纳米金属氧化物催化鲁米诺化学发光新体系的研究及其应用
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摘要
纳米材料是指在三维空间中至少有一维在0.1-100nm范围内的超微细颗粒材料。材料在纳米尺度下往往能够表现出一些独特的效应,包括表面效应、量子尺寸效应、小尺寸效应、宏观量子隧道效应、介电效应等。这些效应使得纳米材料拥有许多奇特的物理和化学性质,具有常规材料所不具备的潜在应用价值。纳米金属氧化物材料是近年来研究的热点,其在光、电、力、磁、声等领域有着广泛的应用和潜在的价值。
化学发光方法因其不需要光源,仪器设备简单,线性范围宽,灵敏度高,易于微型化等优点,已成为最有发展前景的分析方法之一。近年来,贵金属纳米材料的出现拓展了化学发光的研究应用范围,但是关于金属氧化物纳米材料的化学发光催化行为研究的很少。在本研究工作中,我们系统地研究了多种纳米金属氧化材料的合成及其表征,发现它们对鲁米诺-过氧化氢化学发光反应的催化性能,特别是在低鲁米诺和低过氧化氢浓度下的高灵敏和高选择催化作用；实现了催化化学发光高通量现场检测TATP爆炸物,建立了一种新的化学发光法测定人红血球降解过氧化氢速率及超灵敏化学发光方法测定呼吸道疾病病人呼出冷凝物中的氧化应激生物标志物过氧化氢。论文还研究了介孔金属氧化物纳米粒子的双功能探针及传感器高通量快速测定多种生化物质。
本论文首先对金属氧化纳米材料的研究及其在分析化学中的应用作了评述；研究报告分为两部分：
一、金属氧化物纳米材料催化鲁米诺-过氧化氢化学发光新体系的研究及其应用
研究发现金属氧化物纳米材料(Co3O4, Cr2O3, a-Fe2O3, CeO2, NiO, CuO, Y2O3, Nd2O3, Mn2O3)对鲁米诺-过氧化氢化学发光反应都具有强的催化性能,而且远超过常用的金属离子催化剂、过氧化物酶和其他金属纳米粒子的催化性能。基于此建立了一系列测定过氧化物的新化学发光体系,提高了方法的选择性。
(1)金属氧化物纳米粒子催化化学发光高通量现场检测TATP爆炸物
三过氧化三丙酮(Triacetone Triperoxide, TATP)是一种过氧化物型爆炸物,在爆炸中,TATP的每个分子迅速释放出气态4个分子,产生比周围空气高出几百倍的气压,威力与TNT相当。TATP是一种极为敏感的爆炸物,摩擦或敲击就可能引发爆炸,不需要任何起爆装置。TATP可以溶解在很多种溶剂中来增强稳定性,如使用普通的发胶、洗发液、沐浴乳或其他液体和凝胶。由于TATP分子不含硝基,机场传统的安检方法,包括金属探测器,X光机及硝基类爆炸物检查行李的方法都不能检测出TATP。
我们发现Ce02纳米粒子对鲁米诺-过氧化氢化学发光体系的优秀的催化能力,合成了一种在聚苯乙烯上有极强吸附力的Ce02纳米颗粒,在聚苯乙烯96孔板上组装成传感膜。由于TATP有较高的蒸汽压(8×10-2Torr,25℃;而TNT为6×10-6Torr,25℃),即使包装严密,也会在携带物上(皮革、麻布、塑料和金属等)带来痕迹。我们用一小块医用棉球擦拭样品表面,然后将棉球装入10mL带盖的玻璃小瓶中,加入5mL水解液进行提取。依次移取50μL提取液于96微孔板中,放置于Biotech Gen 5微孔板化学发光分析仪器中,依次向各孔自动注入50μL5×10-5M鲁米诺进行定量。我们成功应用于模拟机场现场的安全检查,完全满足机场现场检测的需要。
论文中还合成了一种粒径约20nm的三氧化二铬纳米粒子,研究了其对鲁米诺-过氧化氢化学发光反应的催化机理,也用于TATP的现场检测,与其它测定TATP和H202的方法比较,此法最大的特点之一是有机过氧化物如过氧化氢脲、过氧化氢叔丁醇、过氧化氢异丙苯等均不干扰测定。
(2)四氧化三钴纳米粒子催化鲁米诺-过氧化氢化学发光新体系测定呼出冷凝物中的过氧化氢
系统研究了四氧化三钴纳米颗粒催化鲁米诺-过氧化氢化学发光新体系。比较了Co3O4, NiO, a-Fe2O3及其他普通催化剂的催化性能,四氧化三钴化学发光体系具有高的选择性和灵敏度。在优化条件下,化学发光强度与过氧化氢浓度在1.0×10-9-1.0×10-6M范围内呈良好的线性关系,方法的检出限是3.0×10-10M(3σ)。结合流动注射技术建立了测定呼出冷凝物中的过氧化氢的的化学发光新方法。该测定方法具有灵敏度高,选择性好,简单可行的优点。该方法已成功应用于对呼吸道感染人群和健康人群呼出冷凝物中痕量氧化应激生物标志物过氧化氢的直接测定。实验结果表明两者具有显著性差异。
(3)二氧化铈纳米颗粒催化鲁米诺-过氧化氢化学发光新体系研究测定红血球降解过氧化氢速率
纳米氧化物表面缺陷在催化反应中起到关键作用。二氧化铈表面存在氧空位,具有优异的储氧能力,可应用于氧化还原反应。通过调节制备方法,亲水性二氧化铈纳米粒子也能极大地增敏鲁米诺-过氧化氢化学发光。过氧化氢是在活细胞中的正常代谢物,细胞内多余的过氧化氢会氧化细胞内的组分,控制细胞内的过氧化氢水平很重要。我们结合化学发光微阵列法建立了一种新的化学发光法测定人体血液中红血球降解过氧化氢速率。
二、纳米金属氧化物双功能探针及传感器高通量快速测定生化物质
介孔材料是孔径在2nm到50nm之间的一类材料。作为介孔材料的一种,介孔金属氧化物纳米粒子将介孔材料与纳米材料结合起来。介孔金属氧化物纳米粒子具有良好的单分散性、孔道规则、孔径可调、比表面积大和热稳定性好等特性。超过1000m2.g-1表面积及开孔结构的介孔材料可以给目标物-受体之间提供大量供位点和足够的界面以帮助吸附目标物,从而增强本体浓度。因而适合用做装载或固定各种物质,比如药物、酶和抗体。我们的研究发现这些介孔纳米金属氧化物材料本身又对某些化学发光反应具有很强的催化作用,因而设计的固定酶的介孔金属氧化物纳米粒子所构建的化学发光传感器,将具有双功能探针的特性,具此实现了多种生化物质高通量的快速测定。
