本文關(guān)鍵詞：基于魯米諾及其功能化納米材料的新型電化學(xué)發(fā)光傳感器研究　出處：《西南大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 電化學(xué)發(fā)光傳感器 生物活性分子 魯米諾 聚魯米諾 過(guò)氧化氫

【摘要】：電化學(xué)發(fā)光分析技術(shù)(Electrochemiluminescence,ECL)是在化學(xué)發(fā)光和電化學(xué)基礎(chǔ)上發(fā)展起來(lái)的一種新的分析方法,它是通過(guò)電驅(qū)動(dòng)促使某些物質(zhì)發(fā)生電化學(xué)反應(yīng)形成激發(fā)態(tài)并通過(guò)輻射光子返回基態(tài),并對(duì)發(fā)光強(qiáng)度進(jìn)行測(cè)量的一種分析方法。電化學(xué)發(fā)光分析技術(shù)兼具了化學(xué)發(fā)光和電化學(xué)技術(shù)的一些特點(diǎn),比如靈敏度高、選擇性好、背景信號(hào)低、線性范圍寬,儀器設(shè)備簡(jiǎn)單、易操控。其中以魯米諾為發(fā)光試劑的電化學(xué)發(fā)光傳感器在生物分子的檢測(cè)中表現(xiàn)出巨大的優(yōu)越性,魯米諾的電化學(xué)發(fā)光強(qiáng)度可以被過(guò)氧化氫(H_2O_2)強(qiáng)烈增敏,在生命體系中,多種代謝中間產(chǎn)物在相應(yīng)氧化酶的催化下生成H_2O_2,據(jù)此建立起的基于酶-魯米諾體系的電發(fā)光傳感器可實(shí)現(xiàn)包括葡萄糖、乳酸、膽堿等在內(nèi)的多種活性物質(zhì)的靈敏檢測(cè)。隨著生命科學(xué)的快速發(fā)展,生物活性分子分析逐漸成為生物學(xué)家和分析家的的重要研究?jī)?nèi)容,成為揭開(kāi)生命奧秘、監(jiān)控生命體征、征服疾病的重要手段之一。因此,利用電化學(xué)發(fā)光開(kāi)展對(duì)生物分子的定量分析已然成為研究的熱點(diǎn)。但是,電化學(xué)生物傳感器的蓬勃發(fā)展是近十年的時(shí)間,因此還具有巨大的上升空間。基于魯米諾和酶構(gòu)建的電化學(xué)發(fā)光生物傳感器,其主要構(gòu)建模式目前還比較簡(jiǎn)單,主要通過(guò)一些聚合膜對(duì)酶進(jìn)行簡(jiǎn)單封裝,電阻高,酶活性中心受阻,因而其靈敏度也有待進(jìn)一步提高。同時(shí),由于魯米諾分子是加入到待測(cè)溶液中,也會(huì)對(duì)樣品會(huì)產(chǎn)生一定的干擾和污染,對(duì)活體物質(zhì)可能有毒害作用,操作也相對(duì)復(fù)雜,因此所構(gòu)建的傳感器在使用壽命、穩(wěn)定性、長(zhǎng)效性、靈敏度上均還有待提高。本研究即針對(duì)基于魯米諾電致發(fā)光傳感器的重要性及存在的問(wèn)題,借助納米科技的發(fā)展和應(yīng)用,致力于改善和提高魯米諾電發(fā)光傳感器的性能,一方面開(kāi)發(fā)高效的響應(yīng)界面;另一方面以魯米諾為前驅(qū)體合成魯米諾功能化的電化學(xué)發(fā)光新材料,實(shí)現(xiàn)魯米諾的固定化制備固相電化學(xué)發(fā)光電極,從而對(duì)生物活性分子可以快速、靈敏、無(wú)污染的檢測(cè)。通過(guò)兩方面的改進(jìn),為生物活性分子提供更有效的,充滿前途的分析方法。其主要研究?jī)?nèi)容如下:(1)基于三維響應(yīng)界面的葡萄糖電化學(xué)發(fā)光傳感器構(gòu)建及用于葡萄糖的超靈敏檢測(cè)。葡萄糖是重要的生物活性分子,在近幾年中,采用ECL法構(gòu)建葡萄糖生物傳感器取得了一定進(jìn)展,但是在傳統(tǒng)的葡萄糖ECL傳感器的構(gòu)建當(dāng)中,葡萄糖氧化酶(GOD)主要通過(guò)nafion、殼聚糖等聚合膜封裝,電阻高,酶活性中心被封閉,因此靈敏度并不高。本工作將導(dǎo)電聚合物引入到葡萄糖ECL傳感器的構(gòu)建當(dāng)中,通過(guò)電聚合聚苯胺,在玻碳電極表面形成一層疏松、多孔的三維網(wǎng)狀聚苯胺納米線(PANi),然后制備帶負(fù)電荷的16nm的納米金顆粒,并通過(guò)靜電作用吸附到表面富含氨基的聚苯胺納米線上,然后通過(guò)酶與納米金之間的靜電吸附及共價(jià)鍵合作用將GOD自組裝到納米金,最終構(gòu)建成三維的GOD/Au NPs/PANi響應(yīng)平臺(tái)。由于該結(jié)構(gòu)疏松多孔、導(dǎo)電性好、比表面積高,GOD負(fù)載量大,因此在以魯米諾為發(fā)光試劑進(jìn)行葡萄糖的ECL檢測(cè)時(shí),表現(xiàn)出超靈敏的響應(yīng),其線性范圍為0.1-100μM,檢測(cè)限0.05μM(S/N=3)。通過(guò)比較前人的工作,本實(shí)驗(yàn)所設(shè)計(jì)的電化學(xué)發(fā)光傳感器具有更寬的線性范圍和更低的檢測(cè)限。在對(duì)血清樣品中的葡萄糖進(jìn)行檢測(cè)時(shí),也表現(xiàn)出良好的選擇性、穩(wěn)定性和靈敏度。(2)新型電化學(xué)發(fā)光材料魯米諾還原納米金/還原氧化石墨烯復(fù)合物的合成及用于細(xì)胞釋放H_2O_2的檢測(cè)。在傳統(tǒng)的魯米諾電化學(xué)發(fā)光傳感器構(gòu)建當(dāng)中,魯米諾作為發(fā)光試劑是加入到待測(cè)溶液中,模式雖然簡(jiǎn)單,但是魯米諾會(huì)對(duì)樣品、電極帶來(lái)一定的污染和干擾,對(duì)活體待測(cè)物可能有毒害,同時(shí)也會(huì)增加操作步驟,增大誤差。因此實(shí)現(xiàn)魯米諾在電極上的固定,進(jìn)行無(wú)試劑的檢測(cè),是該類(lèi)傳感器構(gòu)建的重要發(fā)展目標(biāo)。石墨烯具有眾多非比尋常的物理、化學(xué)、光學(xué)性質(zhì),在本實(shí)驗(yàn)中,我們采用魯米諾還原氯金酸的辦法,在水熱條件下制備出魯米諾修飾的納米金粒子,并在制備的過(guò)程中,以片狀的還原氧化石墨烯為載體,一步合成出具有優(yōu)秀電化學(xué)發(fā)光特性的魯米諾還原納米金/還原氧化石墨烯復(fù)合物(Lu-Au NPs/r GO),用以制備性能優(yōu)良的固相電化學(xué)發(fā)光傳感器。過(guò)氧化氫(H_2O_2)是細(xì)胞中重要的生物活性小分子之一,對(duì)其進(jìn)行活細(xì)胞的實(shí)時(shí)檢測(cè)具有非常重要的意義。