基于脂质体纳米粒子的生物传感器在生化分析中的应用
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摘要
霍乱是一种严重的传播广泛的流行性疾病,大多数霍乱病例发生在不发达国家,已成为世界上亟待解决的一个严重公共卫生问题。霍乱毒素是霍乱弧菌分泌的一种蛋白质肠毒素,是霍乱弧菌的一个典型生物标记物,所以对霍乱毒素的快速、超灵敏检测对霍乱流行病的防疫、控制和治疗具有十分重要的意义。近年来,前列腺癌已经成为成年男性群体中一项常见癌症,且危害严重。目前临床上对癌症标记物的检测方法普遍操作复杂、仪器昂贵且需要专业技能。从而需要开发更加简便、灵敏、定量准确且易于临床推广的生物传感技术。此外,地中海贫血又称海洋性贫血,是由于珠蛋白基因的缺失或点突变所致的一组遗传性溶血性贫血。该病以地中海沿岸国家和东南亚各国多见,我国长江以南各省均有报道,以广东、广西、海南、四川、重庆等省区发病率较高,是非常严重的遗传性疾病。故开发灵敏、准确的基因单碱基突变检测方法,对地中海贫血症的诊断有很大意义。
利用化学与生物传感技术检测疾病标志物,进行临床疾病的准确、快速诊断以及流行病的现场筛查与群体监控,是当前医学上既新颖又极具吸引力的热门研究课题之一。近年来,纳米材料得到广泛的研究与应用,研究表明将纳米材料应用于生物传感器的制备可以较大的提高传感器的响应性能。脂质体是一种在水相中由磷脂双分子定向排列而成的直径几纳米至几微米的超微粒子。脂质体纳米粒子可以通过内部包埋或表面修饰方法成为多功能团分子的通用载体且具有良好的生物相容性,本论文将不同功能化的脂质体用作生物传感界面的构建和信号放大探针,并与相适宜的检测手段联用,构建了一系列基于脂质体的新型生物传感器。纳米金制备简单,比表面积大,生物相容性好,是固定生物分子常用的有效载体。有文献报道,电极表面通过自组装纳米金层固定抗体,可以大大增加电极表面积和抗体固定量,同时保持抗体活性,是很好的传感界面修饰方法。另外,碳纳米管具有隧道效应、特殊的电化学、光化学等物化性质,是目前科研领域中最热门的一维纳米材料之一。碳纳米管的功能化修饰是将其应用于生物体系必须解决的一个关键问题。DNA修饰碳纳米管的研究为碳纳米管在基因检测中的应用提供了科学基础和强有力的技术支撑。
本论文以霍乱毒素,甲胎蛋白,前列腺特异性抗原等重大疾病标志物为检测对象,构建了一系列基于脂质体纳米颗粒的生物传感器(第1部分)。具体内容包括:
(1)首次研制了一种基于脂质体的压电免疫凝集检测(PEIA)技术用于对人免疫球蛋白(hIgG)和甲胎蛋白(AFP)的直接定量检测。以脂质体替代传统胶乳颗粒标记抗体,并采用牛血清白蛋白修饰压电探针,直接对脂质体颗粒免疫凝集所引起的溶液粘弹性变化进行响应。其定量检测hIgG的浓度范围为0.05～6μg mL-1;将之用于临床样品分析,定量能力与经典的ELISA法相接近。用所构建的生物传感器对AFP实际临床样品检测,结果证明该方法应用于临床医学检测的可行性。与经典的质量型压电免疫检测技术相比,这种基于脂质体的压电免疫凝集检测(PEIA)无需在石英晶振表面固定抗原或抗体,具有简单、快捷、可再生的检测优势。
(2)开发了一种基于神经节苷脂(GM1)识别和脂质体界面凝集的压电生物传感技术对霍乱毒素进行快速、定量检测。石英晶振传感界面修饰含GM1的支撑磷脂膜,检测介质中含有GM1修饰的脂质体颗粒,这两者构建了压电界面凝集生物传感器。目标物霍乱毒素会引起检测介质中GM1修饰的脂质体在含有GM1的传感界面上发生特异性凝集,同时引起晶体表面巨大的质量负载和溶液粘弹性的变化,结合了压电质量效应和非质量效应,与传统模式的压电频率响应信号相比放大了数倍,具有响应快速、可再生的优势。其定量检测霍乱毒素的浓度范围为0.1～5?μg mL-1,检测下限达到25 ng mL-1。
(3)提出了一种以脂质体为信号放大载体的酶催化增强化学发光传感技术对霍乱毒素的超灵敏检测。用含GM1的支撑磷脂膜为生物传感界面检测目标物霍乱毒素,再用表面修饰了GM1受体和辣根过氧化物(HRP)酶的脂质体为检测探针对目标物夹心检测。最后利用HRP酶为催化剂,对碘苯酚为增强剂的增强化学发光法对霍乱毒素进行定量检测。本传感器实现了对霍乱毒素的超灵敏检测,检测下限可达到0.8 pg mL-1,同时具有简单、可再生的优势。将传感器用于血清样品分析,其检测结果与ELISA试剂盒检测结果相一致。