(1)介孔四氧化三钴双功能探针高通量测定血清中的葡萄糖
基于Co3O4-SiO2介孔复合纳米材料的催化和固定酶的作用,建立了一种新的双功能化学光探针阵列。该化学发光探针阵列由Co3O4-SiO2介孔复合材料和固定的酶组成。Co3O4-SiO2介孔复合材料不仅做酶的载体而且对化学发光体系具有催化作用。这种新的化学发光阵列应用到血液中葡萄糖的检测。测量的葡萄糖线性范围是3-90μM。检出限是0.36μM。
(2)双酶共固定介孔四氧化三钴双功能探针高通量测定牛奶中乳糖
在本研究工作中,我们进一步探究介孔四氧化三钴双功能探针。将葡萄糖氧化酶和β-半乳糖苷酶同时固定在载体介孔四氧化三钴上。用于测定牛奶中乳糖的含量。测量的乳糖线性范围是3.0×10-7到1.0×10-5g.mL-1。检出限是6.9×10-8g.mL-1。
(3)介孔四氧化三钴纳米传感器耦合介孔二氧化硅纳米粒子固定化酶柱高通量测定血清中的葡萄糖
研究将氨基化介孔二氧化硅纳米粒子与海藻酸钙纤维相结合作为载体,将纳米介孔二氧化硅(AMNMS)对酶的吸附作用和催化增强作用与海藻酸钙凝胶对酶的笼蔽效应相结合可以有效的提高固定化酶的催化能力和酶的稳定性。用葡萄糖氧化酶(pI=4.0)为模板制备了海藻酸钙纤维-氨基化介孔二氧化硅(CAF-AMNMS)为载体的微型酶反应器,并将该酶反应器同鲁米诺-H2O2-Co3O4-SiO2介孔催化化学发光体系相耦合,设计了一种阵列传感器,高通量直接测定血清中葡萄糖。
Nanomaterialis between0.1and100nrn in size. Nanomaterials have unique physical and chemical properties due to quantum confinement and exceedingly large surface to volume ratio. They have a superior performance to the materials now existing. Metal oxide nanomaterials have been a hot topic in recent years, which have extensive applications and potential value in optics, electrics, mechanics, and magnetics,
Chemiluminescence (CL) is known as a powerful analytical technique that promises high sensitivity, wide linear range and simple instrumentation. The emergence of noble metal nanoparticles has expanded the application range of CL studies. However, there are only a few reports on the CL of metal oxide nanomaterials. In our work, we systematically studied a variety of metal oxide nanoparticles, and we found that they exhibit high specific catalytic effects on the CL reaction of the luminol-H2O2system in alkaline solution compared with other common catalysts. In this system, luminol with very low concentration can get excellent experimental result with higher selectivity than other luminol system. We have developed a novel CL sensor array to determine Triacetone Triperoxid (TATP) in the suspected items. We have developed a novel CL method to determine the removal rate constant of H2O2by human erythrocytes. We have developed a novel CL approch to determine trace quantities of H2O2in exhaled breath condensate (EBC) where it is a mediator of oxidative stress and a promising biomarker for diagnosing. We also have developed bifunctional CL probe arrays and sensors to determine a variety of biochemical substances, which were based on mesoporous nanocomposite material.
