本文關(guān)鍵詞：基于單粒子分析方法的水體總菌檢測系統(tǒng)關(guān)鍵技術(shù)研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 單粒子檢測 細(xì)菌染色 熒光檢測 峰值處理

【摘要】：目的:近年來伴隨著多種微生物污染問題的爆發(fā),廣義的生物安全問題逐漸成為了研究熱點(diǎn)。水與空氣資源作為微生物污染的主要傳播媒介,其廣泛的分布以及無可替代的重要程度時(shí)刻提醒我們要重視對資源狀況的監(jiān)控與保護(hù)。細(xì)菌總數(shù)作為水中微生物含量的重要指標(biāo)之一,能夠直觀的反映水體受污染的程度,因此在許多水質(zhì)檢測標(biāo)準(zhǔn)中均規(guī)定將細(xì)菌總數(shù)作為一項(xiàng)必要的檢測項(xiàng)目。在日常生活工作中建立快速準(zhǔn)確的水體樣本細(xì)菌檢測方法對生活用水、科研實(shí)驗(yàn)、醫(yī)療衛(wèi)生、精密加工質(zhì)量控制以及水資源的監(jiān)控與保護(hù),甚至是生物恐怖活動(dòng)的防治都具有十分重要的意義。目前在該領(lǐng)域應(yīng)用最多的仍是傳統(tǒng)的檢測方法,尤其以平板培養(yǎng)法為各國檢測的標(biāo)準(zhǔn)方法,新的檢測方法的提出為水體細(xì)菌的檢測提供了許多更快速、更準(zhǔn)確的研究思路,但是總體來說這些方法仍處于研究階段。本文所述課題旨在課題組研究的基礎(chǔ)上,進(jìn)一步掌握單粒子檢測技術(shù)并將其應(yīng)用于水體細(xì)菌總數(shù)的檢測。在系統(tǒng)樣機(jī)研制的過程中積累經(jīng)驗(yàn),助力于單粒子檢測技術(shù)在水體樣本檢測專項(xiàng)應(yīng)用方面的推廣,為單粒子檢測技術(shù)的應(yīng)用提供新的思路。方法和內(nèi)容:通過對現(xiàn)有的流式檢測原理進(jìn)行剖析,在國外已有的檢測技術(shù)和相關(guān)裝備的基礎(chǔ)上,提出單粒子水體總菌檢測系統(tǒng)關(guān)鍵技術(shù)研究和實(shí)驗(yàn)裝置的研制。主要研究內(nèi)容及方法包括:I.樣本前處理方法研究;通過查閱文獻(xiàn)及實(shí)驗(yàn)驗(yàn)證,對不同原理的前處理方法進(jìn)行探索,對比不同染料、不同碳點(diǎn)對細(xì)菌的染色效果,確立課題所使用的染色方案在該檢測原理下的可行性與準(zhǔn)確性;通過前處理方案的確定,為光路系統(tǒng)光源選擇、探測通道鏡片參數(shù)制定提供必要的設(shè)計(jì)參數(shù)。II.光學(xué)模塊設(shè)計(jì)與參數(shù)優(yōu)化;將光路模塊從總體上分為激發(fā)光路與檢測光路兩部分,通過理論仿真分析,設(shè)計(jì)激光光路實(shí)現(xiàn)對原始光斑的整形從而得到理想的檢測光斑;設(shè)計(jì)檢測光路對熒光信號進(jìn)行選擇性接收;在課題中對該系統(tǒng)的光源選擇、鏡片參數(shù)、光路結(jié)構(gòu)三個(gè)方面進(jìn)行系統(tǒng)性優(yōu)化,并依據(jù)前處理方案擬定的染料特性對檢測光路參數(shù)進(jìn)行優(yōu)化處理。III.液流模塊設(shè)計(jì)與參數(shù)優(yōu)化;通過不同設(shè)計(jì)方案對比,使用不同動(dòng)力源控制樣本液和鞘液,使其在檢測區(qū)形成由外部鞘液流包裹內(nèi)部樣本液流的同軸流動(dòng)模式,通過對液流系統(tǒng)效果進(jìn)行評價(jià)分析,優(yōu)化設(shè)計(jì)方案與控制參數(shù)。課題通過實(shí)驗(yàn)探索,確定了具有較高安全性的進(jìn)樣液路及整體流路方案。IV.信號處理模塊設(shè)計(jì)與優(yōu)化;系統(tǒng)在檢測區(qū)由激發(fā)光斑激發(fā)染色后的細(xì)菌,經(jīng)過檢測光路對熒光信號進(jìn)行探測收集,通過光電倍增管將光信號轉(zhuǎn)換為電信號,再經(jīng)過放大、濾波、AD轉(zhuǎn)換后成為數(shù)字信號傳遞給FPGA主控芯片。課題中針對系統(tǒng)的信號特點(diǎn)進(jìn)行了相關(guān)電路設(shè)計(jì)及參數(shù)優(yōu)化,繪制PCB板并完成電路系統(tǒng)調(diào)試。V.峰值處理算法的提出以及驗(yàn)證;對微米級待測樣本粒子經(jīng)熒光染色后熒光信號強(qiáng)度分布進(jìn)行建模仿真,探尋待測樣本熒光信號的特征,結(jié)合系統(tǒng)檢測平臺的檢測需求設(shè)計(jì)具有針對性的信號處理方法,在Verilog語言環(huán)境下實(shí)現(xiàn)算法的應(yīng)用并通過實(shí)驗(yàn)驗(yàn)證算法的準(zhǔn)確性及穩(wěn)定性,對算法進(jìn)行優(yōu)化。VI.系統(tǒng)結(jié)構(gòu)設(shè)計(jì)與優(yōu)化;在系統(tǒng)搭建初期,采用獨(dú)立的分模塊構(gòu)建方案,將總體系統(tǒng)劃分為幾個(gè)子系統(tǒng)模塊,在逐步實(shí)現(xiàn)各個(gè)模塊的擬定功能后,為了提高系統(tǒng)集成度,在控制系統(tǒng)采用以FPGA為核心的控制方案,在硬件電路中圍繞FPGA為核心分別構(gòu)建中心電源板、信號處理板、信號控制板以及微控板為主體的4塊直插式電路控制系統(tǒng),將各獨(dú)立模塊綜合集成為一體,優(yōu)化系統(tǒng)結(jié)構(gòu)。結(jié)果:I.光路系統(tǒng)采用設(shè)計(jì)方案后激光器原始光斑大小約為844*765um,橢圓度約為0.906,經(jīng)過XY雙軸整形透鏡組整形后,在檢測點(diǎn)位置光斑大小77.8*19.8um,在焦點(diǎn)位置附近光斑變化趨勢較小,具有較好的穩(wěn)定性。II.液流系統(tǒng)分別采用柱塞泵與氣壓泵作為動(dòng)力源,解決了樣本“死體積”問題引入的污染,避免了“脈動(dòng)”問題帶來的樣本回流現(xiàn)象;使樣品進(jìn)樣不受樣本管體積限制,實(shí)現(xiàn)連續(xù)檢測;大約0.4s即可在鞘液液瓶中建立穩(wěn)定的氣壓,電壓值約為1.1v,穩(wěn)定效果較好。III.峰值處理算法重復(fù)檢測10組樣本相對標(biāo)準(zhǔn)偏差為5.36%,并且僅需3個(gè)參數(shù)即可實(shí)現(xiàn)系統(tǒng)信號的快速處理,與現(xiàn)有檢測方法相比具有較好的一致性,可滿足系統(tǒng)的檢測需求。IV.整體系統(tǒng)微球測試表明當(dāng)進(jìn)樣速度處于0.5ul/s-1ul/s之間時(shí),樣本檢測結(jié)果間差異較小,檢測過程更穩(wěn)定;針對不同濃度樣本檢測CV值均在2-3.5之間,達(dá)到了較好的檢測精度。V.系統(tǒng)整體測試人工添加金黃色葡萄球菌10倍梯度濃度結(jié)果表明,目前系統(tǒng)最佳檢測限為103-106cfu/ml濃度范圍,在該檢測限內(nèi)與平皿計(jì)數(shù)法檢測結(jié)果具有較好的一致性,在低于該檢測限時(shí)檢測結(jié)果明顯高于平皿計(jì)數(shù)法;與現(xiàn)有流式細(xì)胞儀的統(tǒng)計(jì)結(jié)果始終保持較好的一致性。總結(jié)與展望:I.在現(xiàn)有樣機(jī)的基礎(chǔ)上,通過對子系統(tǒng)各部分分別優(yōu)化,進(jìn)一步提高整體系統(tǒng)的檢測靈敏度。II.增加熒光收集通道,在理論上通過該樣本前處理方法,可實(shí)現(xiàn)對水體細(xì)菌總數(shù)中死菌與活菌數(shù)量分別檢測,通過增加檢測通道,提高系統(tǒng)單次檢測的分析能力。III.優(yōu)化峰值檢測算法,進(jìn)一步提升算法的靈敏度以及多平臺的通用性,提高算法的自適應(yīng)分析能力。IV.對現(xiàn)有系統(tǒng)結(jié)構(gòu)進(jìn)行優(yōu)化,提升系統(tǒng)的集成度與便攜性。
