摘要
金纳米(AuNPs)粒具有优良的电学、光学、热力学与催化性能,已广泛地应用于物理学、化学、生物学、医学和材料科学及其交叉学科。AuNPs毒性小、粒径小,易于被细胞摄取,经功能化修饰后,可以用于生物成像、药物与基因传递等治疗与诊断领域。另外,AuNPs具有卓越的电传导性、等离子体共振特性和高催化性,且其表面易于修饰,已经广泛应用于构建光学、电化学等生物传感器。
化学发光(CL)分析法是根据化学反应产生的辐射光的强度来确定物质含量的分析方法。CL分析技术具有分析速度快、灵敏度高、仪器设备简单、线性范围广且适用于微量分析等特性,已广泛应用于分析化学的各个领域。功能化AuNPs既可用作CL底物的载体；另一方面,由于AuNPs尺寸小、表面效应明显,因此也具有CL催化剂的作用,如AuNPs可以催化银离子形成银原子沉积在AuNPs的表面,同时促进鲁米诺自由基的形成,后者可与溶液中溶解的氧反应产生CL信号。因而,AuNPs可以显著增大CL强度。Luminol-AgNO3-AuNPsCL体系已经用于CL免疫分析、DNA以及有机小分子的含量测定。本论文将AuNPs催化的CL技术与其他技术相结合,发展了一系列具有创新意义的基于金纳米粒的人血浆蛋白、腺苷及端粒酶活性CL分析法。整个论文由以下五个部分组成：
第一章绪论
金纳米粒作为一种具有独特理化性质的贵金属纳米粒子,一直是科学家研究的热点。本章简单介绍了金纳米粒的合成方法、优良性能,并且详细综述了金纳米粒在药物与基因传递、诊断与成像、生物传感器以及CL催化等领域中的应用,着重介绍了金纳米粒作为一种高效的催化剂在鲁米诺CL体系中的重要意义。
第二章基于金纳米粒的适配体体系用于IgE的分析
核酸适配体(Aptamers,适配体)是指利用指数富集配体系统进化技术(SELEX),从随机单链寡聚核苷酸库中筛选得到的能够与靶分子特异性结合的单链DNA或RNA,已经广泛应用于临床治疗、诊断与生物技术研究。人免疫球蛋白E(IgE)是五种免疫球蛋白的一种,对过敏反应有着重要的作用。与其他免疫球蛋白不同的是人血浆中IgE的含量极低,但是过敏性哮喘病人体内的IgE含量会升高。本章发展了基于适配体识别,AuNPs放大体系用于IgE的高选择性、高灵敏CL分析。本策略主要依赖于固定至聚苯乙烯微孔板上的捕获抗体、目标蛋白和适配体功能化金纳米粒(Apt-AuNPs)三者之间形成“三明治”结构的免疫复合物。只有在加入目标蛋白的情况下,金纳米粒才能组装于微孔板上,组装成功的AuNPs进而催化鲁米诺-硝酸银CL体系产生CL信号。为了使CL信号进一步放大,随后在金纳米粒表面进行了简单的氯金酸催化的沉积反应。该CL信号放大技术可使模型蛋白IgE的最低检测限达到50fM,优越于已报道的其他基于适配体的IgE检测技术。该检测方法专属性强,能够较为理想地分辨出IgE,而不受其它血浆蛋白的干扰,如IgG、IgA和IgM等,已成功应用于35个人血样中IgE含量的检测,与对照值相关性良好。综合而言,该检测方法具有显著的优点,如最低检测限能够达到fM级别、线性范围广(跨度达4个数量级),可以拓宽至基于适配体的其它蛋白的含量测定,有望在临床、环境以及生物防御等领域的目标蛋白高灵敏、高选择性检测中获得应用。
第三章基于适配体-金纳米粒识别检测的血小板衍生化生长因子化学发光分析新技术
血小板衍生化生长因子(PDGF)可通过与特异的、高亲和的细胞膜受体结合促进细胞分裂繁殖。因此,PDGF在血管增生中起着重要的作用,并且与细胞转化、肿瘤生长及进展存在着密切联系。作为PDGF的重要亚型,PDGF-BB在正常细胞中的含量极低,甚至检测不到。但是在胶质瘤和肉瘤等肿瘤中,PDGF-BB水平会急剧升高。因此,作为一种肿瘤标志物,PDGF-BB的含量测定在癌症诊断与治疗中有着重要的意义。本章发展了一种新型的、基于适配体识别-金纳米标记的CL分析技术,用于PDGF-BB的高灵敏度检测。该技术首先将PDGF-BB抗体通过氨基-羧基反应结合至表面具有活性羧基的96孔板上；而生物素标记的PDGF-BB适配体序列,预先组装于链霉亲和素金纳米粒上形成Apt-AuNPs复合物；在PDGF-BB作用下发生夹心反应,Apt-AuNPs复合物结合于96孔板上；通过羟胺(NH2OH)与氯金酸(HAuCl4)的氧化还原反应,将96孔板上结合的AuNPs进一步催化放大形成具有更大表面积的粒子,最后采用luminol-AgNO3体系测定其CL信号强度。最优条件下,PDGF-BB的线性范围为0.1～1000pM(LgI=0.9524LgC+1.4995,R2=0.9883),检测限为10fM。此外,专属性实验表明：相同浓度的PDGF-AA、PDGF-AB、干扰素a2b及免疫球蛋白(IgG、IgA、IgM)对PDGF-BB的测定无明显干扰。综合而言,本章建立的CL方法能够简单、灵敏、高效地检测PDGF-BB,并有望扩展到其他蛋白的分析。
