本文關(guān)鍵詞：基于磁分離的現(xiàn)場(chǎng)核酸檢測(cè)平臺(tái)若干關(guān)鍵技術(shù)研究　出處：《東南大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 磁分離 核酸擴(kuò)增 環(huán)介導(dǎo)等溫?cái)U(kuò)增 PID 光電檢測(cè)

【摘要】：隨著微納加工、生物醫(yī)學(xué)、新材料、集成電路和智能控制等高新技術(shù)的快速發(fā)展,使得現(xiàn)場(chǎng)檢測(cè)方法不斷向?qū)崟r(shí)、定量方向發(fā)展；檢測(cè)設(shè)備向小型化、自動(dòng)化和檢測(cè)項(xiàng)目的多樣化方向發(fā)展；檢測(cè)對(duì)象則由生化指標(biāo)、免疫指標(biāo)逐步外延到核酸指標(biāo)。而現(xiàn)場(chǎng)核酸分子檢測(cè)必須具有高特異性和高靈敏度,并且可大大減少疾病檢測(cè)的時(shí)間。長(zhǎng)遠(yuǎn)來說,核酸分子檢測(cè)在疾病的易感性、疾病的分型、分期和預(yù)后判斷及療效檢查等方面都具有重要應(yīng)用前景。本論文以核酸分子為樣本檢測(cè)指標(biāo),以減少樣本檢測(cè)的時(shí)間,減少工作量,降低檢測(cè)成本為目的,結(jié)合磁分離技術(shù),聚合酶鏈?zhǔn)椒磻?yīng)以及環(huán)介導(dǎo)等溫?cái)U(kuò)增等生化技術(shù)構(gòu)建具有自主知識(shí)產(chǎn)權(quán)的小型化,自動(dòng)化的核酸檢測(cè)平臺(tái)；為實(shí)驗(yàn)室自動(dòng)化提供靈活的解決方案；對(duì)高精度、快速的運(yùn)動(dòng)控制技術(shù)和溫度控制技術(shù),微弱光電的檢測(cè)技術(shù)進(jìn)行系統(tǒng)研究與應(yīng)用。首先,設(shè)計(jì)了小型一體化步進(jìn)電機(jī)控制板和高精度的運(yùn)動(dòng)控制算法,研制了基于磁分離的快速全自動(dòng)核酸提取設(shè)備；其次,構(gòu)建了熱電制冷器變溫控制平臺(tái),設(shè)計(jì)了一種高精度快速變溫控制策略,實(shí)現(xiàn)高精度的熱循環(huán)控制,并用于核酸擴(kuò)增裝置的研制；最后,設(shè)計(jì)了重復(fù)性好,精度高的光電檢測(cè)系統(tǒng),對(duì)其光電檢測(cè)電路進(jìn)行計(jì)算、仿真和參數(shù)優(yōu)化,研制了濁度檢測(cè)裝置,實(shí)現(xiàn)了基于環(huán)介導(dǎo)等溫?cái)U(kuò)增的多通道核酸檢測(cè)。整個(gè)平臺(tái)在1小時(shí)左右完成核酸定性檢測(cè)。論文的主要具體內(nèi)容如下：1.基于磁分離的快速小型化核酸提取系統(tǒng)對(duì)基于超順磁性納米顆粒提取核酸方法進(jìn)行了詳細(xì)的過程分析,設(shè)計(jì)了高效的磁分離結(jié)構(gòu)和小型的加熱結(jié)構(gòu)；為了提高磁分離質(zhì)量,避免交叉污染,根據(jù)磁分離和深孔板特點(diǎn),設(shè)計(jì)了8通道一次性磁套；為了體積最小化和控制靈活化,開發(fā)了基于總線控制策略的微型一體化步進(jìn)電機(jī)驅(qū)動(dòng)電路板；同時(shí),為了提高控制精度,根據(jù)步進(jìn)電機(jī)的特性,改進(jìn)了基于脈沖法的等頻率步進(jìn)電機(jī)控制算法,優(yōu)于傳統(tǒng)S型加減速算法；在觸摸屏開發(fā)平臺(tái)上,使用java語言開發(fā)了操作簡(jiǎn)單、參數(shù)可設(shè)置的用戶界面。最后,研制出小型化快速的全自動(dòng)核酸提取設(shè)備；并且結(jié)合實(shí)驗(yàn)室自主研制的磁珠法提取DNA試劑盒,對(duì)設(shè)備的相關(guān)參數(shù)和控制策略進(jìn)一步優(yōu)化,能夠在30分鐘內(nèi)完成高純度的DNA提取和純化。