基于金纳米材料的生物传感新方法研究
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摘要
光学生物传感技术通常是指是利用待测物与识别单元结合过程中产生的光学信号,通过换能器将信号转换与放大处理,直接转换成可读的数据,而待测物的定性或定量分析则可由最终输出的数据直接读取或者换算实现。光学生物传感技术无需分离、操作简便可原位实时活体测定,越来越受到人们的关注。纳米材料,泛指在三维空间中至少有一维处于纳米尺度范围(1-100nm)或者由该尺度范围的物质为基本结构单元所构成的具有特殊效应的纳米级物质。纳米材料因其特殊的结构组成常具有其他一般材料所不具备的特殊性质,包括光学性、导热性、导电性、磁性等等。处于原子簇和宏观物体交界的过渡区域的纳米颗粒材料更是具有量子尺寸效应、微小尺寸效应、表面效应、量子隧道效应、介电限域效应等。纳米材料已经广泛应用于电子、化工、轻工、纺织、军事、医学等领域。纳米材料与光学生物传感技术相结合在蛋白小分子作用、医学临床诊断、药物筛选、靶向治疗等方面的研究和应用更是极为重要和急需的。本论文以此为前提,将蛋白、磷脂肌醇酶、蛋白转录后修饰酶、毒素等与人类肿瘤及重大疾病等相关的重要生物功能标志物作为研究对象,发展了一系列简单快捷、低成本、高通量、线性范围宽、稳定性好,且高灵敏、高选择性的新型生物传感方法。本研究论文的主要内容概括如下：
(1)磷酸肌醇酶的功能紊乱与已知的肿瘤、糖尿病等多种疾病有着密切的关系,其可逆调节磷酸化磷酸肌醇信号检测在分子机制的理解和治疗诊断工具的开发方面具有的强大的意义。第2章,发展了一种新颖的基于酶调控金纳米颗粒自组装均相比色传感方法用于检测磷酸肌醇激酶和去磷酸酶的活性。根据酶改性磷酸酰肌醇的首基达到结合普列克底物同源性细胞质蛋白或膜蛋白的胞质结构域(PH)的目的。支持磷脂膜包覆金纳米颗粒表面被设计提供了膜蛋白的胞质结构域(PH)的底物基质,有效的提高了酶反应效率。在靶标磷酸肌醇的存在下,支持磷脂膜修饰的金纳米颗粒与磷脂酰肌醇结合蛋白的PH结构域修饰的金纳米颗粒形成自组装交联聚合物,引起金纳米颗粒的表面等离子耦合共振效应,引发紫外吸收光谱值的变化,实现对磷酸肌醇信号的快速灵敏、均相高通量的检测。
(2)霍乱弧菌所致的霍乱能导致感染者严重腹泻是流行广泛且古老的烈性传染病,而霍乱毒素(CT)是霍乱弧菌所产生的外毒素,监测霍乱毒素(CT)含量对霍乱的预防、控制工作具有极其重要的意义。人类C反应蛋白(CRP)是与人体内突发性的炎症、或细菌感染、又或者组织损伤、心血管系统疾病(包括冠心病在内)等与发炎相关疾病的指标因子,对于它的评估有着非同寻常的意义。第3章,在前章工作的基础上改进了金纳米颗粒上磷脂膜的修饰方法,使得基于鞘糖脂修饰金纳米颗粒作为可视化生物传感平台用于检测霍乱毒素B亚组分(CTB)及人类C反应蛋白(CRP)更加可靠、灵敏、方便及快速。在此生物传感器的设计上,首先利用带疏水长链烷基的阳离子表面活性剂分子(CTAB)的NH3基端,以Au-NH3键共价作用直接在合成中修饰在金纳米颗粒的表面,紧接着加入鞘糖脂分子。由于鞘糖脂分子含有疏水性的N-脂酰鞘氨醇,它能在合成过程中以范德华(疏水)作用力直接插入到表面活性剂分子(CTAB)的十六烷基疏水链中,在金纳米颗粒表面形成鞘糖脂单分子层,达到将金纳米颗粒表面鞘糖脂功能活化的目的。这种鞘糖脂功能化活性的金纳米颗粒复合物不仅制备过程简单、易操作,而且还具有很好的通用性。利用不同的鞘糖脂及其受体(如霍乱毒素B亚组分(CTB)、人类C反应蛋白(CRP)、破伤风毒素和p糖苷酶等)之间强大的结合力,可以轻松地达到准确定量分析检测毒素及蛋白酶的目的。
(3)PTM酶活性的识别和定量对于细胞调控机制的研究、诊断学的发展以及疾病的治疗来说都是非常急待解决的问题,发展一个简单快捷、可自由标记并能实时监测PTM酶的方法是一个艰巨的挑战。第4章,构建了一个简单的,以多肽为模板直接快速合成高产率荧光AuNCs的方法,并且研究了PTM酶的转录后修饰对AuNCs的光化学特性的影响。以组蛋白去乙酰化酶(HDAC1)和蛋白激酶A(PKA)这两种PTM酶作为模型系统。HDAC1和PKA都被认为是与细胞周期调控、细胞增殖和癌症的发展紧密相连的,是最有潜力的治疗靶点与诊断的生物标志物。研究中,首次发现了多肽—AuNC的纳米复合材料,在保持底物肽活性的同时,AuNCs会在底物肽的酶活性修饰中发生荧光淬灭现象。这种酶响应荧光纳米簇的信号指示开辟了直接检测JPTMs转录后修饰无标记方法的可能,建立了一个简单且有用的生物传感平台,可用于灵敏的、实时的检钡PTMs酶。PTMs酶在生命体中占有十分重要的地位,它们的发展可能会催化带动AuNCs在新的生物医学应用、生物过程以及疾病的诊断和治疗的等领域的理解应用。
(4)MDM2癌基因,参予细胞的基本生理过程,它的突变与扩增与肿瘤(比如胶质瘤、骨肉瘤、软组织肉瘤甚至乳腺癌细胞系)的转移密切相关,是一种与多种肿瘤相关的克隆新基因。gp120表面蛋白,是一种病毒外层的糖蛋白,它对于艾滋病毒进入宿主细胞引起病毒感染等方面至关重要。第5章,在前章工作基础上,提出了一个基于蛋白保护生物活性分子的生物传感分析方法用于小分子结合蛋白的研究。理论上,羧基肽酶Y可以将生物多肽链水解成氨基酸小片段,可是,当生物多肽链与其靶向蛋白绑定结合后,靶向蛋白阻止了羧基肽酶Y的水解作用保证了生物多肽链的完整性。本生物传感平台基于此原理,结合以多肽为模板合成金纳米簇的方法发展出一种新型的靶向研究蛋白及小分子作用的方法。这种方法较之传统的的研究方法不同,无需对生物多肽链或靶向蛋白进行前处理。基于蛋白质保护的多肽模板金纳米簇为研究小分子及其结合蛋白特异性相互作用提供一个新的超灵敏生物传感平台。
Optical biological sensing technology is usually means that optical signal was produced in the object to be measured and the recognition unit combination process, the determinand and identify the object under test unit combination process of optical signal, through the transducer signal conversion and amplification processing, directly into a readable data, and qualitative or quantitative analysis of the object under test can be read directly by the final output data or conversion. Optical sensing technology without biological separation, can be in situ determination of real-time live is easy to operate, so more and