免标记光纤珐—珀生物传感机理与实验研究

发布时间：2019-05-27 01:47

【摘要】：生物传感器在生物医学、临床诊断、药物分析、食品检测、环境监测及军事生化检测等领域具有重要的研究意义和应用价值。随着二十一世纪人们对于医疗保健、食品安全及环境污染等问题的重视,开发高灵敏度、低成本、小型便携、快速易操作的新型生物传感器成为当今社会的迫切需求。光纤传感器具有体积小、灵敏度高、抗电磁干扰、生物相容性好等特点,近二十年来在生物传感领域获得了广泛的研究和迅速的发展。将光纤传感器与免标记生物检测技术相结合,直接利用生物反应引起的光纤中传输光参数的变化对待测物进行检测,可以免去标记过程,节省时间和成本,并且能够对生物反应动态过程进行原位、实时、在线的检测。因此,免标记光纤生物传感器具有极大的发展潜力和应用前景,近年来已成为生物传感器领域的一大研究热点。然而总体来说,目前免标记光纤生物传感器仍处于实验室研究阶段。面对实际应用中外界环境干扰、待测物浓度低和组份复杂等问题,如何进一步提高免标记光纤生物传感器的灵敏度、抗干扰能力、稳定性、重复性和特异性等是急需解决的难题。光纤器件本身具有较高的灵敏度,但同时也容易受到外界环境温度扰动和振动等因素的交叉影响,其抗干扰能力有待提高。生物分子识别元件在传感器表面的固定是制备生物传感器的关键,生物分子在传感器表面的修饰密度和活性的保持等因素,直接影响到生物传感器的灵敏度、稳定性、检测范围、特异性和重复性等性能。近年来,各类新型纳米材料的发展为构建生物传感界面、提高检测性能开创了新的局面。本文主要对基于空芯光子晶体光纤(hollow-core photonic crystal fiber,HCPCF)的光纤珐-珀(Fabry-Perot,F-P)免标记生物传感器进行了理论分析和初步的实验验证。以基于HCPCF的光纤F-P传感器作为生物传感换能器,将固定在其端面的生物分子识别元件与待测物结合时引起的生物分子膜折射率或厚度的变化,转换成输出干涉谱的变化。该传感器具有体积小、结构稳定等特点,探针式结构以及反射式信号探测方式使得检测更为简便灵活,为免标记生物检测提供了一种新的手段。本文进一步提出利用新型碳纳米材料——氧化石墨烯来构建生物传感检测界面,利用氧化石墨烯良好的生物相容性和大的比表面积等特性,来提高生物分子的修饰效果,进而提高生物传感器的检测性能。本文的主要研究内容如下:(1)针对基于HCPCF的光纤F-P传感器,首先采用菲涅尔反射原理和三光束干涉模型对传感器的输出干涉谱进行仿真,分析了传感器结构参数对干涉谱对比度的影响。折射率检测是免标记光纤生物传感的基础,对基于HCPCF的光纤F-P传感器的折射率响应特性进行了理论分析,结果显示,传感器干涉谱的干涉条纹对比度随外界折射率的增大而减小,在较大的折射率范围内呈现良好的线性响应,进一步分析了传感器结构参数对折射率响应灵敏度的影响。同时,对传感器的温度敏感特性进行了理论分析,结果表明,外界环境温度的变化会引起干涉条纹波长的漂移,而对干涉条纹对比度几乎没有影响。因此,通过解调干涉谱条纹对比度和波长,可以实现对外界折射率和温度的同时测量。(2)采用电弧放电熔接法制作了基于HCPCF的光纤F-P传感器,并利用不同浓度的蔗糖溶液对传感器的折射率响应特性进行实验测试。实验结果显示,在1.33-1.44RIU(refractive index unit,RIU)的折射率范围内,传感器检测呈现良好的线性度和重复性,灵敏度约为-136dB/RIU。利用制作的光纤F-P传感器,根据酵母菌生长过程中培养液折射率的变化,对酵母菌的生长过程进行了检测。实验结果验证了该传感器对于微小折射率变化具有较强的分辨能力。此外,提出一种新的基于光谱差分积分(spectrum differential integration,SDI)计算的数据分析方法,有利于提高折射率解调结果的准确性。(3)提出了基于HCPCF的光纤F-P免标记免疫传感器,利用固定在光纤端面的抗体与抗原结合后引起的蛋白质分子膜折射率和厚度的变化,对免疫反应过程进行检测。首先在理论上,建立覆膜光纤F-P传感器四光束干涉模型,对传感器免疫检测原理进行了仿真分析。在实验中,将光纤传感器端面进行硅烷化处理和戊二醛修饰后,采用共价结合的方式固定羊抗兔免疫球蛋白G(immunoglobulin G,IgG),实现了对兔IgG的检测。实验结果初步验证了该传感器在免疫检测中的可行性。(4)提出氧化石墨烯覆膜的光纤F-P免标记免疫传感器,利用氧化石墨烯良好的生物相容性和大的比表面积等特性,来提高传感器端面抗体的固定效果和对抗原检测的灵敏性。首先,采用不同方法在光纤端面制备氧化石墨烯薄膜,并利用扫描电子显微镜对薄膜进行表征和对比分析。在光纤F-P传感器端面涂覆氧化石墨烯薄膜,对氧化石墨烯覆膜光纤F-P传感器的干涉谱进行了仿真,理论分析了覆膜厚度对干涉谱的影响。免疫实验中,在氧化石墨烯覆膜的光纤F-P传感器端面固定羊抗兔IgG,对兔IgG进行检测。实验结果显示,覆膜之后的传感器对于抗体固定以及抗原抗体结合引起的输出干涉谱信号变化更为明显,初步验证了氧化石墨烯覆膜对于传感器检测性能的提升。(5)作为基于HCPCF的光纤F-P传感器在生物医学等领域湿度检测的应用扩展研究,制作了壳聚糖覆膜的光纤F-P湿度传感器,利用壳聚糖膜吸水溶胀特性导致的膜折射率及厚度变化,通过检测传感器干涉谱条纹对比度和波长的变化,实现了对环境相对湿度的检测。
[Abstract]:The biosensor has important research significance and application value in the fields of biomedicine, clinical diagnosis, drug analysis, food detection, environmental monitoring and military biochemical detection. With the attention of people on health care, food safety and environmental pollution in the twenty-first century, the development of a new type of biosensor with high sensitivity, low cost, small portable and fast operation becomes the urgent need of the society. The optical fiber sensor has the characteristics of small volume, high sensitivity, electromagnetic interference resistance and good biocompatibility. the optical fiber sensor and the mark-free biological detection technology are combined, and the detected object is directly detected by the change of the transmission optical parameters in the optical fiber caused by the biological reaction, the marking process can be avoided, the time and the cost can be saved, and the biological reaction dynamic process can be in-situ and real-time, On-line detection. Therefore, the mark-free optical fiber biosensor has great development potential and application prospect, and has become a hotspot in the field of biosensor in recent years. In general, however, that present mar-free fiber biosensor is still in the laboratory research phase. In