我們進(jìn)一步利用合成的新材料、二甲基硅氧烷以及氧化銦錫玻璃構(gòu)建出即可用于直接培養(yǎng)細(xì)胞,又能夠進(jìn)行原位檢測(cè)細(xì)胞釋放H_2O_2的電化學(xué)發(fā)光傳感器,實(shí)現(xiàn)了對(duì)人肝癌細(xì)胞Hep G2所釋放的H_2O_2的定量檢測(cè)。由于細(xì)胞與電極響應(yīng)界面的直接接觸以及電化學(xué)發(fā)光自身的高靈敏響應(yīng)特性,該傳感器對(duì)細(xì)胞釋放H_2O_2的檢測(cè)取得良好效果,而且發(fā)光試劑的固定也避免了魯米諾分子在溶液中對(duì)細(xì)胞的影響。(3)爆米花狀聚魯米諾包裹納米金復(fù)合物的可控合成及用于尿酸的固相電化學(xué)發(fā)光檢測(cè)。雖然魯米諾分子固定化前人做了一定的工作,包括本人合成高效的Lu-Au NPs/r GO材料,但是這些材料在長(zhǎng)效性和穩(wěn)定性上還有待提高,因?yàn)閺谋举|(zhì)上這些方法均是魯米諾分子在納米材料上的吸附,魯米諾分子有限導(dǎo)致在使用幾次后信號(hào)發(fā)生衰減。因此尋找發(fā)光更持久、穩(wěn)定的魯米諾納米材料非常具有吸引力,聚魯米諾是有效的選擇之一。在本實(shí)驗(yàn)中,我們采用水熱法,通過(guò)調(diào)節(jié)魯米諾與氯金酸的反應(yīng)比例,合成出聚魯米諾包裹納米金的核殼結(jié)構(gòu)納米復(fù)合材料(Au@Polyluminol),該復(fù)合物呈爆米花狀,形狀均一,分散度好,直徑在200nm左右。由于聚魯米諾聚合了大量的魯米諾分子,因此比魯米諾吸附的材料具有更穩(wěn)定和持久的發(fā)光性能,而且由于納米金增強(qiáng)電化學(xué)發(fā)光的作用,該材料對(duì)H_2O_2表現(xiàn)出高靈敏的ECL響應(yīng)。我們將該材料與尿酸酶結(jié)合制備用于尿酸檢測(cè)的固相電化學(xué)發(fā)光傳感器,結(jié)果對(duì)尿酸檢測(cè)表現(xiàn)出良好的線性范圍、檢測(cè)限、穩(wěn)定性以及選擇性。可以預(yù)見(jiàn)該復(fù)合物作為一種新的電化學(xué)發(fā)光材料在構(gòu)建其他ECL傳感器方面也將會(huì)發(fā)揮巨大作用。(4)紅毛丹果狀葡萄糖氧化酶/聚魯米諾/納米金復(fù)合物的合成及用于葡萄糖檢測(cè)。在以聚魯米諾包裹納米金為電化學(xué)發(fā)光材料構(gòu)建酶生物傳感器的過(guò)程中,酶與材料之間的比例需要優(yōu)化和調(diào)節(jié),增加了傳感器制備的過(guò)程,也加大了檢測(cè)誤差,如果能夠制備出即含有活性酶,又具有電化學(xué)發(fā)光性能的雙功能材料,那么將能夠直接實(shí)現(xiàn)各種生物分子的檢測(cè),簡(jiǎn)化傳感器制作步驟,增加檢測(cè)的穩(wěn)定性和重復(fù)性,具有非常大的應(yīng)用價(jià)值。在本實(shí)驗(yàn)中,我們發(fā)現(xiàn)即使在常溫下,只要有足夠的時(shí)間,氯金酸也可緩慢氧化魯米諾形成聚魯米諾包裹的納米金粒子。當(dāng)加入適量的葡萄糖氧化酶后,不但沒(méi)有阻止氧化的進(jìn)行,反而在酶的調(diào)節(jié)下生成紅毛丹果狀、形狀均一、分散度好、直徑400nm左右的納米絨球聚合物,經(jīng)成分分析其組成為葡萄糖氧化酶/聚魯米諾/納米金(Au/PLUM/GOD)。由于在常溫下合成,酶仍然保持其活性,基于該復(fù)合物構(gòu)建的生物傳感器后對(duì)葡萄糖表現(xiàn)出顯著的電化學(xué)發(fā)光響應(yīng),并具有較好的靈敏度和很寬的線性范圍,在葡萄糖檢測(cè)上具有很大的利用價(jià)值。
[Abstract]:Electrochemiluminescence Technology (Electrochemiluminescence, ECL) is a new analysis method is developed based on Cl and electrochemistry, it is through the electric drive prompted some substances formed by electrochemical reaction by radiation photon excited state and return to the ground state, and an analysis method for measuring the luminous intensity of electrochemiluminescence technique with some characteristics of chemiluminescence and electrochemical techniques, such as high sensitivity, good selectivity, low background signal, wide linear range, simple equipment, easy operation. The ECL reagent for luminol chemiluminescence sensor shows great advantages in the detection of biological molecules, the electrochemical luminescence intensity can be Lumino hydrogen peroxide (H_2O_2) strong sensitization, in life system, a variety of metabolic intermediates in the catalytic formation of H_2O_2 oxidase under the corresponding, According to the established El Lumino system based on enzyme sensor can be realized including glucose, lactic acid, choline, sensitive detection of various active substances. With the rapid development of life science, molecular biological activity analysis has gradually