more get the attention of people. Nanomaterials, referring in three-dimensional space in at least one dimension in the nanometer scale (1-100nm), or by the scale of the material as the basic structural unit of nanoscale materials with special effects. Because of its special structure, nanomaterials often have special properties, including optical properties, thermal conductivity, electrical conductivity, magnetism and so on. The nanoparticles materials, between the atom clusters and macroscopic object boundary, it has a quantum size effect, small size effect, surface effect, quantum tunnel effect, dielectric confined effect, etc. Nanomaterials have been widely used in electronics, chemical industry, light industry, textile, military, medical and other fields. By combining both nano materials and optical biological sensing technology, applicationing in the small molecular protein, clinical diagnosis, drug screening and target therapy is a very important and urgent. To sum up, put the protein, phospholipids signal inositol, protein post-translational modifications enzymes, toxins and other major diseases, such as human tumor nuclear important biological functions related biomarkers as the research object, development a series of new biological sensing method whit simple and quick, low cost, high flux, wide linear range, good stability, and high sensitivity, high selectivity. The mainly points are as follows:
(1) Enzyme mediated phosphoinositide signaling plays important regulatory roles in diverse cellular processes and has close implication in human diseases. Phosphoinositide signaling system, regulated by reversible phosphorylation of the inositol headgroup by phosphoinositide kinases and phosphatases, plays important roles in diverse fundamental cellular processes. In chapter2, we have developed a novel enzyme-activated AuNPs assembly strategy as a homogeneous colorimetric biosensor for activity detection of phosphoinositide kinases and phosphatases. This strategy utilizes a biomimetic mechanism of phosphoinositide signaling, in which AuNP supported phospholipid membranes was constructed to mimic the cellular membrane substrate, and AuNP modified binding PH domain was designed for specific recognition of the phosphorylated phosphoinositides. The biomimetic strategy enables efficient enzymatic reactions of the substrate and highly selective detection of the target enzyme with minimized interference. The developed biosensor strategy allows convenient, rapid, sensitive visual detection of phosphoinositide enzymes with a pM detection limit and a four-decade wide dynamic range. The biosensor is also demonstrated for the utility for enzyme assay in real cell lysate samples. Moreover, the biosensor is configured into a homogeneous format that increases the potential for high-throughput analysis. In view of these advantages, the developed strategy might create a general biosensor platform for visual detection of phosphoinositide signaling with high sensitivity and selectivity in biomedical research and clinical diagnostics.
