【作者】 王弋嘉；

【导师】 袁小聪；

【作者基本信息】 南开大学 ， 光学工程， 2013， 博士

【摘要】 表面等离子体共振(Surface Plasmon Resonance, SPR)传感器是一种用于探测传感芯片表面折射率变化的传感器,它主要适用于对大分子结合反应的实时测量,自从上世纪80年代开始,SPR传感器已经广泛应用于生物医学领域,并有一系列商用传感器面世。SPR传感器最重要的性能指标是分辨率和动态范围,在SPR的四种检测方式(强度、角度、波长、相位)之中,相位检测方法具有最高的灵敏度,但由于其光路复杂、易受环境噪声影响以及动态范围较窄,限制了它的使用。本研究搭建的基于马赫泽德结构的差分干涉型SPR传感系统是一种新型的相位调制型SPR系统,它在拥有高灵敏度优点的同时,可大幅降低环境噪声,从而达到很高的分辨率,并且由于该系统从时间轴提取相位信息,在进行SPR成像传感时不损失空间分辨率,与传统的相位调制型SPR成像技术相比具有很大优势。本文首先介绍了SPR技术的基本理论,并在该理论基础上分析了影响相位调制型SPR传感器灵敏度和动态范围的四大因素：金属介电常数、入射光波长、金膜厚度和入射角度,其中金膜厚度是一个易于调节的因素,可以在动态范围满足测量要求的基础上尽可能提高灵敏度,并进行了实验验证。搭建了基于马赫泽德结构的差分干涉型SPR传感器,介绍了本系统提取相位信息的方法和差分干涉降低噪声的原理,编写了相位提取程序,在程序中使用非线性拟合及取平均的方法降低了系统噪声。制作了微流控系统,使用轮廓仪测量高真空镀膜机蒸镀所得金膜的厚度,从而选取厚度为45nm左右的金膜进行传感芯片的制作,将使用模塑法制作的PDMS基片和传感芯片进行氧离子键合以制成微流槽。通过精确调节入射角,尽可能减小入射角与SPR角的误差以发挥相位调制SPR传感器高灵敏度的优势,确定系统达到最高分辨率所需测量次数为15次,通过测量一系列折射率的NaCl溶液,确定了系统的分辨率为10(?)折射率单位。进行了基于马赫泽德结构的差分干涉SPR成像传感器的研究工作,虽然差分干涉SPR传感器提取相位的方式不损失空间分辨率,但该方法需要将p光和s光两幅干涉图进行对准后提取相位,本研究使用光阑增加参考光图样的相似性,并进行互相关运算来优化对准效果。由于硬件性能的限制,系统中使用CCD采集图像数据的频率为30帧／s,在程序中采取局部多项式最小二乘曲线拟合法尽量减小由采集频率较低造成的误差。调节压电陶瓷的移动速度至0.6um／s,以弥补较低采集频率带来的误差,程序计算所得相变值的误差为±0.65,通过测量一系列折射率的NaCl溶液,确定了系统的分辨率为10-4叫折射率单位,系统的空间分辨率为9.77um×15.63um。将本系统应用于生物医学检测的研究工作包括：传感芯片表面的活化、实时检测牛血清白蛋白与抗体的结合反应、检测低浓度乙肝表面抗原溶液、实时检测结肠癌细胞系与表皮生长因子的反应。本系统使用以硫醇体系为基础的自组装膜技术活化传感芯片,可以使生物分子定向结合在芯片表面。通过实验验证,本系统可以实时检测牛血清白蛋白与特异性抗体的结合反应,在整个测量过程中,一共增加了2.5×10-3折射率单位,对应IgG免疫球蛋白结合密度为160个／um2。从血清中检测乙肝表面抗原是否存在是判断乙肝感染的指标,因此使用高灵敏度的检测手段,进行准确的定性测量是具有重要意义的,使用本系统能够在30分钟之内测量出1-10ng/ml浓度的乙肝表面抗原,在检测分辨率上相当于目前常用的ELISA和RIA检测方法,而检测时间则短于上述两种方法。对三种具有不同表皮生长因子受体表达水平的结肠癌细胞系与表皮生长因子的结合过程以及接下来的信号转导过程进行了实时检测,结果表明,受体表达水平越高,两个信号转导过程所产生的信号峰越陡峭,使用本系统能够确定癌细胞某项肿瘤标志物或细胞表面受体的表达水平,并能够实时检测信号转导途径的进行。更多还原

