免標(biāo)記光纖琺—珀生物傳感機(jī)理與實(shí)驗(yàn)研究
發(fā)布時(shí)間：2019-05-27 01:47
【摘要】：生物傳感器在生物醫(yī)學(xué)、臨床診斷、藥物分析、食品檢測(cè)、環(huán)境監(jiān)測(cè)及軍事生化檢測(cè)等領(lǐng)域具有重要的研究意義和應(yīng)用價(jià)值。隨著二十一世紀(jì)人們對(duì)于醫(yī)療保健、食品安全及環(huán)境污染等問(wèn)題的重視,開(kāi)發(fā)高靈敏度、低成本、小型便攜、快速易操作的新型生物傳感器成為當(dāng)今社會(huì)的迫切需求。光纖傳感器具有體積小、靈敏度高、抗電磁干擾、生物相容性好等特點(diǎn),近二十年來(lái)在生物傳感領(lǐng)域獲得了廣泛的研究和迅速的發(fā)展。將光纖傳感器與免標(biāo)記生物檢測(cè)技術(shù)相結(jié)合,直接利用生物反應(yīng)引起的光纖中傳輸光參數(shù)的變化對(duì)待測(cè)物進(jìn)行檢測(cè),可以免去標(biāo)記過(guò)程,節(jié)省時(shí)間和成本,并且能夠?qū)ι锓磻?yīng)動(dòng)態(tài)過(guò)程進(jìn)行原位、實(shí)時(shí)、在線的檢測(cè)。因此,免標(biāo)記光纖生物傳感器具有極大的發(fā)展?jié)摿蛻?yīng)用前景,近年來(lái)已成為生物傳感器領(lǐng)域的一大研究熱點(diǎn)。然而總體來(lái)說(shuō),目前免標(biāo)記光纖生物傳感器仍處于實(shí)驗(yàn)室研究階段。面對(duì)實(shí)際應(yīng)用中外界環(huán)境干擾、待測(cè)物濃度低和組份復(fù)雜等問(wèn)題,如何進(jìn)一步提高免標(biāo)記光纖生物傳感器的靈敏度、抗干擾能力、穩(wěn)定性、重復(fù)性和特異性等是急需解決的難題。光纖器件本身具有較高的靈敏度,但同時(shí)也容易受到外界環(huán)境溫度擾動(dòng)和振動(dòng)等因素的交叉影響,其抗干擾能力有待提高。生物分子識(shí)別元件在傳感器表面的固定是制備生物傳感器的關(guān)鍵,生物分子在傳感器表面的修飾密度和活性的保持等因素,直接影響到生物傳感器的靈敏度、穩(wěn)定性、檢測(cè)范圍、特異性和重復(fù)性等性能。近年來(lái),各類(lèi)新型納米材料的發(fā)展為構(gòu)建生物傳感界面、提高檢測(cè)性能開(kāi)創(chuàng)了新的局面。本文主要對(duì)基于空芯光子晶體光纖(hollow-core photonic crystal fiber,HCPCF)的光纖琺-珀(Fabry-Perot,F-P)免標(biāo)記生物傳感器進(jìn)行了理論分析和初步的實(shí)驗(yàn)驗(yàn)證。以基于HCPCF的光纖F-P傳感器作為生物傳感換能器,將固定在其端面的生物分子識(shí)別元件與待測(cè)物結(jié)合時(shí)引起的生物分子膜折射率或厚度的變化,轉(zhuǎn)換成輸出干涉譜的變化。該傳感器具有體積小、結(jié)構(gòu)穩(wěn)定等特點(diǎn),探針式結(jié)構(gòu)以及反射式信號(hào)探測(cè)方式使得檢測(cè)更為簡(jiǎn)便靈活,為免標(biāo)記生物檢測(cè)提供了一種新的手段。本文進(jìn)一步提出利用新型碳納米材料——氧化石墨烯來(lái)構(gòu)建生物傳感檢測(cè)界面,利用氧化石墨烯良好的生物相容性和大的比表面積等特性,來(lái)提高生物分子的修飾效果,進(jìn)而提高生物傳感器的檢測(cè)性能。本文的主要研究?jī)?nèi)容如下:(1)針對(duì)基于HCPCF的光纖F-P傳感器,首先采用菲涅爾反射原理和三光束干涉模型對(duì)傳感器的輸出干涉譜進(jìn)行仿真,分析了傳感器結(jié)構(gòu)參數(shù)對(duì)干涉譜對(duì)比度的影響。折射率檢測(cè)是免標(biāo)記光纖生物傳感的基礎(chǔ),對(duì)基于HCPCF的光纖F-P傳感器的折射率響應(yīng)特性進(jìn)行了理論分析,結(jié)果顯示,傳感器干涉譜的干涉條紋對(duì)比度隨外界折射率的增大而減小,在較大的折射率范圍內(nèi)呈現(xiàn)良好的線性響應(yīng),進(jìn)一步分析了傳感器結(jié)構(gòu)參數(shù)對(duì)折射率響應(yīng)靈敏度的影響。