The application of metal oxide nanomaterials in analysis is summarized in Chapter
1. The research work of the dissertation is made up of two sections:
(I) The study of metal oxide nanomaterials used as catalyter in luminol-H2O2CL system and its application
Metal oxide nanomaterials (Co3O4, Cr2O3, CeO2, NiO, a-Fe2O3,CuO, Y2O3, Nd2O3, possess more excellent catalysis in H2O2CL system than previously reported (such as Co2+, Cu2+, Cr3+, HRP and so on). The selectivity CL reaction of luminol-H2O2metal oxide nanomaterials has been proposed.
(1) New CL techniques based on metal oxide nanomaterials catalysis for high-throughput determination of the explosive Triacetone Triperoxide at the scene
TATP is a new organic peroxide based explosive, which decomposes via an entropically favorable mechanism to form4gas phase molecules (a10g sample gave250cm3expansion in the trauzl test as compared to300cm3for TNT). TATP is a highly unstable compound. Explosions may occur by friction, impact or temperature changes, especially in solid phase samples. TATP can be dissolved in a variety of solvents to enhance the stability, such as using an ordinary hair gel, shampoo, shower gel, or other liquids and gels. Metal detector, X-ray baggage scanner, and many detection devices that readily detect tradition explosives made of organic nitro and nitrate compounds fail to detect TATP.
Here we report on the finding that CeO2nanoparticles are capable of strongly enhancing the CL of the luminol-H2O2system. CeO2nanoparticles can be firmly adsorbed on the96-well plates made of polystyrene to form a very solid sensing membrane. The vapor pressure of TATP is8×10-2Torr at25℃. Its vapor can be absorbed onto suitcases and rapidly hydrolyze into H2O2in wet air. Soaked swabs were used to sample the suspected items (pen, plastic bag, banknotes, paper, card), which may be possible carried on an airplane. Immediately after collection, samples were transferred to10mL centrifuge tubes with5mL ultrapure water, which were then sealed for storage.50μL sample solution and50μL (5×10-5M) luminol were injected into the CL sensor array and readings were acquired. The integrated kinetic curve for each sensor can be obtained within5s. The process is easy to perform. It has been successfully applied to the detection of TATP in the suspected items, which may be possible carried on an airplane.
In this paper, we also synthesized Cr2O3nanoparticles (about20nm). Cr2O3nanoparticles were found to greatly enhance the CL intensity of luminol-H2O2system. A reaction mechanism was discussed. The novel CL method also has been successfully applied to the detection of TATP at the scene. Especially, some organic hydroperoxides such as carbamide peroxide, tert-butyl hydroperoxide and cumene hydroperoxide do not interfere with the determination.
