【作者】 邓婷；

【导师】 沈国励； 俞汝勤；

【作者基本信息】 湖南大学 ， 分析化学， 2007， 博士

【摘要】 染料掺杂荧光纳米颗粒相对于量子点、荧光染料和等离子体共振颗粒而言具有诸如高量子产率、光稳定性、亲水性及易于使各种功能团在其表面修饰等显著的优点,所以近年来关于染料掺杂荧光纳米颗粒的研究报道时有增加,也引起了众多研究者的关注。但目前几乎所有的研究报道都集中于那些在可见区域激发的染料的包埋,这些染料掺杂的荧光纳米颗粒易于受自身荧光或细胞和体内测量的内滤效应的影响,从而限制了荧光纳米颗粒在生物分析、生物医学领域的进一步应用。相对于常规荧光检测而言,在近红外光区的荧光检测,生物基体光吸收或荧光强度很小,且致密介质(如组织)的光散射明显降低,激发光的穿透性更强,因而自发荧光的背景干扰显著降低,可以高灵敏和选择性地检测复杂生物分子及活细胞中的特定成分。而且,由于全血在近红外区域具有很弱的吸收,所以有望发展一种基于近红外荧光纳米颗粒对全血样品无需分离的直接检测方法。本论文前半部份报告有关近红外核壳荧光纳米颗粒的生化分析应用研究结果。针对目前荧光纳米颗粒的研究现状,发展了一种新型近红外核壳荧光纳米颗粒,并将其进行生物标记应用于全血样品中肿瘤标志物、白血病细胞及单碱基突变的直接检测。此外,利用纳米金的凝集变色效应,尚建立了一种新的等温滚环扩增反应的单碱基突变检测方法。主要内容如下:(1)基于一种改进的合成方法制备了新型近红外核壳荧光纳米颗粒。通过引入疏水性的硅醇盐作为纳米粒子内核,接着在油包水的微乳液中通过氨水催化正硅酸乙酯水解形成一层亲水的硅外壳,合成了核壳型的亚甲基兰掺杂荧光纳米颗粒,并对纳米颗粒的合成条件进行了优化。这种纳米颗粒的荧光明显强于用常规方法合成的纳米颗粒,且纳米颗粒的光稳定性明显增强,在水中几乎没有染料泄漏。纳米颗粒呈均匀的圆球型,大小一致,直径大约为65 nm。由于荧光分子在硅基质中得到了很好的保护,从而可免于被环境所污染。同时,基于近红外荧光的特性构建了一种新型近红外荧光纳米粒子的荧光各向异性免疫凝集分析方法,用于直接检测全血样品中的肿瘤标记物——甲胎蛋白(AFP),而不需经过分离步骤。结果表明,近红外荧光纳米探针在这种复杂的生物体系中不受背景的干扰,因此可以快速进行全血样品中AFP的直接检测。(2)结合cell-SELEX技术筛选得到能特异性识别人急性淋巴白血病细胞的核酸适体的高特异性和核壳型近红外荧光纳米颗粒的优点,发展了一种能快速实现全血样品中肿瘤细胞的识别新技术,该方法简便、快速、灵敏,可望用于癌症的早期诊断、治疗方案的选择及预后的判断。(3)结合运用连接酶反应和核壳型荧光纳米颗粒,提出了一种基于荧光各向异性技术与DNA连接酶的高保真性的检测方法,用于单碱基突变的直接检测。该方法使用一个等位特异的识别探针和一个普通的探针,这两条探针都标记在荧光纳米颗粒的表面。当这两条DNA探针与目标链发生杂交反应时,就引起荧光纳米颗粒的凝集,从而引起荧光探针的荧光各向异性值的增加。当识别探针3’端的碱基与目标链完全匹配时,DNA连接酶就会把这两条探针之间的缺口封闭,反之,当识别探针3’端的碱基与目标链不匹配时,缺口就不能被封闭。对于不匹配的情况,当加热解链形成的双链时,凝集的荧光纳米颗粒就会彼此分开,溶液的荧光各向异性值将恢复到初始值;而对于完全匹配的情况,溶液的荧光各向异性值仍保持纳米粒子团聚后的值。因此,通过灵敏的荧光各向异性检测就能很好地实现单碱基突变的检测。用该方法对结肠癌k-ras基因的第12位密码子的突变情况进行了检测,使突变型和野生型得到了很好的区分。(4)结合本实验室在基因单碱基突变检测技术方面已有的研究成果,利用核酸修饰纳米金的这一凝集变色特性,提出了一种新的等温滚环扩增反应的检测方法,并用于人β地中海贫血基因-28位点的点突变灵敏定量检测。该方法能够区分突变的杂合子或纯合子,为DNA目标链单碱基突变的临床医学诊断提供了一种新的途径。本论文的余下部分报告有关新型压电免疫传感器的研究成果。针对当前免疫传感器传感界面构建研究中存在的问题,在实验室原有工作的基础上,以压电石英晶体微天平为换能器,结合自组装膜技术、纳米生物修饰技术及聚电解质吸附组装技术,设计了一系列免疫活性材料固定化新方法,以期提高压电免疫传感技术用于临床实际诊断的可行性。同时,由于压电石英晶体微天平很难实现对小分子物质的直接检测,所以利用大分子亲和素与生物素之间的亲和力,设计了一种石英晶体微天平实现对小分子生物素的检测的方法。(5)结合电聚合膜和纳米金自组装技术,提出了一种新的生物分子固定化方法,研制成一种检测抗胰蛋白酶的压电免疫传感器。通过在石英晶振金电极表面电聚合邻苯二胺膜,再在膜表面自组装一层纳米金粒,以静电吸附作用固定抗体(抗原),实现对相应抗原(抗体)的检测。利用扫描电镜技术,从形态上考察了晶振金电极上自组装纳米金后的表面形貌。考察了抗体的固定化条件,探讨了传感器的响应与再生性能。结果表明,这种固定化方法对所固定的生物分子的生物活性影响小,传感器的测定灵敏度高,响应性能和再生性能较好。(6)基于Nafion膜界面构建了一种石英晶体微天平免疫传感器,用于血清中补体C4的检测。Nafion膜界面的表面形貌通过扫描电子显微镜进行表征。考察了抗体静电吸附过程,抗体固定的最佳条件以及免疫反应过程,所构建的免疫传感器测定补体C4的线性浓度范围为0.08-1.6μg/mL,检测相对标准偏差低于5.3%。该传感器制作简便,也易于使用后再生。(7)通过带相反电荷聚电解质可交替吸附的自组装技术,研制了一种新型的生物传感界面。在压电石英晶体微天平(QCM)的金电极表面,修饰巯基乙酸(MAA)自组装单层膜(SAM)和带相反电荷聚电解质的多层自组装膜。然后将具有双面胶功能的带正电荷的壳聚糖分子连接带负电荷的褐藻酸钠-HSA抗体分子到负电性的MAA-SAM层。并实时考察了传感界面的组装过程和组装条件,运用原子力显微镜(AFM)考察了每层组装膜的表面形貌。与传统的抗体直接固定方法相比,本文提出的新型免疫传感技术能很好地保持抗体的活性,使灵敏度显著提高,也使传感器检测的线性范围明显变宽。尤其这种传感器具有简便而快速的再生性能。因此这种新的生物传感界面,在开发新的生物传感器件上具有潜在的优势。(8)结合自组装技术和聚电解质间的静电吸附作用,在石英晶振的金电极表面构建了半胱胺自组装膜和壳聚糖/褐藻酸钠相反电荷聚电解质的多层自组装膜的传感界面,研制成一种检测人血清中B因子的压电免疫传感器。考察了标记抗体的固定化条件,探讨了传感器的响应与再生性能。与戊二醛共价交联固定方法相比较,这种优化的固定化方法所获得的传感器响应的频移值大,检测限低,检测范围宽,灵敏度高,传感器能够简便地实现再生。而且这种固定化方法有利于传感器上所固定的蛋白质分子的生物活性的保持。特异性实验和回收率结果表明,该传感系统可发展成临床检测B因子的一种有效工具。(9)提出了一种新型的石英晶体微天平生物传感器用于小分子生物素的测定,该传感器是基于巯基化合物在金基底上稳固的混合自组装单层膜以及生物素和生物素类似化合物HABA与亲和素之间生物亲和力的不同。亲和素与固定于电极表面的HABA形成一种亚稳态分子复合物,当该传感器接触到包含生物素的样品溶液时,亚稳态分子复合物中的亲和素由于与溶液中生物素形成更为稳定的复合物而被拉离传感器表面,从而引起晶振频率的变化。该频率的变化与脱落的亲和素量成一定的比例关系,而且与溶液中生物素的浓度成一种明确的数学关系。传感器的制备过程中,混合自组装单层膜一方面有利于生物分子在电极表面的牢稳固定,另一方面,由于混合自组装单层膜较长的空间“手臂”更有利于俘获生物分子,有效提高了生物分子的固定量。实验证明,本章提出的生物传感器对浓度范围在0.017– 1.67μg/mL的小分子生物素有很好的响应,而且该传感器的再生非常简单方便。更多还原

