贵金属纳米材料在元素标记、生物传感和成像中的应用

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英文题名：Application of Noble Metal Nanomaterials in Element-Tagging ICP-MS Protein Detection, Biosensing and Bioimaging 作者：刘敬民 论文级别：博士 学科专业名称：分析化学 中文关键词：贵金属纳米材料 ; 电感耦合等离子体质谱 ; 元素标记 ; 光学传感 ; 靶标生物成像 英文关键词：noble metal nanomaterials ; ICP-MS ; elemental tagging ; optical biosensing ; targeted bioimaging 学位年度：2013 导师：严秀平 学科代码：070302 学位授予单位：南开大学 论文提交日期：2013-05-01 答辩委员会主席：万谦宏

摘要

贵金属纳米材料由于其独特的表面等离子体共振(SPR)性质引起了科研工作者的巨大兴趣。随着多种形貌贵金属纳米材料的可控合成及其功能化表面化学技术的日臻成熟,贵金属纳米材料已广泛应用于生物标记、传感成像、分析分离及生物医学领域。本论文旨在利用贵金属纳米材料电感耦合等离子体质谱(ICP-MS)信号放大、SPR和荧光等特性,将不同形貌的贵金属纳米材料应用于生物标记、生物传感和成像。本论文的主要内容与创新点如下：
     (1)基于毛细管电泳(CE)电感耦合等离子体质谱-(ICP-MS)联用技术和纳米金(AuNPs)信号放大及其对于生物分子的静电吸附作用,发展了一种简单、快速、经济、高效的元素标记方法,实现了对尿白蛋白的超高灵敏度高选择性检测。采用柠檬酸钠还原法合成粒径分散均匀的纳米金颗粒,将白蛋白(albumin)与过量的纳米金通过静电吸附作用结合,然后通过毛细管电泳将白蛋白-纳米金结合物和反应剩余的纳米金分离,最后同时进入ICP-MS检测。金元素在ICP-MS上具有高灵敏度,单个纳米金颗粒中含有几十万个金原子,因此将纳米金标记到白蛋白上可以实现理想的信号放大效果。实验中采用自行设计的四通联用接口,并详细优化了纳米金和白蛋白的结合条件以及毛细管电泳的分离条件。在优化的条件下,纳米金颗粒可以特异性地结合白蛋白并能与剩余纳米金颗粒得到良好的CE分离。本方法可以实现尿液中白蛋白的高灵敏度高选择性准确定量,线性范围为1.8pM-18nM,检出限(S/N=3)为0.5pM。应用该方法测得人尿液样品中白蛋白的含量在270～330nM之间,加标回收率在93%-100%之间。
     (2)基于适配体(aptamer)和抗体(antibody)的特异性识别目标蛋白,纳米金(AuNPs)和纳米银(AgNPs)的ICP-MS元素标记效果,借助适配体修饰的纳米金颗粒(apt-AuNPs)和抗体修饰的纳米银颗粒(ab-AgNPs)分别作为目标物细胞色素C和胰岛素的特异性识别探针,构建了一种多元素标记贵金属纳米颗粒信号放大的ICP-MS生物检测方法,实现了对于两种目标蛋白的高灵敏度高选择性的同时准确定量。制备了醛基表面功能化的磁性微球(MMPs)作为磁性分离载体,可以保证快速分离过量的元素标记探针。对实验中的表面功能化的MMPs合成条件、MMPs的用量、apt-AuNPs和ab-AgNPs探针的用量以及反应时间等条件进行了详细优化。在优化条件下,该方法对细胞色素C和胰岛素检测的线性范围分别为0.1～20nM和0.2～40nM,检出限(3s)分别为30pM和110pM。该方法对细胞色素C和胰岛素具有很好的选择性,并成功应用于人血清样品中的两种目标蛋白的同时检测,加标回收率在87%-98%之间。
     (3)以半胱氨酸(cys)修饰的金纳米棒作为比色探针,借助金纳米棒长轴SPR峰的高灵敏度和半胱氨酸与铜离子的特异性配位结合作用,实现了铜离子的高灵敏度高选择性快速检测。金纳米棒的SPR峰的强度和位置对于金纳米棒微环境介质的折光率异常敏感。首先在金纳米棒的短轴端通过Au-S共价键修饰上半胱氨酸,铜离子存在时与半胱氨酸发生强烈的配位结合形成cys-Cu-cys三元结合态,使得金纳米棒发生头尾相接的自组装过程,从而引起金纳米棒SPR峰红移,同时金纳米棒溶液颜色由蓝绿色变为暗灰色,据此过程产生对于铜离子的特异性响应,实现比色探针快速检测铜离子。对可能影响金纳米棒比色探针检测效率的实验条件进行了详细优化,包括缓冲介质的pH值、半胱氨酸的浓度、体系盐浓度以及半胱氨酸和铜离子配位结合的动力学过程。在优化的条件下,该方法对铜离子的检测线性范围为1～100μM,检出限(3s)为0.34μM。该方法对铜离子具有很好的选择性,并成功应用于环境水样中铜离子的准确检测,加标回收率在90%-107%之间。
     (4)基于碱基T与金属配对结构(T-Hg-T)和适配体(aptamer)构建了一种分子构型开关(MCS)生物传感器用于选择性检测三磷酸腺苷(ATP),这种传感器结合了适配体的特异性、单链DNA的序列可调性、T-Hg-T配对的分子构型开关以及巯基十一烷酸修饰的金纳米簇(MUA-AuNCs)对于汞离子的高选择性高灵敏度的定量能力。ATP的适配体与汞离子竞争结合富含碱基T序列的分子构型开关,目标物ATP与适配体结合后打破竞争反应从而影响到游离态汞离子的量,释放出的汞离子由MUA-AuNCs的荧光猝灭程度定量,最终实现信号转导。对实验中荧光纳米簇的种类、反应动力学、缓冲体系的pH值、以及汞离子和单链DNA的浓度进行了详细优化。在优化的条件下,该方法对ATP的检测线性范围为100～2000nM,检出限(3s)为48nM。该方法对ATP有很好的选择性,并成功应用于人尿液样品中腺苷的准确检测,加标回收率在89%-105%之间。
     (5)基于胰蛋白酶修饰的金纳米簇(try-AuNCs)多功能荧光探针,构建表面等离子体增强能量转移(SPEET)生物传感器用于选择性检测肝素钠,进一步通过叶酸表面功能化的try-AuNCs构建了一种近红外荧光探针用于生物活体靶标荧光成像。SPEET/try-AuNCs生物传感器是基于try-AuNCs和巯基乙胺修饰的纳米金之间的能量转移过程,目标物肝素钠的存在可以使得纳米金SPR吸收红移并且拉远纳米金与纳米簇之间的距离,从而打破能量转移过程,使得体系荧光恢复。在优化的条件下,该方法对肝素钠的检测线性范围为0.1-4.0μgmL-1,检出限(3s)为0.05μg mL-1。该方法对肝素钠有很好的选择性,并成功应用于人血清样品中肝素钠的准确测定,加标回收率在97%-100%之间。进一步的叶酸表面功能化try-AuNCs得到具有靶标肿瘤能力的近红外荧光成像探针,并应用于叶酸受体过表达的hela肿瘤的活体靶标成像。这种探针表现了良好的生物相容性和低细胞毒性,并且对于hela冲瘤具有较强的特异性靶标能力。
Noble metal nanomaterials (NMNMs) have been proven to be highly versatile and tunable materials for a range of bioapplications including biosensing, bioimaging, biophysical research, medical diagnostics, and cancer therapy. Noble metal nanostructures possess significant ICP-MS signal amplification effect and optical field enhancements, unique surface plasmon resonance (SPR) and fluorescence property, controllable size and morphology in preparation, excellent biocompatibility and stability, and the availability of surface modification of diverse bioactive molecules. This dissertation aims at developing NMNMs-based elemental tagging ICP-MS ultrasensitive assay, biosensing and bioimaging assays. The main contents and novelty of the dissertation are summarized as follows:
     (1) A strategy based on capillary electrophoresis with on-line inductively coupled plasma mass spectroscopic detection (CE-ICP-MS) in conjunction with gold nanoparticles (AuNPs) amplification was constructed for ultrasensitive quantification of human urinary protein. The albumin in the sample solution was incubated with excess AuNPs to form the AuNP-albumin adduct. The excess AuNPs and the AuNP-albumin adduct were then effectively separated by CE for on-line ICP-MS detection. As a result of AuNPs-tagging, more than2000gold atoms on average were attached to each albumin molecule to successfully achieve a significant amplification of ICP-MS signal with extremely low limit of detection (0.5pM for280nL of sample injection, corresponding to0.1amol) and a wide linear response over4orders of magnitude. The relative standard deviations of the migration time, peak area, and peak height for seven replicate injections of a mixture of0.4pM AuNPs and9.0pM albumin ranged from1.8%to4.4%. The developed method was successfully applied for detecting albumin in human urine samples with quantitative recoveries in the range of93.0-99.7%. The methodology demonstrated here has potential for simultaneous determination of low-abundance multiple biomarkers of interest via multiple nanomaterials tags because of high-resolution CE separation and ultrasensitive ICP-MS detection.
     (2) An ultrasensitive method for simultaneous determination of cytochrome c (cyt-c) and insulin was developed by combining aptamer-based bioassay and immunoassay, multielement-tagging and ICP-MS. Aptamer-modified gold nanoparticles (apt-AuNPs) and antibody-modified silver nanoparticles (ab-AgNPs) were employed as specific element tags for cyt-c and insulin, respectively. The prepared surface-functionalized magnetic microparticles (MMPs) were used for efficient and fast magnetic separation. The bioassay conditions were carefully optimized, including the amount of MMPs, the concentration of AuNPs and AgNPs, and the reaction time. Under optimal conditions, the developed method gave a linear range of0.1-20nM for cyt-c and0.2-40nM for insulin, a detection limit (3s) of1.5fmol (30pM in50mL) for cyt-c and5.5fmol (110pM in50mL) for insulin. The precision (relative standard deviation) for six replicate determinations of cyt-c (0.6nM) and insulin (2.0nM) was6.6%and6.0%, respectively. The present method exhibits good specificity with recoveries from87%to98%for spiked cyt-c and insulin in human serum samples. The methodology demonstrated here provides a new possibility for bioassays and clinical diagnoses, which has potential for simultaneous determination of two or more low-abundance biomarkers of interest via multi-element tags.
     (3) A gold nanorod (AuNR) based colorimetric probe was reported for the rapid and selective detection of Cu2+ions. The probe was fabricated by functionalizing cysteine (Cys) onto AuNR (Cys-AuNR) with an aspect ratio of2.3. The strong coordination of Cu2+with cysteine resulted in a stable Cys-Cu-Cys complex, and induced the aggregation of the colloidal nanorods along with a rapid colour change from blue-green to dark gray. Potential factors affecting the performance of the probe for the detection of Cu2+were carefully optimized, including the pH value of the buffer media, the concentration of cysteine, and the kinetics for the coordination of Cu2+with Cys-AuNR. Under optimal conditions, the developed colorimetric method gave a linear range of1-100mM for Cu2+, and a detection limit (3s) of0.34mM. Moreover, the developed method exhibited excellent selectivity for Cu2+, and quantitative spike-recoveries from90%to107%in environmental water samples. The proposed colorimetric approach can in principle be used to detect other metal ions by functionalizing various specific ligands onto the AuNR that can selectively bind the other target metal ions.
