本文選題：自動(dòng)交叉配血實(shí)驗(yàn)　切入點(diǎn)：臨床免疫檢測　出處：《大連醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:輸血現(xiàn)已成為臨床治療的重要組成部分,臨床輸血工作的最重要原則為保證每一例輸血的安全性。在現(xiàn)代臨床醫(yī)學(xué)檢驗(yàn)方法中,交叉配血試驗(yàn)是保證輸血安全的重要手段,它是將供血者紅細(xì)胞和血清分別與受血者血清和紅細(xì)胞交叉混合,檢查受血者血清中有無破壞供血者紅細(xì)胞的抗體,當(dāng)受血者和供血者的血液間沒有可測的不相配的抗原、抗體成分時(shí),即試驗(yàn)結(jié)果無溶血無凝集時(shí),方可將供者血液成分輸注給受血者。臨床中通常采用的配血方式存在一些問題,如依賴人工操作、自動(dòng)化程度低、試劑和樣品的消耗較多;另一方面,自動(dòng)配血儀的儀器成本和運(yùn)行費(fèi)用高,對疑難案例仍然需要人工判斷,降低了篩測效率和準(zhǔn)確性。在近年的免疫診斷的技術(shù)發(fā)展中,床邊診斷因其人工參與度低、可自動(dòng)化運(yùn)行和記錄試驗(yàn)結(jié)果、試劑與樣品消耗小、檢測時(shí)間縮短而受到關(guān)注,而免疫檢測平臺(tái)的縮微化有利于床邊診斷的實(shí)現(xiàn)。其中,微流控芯片是一種新型的分離分析技術(shù),目前已經(jīng)成為分析化學(xué)和生化檢測的重要平臺(tái)技術(shù)。引入微流控等縮微化的新方法,將有利于交叉配血實(shí)驗(yàn)的自動(dòng)化,這也成為免疫檢測中的新思路。超磁磁珠因其比表面積大、可以被外加磁場操控,已經(jīng)成為免疫檢測,如酶聯(lián)免疫吸收檢測和化學(xué)發(fā)光免疫分析等方法中的重要固相樣品載體,但檢測過程中需要反應(yīng)、封閉、分離和洗滌等多步復(fù)雜操作,且自動(dòng)化程度低。因此,建立一種能夠自動(dòng)實(shí)現(xiàn)免疫檢測樣品處理步驟中多步復(fù)雜操作的平臺(tái)和載體,將對提高免疫檢測診斷的效率,具有重要的實(shí)用意義。目的:本課題擬構(gòu)建基于微流控芯片的免疫檢測和樣品處理平臺(tái),首先通過將傳統(tǒng)配血實(shí)驗(yàn)中采用的各種血液樣品和免疫試劑裝載于單個(gè)微液滴中,利用數(shù)字液滴芯片對液滴的自動(dòng)操控功能,在芯片上實(shí)現(xiàn)血紅細(xì)胞和血清間的交叉反應(yīng)實(shí)驗(yàn),在概念上驗(yàn)證數(shù)字液滴芯片作為免疫床邊檢測平臺(tái)的可行性。以超磁磁珠作為免疫樣品的固相載體裝載于液滴中,結(jié)合數(shù)字液滴的操控功能與磁珠的載體功能,并輔以磁珠固定模塊,探索建立免疫檢測樣品處理步驟中自動(dòng)實(shí)現(xiàn)多步復(fù)雜操作體系的可能性。方法:1.構(gòu)建基于數(shù)字液滴微流控微反應(yīng)器平臺(tái),取得適合配血試驗(yàn)中實(shí)際樣品液滴輸運(yùn)的芯片絕緣工藝和液滴改性方案。2.選取2016年4~5月間大連市中心醫(yī)院收治的需要輸注紅細(xì)胞治療的住院患者,排除輸血前檢測抗體篩查陽性者,共32例,其中男性21例,女性11例,年齡28~78歲;A型8份,B型12份,O型10份,AB型2份,Rh D均為陽性。與獻(xiàn)血員血液同型進(jìn)行鹽水法交叉配血,同時(shí)利用數(shù)字液滴芯片對液滴的自動(dòng)操控功能,在芯片上實(shí)現(xiàn)交叉配血,觀察融合液滴中是否出現(xiàn)抗原-抗體的凝集反應(yīng)與鹽水法結(jié)果比較。3.構(gòu)建固相免疫載體磁珠的固定模塊,探索在數(shù)字液滴芯片上進(jìn)行磁珠反應(yīng)、封閉、分離和洗滌的條件。結(jié)果:1.構(gòu)建了包括數(shù)字微流控芯片內(nèi)的數(shù)字微流控微反應(yīng)器。得到了適合實(shí)際樣品的芯片制作配方和液滴改性配方,消除了含蛋白質(zhì)液滴對芯片表面發(fā)生生物污損的機(jī)會(huì)。2.在數(shù)字微流控芯片表面上嘗試了交叉配血實(shí)驗(yàn),實(shí)驗(yàn)結(jié)果與傳統(tǒng)方法完全一致。而芯片法實(shí)驗(yàn)呈現(xiàn)陽性時(shí),第8秒就可以觀察到紅細(xì)胞凝集的發(fā)生,而且樣品消耗量僅為1.2微升,為傳統(tǒng)方法的1/20-1/40。3.通過將磁珠置于液滴中,并構(gòu)建強(qiáng)磁體和磁導(dǎo)構(gòu)成的磁珠鉗制模塊,將此模塊與數(shù)字液滴操控平臺(tái)結(jié)合,實(shí)現(xiàn)了對液滴中磁珠樣品的有效分離、與反應(yīng)物結(jié)合、利用緩沖液對磁珠進(jìn)行洗滌等,在此基礎(chǔ)上進(jìn)行了樣品和試劑的多步反應(yīng)、分離與清洗的自動(dòng)化操作,初步滿足了床邊免疫診斷中樣品操控的需求。這些步驟均為磁珠酶聯(lián)免疫及化學(xué)發(fā)光免疫分析檢測的基礎(chǔ)操作單元。結(jié)論:1.成功構(gòu)建了以數(shù)字微流控芯片為核心的數(shù)字微流控微反應(yīng)器,實(shí)現(xiàn)了液滴的移動(dòng)、融合、分離與等份分裂等基本操控功能。2.在數(shù)字微流控芯片上進(jìn)行的交叉配血實(shí)驗(yàn),其實(shí)驗(yàn)結(jié)果與傳統(tǒng)方法完全一致,反應(yīng)時(shí)間和樣品消耗得到大幅度減少,芯片法中反應(yīng)時(shí)間和樣品消耗的減少得益于液滴體系的縮微化,在概念上驗(yàn)證了數(shù)字液滴芯片作為免疫凝集反應(yīng)實(shí)驗(yàn)平臺(tái)的可行性。3.將免疫反應(yīng)的重要樣品固相載體-磁珠裝載與液滴中,實(shí)現(xiàn)了將磁珠鉗制模塊與數(shù)字液滴操控平臺(tái)結(jié)合,初步進(jìn)行了芯片上的多步反應(yīng)、分離與清洗的自動(dòng)化操作,建立了一種能夠自動(dòng)實(shí)現(xiàn)免疫檢測樣品處理步驟中多步復(fù)雜操作的體系。