【Abstract】 Nanoparticle-based bionanotechnology is a rapidly growing field that deals with particulate systems for bioanalytical, biotechnological, and biomedical applications. Recent years have seen proliferated studies of fluorescent organic nanoparticles, among which dye-doped silica nanoparticles attracted research interest due to their potential applications as fluorescence probes for various biological detection. The dye-doped silica nanoparticles entrap a large number of fluorophores in the silica matrix, which produce a strong fluorescence signal when excited properly. Incorporation of dye molecules inside the silica matrix protects the dye from the surroundings, making the fluorescence very stable and thereby offering accurate measurements for bioanalysis. Moreover, the silica surface serves as a universal biocompatible and versatile substrate for the immobilization of biomolecules. Nevertheless, almost all these studies are focused on the entrapment of dyes with excitation in the visible region. These dye-doped silica nanoparticles are susceptible to the interference from autofluorescence and inner filtration effect in cellular or in vivo measurement implementations.Spectrofluorimetry in the near-infrared region (about 650 - 1000 nm) is an area of increasing interest. In comparison to more conventional measurements made in the ultraviolet and visible regions, near-infrared (NIR) fluorescence has many advantages. For instance, NIR fluorescence generally exhibit relatively low levels of background interference, since few naturally occurring molecules undergo electronic transitions in such a low-energy region of the electromagnetic spectrum. Moreover, the penetration of NIR light through skin and overlaying tissue is as deep as a few millimeters, NIR fluorescence holds considerable promise for the development of noninvasive diagnostic techniques. For these reasons, NIR fluorescence imaging is potentially very attractive for in vivo imaging. In addition, as the whole blood shows very weak absorption in the NIR region, there is potential to use NIR fluorescence for direct analysis of whole blood samples without separation steps. The first-part of this thesis focused on the research topics of fluorescent nanoparticles. We synthesized a class of novel core-shell near-infrared fluorescent nanoparticles and used them as highly sensitive and photostable label in the detection of alpha fetoprotein (AFP) and leukemia cancer cells recognition in whole blood samples as well as the detection of single nucleotide polymorphisms (SNPs). Moreover, based on the optical properties of aggregated Au nanoparticles, a colorimetric method for the identification of point mutation based on isothermal rolling-circle amplification has been developed.