胶体金LSPR传感膜的制备及其在免疫检测中的应用
摘要
贵金属纳米粒子较强的局域表面等离子共振(LSPR)使其具有独特的光学性质,由于这种性质在理论和应用方面具有很好的前景,而备受世人瞩目。特别是其传感性(能将周围介质折射率变化转变为光学信号),引起了材料化学家和物理化学家广泛的兴趣。近年来,纳米材料的制备技术发展迅速,但构筑粒子分布均匀、单分散性好、重现性高、稳定性好、制备过程简单的纳米粒子膜层材料仍是科学家们热衷的课题。本论文针对这一课题构筑了符合上述要求的金纳米粒子单层膜,并对其传感性能进行研究,最终将其应用于免疫识别的检测。主要内容为:一、采用传统的聚电解质静电组装方法制备了12种膜层结构,并对膜中胶体金纳米粒子单分散性、粒子分布、稳定性及重现性等方面进行了系统地研究,得到符合上述纳米粒子膜层材料要求的组装方法。二、利用优化的组装方式制备金纳米粒子单层膜,并对其传感性能以及LSPR传感膜粒径参数进行优化。实验结果表明,在膜传感性能的研究中,金纳米粒子膜能将周围介质折射率(“体相”折射率和“局域”折射率)变化转变为光学信号,且表现出较高的灵敏度,具备传感器的要求,故称之为LSPR传感膜。在此基础上,通过改变LSPR传感膜中粒子的粒径来优化其传感性能,并从中选择检测灵敏度最高时膜中纳米粒子粒径。得到对于波长、峰强对折射率变化最敏感的膜分别是LSPR_(Au41nm)和LSPR_(Au69nm)膜。三、将LSPR_(Au41nm)和LSPR_(Au69nm)膜应用于免疫检测中。实验结果表明,二者均能用于生物传感,而且LSPR_(Au69nm)膜比LSPR_(Au41nm)膜具有更高的灵敏度。对于羊抗人IgG与人IgG体系(固定抗体以及识别相应抗原整个过程限定为2h),抗体浓度选择100μg/mL的条件下,抗原的最低检出限为1μg/mL。
Immunoassay is a powerful analytical tool for applications in the field of biochemical analysis and clinical diagnosis that based on the specific interaction between antigen and its complementary antibody. Several types of immunoassay techniques have been developed, such as enzyme-linked immunoassays, radioimmunoassay, immunoagglutination assays, and fluorescent immunoassays. These immunoassay techniques display important roles in clinical analyses, medical diagnostics, and environmental analyses. One common feature of these immunoassay technique listed above is the requirement of a label molecule for detecting bio-interactions. Great efforts have been made in the last two decades in development of several new types of optical biosensors, and their corresponding applications in the field of environment protection, biotechnology, medical diagnostics, drug screening, food safety, and security. Surface Plasmon Resonance (SPR), a phenomenon describes the interaction between the incident light wave and the surface electrons of metal film or particle that give rise to electromagnetic field (EF), is very sensitive to the changes of refractive index (RI). The potential of SPR for applications in the field of sensing was realized in the early 1980s by Liedberg et al., who were able to sense immunoglobulin antibodies by measuring the changes in critical angle when the antibodies bound selectively to a gold film. Generally, there are two types of SPR sensors: the propagating SPR (PSPR) sensor based on the response of EF to the changes of RI on a continuous metal film, and the localized SPR (LSPR) sensor based on sensing of the local changes of RI on the surface of metal nanoparticles. In these years, SPR technique, especially LSPR technique, has been applied in the label-free sensing on the detection of biomolecular interactions in real time. Self-assemble is a platform to extend the employment of LSPR for biosensing. Self-assemble was developed by G. Decher in 1991, who found a route to fabricate a multilayer structure by using the electrostatic interaction as the driving force. The self-assemble technique has been proven to be a rapid and experimentally simple way to produce complex multilayer structure with precise control over the composition and thickness. Although much work has been reported on self-assemble of monolayer gold nanoparticle films, few of them were used as the biosensing film. In this thesis, we fabricate the biosensor by self-assemble gold nanoparticles on polyelectrolyte films. All the relative studies are outlined as follows:
1. Investigations on the preparation of colloidal gold nanoparticle monolayer film by electrostatic self-assembly.
A serials of gold nanoparticle monolayer films were fabricated by self-assemble of gold nanparticles on the substrates which contained monolayer or trilayer of polyelectrolytes. Several types polyelectrolytes were chosen: poly(vinylbenzyl) trimethylammonium chloride (PVTC), poly(diallyl-dimethylammonium) (PDDA), and poly(allylamine) hydrochloride (PAH) as the cationic polyelectrolytes; polystyrenesulfonate (PSS) as the anionic polyelectrolyte. The assembled gold nanoparticle films were characterized by the UV-vis spectroscopy and SEM. The result showed that the PDDA/PSS/PVTC/AuNP assembly film owes good reproducibility, high intensity and excellent stability.
We investigated the sensing performance of PDDA/PSS/PVTC/AuNP film by changing dielectric environment (“bulk”refractive index and“local”refractive index) around the assemble film. The result showed that the LSPR peak intensity, band intensity at 575nm, and band position of the LSPR peak of the gold assemble film were linear to the changes of RI of surrounding medium. It proved that the PDDA/PSS/PVTC/AuNP assembly film can be employed as a good LSPR sensing flim.