the face of the problems such as external environment interference, low concentration of the object to be measured and complex components, how to further improve the sensitivity, the anti-interference ability, the stability, the repeatability and the specificity of the label-free optical fiber biosensor is the difficult problem to be solved. The fiber optic device itself has a high sensitivity, but at the same time it is also susceptible to the cross-influence of external ambient temperature disturbance and vibration, and the anti-interference ability of the optical fiber device is to be improved. The immobilization of the biological molecule recognition element on the surface of the sensor is the key to the preparation of the biosensor, the modification density and the activity of the biological molecule on the surface of the sensor and the like can directly affect the sensitivity, the stability, the detection range, the specificity and the repeatability of the biosensor. In recent years, the development of new type of nano-materials has made a new situation for building the bio-sensing interface and improving the detection performance. The optical fiber Fabry-Perot (F-P)-free biosensor based on the hollow-core photonic crystal fiber (HCPCF) is analyzed theoretically and experimentally. The optical fiber F-P sensor based on the HCPCF is used as a biological sensing transducer, and the change of the refractive index or the thickness of the biological molecule film which is caused when the biological molecule identification element fixed on the end surface of the biological sensing transducer is combined with the object to be measured is converted into the change of the output interference spectrum. The sensor has the characteristics of small volume, stable structure and the like, and the probe type structure and the reflection type signal detection mode enable the detection to be more convenient and flexible, and a new method is provided for the mark-free biological detection. In this paper, the new carbon nano-material _ oxidized graphene is used to construct the biological sensing detection interface, and the modification effect of the biological molecule is improved by utilizing the characteristics of good biocompatibility and large specific surface area of the graphene oxide, and the detection performance of the biological sensor is further improved. The main contents of this paper are as follows: (1) For the HCPCF-based optical fiber F-P sensor, firstly, the Fresnel reflection principle and the three-beam interference model are adopted to simulate the output interference spectrum of the sensor, and the influence of the sensor structure parameters on the contrast of the interference spectrum is analyzed. In this paper, the refractive index of the fiber F-P sensor based on the HCPCF is theoretically analyzed. The results show that the contrast of the interference fringe of the sensor interference spectrum decreases with the increase of the external refractive index. The influence of the sensor structure parameters on the response sensitivity of the refractive index is further analyzed. At the same time, the temperature-sensitive characteristic of the sensor is analyzed. The result shows that the change of the ambient temperature can cause the drift of the interference fringe wavelength, and the contrast of the interference fringe has little effect. Therefore, the simultaneous measurement of the external refractive index and the temperature can be achieved by demodulating the contrast and the wavelength of the interference spectrum. (2) The F-P sensor