(2) Vibrio cholerae causes cholera, cholera can cause infections caused by severe diarrhea is widespread and ancient deadly infectious diseases, the cholera toxin (CT) is the external toxin produced by the bacterium vibrio cholera, so, monitoring the cholera toxin (CT) content on the cholera prevention and control work has very important significance. Human c-reactive protein (CRP) is the index factors of inflammation related diseases in the human body, these diseases include bacterial infection, or tissue damage and cardiovascular system diseases (including coronary heart disease), etc. For assessment of human c-reactive protein (CRP) has extraordinary significance. In chapter3, based on the previous chapter, improved the modification methods of phospholipids membrane on AuNPs, use with hydrophobic long chain alkyl NH3of cationic surfactant molecules (CTAB), Au-covalent NH3key role in the synthesis of modified directly on the surface of AuNPs. Glycosphingolipids N-end is inserted into the gold nanoparticle surface,forming a single molecular layer of glycosphingolipids. Using the Powerful combination between different scabbard glycolipids glycolipids and its receptor (such as the cholera toxin (CTB), human c-reactive protein (CRP), tetanus toxin and beta glycosidase, etc.), it can easily achieve the detection of toxins and proteinase accurate quantification of purpose.
(3) Protein post-translational modifications (PTMs) play key roles in functional proteomics, via regulating the activity, localization, interactions with other biomolecules, and degradation of proteins. There is a grand need but a daunting challenge in the development of simple and label-free methods that allow rapid and real-time detection of PTM enzymes. In chapter4, we developed a simple, rapid method for highly fluorescent AuNCs synthesis with different peptide templates and discovered that PTM enzymes were able to exert chemical modifications on the peptide-templated AuNCs and quench the AuNCs'fluorescence. The peptide-templated synthesis and the enzyme-responsive property of AuNCs were successfully demonstrated using two PTM enzymes, HDAC1and PKA. It was found that the synthetic method produced highlyfluorescent Au8clusters with compact peptide coating. Enzymatic modification of the peptides could largely quench thefluorescence of the AuNCs, presumably because chemical modifications destroyed the protective peptide coating on AuNCs and induced O2-mediated quenching and oxidation of thefluorescent clusters. This enzyme-responsivefluorescent nanocluster beacon was shown to be highly sensitive and selective for label-free and real-time quantification of HDAC1and PKA with wide linear detection ranges. In virtue of the important biological roles of PTM enzymes and the advantages of the enzyme-responsive nanocluster beacon, this technology indeed created a useful label-free biosensor platform for the detection of PTM enzymes and their inhibitors, implying its great potential in proteomics, biomedical imaging, and clinical theranostics.
