發(fā)布時(shí)間：2018-01-07 19:34

  本文關(guān)鍵詞：唾液sIgA納米免疫生物傳感器的實(shí)驗(yàn)研究　出處：《重慶醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 分泌性免疫球蛋白A 甘氨酸 多壁碳納米管 層層自組裝 免疫生物傳感器

【摘要】：唾液是人體重要體液之一,其成分相對(duì)穩(wěn)定,人類唾液蛋白質(zhì)組圖發(fā)現(xiàn)唾液中有20%的蛋白質(zhì)與血液中所含蛋白質(zhì)相同。同時(shí)唾液也受全身各種因素和狀態(tài)的影響,能夠很好反映血液中各種蛋白質(zhì)水平的變化,可作為疾病診斷、療效判斷、藥物監(jiān)測(cè)的參考指標(biāo),在疾病診治中具有很好的應(yīng)用前景。而且與血清標(biāo)本相比,唾液采集無(wú)創(chuàng)傷,安全方便,無(wú)血源性疾病傳染的危險(xiǎn),因此唾液診斷方法是當(dāng)前檢測(cè)方法的一個(gè)補(bǔ)充。但由于很多成分在唾液中的濃度遠(yuǎn)低于血液,以往的技術(shù)難以將其檢測(cè)出來(lái)。隨著生物傳感技術(shù)迅速發(fā)展,電化學(xué)免疫傳感技術(shù)因其方法簡(jiǎn)單、靈敏度高、選擇性強(qiáng)等優(yōu)點(diǎn)而被廣泛應(yīng)用于微量物質(zhì)的測(cè)定。 電化學(xué)免疫傳感技術(shù)的基礎(chǔ)是氧化還原活性中心與電極間的電子傳遞。它可以通過(guò)兩種不同的途徑實(shí)現(xiàn),一方面活性中心與電極間的電子傳遞的介質(zhì)非常重要;另一方面,生物活性分子的固定化對(duì)構(gòu)筑生物傳感器作用重大,這是影響生物傳感器的穩(wěn)定性、靈敏度和選擇性的兩個(gè)關(guān)鍵因素。 本文選擇唾液中高豐度蛋白質(zhì)sIgA作為標(biāo)志物,利用比表面積大、表面活性高的納米材料和電子介體硫堇層層組裝,通過(guò)靜電吸附和金-氮(Au-N)、金-硫(Au-S)共價(jià)鍵作用實(shí)現(xiàn)了電子介體的有效固定,從而構(gòu)建了性能優(yōu)良的介體型唾液免疫傳感器。同時(shí),還對(duì)Nafion與納米金、碳納米管與納米金結(jié)合作為固定基質(zhì)在唾液免疫傳感器領(lǐng)域的應(yīng)用做了探索和研究。具體研究?jī)?nèi)容如下: 1.基于金納米粒子/硫堇/Nafion膜修飾的可再生唾液sIgA免疫傳感器的研制 通過(guò)層層自組裝將硫堇(ThI)和納米金(GNPs)修飾到Nafion修飾的玻碳(GCE)電極表面,利用納米金單層吸附唾液分泌性免疫球蛋白A(sIgA),最后用辣根過(guò)氧化物酶(HRP)封閉電極上的非特異性吸附位點(diǎn),構(gòu)建了一種檢測(cè)唾液sIgA的新型電流型納米免疫生物傳感器。該生物傳感器靈敏度高、特異性好、測(cè)試方便,檢測(cè)線性范圍為6.5-300 ng/mL,檢出限為3.0 ng/mL;電流值達(dá)到95%穩(wěn)態(tài)時(shí)間小于20 s。探討了抗體及底物濃度、pH值和溫度、孵育時(shí)間、干擾物對(duì)傳感器的影響。該傳感器與ELISA法相關(guān)性良好(r=0.98932,P0.001),可用于唾液sIgA的快速檢測(cè),從而判斷人體局部免疫狀況。 2.基于功能化碳納米管/聚硫堇/金納米粒子層層自組裝修飾的可再生唾液sIgA免疫傳感器的研制 將殼聚糖(CHI)分散的羧基化多壁碳納米管(MWNTs)吸附到GCE表面,形成負(fù)電荷界面,利用靜電吸附和金-氮(Au-N)、金-硫(Au-S)共價(jià)鍵作用將陽(yáng)離子電子媒介體THI和GNPs層層自組裝到電極表面,通過(guò)GNPs單層吸附唾液sIgA,最后用HRP封閉電極上的非特異性吸附位點(diǎn),同時(shí)放大電流信號(hào),構(gòu)建了一種檢測(cè)唾液sIgA的新型納米免疫生物傳感器。實(shí)驗(yàn)結(jié)果表明,該傳感器具有良好的準(zhǔn)確性和穩(wěn)定性;檢測(cè)線性范圍為3.0-350 ng/ml,r=0.98967(P0.001);檢出限為1.5 ng/mL(S/N=3);電流值達(dá)到95%穩(wěn)態(tài)時(shí)間小于20 s;可用0.1 mol/L的甘氨酸再生8次。探討了底物濃度、抗體濃度、孵育時(shí)間、溫度和pH值及干擾物質(zhì)對(duì)該傳感器的影響。該免疫傳感器靈敏度高、特異性好、方便可再生,可用于唾液sIgA的快速檢測(cè),從而判斷人體局部免疫狀況。
[Abstract]:Saliva is one of the important components of the human body, relatively stable, human salivary proteins found in saliva protein and photos of 20% blood contained in the same protein. At the same time saliva is also affected by the various factors and systemic state, can well reflect the changes of protein levels in the blood, as disease diagnosis, curative effect evaluation, reference drug monitoring index, has a good application prospect in the diagnosis of disease. But compared with the serum samples of saliva collection without trauma, safe and convenient, without the risk of blood borne diseases infection, so saliva diagnosis method is a supplement to the current detection method. But because many of the components of concentration in saliva is far lower than that of blood in the past, the technology is difficult to be detected. With the rapid development of bio sensing technology, electrochemical immunosensor technology because of its simplicity, high sensitivity, adaptability and selection It is widely used for the determination of trace substances.