(4)发展了一种基于包酶脂质体的化学发光生物传感技术对前列腺癌特异性抗原的超灵敏检测。以表面修饰了抗体、内部包被HRP酶的脂质体替代传统酶标抗体对界面识别的抗原夹心检测,并采用表面活性剂释放所固定脂质体中的酶分子,用增强化学发光试剂盒放大酶催化化学发光强度,间接检测前列腺特异性抗原。实现了对目标物的超灵敏检测,检测下限可达到4 pg mL-1。在第一部分中,不同形式和性能的功能化脂质体被合理运用,并结合使用多种检测方法,提高了生物传感器的分析性能。此外,我们还构建了几种基于其他纳米粒子的电化学生物传感技术,其中纳米粒子被用于生物分子的固定或检测探针的制备(第2部分)。纳米金比表面积大,生物相容性好,是固定生物分子常用的有效载体。具体内容包括:
(5)构建了一种以纳米金颗粒为固定化材料和多步信号放大探针的电化学阻抗免疫传感器对hIgG的超灵敏检测。将纳米金颗粒自组装于己二硫醇修饰的金电极表面,利用纳米金层吸附抗体,构建了一种纳米金界面吸附的生物分子固定化方法。信号放大步骤为:用纳米金标记的抗体做为第一步放大探针,然后分别用纳米金标记的二抗和纳米金标记的抗体交替反应,进行多步信号放大。基于纳米金界面固定抗体,具有抗体固定量大、免疫活性强、操作稳定性高等优点。基于纳米金的信号放大技术极大的提高了传感器的灵敏度,对目标物hIgG的线性检测范围为15.3～328.3 ng L-1。
(6)通过自组装纳米金层固定具有碱基突变识别能力的MutS蛋白,MutS蛋白能捕获含碱基突变的DNA双链并以亚甲基兰在DNA双链中的嵌入作为电化学响应信号来检测单碱基突变核酸链。该方法成功地实现了对β-地中海贫血症基因的-28位点的单个碱基突变的识别,检测限达到5.66×10-13 M,对点突变的检测具有快速、灵敏度高、成本低的特点。对实际样品的两步检测方法可实现样品的基因分型,即将样品分类为变异杂合子,野生纯合子和变异纯合子。
(7)单壁碳纳米管具有隧道效应、特殊的电化学、光化学等物化性质,将其作为纳米材料探针与电化学方法联用可以超灵敏的检测核酸单链,构建了新型的核酸检测方法。检测原理:将单壁碳纳米管与核酸检测探针混合机械超声,制备核酸检测探针修饰的水溶液分散的单壁碳纳米管。与目标DNA链的杂交反应会使检测探针解离离开碳管表面,裸露的单壁碳纳米管继而吸附在疏水的电极表面,由于其特有的隧道效应使原本绝缘的电极导通,从而检测到氧化还原电子对在电极表面实现电子传递的电化学信号。背景非特性吸附不能产生隧道效应,故检测背景极低,实现了对目标核酸单链的超灵敏测定,线性范围为6.9 pM～50 nM。
Cholera toxin (CT) is a protein enterotoxin secreted by the bacterium Vibrio cholerae that can cause an epidemic disease leading to rapid dehydration, acidosis, and even death in several hours without appropriate treatment. Most cholera cases are reported in many underdeveloped countries, and it is estimated that cholera causes approximately 120,000 deaths annually. As a typical biomarker of Vibrio cholerae, CT has become a very important target in biological detection, and there has been increasing interest in the development of rapid and sensitive methods for the determination of CT. In addition, prostate cancer (PCa) has become a most widespread and stubborn diseases and a major cause of death in the old age male population nowadays. It admits of no delay for the sensitive diagnosis and efficient treatment of PCa. Porstate-specific antigen (PSA), a 33-kD single-chain glycoprotein with chymotrypsin-like protease activity, has been used as the most validated marker for the detection of PCa in screening, diagnosis and monitor of disease recurrence after surgical prostatectomy.