【Abstract】 Surface Plasmon Resonance (SPR) sensors can be used to detect change of refractive index near sensor chips. It is mainly applied to real-time measurement of macromolecules binding reactions. Since the1980s, SPR sensors have been widely used in the biomedical field and a series of commercial sensors have been produced. The most important performance index of SPR sensors are resolution and dynamic range. Among the four detection methods (intensity, angle, wavelength, phase) of SPR sensors, the phase modulation method has the highest sensitivity. However, phase modulation SPR sensors have a limited range of applications because its optical system is complicated, sensitive to environmental noise influence, and its dynamic range is narrow. A differential interference SPR sensing system based on Mach-Zehnder configuration in this study is a new kind of phase modulation SPR system. It not only has the advantage of high sensitivity, but also can significantly reduce the influence of enviromental noise to achieve a higher resolution. On the other hand, it has not loss of spatial resolution during SPR imaging because it extract phase information from the time axis. In this performance index, it has a great advantage compared with the conventional phase modulation SPR imaging.This paper first introduces the basic theory of SPR technology. From this, the four factors which effect sensitivity and dynamic range of SPR sensors are analyzed: the dielectric constant of metal, the wavelength of incident light, the thickness of gold film and the incident angle. The thickness of gold film can be adjusted easily among these factors to meet the measurement requirements of the dynamic range on the basis of improving sensitivity as much as possible. The above has been verified by experiment. A differential interference SPR sensor based on Mach-Zehnder configuration is set up. The method of extracting phase information and the principle of reduction noise by differential interference are described. The phase extraction procedures are programmed in which nonlinear fitting and averaging method are used to reduce the system noise. In the microfluidic system production, the profilometer is used to measure the gold membrane thickness vapor-deposited by the high vacuum coating machine in order to select film with about45nm thickness of gold membrane to make the sensor chip. Sensor chip and PDMS substrate made by molding method are used to make microfluid groove, which are produced by PDMS oxygen ions bonding method. The incident angle is adjusted precisely to minimize the error between the incident angle and the SPR angle to utilize high sensitivity advantage of phase modulation SPR sensors.15measurement times are needed to achieve the highest resolution of the system. The resolution of the system is determined to10’5refractive index units by measuring a series refractive index of NaCl solution.The research work of the differential interference SPR imaging sensor based on Mach-Zehnder configuration is described. Although there is no loss of spatial resolution in the method of phase extraction of differential interference SPR sensors, but the method requires to align two interferogram of p-and s-polarization light to extract phase. Diaphragm is used to increase the similarity of the reference light pattern, and correlation algorithm is carried on to optimize the alignment effect. Due to the limitations of the hardware performance, acquisition frequency of the CCD used in the system is30frame/s. the method of local polynomial least squares curve fitting is applied to minimize the error caused by the low acquisition frequency of the CCD. The moving speed of the piezoelectric ceramic is adjusted to0.6μm/s in order to compensate the error caused by the lower acquisition frequency. The error of the phase change calculated by the program is±0.65°. The resolution of the system is determined to be10-4refractive index units and the spatial resolution is0.77μm×15.63μm by measuring a series refractive index of NaCl solution.The system is applied to biomedical detection including the following research works:activation of the sensor chip surface, real-time detecting binding reaction between bovine serum albumin and antibody, detecting low concentration solution of hepatitis B surface antigen. Self assembled monolayer technology based on mercaptan system is used to activate sensor chip which can make biological moleculars coated to the chip surface directed. Verified by experiment, the system can real-time detect binding reaction between bovine serum albumin and its specific antibody. In the whole process, the increase of refractive index is2.5×10-3units which is corresponded to160number/μm2binding density of IgG immunoglobulin. Because hepatitis B surface antigen detected from serum is the indicator of hepatitis B infection, it has important significance to qualitative measure accurately by high sensitivity method. Concentration of hepatitis B surface antigen low to1-10ng/ml can be detected by the system in30minutes. The resolution of the system is equivalent to the currently used methods, such as enzyme linked immunosorbent and radioimmunoassay, while the detection time is shorter than the above two methods. The binding process between three colorectal cancer cell lines with different epidermal growth factor receptor expression level and epidermal growth factor are measured. Then the subsequent signal transduction process is detected in real-time. The results show that the signal peaks generated by two signal transduction process become steeper when the receptor expresstion level is higher. The system can determine expression level of a tumor marker or cell surface receptor in cancer cell and real-time detect signal transduction pathway progress.更多还原