[Abstract]:Background: blood transfusion has become an important part of clinical treatment, the most important principle of clinical transfusion work to ensure the safety of every case of blood transfusion. In modern clinical medicine inspection method, cross matching test is an important means to ensure the safety of blood transfusion, it is the blood red blood cells and serum and recipient serum respectively. And the red cross cell mixed antibody test in serum of blood recipients without destruction of red blood cells when blood donors, recipients and donors were not matched between blood antigen no measurable antibody components, namely, the test results of hemolysis without agglutination, can the donor blood component transfusion to blood recipients. There are some problems with the commonly used method in clinical blood, such as rely on manual operation, low degree of automation, reagent and sample consumption more; on the other hand, equipment cost and operation cost of automatic matching for high, the case is still difficult You need artificial judgment, reduce the screening efficiency and accuracy. In the development of technology in recent years in the immunological diagnosis, bedside diagnosis because of its artificially low participation, it can run automatically and record test results, reagent and sample consumption, shorten the detection time and attention, and the immune detection platform is conducive to the realization of microcopying bedside diagnosis. Among them, the microfluidic chip analysis technique is a new separation technology, has become an important platform for the analysis of chemical and biochemical detection. The introduction of new methods such as microfluidic microcopying, there will be conducive to automated blood cross matching test, it has also become a new idea in the immunoassay ultra magnetic beads. Because of its large surface area, the magnetic field can be manipulated, has become an important carrier of solid phase immunoassay, enzyme-linked immunosorbent assay as sample absorption measurement and chemiluminescence immune analysis method, but the detection process Need to be closed, reaction, separation and washing steps such as complex operation, and low degree of automation. Therefore, to build a automatic immune detection sample processing steps in step complex operation platform and carrier, will be to improve the efficiency of immune detection and diagnosis, has important practical significance. Objective: This study intends to build immunity microfluidic chip detection and sample processing platform based on the first by using the traditional matching experiments in various blood samples and immune reagent loaded on single droplet, automatic control of droplet droplet using digital chip, realizes the cross reaction experiment of red blood cells and serum in the chip. The concept of verification of digital droplet chip as the feasibility of immune bedside testing platform. With super magnetic beads as solid carrier immune sample loading in the liquid droplets, combined with the control function and magnetic digital droplet The carrier function of beads, and beads with fixed module, to explore the possibility of multi-step operation system automatically establish immunoassay sample processing steps. Methods: 1. construction of digital microfluidic droplet based micro reactor platform, made suitable