(1) A class of novel core-shell near-infrared fluorescent nanoparticles was prepared through co-hydrolysis of a hydrophobic silicon alkoxide, hexadecyltrimethoxysilane, and tetraethyl orthosilicate as the dye-doped core followed by the formation of a hydrophilic shell via hydrolysis of tetraethyl orthosilicate in water-in-oil microemulsion. The co-hydrolysis of hexadecyltrimethoxysilane and tetraethyl orthosilicate produced a highly hydrophobic core for the entrapment of a low-cost near-infrared fluorescence dye methylene blue. Experimental investigation of this particular core-shell nanoparticles in comparison with conventional dye-doped silica nanoparticles demonstrated that the hydrophobic core enabled the doped dye to exhibit enhanced fluorescence and show improved stability to dye leaching and exogenous quenchers. In contrast to rhodamine B-doped silica nanoparticles, the near-infrared fluorescent nanoparticles also showed negligible background fluorescence and low inner filtration interference in complex biologic systems such as whole blood. This advantage was utilized for the development of an immunoagglutination assay method based on fluorescence anisotropy measurement for the detection of alpha fetoprotein (AFP) in whole blood samples. The results revealed that the fluorescence anisotropy increases were linearly correlated to the AFP concentration in the range from 1.9 to 51.9 ng/mL.(2) A method for the rapid detection of leukemia cells in whole blood samples has been developed based on a group of aptamers for the specific recognition of leukemia cells and the core-shell near-infrared fluorescent nanoparticles. The results demonstrate the potential application of this method for medical diagnostics.(3) A proof-of-principle has been reported for a sensitive genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) based on the sensitive fluorescence anisotropy measurement through core-shell fluorescent nanoparticles assembly and the ligase reaction. By incorporating the core-shell fluorescent nanoparticles into the fluorescence anisotropy measurement, this assay provided a convenient and sensitive detection assay that enabled a straightforward single-base discrimination without the need of the complicated operational steps. The assay could be implemented via two steps: firstly, the hybridization reaction that allowed two nanoparticle-tagged probes to hybrid with the target DNA strand and the ligase reaction that generated the ligation between perfectly matched probes while no ligation occurred between mismatched ones were implemented synchronously in the same solution. Then, a thermal treatment at a relatively high temperature discriminated the ligation of probes. When the reaction mixture was heated to denature the formed duplex, fluorescence anisotropy value of the perfect-match solution does not revert to the initial value, while that of the mismatch again comes back as the assembled fluorescent nanoparticles dispart. The present approach has been demonstrated with the discrimination of a single base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild type and mutant type were successfully scored. Due to its ease of operation and high sensitivity, it was expected that the proposed detection approach might hold great promise in practical clinical diagnosis.