Use of chemo/biosensing techniques to detect the meaningful bio-markers for the accurate and rapid diagnosis of clinical diseases as well as for the field screening and mass monitoring of the epidemic diseases has been a novel, attractive and hot topic in the current medical studies. In the past few years, nanomaterials have been widely researched and used. Applying nanomaterials for the fabrication of biosensors will greatly improve the performance of the resulting biosensor.
liposomes are formed spontaneously when phospholipids are dispersed into water. Liposomes can simply be portrayed as spherical vesicles consisting of one or more phospholipid bilayers surrounding an aqueous cavity. Molecules such as fluorophores, enzymes or drugs, present in the aqueous phase during the preparation, can be encapsulated in the liposome. Liposomes have been widely used as analytical tools in immunochemistry and can be implemented with hardly any modifications. Since colloidal Au has a very large surface area and good bio-compatibility, the use of colloidal particles as versatile and efficient templates for the immobilization of biomolecules has been recognized since the early 1980s. As a new kind of carbon materials, carbon nanotubes (CNTs) have attracted considerable interest because of their unique structural, mechanic, electronic, magnetic and optical characteristics. Biomolecule-functionalized carbon nanotubes have rapidly attracted substantial research interests for its application in the biosensor and electrochemistry detection.
This dissertation focuses on developing a series of nanoparticules-based biosensors for the diagnosis of some clinical serious diseases including prostate cancer, Vibrio cholera andβ-thalassemia diseases etc. by probing their specific markers (sometimes as the model test reagents). The detailed materials are shown as follows:
(1) A piezoelectric immunoagglutination assay has been proposed for rapid detection of human immunoglobulin G (hIgG) using antibody-modified liposoms (in Chapter 2). liposomes are employed as replacements for the traditional latex to be labeled with goat anti-human IgG antibody, the specific agglutination event in the presence of corresponding antigen was monitored by the sensing probe modified with bovine serum albumin (BSA). It is found that the frequency responses of the liposome-based PEIA are linearly correlated to hIgG concentration in the range of 0.05～6μg mL?1 with a detection limit of 50 ng mL?1, without necessity for immobilization of immunoactive entities and purification of samples. Results of evaluating practical specimens show that the analytical ability of the developed piezoelectric technique is comparable to that of the ELISA method. (2) A novel reusable piezoelectric biosensor has been developed for the detection of cholera toxin (CT) based on analyte-specific surface agglutination of ganglioside GM1-functionalized liposomes on supported lipid membrane incorporated with ganglioside GM1 (in Chapter 3). Compared with traditional piezoelectric biosensor, the proposed technique combines the detection of both the gravimetric and the viscoelastic effects, thereby enabling vast sensitive enhancement in protein determination. Besides the use of stabilized response for the quantification of CT, the initial response rate, defined by the slope of the frequency change curve at the initial stage, could also be utilized for the detection of CT, which might be of particular significance in rapid assay practices. This method had an analytical interval of 0.1～5μg mL-1, with a detection limit of 25 ng mL-1. (3) An ultrasensitive chemiluminescence biosensor was developed for the detection of CT based on a supported lipid membrane as sensing surface and the HRP/GM1-functionalized liposome as detection probe (in Chapter 4). The supported lipid based biosensing surface could be renewed easily and rapidly. The application of enhanced chemiluminescence reaction in the detection of HRP-bearing liposome afforded a further signal amplification and background alleviation. The developed biosensor was shown to give chemiluminescence signal in linear correlation to CT concentration within the range from 1 pg mL-1 to 1 ng mL-1 with readily achievable detection limit of 0.8 pg mL-1. (4) Another highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome (in Chapter 5). The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed lumino/peroxide/enhancer system. The chemiluminescence intensity was directly proportional to the concentration of PSA in sample solution in the range from 7.4 pgmL-1 to 74 ng mL-1 with a detection limit of 4 pg mL-1. In this part, the application of several kinds of functionalized liposome for the construction of biosensor have greatly improved the performance of the resulting biosensor.
