發(fā)布時(shí)間：2018-04-18 18:53

  本文選題：細(xì)胞阻抗傳感器 + 聲表面波傳感器��； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：海洋生物毒素是一類(lèi)由赤潮藻產(chǎn)生的小分子化合物,通過(guò)貝類(lèi)濾食后進(jìn)入貝肉體內(nèi)并且完成生物富集。一旦人們誤食了被毒素污染的貝肉,就會(huì)引起中毒,嚴(yán)重時(shí)甚至?xí)䦟?dǎo)致死亡。近年來(lái),隨著環(huán)境污染的加劇,由于誤食被污染的貝類(lèi)而引起的人類(lèi)死亡事件也常有報(bào)道。目前用來(lái)檢測(cè)海洋毒素的方法主要有小鼠生物法(MBA)、酶聯(lián)免疫吸附法(ELISA)、高效液相色譜法(HPLC)和液相色譜質(zhì)譜聯(lián)用法(LC-MS/MS)等。然而,這些方法在實(shí)際應(yīng)用中都有一定的局限性,例如重復(fù)性差、設(shè)備昂貴以及涉及動(dòng)物倫理等問(wèn)題。作為一門(mén)新興的檢測(cè)技術(shù),生物傳感器是一種以細(xì)胞分子等生物材料為敏感元件,結(jié)合了物理化學(xué)等二級(jí)傳感器來(lái)實(shí)現(xiàn)對(duì)于各種物質(zhì)進(jìn)行檢測(cè)的分析系統(tǒng)。由于其具有操作簡(jiǎn)單、分析速度快、靈敏度高、小型化和容易實(shí)現(xiàn)自動(dòng)化等優(yōu)點(diǎn),生物傳感器在細(xì)胞生理、藥物篩選以及食物檢測(cè)等領(lǐng)域都有著廣泛的應(yīng)用。本論文基于細(xì)胞分子水平上檢測(cè)海洋毒素的機(jī)理不同,結(jié)合了特定類(lèi)型的二級(jí)傳感器與檢測(cè)系統(tǒng),分別從細(xì)胞、抗體和適配體三個(gè)方面分別研究了生物傳感器在海洋毒素檢測(cè)中的應(yīng)用潛力。本論文的主要的創(chuàng)新點(diǎn)工作包括：1.提出了一種基于細(xì)胞阻抗傳感器的用于檢測(cè)貝類(lèi)腹瀉性毒素的新方法構(gòu)建了一種基于人類(lèi)細(xì)胞株的細(xì)胞阻抗傳感器,并將其應(yīng)用到腹瀉性毒素的檢測(cè)中。當(dāng)毒素濃度在10-100μg/L的范圍內(nèi),該傳感器檢測(cè)到的細(xì)胞阻抗值與毒素的濃度呈良好的線性關(guān)系。并且通過(guò)檢測(cè)其他毒素與貝肉基質(zhì)的干擾實(shí)驗(yàn),可以證明該方法具有很好的特異性與重復(fù)性,且與小鼠生物法有著很好的相關(guān)性。說(shuō)明該方法對(duì)于貝肉中腹瀉性毒素的檢測(cè)方面具有很好的應(yīng)用潛力。2.提出了一種基于神經(jīng)瘤細(xì)胞的阻抗傳感器的麻痹性毒素檢測(cè)的改進(jìn)方法基于麻痹性毒素能夠特異性結(jié)合細(xì)胞膜上鈉離子通道的特性,建立了一種新的基于小鼠神經(jīng)瘤細(xì)胞的阻抗傳感器檢測(cè)麻痹性毒素的方法。采用了細(xì)胞鈉離子通道激活劑的藜蘆定和鈉鉀泵抑制劑的烏本苷共同作用小鼠神經(jīng)瘤細(xì)胞,細(xì)胞會(huì)因?yàn)殁c離子持續(xù)性內(nèi)流而引起死亡,但是麻痹性毒素可以通過(guò)阻礙鈉離子內(nèi)流來(lái)延緩藜蘆定和烏本苷引起的細(xì)胞死亡。當(dāng)麻痹性毒素的代表性毒素石房蛤毒素(Saxitoxin, STX)濃度在0.1-1000nM的范圍內(nèi),細(xì)胞的相對(duì)存活率與STX濃度的對(duì)數(shù)呈良好的線性關(guān)系。且該方法具有很好的特異性與靈敏度,可以用于麻痹性毒素的實(shí)際樣品檢測(cè)。3.提出了一種基于納米金修飾的聲表面波免疫傳感器,實(shí)現(xiàn)了大田軟海綿酸(OA)的特異性檢測(cè)由于OA的分子量很小,因此本章采用免疫競(jìng)爭(zhēng)分析法來(lái)檢測(cè)貝肉中OA的含量。納米金標(biāo)記的二抗能夠放大傳感器的檢測(cè)信號(hào),且傳感器檢測(cè)信號(hào)與OA濃度在10-150ng/mL之間有著很好的線性關(guān)系,檢測(cè)限為5.45ng/mL。并且通過(guò)與ELISA方法的對(duì)比實(shí)驗(yàn),說(shuō)明該方法具有很好的特異性和準(zhǔn)確性,且能夠用于貝肉實(shí)際樣品中OA的特異性檢測(cè)。4.設(shè)計(jì)了一種基于核酸適配體的聲表面波傳感器用于OA的高靈敏檢測(cè)基于核酸適配體能夠特異性結(jié)合OA分子以及DNA雙鏈互補(bǔ)雜交的特性,設(shè)計(jì)一種基于核酸適配體的三明治結(jié)構(gòu)用來(lái)檢測(cè)樣品中的OA分子。當(dāng)存在OA分子時(shí),適配體鏈便會(huì)與OA形成特異的結(jié)構(gòu),從而從傳感器表面脫落下來(lái),引起傳感器的相位幅值增大。當(dāng)OA濃度在1-100ng/mL范圍內(nèi),傳感器相位幅值的增大與OA濃度之間呈一定的線性關(guān)系,且檢測(cè)限為0.3ng/mL。該方法為生物毒素的檢測(cè)提供了一個(gè)新的思路,且三明治結(jié)構(gòu)的設(shè)計(jì)也具有一定的通用性。此外,適配體作為敏感元件不僅降低了檢測(cè)成本也縮短了檢測(cè)時(shí)間。
[Abstract]:Marine biological toxins are small molecular compounds produced by algae and shellfish by Filter Feeding Shellfish after entering the body and complete biological enrichment. Once people are eating the contaminated shellfish toxins, can cause poisoning, even death. In recent years, with the aggravation of environmental pollution, due to the ingestion of the event the contaminated shellfish caused human death are often reported. Current methods for detecting marine toxins include mouse bioassay (MBA), enzyme linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS/MS). However, these methods are there are some limitations in the practical application, such as poor repeatability, expensive equipment and relates to animal ethics problems. As a new detection technology, biosensor is a kind of cell molecular biological materials as a sensitive element, combined with The physical and chemical sensors to achieve level two for all kinds of material for detection and analysis system. Because of its simple operation, fast analysis, high sensitivity, miniaturization and easy to realize the advantages of automated biosensors in the field of cell physiology, drug screening and detection of food have extensive application. The mechanism of cell