本文關(guān)鍵詞： 免疫傳感器 離子液體 多壁碳納米管 阪崎腸桿菌 快速檢測　出處：《浙江工商大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：阪崎腸桿菌曾名為黃色陰溝腸桿菌,阪崎腸桿菌被認(rèn)為是嚴(yán)重危害特定人群,危害生命或?qū)е侣詫?shí)質(zhì)性后遺癥的致病菌,其致死率為40%-80%。 電化學(xué)免疫傳感器具有高特異性、簡易低廉、易于攜帶等優(yōu)點(diǎn)而被廣泛應(yīng)用于臨床診斷、環(huán)境分析和食品產(chǎn)業(yè)等領(lǐng)域。應(yīng)用新材料,例如多壁碳納米管(multi-wall carbon nanotubes, MWCNTs)和離子液體(i)nic liquid,IL)制作傳感器是當(dāng)前生物傳感器研究領(lǐng)域的熱點(diǎn)。本論文主要圍繞以下幾個(gè)方面開展了研究工作： 1阪崎腸桿菌抗原的制備 本試驗(yàn)成功制備了以0.4%(v/v)甲醛滅活12h的阪崎腸桿菌抗原,此抗原具有良好的生物活性和特異性,為后續(xù)研究工作的順利開展奠定了基礎(chǔ)。 2基于多壁碳納米管/硫堇阪崎腸桿菌免疫傳感器的研究 為了研制一種靈敏度高、電化學(xué)性能穩(wěn)定的免疫傳感器,構(gòu)建準(zhǔn)確檢測阪崎腸桿菌的電化學(xué)方法,將硫堇、辣根過氧化物酶標(biāo)記的阪崎腸桿菌抗體依次自組裝固定于多壁碳納米管/十二烷基苯磺酸鈉修飾的四通道絲網(wǎng)印刷電極上,制得一次性免疫傳感器。采用原子力顯微鏡表征電極修飾和孵育抗原后的表而形態(tài),用循環(huán)伏安法考察不同修飾電極的電化學(xué)特性,根據(jù)還原峰電流的變化對阪崎腸桿菌進(jìn)行測定。在優(yōu)化的試驗(yàn)條件下,該方法檢測阪崎腸桿菌的線性范圍為102～108CFU/mL,檢測限為5.7×101CFU/mL(S/N=3)。該免疫傳感器靈敏度高,具有較好的特異性、重現(xiàn)性(RSD=6.3%)、穩(wěn)定性(4℃無菌容器中放置15d后電流響應(yīng)為初始值的93.24%)和準(zhǔn)確性(與GB/T4789.40-2010符合率為96.67%),具有一定的應(yīng)用潛力。 3基于多壁碳納米管/離子液體/硫堇的阪崎腸桿菌免疫傳感器 首次研發(fā)了基于多壁碳納米管/離子液體/硫堇修飾絲網(wǎng)印刷電極的免疫傳感器,提高了免疫傳感器的貯存穩(wěn)定性。用原子力顯微鏡表征電極各層修飾后的表面形態(tài),用循環(huán)伏安法考察修飾電極的電化學(xué)特性,十二烷基苯磺酸鈉降低了多壁碳納米管/離子液體膠體的背景電流。包埋在離子液體中的阪崎腸桿菌酶標(biāo)抗體表現(xiàn)出較高的活性和穩(wěn)定性((4℃無菌容器中放置30d后電流響應(yīng)為初始值的96.30%)。在最優(yōu)條件下,該方法檢測阪崎腸桿菌的線性范圍為103～109CFU/mL,在磷酸鹽緩沖溶液檢出限為7.7×101CFU/mL(S/N=3),模擬樣品檢出限為8.3×102CFU/mL 4基于MWCNT/Nafion和離子液體[BMIM]PFF6的免疫傳感器研究 為改善基于MWCNT/Nafion生物傳感器電化學(xué)信號及儲存的穩(wěn)定性,采用[BMIM]PF6/Nafion復(fù)合物將辣根過氧化物酶標(biāo)抗體包埋固定于MWCNT/Nafion修飾的絲網(wǎng)印刷碳電極上,構(gòu)建了一種新的免疫傳感器。用原子力顯微鏡表征電極各層修飾后的表面形態(tài),用循環(huán)伏安法和交流阻抗法考察修飾電極的電化學(xué)特性,利用還原峰電流變化對被檢物進(jìn)行測定。本試驗(yàn)選用阪崎腸桿菌為目標(biāo)抗原,在優(yōu)化的檢測條件下,該方法檢測阪崎腸桿菌的線性范圍為103～109CFU/mL,檢出限為3.4×102CFU/mL(S/N=3)。該免疫傳感器的儲存穩(wěn)定性有明顯改善,4℃放置30d后電流響應(yīng)值仍為初始值的95.50%,且具有較高的靈敏度,較好的重現(xiàn)性(RSD6%)、特異性(非目標(biāo)抗原的△Ipc0.3μA)和準(zhǔn)確性(與GB/T4789.40-2010符合率為100%),操作簡便、成本低,有很好的潛在應(yīng)用價(jià)值。
[Abstract]:Sakazakii Enterobacteriaceae, once known as yellow cloaca, Enterobacter sakazakii, is considered to be a serious risk to specific populations, life-threatening or chronic sequelae, and its mortality is 40%-80%..
The electrochemical immunosensor with high specificity, simple and inexpensive, easy to carry advantages and is widely used in clinical diagnosis, environmental analysis and food industry and other fields. The application of new materials, such as multi walled carbon nanotubes (multi-wall carbon, nanotubes, MWCNTs) and ionic liquid (I) nic liquid, IL) is a hotspot in biological sensor the sensor research field. This thesis mainly focuses on the following research work has been carried out:
1 Preparation of Enterobacter sakazakii antigen
In this experiment, we successfully prepared the antigen of Enterobacter sakazakii, which was inactivated by 0.4% (v/v) formaldehyde. The antigen has good biological activity and specificity, which laid the foundation for the subsequent research work to carry out smoothly. The 12h antigen was successfully prepared.
2 based on multi wall carbon nanotube / Enterobacter osakazaki immunosensor