In this dissertation, the Au nanoparticles and the carbon nanotubes have also been used for the fabrication of biosensor and combined with electrochemical detection methods:
(5) A highly sensitive electrochemical impedance immunosensor was developed by using a novel amplification procedure with the application of an Au-colloid labeled antibody as the primary amplifying probe and a multistep amplification by alternating treatment of the resulting assembly with an Au-colloid labeled secondary antibody and an Au-colloid labeled antibody(in Chapter 6). Colloidal Au was used as a versatile and efficient template for the immobilization of antibody due to its relatively large surface and good biocompatibility. A novel amplification method was developed with the application of a Au-colloid labeled antibody as the primary amplifying probe and a multistep amplification by alternating treatment of the resulting assembly with a Au-colloid labeled secondary antibody and a Au-colloid labeled antibody. The results obtained revealed that the sensitivity could be substantially improved via the amplification step and a detection limit as low as 4.1 ng L?1 could be reached for a model analyte of human immunoglobulin G (hIgG). (6) A novel approach for scanning of unknown gene mutations was developed based on the utilization of MutS protein for the mutation recognition and spontaneously intercalated MB markers for electrochemical signal generation(in Chapter 7). The occurrence of mutation in target genes resulted in the formation of heteroduplex, which could specifically bind to the immobilized MutS protein on the electrode. The adsorption of heteroduplex on the electrode surface could then be probed electrochemically by a certain redox indicator as methylene blue (MB) that was selectively intercalated in nucleic acid duplexes. The proposed approach has been successfully implemented for the identification of single-base mutation in ?28 site of theβ-thalassemia gene with a detection limit of 5.6×10-13M, providing a highly specific and cost-efficient approach for point mutation detection. (7) An electrochemical label-free detection technique was fabricated for the detection of nucleic acids based on the precipitation of SWNTs by DNA hybridization (in Chapter 8). The DNA hybridization between target DNA and the DNA probe coating on the nanotubes could actively remove DNA probe from the SWNTs surface. The precipitation of SWNTs onto the n-octadecyl mercaptan (C18H37SH) modified Au electrode substantially restores heterogeneous electron transfer between bare Au electrode and redox species in solution phase which was almost totally blocked by the SAM of C18H37SH, and as a result, the electrical signal of the electrode was correlated with the concentration of target DNA in the range from 6.4pM～50nM with a detection limit of 3.2pM.
