本文關(guān)鍵詞：檢測(cè)腹瀉性貝類毒素大田軟海綿酸的免疫傳感器的研究　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 免疫傳感器 貝類毒素 腹瀉性毒素 納米金 級(jí)聯(lián)放大

【摘要】：隨著世界范圍內(nèi)環(huán)境惡化,海洋赤潮頻發(fā),赤潮藻類會(huì)產(chǎn)生多種海洋生物毒素,其中主要包括腹瀉性、麻痹性和神經(jīng)性毒素。其中,腹瀉性毒素經(jīng)過動(dòng)物實(shí)驗(yàn)證明具有潛在的致癌性、致畸性和致突變性等,而短期內(nèi)會(huì)出現(xiàn)腹瀉、嘔吐、暈眩等癥狀,對(duì)人體健康具有極大得威脅。因此急需快速、準(zhǔn)確、靈敏的方法來(lái)檢測(cè)腹瀉性毒素中的主要種類-大田軟海綿酸。本文首先簡(jiǎn)單介紹了腹瀉性貝類毒素以及針對(duì)大田如海綿酸的檢測(cè)方法,并簡(jiǎn)單闡述生物素-親和素系統(tǒng)及納米金在免疫傳感器中的應(yīng)用。然后設(shè)計(jì)研發(fā)了三種免疫傳感器,包括將簡(jiǎn)單的免疫原理應(yīng)用到研發(fā)試劑盒,將生物素-親和素系統(tǒng)引入傳統(tǒng)的免疫傳感器實(shí)現(xiàn)信號(hào)放大,以及將納米金和基于生物素-親和素系統(tǒng)以及辣根辣根過氧化物酶抗原抗體的循環(huán)放大系統(tǒng)應(yīng)用于構(gòu)建更為靈敏的生物免疫傳感器。三種傳感器都能有效的應(yīng)用于檢測(cè)大田軟海綿酸。本論文完成的主要工作和創(chuàng)新點(diǎn)如下:1.研制了新型的快速檢測(cè)大田軟海綿酸貝類毒素的試劑盒,設(shè)計(jì)了基于試劑盒的現(xiàn)場(chǎng)檢測(cè)儀器。利用競(jìng)爭(zhēng)性免疫反應(yīng),設(shè)計(jì)了一種用于檢測(cè)大田軟海綿酸(OA)的試劑盒。通過優(yōu)化實(shí)驗(yàn),該試劑盒的檢測(cè)限為0.19 μg/L、線性范圍0.2-10 μg/L、與鰭藻毒素(DTX)的交叉反應(yīng)率為23.6%,對(duì)其他毒素的交叉反應(yīng)率幾乎為0,加標(biāo)回收實(shí)驗(yàn)說明該試劑盒具有較好的回收率。該試劑盒對(duì)重金屬離子具有較好的抗干擾性,且目前該試劑盒能保存到6個(gè)月。該試劑盒的檢測(cè)時(shí)間在90min內(nèi),且成本是Beacon試劑盒的1/30。基于開發(fā)的試劑盒,完成了現(xiàn)場(chǎng)光學(xué)檢測(cè)儀器Bionic e-Eye的設(shè)計(jì),該儀器操作簡(jiǎn)單,直接顯示結(jié)果,結(jié)合試劑盒非常適用于現(xiàn)場(chǎng)快速檢測(cè)和篩查,實(shí)際樣品的檢測(cè)結(jié)果與高效液相-質(zhì)譜聯(lián)用((HPLC-MS)和酶標(biāo)儀都有很好的一致性。2.提出了結(jié)合生物素-親和素的免疫傳感器設(shè)計(jì)方法,提高檢測(cè)OA靈敏度的設(shè)計(jì)方法在試劑盒的基礎(chǔ)上,引入生物素-親和素系統(tǒng),設(shè)計(jì)了信號(hào)放大的生物傳感器。該傳感器的檢測(cè)限為0.04 μg/L,線性范圍為0.01-10 μg/L,相比于試劑盒,該傳感器的檢測(cè)限降低了 5倍,線性范圍的下限降低了 20倍;另外,該傳感器對(duì)DTX1的交叉反應(yīng)率為44.14%,對(duì)其他毒素的交叉反應(yīng)率幾乎為0,加標(biāo)回收實(shí)驗(yàn)證明該傳感器用于檢測(cè)OA的回收率范圍在91.09%-111.86%之間,變異系數(shù)CV低于15%。檢測(cè)OA與DTX1混合加標(biāo)的回收率范圍在81.98-132.61%,說明該傳感器對(duì)于檢測(cè)OA與DTX1都具有較好的準(zhǔn)確性。3、提出了結(jié)合納米金和生物素-親和素的級(jí)聯(lián)放大的免疫傳感器,實(shí)現(xiàn)了對(duì)OA超低濃度的檢測(cè)。納米金具有很大的比表面積且與生物分子具有較高的親和力,因此,使其表面結(jié)合二抗-辣根過氧化物酶(SAb-HRP)和HRP,有了很大程度的信號(hào)放大。另外,通過引入生物素-親和素系統(tǒng)以及辣根過氧化物酶及其抗體形成的免疫復(fù)合物構(gòu)成三級(jí)級(jí)聯(lián)放大,形成具有更強(qiáng)放大作用的生物傳感器,該級(jí)聯(lián)放大免疫傳感器的檢測(cè)限為0.0025 μg/L,線性范圍為0.002-5 μg/L,線性區(qū)間的下限0.002μg/L比試劑盒0.2μg/L低了 100倍,并且,該傳感器對(duì)DTX1的交叉反應(yīng)率為19.79%,對(duì)其他毒素的交叉反應(yīng)率幾乎為0;該傳感器的回收率的范圍約在76.07%-94.63%之間,變異系數(shù)CV也低于15%,實(shí)現(xiàn)了對(duì)大田軟海綿酸的高靈敏檢測(cè)。
[Abstract]:With the worldwide environmental deterioration, frequent red tide, red tide algae will produce a variety of marine biological toxins, including diarrhea, paralysis and neurogenic toxin. The diarrhea of toxins through animal experiments that are potentially carcinogenic, teratogenic and mutagenic, but in the short term there will be diarrhea, vomiting. Dizziness and other symptoms, has a great threat to human health. So it is urgent to fast, accurate and sensitive method to detect the main field types diarrhetic toxin in okadaic acid. This paper introduces the diarrhetic shellfish toxoid and on the field detection method such as sponge acid, and briefly expounds the application of biotin avidin gold nanoparticles in the immune system and sensor. Then the design and development of three kinds of immune sensor, including the simple immune principle is applied to the development kit, the biotin avidin system The introduction of the traditional immune sensor signal amplification, and the gold nanoparticles and based on biotin avidin system and horseradish HRP