD-Pdots)作为供体，罗丹明B(rhodamine B，RB)作为受体，利用主-客体的包结作用建立荧光共振能量转移(fluorescence resonance energy transfer,FRET)体系。当加入Cho时，Cho和RB与CD间的竞争包结作用一定程度上抑制了上述FRET过程。基于这种抑制作用，构筑了一种灵敏的比率型Cho传感器。该传感器线性检测范围为25-350 nM，检出限为4.9 nM。同时该传感器也可以用于人血清中Cho的测定，结果令人满意，回收率在97.41～103.52％之间。 2)基于可选择性结合铜离子的罗丹明B-酰肼(rhodamine B hydrazide，RB-hy)功能化的PFBT Pdots构筑了一种可同时测定铜离子和碱性磷酸酶(alkaline phosphatase， ALP)的比率型荧光传感器。在功能化Pdots(Pdots@RB-hy)中，以PFBT Pdots作为供体，RB-hy与铜离子络合物(RB-hy-cu(II))作为受体，基于二者间的FRET作用构筑了比率型Cu(II)传感器。该传感器检测Cu(II)的线性范围为0.05-5 μM，检出限为15 nM。同时，以Pdots@RB-hy-Cu(II)体系为信号传导单元，结合碱性磷酸酶(ALP)对焦磷酸盐(pyrophosphate，PPi)的催化水解作用，我们将上述传感体系拓展为比率型ALP传感体系，检测ALP的线性范围为0.005-15 U L⁻¹，检出限为0.0018 U L⁻¹。采用标准加入法对人血清中ALP的活性进行了测定，回收率在95.9～102.0％之间。 3)通过聚合物掺杂和纳米沉淀法制备了一种双组份(聚(9,9-二正辛基芴基) (poly(9,9-dioctylfluorenyl-2，7-diyl)，PFO)和聚(5-(2-乙基己氧基)-2-甲氧基-氰基对苯二亚甲基)(poly-[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1，4-phenylene)]， CN-PPV))、双发射带(蓝光和橙光)的Pdots。利用环境稳定的酶底物-酪氨酸甲酯(L-tyrosine methyl ester，Tyr-OMe)对所制备的纳米材料进行功能化得到纳米复合物(Pdots@Tyr-OMe)。酪氨酸酶(tyrosinase，TR)对Tyr-OMe的催化氧化作用能够通过选择性电子转移作用来完成双发射带Pdots荧光的有效调制，从而实现对TR的比率型测定。Pdots具有的双光子特性使得所构筑的探针可用于目标物的零背景检测和成像，在生物医学领域具有潜在的应用价值。 4)基于功能化Pdots的竞争性FRET作用构筑了一种用于线粒体内pH监控与成像的比率型、靶向探针。首先利用PFO，聚(9,9-二辛基芴-4,7-二噻吩-2,1,3-苯并噻二唑)(poly(9,9-dioctylfluorene)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)，PF-DBT5)、聚苯乙烯马来酸酐共聚物(poly(styrene-co-maleic anhydride)，PSMA)和含三苯基磷单元的聚氧代乙烯(5)壬基苯基醚(triphenylphosphonium-modified polyoxyethylene nonylphenylether，TPP-CO-520)制备了表面含有TPP和COOH的双发射Pdots，使用刚果红(congo red，CR)对其进行修饰得到了具有线粒体靶向功能的pH探针(CR／TPP@Pdots)。所制备探针中不同荧光组分(供体)与受体CR间的FRET效率可以通过pH变化来进行有效调节而实现pH的比率型传感。在pH值2.57～8.96的范围内，探针表现出良好的生物相容性、可逆性、选择性和光稳定性。细胞实验结果表明CR／TPP@Pdots具有较好的线粒体靶向能力，可用于活细胞中线粒体pH的监控与成像。 关键词：半导体聚合物量子点；比率型荧光传感；荧光成像；胆固醇；Cu(II)；碱性磷酸酶；酪氨酸酶；pH
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| 英文摘要 |
Semiconducting polymer dots (Pdots) are water-soluble nanomaterials formed by π-conjugated polymers and have the common characteristics of fluorescence polymer and nanomaterials. Pdots, as the excellent fluorescence probe, have been used in biosensing, bioimaging and photodymamics therapy. However, the recognition group of Pdots-based fluorescent probes is relatively single in content, and designing and synthesizing Pdots fluorescent probes with ratiometric, excited with near-infrared wavelength or specific targeted for biosensing and bioimaging have rarely reported. This paper is focused on the development of ratiometric fluorescent sensing platform with specific function by using functionalized Pdots as fluorophore, and further exploring its application in fluorescence sensing and imaging. These sensors show excellent sensing and imaging performance due to the fascinating merits of Pdots, such as extraordinary fluorescence brightness, amplified energy transfer, easy functionalization and non-toxicity. The main work is as follows: 1)Carboxyl-functionalizedpoly[(9,9-dioctylfluorenyl-2,7-diyl)-co(l,4-benzo-{2,1′,3}-thiadiazole)] dots covalently tagged with aminated β-cyclodextrin (NH₂-CD) have been designed to construct a ratiometric sensor for cholesterol (Cho). Using CD-Pdots as energy donors with rhodamine B (RB) as energy acceptors, a fluorescence resonance energy transfer (FRET) pair has been built because the host-guest interaction between RB and CD attached to Pdots brings donors and acceptors into close proximity. In the presence of Cho, the acceptors will depart from the donors because of the competitive inclusion interaction between Cho and RB with CD, resulting in the hindering of the FRET process between CD-Pdots and RB. On the basis of the turn-on fluorescence of CD-Pdots and turn-off fluorescence of RB, a sensitive ratiometric method for the determination of Cho in the concentration range from 25 to 350 nM with a detection limit of 4.9 nM was achieved. The proposed method was validated to determine free Cho in human serum samples with satisfactory results. 