for blood test in actual sample droplet transport process and droplet change chip insulation for the month of 2016 4~5.2. selected from Dalian central hospital to red blood cell transfusion patients, detection of antibody positive were excluded before blood transfusion, a total of 32 cases, including 21 cases of male, female 11 cases, age 28~78; 8 A, 12 B, 10 O. 2 AB, Rh and D were positive. Blood donors were saline crossmatching of blood with the same type, using digital chip automatic control function of droplet droplet, realize the cross matching of blood on the chip, to observe the fusion antigen antibody is the emergence of the liquid drop in anti agglutination With the saline method compared the results of.3. fixed modules to build a solid phase immune carrier beads, beads of reaction in the digital droplet chip sealing, separation and washing conditions. Results: 1. constructs including digital microfluidic chip digital microfluidic reactor. The suitable sample chip production formula and the droplet modified formula, eliminating the protein droplet on the surface of the chip biofouling happening.2. in the digital microfluidic chip on the surface to blood cross matching test, the experimental results with the traditional method of exactly the same. While the chip method experiment were positive, eighth seconds can observe the agglutination of red blood cells, and sample consumption is only 1.2 L, as the traditional method of 1/20-1/40.3. by magnetic bead droplet placed, and build a strong magnet and magnetic bead clamping module, this module and digital droplet manipulation The platform combined, realize effective separation of bead sample droplets, combined with reactants, washing the beads using buffer, based on the reaction of samples and reagents, automation of separation and cleaning, to meet the initial sample manipulation in bedside immune diagnosis demand. These steps are as the bead ELISA and chemiluminescence immunoassay detection. Conclusion: the 1. basic operation unit is successfully constructed with digital micro fluidic chip as the core of the digital microfluidic reactor, the moving droplet fusion, blood cross matching test and separation of equal division basic operation functions of.2. in digital microfluidics on chip, the experimental results and the traditional method is completely consistent, reaction time and sample consumption is greatly reduced, the chip method reaction time and sample consumption reduced due to the system of micro droplet In concept, to verify the feasibility of.3. chip as digital droplet immune agglutination reaction experiment platform will carry important samples of solid carrier - immunoreactive beads and droplets, the magnetic clamping module and digital droplet manipulation platform with a preliminary multi-step reaction on a chip, automatic operation and separation cleaning, to establish a new automatic immunoassay sample processing steps in step complex operating system.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.6

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本文編號：1605077