波长检测型表面等离子体子共振传感器性能改进的研究
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摘要
本文系统、深入地研究了改进多波长同时检测型表面等离子体子共振传感器性能的一些方法,以及这些方法在免疫分析方面的应用。详细介绍了多波长同时检测型表面等离子体子共振传感器的仪器构造,概述了SPR传感器的性能以及在提高SPR传感器灵敏度方面的研究进展。
表面等离子体子共振(SPR)传感器通用性强、无需标记、可同时得到反应的一些热力学和动力学常数,在研究分子间的相互作用方面有优势,但其灵敏度有限。因此,有关SPR研究的一个重要内容是提高传感器的灵敏度。本论文通过改进SPR传感器表面结构和试样溶液组成来提高灵敏度。研究了四种方法:1、改进了传统的SPR传感膜结构,应用磁粒子修饰SPR传感膜。与传统的SPR免疫检测传感膜上固定抗体的方法相比,这种方法简化了抗体的固定,加速了待测物的检测,并进一步改进这一方法,通过层层自组装聚电解质和气相沉积二氧化钛两种方法修饰金膜表面,对金膜起到了保护的作用,避免了磁纳米粒子与金膜表面的直接接触而产生的沉积,同时增加了传感器的灵敏度和使用的寿命;2、应用不同浓度的基底溶液检测热休克蛋白(Hsp70);3、抗体-抗原结合法测定补体B因子和纤维蛋白Fn;4、基于电沉积Au-Ag膜和Au-Hg膜修饰金膜的方法测定人免疫球蛋白(hIgG)和兔免疫球蛋白(rIgG)。这几种提高SPR传感器灵敏度的方法操作简单,检测的最低浓度降低了5-10倍。
The most important characteristics of surface plasmon resonance (SPR) sensor are its versatility and capability in real time monitoring the association or dissociation of biomolecules on the surface of the sensor, no label and high selectivity. These characteristics made the SPR technique an easy, convenient and reliable one for determining the concentration and molecular weight, monitoring change in structure, measuring kinetic constant and binding specificity of individual biomolecules. Since the first SPR sensor was developed in 1983 by Liedberg et al, the research and application of SPR technology have shown extensive growth and gradually become the hot spot and research frontier in the biosensor field in the world. In recent years, the SPR sensors have been developed very rapidly and applied to such function research based on molecular level. After the SPR technology was first commercially developed to dominate this market, SPR biosensors have become standard bio-tools in both academic and industrial research laboratories. The popularity of the technology continues to grow, as numbers of scientific publications immerged that describing experiments using SPR.
In addition, new methods of SPR are studied mostly for the sensitivity enhancement of SPR sensor, except for enlarging application range of SPR. So, exploring various analytical methods actively for enhancing SPR sensitivity is also a mainly topic. The work includes two aspects for sensitivity enhancement of SPR sensor. On the one hand, the research system is improved. Such as, improving SPR sensor surface structure and changing component of reagent solution. On the other hand, the SPR instrument is improved including optimizing wave-guide element, renovating detection system and combining with other high sensitivity instrument et al. The element theory and technical characteristics of SPR sensor are introduced. Several types of wavelength modulation SPR sensors have been employed and some applications of these sensors presented. The configurations of wavelength modulation SPR sensors installed in our lab are outlined and the latest developments in the application of sensitivity enhancement of SPR sensor were summarized.
Dextran coated magnetic nanocomposites were prepared. A periodate oxidation method was used to further activate the magnetic dextran, forming magnetic polyaldehyde-dextran, which could be conjugated to antibodies. This biological probe could be trapped on the Au film firmly due to the magnetic force. Since the magnetic bead is coated with dextran, the antibodies and some specific biomolecular receptors can be immobilized using a variety of chemical reactions. And the conjugates of magnetic microbeads coupling with antibody could be dissociated from the Au film due to the absence of the magnetic field. Compared to traditional antibody immobilization on the sensing film, there is not a covalent link between the Au film and the antibody. There is a great advantage in that sensor can be stripped and reused. After the magnetic pillar was above the prism, the sensing film of the conjugates was structured on the gold surface for the immunoassay of Hsp70. Different dilution ratios (V/V) of the conjugates result in different detectable ranges. When the dilution ratio of the conjugates was 1:10, 1:8 and 1:5, the SPR biosensor yields good response to Hsp70 in the concentration range of 1.50-30.00μg mL~(-1), 0.75-30.00μg mL~(-1) and 0.30-30.00μg mL~(-1), respectively. In addition, the stability of conjugates trapped on the Au film was investigeted and the immobilization of conjugates was analyzed by UV-Visible spectrum.