第四章基于核酸外切酶辅助循环放大的高灵敏化学发光传感器
腺苷作为一种嘌呤核苷,在生物化学上扮演重要角色,包括以腺苷三磷酸或腺苷双磷酸形式转移能量,或是以环状腺苷单磷酸进行信号传递等,此外腺苷也是一种抑制性神经传导物,可能会促进睡眠。因此,监测生理条件下的腺苷水平有着重大的意义。核酸外切酶Ⅲ(Exo Ⅲ)对双链DNA具有高度特异性,能降解平滑末端、3’凹陷末端及有切口的双链DNA,但是不能降解单链DNA与3’-突出末端双链DNA。本章我们利用Exo Ⅲ辅助循环信号放大技术,构建了新型的AuNPs催化CL适配体传感器用于腺苷的高灵敏检测分析。腺苷适配体通过与96孔板上的捕获探针杂交固定于96孔板上；同时合成报告探针功能化的金纳米粒,此AuNPs表面的报告探针与上述腺苷适配体末端部分碱基互补。在腺苷存在的情况下,腺苷适配体会折叠形成双链DNA将腺苷包裹起来,加入Exo Ⅲ,双链DNA从3’-OH端开始逐渐降解直到腺苷释放出来,释放出来的腺苷再次与96孔板上的腺苷适配体结合,降解得到的单链DNA由于3’-端降解失去四个碱基,不能够再与AuNPs表面的DNA碱基形成稳定配对,从而影响AuNPs在96孔板上的结合,进而降低所测得的CL信号强度。CL信号减弱强度与腺苷浓度线性相关良好,此法测得腺苷的最低检测限为0.5nM,低于文献报道的其他基于适配体的腺苷检测方法。该新型CL检测方法为大量目标物提供了新的检测方法,为临床诊断、环境监测、药物分析以及生物医学研究等提供了一个高效多能的平台。
第五章基于分子信标-金纳米粒的端粒酶活性化学发光放大检测技术
端粒酶是一种与许多肿瘤相关的敏感性和特异性的肿瘤标志物。因此,端粒酶活性的快速、灵敏分析对于肿瘤的检测以及治疗方案功效的评价显得至关重要。本章我们将基于分子信标功能化金纳米粒(MB-AuNPs)的CL放大技术应用到人非小细胞肺癌A549细胞中端粒酶活性的检测。端粒引物在端粒酶与脱氧三磷酸核苷的作用下复制得到靶向DNA。将一端可与96孔板上捕获DNA发生碱基配对、另一端生物素修饰的分子信标偶联至链霉亲和素标记的金纳米粒上得到MB-AuNPs,此MB-AuNPs上的MB在上述靶向DNA的作用下产生开环现象,从而将AuNPs连接至96孔板上。连接到96孔板的AuNPs经NH2OH-HAuCl4氧化还原反应沉积放大后采用luminol-AgNO3CL体系测定CL强度,从而间接检测端粒酶的活性。本法可检测出200个A549细胞中的端粒酶活性,以合成的靶向DNA浓度或人肿瘤细胞数目与CL强度作标准曲线,获得了良好的线性,证明此方法可用于肿瘤细胞中端粒酶活性的简单、灵敏、直观检测分析。
The unique electric, optical, thermodynamic and catalytic properties of gold nanoparticles (AuNPs) have stimulated the increasing interest in the application of AuNPs in physics, chemistry, biology, materials science and cross subjects. Due to their less toxicity, small size and ease of cellular uptake, AuNPs have been an obvious choice in gene delivery, drug delivery, bioimaging and other therapeutic and diagnostic applications. The excellent conductivity, surface plasmon resonance (SPR) and high catalytic properties of AuNPs facilitate extensive application of AuNPs in construction of optical, electrochemical and piezoelectric biosensors with enhanced analytical performance.