(4) Using the distance-dependent optical properties of aggregated Au nanoparticles functionalized with oligonucleotides, we developed a detection method for identification of point mutation based on the isothermal rolling-circle amplification and the agglutination aggregation of Au nanoparticles.The remaining sections of the present thesis repart the research results concerning fabrication of new-type biosensing interfaces for pizeoelectric immunosensor. Pizeoelectric immunosensor are widely used for the assay of biological analytes due to the advantages of this approach including simple-design, high-sensitivity and low-cost. However, the method of immobilization, the reproducibility and the reusability still remain to be improved in the design and applications of these sensors. Among these problems presented, the key step for the fabrication of biosensors with excellent property is the immobilization of bio-species on the surface of transducers. Focused on these topics in the formation of the biosensing interface, several new procedures for immobilizing bio-species to construct biosensor have been developed.(5) Electropolymerized films (Eps) and the self-assembled technology are combined for a novel immobilization of biomolecules applied to an immunosensor for detecting the antitrypsin (α1-AT) in human serum. The o-phenylenediamine electropolymerized films are immobilized on the surface of quartz crystal microbalance (QCM). After self-assembling a nano-gold layer on the Eps, the antibodies of the antitrypsin (Anti-α1-AT) are electrostatically adsorbed onto the QCM for immunoreacting Antitrypsin. The surface morphologies of the QCM are investigated by scanning electron microscopy (SEM) after being modified with o-phenylenediamine Eps and nano-gold layer. The conditions of immobilizing anti-α1-AT are optimized in detail. Compared to the glutaraldehyde binding approach, the antibodies immobilized by the nano-gold self-assembly procedure present higher bioactivity and greater frequency response to immunoreaction. It is concluded that this immunosensing system provides the advantages of improved sensitivity, selectivity and reusability.(6) A piezoelectric immunosensor based on Nafion membrane interface with a simple immobilization procedure has been developed for the determination of complement C4 in human serum. The polyanionic perfluorosulfonated Nafion polymer was used to modify the electrode surface of QCM as a platform for the immobilization of complement C4 antibodies. The surface morphology of QCM modified with Nafion membrane was investigated using scanning electron microscopy. The system was optimized with regard to parameters involved in the preparation of immunosensor and the assay process. The proposed immunosensor possesses nice response to C4 in the range of 0.08-1.6μg/mL with a relative standard deviation of below 5.3%. Moreover, the proposed immunosensor shows the advantage in terms of the speed and ease in the immobilization procedure as well as the simple and advantageous regeneration process. Experimental results obtained with regards of non-specific adsorption and recoveries indicated that the proposed immunosensor offers a promising alternative tool for clinical diagnosis of complement C4.