based on HCPCF was fabricated by arc discharge welding, and the response characteristics of the refractive index of the sensor were tested by different concentration of sucrose solution. The results show that in the range of the refractive index of 1.33-1.44 RIU, the sensor has good linearity and repeatability, and the sensitivity is -136 dB/ RIU. The growth process of the yeast is detected according to the change of the refractive index of the culture medium during the growth of the yeast according to the change of the refractive index of the culture medium during the growth of the yeast. The experimental results show that the sensor has strong resolving power to the change of the micro-refractive index. In addition, a new method of data analysis based on spectral differential integral (SDI) is proposed to improve the accuracy of the refractive index demodulation. (3) An optical fiber F-P free-label immune sensor based on HCPCF is proposed, and the immunological reaction process is detected by the change of the refractive index and the thickness of the protein molecule caused by the binding of the antibody immobilized on the end surface of the optical fiber and the antigen. Firstly, the four-beam interference model of the F-P sensor of the coated optical fiber is established, and the principle of the detection of the sensor is simulated and analyzed. In the experiment, after the end face of the optical fiber sensor was subjected to silanization treatment and glutaraldehyde modification, the rabbit anti-rabbit immunoglobulin G (IgG) was fixed in a covalently bound manner, and the detection of rabbit IgG was realized. The feasibility of the sensor in the immunoassay is preliminarily verified by the experimental results. (4) The fiber F-P free-label immune sensor with graphene oxide film is proposed, and the fixing effect of the antibody on the end face of the sensor and the sensitivity to the detection of the antigen are improved by using the characteristics of good biocompatibility and large specific surface area of the graphene oxide. First, an oxide-graphene film was prepared on the end of the fiber by different methods, and the film was characterized and compared by a scanning electron microscope. The interference spectrum of the F-P sensor of the graphene-coated optical fiber is simulated by coating the graphene film on the end surface of the optical fiber F-P sensor, and the influence of the film thickness on the interference spectrum is theoretically analyzed. In the immune experiment, goat anti-rabbit IgG was fixed on the end surface of the F-P sensor of the graphene oxide film, and the rabbit IgG was detected. The results of the experiment show that the change of the output interference spectrum caused by the combination of the antibody and the antigen-antibody is more obvious for the sensor after the coating, and the improvement of the detection performance of the graphene oxide film on the sensor is preliminarily verified. (5) As an extension of the application of the HCPCF-based fiber F-P sensor in the fields of biomedicine and the like, the fiber F-P humidity sensor with the chitosan coating film is manufactured, and the film refractive index and the thickness change caused by the water-absorbing and swelling characteristics of the chitosan film are utilized, And the detection of the relative humidity of the environment is realized by detecting the change of the contrast and the wavelength of the interference spectrum of the sensor.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TP212

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