同時(shí),對(duì)傳感器的溫度敏感特性進(jìn)行了理論分析,結(jié)果表明,外界環(huán)境溫度的變化會(huì)引起干涉條紋波長(zhǎng)的漂移,而對(duì)干涉條紋對(duì)比度幾乎沒(méi)有影響。因此,通過(guò)解調(diào)干涉譜條紋對(duì)比度和波長(zhǎng),可以實(shí)現(xiàn)對(duì)外界折射率和溫度的同時(shí)測(cè)量。(2)采用電弧放電熔接法制作了基于HCPCF的光纖F-P傳感器,并利用不同濃度的蔗糖溶液對(duì)傳感器的折射率響應(yīng)特性進(jìn)行實(shí)驗(yàn)測(cè)試。實(shí)驗(yàn)結(jié)果顯示,在1.33-1.44RIU(refractive index unit,RIU)的折射率范圍內(nèi),傳感器檢測(cè)呈現(xiàn)良好的線性度和重復(fù)性,靈敏度約為-136dB/RIU。利用制作的光纖F-P傳感器,根據(jù)酵母菌生長(zhǎng)過(guò)程中培養(yǎng)液折射率的變化,對(duì)酵母菌的生長(zhǎng)過(guò)程進(jìn)行了檢測(cè)。實(shí)驗(yàn)結(jié)果驗(yàn)證了該傳感器對(duì)于微小折射率變化具有較強(qiáng)的分辨能力。此外,提出一種新的基于光譜差分積分(spectrum differential integration,SDI)計(jì)算的數(shù)據(jù)分析方法,有利于提高折射率解調(diào)結(jié)果的準(zhǔn)確性。(3)提出了基于HCPCF的光纖F-P免標(biāo)記免疫傳感器,利用固定在光纖端面的抗體與抗原結(jié)合后引起的蛋白質(zhì)分子膜折射率和厚度的變化,對(duì)免疫反應(yīng)過(guò)程進(jìn)行檢測(cè)。首先在理論上,建立覆膜光纖F-P傳感器四光束干涉模型,對(duì)傳感器免疫檢測(cè)原理進(jìn)行了仿真分析。在實(shí)驗(yàn)中,將光纖傳感器端面進(jìn)行硅烷化處理和戊二醛修飾后,采用共價(jià)結(jié)合的方式固定羊抗兔免疫球蛋白G(immunoglobulin G,IgG),實(shí)現(xiàn)了對(duì)兔IgG的檢測(cè)。實(shí)驗(yàn)結(jié)果初步驗(yàn)證了該傳感器在免疫檢測(cè)中的可行性。(4)提出氧化石墨烯覆膜的光纖F-P免標(biāo)記免疫傳感器,利用氧化石墨烯良好的生物相容性和大的比表面積等特性,來(lái)提高傳感器端面抗體的固定效果和對(duì)抗原檢測(cè)的靈敏性。首先,采用不同方法在光纖端面制備氧化石墨烯薄膜,并利用掃描電子顯微鏡對(duì)薄膜進(jìn)行表征和對(duì)比分析。在光纖F-P傳感器端面涂覆氧化石墨烯薄膜,對(duì)氧化石墨烯覆膜光纖F-P傳感器的干涉譜進(jìn)行了仿真,理論分析了覆膜厚度對(duì)干涉譜的影響。免疫實(shí)驗(yàn)中,在氧化石墨烯覆膜的光纖F-P傳感器端面固定羊抗兔IgG,對(duì)兔IgG進(jìn)行檢測(cè)。實(shí)驗(yàn)結(jié)果顯示,覆膜之后的傳感器對(duì)于抗體固定以及抗原抗體結(jié)合引起的輸出干涉譜信號(hào)變化更為明顯,初步驗(yàn)證了氧化石墨烯覆膜對(duì)于傳感器檢測(cè)性能的提升。(5)作為基于HCPCF的光纖F-P傳感器在生物醫(yī)學(xué)等領(lǐng)域濕度檢測(cè)的應(yīng)用擴(kuò)展研究,制作了殼聚糖覆膜的光纖F-P濕度傳感器,利用殼聚糖膜吸水溶脹特性導(dǎo)致的膜折射率及厚度變化,通過(guò)檢測(cè)傳感器干涉譜條紋對(duì)比度和波長(zhǎng)的變化,實(shí)現(xiàn)了對(duì)環(huán)境相對(duì)濕度的檢測(cè)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TP212
本文編號(hào)：2485805
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TP212
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