Chemiluminescence (CL) is the production of electromagnetic radiation by a chemical reaction. The CL intensity is directly proportional to the concentration of a limiting reactant involved in the CL reaction. CL has been combined with other technologies for a wide range of applications in various fields owing to its extremely hypersensitivity, simple instrumentation, wide calibration ranges and suitability for miniaturization in analytical chemistry. Due to their ease of modification, functionalized AuNPs are suitable for the carriers of CL reagents, such as AuNPs functionalized with luminol can effectively improve the CL sensitivity of analyte. Owing to their small size and prominent surface effect, AuNPs have been used as CL enhancer. For instance, AuNPs as nucleation centers catalyzed the reduction of AgN03to Ag atoms by luminol to yield Au/Ag core/shell nanoparticles. Meanwhile, luminol was oxidized to luminol radical, which further reacted with the dissolved oxygen, giving rise to light emission. The luminol-AgNO3-AuNPs CL system has the advantages of low background and good stability, which may be of great potential for the CL immunoassay, DNA and small molecules analysis. Hence, combining with other technologies, a series of CL analysis methods based on AuNPs have been developed to detect human plasma proteins, adenosine and telomerase activity. Description of research in the thesis is presented as follows:
Chapter1:Introduction
Precious metal nanoparticles, especially AuNPs are being one of the hot research areas due to their unique physical and chemical properties. This chapter includes the following parts:the synthesis methods and excellent properties of AuNPs; the applications of AuNPs in bioimaging, diagnostics, biosensors, CL catalyst, drug and gene delivery; the important application of AuNPs in luminol-AgNO3CL system.
Chapter2:Aptameric system for detection of IgE based on gold nanoparticles
Aptamers are small oligonucleotides that bind with affinity and specificity to a large number of target molecules, which are selected from a large combinatorial oligonucleotides library through an in vitro evolution process termed SELEX (systematic evolution of ligands by exponential enrichment). Aptamers have possessed a broader range of applications in therapeutics, diagnostics and biotechnology. IgE is one of the five classes of immunoglobulins, which plays a key role in allergic responses. Unlike other immunoglobulins, the circulating concentration of IgE is very low, however, elevated in patients afflicted with allergic asthma. A novel approach is proposed in this chapter for the development of an aptameric assay system for protein based on non-stripping gold nanoparticles-triggered CL upon target binding. The strategy chiefly depends on the formation of a sandwich-type immunocomplex among the capture antibody immobilized on the polystyrene microwells, target protein and aptamer-functionalized AuNPs. Introduction of target protein into the assay system leads to the attachment of AuNPs onto the surface of the microwells and thus the assembled AuNPs could trigger the reaction between luminol and AgNO3with a CL emission. Further signal amplification was achieved by a simple gold metal catalytic deposition onto the AuNPs. Such an amplified CL transduction allowed for the detection of model target IgE down to50fM, which is better than most existing aptameric methods for IgE detection. This new protocol also provided a good capability in discriminating IgE from nontarget proteins such as IgG, IgA, IgM and interferon. The practical application of the proposed AuNPs-based immunoassay was successfully carried out for the determination of IgE in35human serum samples. Overall, the proposed assay system exhibits excellent analytical characteristics (e.g., a detection limit on the attomolar scale and a linear dynamic range of4orders of magnitude), and it is also straightforward to adapt this strategy to detect a spectrum of other proteins by using different aptamers. This new CL strategy might create a novel technology for developing simple biosensors in the sensitive and selective detection of target protein in a variety of clinical, environmental and biodefense applications.
