【作者】 薛庆旺；

【导师】 姜玮；

【作者基本信息】 山东大学 ， 分析化学， 2012， 博士

【摘要】 近年来,人们一直致力于发展和完善生物大分子的研究和分析方法,使其更加灵敏、精确、便捷、经济,以满足各个领域对其越来越高的要求。生物分子信号放大检测技术是近年来发展起来的一种利用各种工具酶和纳米颗粒等手段,并结合电化学、光学等技术建立起来的高灵敏检测方法,可实现对生物分子的直接高灵敏检测,是生物分析科学领域中的一个研究重点和热点。如何利用现有分子生物学技术和手段将生物分子的含量、相互作用等信息实时、灵敏、特异地转化为易于检测的物理量并拓展其在分子生物学及生物医学中的应用,如何设计新的信号转换手段和建立新的生物分子信号放大检测技术平台来解决生命研究过程中的新问题,仍然是生物医学和分析化学工作者所面临的重大研究科题。本工作以上述科学问题为目标,以蛋白质和核酸这两类组成生命的最主要生物大分子为研究对象,利用多种核酸工具酶、纳米颗粒和荧光核酸探针技术,建立了一系列的生物大分子荧光信号放大检测技术平台,在蛋白质和核酸定量分析方面得到了重要的应用。主要内容归纳如下：1、单球体积荧光放大法检测生物大分子近年来,随着纳米材料科学的发展,不同组成、大小、形状的磁性纳米颗粒已经广泛应用于生物分析研究领域。功能化的磁性纳米颗粒作为标记探针,已经越来越引起人们的关注。磁性纳米颗粒具有磁性、粒径小、表面积大且表面可修饰等特点。功能化的纳米磁性纳米颗粒标记生物分子时,一方面能够作为载体起到富集、固定的作用,同时还保持了标记生物分子的生物化学活性。另一方面,纳米磁性纳米颗粒在外加磁场的作用下,能够快速有效地实现免疫复合物与未结合物的分离。所以,在免疫测定中,应用纳米磁性纳米颗粒和磁分离技术能够降低假阳性信号,提高信噪比,从而提高蛋白质检测方法的灵敏度。在本文中,我们基于信号双重扩增(DNA扩增、SYBR Green I/BT-DNA结合物荧光信号扩增)结合磁分离技术,利用落射荧光显微术建立了一种超灵敏的靶蛋白荧光免疫测定方法。在高浓度时,荧光强度与靶抗原在5.0×10-13～8.0×10-15mol L-1的浓度对数范围内呈良好的线性关系。在低浓度时,一个功能化得磁纳米球对应一个蛋白分子,这时我们通过计数单个磁纳米球定量检测单个生物分子。磁纳米球的数目和抗体的浓度在8.0×10-14～3.0×10-15mol L-1的范围内呈良好的线性关系。2、多重标记结合RCA技术构建免疫分析新方法用于蛋白质定量检测为了提高蛋白检测的灵敏度,人们已经努力发展了一些新的蛋白检测技术。其中,荧光检测技术联合信号扩增技术是最流行的检测技术。特定核酸序列的扩增是分子生物学的一项基本技术,随着分子生物学的发展与生物化学、生物物理学等交叉学科在该领域的广泛应用,新的核酸扩增技术不断涌现。其中,滚环扩增(Rolling Circle Amplification, RCA)技术凭借其高特异性、高灵敏度和易操作性在近几年中逐渐引起人们的注意,并越来越多的用于基础研究和临床诊断分析及医药领域中。与其他核酸扩增技术相比,RCA技术具有高灵敏度、高特异性、多元性和高通量、等温反应和操作简单等优势。为了满足低浓度蛋白检测的需要和解决多重荧光标记和纳米探针的信号放大存在的问题,本章提出了一种基于链霉亲和素-生物素多重结合和滚环扩增辅助组装的级联荧光DNA纳米标签作为信号标记的新型级联荧光信号放大技术超灵敏检测靶蛋白目标分子。荧光强度与人IgG浓度的对数成线性关系,并且动态范围从1×10-12molL-1-1×10-15mol L-1。超过3个数量级。线性相关系数为0.995。检测限达到0.9×10-15mol L-1(采用3倍的信噪比)。实验结果出示了该方法具有较宽的动态范围,超高的灵敏度、可接受的重现性、好的特异性和较低的基质效应影响。另外更重要的是,该方法不需要复杂的接合化学并且该方法出示了快的反应动力学和简单的操作在组装荧光DNA标签。与功能化纳米粒子的标签相比,该策略标记物避免了复杂和繁琐的合成工艺。因此该策略将在免疫靶蛋白的超灵敏检测中会成为一个强有力的检测工具。3、磁性纳米颗粒结合RCA技术构建免疫分析新方法用于蛋白质定量检测在荧光免疫测定法中,为了定量检测样品中低浓度的靶蛋白,实现超灵敏分析,一个关键因素在于获取强的、稳定的光信号强度。但由于有机染料荧光强度低、易发生光漂白,限制了单个有机染料标记靶目标检测时分析灵敏度的提高。为了解决这一问题,采用信号扩增技术,即多个荧光探针标记物对单个靶分子同时识别。这样能够增强荧光信号的强度,提高灵敏度,实现超灵敏分析。典型的高灵敏的分析标记物包括酶、或者它们的聚合形式、以及功能化的纳米粒子比如染料填充的纳米粒子、金纳米粒子、量子点(QD)。尽管这些技术起了很重要的作用,但是这些技术标记的信号放大能力仍然是有限的。特定核酸序列的扩增是分子生物学的一项基本技术。其中,滚环扩增(RCA)技术凭借其高特异性、高灵敏度和易操作性在近几年中逐渐引起人们的注意,并越来越多的用于基础研究和临床诊断分析及医药领域中。与其他核酸扩增技术相比,RCA技术具有高灵敏度、高特异性、多元性和高通量、等温反应和操作简单等优势。随着核酸序列越来越多的被应用于生物蛋白质标记物,本章中,为了发展具有最大信号放大功能和最小非特异性吸附的信号标记物来高灵敏的检测蛋白。我们首次报道了一种基于磁纳米颗粒富集DNA的RCA扩增免疫分析新方法用于蛋白检测,磁纳米颗粒-RCA免疫分析方法,该方法出示了很多优点：(1)磁分离技术的运用明显的降低了磁纳米颗粒免疫探针的非特异性吸附：(2)三次信号放大技术(磁纳米颗粒富集DNA引物、RCA放大、荧光DNA纳米标签荧光放大)的联用极大的提高了方法的灵敏度。另外,该技术方法可以比较容易的制备卸载DNA的抗体检测试剂和并且操作比较简单。因此,发展的该免疫分析方法将成为一种强有力的工具用于高灵敏的蛋白检测。在对数范围内,荧光强度与人IgG浓度的成线性关系,并且动态范围从1×10-12mol L-1-1×10-17molL-1超过6个数量级。线性相关系数为0.997。检测限达到8.3×10mol L-1(采用3倍的信噪比)。实验证明,该方法具有较宽的动态范围,超高灵敏度、可接受的重现性、好的特异性和较低的基质效应影响。该技术作有望成为一种超灵敏的蛋白质检测技术用于蛋白质组学和临床诊断。4、基于核酸外切酶辅助的免分离和内切酶介导的循环放大荧光法检测凝血酶核酸适配体作为一种分子识别元件,越来越受到人们的关注,目前,应用核酸适配体荧光检测蛋白主要是基于适配体与目标蛋白作用后产生的荧光偏振度或荧光强度的改变来检测蛋白。这类方法也是信号变化范围较小,荧光背景较大,线性范围不宽,对于低浓度的蛋白测定存在一定困难。核酸外切酶Exonucleasel (Exol)可以按照3’→5’方向水解单链DNA核苷酸,产生5’单磷酸脱氧核苷和末端二磷酸二核苷。核酸内切酶可以对双联DNA中某一段核苷酸基因位点进行特异识别切割,从而释放出靶目标物。本章基于此巧妙的设计了一段凝血酶的核酸适配体、将核酸外切酶和切割内切酶连用,发展了一种快速、高灵敏的基于核酸外切酶辅助的免分离和核酸内切酶介导的循环放大荧光法检测凝血酶。在这个方法中,我们首先合成一段含有凝血酶适体和一段含有识别序列的寡核苷酸即适体检测探针,当把探针与凝血酶一起孵育时,其可以识别凝血酶并与其结合。然后加入ExoI酶切除未结合的适体探针,而与凝血酶结合的探针则被保护下来；进而对被保护的探针的其中一段序列进行与分子灯塔MB杂交,从而打开分子灯塔发出荧光,然后加入切割内切酶去特定识别双联核苷酸的特定位点从而进行切割,释放出被保护的探针,在进行下一轮的循环放大。