(2) CL assay for hydrogen peroxide in exhaled breath condensate using Co3O4 nanoparticle-based catalysis
We systematically studied the nanoparticles-luminol-H2O2CL system. The catalytic activity of three nanoparticles (Co3O4, NiO, a-Fe2O3,) were investigated using microarray method. The CL method based on the use of the Co3O4nanoparticles is ultrasensitive and particularly selective. Under the optimum conditions described above, the linear calibration range prolonged over3orders of magnitude from1.0nm to1000nM. The limit of detection (LOD,3σ) for hydrogen peroxide was0.3n M. A new CL method for the determination of H2O2in exhaled breath condensate (EBC) was built by combining with flow injection technology. The method was successfully applied to the determination of trace quantities of H2O2in EBC where it is a mediator of oxidative stress and a promising biomarker for diagnosing (healthy subjects, rheum subjects, and feverish subjects). Our data suggested that the average H2O2concentration of EBC from feverish subjects was significantly higher than healthy subjects and rheumatic subjects.
(3) A CL microarray based on catalysis by CeO2nanoparticles, and its application to determine the rate of removal of hydrogen peroxide by human erythrocytes
It is important that nano-oxide surface defects in the catalytic reactions. CeO2nanoparticles vacancies present on the surface. It has excellent oxygen storage capacity. Hydrophilic CeO2nanoparticles were also shown highly active and selective for the luminol-H2O2system. H2O2is a normal metabolite in living cells. It is important for cells to remove hydrogen peroxide due to superfluous H2O2may oxidize cellular components. Tight control of cellular H2O2levels is important to avoid oxidative damage of cellular components and to maintain the regulation of various cell responses. The microarray CL method based on the use of the CeO2nanoparticles is ultrasensitive and particularly selective. It has been successfully applied to the measurement of removal rate constant of hydrogen peroxide by human erythrocytes.
(II) The study of metal oxide nanomaterials used as bifunctional CL probe arrays and sensors and its application in analysis a variety of biochemical substances.
Mesoporous material is a material containing pores with diameters between2and50nm. Mesoporous metal oxide nanoparticles have good monodisperse pore rules, adjustable aperture, large surface area, good thermal stability and other characteristics. Mesoporous metal material with over1000m2.g-1surface area and pore structures can provide the target-receptor a lot sites and sufficient interface to help adsorbed target and enhance the concentration of the target. Mesoporous material is suitable for immobilization of various substances, such as drugs, enzymes and antibodies. Our research found that mesoporous metal oxide nanomaterial itself has an excellent catalytic effect on the CL reactions. The novel bifunctional CL probe array has been applied to the high-throughput determination of a variety of biochemical substances.
(1) A novel bifunctional CL probe array for the determination of glucose in human serum.
A new CL probe array assay approach was first developed. The new CL probe array was based on Co3O4-SiO2mesoporous nanocomposite material, which not only has an excellent catalytic effect on the luminol-H2O2CL reaction in alkaline medium but also can be used for the immobilization of enzymes. As a model, the novel bifunctional CL probe array has been applied to the high-throughput determination of glucose in human. The linear range of the glucose concentration was3-90μM and the detection limit was0.36μM.
(2) A novel bifunctional CL probe array for the determination of lactose in milk was established.
In our work, we further explored the mesoporous Co3O4-SiO2bifunctional CL probe array. The new type CL probe array was also based on enzyme mimics of Co3O4-SiO2mesoporous nanocomposite material, which not only have an excellent catalytic effect on the luminol-H2O2CL reaction in alkaline medium but also can be used for the immobilization of enzymes.β-galactosidase and glucose oxidase were selected as a model for enzyme assays to demonstrate the applicability of Co3O4-SiO2mesoporous nanocomposite material in multienzyme immobilization. It is different from the traditional probe. The linear range of the lactose concentration was3.0×10-7to1.0×10-5g·mL-1and the detection limit was6.9×10-8g·mL-1. It has been successfully applied to determine lactose in milk.