利用化学与生物传感技术检测疾病标志物,进行临床疾病的准确、快速诊断以及流行病的现场筛查与群体监控,是当前医学上既新颖又极具吸引力的热门研究课题之一。近年来,纳米材料得到广泛的研究与应用,研究表明将纳米材料应用于生物传感器的制备可以较大的提高传感器的响应性能。脂质体是一种在水相中由磷脂双分子定向排列而成的直径几纳米至几微米的超微粒子。脂质体纳米粒子可以通过内部包埋或表面修饰方法成为多功能团分子的通用载体且具有良好的生物相容性,本论文将不同功能化的脂质体用作生物传感界面的构建和信号放大探针,并与相适宜的检测手段联用,构建了一系列基于脂质体的新型生物传感器。纳米金制备简单,比表面积大,生物相容性好,是固定生物分子常用的有效载体。有文献报道,电极表面通过自组装纳米金层固定抗体,可以大大增加电极表面积和抗体固定量,同时保持抗体活性,是很好的传感界面修饰方法。另外,碳纳米管具有隧道效应、特殊的电化学、光化学等物化性质,是目前科研领域中最热门的一维纳米材料之一。碳纳米管的功能化修饰是将其应用于生物体系必须解决的一个关键问题。DNA修饰碳纳米管的研究为碳纳米管在基因检测中的应用提供了科学基础和强有力的技术支撑。
本论文以霍乱毒素,甲胎蛋白,前列腺特异性抗原等重大疾病标志物为检测对象,构建了一系列基于脂质体纳米颗粒的生物传感器(第1部分)。具体内容包括:
(1)首次研制了一种基于脂质体的压电免疫凝集检测(PEIA)技术用于对人免疫球蛋白(hIgG)和甲胎蛋白(AFP)的直接定量检测。以脂质体替代传统胶乳颗粒标记抗体,并采用牛血清白蛋白修饰压电探针,直接对脂质体颗粒免疫凝集所引起的溶液粘弹性变化进行响应。其定量检测hIgG的浓度范围为0.05～6μg mL-1;将之用于临床样品分析,定量能力与经典的ELISA法相接近。用所构建的生物传感器对AFP实际临床样品检测,结果证明该方法应用于临床医学检测的可行性。与经典的质量型压电免疫检测技术相比,这种基于脂质体的压电免疫凝集检测(PEIA)无需在石英晶振表面固定抗原或抗体,具有简单、快捷、可再生的检测优势。
(2)开发了一种基于神经节苷脂(GM1)识别和脂质体界面凝集的压电生物传感技术对霍乱毒素进行快速、定量检测。石英晶振传感界面修饰含GM1的支撑磷脂膜,检测介质中含有GM1修饰的脂质体颗粒,这两者构建了压电界面凝集生物传感器。目标物霍乱毒素会引起检测介质中GM1修饰的脂质体在含有GM1的传感界面上发生特异性凝集,同时引起晶体表面巨大的质量负载和溶液粘弹性的变化,结合了压电质量效应和非质量效应,与传统模式的压电频率响应信号相比放大了数倍,具有响应快速、可再生的优势。其定量检测霍乱毒素的浓度范围为0.1～5?μg mL-1,检测下限达到25 ng mL-1。
(3)提出了一种以脂质体为信号放大载体的酶催化增强化学发光传感技术对霍乱毒素的超灵敏检测。用含GM1的支撑磷脂膜为生物传感界面检测目标物霍乱毒素,再用表面修饰了GM1受体和辣根过氧化物(HRP)酶的脂质体为检测探针对目标物夹心检测。最后利用HRP酶为催化剂,对碘苯酚为增强剂的增强化学发光法对霍乱毒素进行定量检测。本传感器实现了对霍乱毒素的超灵敏检测,检测下限可达到0.8 pg mL-1,同时具有简单、可再生的优势。将传感器用于血清样品分析,其检测结果与ELISA试剂盒检测结果相一致。
(4)发展了一种基于包酶脂质体的化学发光生物传感技术对前列腺癌特异性抗原的超灵敏检测。以表面修饰了抗体、内部包被HRP酶的脂质体替代传统酶标抗体对界面识别的抗原夹心检测,并采用表面活性剂释放所固定脂质体中的酶分子,用增强化学发光试剂盒放大酶催化化学发光强度,间接检测前列腺特异性抗原。实现了对目标物的超灵敏检测,检测下限可达到4 pg mL-1。在第一部分中,不同形式和性能的功能化脂质体被合理运用,并结合使用多种检测方法,提高了生物传感器的分析性能。此外,我们还构建了几种基于其他纳米粒子的电化学生物传感技术,其中纳米粒子被用于生物分子的固定或检测探针的制备(第2部分)。纳米金比表面积大,生物相容性好,是固定生物分子常用的有效载体。具体内容包括:
(5)构建了一种以纳米金颗粒为固定化材料和多步信号放大探针的电化学阻抗免疫传感器对hIgG的超灵敏检测。将纳米金颗粒自组装于己二硫醇修饰的金电极表面,利用纳米金层吸附抗体,构建了一种纳米金界面吸附的生物分子固定化方法。信号放大步骤为:用纳米金标记的抗体做为第一步放大探针,然后分别用纳米金标记的二抗和纳米金标记的抗体交替反应,进行多步信号放大。基于纳米金界面固定抗体,具有抗体固定量大、免疫活性强、操作稳定性高等优点。基于纳米金的信号放大技术极大的提高了传感器的灵敏度,对目标物hIgG的线性检测范围为15.3～328.3 ng L-1。
(6)通过自组装纳米金层固定具有碱基突变识别能力的MutS蛋白,MutS蛋白能捕获含碱基突变的DNA双链并以亚甲基兰在DNA双链中的嵌入作为电化学响应信号来检测单碱基突变核酸链。该方法成功地实现了对β-地中海贫血症基因的-28位点的单个碱基突变的识别,检测限达到5.66×10-13 M,对点突变的检测具有快速、灵敏度高、成本低的特点。对实际样品的两步检测方法可实现样品的基因分型,即将样品分类为变异杂合子,野生纯合子和变异纯合子。
(7)单壁碳纳米管具有隧道效应、特殊的电化学、光化学等物化性质,将其作为纳米材料探针与电化学方法联用可以超灵敏的检测核酸单链,构建了新型的核酸检测方法。检测原理:将单壁碳纳米管与核酸检测探针混合机械超声,制备核酸检测探针修饰的水溶液分散的单壁碳纳米管。与目标DNA链的杂交反应会使检测探针解离离开碳管表面,裸露的单壁碳纳米管继而吸附在疏水的电极表面,由于其特有的隧道效应使原本绝缘的电极导通,从而检测到氧化还原电子对在电极表面实现电子传递的电化学信号。背景非特性吸附不能产生隧道效应,故检测背景极低,实现了对目标核酸单链的超灵敏测定,线性范围为6.9 pM～50 nM。