通过建立荧光强度与凝血酶浓度的相关曲线,我们可以实现对凝血酶的定量。检测限的灵敏度达到了5×10-11mol L-1。实验证明,该方法具有高的灵敏度、可接受的重现性、好的特异性和较低的基质效应影响。该技术在改变相应蛋白的核酸适体情况下,有望用于对多种蛋白进行高灵敏和高特异性的检测。5、基于RCA辅助诱导的G四倍体构型免标记荧光信号放大策略超灵敏检测DNAG-四倍体DNA是由富含G的单链DNA,在特定的离子强度和pH条件下,单链内或单链之间对应的G碱基通过Hoogsteen氢键的配对,使4段或4条富G的DNA片段相互结合形成一段四倍体DNA。卟啉衍生物(NMM),是一种阴离子卟啉化合物。能选择性的与G-四倍体DNA结合。游离的NMM发出很弱的光,但是在与G-四倍体DNA结合后展现出很强的荧光信号增强。磁球是指含有磁性金属或金属氧化物的超细粉末而具有磁响应的高分子微球,它除了具有纳米颗粒的特性外,还可以通过表面的功能基团进行功能修饰。并因具有磁性,可在外加磁场的作用下方便迅速地分离。滚环复制(rolling circle replication)为噬菌体感染细菌后进行自我复制而普遍采取的一种形式。这种复制形式可在等温下以环状单链DNA为模板进行相对无限单链扩增,产生多个单元拷贝的长单链DNA分子。滚环扩增(RCA)即是基于这样一个原理的信号放大检测技术,有线性扩增和指数扩增两种形式。为了设计方便、便宜、快速、易操作、灵敏度高、选择性好的核苷酸检测方法,本章基于卟啉衍生物(NMM)与G-四倍体DNA的相互作用,将其与RCA技术连用,巧妙的设计了两种免标记信号放大方法检测DNA,第一种：我们发展了一种基于磁纳米颗粒,核酸外切酶辅助循环放大和RCA扩增辅助的G-四倍体与卟啉衍生物(NMM)相互作用联用的的功能化传感器用于信号放大检测DNA。该方法应用了三次信号放大技术(磁纳米球富集、核酸外切酶辅助循环放大、RCA扩增放大),一方面提高了检测的灵敏度,一方面提高了方法的选择性。第二种：为了进一步简化实验步骤,缩短反应时间,避免污染,我们巧妙的设计了一种哑铃型的RCA模版、将卟啉衍生物(NMM)与RCA的产物(双向G-四倍体DNA)相互作用,发展了一种快速、简便、高灵敏的基于哑铃型探针诱导RCA扩增辅助的G-四倍体免标记荧光信号放大策略超灵敏检测DNA。更多还原

【Abstract】 With the development of genome project and protein project, more sensitive, accurate, convenient and economic detection methods for biological macromolecules have been the focus of biology, medicine and chemistry. In recent years, signal amplification detection technologies based on electrochemical, optical, piezoelectric technologies and the use of various enzymes and nanoparticles, have realized highly sensitive detections of biological macromolecules. How to utilize the existing molecular biology techniques and tools to convert the information of biological molecules to detectable signals with high sensitivity and specificity, how to further expand the use of signal amplifying methods in molecular biology and biomedical application, and how to design novel signal conversion means and develop novel signal amplification detection technology platform to address the research process to life in the new issue, are still significant topics for researches in biomedical and analysis chemical area.In this thesis, a series of fluorescence signal amplifying detection technology platforms based on a variety of nucleic acid enzymes, nanoparticles and fluorescent nucleic acid probes have been developed for quantitative analysis of protein and DNA detection. The main researches included in this dissertation are presented as following:1. Ultrasensitive Fluorescence immunosensor for Detection of Protein Based on a Combination of the Magnetic Nanobeads and the assembly of fluorescent DNA nanotagsAlong with the efficient magnetic nonparticle amplifying strategy for sensitive detecting biomolecules, magnetic nanoparticle-based label sensors toward targets have been developed with its enrichment and magnetic separation properties. Sensor based on functionalized magnetic nonparticle has gained much attention as a new type tool of non-isotopic immunoassay labels. Magnetic particles act as a carrier and a bridge when functionalized magnetic particles are used to be as signal labels. On one hand, the significant signal intensity could be gained due to the application of amplification strategy. On the other hand, the background signals from labels nonspecific adsorption could be reduced due to the application of magnetic