Meanwhile, the surface of the sensing film was modified with self-assembly of polyelectrolyte and vapor deposition of titania sol-gel matrix. With self-assembly of polyelectrolyte, different layers of polyelectrolyte can be assembled onto the Au film based on an electrostatic force between polycations and polyanions. The polyelectrolyte layers with appropriate thickness could effectively prevent the magnetic beads from depositing on the Au film and increase the life expectancy of this biosensor that favors the sensitivity of the biosensor and the regeneration of the sensing membrane. The assay of human IgG with standard SPR sensors by traditional method without magnetic beads was performed, and the lowest concentration that could be detected was 2.00μg mL~(-1). When the polyelectrolyte layer of (PAH/PSS)_1, (PAH/PSS)_2 and (PAH/PSS)_3 were separately constructed on the Au film, human IgG was determined in the concentration range of 2.00-30.00μg mL~(-1), 0.50-30.00μg mL~(-1) and 0.25-30.00μg mL~(-1), respectively. The sensing film with (PAH/PSS)_3 layer exhibits a larger dynamic range since it results in up to about 8 times lower determination limit than that obtained with (PAH/PSS)1 layer. The rational explanation was given for every experiment phenomenon. With the increase of thickness for the polyelectrolyte layers, the space of the surface was expanded producing more binding points, following with the resonant wavelength moving to longer wavelength and enhancing sensitivity of the wavelength modulation SPR biosensor. Also the effects of different polyelectrolyte layers on the regeneration of the biosensor were investigated. Meanwhile, the association constant of human IgG with antibody is 3.195×107 L mol~(-1). The surface of the Au film modified with (PAH/PSS)3 was measured by atomic force microscope (AFM).
A facile vapor deposition method has been developed for the preparation of sol-gel matrix on the Au film by determining Hsp70. TiO_2 sol-gel matrix was constructed on the thin Au films by vapor deposition method with proper temperature. The vapor deposition method simplified the traditional sol-gel process and prevented the cracking of conventional sol-gel derived glasses. The sol-gel derived titania-modified Au film retained the bioactivity and provided for long-term stability of the biomaterials in storage. In the magnetic field, the conjugates of magnetic beads coupled with rabbit anti-Hsp70 were immobilized on the Au film modified with TiO_2 sol-gel membrane and the analysis of the immunoassay was done by SPR biosensor. Effects of the vapor deposition time and operating potential for obtaining the TiO_2 sol-gel matrix were explored. When titania sol-gel membrane obtained for deposition time of 30, 45 and 60 min, the SPR biosensor yields good response to Hsp70 in the concentration range of 1.00-30.00μg mL~(-1), 0.50-30.00μg mL~(-1) and 0.20-30.00μg mL~(-1), respectively. The limit of detection of the immunoassay is 0.062μg mL~(-1). Meanwhile, the effects of TiO_2 sol-gel membrane on the stability and regeneration of the SPR sensor were discussed.