     (4) A competitive aptamer bioassay was developed for the selective detection of adenosine triphosphate (ATP). The proposed bioassay employed the T-Hg-T induced hairpin-structure as the molecule conformational switch (MCS), aptamer as a specific recognizer, and mercaptoundecanoic acid modified gold nanoclusters (MUA-AuNCs) as a sensitive signal reporter. The T-rich MCS ssDNA with the sequence complementary with that for the aptamer of ATP was bound with Hg2+to form the metal-paired hairpin-structure. Addition of the aptamer and its target biomolecule ATP resulted in a competitive aptamer bioassay. The aptamer competed with Hg2+to hybridize with T-rich MCS ssDNA, thereby destroyed the hairpin-structure. As a result, the Hg2+was released and the signal transduction was achieved. The ATP affected the interaction between aptamer and hairpin-structure, thus mediated the release of Hg2+, which was sensitively quantified by fluorescent MUA-AuNCs. Under selected conditions, the developed method allowed sensitive and selective detection of ATP with a linear range of100-2000nM and a detection limit (3s) of48nM. The relative standard deviation for sixty replicate detections of200nM ATP was2.1%, and the recoveries of the spiked ATP in urine samples ranged from89%to105%. The developed metal-paired MCS can be easily extended to the sensitive and selective detection of other biomolecules by changing the base sequence of hairpin structure and choosing the corresponding aptamer for the target biomolecule.
     (5) A multifunctional fluorescence probe was fabricated by the preparation of trypsin stabilized gold nanoclusters (try-AuNCs) with near-infrared fluorescence for biosensing heparin based on surface plasmon enhanced energy transfer (SPEET), and folic acid (FA) modified try-AuNCs for in vivo cancer bioimaging. The SPEET/try-AuNCs fluorescence biosensor was designed via heparin mediated energy transfer between try-AuNCs and cysteamine modified gold nanoparticles (cyst-AuNPs). The developed SPEET/try-AuNCs fluorescence biosensor allowed sensitive and selective detection of heparin with a linear range of0.1-4.0μg mL-1and a detection limit (3s) of0.05μg mL-1. The relative standard deviation for eleven replicate detections of2.5μg mL-1heparin was1.1%, and the recoveries of the spiked heparin in human serum samples ranged from97%to100%. In addition, folic acid was immobilized on the surface of try-AuNCs to ameliorate the specific affinity of AuNCs for tumors, and the near-infrared fluorescent FA-try-AuNCs were applied for in vivo cancer imaging of high folate receptor (FR) expressing Hela tumor. In vivo study of the dynamic behavior and targeting ability of FA-try-AuNCs probe to Hela tumor bearing mice and normal nude mice validated the high specific affinity of FA-try-AuNCs probe to FR positive tumors. The results show that the prepared try-AuNCs have great potential as multifunctional biomaterials for biosensing biomolecules with SPEET mode and in vivo cancer imaging with high targeting ability.

引文

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