particles. In immunoassay, the application of the sensor based on magnetic nanoparticles can improve the sensitivity by reducing false positive signals with magnetic separation technology and increasing label signal intensity by its enrichment as a carrier.In this paper, aiming at the detection need of low abundant proteins, we report an ultrasensitive fluorescence immunoassay based on a novel and sensitive fluorescence biosensor by the combination of MNB and assembly of fluorescent DNA nanotags (SYBR Green I/BT-DNA conjugates) for detection of target protein. Highly amplified fluorescence signal and low background signal are achieved by using a double amplification (DNA amplification and the signal amplification feature of SYBR Green I/BT-DNA) and magnetic separation. In the high concentration, on logarithmic scales, the fluorescence intensity was found to exhibit a linear correlation to human IgG concentration from5.0×10-13mol L-1to8.0×10-15mol L-1with a linear correlation coefficient of0.995. In the low concentration, one protein brings one MNB onto the substrate. The linear relationship between the number of beads and human IgG concentration is in the range of50to3.0fM. The linear regression equation for human IgG in the range of50to3.0fM was determined to be y=13.135+7.684×1015x(R=0.996).2. Sensitive Detection of Proteins Using Assembled Cascade Fluorescent DNA Nanotags Based on Rolling Circle AmplificationSensitive proteins detection is critically important in basic discovery research and clinical practice because a few molecules of protein are sufficient to affect the biological functions of cells and trigger pathophysiological processes. To improve the sensitive, Great efforts have been made to develop some new protein detection techniques. Among them, fluorescence detection techniques combined with signal amplification are one of the most popular techniques. Specific nucleic acid sequences amplification is a basic technology of molecular biology. With the development of molecular biology and the application of the cross subject biochemistry and biophysics in the field, new nucleic acid amplification technologys have emerged. Among them, RCA (Rolling Circle Amplification) technology with high specificity, high sensitivity and simple operational gradually draw people’s attention in recent years. And RCA is more and more used in basic research, clinical diagnosis analysis and the field of medicine. Compared with other nucleic acid amplification technology, RCA technology has the following advantages:high sensitivity, high specificity, diversity and high throughput, isothermal reaction and simple operation and so on.Aiming at the detection need of low abundant proteins and the problems existing in the amplification techniques by multiple fluorophores and nanoparticle probes, this work proposed a novel cascade fluorescence signal amplification strategy for ultrasensitive detection of protein, which based on the rolling circle amplification (RCA)-aided and assembled cascade fluorescent DNA nanotags as signal label and multiplex binding of the biotin-streptavidin system. The designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a3-decade concentration range from1.0pM to1.0fM