The sensitivity enhancement of wavelength modulation SPR biosensor was described by improving the baseline solution. The baseline solutions used are ethanol solutions, and all reagents in the assay were diluted with ethanol solution. Molecular self-assembling on the surface of the sensor is applied to form the sensing membrane on the gold substrate and detect Hsp70 quantitatively. The resonant wavelength changes with the change of baseline solutions. For the baseline solution of higher ethanol concentration, the resonant wavelength moved to the longer wavelength and the change of refractive index at longer wavelength could made larger shift of the resonant wavelength. When 1.0%, 2.0%, 4.0% and 6.0% ethanol solutions were used as the baseline solutions, the SPR biosensor has good response to Hsp70 in the concentration range 1.00-10.00μg mL~(-1) , 0.50-10.00μg mL~(-1) , 0.20-10.00μg mL~(-1) and 0.10-10.00μg mL~(-1), respectively. For the detection using 6.0% ethanol solution as the baseline solution, the lowest detectable concentration (0.10μg mL~(-1)) is 10-fold lower than that using 1.0% ethanol solution as the baseline solution (1.00μg mL~(-1)). The non-specific binding and the activity of antibody due to the ethanol solution were also discussed in detail. Meanwhile, it was proved with theoretical calculation that the sensitivity enhancement is not only relative to the increase of binding constant KA, but also to the shift of resonant wavelength towards longer wavelength. The method of changing the baseline solution that made the resonant wavelength move to the longer wavelength is authentic and feasible to enhance the sensitivity of SPR biosensor.
A method of the sensitivity enhancement for the SPR biosensor is presented using antibody-antigen coupling assay. The human factor B (Bf) and fibronectin (FN) were determined and analyzed with kinetic process. The sensor surface was prepared by self-assembling monolayer (SAM), and regenerated by rising with citrate buffer. Thoil terminal of MPA is easily adsorbed to gold surface and–COOH is an active group that can bind to antibody. The antibody in the solution first bound to antigen. Then the binding of antigen to antibody in the solution leads to the formation of large couplers that could bind to the antibody immobilized on the Au film. The lowest determination concentration of Bf (0.05μg mL~(-1)) obtained by antibody-antigen coupling method is 10-fold lower than that by direct immunoreaction (0.5μg mL~(-1) ). The lowest determination concentration of FN (0.25μg mL~(-1)) obtained by antibody-antigen coupling assay is 8-fold lower than that by direct immunoreaction (2.00μg mL~(-1) ). The binding constants KA for Bf and FN are 3.72×107 L mol~(-1) and 1.92×107 L mol -1, respectively. Due to the coupling of antibody to antigen, the binding of antigen to antibody on the Au film could cause a large shift of the resonant wavelength. The sensitivity of the assay can be optimized with adjusting the concentration of the antigen and antibody preparations. Meanwhile, the selectivity, stability and the linear ranges of the SPR biosensor were investigated and the rational explanation was given for the experiment phenomenon.
The sensitivity of the SPR biosensor was enhanced with the combination of electrochemical technique which is very accurate and advantageous. Surface functionality with element Hg or Ag is used as precursor for depositing the sensing layer onto the Au glass substrates. Electrochemical experiments were performed with a CHI660A electrochemical station. Hg or Ag was electrodeposited on the Au surface to form the Au-Hg or Au-Ag film. The SPR biosensors based on the Au-Hg or Au-Ag film were used to determine the human IgG and rabbit IgG. MPA is easily adsorbed on the Au-Hg or Au-Ag film. Then the COO- terminal group is an active group that can bind to antibody. When Hg or Ag overlayer was formed on the gold film, the surface of the film becomes rough that results in more molecules immobilized on the surface. Meanwhile, the electrodeposition time of Hg and Ag also has effect on the IgG determination. When the electrodeposition time of Hg on the Au film was 900 s and 1200 s, the concentration ranges for determination of human IgG are 1.00-40.00μg mL~(-1) and 0.50-40.00μg mL~(-1), respectively. When Ag was electrodeposited on the Au film for 1000 s and 1500 s, the concentration ranges for human IgG are 0.50-20.00μg mL~(-1) and 0.25-20.00μg mL~(-1), respectively. For the determination of rabbit IgG, the Au-Hg film is the most sensitive resulting in up to 4 times lower determination limits than that obtained by Au film and the Au-Ag film is 6 times than by Au film. With the electrodeposition time increasing, the thickness of Hg or Ag overlayer increases. These make the resonant wavelength move to longer wavelength in some wavelength range that results in higher sensitivity of the wavelength modulation SPR biosensor. Meanwhile, Hg and Ag film themselves also contributed to the higher sensitivity.