The basis of electrochemical immunosensors is redox active center and electron transfer between the electrode. It can be achieved through two different pathways, one electron transfer between electrode and the active center of the medium is very important; on the other hand, the immobilization of bioactive molecules on the construction of major biological sensor, which is affecting the stability biosensor, two key factors of sensitivity and selectivity.
The high abundance of protein sIgA in saliva as a marker, with large surface area, nano materials and electron mediator thionine high surface activity layer by layer, through electrostatic adsorption and gold - nitrogen (Au-N), gold sulfur (Au-S) covalent interaction to achieve the electronic mediator effectively fixed, so as to construct the excellent performance of the medium saliva immune sensor. At the same time, the Nafion and gold nanoparticles, carbon nanotubes and nano gold as a combination of application in the field of saliva immune sensor immobilization matrix makes the exploration and research. The main research contents are as follows:
1. development of a renewable saliva sIgA immunosensor based on gold nanoparticles / thio /Nafion membrane
Through the layers of self-assembly of Thionine (ThI) and gold nanoparticles (GNPs) modified glassy carbon electrode modified with Nafion (GCE) surface by using gold nanoparticle monolayer adsorption of salivary secretory immunoglobulin A (sIgA), finally using horseradish peroxidase (HRP) blocking non-specific adsorption sites on the electrode, construct a new current type nano immunosensor for detection of salivary sIgA. The biosensor has high sensitivity, good specificity, convenient testing, detection of the linear range of 6.5-300 ng/mL, the detection limit was 3 ng/mL; the current value reached 95% steady state time is less than 20 s. of antibody and substrate concentration, pH value and temperature, incubation time effect, disruptors on the sensor. The sensor and ELISA good correlation (r=0.98932, P0.001), can be used for rapid detection of sIgA in saliva, so as to determine the human body local immune status.
2. development of sIgA immunosensor based on functional carbon nanotube / polythio / gold nanoparticle self redecorating and decorating regenerative saliva
The chitosan (CHI) dispersed carboxylated multiwalled carbon nanotubes (MWNTs) adsorbed on the surface of GCE, forming a negative charge interface, using electrostatic adsorption and gold - nitrogen (Au-N), gold sulfur (Au-S) covalent interaction of cationic mediator THI and GNPs self-assembly on the electrode surface by GNPs monolayer adsorbed salivary sIgA, finally used HRP to block the non-specific adsorption site on the electrode, and the amplified current signals, constructed a sialic sIgA nano immunosensor. The experimental results show that the sensor has good accuracy and stability; the linear range of detection for 3.0-350 ng/ml r=0.98967 (P0.001); detection the limit is 1.5 ng/mL (S/N=3); current value reached 95% steady state time is less than 20 s; available 0.1 mol/L glycine 8 times of regeneration were discussed. The concentration of substrate, antibody concentration, incubation time, temperature and pH value of the sensor and interfering substances on the. The immunosensor has high sensitivity, good specificity, convenient and renewable, and can be used for rapid detection of saliva sIgA, so as to judge the local immune status of the human body.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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