2.熱電制冷器快速變溫核酸擴(kuò)增系統(tǒng)通過對(duì)核酸擴(kuò)增的原理進(jìn)行過程分析,提高熱循環(huán)系統(tǒng)性能可直接影響核酸擴(kuò)增的效率。為研制性能優(yōu)良的快速變溫核酸擴(kuò)增系統(tǒng),本論文對(duì)熱循環(huán)控制中的核心執(zhí)行部件(熱電制冷器)的變溫機(jī)理進(jìn)行詳細(xì)的研究,包括理論分析、計(jì)算與器件選型；采用高速場(chǎng)效應(yīng)晶體管組成全電橋功率放大器,包括全橋驅(qū)動(dòng)電路和邏輯控制電路,以提高熱電制冷器工作效率；對(duì)基于恒流源的鉑電阻溫度采集方法設(shè)計(jì)了簡(jiǎn)單的信號(hào)調(diào)理電路,對(duì)溫度-電阻分度表進(jìn)行了曲線擬合,在0℃～140℃范圍內(nèi)尋找出誤差小于0.1℃的分段公式,在理論誤差分析和實(shí)驗(yàn)驗(yàn)證中,均表明其誤差小于0.1℃。在軟件方面,為了提高溫度控制的精度、魯棒性以及減少超調(diào),我們開發(fā)了一種自適應(yīng)熱循環(huán)控制策略：根據(jù)繼電器反饋模型推導(dǎo)出階躍響應(yīng)產(chǎn)生等幅振蕩曲線,進(jìn)而計(jì)算出系統(tǒng)的臨界參數(shù)和系統(tǒng)PID參數(shù)值,再結(jié)合模糊PID的控制思想,系統(tǒng)自動(dòng)尋找最合適的PID參數(shù)；最后,研制出核酸擴(kuò)增設(shè)備,實(shí)現(xiàn)了高效核酸擴(kuò)增,溫度控制超調(diào)量小于0.5℃,溫控精度小于0.2℃,具有良好穩(wěn)定性和魯棒性。與國(guó)外儀器相比實(shí)驗(yàn)結(jié)果相差不大。3.實(shí)時(shí)等溫?cái)U(kuò)增濁度檢測(cè)系統(tǒng)對(duì)環(huán)介導(dǎo)等溫?cái)U(kuò)增濁度法檢測(cè)方法進(jìn)行分析,構(gòu)建實(shí)時(shí)等溫?cái)U(kuò)增濁度檢測(cè)系統(tǒng)。首先,對(duì)濁度法多通道光電檢測(cè)原理進(jìn)行了理論研究,對(duì)關(guān)鍵部件光源和探測(cè)器進(jìn)行對(duì)比與選型；設(shè)計(jì)了恒功率激光二極管驅(qū)動(dòng)電路,以提高光電檢測(cè)的強(qiáng)度和光源的穩(wěn)定性；其次,以提高抗干擾和抗噪聲能力為出發(fā)點(diǎn),實(shí)現(xiàn)從微弱的生物信號(hào)提取有效的光電信號(hào),我們對(duì)光電二極管模型進(jìn)行了分析、計(jì)算與仿真驗(yàn)證,設(shè)計(jì)了微弱信號(hào)調(diào)理電路,并且對(duì)調(diào)理電路關(guān)鍵參數(shù)進(jìn)行了優(yōu)化；此外,我們對(duì)噪聲的來源進(jìn)行了詳細(xì)分析,通過各種濾波,屏蔽等方法抑制噪聲的干擾,并且通過變異系數(shù)來表征系統(tǒng)具有較好重復(fù)性和穩(wěn)定性。軟件方面,從提取的多個(gè)光電信號(hào)進(jìn)行數(shù)字濾波與識(shí)別,并且繪制曲線圖；最后,搭建出基于LAMP的實(shí)時(shí)濁度檢測(cè)平臺(tái),并且對(duì)系統(tǒng)進(jìn)行定標(biāo),使用軍團(tuán)菌和H7N9病毒商用試劑盒檢驗(yàn)了系統(tǒng)的性能。實(shí)驗(yàn)表明系統(tǒng)具有良好的特異性,能在1小時(shí)左右完成核酸的定性檢測(cè)。
[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.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q503

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