become an important research content of biologists and analysts, is to open the mystery of life, monitoring of vital signs, one of the most important means to conquer disease. Therefore, to carry out the quantitative analysis by using electrochemiluminescence of biological molecules have become a hot research topic. However, the vigorous development of electrochemical biosensor is nearly ten years, it also has a huge rise in space. Lumino and the construction of electrochemical enzyme biosensor based on light, its main mode is simple, mainly through some simple encapsulation of polymeric membrane, enzyme resistance of a high center of enzyme activity is blocked, because of its sensitivity Also needs to be further improved. At the same time, because the Lumino elements are added to the test solution, will also have certain interference and pollution to the sample, may have toxic effects on living matter, the operation is relatively complicated, therefore the service life of the sensor, long-term stability, sensitivity, both need to be improved. This study focuses on the importance of Lumino electric luminescence sensor and based on the existing problems, with the development and application of nano science and technology, is committed to improve and enhance the performance of Lumino electric light sensor, a high efficiency response interface development; on the other hand, the electrochemical synthesis of precursor Lumino functional luminescent new material by Lumino. The immobilized system of Lumino using solid state electrochemiluminescence, and biological activity of molecules can be rapid, sensitive detection, no pollution. The improvement of two aspects, for the students Active molecules provide more effective and promising analysis method. The main research contents are as follows: (1) ultra sensitive detection of glucose electrochemical luminescence sensor response three-dimensional interface construction and for glucose based on glucose is the important bioactive molecule, in recent years, using the ECL method to construct glucose biosensor has been some progress, but in the construction of the traditional ECL sensor of glucose, glucose oxidase (GOD) by Nafion, chitosan polymer film packaging, high resistance, enzyme active center was closed, so the sensitivity is not high. The conductive polymer is introduced to construct ECL glucose sensor, formed by electropolymerization of polyaniline. A layer of loose on the surface of glassy carbon electrode, three-dimensional porous polyaniline nanowires (PANi), gold nanoparticles and preparation of negatively charged 16nm, and through the static Electric adsorption to polyaniline nano line surface rich in amino, followed by electrostatic adsorption and covalent bond cooperation between enzyme and gold nanoparticles with GOD self-assembled onto gold nanoparticles, to build up the 3D GOD/Au NPs/PANi response platform. Due to the porous