In order to develop a kind of high sensitivity, stable performance of the electrochemical immunosensor, electrochemical method to construct accurate detection of Enterobacter sakazakii, the thionine, Enterobacter sakazakii horseradish peroxidase labeled antibody were fixed on the self-assembled multi wall carbon nanotubes / twelve benzene sulfonic acid sodium channel four modified screen printed electrode, preparation have disposable immunosensor. By using atomic force microscopy characterization of modified electrode and incubated with antigen after surface morphology, electrochemical properties of different modified electrode by cyclic voltammetry, according to the change of the reduction peak current was measured for Enterobacter sakazakii. Under the optimum conditions, the linear range of the method for detection of Enterobacter sakazakii for 102 ~ 108CFU/mL, the detection limit is 5.7 * 101CFU/mL (S/N=3). The immune sensor has high sensitivity and has good specificity and reproducibility (RSD=6.3%), stability (4 C sterile container The current response after 15d is 93.24% of the initial value and the accuracy (the coincidence rate of GB/T4789.40-2010 is 96.67%). It has a certain application potential.
3 immunological sensors based on multi walled carbon nanotubes / ionic liquids / thio
For the first time developed immunosensor of multi walled carbon nanotube / ionic liquid / thionine modified screen printed electrode based on improved storage stability of immunosensor. The surface morphology of each layer were characterized by atomic force modified electrode, electrochemical properties of the modified electrode by cyclic voltammetry, twelve sodium reduced the background current multi wall carbon nanotube / ionic liquid gel. Enterobacter sakazakii enzyme buried in ionic liquid antibody showed higher activity and stability ((placed 4 C 30d sterile container after the current response of the initial value is 96.30%). Under optimal conditions, the linear range for the detection of Enterobacter sakazakii 103 ~ 109CFU/mL in phosphate buffer solution detection limit of 7.7 * 101CFU/mL (S/N=3), the simulated sample detection limit of 8.3 * 102CFU/mL
4 immune sensor based on MWCNT/Nafion and ionic liquid [BMIM]PFF6
In order to improve the stability of electrochemical MWCNT/Nafion biosensor based on signal and storage, using [BMIM]PF6/Nafion compound to HRP screen printed carbon electrode antibody immobilized on the MWCNT/Nafion modified on the construction of a new immune sensor. The surface morphology of the modified electrode layer was characterized by atomic force, electrochemical characteristics on modified electrode by using cyclic voltammetry and AC impedance method, the reduction peak current change of the detected objects were determined. The experiment of Enterobacter sakazakii as target antigen, detected in the optimum conditions, the linear range of the method for detection of Enterobacter sakazakii was 103 ~ 109CFU/mL, the detection limit of 3.4 * 102CFU/mL (S/N=3). Improved the storage stability of the immunosensor, 4 DEG C after placing 30d response is still the initial value of 95.50%, and has high sensitivity, good Reproducibility (RSD6%), specificity (non target antigen Ipc0.3 A (GB/T4789.40-2010) and the accuracy and the coincidence rate is 100%), simple operation, low cost, has good potential application value.

【學(xué)位授予單位】：浙江工商大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1;TP212

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 夏詠梅;吳紅平;張s，

本文編號：1464510