基于光学读出的微悬臂梁生化传感技术研究
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摘要
微悬臂梁生化传感技术是近年发展起来的新兴技术。当有生化反应在微悬臂梁的单侧表面发生时,其表面应力的改变会导致微悬臂梁产生弯曲变形,通过光学或电学读出方法可以测量变形值。由于其具有大比表面积,表面效应强,容易实现阵列集成等优点,为生化传感器的研究提供了新的思路,成为研究的热点。
本文以微悬臂梁生化传感技术发展的两大核心——提高检测灵敏度和高通量检测为出发点,对基于光杠杆原理的单悬臂梁检测系统进行改进,利用改进后的实验系统研究了大分子在界面的自组装过程以及在不同的抗体修饰方法下,抗原抗体的特异性结合,并提出了两种微悬臂梁阵列检测的方案。
通过使用聚焦处半径较小的激光器,稳定激光器的输出强度,使实验系统的可重复性得到提高。通过实验和理论分析,在反射光路中加入凸透镜以及优化微悬臂梁尺寸,可以使系统检测灵敏度分别提高4倍和20倍。通过使用高精度的恒温器(温控精度±0.01K),消除了微悬臂梁的热变形影响,同时使本底噪音保持在合理的范围,而且确保样品温度和容器池的温差不会对系统检测造成影响,提高了系统的信噪比。通过对传统光路参数的理论分析,得到在特定的光路参数下,可以完全消除溶液折射率改变对系统检测的影响,而且不改变系统的检测灵敏度。理论分析得到实验结果的验证。利用溶液折射率变化对系统检测的影响,提出了一种通过检测液体折射率来检测液体浓度的方案。
研究了不同分子量的巯基化的聚N-异丙基丙烯酰胺(HS-PNIPAM)在金表面自组装的动力学过程。实验结果表明HS-PNIPAM在自组装过程中存在三个阶段,分别对应不同的分子构象。第一阶段为物理吸附阶段,第二、三阶段为伴随着分子构象变化的化学吸附阶段。化学吸附曲线符合Langmuir等温吸附。分析结果还显示HS-PNIPAM的表面吸附速率κ远小于小分子的吸附速率,并与分子量成负指数关系;HS-PNIPAM的自组装时间远大于小分子的自组装时间,并与分子量成正比;底物表面应力的改变与分子量成正线性关系。
研究了在三种抗体修饰方法下,抗原抗体的特异性结合。将抗体修饰在微悬臂梁的金面,检测不同浓度的标准样品下微悬臂梁的弯曲变形,包括抗体巯基化修饰方法检测瘦肉精、SPA修饰抗体的方法检测瘦肉精和氯霉素以及二抗修饰抗体的方法检测青蒿素和马兜铃酸。抗体的活性以及在微悬臂梁上进行抗原抗体结合的可行性得到了ELISA实验的验证。实验结果表明:抗原抗体的结合具有很强的选择性。结合产生了压应力,且与浓度成正比。二抗修饰方法是三种方法中效果最差的,检测极限比标准曲线对应的IC_(20)低于两个数量级。SPA修饰方法与巯基化修饰方法的检测效果大体一致,检测极限都要优于1ng/ml,与标准曲线对应的IC_(20)相当。分析认为,检测极限与抗体活性和抗原抗体结合位置到微悬臂梁表面的距离有直接的关系。
提出了两种检测微悬臂梁阵列变形的光路方案。第一,在UC Berkeley的光路基础之上进行改进,将偏振光引入检测系统,通过对光束偏振态的控制,降低了测量信号的背景噪声,提高了系统信噪比;通过对微悬臂梁的优化设计(初始转角和反光板),提高光学检测灵敏度。第二,将光学滤波方法引入到生化检测当中。
In recent years,as a new technology,microcantilever biochemical sensing technique(MCS) is developing quickly.When specific bio-molecular interactions occur on one surface of microcantilever,a differential stress between the top and bottom surfaces of the cantilever will cause the microcantilever bending.The deflection can be measured by both optical and electrical readout methods.It has brought some new ideas for biochemical sensing technique and becomes a hotspot of research because of its advantages,such as large surface-area-to-volume ratio,very sensitive to surface effect,easy to realize array compact.
In this paper,two core of development of MCS- improving detecting sensitivity and high-throughput detection were taken as the starting point.First,a single cantilever detection system based on optical lever technique was improved. Second,the improved experimental system was used to study self-assembly of macromolecules on gold surface and the specificity of antigen-antibody binding in the different antibody-modified methods.Finally,two readout methods based on microcantilever array were proposed.