表面等离子体子共振(SPR)传感器通用性强、无需标记、可同时得到反应的一些热力学和动力学常数,在研究分子间的相互作用方面有优势,但其灵敏度有限。因此,有关SPR研究的一个重要内容是提高传感器的灵敏度。本论文通过改进SPR传感器表面结构和试样溶液组成来提高灵敏度。研究了四种方法:1、改进了传统的SPR传感膜结构,应用磁粒子修饰SPR传感膜。与传统的SPR免疫检测传感膜上固定抗体的方法相比,这种方法简化了抗体的固定,加速了待测物的检测,并进一步改进这一方法,通过层层自组装聚电解质和气相沉积二氧化钛两种方法修饰金膜表面,对金膜起到了保护的作用,避免了磁纳米粒子与金膜表面的直接接触而产生的沉积,同时增加了传感器的灵敏度和使用的寿命;2、应用不同浓度的基底溶液检测热休克蛋白(Hsp70);3、抗体-抗原结合法测定补体B因子和纤维蛋白Fn;4、基于电沉积Au-Ag膜和Au-Hg膜修饰金膜的方法测定人免疫球蛋白(hIgG)和兔免疫球蛋白(rIgG)。这几种提高SPR传感器灵敏度的方法操作简单,检测的最低浓度降低了5-10倍。
The most important characteristics of surface plasmon resonance (SPR) sensor are its versatility and capability in real time monitoring the association or dissociation of biomolecules on the surface of the sensor, no label and high selectivity. These characteristics made the SPR technique an easy, convenient and reliable one for determining the concentration and molecular weight, monitoring change in structure, measuring kinetic constant and binding specificity of individual biomolecules. Since the first SPR sensor was developed in 1983 by Liedberg et al, the research and application of SPR technology have shown extensive growth and gradually become the hot spot and research frontier in the biosensor field in the world. In recent years, the SPR sensors have been developed very rapidly and applied to such function research based on molecular level. After the SPR technology was first commercially developed to dominate this market, SPR biosensors have become standard bio-tools in both academic and industrial research laboratories. The popularity of the technology continues to grow, as numbers of scientific publications immerged that describing experiments using SPR.
In addition, new methods of SPR are studied mostly for the sensitivity enhancement of SPR sensor, except for enlarging application range of SPR. So, exploring various analytical methods actively for enhancing SPR sensitivity is also a mainly topic. The work includes two aspects for sensitivity enhancement of SPR sensor. On the one hand, the research system is improved. Such as, improving SPR sensor surface structure and changing component of reagent solution. On the other hand, the SPR instrument is improved including optimizing wave-guide element, renovating detection system and combining with other high sensitivity instrument et al. The element theory and technical characteristics of SPR sensor are introduced. Several types of wavelength modulation SPR sensors have been employed and some applications of these sensors presented. The configurations of wavelength modulation SPR sensors installed in our lab are outlined and the latest developments in the application of sensitivity enhancement of SPR sensor were summarized.
Dextran coated magnetic nanocomposites were prepared. A periodate oxidation method was used to further activate the magnetic dextran, forming magnetic polyaldehyde-dextran, which could be conjugated to antibodies. This biological probe could be trapped on the Au film firmly due to the magnetic force. Since the magnetic bead is coated with dextran, the antibodies and some specific biomolecular receptors can be immobilized using a variety of chemical reactions. And the conjugates of magnetic microbeads coupling with antibody could be dissociated from the Au film due to the absence of the magnetic field. Compared to traditional antibody immobilization on the sensing film, there is not a covalent link between the Au film and the antibody. There is a great advantage in that sensor can be stripped and reused. After the magnetic pillar was above the prism, the sensing film of the conjugates was structured on the gold surface for the immunoassay of Hsp70. Different dilution ratios (V/V) of the conjugates result in different detectable ranges. When the dilution ratio of the conjugates was 1:10, 1:8 and 1:5, the SPR biosensor yields good response to Hsp70 in the concentration range of 1.50-30.00μg mL~(-1), 0.75-30.00μg mL~(-1) and 0.30-30.00μg mL~(-1), respectively. In addition, the stability of conjugates trapped on the Au film was investigeted and the immobilization of conjugates was analyzed by UV-Visible spectrum.