[Abstract]:Objective: in recent years, accompanied by a variety of microbes pollution outbreak, bio safety problem of generalized gradually becomes a hot research topic. The main media of water and air resources as microbial contamination, the importance of time its wide distribution and it reminds us to attach importance to the monitoring and protection of resources. As an important index of the total number of bacteria the microbial content in water, is able to reflect the degree of water pollution, so in many water quality testing standards are provisions of the total number of bacteria as a necessary test items. In daily life, water for life, scientific research, medical and health to establish a method for detection of bacteria in water samples quickly and accurately in precision machining quality control and the water resources monitoring and protection, and even has a very important significance for prevention and treatment of bioterrorism. Currently in the lead The detection method is still the most widely used traditional domain, especially with plate culture method as the standard method of national detection, a new detection method is proposed for the detection of water bacteria provides a lot more rapid, more accurate research ideas, but overall these methods are still in the research stage. Based on the topic of this paper aims at research group on the further mastery of single particle detection technology is applied to detect the total number of bacteria in the water. The experience gained in the process of system prototype, and help the single particle detection technology in the application of water samples of the special promotion, to provide new ideas for the application of single particle detection technology. The methods and contents: through the flow detection principle of the existing analysis, based on the existing detection technology and related equipment, the key technologies of detection system of total bacteria in single particle water research and The development of experimental equipment. Including the main research contents and methods: Study on pretreatment method of I. samples; through the literature and experiment, explored the pretreatment method of different principle, comparison of different dyes, the dyeing effect of different carbon on bacteria, establish the accuracy and feasibility of dyeing scheme used in this topic detection principle; determined by pretreatment scheme, selection of light source system, detecting channels provide lens parameters and parameter optimization of design parameters of.II. optical module design will be necessary; optical module includes excitation light path and optical detection part two, through the simulation analysis, the design of laser beam shaping the original spot so as to obtain the ideal spot detection; design of optical detection of fluorescence signal for selectively receiving; selection of the source of the system in the paper, lens parameters, optical path The three aspects of structure system optimization, and based on the characteristics of dye processing scheme to develop the detection optical path to optimize the parameters of.III. flow and parameter optimization design module; through the comparison of different design schemes, using different power control sample solution and the sheath liquid, the coaxial flow mode by external sheath fluid inclusions inside the sample the liquid flow formed in the detection area, were analyzed by flow effect on liquid, optimization design and control parameters. Research by experiments, determined with high safety injection liquid road and road scheme of.IV. signal processing module design and optimization of the overall flow; system in the detection area by the excitation beam excited after dyeing the bacteria, after testing the optical path of the fluorescence signal detected by the photomultiplier tube to collect, convert optical signals into electrical signals, after amplification, filtering, AD converted into digital signal Signal for the FPGA main control chip. The signal characteristics of the system the