with a limit of detection as low as0.9fM. It was demonstrated that the proposed strategy shows a broad dynamic range, ultra-high sensitivity, acceptable reproducibility, excellent specificity and a low matrix effect. Futhermore, the method does not require complicated conjugation chemistry and shows fast reaction kinetics and simply manipulating in assembly of fluorescent DNA nanotags. Compared with functionalized nanoparticles labels, it avoids the complicated and tedious synthesis process. The proposed strategy would become a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.3. A Versatile Platform for Ultrasensitive Detection of Protein:DNA enriching magnetic nanoparticles-Based Rolling Circle Amplification ImmunoassayIn fluorescence immunoassay, for the detection of trace amounts of biological analytes, a key factor of the detection and quantification of trace levels of protein analytes is to increase label fluorescence signal intensity to improve sensitivity. Great efforts have been made to develop some new protein detection techniques relying on labeling with different probes to improve sensitivity. In general, high-sensitivity assay labels involve the use of certain signal amplification strategies by which a single signal-reporting tag is able to incorporate numerous detectable elements. Typical high-sensitivity assay labels include enzymes or their polymeric forms as well as nanoparticles such as dye-doped nanoparticles, gold nano-particles and quantum dots. Notwithstanding the importance of these techniques, the signal amplification capacity of these assay labels is still limited.With the development of molecular biology and the application of the cross subject biochemistry and biophysics in the field, new nucleic acid amplification technologys have emerged. Among them, RCA (Rolling Circle Amplification) technology with high specificity, high sensitivity and simple operational gradually draw people’s attention in recent years. And RCA is more and more used in basic research, clinical diagnosis analysis and the field of medicine. Compared with other nucleic acid amplification technology, RCA technology has the following advantages: high sensitivity, high specificity, diversity and high throughput, isothermal reaction and simple operation and so on.Along the direction of implementation of oligonucleotide-based immunoassay labels, to develop signal-reporting labels with maximized signal amplification and minimized nonspecific adsorption for highly sensitive proteins detection, we reported for the first time a DNA enriching magnetic nanoparticles and assembled fluorescent DNA nanotags based RCA immunoassay, magnetic nanoparticles-RCA immunoassay, as an alternative strategy. On logarithmic scales, the fluorescence intensity at the maximum emission wavelength was found to exhibit a linear correlation to human igG concentration over a6-decade concentration range from1pM to10aM with a linear correlation coefficient of0.997. The calculated limit of lower detection is8.3aM in a3s rule. The ultrahigh sensitivity and the broad dynamic range were attributed to the following reasons:(ⅰ) the primary amplification via functionalized magnetic nanoparticle detection probe attaching