Chapter3:Aptamer-based biosensor for chemiluminescence immunoassay of platelet-derived growth factor using gold nanoparticles
Platelet-derived growth factor (PDGF) is a growth factor protein that has growth-promoting activity to stimulate the division and proliferation of the cells through binding its receptors on the cell membrane surface. Therefore, PDGF plays an important role in angiogenesis and is linked to cell transformation and tumor growth and progression. PDGF-BB, one of the important isoforms of PDGF, is expressed at low level or undetectable in normal cells, but is found to be overexpressed in some human tumors, including gliboblastomas and sarcomas. Detection of PDGF is significant for its potential use as a protein marker in cancer diagnosis. In this chapter, a novel aptamer-based CL immunoassay coupling with AuNPs as the tag was developed for the rapid, sensitive detection of PDGF-BB. Typically, PDGF-BB antibodies were immobilized on the surface of96-well plate to capture target PDGF-BB, and then sandwiched with the aptamer conjugates which were prepared by assembling AuNPs with PDGF-BB aptamer. The captured AuNPs on the96-well plate was further enlarged in the presence of hydroxylamine and chlorauric acid. Thus the enlarged AuNPs triggered the reaction between luminol and silver nitrate for the generation of a CL signal. Under the optimal conditions, a good linear relation was achieved in the range of0.1-1000pM PDGF-BB with a detection limit down to10fM. Other PDGF isoforms (PDGF-AA, PDGF-AB), IgG, IgA, IgM and IFN-α2b showed no obvious interference for the determination of PDGF-BB. Therefore, our strategy provided a simple and sensitive detection of PDGF-BB, and with high specificity, which could find wide applications in protein assay.
Chapter4:Ultrasensitive chemiluminescence aptasensor based on Exonuclease-assisted recycling amplification
Adenosine is a nucleoside with value role in biochemistry, including the energy translation in the form of adenosine triphosphate or adenosine diphosphate, and the signal transmission in adenosine monophasphate form. Adenosine has received much attention due to its crucial signaling functions in both the peripheral and central nervous system. Elevated levels of adenosine in the brain appear to promote sleep. The monitor of adenosine under physiological conditions therefore is of great value. Exonuclease-III (Exo-III) could catalyze the stepwise removal of mononucleotides from3'-OH termini of double-stranded DNA when the substrates are blunt or recessed3'-terminus. However, its activity on single-strand DNA and protruding3'-terminus of double-stranded DNA is limited. In this chapter, we develop an Exo Ⅲ-assisted aptamer-based target recycling amplification strategy for sensitive and selective CL determination of small molecules, employing adenosine as the model target analyte. The system contained an amino DNA sequence as capture probe, aptamer A as detection probe, AuNPs-linked DNA sequence as reporter probe. Aptamer A consisted of the whole adenosine aptamer and an extension sequence which hybridized with capture probe immobilized on the wells, whereas12bases of the adenosine aptamer on the3'-terminal hybridized to the reporter probe functionalized with AuNPs. When adenosine was added, aptamer A turned into a duplex DNA with3'-hydroxyl termini relied on the structure-switching properties of aptamers binding to their target molecules. Therefore, adenosine could reduce the gold probes hybridized with aptamer A, then minus CL signal could be detected as a readout signal for the quantitative detection of adenosine. As the Exo Ⅲ catalyzed the stepwise removal of mononucleotides from3'-hydroxyl termini of double stranded DNA when the substrates are recessed3'-terminus or blunt, adenosine was released and hybridized with the second aptamer A. Thus Exo Ⅲ could catalyze a new cycle of probe transformation and led to enlarged unusual high sensitivity. Experimental results revealed that the enzymatic-assisted recycling strategy enabled the monitoring of adenosine with low detection limits of0.5nM, which is much lower than other analysis methods previously reported. This unique property of Exo Ⅲ makes the proposed sensor hold great potential for highly sensitive, selective and simple detection of a wide range of target molecules. The approach thus provides a versatile platform for clinical diagnosis, environmental monitoring, pharmaceutical and biomedical analysis.
Chapter5:Amplified detection of telomerase activity using chemiluminescence analysis based on molecular beacon-gold nanoparticles
Telomerase has been regarded as an important sensitive and specific cancers marker, which is closely linked to malignancy and tumor progression. Rapid and ultrasensitive detection of telomerase activity plays a vital role in measure of malignancy and evaluation of treatment plans. In this chapter, a CL methods based on molecular beacon-gold nanoparticles (MB-AuNPs) for amplified detection telomerase activity originating from non-small-cell lung cancer A549cell is described. This strategy involves the telomerization of primer, in the presence of telomerase from A549cell extract and dNTP, followed linked to molecular beacon functionalized AuNPs by hybridization. MB could be open to linear structure and hybridize with the capture probe immobilized on the surface of DNA-binding96-well plate. As a consequence, AuNPs were captured on the plate-surface, which were further enlarged by HAuCl4-NH2OH. Then CL signals were considerably enhanced. The methods enable the detection telomerase activity that was extracted from200A549cancer cells, and good linear relationship between CL intensity and the concentrations of synthetic targeted DNA and human cancer cells were obtained. These easily fabricated MB-AuNPs biosensors show excellent promise for simple, sensitive and visible detection of telomerase activity in cancer cells.
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