Meanwhile, the surface of the sensing film was modified with self-assembly of polyelectrolyte and vapor deposition of titania sol-gel matrix. With self-assembly of polyelectrolyte, different layers of polyelectrolyte can be assembled onto the Au film based on an electrostatic force between polycations and polyanions. The polyelectrolyte layers with appropriate thickness could effectively prevent the magnetic beads from depositing on the Au film and increase the life expectancy of this biosensor that favors the sensitivity of the biosensor and the regeneration of the sensing membrane. The assay of human IgG with standard SPR sensors by traditional method without magnetic beads was performed, and the lowest concentration that could be detected was 2.00μg mL~(-1). When the polyelectrolyte layer of (PAH/PSS)_1, (PAH/PSS)_2 and (PAH/PSS)_3 were separately constructed on the Au film, human IgG was determined in the concentration range of 2.00-30.00μg mL~(-1), 0.50-30.00μg mL~(-1) and 0.25-30.00μg mL~(-1), respectively. The sensing film with (PAH/PSS)_3 layer exhibits a larger dynamic range since it results in up to about 8 times lower determination limit than that obtained with (PAH/PSS)1 layer. The rational explanation was given for every experiment phenomenon. With the increase of thickness for the polyelectrolyte layers, the space of the surface was expanded producing more binding points, following with the resonant wavelength moving to longer wavelength and enhancing sensitivity of the wavelength modulation SPR biosensor. Also the effects of different polyelectrolyte layers on the regeneration of the biosensor were investigated. Meanwhile, the association constant of human IgG with antibody is 3.195×107 L mol~(-1). The surface of the Au film modified with (PAH/PSS)3 was measured by atomic force microscope (AFM).
A facile vapor deposition method has been developed for the preparation of sol-gel matrix on the Au film by determining Hsp70. TiO_2 sol-gel matrix was constructed on the thin Au films by vapor deposition method with proper temperature. The vapor deposition method simplified the traditional sol-gel process and prevented the cracking of conventional sol-gel derived glasses. The sol-gel derived titania-modified Au film retained the bioactivity and provided for long-term stability of the biomaterials in storage. In the magnetic field, the conjugates of magnetic beads coupled with rabbit anti-Hsp70 were immobilized on the Au film modified with TiO_2 sol-gel membrane and the analysis of the immunoassay was done by SPR biosensor. Effects of the vapor deposition time and operating potential for obtaining the TiO_2 sol-gel matrix were explored. When titania sol-gel membrane obtained for deposition time of 30, 45 and 60 min, the SPR biosensor yields good response to Hsp70 in the concentration range of 1.00-30.00μg mL~(-1), 0.50-30.00μg mL~(-1) and 0.20-30.00μg mL~(-1), respectively. The limit of detection of the immunoassay is 0.062μg mL~(-1). Meanwhile, the effects of TiO_2 sol-gel membrane on the stability and regeneration of the SPR sensor were discussed.