2. Size optimization of the gold nanoparticles in the PDDA/PSS/PVTC/AuNP assembly film
The effect of the assembled gold nanoparticle size on the response character of the sensing film was investigated. Several sizes of gold nanoparticles were chosen. The investigations were achieved by evaluating sensitivities of the LSPR peak intensity and LSPR band position of the assembly gold nanoparticles film to the changes in RI of surrounding medium. The result showed that the 41-nm assembly film exhibited maximum sensitivity on the response of LSPR band position to the changes of RI, and the 69-nm assembly film displayed maximum sensitivity on the LSPR band intensity.
3. Biosensing by LSPR of nanoparticles on quartz
Both 41-nm and 69-nm gold nanoparticle assembly films were employed as the LSPR sensing film for biodetection. The changes inλ_(max) of LSPR_(41 nm) and Ext_(575 nm) of LSPR_(69 nm) were monitored during the adsoption of different concentrations of human IgG, and used to characterize the sensing properties of these films. The result showed that the LSPR_(69 nm) film was more sensitive, and it was applied in immunorecognition. The applicability of the biosensor in the presence of interference agents was verified by introducing rabbit IgG and bovine IgG in the detection of human IgG. The biosensing film exhibited a limit of detection of 1μg/mL for the human IgG interaction after immersed in goat-anti-human IgG for 30 min.
Immunoassay is a powerful analytical tool for applications in the field of biochemical analysis and clinical diagnosis that based on the specific interaction between antigen and its complementary antibody. Several types of immunoassay techniques have been developed, such as enzyme-linked immunoassays, radioimmunoassay, immunoagglutination assays, and fluorescent immunoassays. These immunoassay techniques display important roles in clinical analyses, medical diagnostics, and environmental analyses. One common feature of these immunoassay technique listed above is the requirement of a label molecule for detecting bio-interactions. Great efforts have been made in the last two decades in development of several new types of optical biosensors, and their corresponding applications in the field of environment protection, biotechnology, medical diagnostics, drug screening, food safety, and security. Surface Plasmon Resonance (SPR), a phenomenon describes the interaction between the incident light wave and the surface electrons of metal film or particle that give rise to electromagnetic field (EF), is very sensitive to the changes of refractive index (RI). The potential of SPR for applications in the field of sensing was realized in the early 1980s by Liedberg et al., who were able to sense immunoglobulin antibodies by measuring the changes in critical angle when the antibodies bound selectively to a gold film. Generally, there are two types of SPR sensors: the propagating SPR (PSPR) sensor based on the response of EF to the changes of RI on a continuous metal film, and the localized SPR (LSPR) sensor based on sensing of the local changes of RI on the surface of metal nanoparticles. In these years, SPR technique, especially LSPR technique, has been applied in the label-free sensing on the detection of biomolecular interactions in real time. Self-assemble is a platform to extend the employment of LSPR for biosensing. Self-assemble was developed by G. Decher in 1991, who found a route to fabricate a multilayer structure by using the electrostatic interaction as the driving force. The self-assemble technique has been proven to be a rapid and experimentally simple way to produce complex multilayer structure with precise control over the composition and thickness. Although much work has been reported on self-assemble of monolayer gold nanoparticle films, few of them were used as the biosensing film. In this thesis, we fabricate the biosensor by self-assemble gold nanoparticles on polyelectrolyte films. All the relative studies are outlined as follows:
1. Investigations on the preparation of colloidal gold nanoparticle monolayer film by electrostatic self-assembly.
A serials of gold nanoparticle monolayer films were fabricated by self-assemble of gold nanparticles on the substrates which contained monolayer or trilayer of polyelectrolytes. Several types polyelectrolytes were chosen: poly(vinylbenzyl) trimethylammonium chloride (PVTC), poly(diallyl-dimethylammonium) (PDDA), and poly(allylamine) hydrochloride (PAH) as the cationic polyelectrolytes; polystyrenesulfonate (PSS) as the anionic polyelectrolyte. The assembled gold nanoparticle films were characterized by the UV-vis spectroscopy and SEM. The result showed that the PDDA/PSS/PVTC/AuNP assembly film owes good reproducibility, high intensity and excellent stability.
We investigated the sensing performance of PDDA/PSS/PVTC/AuNP film by changing dielectric environment (“bulk”refractive index and“local”refractive index) around the assemble film. The result showed that the LSPR peak intensity, band intensity at 575nm, and band position of the LSPR peak of the gold assemble film were linear to the changes of RI of surrounding medium. It proved that the PDDA/PSS/PVTC/AuNP assembly film can be employed as a good LSPR sensing flim.
2. Size optimization of the gold nanoparticles in the PDDA/PSS/PVTC/AuNP assembly film
The effect of the assembled gold nanoparticle size on the response character of the sensing film was investigated. Several sizes of gold nanoparticles were chosen. The investigations were achieved by evaluating sensitivities of the LSPR peak intensity and LSPR band position of the assembly gold nanoparticles film to the changes in RI of surrounding medium. The result showed that the 41-nm assembly film exhibited maximum sensitivity on the response of LSPR band position to the changes of RI, and the 69-nm assembly film displayed maximum sensitivity on the LSPR band intensity.
3. Biosensing by LSPR of nanoparticles on quartz
Both 41-nm and 69-nm gold nanoparticle assembly films were employed as the LSPR sensing film for biodetection. The changes inλ_(max) of LSPR_(41 nm) and Ext_(575 nm) of LSPR_(69 nm) were monitored during the adsoption of different concentrations of human IgG, and used to characterize the sensing properties of these films. The result showed that the LSPR_(69 nm) film was more sensitive, and it was applied in immunorecognition. The applicability of the biosensor in the presence of interference agents was verified by introducing rabbit IgG and bovine IgG in the detection of human IgG. The biosensing film exhibited a limit of detection of 1μg/mL for the human IgG interaction after immersed in goat-anti-human IgG for 30 min.
引文
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