The detecting reproducibility of system can be improved by way of using the laser with the smaller focus radius and steady output intensity.Through experiments and theoretical analysis,by adding convex lens in the path of reflected light and optimizing the dimension parameters of microcantilever,the detecting sensitivity of system can be improved 4 and 20 times,separately.Through the use of high-precision thermostat,the thermal deformation of microcantilever can be eliminated,while the background noise is kept within reasonable scope,and the temperature difference between the sample and the cell will not impact on the system detection.The theoretical analysis showed that by conditioning the experimental parameter,the influence of refractive index change on the detection signal can be effectively eliminated without reducing the detecting sensitivity of the system.The theoretical results were validated by the experiments.Based on this,a method of detecting liquid concentration by detecting the refractive index of liquid was proposed.
The self-assembly of different molecular weight of Poly(N-isopropylacrylamide) (HS-PNIPAM) on gold surface was investigated.The results showed that the kinetic curves can be divided into three stages corresponding to different conformations, respectively.The first stage responded to physical adsorption of HS-PNIPAM to gold-coated side.The second and third stages were responsible for chemical adsorption to gold-coated side with conformation transition.The kinetic curves fits Langmuir adsorption isotherm well.The results also show that the reaction rateκof HS-PNIPAM is far less than that of small molecules and decreases exponentially with the molecular weight;while the time of HS-PNIPAM's self-assembly is far more than that of small molecules and proportional to the molecular weight.The change of the surface stress is linear to the molecular weight of HS-PNIPAM.
The specificity of antigen-antibody binding in three different antibody-modified methods was investigated.Antibody was immobilized on the gold-coated side of the microcantilever and the deflections of the microcantilever corresponding to the process of injecting different concentration of antigen were real-time monitored, including the clenbuterol was detected in the modified method of thiolated antibody, the clenbuterol and chloramphenicol were detected in the modified method of Staphylococcus aureus protein A(SPA),the artemisinin and aristolochic acid were detected in the modified method of second antibody(goat anti-mouse IgG).The activity of antibody and antigen-antibody binding on the gold-coated side of the microcantilever were confirmed by an Enzyme-Linked Immunosorbent Assay (ELISA).The results showed that antigen-antibody binding on microcantilever generates a compressive surface stress and the surface stress is linear to logarithm of concentration.The detecting sensitivity in the modified method of second antibody is the worst in three methods and the detecting limit is two orders of magnitude lower than IC_(20).The detecting sensitivity in the modified method of SPA and thiolated antibody is the same.The detecting sensitivity in both modified methods can reach at least 1ng/ml and are equivalent with IC_(20).By analysis,the detecting sensitivity is directly related to the activity of antibody and the distance from the position where antigen-antibody binding to gold-coated side.
Two readout methods based on microcantilever array were proposed.First,the optical path of UC Berkeley was improved.The signal-to-noise ratio of system was increased by controlling polarization of the laser.The optical detection sensitivity can be increased by optimal design of microcantilever.Second,optical filter readout technique was introduced to MCS.
本文以微悬臂梁生化传感技术发展的两大核心——提高检测灵敏度和高通量检测为出发点,对基于光杠杆原理的单悬臂梁检测系统进行改进,利用改进后的实验系统研究了大分子在界面的自组装过程以及在不同的抗体修饰方法下,抗原抗体的特异性结合,并提出了两种微悬臂梁阵列检测的方案。
通过使用聚焦处半径较小的激光器,稳定激光器的输出强度,使实验系统的可重复性得到提高。通过实验和理论分析,在反射光路中加入凸透镜以及优化微悬臂梁尺寸,可以使系统检测灵敏度分别提高4倍和20倍。通过使用高精度的恒温器(温控精度±0.01K),消除了微悬臂梁的热变形影响,同时使本底噪音保持在合理的范围,而且确保样品温度和容器池的温差不会对系统检测造成影响,提高了系统的信噪比。通过对传统光路参数的理论分析,得到在特定的光路参数下,可以完全消除溶液折射率改变对系统检测的影响,而且不改变系统的检测灵敏度。理论分析得到实验结果的验证。利用溶液折射率变化对系统检测的影响,提出了一种通过检测液体折射率来检测液体浓度的方案。
研究了不同分子量的巯基化的聚N-异丙基丙烯酰胺(HS-PNIPAM)在金表面自组装的动力学过程。实验结果表明HS-PNIPAM在自组装过程中存在三个阶段,分别对应不同的分子构象。第一阶段为物理吸附阶段,第二、三阶段为伴随着分子构象变化的化学吸附阶段。化学吸附曲线符合Langmuir等温吸附。分析结果还显示HS-PNIPAM的表面吸附速率κ远小于小分子的吸附速率,并与分子量成负指数关系;HS-PNIPAM的自组装时间远大于小分子的自组装时间,并与分子量成正比;底物表面应力的改变与分子量成正线性关系。
研究了在三种抗体修饰方法下,抗原抗体的特异性结合。将抗体修饰在微悬臂梁的金面,检测不同浓度的标准样品下微悬臂梁的弯曲变形,包括抗体巯基化修饰方法检测瘦肉精、SPA修饰抗体的方法检测瘦肉精和氯霉素以及二抗修饰抗体的方法检测青蒿素和马兜铃酸。抗体的活性以及在微悬臂梁上进行抗原抗体结合的可行性得到了ELISA实验的验证。实验结果表明:抗原抗体的结合具有很强的选择性。结合产生了压应力,且与浓度成正比。二抗修饰方法是三种方法中效果最差的,检测极限比标准曲线对应的IC_(20)低于两个数量级。SPA修饰方法与巯基化修饰方法的检测效果大体一致,检测极限都要优于1ng/ml,与标准曲线对应的IC_(20)相当。分析认为,检测极限与抗体活性和抗原抗体结合位置到微悬臂梁表面的距离有直接的关系。
提出了两种检测微悬臂梁阵列变形的光路方案。第一,在UC Berkeley的光路基础之上进行改进,将偏振光引入检测系统,通过对光束偏振态的控制,降低了测量信号的背景噪声,提高了系统信噪比;通过对微悬臂梁的优化设计(初始转角和反光板),提高光学检测灵敏度。第二,将光学滤波方法引入到生化检测当中。