numerous primer DNAs for RCA, that is to say, a high ratio of primer DNAs are attached to an antigen;(ⅱ) the released numerous primer DNAs initiate a secondary RCA amplification;(ⅲ) a tertiary "assembled fluorescent DNA nanotags" amplification;(iv) a low nonspecific adsorption of functionalized magnetic nanoparticle detection probe by magnetic separation. This technique also enabled easy preparation of the detection probe, and isothermal end-point detection with simple instrumentation. It was demonstrated that the technique gave high specificity, low matrix effect and dose-response sensitivity. This technique holds great promise as an ultrasensitive, specific, powerful technology for protein detection in proteomics and clinical diagnostics.4. Sensitive and Homogeneous protein detection based on Exo I assisted separation-free and Nicking Enzyme assisted fluorescence signal amplificationAptamers, which are novel in vitro selected functional DNA or RNA structures from random-sequence nucleic acids libraries, possess high recognition ability to specific targets. Due to their inherent selectivity, affinity and their multifarious advantages over the traditional recognition elements, aptamers can rival antibodies for molecular recognition and detection. Compared with antibodies, the biggest advantage of aptamer-based methods is that oligonucleotides can be synthesized chemically with ease and extreme accuracy at low cost nowadays. Furthermore, they are thermally stable, reusable, and show good stability during long-term storage. At present, aptamers sensor for fluorescent detecting protein is mainly based on the change of fluorescence polarization degree or the fluorescence signal intensity after aptamer and the target protein binding. In this method, signal change is in the small areas, fluorescence background is bigger, linear range is not wide. It is difficult for low concentration of protein determination in some degree.Exonuclease I catalyzes the removal of nucleotides from single-stranded DNA in the3’to5’direction. The single-stranded DNA chains would be degraded to5’-terminal dinucleotides with the release of deoxyribonucleoside5’-monophosphates from the3’-termini of the single-stranded DNA chains. Nicking Enzyme can identify and cleave double-stranded nucleotides specific sites. In this work, we developed a sensitive and homogeneous protein detection based on Exo I assisted separation-free and Nicking Enzyme assisted fluorescence signal amplification. In this study, we firstly synthesize a protein detection probe containing a thrombin aptamer sequence and an ex-DNA recognition probe sequence. The ex-DNA was designed to be complementary to the MB-quenching fluorescence (MB). The MB probe is a short oligo-DNA, which carried the recognition sequences and cleavage site for the nicking endonuclease Nb.BbvcI, and was labeled with the fluorescent dye6-carboxyfluorescein (6-FAM) and its quencher DABCYL at the5’-and3’-ends, respectively. Nicking endonuclease is an enzyme that binds to its asymmetrical recognition sequence in double-stranded DNA and nicks only one specific strand of the duplex. When thrombin and its aptamer incubated together, the thrombin bound an aptamer and thereby was