基于磁分离的现场核酸检测平台若干关键技术研究

发布时间：2018-01-08 22:32

本文关键词：基于磁分离的现场核酸检测平台若干关键技术研究　出处：《东南大学》2016年博士论文　论文类型：学位论文

【摘要】：随着微纳加工、生物医学、新材料、集成电路和智能控制等高新技术的快速发展,使得现场检测方法不断向实时、定量方向发展；检测设备向小型化、自动化和检测项目的多样化方向发展；检测对象则由生化指标、免疫指标逐步外延到核酸指标。而现场核酸分子检测必须具有高特异性和高灵敏度,并且可大大减少疾病检测的时间。长远来说,核酸分子检测在疾病的易感性、疾病的分型、分期和预后判断及疗效检查等方面都具有重要应用前景。本论文以核酸分子为样本检测指标,以减少样本检测的时间,减少工作量,降低检测成本为目的,结合磁分离技术,聚合酶链式反应以及环介导等温扩增等生化技术构建具有自主知识产权的小型化,自动化的核酸检测平台；为实验室自动化提供灵活的解决方案；对高精度、快速的运动控制技术和温度控制技术,微弱光电的检测技术进行系统研究与应用。首先,设计了小型一体化步进电机控制板和高精度的运动控制算法,研制了基于磁分离的快速全自动核酸提取设备；其次,构建了热电制冷器变温控制平台,设计了一种高精度快速变温控制策略,实现高精度的热循环控制,并用于核酸扩增装置的研制；最后,设计了重复性好,精度高的光电检测系统,对其光电检测电路进行计算、仿真和参数优化,研制了浊度检测装置,实现了基于环介导等温扩增的多通道核酸检测。整个平台在1小时左右完成核酸定性检测。论文的主要具体内容如下：1.基于磁分离的快速小型化核酸提取系统对基于超顺磁性纳米颗粒提取核酸方法进行了详细的过程分析,设计了高效的磁分离结构和小型的加热结构；为了提高磁分离质量,避免交叉污染,根据磁分离和深孔板特点,设计了8通道一次性磁套；为了体积最小化和控制灵活化,开发了基于总线控制策略的微型一体化步进电机驱动电路板；同时,为了提高控制精度,根据步进电机的特性,改进了基于脉冲法的等频率步进电机控制算法,优于传统S型加减速算法；在触摸屏开发平台上,使用java语言开发了操作简单、参数可设置的用户界面。最后,研制出小型化快速的全自动核酸提取设备；并且结合实验室自主研制的磁珠法提取DNA试剂盒,对设备的相关参数和控制策略进一步优化,能够在30分钟内完成高纯度的DNA提取和纯化。2.热电制冷器快速变温核酸扩增系统通过对核酸扩增的原理进行过程分析,提高热循环系统性能可直接影响核酸扩增的效率。为研制性能优良的快速变温核酸扩增系统,本论文对热循环控制中的核心执行部件(热电制冷器)的变温机理进行详细的研究,包括理论分析、计算与器件选型；采用高速场效应晶体管组成全电桥功率放大器,包括全桥驱动电路和逻辑控制电路,以提高热电制冷器工作效率；对基于恒流源的铂电阻温度采集方法设计了简单的信号调理电路,对温度-电阻分度表进行了曲线拟合,在0℃～140℃范围内寻找出误差小于0.1℃的分段公式,在理论误差分析和实验验证中,均表明其误差小于0.1℃。在软件方面,为了提高温度控制的精度、鲁棒性以及减少超调,我们开发了一种自适应热循环控制策略：根据继电器反馈模型推导出阶跃响应产生等幅振荡曲线,进而计算出系统的临界参数和系统PID参数值,再结合模糊PID的控制思想,系统自动寻找最合适的PID参数；最后,研制出核酸扩增设备,实现了高效核酸扩增,温度控制超调量小于0.5℃,温控精度小于0.2℃,具有良好稳定性和鲁棒性。与国外仪器相比实验结果相差不大。3.实时等温扩增浊度检测系统对环介导等温扩增浊度法检测方法进行分析,构建实时等温扩增浊度检测系统。首先,对浊度法多通道光电检测原理进行了理论研究,对关键部件光源和探测器进行对比与选型；设计了恒功率激光二极管驱动电路,以提高光电检测的强度和光源的稳定性；其次,以提高抗干扰和抗噪声能力为出发点,实现从微弱的生物信号提取有效的光电信号,我们对光电二极管模型进行了分析、计算与仿真验证,设计了微弱信号调理电路,并且对调理电路关键参数进行了优化；此外,我们对噪声的来源进行了详细分析,通过各种滤波,屏蔽等方法抑制噪声的干扰,并且通过变异系数来表征系统具有较好重复性和稳定性。软件方面,从提取的多个光电信号进行数字滤波与识别,并且绘制曲线图；最后,搭建出基于LAMP的实时浊度检测平台,并且对系统进行定标,使用军团菌和H7N9病毒商用试剂盒检验了系统的性能。实验表明系统具有良好的特异性,能在1小时左右完成核酸的定性检测。
[Abstract]:With the micro nano processing, biomedicine, new materials, the rapid development of high-tech integrated circuit and intelligent control, making the scene detection method to real-time, quantitative direction; testing equipment to miniaturization, automation and detection of the direction of diversification project development; object detection by the biochemical index, immune index gradually extended to nucleic acid targets. The on-site detection of nucleic acid molecules must have high specificity and high sensitivity, and can greatly reduce the disease detection time. In the long run, nucleic acid detection in susceptibility to disease, type of disease, have important application prospects of staging and prognosis and curative effect of examination. In this paper, nucleic acid molecules as sample indexes. In order to reduce the detection time, reduce the workload, reduce the cost of testing for the purpose of combining the magnetic separation technique, polymerase chain reaction and loop mediated isothermal expansion Building small with independent intellectual property rights by biochemical technology, nucleic acid detection platform of automation; provide a flexible solution for laboratory automation; of high precision motion control technology and rapid temperature control technology, detection technology of weak photoelectric system research and application. Firstly, the design of small integrated stepper motor control in high precision and motion control algorithm, developed fast automatic nucleic acid extraction equipment based on magnetic separation; secondly, construct a thermoelectric temperature control platform, the design of a high precision and fast temperature control strategy, realize thermal cycle control with high precision, and for the development of nucleic acid amplification device; finally, design good repeatability, high precision photoelectric detection system, the photoelectric detection circuit calculation, simulation and parameter optimization is developed, the turbidity detection device is realized based on the ring Multi channel mediated isothermal amplification of nucleic acid detection. The whole platform to complete the nucleic acid qualitative detection in about 1 hours. The main contents of this paper are as follows: 1. based on the magnetic separation of miniaturizing nucleic acid extraction system of superparamagnetic nanoparticles for nucleic acid extraction methods are analyzed in detail based on the design of magnetic separation structure and high efficiency the heating structure of small; in order to improve the quality of magnetic separation, to avoid cross contamination, according to