(7) A novel biosensing interfacial design strategy has been produced by the alternate adsorption of the oppositely charged polyelectrolytes. A quartz-crystal microbalance as a model transducer was modified by use of mercaptoacetic acid (MAA) self-assembled monolayer (SAM) and the adsorption multilayers of the oppositely charged polyelectrolytes. MAA-SAM was first applied to the gold electrode surface of the crystal, and the positively charged chitosan was used as a double-sided linker to attach the negatively charged alginate-HSA antibodies to the negatively charged MAA-SAM layer. The assembly process and conditions were studied using the real-time-output device and the surface topologies of the resulting crystals were characterized by atomic force microscopy (AFM) imaging. The proposed immunosensor in optimal conditions has a linear detection range of 12.3-184.5μg/mL for HSA detection. Comparing with the direct immobilization method of antibodies, the immunosensor with the proposed immobilization procedure shows some advantages, such as improved sensitivity due to the well-retained antibody activity and the significantly extended detection range. In particular, the regeneration of the developed immunosensor was simple and fast. Analytical results indicate that the developed immobilization procedure is a promising alternative for the immobilization of biorecognition element on the electrode surface.(8) A piezoelectric immunosensor based on a novel immobilization strategy combining cysteamine self-assembled monolayer and chitosan/alginate adsorption procedure has been developed for the determination of factor B in human serum. Cysteamine SAM was first applied to the gold electrode surface of the crystal, and a strong positively-charged chitosan monolayer was posed on the cysteamine SAM by using glutaraldehyde cross-linking. Alginate-factor B was then electrostatically immobilized on the crystal with the aid of the negatively-charged alginate layer. The conditions of immobilizing antibodies were optimized in detail. Comparing with the conventional glutaraldehyde covalent immobilization, the immunosensor with the proposed immobilization procedure shows an improved performance in terms of the magnitude of the response and sensitivity. Moreover, the proposed immobilization procedure results in immobilized entities with relatively high biological activity and favorable immunosensing characteristics. The QCM devices are readily regenerated. Analytical results obtained from the tests of non-specific adsorption and recoveries indicated that the immunosensor prepared by use of the chitosan/alginate adsorption procedure is a useful tool for the determination of factor B in plasma in clinical diagnosis.(9) A quartz crystal microbalance sensor was proposed for the detection of small molecule biotin based on the mixed self-assembled monolayer of thiols on gold substrate and the bioaffinity difference between an analyte (biotin) and an analogue compound 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) in binding avidin. Avidin formed a metastable complex with HABA immobilized on the crystal surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The frequency change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the frequency change and the biotin concentration. The use of mixed SAMs allows the stable attachment of bioreceptor molecules on the QCM and enhances the amount of the immobilized molecules on the QCM, as a longer“space arm”in the mixed SAMs makes this monolayer membrane more accessible to capture the immobilized molecules. The proposed bioaffinity sensor has nice response to biotin in the range of 0.017– 1.67μg/mL. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.更多还原