2)A novel ratiometric fluorescent sensing platform using functionalized Pdots as probes for fluorescence signal transmission has been designed for sensing Cu( II) and activity of alkaline phosphatase (ALP) with high selectivity and enhanced sensitivity. The highly fluorescent Pdots were firstly prepared with Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-( 1,4-benzo- {2,1 ',3} -thiadiazole)] (PFBT) via nanoprecipitation method, and then assembled with non-fluorescent rhodamine B hydrazide (RB-hy), which shows special binding activity to Cu( II), through adsorption process to obtain functionalized nanohybrids, Pdots@RB-hy. As thus, a FRET donors/acceptors pair, in which PFBT Pdots act as energy donors while RB-hy-Cu(II) complexes act as energy acceptors were constructed. On the basis of the varies in fluorescence intensities of donors/acceptors in the presence of different amounts of Cu(II), a ratiometric method for sensing Cu(II) has been proposed. The proposed ratiometric Cu(II) sensor shows a good linear detection range from 0.05 to 5 μM with a detection limit of 15 nM. Furthermore, using the Pdots@RB-hy-Cu(II) system as signal transducer, a ratiometric sensing for alkaline phosphatase (ALP) activity has also been established with pyrophosphate (PPi) as substrates. The constructed ratiometric sensor of ALP activity displays a linear detection range from 0.005 to 15 U L⁻¹ with a detection limit of 0.0018 U L⁻¹. The sensor was further successfully used for ALP activity detection in human serum with satisfactory results. 3)Semiconducting polymer dots (Pdots) with one-, two-photon excitation and dual-emission have been synthesized by coprecipitation of two conjugated polymers including poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly [2-methoxy-5-(2-ethylhexyloxy)-1,4-(l-cyanovinylene-1,4-phenylene)] (CN-PPV) and have been further functionalized with L-tyrosine methyl ester (Tyr-OMe) via electrostatic assembly for ratiometric fluorescent sensing and bioimaging of tyrosinase activity. Tyrosinase-catalyzed oxidation of TyrOMe effectively modulate the dual-emission fluorescence of PFO/CN-PPV@Tyr-OMe Pdots from orange to blue through a selective photoinduced electron transfer (PET) process. A two-photon ratiometric sensor at almost zero-background interference and bioimaging of tyrosinase activity have been demonstrated, suggesting the potential biomedical applications of the prepared functionalized Pdots. 4)We design a novel ratiometric fluorescent chemical probe for monitoring and imaging pH of mitochondria in living cells based on congo-red (CR)-modified dual-emission semiconducting polymer dots (Pdots) via a competitive fluorescence resonance energy transfer (FRET) mechanism. The Pdots are synthesized by triphenylphosphonium (TPP)-modified polyoxyethylene nonylphenylether(CO-520), Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), poly(9,9-dioctylfluorene)-co-(4,7-di-2-thienyl -2,1,3-benzothiadiazole) (PF-DBT5), and poly(styrene-co-maleic anhydride) (PSMA) via nanoprecipitation method, and the prepared Pdots are further chemically linked with pH-sensitive, non-fluorescent CR to obtain the mitochondria-targeted pH fluorescent probes. This Pdots-based probe consists of two luminescent components including PFO and PF-DBT5 as fluorescence donors, a acid-base indicator CR as energy acceptor, and TPP as mitochondria-targeting group. At different pH region, the FRET efficiency between CR and PFO, or CR and PF-DBT5 can be modulated. This probe exhibits good biocompatibility, wide pH detection range from 2.57 to 8.96, good reversibility, high selectivity and excellent photostability for pH monitoring. This probe allows for detecting and imaging of mitochrondrial pH in the living cells with satisfactory results. Keywords: Semiconducting polymer dots; Ratiometric fluorescence sensor; Bioimaging; Cholesterol; Cu( II); Alkaline phosphatase; Tyrosinase; pH
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