(3)A novel CL sensor array combined with mesoporous enzyme reactor for high-throughput determination of glucose in human serum
A novel enzyme reactor was prepared by calcium alginate fiber and amino modified nanosized mesoporous silica (CAF-AMNMS) as support. Combination the adsorption of enzyme on AMNMS with the cage effect of the polymer greatly increases catalytic activity and stability of immobilized enzyme. It was showed that the lifetime, stability and catalytic activity of enzyme reactor greatly improved by incorporating AMS into CAF to efficiently encapsulate enzyme. Glucose oxidase ((pI=4.0)) was chosen as a model enzyme to explore the possibility of CAF-AMNMS as a matrix for enzyme immobilization in the design of a CL microreactor. The unique sensor array combined with the enzyme reactor has been successfully applied to the high-throughput determination of glucose in human
化学发光方法因其不需要光源,仪器设备简单,线性范围宽,灵敏度高,易于微型化等优点,已成为最有发展前景的分析方法之一。近年来,贵金属纳米材料的出现拓展了化学发光的研究应用范围,但是关于金属氧化物纳米材料的化学发光催化行为研究的很少。在本研究工作中,我们系统地研究了多种纳米金属氧化材料的合成及其表征,发现它们对鲁米诺-过氧化氢化学发光反应的催化性能,特别是在低鲁米诺和低过氧化氢浓度下的高灵敏和高选择催化作用；实现了催化化学发光高通量现场检测TATP爆炸物,建立了一种新的化学发光法测定人红血球降解过氧化氢速率及超灵敏化学发光方法测定呼吸道疾病病人呼出冷凝物中的氧化应激生物标志物过氧化氢。论文还研究了介孔金属氧化物纳米粒子的双功能探针及传感器高通量快速测定多种生化物质。
本论文首先对金属氧化纳米材料的研究及其在分析化学中的应用作了评述；研究报告分为两部分：
一、金属氧化物纳米材料催化鲁米诺-过氧化氢化学发光新体系的研究及其应用
研究发现金属氧化物纳米材料(Co3O4, Cr2O3, a-Fe2O3, CeO2, NiO, CuO, Y2O3, Nd2O3, Mn2O3)对鲁米诺-过氧化氢化学发光反应都具有强的催化性能,而且远超过常用的金属离子催化剂、过氧化物酶和其他金属纳米粒子的催化性能。基于此建立了一系列测定过氧化物的新化学发光体系,提高了方法的选择性。
(1)金属氧化物纳米粒子催化化学发光高通量现场检测TATP爆炸物
三过氧化三丙酮(Triacetone Triperoxide, TATP)是一种过氧化物型爆炸物,在爆炸中,TATP的每个分子迅速释放出气态4个分子,产生比周围空气高出几百倍的气压,威力与TNT相当。TATP是一种极为敏感的爆炸物,摩擦或敲击就可能引发爆炸,不需要任何起爆装置。TATP可以溶解在很多种溶剂中来增强稳定性,如使用普通的发胶、洗发液、沐浴乳或其他液体和凝胶。由于TATP分子不含硝基,机场传统的安检方法,包括金属探测器,X光机及硝基类爆炸物检查行李的方法都不能检测出TATP。
我们发现Ce02纳米粒子对鲁米诺-过氧化氢化学发光体系的优秀的催化能力,合成了一种在聚苯乙烯上有极强吸附力的Ce02纳米颗粒,在聚苯乙烯96孔板上组装成传感膜。由于TATP有较高的蒸汽压(8×10-2Torr,25℃;而TNT为6×10-6Torr,25℃),即使包装严密,也会在携带物上(皮革、麻布、塑料和金属等)带来痕迹。我们用一小块医用棉球擦拭样品表面,然后将棉球装入10mL带盖的玻璃小瓶中,加入5mL水解液进行提取。依次移取50μL提取液于96微孔板中,放置于Biotech Gen 5微孔板化学发光分析仪器中,依次向各孔自动注入50μL5×10-5M鲁米诺进行定量。我们成功应用于模拟机场现场的安全检查,完全满足机场现场检测的需要。
论文中还合成了一种粒径约20nm的三氧化二铬纳米粒子,研究了其对鲁米诺-过氧化氢化学发光反应的催化机理,也用于TATP的现场检测,与其它测定TATP和H202的方法比较,此法最大的特点之一是有机过氧化物如过氧化氢脲、过氧化氢叔丁醇、过氧化氢异丙苯等均不干扰测定。
(2)四氧化三钴纳米粒子催化鲁米诺-过氧化氢化学发光新体系测定呼出冷凝物中的过氧化氢