design and optimization of circuit drawing PCB and complete the circuit board system debugging.V. peak processing algorithm is proposed and verified; on micron sample particles by fluorescent staining after fluorescence signal intensity distribution simulation, to explore the characteristics of sample fluorescence the signal to be measured, combined with the test requirements of design system testing platform is for signal processing methods, algorithms used and the accuracy and stability of the algorithm in the Verilog language environment, optimize the design and optimization of.VI. system structure of the algorithm; in early stage of building the system, the construction scheme of independent modules overall, the system is divided into several sub modules of the system, to gradually realize the function of each module, in order to improve the degree of system integration, in the control system The control scheme based on FPGA as the core in the hardware as the core center were constructed around the FPGA power board, signal processing board, signal control board and control system of the 4 block in-line circuit of micro control board as the main body, each independent module integrated into one, optimize the system structure. Results: the design scheme based on I. the optical system of laser original spot size is about 844*765um, the ellipticity is about 0.906, after the plastic lens group after XY biaxial plastic, in the detection of the position of light spot size 77.8*19.8um, in a small spot change trend of focal position, with using piston pump and pneumatic pump as a power source for better stability of.II. liquid flow system, solution a sample of "dead volume" problems of introducing pollution, avoid the sample ripple problem caused by the reflux phenomenon "; the sample from the sample tube volume limit, realize the continuous detection; about 0.4s can establish a stable pressure in the sheath liquid bottle, the voltage value is about 1.1V, the peak value of.III. stabilization processing algorithm repeated detection of 10 samples the relative standard deviation is 5.36%, and only 3 parameters to realize fast signal processing system, and the existing detection method compared with good consistency, and can meet the the requirements of.IV. system testing system testing shows that when the microsphere injection speed is between 0.5ul/s-1ul/s, the difference between the small sample test results, the detection process is more stable; with different concentration sample CV values were between 2-3.5, achieve the detection accuracy of.V. system better overall test added Staphylococcus aureus 10 times the concentration gradient the current system shows that the best detection limit of 103-106cfu/ml concentration range, the detection results within limits and plate count method have good consistency, low in the The detection limit of detection results were significantly higher than the plate count method; statistics and current flow cytometry results remain consistent. Summary and Outlook: I. based on the existing prototype, respectively through the optimization of each part of the sub system, and further improve the overall system's sensitivity to increase.II. fluorescence collection channel, through processing methods the sample in theory, can respectively realize detection of viable and dead bacteria bacteria in water by increasing the number of detection channels, improve the system of single detection of.III. peak detection algorithm optimization ability, to further improve the algorithm sensitivity and versatility of multi platform, improve the analysis of the existing system structure optimization of.IV. algorithm in adaptive ability, enhance the integration and portability of the system.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R123.1

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