| Along with the continuous development of micro-nano processing, biomedicine, new materials, integrated circuits, intelligent control and other high-tech, on-site sample testing methods are moving in real-time and quantitative direction and testing equipment is going toward miniaturization, automation and diversified testing index. The detected objection of biochemical or immune targets is developed to nucleic acid molecules. On-site nucleic acid testing (NAE) has characteristic of high specificity and sensitivity and greatly shorten testing time of disease. In the long run, it has great potential in investigation of diseases susceptibility, genotyping, clinical staging, prognosis estimation and curative effect, etc.In this paper, nucleic acid molecules are the testing targets of samples. For reducing the sample testing time and costs, and the workload and health risk, combining the technology of magnetic separation (MS), polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) and other biochemistry, we have built a miniaturization and automation nucleic acid testing technology platform by domestic technology. Besides that, the motion control and temperature control system with high accuracy and speed, and the weak photoelectric signal measuring system were researched and applied, so it also provided a flexible and reliable solution to lab automation. First, a small stepper motor control hardware and motion control algorithm is designed and realized. With magnetic separation technology, a quick nucleic acid extractor (NAE) was developed. Next, a high precision and rapid cooling and heating control hardware and PID algorithm are also designed and realized. With the research of thermoelectric refrigerator, a thermal cycler device for PCR was developed. Finally, a turbidity measuring device with better repeatability and reliability was designed and realized, and is successfully used in LAMP. The entire platforms can realize qualitative analysis of nucleic acid around 1 hours. The main concrete content of the thesis is as follows:1. Quickly and miniaturization nucleic acid extraction system based on MSThe experimenting process of nucleic acid extraction with magnetic beads was analyzed and modeled. And an efficient MS structure and small size heating structure were designed and processed. For improving the quality of MS and avoiding cross-contamination, an 8-disposable magnetic sleeve was custom-designed according to the characteristic of MS and deep-hole well. For the minimal volume and control flexibility, a micro intelligent stepping motor driver was developed. Besides, according to the stepper motor features, an improved equal-frequency acceleration and deceleration algorithm was applied in step motor control and is superior to conventional S-shape curve. In the touch screens platform, we used the java language to develop a user interface with simple operation and configurable parameters. At last the device with the function of miniaturization and rapidity was developed. Moreover, we optimized related equipment parameters and control methods in accordance with the demands of our own developed NAE kit, and obtained DNA extracted and purified in 30 minutes with high purity.2. Thermoelectric cooler quickly temperature-varying control system for PCRBased on the analysis of the principle of PCR, the control performance of thermal cyclers was found to directly affect PCR efficiency, while the key component was the thermoelectric cooler (TEC). So, we needed to analyze and calculate the heating and cooling mechanism of TEC for a better selection. For increasing cooling system efficiency, A H-whole bridge power amplification circuit based on metal oxide semiconductor (MOS) was developed, which was consist of full-bridge driver circuit and logic control circuit We design the signal conditioning circuit for high precision temperature acquisition system, and using constant current source methods to measure platinum resistance value and calculating the temperature result by piecewise curve fitting by matlab. With theoretical analyses and experimental proof, the error of temperature measuring is less than 0.1℃. On the software side, the adaptive thermal cycler control strategy was proposed for improving the robustness and precision and reducing overshoot. Based on the relay-feedback model, we derived that the step response can generate the equal amplitude oscillations curve, so we can obtain the critical parameters and PID parameters in current system. Otherwise, combining the advantage of fuzzy PID controller, the system can automatically find the perfect parameters to control. At last, the device with the function of high-precise and quickly temperature-varying control was developed, and efficiently accomplished PCR. By many experiments of temperature control, it was found to be a practical system with high precision (<0.2℃) and small extravasation (<0.5℃). Through the practical validation, compared with the foreign instruments, the result of our PCR is the same.3. Real-time turbidity test system for isothermal amplificationAfter analyzing the process of LAMP, we built a real-time turbidity test system. First, photoelectric detection principle was studied in theoretically and applied to liquid turbidity measuring. Besides, the critical components of light sources and photoelectric detector were contrasted in detail and the laser diodes (LD) and silicon photodiode (PD) were selected. So the LD driver circuit with constant power control was designed and realized to improve the intensity and the stability. Furthermore, the photodiode detected signal is very faint and the detection is very difficult, so we need to improve anti-jamming and anti-noise performance. By the analysis, computation and simulation of the PD model, we design a weak optic signal test circuit and optimized the circuit parameters according to actual situations. Then, we analyzed the sources of noise in detail and used a series of filters and shielding methods to effectively suppressed noises. Then, we used the coefficient of variation to test the system. Finally, we built a turbidity test platform and it was calibrated using standard turbidity solution. Meanwhile we used the test kit of legionella and H7N9 to test the system performance. Experiments showed that the system perfomed well with good specificity, and nucleic acid qualitative test could be completed about 1 hour. |
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