Cholera toxin (CT) is a protein enterotoxin secreted by the bacterium Vibrio cholerae that can cause an epidemic disease leading to rapid dehydration, acidosis, and even death in several hours without appropriate treatment. Most cholera cases are reported in many underdeveloped countries, and it is estimated that cholera causes approximately 120,000 deaths annually. As a typical biomarker of Vibrio cholerae, CT has become a very important target in biological detection, and there has been increasing interest in the development of rapid and sensitive methods for the determination of CT. In addition, prostate cancer (PCa) has become a most widespread and stubborn diseases and a major cause of death in the old age male population nowadays. It admits of no delay for the sensitive diagnosis and efficient treatment of PCa. Porstate-specific antigen (PSA), a 33-kD single-chain glycoprotein with chymotrypsin-like protease activity, has been used as the most validated marker for the detection of PCa in screening, diagnosis and monitor of disease recurrence after surgical prostatectomy.
Use of chemo/biosensing techniques to detect the meaningful bio-markers for the accurate and rapid diagnosis of clinical diseases as well as for the field screening and mass monitoring of the epidemic diseases has been a novel, attractive and hot topic in the current medical studies. In the past few years, nanomaterials have been widely researched and used. Applying nanomaterials for the fabrication of biosensors will greatly improve the performance of the resulting biosensor.
liposomes are formed spontaneously when phospholipids are dispersed into water. Liposomes can simply be portrayed as spherical vesicles consisting of one or more phospholipid bilayers surrounding an aqueous cavity. Molecules such as fluorophores, enzymes or drugs, present in the aqueous phase during the preparation, can be encapsulated in the liposome. Liposomes have been widely used as analytical tools in immunochemistry and can be implemented with hardly any modifications. Since colloidal Au has a very large surface area and good bio-compatibility, the use of colloidal particles as versatile and efficient templates for the immobilization of biomolecules has been recognized since the early 1980s. As a new kind of carbon materials, carbon nanotubes (CNTs) have attracted considerable interest because of their unique structural, mechanic, electronic, magnetic and optical characteristics. Biomolecule-functionalized carbon nanotubes have rapidly attracted substantial research interests for its application in the biosensor and electrochemistry detection.