(4) MDM2oncogene, is a clone of new genes associated with wide variety of tumor, basic physiological processes in the cell, its mutation andamplification and tumor (such as glioma, osteosarcoma, soft tissue sarcomaand breast cancer cell line) of metastasis. Gp120surface protein, is a glycoprotein virus outer, it for HIV entry into host cells caused by viral infection is very important. In chapter5, based on the previous chapter, proposed a method of biological sensor protein protection of biologically active molecules for protein analysis based on the study of small molecule. In theory, polypeptide by carboxypeptidase Y hydrolyzed into amino acid fragments, but, when the biological polypeptide chain and its target protein binding, theprotein prevents hydrolysis. Based on this principle, combined with the synthesis method of peptide-template AuNCs, this paper presents a new label-free biosensing platform method for rapid, sensitive detection MDM2and gp120. Also, the new method provides a new ultra sensitive biosensing platform for the study of small molecules and protein binding of specific interaction.
(1)磷酸肌醇酶的功能紊乱与已知的肿瘤、糖尿病等多种疾病有着密切的关系,其可逆调节磷酸化磷酸肌醇信号检测在分子机制的理解和治疗诊断工具的开发方面具有的强大的意义。第2章,发展了一种新颖的基于酶调控金纳米颗粒自组装均相比色传感方法用于检测磷酸肌醇激酶和去磷酸酶的活性。根据酶改性磷酸酰肌醇的首基达到结合普列克底物同源性细胞质蛋白或膜蛋白的胞质结构域(PH)的目的。支持磷脂膜包覆金纳米颗粒表面被设计提供了膜蛋白的胞质结构域(PH)的底物基质,有效的提高了酶反应效率。在靶标磷酸肌醇的存在下,支持磷脂膜修饰的金纳米颗粒与磷脂酰肌醇结合蛋白的PH结构域修饰的金纳米颗粒形成自组装交联聚合物,引起金纳米颗粒的表面等离子耦合共振效应,引发紫外吸收光谱值的变化,实现对磷酸肌醇信号的快速灵敏、均相高通量的检测。
(2)霍乱弧菌所致的霍乱能导致感染者严重腹泻是流行广泛且古老的烈性传染病,而霍乱毒素(CT)是霍乱弧菌所产生的外毒素,监测霍乱毒素(CT)含量对霍乱的预防、控制工作具有极其重要的意义。人类C反应蛋白(CRP)是与人体内突发性的炎症、或细菌感染、又或者组织损伤、心血管系统疾病(包括冠心病在内)等与发炎相关疾病的指标因子,对于它的评估有着非同寻常的意义。第3章,在前章工作的基础上改进了金纳米颗粒上磷脂膜的修饰方法,使得基于鞘糖脂修饰金纳米颗粒作为可视化生物传感平台用于检测霍乱毒素B亚组分(CTB)及人类C反应蛋白(CRP)更加可靠、灵敏、方便及快速。在此生物传感器的设计上,首先利用带疏水长链烷基的阳离子表面活性剂分子(CTAB)的NH3基端,以Au-NH3键共价作用直接在合成中修饰在金纳米颗粒的表面,紧接着加入鞘糖脂分子。由于鞘糖脂分子含有疏水性的N-脂酰鞘氨醇,它能在合成过程中以范德华(疏水)作用力直接插入到表面活性剂分子(CTAB)的十六烷基疏水链中,在金纳米颗粒表面形成鞘糖脂单分子层,达到将金纳米颗粒表面鞘糖脂功能活化的目的。这种鞘糖脂功能化活性的金纳米颗粒复合物不仅制备过程简单、易操作,而且还具有很好的通用性。利用不同的鞘糖脂及其受体(如霍乱毒素B亚组分(CTB)、人类C反应蛋白(CRP)、破伤风毒素和p糖苷酶等)之间强大的结合力,可以轻松地达到准确定量分析检测毒素及蛋白酶的目的。
(3)PTM酶活性的识别和定量对于细胞调控机制的研究、诊断学的发展以及疾病的治疗来说都是非常急待解决的问题,发展一个简单快捷、可自由标记并能实时监测PTM酶的方法是一个艰巨的挑战。第4章,构建了一个简单的,以多肽为模板直接快速合成高产率荧光AuNCs的方法,并且研究了PTM酶的转录后修饰对AuNCs的光化学特性的影响。以组蛋白去乙酰化酶(HDAC1)和蛋白激酶A(PKA)这两种PTM酶作为模型系统。HDAC1和PKA都被认为是与细胞周期调控、细胞增殖和癌症的发展紧密相连的,是最有潜力的治疗靶点与诊断的生物标志物。研究中,首次发现了多肽—AuNC的纳米复合材料,在保持底物肽活性的同时,AuNCs会在底物肽的酶活性修饰中发生荧光淬灭现象。这种酶响应荧光纳米簇的信号指示开辟了直接检测JPTMs转录后修饰无标记方法的可能,建立了一个简单且有用的生物传感平台,可用于灵敏的、实时的检钡PTMs酶。PTMs酶在生命体中占有十分重要的地位,它们的发展可能会催化带动AuNCs在新的生物医学应用、生物过程以及疾病的诊断和治疗的等领域的理解应用。
(4)MDM2癌基因,参予细胞的基本生理过程,它的突变与扩增与肿瘤(比如胶质瘤、骨肉瘤、软组织肉瘤甚至乳腺癌细胞系)的转移密切相关,是一种与多种肿瘤相关的克隆新基因。gp120表面蛋白,是一种病毒外层的糖蛋白,它对于艾滋病毒进入宿主细胞引起病毒感染等方面至关重要。第5章,在前章工作基础上,提出了一个基于蛋白保护生物活性分子的生物传感分析方法用于小分子结合蛋白的研究。理论上,羧基肽酶Y可以将生物多肽链水解成氨基酸小片段,可是,当生物多肽链与其靶向蛋白绑定结合后,靶向蛋白阻止了羧基肽酶Y的水解作用保证了生物多肽链的完整性。本生物传感平台基于此原理,结合以多肽为模板合成金纳米簇的方法发展出一种新型的靶向研究蛋白及小分子作用的方法。这种方法较之传统的的研究方法不同,无需对生物多肽链或靶向蛋白进行前处理。基于蛋白质保护的多肽模板金纳米簇为研究小分子及其结合蛋白特异性相互作用提供一个新的超灵敏生物传感平台。