In recent years,as a new technology,microcantilever biochemical sensing technique(MCS) is developing quickly.When specific bio-molecular interactions occur on one surface of microcantilever,a differential stress between the top and bottom surfaces of the cantilever will cause the microcantilever bending.The deflection can be measured by both optical and electrical readout methods.It has brought some new ideas for biochemical sensing technique and becomes a hotspot of research because of its advantages,such as large surface-area-to-volume ratio,very sensitive to surface effect,easy to realize array compact.
In this paper,two core of development of MCS- improving detecting sensitivity and high-throughput detection were taken as the starting point.First,a single cantilever detection system based on optical lever technique was improved. Second,the improved experimental system was used to study self-assembly of macromolecules on gold surface and the specificity of antigen-antibody binding in the different antibody-modified methods.Finally,two readout methods based on microcantilever array were proposed.
The detecting reproducibility of system can be improved by way of using the laser with the smaller focus radius and steady output intensity.Through experiments and theoretical analysis,by adding convex lens in the path of reflected light and optimizing the dimension parameters of microcantilever,the detecting sensitivity of system can be improved 4 and 20 times,separately.Through the use of high-precision thermostat,the thermal deformation of microcantilever can be eliminated,while the background noise is kept within reasonable scope,and the temperature difference between the sample and the cell will not impact on the system detection.The theoretical analysis showed that by conditioning the experimental parameter,the influence of refractive index change on the detection signal can be effectively eliminated without reducing the detecting sensitivity of the system.The theoretical results were validated by the experiments.Based on this,a method of detecting liquid concentration by detecting the refractive index of liquid was proposed.
The self-assembly of different molecular weight of Poly(N-isopropylacrylamide) (HS-PNIPAM) on gold surface was investigated.The results showed that the kinetic curves can be divided into three stages corresponding to different conformations, respectively.The first stage responded to physical adsorption of HS-PNIPAM to gold-coated side.The second and third stages were responsible for chemical adsorption to gold-coated side with conformation transition.The kinetic curves fits Langmuir adsorption isotherm well.The results also show that the reaction rateκof HS-PNIPAM is far less than that of small molecules and decreases exponentially with the molecular weight;while the time of HS-PNIPAM's self-assembly is far more than that of small molecules and proportional to the molecular weight.The change of the surface stress is linear to the molecular weight of HS-PNIPAM.
The specificity of antigen-antibody binding in three different antibody-modified methods was investigated.Antibody was immobilized on the gold-coated side of the microcantilever and the deflections of the microcantilever corresponding to the process of injecting different concentration of antigen were real-time monitored, including the clenbuterol was detected in the modified method of thiolated antibody, the clenbuterol and chloramphenicol were detected in the modified method of Staphylococcus aureus protein A(SPA),the artemisinin and aristolochic acid were detected in the modified method of second antibody(goat anti-mouse IgG).The activity of antibody and antigen-antibody binding on the gold-coated side of the microcantilever were confirmed by an Enzyme-Linked Immunosorbent Assay (ELISA).The results showed that antigen-antibody binding on microcantilever generates a compressive surface stress and the surface stress is linear to logarithm of concentration.The detecting sensitivity in the modified method of second antibody is the worst in three methods and the detecting limit is two orders of magnitude lower than IC_(20).The detecting sensitivity in the modified method of SPA and thiolated antibody is the same.The detecting sensitivity in both modified methods can reach at least 1ng/ml and are equivalent with IC_(20).By analysis,the detecting sensitivity is directly related to the activity of antibody and the distance from the position where antigen-antibody binding to gold-coated side.
Two readout methods based on microcantilever array were proposed.First,the optical path of UC Berkeley was improved.The signal-to-noise ratio of system was increased by controlling polarization of the laser.The optical detection sensitivity can be increased by optimal design of microcantilever.Second,optical filter readout technique was introduced to MCS.
引文
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