摘要
免疫凝集技术是一项广泛应用于医药、农业、食品等众多领域的生化检测技术。基于微流控芯片的免疫凝集定量检测技术具有耗材量小、检出限低及自动化程度高等优点,是生化检测领域的一项国际前沿技术。
然而该项技术主要存在以下关键问题：1、在微尺度条件下如何摆脱层流系统的束缚,实现致敏乳胶试剂和抗体(抗原)间的充分混合；2、如何选定检测参量、减小检测干扰和误差,实现微尺度条件下的高精度定量检测,这两大关键问题也是国内外研究热点。
根据上述关键问题,文章对国内外关于微混合器和光电检测技术的相关研究进行归纳、总结的基础上,依据免疫凝集的特点,提出了一种电控混合微流控芯片免疫凝集定量检测方法,并针对实现该方法的关键技术作出了深入的讨论。论文的主要工作体现在：1、阐述了所提出的电控混合微流控芯片免疫凝集定量检测方法的基础理论,证明了所提出方法的理论可行性。2、分析了动态壁面电势驱动下的微流体运动状态,证明了通过驱动电极产生动态电势来促进流体混合的可行性,并建立了能够促进微混合所需要的物理模型、相应的控制方程和边界条件。筛选了壁面电极可施加的混沌反控制算法,并确立了完成混合混沌反控制的评价体系。3、根据微尺度条件下光电检测对粒度以及环境参数的敏感度较高的特征,在微尺度条件下对高精度光散射检测所需要的检测模型、工艺参数和误差补偿方法等进行了研究。4、为了验证所述电控混合微流控芯片免疫凝集定量检测方法的检测效果,设计了专用的电控混合微流控芯片以及混沌电场控制器。创建了从进样到混合再到光电检测的完整的实验平台。对于整机实验中混合过程与光电检测过程进行耦合使用所需参数,进行了设置并优化处理。同时将常规尺度下免疫凝集比浊法与论文所设计的电控混合微流控芯片免疫凝集定量检测法进行了对比实验研究。
论文的创新点归纳成如下几点：
1、对于电动混合方法,论文提出了混合混沌反控制概念,用混沌电场施加到微流控芯片的微电极来驱动控制流体进行混沌混合,并针对不同混沌算法对流体的控制进行了区别比较。
2、针对混沌电场系统与混沌流体系统的特性,分别引入混沌尺度定量评价方法,针对流体系统的特殊性引入粒子追踪模拟仿真来实现其混沌尺度的评价问题,并在此基础上验证了混沌电场系统与混沌流场系统的广义同步关系。通过研究混沌尺度与混合效率之间的关系,得到大尺度混沌电场系统能够有效提升混合效率的结论。
3、针对传统方法难以有效测量免疫凝集微颗粒的粒度参数的难点,提出一种基于显微图像处理技术的有效测量免疫凝集颗粒粒度的方法,并将该方法有效地用于凝集颗粒粒度大小与散射模型关系的研究中。论文对于各散射检测模型的适用范围进行了讨论,并对微尺度条件下免疫凝集检测的最佳致敏乳胶颗粒粒度的进行了优化选择。针对微尺度条件下光散射检测的特殊性,探讨了粒度接近检测光波长的凝集颗粒所产生的散射模型渡越特性,并在此基础上筛选出最佳检测角度。
4、分析了微尺度条件下短光程且恒定时,待测物随浓度变化所引起的检测误差变化,并建立了误差补偿模型,实验结果表明该误差补偿模型能够有效提高浓度的检测范围。
5、设计出电控混合类型的微流控芯片,以及与之配套使用的混沌电场控制器；首次创建出基于电控混合式的微流控芯片测试系统,并建立了免疫凝集条件下的混合尺度验证模式。而目前国内外电控混合研究仅局限于仿真研究。
采用优化后的各项工艺参数,在所设计的电控混合微流控芯片免疫凝集定量检测实验平台上进行了定量检测实验并与常规尺度下的定量检测结果进行了比较。结果表明,所提出的电控混合免疫凝集定量检测方法实现了从进样到检测的全自动化过程,对于类风湿因子的检测精度也接近于常规尺度,而检出限则比常规尺度降低了60%～85%,且耗材量约为常规尺度的千分之一。该项研究为微流控芯片电控式微混合器开发,也为微尺度条件下一体化高精度光电检测设备的研发以及基于微流控芯片的免疫凝集检测微型自动化整机系统的设计与开发,提供了理论基础和实验指导。
Immunoagglutination technique is a biochemical detection technology and has been widely used in the area of pharmaceutical, agriculture, food, etc. As an advanced research task of present, immunoagglutination quantitative determination technique based on microfluidic chip is to achieve immune agglutination detection in micronano scale, and has been demonstrated to have virtues of less consumption of reagent, low detection limit, high automation degree, etc.
