【作者】 刘玲芝；

【导师】 刘志洪； 何治柯；

【作者基本信息】 武汉大学 ， 分析化学， 2010， 博士

【摘要】 荧光共振能量转移(FRET)被认为是1.0-10.0nm距离范围内有效的光学分子尺,在杂交、免疫分析、以及生物大分子相互作用的研究中具有广泛的应用。然而,传统的单光子激发-荧光共振能量转移(OPE-FRET)模式下,当用紫外光或可见光激发能量供体时,常常会受到生物样品自身荧光和散射光的干扰；而且在供体的有效激发波长下常常会造成受体的直接激发。因此,FRET方法的发展,迫切需要选择新的能量供—受体对,尤其是能量供体,以克服上述缺陷。双光子激发(TPE)属于三阶非线性光学效应,具有长波激发(近红外光),短波发射(可见光)的特点,其本质是一种反斯托克斯发光。以近红外光为激发光,可以有效地消除生物样品本底荧光和散射光的干扰,减小对生物样品的损伤和光漂白,避免对能量受体的直接激发。因此,具有双光子激发性质的荧光团是一类潜在的新型能量供体,基于双光子激发的TPE-FRET能够克服复杂生物样品自发荧光和散射光的干扰,提高分析检测的信噪比和灵敏度。鉴于TPE-FRET在复杂生物样品分析中的优越性以及目前国内外在此方向研究的空白,本论文以反斯托克斯荧光团为能量供体(包括有机小分子和无机纳米晶(QDs)),系统地研究了TPE-FRET技术在复杂生物样品分析中的优势和适用性。1.合成具有双光子激发特性的有机小分子反式-4-(N-2-羟乙基-N-乙基胺基)-4’-(二甲基胺基)二苯乙烯(简写为DMAHAS),将其作为能量供体,通过其末端活性羟基与生物素的羧基进行偶联。以暗猝灭剂4-(二甲基氨基)偶氮苯磺酰氯(DABS-C1)作为能量受体,用于标记亲和素。利用生物素与亲和素之间的特异性相互作用,构建了一个基于TPE-FRET的竞争法检测亲和素模型,用于阐述和证明双光子激发在FRET中的优势。结果显示,在单光子激发条件下,生物分子具有较强的背景信号。但是,在双光子激发模式下生物分子无背景信号。说明双光子模式下以近红外光激发样品时,可以避免生物样品自身荧光和散射光的干扰,提高分析检测的灵敏度。2.在以DMAHAS为能量供体的TPE-FRET模型成功构建的基础上,合成了一个新的具有氨基活性基团的双光子激发分子2-((E)-4-(二甲基胺基)二苯乙烯)-5-((E)-4-胺基二苯乙烯)对苯二腈(缩写为TP-NH2)。相对于DMAHAS, TP-NH2的光物理性质有大幅度改进,具有较大的双光子吸收截面。以TP-NH2为能量供体,通过分子末端的氨基将其共价结合到牛血清白蛋白(BSA)上,从而构建了一个基于TPE-FRET的均相免疫分析体系,将其用于抗体蛋白的定量分析。实验中直接使用抗血清为样品,获得了较高的灵敏度和较宽的线性范围(0.05-2.5nM)。然而,传统的OPE-FRET技术由于血清样品背景信号的干扰完全不能工作,证明TPE-FRET能够克服生物样品自发荧光和散射光的干扰。数据显示TPE-FRET技术在均相免疫分析中具有很大的优势,尤其是应用于复杂生物样品的分析。3.将TPE-FRET方法的适用性进一步扩展到均相核酸杂交分析。通过优化分子结构,设计并合成了一种具有活性羧基的双光子激发有机分子2-(2,5-二(4-(二甲基胺基)苯乙烯基)-1H-吡咯-1-基)乙酸(简称TP-COOH),通过生物素-链霉亲和素桥连技术,建立了一个基于TPE-FRET的均相竞争法,用于检测单链靶DNA。该方法在磷酸盐缓冲溶液中获得了较好的线性关系。并通过加标回收实验进一步检验了所建立的TPE-FRET方法对实际生物样品的适用性,在血清样品和尿样中得到了满意的回收率。而OPE模式下在血清样品和尿样中不能得到合理的回收率。这可归因于复杂生物基质中生物分子的背景信号干扰。这部分工作进一步证实TPE-FRET方法对于复杂生物样品的分析检测比OPE-FRET更具优势。同时也表明由于分子结构上高度的灵活性,通过分子设计和裁剪,有可能将TPE-FRET用于不同的分析对象,扩展该方法的应用领域。4.在以有机小分子为供体,构建TPE-FRET方法模型并成功用于复杂生物基质中均相免疫分析、杂交分析的基础上,本章考察了无机纳米量子点作TPE供体的能力。鉴于QDs较大的双光子活性截面以及分子信标发夹结构高度的特异性,作者设计了一种新的双光子激发分子信标(MB),用链霉亲和素修饰的QDs作为供体,染料6-羧基-X-罗丹明作为受体,利用链霉亲和素与生物素之间的特异性相互作用,建立了一种特异性识别靶DNA链、完全不互补DNA链以及单碱基错配DNA链的新方法,并探讨了这种新技术在复杂样品基质中对靶DNA的特异性识别性能。结果显示,在双光子激发模式下,观察不到任何复杂生物基质自身荧光和散射光的干扰,在血清中该TPE-MB探针仍能特异性地识别靶序列。然而,在单光子激发模式下,供体QDs的发光却受到了复杂基质背景荧光的干扰。同时也验证了QDs由于其较大的双光子活性截面可以克服TPE模式下供受体同时激发所产生的负面效应。本章工作中所构建的TPE-MB探针有望用于复杂生物基质中靶序列的定量分析和检测,以及荧光原位杂交。更多还原