Optical biological sensing technology is usually means that optical signal was produced in the object to be measured and the recognition unit combination process, the determinand and identify the object under test unit combination process of optical signal, through the transducer signal conversion and amplification processing, directly into a readable data, and qualitative or quantitative analysis of the object under test can be read directly by the final output data or conversion. Optical sensing technology without biological separation, can be in situ determination of real-time live is easy to operate, so more and more get the attention of people. Nanomaterials, referring in three-dimensional space in at least one dimension in the nanometer scale (1-100nm), or by the scale of the material as the basic structural unit of nanoscale materials with special effects. Because of its special structure, nanomaterials often have special properties, including optical properties, thermal conductivity, electrical conductivity, magnetism and so on. The nanoparticles materials, between the atom clusters and macroscopic object boundary, it has a quantum size effect, small size effect, surface effect, quantum tunnel effect, dielectric confined effect, etc. Nanomaterials have been widely used in electronics, chemical industry, light industry, textile, military, medical and other fields. By combining both nano materials and optical biological sensing technology, applicationing in the small molecular protein, clinical diagnosis, drug screening and target therapy is a very important and urgent. To sum up, put the protein, phospholipids signal inositol, protein post-translational modifications enzymes, toxins and other major diseases, such as human tumor nuclear important biological functions related biomarkers as the research object, development a series of new biological sensing method whit simple and quick, low cost, high flux, wide linear range, good stability, and high sensitivity, high selectivity. The mainly points are as follows:
(1) Enzyme mediated phosphoinositide signaling plays important regulatory roles in diverse cellular processes and has close implication in human diseases. Phosphoinositide signaling system, regulated by reversible phosphorylation of the inositol headgroup by phosphoinositide kinases and phosphatases, plays important roles in diverse fundamental cellular processes. In chapter2, we have developed a novel enzyme-activated AuNPs assembly strategy as a homogeneous colorimetric biosensor for activity detection of phosphoinositide kinases and phosphatases. This strategy utilizes a biomimetic mechanism of phosphoinositide signaling, in which AuNP supported phospholipid membranes was constructed to mimic the cellular membrane substrate, and AuNP modified binding PH domain was designed for specific recognition of the phosphorylated phosphoinositides. The biomimetic strategy enables efficient enzymatic reactions of the substrate and highly selective detection of the target enzyme with minimized interference. The developed biosensor strategy allows convenient, rapid, sensitive visual detection of phosphoinositide enzymes with a pM detection limit and a four-decade wide dynamic range. The biosensor is also demonstrated for the utility for enzyme assay in real cell lysate samples. Moreover, the biosensor is configured into a homogeneous format that increases the potential for high-throughput analysis. In view of these advantages, the developed strategy might create a general biosensor platform for visual detection of phosphoinositide signaling with high sensitivity and selectivity in biomedical research and clinical diagnostics.