However, there are two challenges remain in immunoagglutination technique: First, how to break free of the shackles of the laminar flow system in microscale thus realize sufficient mixing of the sensitized latex reagent and antibody(antigen) in the microchannel; Second, how to select parameters, reduce the interference and error of the detection, to achieve high-precision quantitative detection in microscale. All of these are key technologies and research focus for immunoagglutination quantitative determination technique.
We summarized correlation research of the micromixer and photoelectric detection technology, and improved understanding of immunoagglutination characteristics, then introduce an microfluidic chip with electric control micro mixer for immunoagglutination quantitative detection, and give a further discussion on the key technology. The main contributions of this dissertation are as follows:Firstly, we introduce the theoretical basis of the method, and demonstrate the theoretical feasibility for microfluidic chip with electric control micro mixer for immunoagglutination quantitative detection. Secondly, the microfluids flow states that driven by the dynamic wall electromotive force are analyzed, and we have demonstrated that driving electrodes yield dynamic potential to promote flow mixing is feasible. And the physical model of promoting micro-hybrid was established, as well as corresponding governing equations and boundary conditions. The chaotic anti-control algorithm can be applied to the wall electrode were screened, and then we established the mixed chaotic anti-control evaluation system. Thirdly, the Granularity and environment parameter showed a high sensitivity for the photodetector in conditions of the microscale. We study the detection model, process parameter of the high-precision scattering detection, as well as environmental parameters, error compensation method of the absorbance detection in corresponding condition. Finally, to demonstrate the designed method, we developed a microfluidic chip with electric control micro mixer and a chaos controller. An integrated experiment platform is founded for sample introduction, mixing and photoelectric detector. The needed parameters is setted coupling the mixing process is optimized in the whole experiment with photoelectric detection process. The performance is compared with the conventional scale immune agglutination turbidimetry.
The dissertation contains the following innovations:
1. To electric driving mixing, the concept of mixed chaotic anti-control is presented in this thesis, namely apply the chaos electric field to the microelectrode of microfluidic chip to drive the control of fluid chaotic mixing, and the kinds of flow control methods via different chaos algorithms are compared.
2. To the characteristics of the chaotic farm system and chaos fluid system, we introduce chaos scale quantitative evaluation methods respectively, and for the particularity of the flow system, we employ the particle tracking simulation method and via it to evaluate the chaotic scale. On this basis, the generalized synchronization relation of the chaotic farm system and the chaotic flow field system is validated. And based on the research of relations between the chaotic scale and mixing efficiency, our findings indicate that the large scale chaos electric field system can effectively improve the efficiency of mixing.
3. The difficulty of the traditional methods can not effectively measure the size of the immune agglutination micro particles, we proposed a method that can effectively measure the size of the immune agglutination particle based on microscopic image processing technology, and the method is effective used into study the relationship between the size of the aggregation particle and the scattering model. We have also proposed for the first time, the scope of application of each scattering detector model and optimized selection of the sensitized latex particle size for immunoagglutination detection under the microscale condition. To light scattering detector particularly under microscale conditions, we first discussed the scattering model transit characteristic that generate from the aggregation particles which granularity scale is proximity detection optical wavelength. And on this basis, we screened out the best detection angle.
4. We also analyzed the short optical path and constant under the microscale conditions, with the change of detection error that caused by to-be-detected changing of the concentration, and to establish the error compensation model.
5. Here we achieved an electronically hybrid controlled microfluidic, and its accompanied chaotic electric field controller. First time we fabricated microfluidic test system based on electronically hybrid controlled, and created the mixed-scale validation mode under the immune agglutination condition. Several studies on electronically hybrid controlled were limited to simulation at present.
In the final section, we using the optimized synthetic parameters, the quantitation detection experiment is processed on the immune agglutination quantitative detection experiment platform of microfluidic chip with electric control micro mixer, and comparing with the quantitative test results under the conventional scale. The results demonstrate that our method achieved the fully automated process from the reaction to the detection, to the detection accuracy of the rheumatoid factor is also close to the conventional scale, but the detection limit is about60%～85%lower than the conventional scale, and the liquid consumption is about one-thousandth of the conventional scale. This work provides theoretical and experimental guidance for developing the electrical control micromixer based on the microfluidic chip, for researching and developing the microscale high-precision optical testing equipment, and designing the immune agglutination detection of micro-automation machine system based on the microfluidic chip.
引文
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