This dissertation focuses on developing a series of nanoparticules-based biosensors for the diagnosis of some clinical serious diseases including prostate cancer, Vibrio cholera andβ-thalassemia diseases etc. by probing their specific markers (sometimes as the model test reagents). The detailed materials are shown as follows:
(1) A piezoelectric immunoagglutination assay has been proposed for rapid detection of human immunoglobulin G (hIgG) using antibody-modified liposoms (in Chapter 2). liposomes are employed as replacements for the traditional latex to be labeled with goat anti-human IgG antibody, the specific agglutination event in the presence of corresponding antigen was monitored by the sensing probe modified with bovine serum albumin (BSA). It is found that the frequency responses of the liposome-based PEIA are linearly correlated to hIgG concentration in the range of 0.05～6μg mL?1 with a detection limit of 50 ng mL?1, without necessity for immobilization of immunoactive entities and purification of samples. Results of evaluating practical specimens show that the analytical ability of the developed piezoelectric technique is comparable to that of the ELISA method. (2) A novel reusable piezoelectric biosensor has been developed for the detection of cholera toxin (CT) based on analyte-specific surface agglutination of ganglioside GM1-functionalized liposomes on supported lipid membrane incorporated with ganglioside GM1 (in Chapter 3). Compared with traditional piezoelectric biosensor, the proposed technique combines the detection of both the gravimetric and the viscoelastic effects, thereby enabling vast sensitive enhancement in protein determination. Besides the use of stabilized response for the quantification of CT, the initial response rate, defined by the slope of the frequency change curve at the initial stage, could also be utilized for the detection of CT, which might be of particular significance in rapid assay practices. This method had an analytical interval of 0.1～5μg mL-1, with a detection limit of 25 ng mL-1. (3) An ultrasensitive chemiluminescence biosensor was developed for the detection of CT based on a supported lipid membrane as sensing surface and the HRP/GM1-functionalized liposome as detection probe (in Chapter 4). The supported lipid based biosensing surface could be renewed easily and rapidly. The application of enhanced chemiluminescence reaction in the detection of HRP-bearing liposome afforded a further signal amplification and background alleviation. The developed biosensor was shown to give chemiluminescence signal in linear correlation to CT concentration within the range from 1 pg mL-1 to 1 ng mL-1 with readily achievable detection limit of 0.8 pg mL-1. (4) Another highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome (in Chapter 5). The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed lumino/peroxide/enhancer system. The chemiluminescence intensity was directly proportional to the concentration of PSA in sample solution in the range from 7.4 pgmL-1 to 74 ng mL-1 with a detection limit of 4 pg mL-1. In this part, the application of several kinds of functionalized liposome for the construction of biosensor have greatly improved the performance of the resulting biosensor.
In this dissertation, the Au nanoparticles and the carbon nanotubes have also been used for the fabrication of biosensor and combined with electrochemical detection methods:
(5) A highly sensitive electrochemical impedance immunosensor was developed by using a novel amplification procedure with the application of an Au-colloid labeled antibody as the primary amplifying probe and a multistep amplification by alternating treatment of the resulting assembly with an Au-colloid labeled secondary antibody and an Au-colloid labeled antibody(in Chapter 6). Colloidal Au was used as a versatile and efficient template for the immobilization of antibody due to its relatively large surface and good biocompatibility. A novel amplification method was developed with the application of a Au-colloid labeled antibody as the primary amplifying probe and a multistep amplification by alternating treatment of the resulting assembly with a Au-colloid labeled secondary antibody and a Au-colloid labeled antibody. The results obtained revealed that the sensitivity could be substantially improved via the amplification step and a detection limit as low as 4.1 ng L?1 could be reached for a model analyte of human immunoglobulin G (hIgG). (6) A novel approach for scanning of unknown gene mutations was developed based on the utilization of MutS protein for the mutation recognition and spontaneously intercalated MB markers for electrochemical signal generation(in Chapter 7). The occurrence of mutation in target genes resulted in the formation of heteroduplex, which could specifically bind to the immobilized MutS protein on the electrode. The adsorption of heteroduplex on the electrode surface could then be probed electrochemically by a certain redox indicator as methylene blue (MB) that was selectively intercalated in nucleic acid duplexes. The proposed approach has been successfully implemented for the identification of single-base mutation in ?28 site of theβ-thalassemia gene with a detection limit of 5.6×10-13M, providing a highly specific and cost-efficient approach for point mutation detection. (7) An electrochemical label-free detection technique was fabricated for the detection of nucleic acids based on the precipitation of SWNTs by DNA hybridization (in Chapter 8). The DNA hybridization between target DNA and the DNA probe coating on the nanotubes could actively remove DNA probe from the SWNTs surface. The precipitation of SWNTs onto the n-octadecyl mercaptan (C18H37SH) modified Au electrode substantially restores heterogeneous electron transfer between bare Au electrode and redox species in solution phase which was almost totally blocked by the SAM of C18H37SH, and as a result, the electrical signal of the electrode was correlated with the concentration of target DNA in the range from 6.4pM～50nM with a detection limit of 3.2pM.
引文
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