antibody cycle amplification system was used to build more sensitive immunobiosensor. Three kinds of sensors can effectively be used for detecting okadaic acid. This paper completed the main work and innovation are as follows: 1. the development kit for rapid detection of okadaic acid shellfish toxin model, designed the instrument kit based on using competitive immune response, a design for detection of okadaic acid (OA) kit. Through the optimization experiments, the detection kit the limit is 0.19 g/L, the linear range of 0.2-10 g/L, and dinophysistoxin (DTX) of the cross reaction rate was 23.6% and the cross reaction with other toxins rate is almost 0, recovery experiments that the reagent The box has a better recovery. The kit has better anti-interference of heavy metal ions, and the kit can save up to 6 months. The detection time of the kit in the 90min, and the cost is 1/30. Beacon kit kit based on the development of the designed field optical detector Bionic e-Eye, the instrument has the advantages of simple operation, direct display results, combined with the kit is very suitable for on-site rapid detection and screening, detection results of actual samples with high performance liquid chromatography - mass spectrometry ((HPLC-MS) and eliasa have good consistency with.2. made with the avidin immune sensor design in the method, improve the design method of OA based on sensitivity detection kit, the introduction of a biotin avidin system designed biosensor signal amplification. The detection limit of the sensor is 0.04 g/L, the linear range of 0.01-10 g/ Compared to the L kit, the detection limit of the sensor is reduced by 5 times, the lower limit of the linear range is reduced by 20 times; in addition, the sensor cross reaction rate of DTX1 was 44.14%. The cross reaction with other toxins rate is almost 0, recovery experiments show that the detection rate of OA sensor for the recovery in the range of 91.09%-111.86% standard addition, variation coefficient of CV is lower than that of the 15%. detection of OA and DTX1 mixed with standard recovery rate in the range of 81.98-132.61%, indicating that the sensor has good accuracy for the detection of.3 OA and DTX1, put forward a combination of gold nanoparticles and avidin immune sensor in cascade, achieves the detection of low concentration OA the nano fittings with great specific surface area and biological molecules have high affinity, so that its surface with two anti horseradish peroxidase (SAb-HRP) and HRP, a large degree of signal amplification. In addition, Through the introduction of biotin avidin system and horseradish peroxidase antibody and the formation of immune complexes composed of three cascaded amplification, the formation of biosensor has stronger amplification and detection of the immune cascade limit of the sensor is 0.0025 g/L, the linear range of 0.002-5 g/L, the lower limit of linear range of 0.002 g/L than kit 0.2 g/L 100 times lower, and the sensor cross reaction rate of DTX1 was 19.79%. The cross reaction with other toxins rate of almost 0; the recovery rate of the sensor range is about 76.07%-94.63%, the variation coefficient of CV is less than 15%, to achieve a high sensitive detection of okadaic acid on the field.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R155.3;TP212.3

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