| 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. |
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