molecular detection of marine toxin based on the different combination of two level sensor and detection of specific types of systems, respectively, from the three aspects of cells, antibodies and aptamers were studied using potential biosensor in marine toxin detection. The main innovation work of this dissertation includes: 1. propose a new the method of cell impedance sensor for the detection of diarrhetic shellfish toxin was constructed based on a cell impedance sensor based on human cell lines, and its application to the diarrhea of Toxin detection. When the toxin concentration in the range of 10-100 g/L, the cell impedance sensor detects the value of a good linear relationship between concentration and toxin. And through the experiment of other shellfish toxins and interference detection matrix, it can be proved that the method has good specificity and repeatability, and a good correlation with mouse bioassay. The method presents an improved method based on the paralytic toxin detection of the cell impedance sensor neuroma paralysis toxin binds to the cell membrane properties of sodium channels based on the detection of diarrhetic shellfish meat toxin.2. has very good application potential, established a new method for impedance sensor Neuro-2a cells based on the detection of paralytic toxin. The cell sodium channel inhibitor and veratridine activated sodium potassium pump inhibitor ouabain In the interaction of Neuro-2a cells, cells because of persistent sodium ion influx and cause death, but the PSP can prevent the deaths to delay veratridine and ouabain induced cell flow of sodium ions. Representative toxin saxitoxin as paralytic toxin (Saxitoxin, STX) concentration in 0.1-1000nM within the range of relative log survival rate and STX concentration showed a good linear relationship between the cells. And this method has good specificity and sensitivity, can be used for real sample detection of.3. paralytic toxin presents a surface acoustic wave immunosensor based on gold nanoparticles, the okadaic acid (OA) the specificity of detection due to the molecular weight of OA is very small, so this chapter uses immune competition to detect content of shellfish meat OA analysis method. Nano gold labeled anti two can amplify the detection signal and the sensing sensor. For detecting signal and the concentration of OA has a good linear relationship between 10-150ng/mL, the detection limit is 5.45ng/mL. and by comparison with the ELISA method, this method has good specificity and accuracy, and can be used for specific detection of.4. OA in actual samples of shellfish meat to design a surface acoustic wave sensor nucleic acid aptamer based on OA for highly sensitive detection of nucleic acid aptamer capable of specific binding of OA molecules and DNA double strand complementary hybridization based on the characteristics of the design of a sandwich structure of the aptamer for detection of OA molecules. Based on the presence of OA molecules, the aptamer chain will form a specific structure and OA, which dropped from the sensor surface, caused by phase amplitude of the sensor increases. When the concentration of OA in the range of 1-100ng/mL, the phase and amplitude increase between sensor and OA concentration showed a linear relationship The detection limit is 0.3ng/mL.. This method provides a new idea for the detection of biological toxins, and the design of sandwich structure is also universal. In addition, aptamers as sensitive components not only reduce the cost of detection, but also shorten the detection time.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R99;TP212.3

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