protected from degraded by exonuclease I. The protected protein detection probes hybridize with MB probe. Therefore, a fluorescence signal appears only when the MB probe was cleaved by Nb.BbvCI. After nicking, the hybrid MB probe became less stable, and the cleaved strand dissociated from the target, thus resulting in the complete disconnection of the fluorophore from the quencher. The released target strand could then hybridize to another MB probe and initiated the second cycle of cleavage. Finally each target strand could go through many cycles, resulting in cleavage of many probes. The detect limit was estimated to be PM. It was demonstrated that the proposed strategy shows an ultra-high sensitivity, acceptable reproducibility, excellent specificity and a low matrix effect. In addition, the strategy has promising application in the monitoring of many other proteins with high sensitivity and excellent specificity.5. A facile, label-free amplified DNA detection system based on rolling circle amplification strategy assisted quadruplex formationIn particular ionic strength and pH conditions, G-quadruplexes is comprised of single nucleotide with rich G basic groups through the counterpart of the Hoogsteen hydrogen bonding. N-methyl mesoporphyrin IX (NMM) is an anionic porphyrin characterized by a pronounced structural selectivity for G-quadruplexes. It is weakly fluorescent by itself, but exhibits a dramatic fluorescence enhancement upon binding to quadruplex DNA.Magnetic nanoparticle-based sensors have gained much attention with its enrichment and magnetic separation properties as a new type tool of assay sensor. RCA (Rolling Circle Amplification) technology with high specificity, high sensitivity and simple operational gradually draw people’s attention in recent years. In a typical RCA process, DNA polymerase replicates the circular template hundreds to thousands of times. Therefore, the end products of an RCA reaction are extremely long ssDN A molecules. RCA has a wide range of applications in the biochemical analysis and clinical diagnosis due to its high sensitivity and specificity, isothermal operation and many other advantages.In order to design convenient, cheap, fast, easy operation, high sensitivity, good selectivity nucleotide detection method, in this work, we developed two facile, label-free amplified DNA detection systems.(1) A magnetic bead-based label-free fluorescent turn-on enzymatic amplification assay for DNA detection using RCA assistant ligand-responsive G-quadruplex formation. The developed assay exploits three amplifications (Exo III cleavage cycle amplification, RCA, a dramatic fluorescence enhancement of G-quadruplex/NMM). Here, the integration of a series of amplification techniques further improves the sensitivity. In addition, the developed assay realizes label-free DNA detection, which is important for retaining the high target binding activity.(2) In order to further simplified experimental steps, shorten the reaction time, avoid vulnerability to contamination, we developed a facile, label-free amplified DNA detection system based on a dumbbell probe-mediated rolling circle amplification assisted quadruplex formation for highly sensitive DNA detection.更多还原