structure, good conductivity, high surface area, large load capacity GOD therefore, the ECL for the detection of glucose reagent in response to Lumino, showed a super sensitive, the linear range is 0.1-100 ~ M, the detection limit of 0.05 M (S/N=3). Through the comparison of previous work, detection of electrical chemiluminescence sensor designed in this experiment has a wider linear range and lower limit in the detection of glucose in blood serum samples, also exhibited good selectivity, stability and sensitivity. (2) the new type of electrochemical luminescence materials Lumino synthesis of reduced gold nanoparticles / graphene Composites And for the detection of cells to release H_2O_2. Lumino in the traditional electrochemical sensor light construction, Lumino as a light reagent is added to the test solution, although the mode is simple, but Lumino will bring some of the sample, the electrode pollution and interference on the in vivo analyte may be toxic, will also increase the operation steps. Increase the error. So the realization of Lumino on the electrode fixed, no detection reagent, is an important goal of this kind of sensor construction. Physics, graphene has many unusual chemical and optical properties, in this experiment, we use the Lumino reduction of HAuCl4 to prepare gold nanoparticles Lumino modified under hydrothermal conditions and in the preparation process, the reduction of graphene oxide sheets as the carrier, one step synthesis has excellent electrochemical luminescence properties of Lumino Na reduction Nano / reduced graphene oxide (Lu-Au NPs/r GO), with excellent performance by using solid state electrochemical luminescent sensor. Hydrogen peroxide (H_2O_2) is one of the important biological activity of small molecules in the cell, real-time detection of living cells on it has very important significance. We further use of new synthetic materials. Two methyl siloxane and indium tin oxide glass can be used to construct directly cultured cells, but also be able to detect in situ electrochemical cells release H_2O_2 luminescence sensor, realizes the quantitative detection of the release of human hepatocellular carcinoma cell line Hep G2 H_2O_2. Due to the direct contact with the electrode cell response interface and ECL its high sensitive response characteristics detection of the sensor on the release of H_2O_2 cells achieved good results, but also avoid the fixed reagent Lumino molecule in solution on the cell. Ring. (3) for solid phase controlled synthesis and electrochemical luminescence detection of uric acid popcorn poly Lumino wrapped gold nanocomposites. Although Lumino molecules immobilized predecessors have done some work, including the Lu-Au NPs/r GO I synthesis of highly efficient materials, but these materials in the long-term and stability need to be improved, because from essentially, these methods are Lumino adsorption molecules in nano materials, Lumino