(2) Vibrio cholerae causes cholera, cholera can cause infections caused by severe diarrhea is widespread and ancient deadly infectious diseases, the cholera toxin (CT) is the external toxin produced by the bacterium vibrio cholera, so, monitoring the cholera toxin (CT) content on the cholera prevention and control work has very important significance. Human c-reactive protein (CRP) is the index factors of inflammation related diseases in the human body, these diseases include bacterial infection, or tissue damage and cardiovascular system diseases (including coronary heart disease), etc. For assessment of human c-reactive protein (CRP) has extraordinary significance. In chapter3, based on the previous chapter, improved the modification methods of phospholipids membrane on AuNPs, use with hydrophobic long chain alkyl NH3of cationic surfactant molecules (CTAB), Au-covalent NH3key role in the synthesis of modified directly on the surface of AuNPs. Glycosphingolipids N-end is inserted into the gold nanoparticle surface,forming a single molecular layer of glycosphingolipids. Using the Powerful combination between different scabbard glycolipids glycolipids and its receptor (such as the cholera toxin (CTB), human c-reactive protein (CRP), tetanus toxin and beta glycosidase, etc.), it can easily achieve the detection of toxins and proteinase accurate quantification of purpose.
(3) Protein post-translational modifications (PTMs) play key roles in functional proteomics, via regulating the activity, localization, interactions with other biomolecules, and degradation of proteins. There is a grand need but a daunting challenge in the development of simple and label-free methods that allow rapid and real-time detection of PTM enzymes. In chapter4, we developed a simple, rapid method for highly fluorescent AuNCs synthesis with different peptide templates and discovered that PTM enzymes were able to exert chemical modifications on the peptide-templated AuNCs and quench the AuNCs'fluorescence. The peptide-templated synthesis and the enzyme-responsive property of AuNCs were successfully demonstrated using two PTM enzymes, HDAC1and PKA. It was found that the synthetic method produced highlyfluorescent Au8clusters with compact peptide coating. Enzymatic modification of the peptides could largely quench thefluorescence of the AuNCs, presumably because chemical modifications destroyed the protective peptide coating on AuNCs and induced O2-mediated quenching and oxidation of thefluorescent clusters. This enzyme-responsivefluorescent nanocluster beacon was shown to be highly sensitive and selective for label-free and real-time quantification of HDAC1and PKA with wide linear detection ranges. In virtue of the important biological roles of PTM enzymes and the advantages of the enzyme-responsive nanocluster beacon, this technology indeed created a useful label-free biosensor platform for the detection of PTM enzymes and their inhibitors, implying its great potential in proteomics, biomedical imaging, and clinical theranostics.
(4) MDM2oncogene, is a clone of new genes associated with wide variety of tumor, basic physiological processes in the cell, its mutation andamplification and tumor (such as glioma, osteosarcoma, soft tissue sarcomaand breast cancer cell line) of metastasis. Gp120surface protein, is a glycoprotein virus outer, it for HIV entry into host cells caused by viral infection is very important. In chapter5, based on the previous chapter, proposed a method of biological sensor protein protection of biologically active molecules for protein analysis based on the study of small molecule. In theory, polypeptide by carboxypeptidase Y hydrolyzed into amino acid fragments, but, when the biological polypeptide chain and its target protein binding, theprotein prevents hydrolysis. Based on this principle, combined with the synthesis method of peptide-template AuNCs, this paper presents a new label-free biosensing platform method for rapid, sensitive detection MDM2and gp120. Also, the new method provides a new ultra sensitive biosensing platform for the study of small molecules and protein binding of specific interaction.
引文
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