The sensitivity enhancement of wavelength modulation SPR biosensor was described by improving the baseline solution. The baseline solutions used are ethanol solutions, and all reagents in the assay were diluted with ethanol solution. Molecular self-assembling on the surface of the sensor is applied to form the sensing membrane on the gold substrate and detect Hsp70 quantitatively. The resonant wavelength changes with the change of baseline solutions. For the baseline solution of higher ethanol concentration, the resonant wavelength moved to the longer wavelength and the change of refractive index at longer wavelength could made larger shift of the resonant wavelength. When 1.0%, 2.0%, 4.0% and 6.0% ethanol solutions were used as the baseline solutions, the SPR biosensor has good response to Hsp70 in the concentration range 1.00-10.00μg mL~(-1) , 0.50-10.00μg mL~(-1) , 0.20-10.00μg mL~(-1) and 0.10-10.00μg mL~(-1), respectively. For the detection using 6.0% ethanol solution as the baseline solution, the lowest detectable concentration (0.10μg mL~(-1)) is 10-fold lower than that using 1.0% ethanol solution as the baseline solution (1.00μg mL~(-1)). The non-specific binding and the activity of antibody due to the ethanol solution were also discussed in detail. Meanwhile, it was proved with theoretical calculation that the sensitivity enhancement is not only relative to the increase of binding constant KA, but also to the shift of resonant wavelength towards longer wavelength. The method of changing the baseline solution that made the resonant wavelength move to the longer wavelength is authentic and feasible to enhance the sensitivity of SPR biosensor.
A method of the sensitivity enhancement for the SPR biosensor is presented using antibody-antigen coupling assay. The human factor B (Bf) and fibronectin (FN) were determined and analyzed with kinetic process. The sensor surface was prepared by self-assembling monolayer (SAM), and regenerated by rising with citrate buffer. Thoil terminal of MPA is easily adsorbed to gold surface and–COOH is an active group that can bind to antibody. The antibody in the solution first bound to antigen. Then the binding of antigen to antibody in the solution leads to the formation of large couplers that could bind to the antibody immobilized on the Au film. The lowest determination concentration of Bf (0.05μg mL~(-1)) obtained by antibody-antigen coupling method is 10-fold lower than that by direct immunoreaction (0.5μg mL~(-1) ). The lowest determination concentration of FN (0.25μg mL~(-1)) obtained by antibody-antigen coupling assay is 8-fold lower than that by direct immunoreaction (2.00μg mL~(-1) ). The binding constants KA for Bf and FN are 3.72×107 L mol~(-1) and 1.92×107 L mol -1, respectively. Due to the coupling of antibody to antigen, the binding of antigen to antibody on the Au film could cause a large shift of the resonant wavelength. The sensitivity of the assay can be optimized with adjusting the concentration of the antigen and antibody preparations. Meanwhile, the selectivity, stability and the linear ranges of the SPR biosensor were investigated and the rational explanation was given for the experiment phenomenon.
The sensitivity of the SPR biosensor was enhanced with the combination of electrochemical technique which is very accurate and advantageous. Surface functionality with element Hg or Ag is used as precursor for depositing the sensing layer onto the Au glass substrates. Electrochemical experiments were performed with a CHI660A electrochemical station. Hg or Ag was electrodeposited on the Au surface to form the Au-Hg or Au-Ag film. The SPR biosensors based on the Au-Hg or Au-Ag film were used to determine the human IgG and rabbit IgG. MPA is easily adsorbed on the Au-Hg or Au-Ag film. Then the COO- terminal group is an active group that can bind to antibody. When Hg or Ag overlayer was formed on the gold film, the surface of the film becomes rough that results in more molecules immobilized on the surface. Meanwhile, the electrodeposition time of Hg and Ag also has effect on the IgG determination. When the electrodeposition time of Hg on the Au film was 900 s and 1200 s, the concentration ranges for determination of human IgG are 1.00-40.00μg mL~(-1) and 0.50-40.00μg mL~(-1), respectively. When Ag was electrodeposited on the Au film for 1000 s and 1500 s, the concentration ranges for human IgG are 0.50-20.00μg mL~(-1) and 0.25-20.00μg mL~(-1), respectively. For the determination of rabbit IgG, the Au-Hg film is the most sensitive resulting in up to 4 times lower determination limits than that obtained by Au film and the Au-Ag film is 6 times than by Au film. With the electrodeposition time increasing, the thickness of Hg or Ag overlayer increases. These make the resonant wavelength move to longer wavelength in some wavelength range that results in higher sensitivity of the wavelength modulation SPR biosensor. Meanwhile, Hg and Ag film themselves also contributed to the higher sensitivity.
引文
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