- 學位論文
物聯網於基因複製裝置之應用
Application of Internet of Things on Gene Replication Devices
摘要
聚合酶連鎖反應(PCR)是一種在短時間複製大量DNA的技術，原理是讓DNA重複裂解、黏合和延伸三個步驟，完成三個步驟，稱ㄧ次熱循環，進行n個循環後，可得到2n個DNA片段結果。近年來物聯網的技術成長發展快速，本研究以腔體式PCR進行研究並開發成可攜式裝置，以樹莓派3B+為開發平台，並結合PCR溫控系統、螢光檢測系統、電源系統、人機介面、資料庫系統，整合成可攜式腔體式聚合酶連鎖反應裝置，PCR溫控系統以溫度感測器偵測反應槽溫度，將溫度結果由PI控制器程式運算，輸出電壓至L298N模組，驅動致冷晶片與風扇，建立穩定的加熱平台;螢光檢測系統以UV燈做為激發樣本的光源，搭配樹莓派相機拍照，經由Open CV運算分析螢光訊號; 電源系統選用18650鋰電池提供電源; 人機介面系統以網頁呈現溫度、循環數、開始與停止按鈕、影像分析結果圖片畫廊、螢光平均值曲線圖，可利用手機與電腦連上網頁進行控制; 資料庫系統以phpMyAdmin雲端資料庫為儲存數據的平台，將溫度與螢光平均值以及循環數上傳至資料庫，方便後續的資料分析;透過k-type熱電偶與溫度感測器比較結果水的溫度是否達到PCR的反應溫度條件;測試自製PCR裝置實驗以不同濃量實驗，完成PCR實驗收集樣本後進行凝膠電泳測試，觀察電泳圖的結果分析比較，並成功在25 μl與15 μl擴增219 bp薑黃DNA，PCR總執行時間1小時13分鐘;最後進行螢光檢測將樣本混合螢光偵測試劑進行檢測，每一循環進行圖片拍照分析處理，將含有螢光範圍進行平均值運算，上傳至資料庫，由網頁取得資料，以曲線圖方式呈現，最後從曲線圖與結果圖觀察結果在25 μl、15 μl、10 μl的DNA樣本，都有螢光的結果。
並列摘要
Polymerase chain reaction (PCR) is a technique that replicates large amounts of DNA in a short time，the principle is to repeat three steps of DNA denaturation, annealing and extension, Complete three steps,called one thermal cycle,completing n thermal cycle, can Get 2n DNA fragment results.The technology of the Internet of Things has grown rapidly in recent years.In study,was performed using cavity PCR and developed into a portable device,Based on Raspberry Pi 3B +,Combined with PCR temperature control system, fluorescent detection system, power supply system, human-machine interface, database system, integrated into a portable cavity polymerase chain reaction device,In the PCR temperature control system,one temperature sensors measure values at specific points,then the raspberry pi outputs the PWM signal computed from the PI program to the L298N module to drive the Peltier element and fan to establish a stable heating platform. The fluorescence detection system uses UV light as a light source for exciting the sample, and takes pictures with a Raspberry Pi camera, and analyzes the fluorescent signal through Open CV calculation. Eight 18650 lithium batteries provide the electric power to the whole device. Human machine interface system displays temperature, cycle number, start and stop buttons, image analysis result picture gallery, fluorescence average curve graph on web page, which can be controlled by mobile phone and computer through web. In database system ,uses the phpMyAdmin cloud database as a platform for storing data, and uploads the temperature, average value and cycle number to the database for subsequent data analysis. Use a k-type thermocouple and temperature sensor to test whether the temperature in the water reaches the reaction temperature conditions of the PCR. Test the home-made PCR device experiment at different concentrations,Complete the PCR experiment and collect the samples for gel electrophoresis,observe the results of the electrophoresis analysis and comparison,the 219 bp turmeric DNA was successfully amplified in 25 μl、15 μl DNA concentration,the total PCR execution time is 1 hour and 13 minutes. Finally, the fluorescent detection is performed. The sample is mixed with the fluorescent reagent for detection, in each cycle is performed to take pictures and analyze the image,the average value of the fluorescent range is calculated and uploaded to the database,in the end, the turmeric DNA with fluorescence results in 25 μl、15 ul、10 μl was successfully analyzed.