【Abstract】 Fluorescence resonance energy transfer (FRET) is considered as a sensitive and reliable "ruler" over distances of 10-100 A and has broad applications in studying interactions of biological macromolecules, in immunoassay, and so on. However, in the application of conventional one-photon excitation (OPE)-FRET, where the energy donor is excited with ultraviolet or visible light, autofluorescence or scattering light always arises from biomolecules upon excitation of the energy donor. In addition, the energy acceptor is often coexcited with the energy donor because of the overlap of their excitation spectra. Therefore, new energy donor-acceptor pairs are desired to overcome the problem. Recently, two-photon excitation (TPE) has attracted a lot of attention in biological applications. In such a nonlinear optical process, two low-energy photons are simultaneously absorbed to reach the excited state. TP excitable materials can be excited in IR region to give emission in the visible region. Under TP excitation with IR light, the autofluorescence of biomolecules and the scattered excitation light could be eliminated, and the coexcitation of energy acceptor with energy donor could be avoided. Besides, the IR light causes much less photobleaching off the focal plane and less photodamage to biological samples. Therefore, TP excitable fluorophores would be a promising alternative as the FRET donor. TPE-FRET based bioassays would be free of interference from autofluorescence of biomolecules and the scattered excitation light in real biological systems. Owing to the advantages of TPE-FRET in complicated biological sample matrix and the originality of such idea, in this dissertation, several TPE-FRET models had been constructed with TP excitable small organic molecules and QDs as donors to demonstrate the abilities and advantages of TPE-FRET in complicated biological sample matrix.1. A FRET model using two-photon excitable small organic molecule trans-4-(N-2-hydroxyethyl-N-ethyl amino)-4’-(dimethyl amino) stilbene (DMAHAS) as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-C1. Binding of DABS-C1 labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor.2. A two-photon excitable small organic molecule 2-((E)-4-(dimethylamino) styryl)-5-((E)-4-aminostyrul) terephthalonitrile (abbreviated as TP-NH2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared. Using the TP-NH2 molecule as an energy donor, a TPE-FRET based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-C1) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.3. The newly emerging TPE-FRET technique was extended to the determination of oligonucleotide. A new TPE molecule with favorable two photon action cross section was synthesized [2-(2,5-bis(4-(dimethylamino)styryl)-1H-pyrrol-1-yl)acetic acid, abbreviated as TP-COOH], with the tagged reactive carboxyl group allowing facile conjugation with streptavidin (SA). Employing the TP-COOH molecule as energy donor and black hole quencher 1 (BHQ-1) as acceptor, a TPE-FRET based homogeneous competitive hybridization model was constructed via a biotin-streptavidin bridge. Through the hybridization between a biotinylated single-stranded DNA (ssDNA) and a BHQ-1-linked ssDNA, and the subsequent capture of the as-formed hybrid by TP-COOH labeled SA, the donor fluorescence was quenched due to the FRET between TP-COOH and BHQ-1. Upon the competition between a target ssDNA and the quencher-linked ssDNA toward the biotinylated oligonucleotide, the donor fluorescence was recovered in a target-dependent manner. Good linearity was obtained with the target oligonucleotide ranging from 0.08 to 1.52μM. The method was applied to spiked serum and urine samples with satisfying recoveries obtained. The results of this work verified the applicability of TPE-FRET technique in hybridization assay and confirmed the advantages of TPE-FRET in complicated matrix.4. FRET performance driven by a two-photon excitation process has been limited by the photophysical properties of organic dyes and fluorescent proteins. Considering the large two-photon action cross-section of quantum dots (QDs) and the high specificity of hairpin shaped molecular beacons (MB), a new MB driven by two-photon excitation was developed with QDs as donor,6-carboxy-X-rhodamine (ROX) as acceptor via a biotin-streptavidin bridge. The TPE-MB can successfully recognize the complementary target DNA, non-complementary target DNA, and discriminate single mismatched target DNA in assay buffer. Furthermore, the method was applied to spiked serum with satisfying recognition of complementary target DNA with no interference from background fluorescence or scattering light. The results of this work suggest that the TPE-MB can be applied to quantitative detection of complementary target DNA in biological matrix and fluorescence in situ hybridization.更多还原