molecular Co. resulted in the use of several times after the signal attenuation. So looking for more durable luminescent nano materials, Lumino stability is very attractive, poly Lumino is one of the effective options. In this experiment, we used the hydrothermal method. Lumino and the proportion of adjusting the reaction of chloroauric acid, synthesis of core-shell structure nano composite material poly Lumino wrapped gold nanoparticles (Au@Polyluminol), the compound is popcorn shaped shape. The shape of uniform, good dispersion degree, the diameter of around 200nm. The Lumino Lumino molecular mass poly polymerization, so the adsorption material than Lumino emission is more stable and persistent, and because the effect of gold nanoparticles enhanced electrochemical luminescence, the material of H_2O_2 exhibits a high sensitive ECL response. We use the materials and uricase combined with the preparation for the electrochemical detection of uric acid solid luminescence sensor, the determination of uric acid showed a good linear range, detection limit, stability and selectivity. The composite can be predicted as a new electrochemical luminescent materials in the construction of other ECL sensors will also play a significant role. (4) the fruiting of glucose oxidase Lumino / / poly rambutan complexes and synthesis of gold nanoparticles for glucose detection. On poly Lumino wrapped gold nanoparticles as electrochemical luminescence materials to construct enzyme The sensor in the process between the enzyme and the proportion of materials need to be optimized and adjusted, increasing the process of sensor preparation, but also increased the detection error, if can be prepared with enzyme activity, and has the dual function materials of electrochemiluminescence detection, then it will be able to directly realize all kinds of biological molecules, simplified sensor the production steps, increase the detection stability and repeatability, has great application value. In this experiment, we found that even at room temperature, as long as there is enough time, chloroauric acid can also slow oxidation of luminol formation of gold nanoparticles Polyluminol package. When the adding amount of glucose oxidase, not only did not stop oxidation, but produced rambutan, in the regulation of the enzyme under the uniform shape, good dispersion, nano polymer ball diameter of about 400nm, the composition is composed of glucose and oxygen Enzyme / Polyluminol / gold nanoparticles (Au/PLUM/GOD). The synthesis at room temperature, the enzyme still maintain its biological activity, the compound sensor constructed of glucose showed a significant response based on electrochemiluminescence, and has good sensitivity and linear range is very wide, has great application value in the detection of glucose..

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O657.3;TP212

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