the characteristics of magnetic separation and deep plate, 8 channel disposable magnetic sleeve is designed; in order to minimize the volume and control flexibility, the development of the micro step control strategy integration bus stepper motor drive circuit board based on; at the same time, in order to improve the control precision, according to the characteristics of stepping motor, pulse method, frequency stepper motor control algorithm based on improved, better than the traditional S type deceleration algorithm; the touch screen development platform, the Using java language to develop simple user interface, parameters can be set. Finally, developed automatic nucleic acid extraction equipment miniaturization fast; and combining the magnetic beads method developed by our laboratory DNA extraction kit, related parameters of the equipment and the control strategy is optimized further, to complete the extraction and purification of high purity DNA.2. power refrigerator temperature rapid nucleic acid amplification system through the analysis of the process principle of nucleic acid amplification in 30 minutes, to improve the performance of thermal cycling system can directly influence the efficiency of nucleic acid amplification. In order to develop excellent performance in variable temperature nucleic acid amplification system, the execution unit of the core control in thermal cycling (thermoelectric cooler) temperature mechanism the detailed research, including theoretical analysis, calculation and selection of devices; consisting of full bridge power amplifier using high-speed field-effect transistors, including full bridge driver Circuit and logic control circuit, to improve the efficiency of thermoelectric cooler; the temperature acquisition method of constant current source of platinum resistance is designed based on the simple signal conditioning circuit for temperature - resistance indexing table curve fitting was conducted in 0 to 140 DEG C range for piecewise formula error is less than 0.1 DEG C, in the theory of error analysis and experimental verification, the error is less than 0.1 degrees. In terms of software, in order to improve the accuracy of temperature control, robustness and reduce the overshoot, we developed an adaptive thermal cycle control strategy: Based on relay feedback model is derived from the oscillation amplitude step response curve, and then calculate the critical parameters and the PID parameter value of the system, combined with the control theory of fuzzy PID, PID automatically find the most suitable parameters of the system; finally, developed nucleic acid amplification equipment, to achieve efficient nucleic acid amplification, temperature control The overshoot is less than 0.5 DEG C, the temperature control accuracy of less than 0.2 DEG C, has good stability and robustness. Compared with the experimental results of similar foreign equipment.3. real-time turbidity detection system by isothermal isothermal amplification of ring mediated amplification turbidimetric assay methods are analyzed. The construction of real-time isothermal amplification turbidity detection system. Firstly, the turbidity method channel photoelectric detection principle is studied and compared with the selection of the key components of the light source and detector; design the constant power driving circuit of laser diode, in order to improve the strength and stability of photoelectric detection light source; secondly, in order to improve the anti-jamming and anti noise ability as a starting point, to achieve effective photoelectric signal extraction from biological signal and we analyze the photodiode model, calculation and simulation, the design of weak signal conditioning circuit, and the circuit parameters In addition, we optimized; sources of noise are analyzed, through various methods such as filtering, shielding noise suppression, and the coefficient of variation to characterize the system has good repeatability and stability. The software, digital filtering and identification from a plurality of photoelectric signal extraction, and finally draw the graph; to build a real-time turbidity detection platform based on LAMP, and the system is calibrated, the system performance test using Legionella bacteria and virus H7N9 commercial kit. The experiment shows that the system has good specificity and can complete the qualitative detection of nucleic acids in about 1 hours.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q503

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