系统研究了四氧化三钴纳米颗粒催化鲁米诺-过氧化氢化学发光新体系。比较了Co3O4, NiO, a-Fe2O3及其他普通催化剂的催化性能,四氧化三钴化学发光体系具有高的选择性和灵敏度。在优化条件下,化学发光强度与过氧化氢浓度在1.0×10-9-1.0×10-6M范围内呈良好的线性关系,方法的检出限是3.0×10-10M(3σ)。结合流动注射技术建立了测定呼出冷凝物中的过氧化氢的的化学发光新方法。该测定方法具有灵敏度高,选择性好,简单可行的优点。该方法已成功应用于对呼吸道感染人群和健康人群呼出冷凝物中痕量氧化应激生物标志物过氧化氢的直接测定。实验结果表明两者具有显著性差异。
(3)二氧化铈纳米颗粒催化鲁米诺-过氧化氢化学发光新体系研究测定红血球降解过氧化氢速率
纳米氧化物表面缺陷在催化反应中起到关键作用。二氧化铈表面存在氧空位,具有优异的储氧能力,可应用于氧化还原反应。通过调节制备方法,亲水性二氧化铈纳米粒子也能极大地增敏鲁米诺-过氧化氢化学发光。过氧化氢是在活细胞中的正常代谢物,细胞内多余的过氧化氢会氧化细胞内的组分,控制细胞内的过氧化氢水平很重要。我们结合化学发光微阵列法建立了一种新的化学发光法测定人体血液中红血球降解过氧化氢速率。
二、纳米金属氧化物双功能探针及传感器高通量快速测定生化物质
介孔材料是孔径在2nm到50nm之间的一类材料。作为介孔材料的一种,介孔金属氧化物纳米粒子将介孔材料与纳米材料结合起来。介孔金属氧化物纳米粒子具有良好的单分散性、孔道规则、孔径可调、比表面积大和热稳定性好等特性。超过1000m2.g-1表面积及开孔结构的介孔材料可以给目标物-受体之间提供大量供位点和足够的界面以帮助吸附目标物,从而增强本体浓度。因而适合用做装载或固定各种物质,比如药物、酶和抗体。我们的研究发现这些介孔纳米金属氧化物材料本身又对某些化学发光反应具有很强的催化作用,因而设计的固定酶的介孔金属氧化物纳米粒子所构建的化学发光传感器,将具有双功能探针的特性,具此实现了多种生化物质高通量的快速测定。
(1)介孔四氧化三钴双功能探针高通量测定血清中的葡萄糖
基于Co3O4-SiO2介孔复合纳米材料的催化和固定酶的作用,建立了一种新的双功能化学光探针阵列。该化学发光探针阵列由Co3O4-SiO2介孔复合材料和固定的酶组成。Co3O4-SiO2介孔复合材料不仅做酶的载体而且对化学发光体系具有催化作用。这种新的化学发光阵列应用到血液中葡萄糖的检测。测量的葡萄糖线性范围是3-90μM。检出限是0.36μM。
(2)双酶共固定介孔四氧化三钴双功能探针高通量测定牛奶中乳糖
在本研究工作中,我们进一步探究介孔四氧化三钴双功能探针。将葡萄糖氧化酶和β-半乳糖苷酶同时固定在载体介孔四氧化三钴上。用于测定牛奶中乳糖的含量。测量的乳糖线性范围是3.0×10-7到1.0×10-5g.mL-1。检出限是6.9×10-8g.mL-1。
(3)介孔四氧化三钴纳米传感器耦合介孔二氧化硅纳米粒子固定化酶柱高通量测定血清中的葡萄糖
研究将氨基化介孔二氧化硅纳米粒子与海藻酸钙纤维相结合作为载体,将纳米介孔二氧化硅(AMNMS)对酶的吸附作用和催化增强作用与海藻酸钙凝胶对酶的笼蔽效应相结合可以有效的提高固定化酶的催化能力和酶的稳定性。用葡萄糖氧化酶(pI=4.0)为模板制备了海藻酸钙纤维-氨基化介孔二氧化硅(CAF-AMNMS)为载体的微型酶反应器,并将该酶反应器同鲁米诺-H2O2-Co3O4-SiO2介孔催化化学发光体系相耦合,设计了一种阵列传感器,高通量直接测定血清中葡萄糖。
Nanomaterialis between0.1and100nrn in size. Nanomaterials have unique physical and chemical properties due to quantum confinement and exceedingly large surface to volume ratio. They have a superior performance to the materials now existing. Metal oxide nanomaterials have been a hot topic in recent years, which have extensive applications and potential value in optics, electrics, mechanics, and magnetics,
Chemiluminescence (CL) is known as a powerful analytical technique that promises high sensitivity, wide linear range and simple instrumentation. The emergence of noble metal nanoparticles has expanded the application range of CL studies. However, there are only a few reports on the CL of metal oxide nanomaterials. In our work, we systematically studied a variety of metal oxide nanoparticles, and we found that they exhibit high specific catalytic effects on the CL reaction of the luminol-H2O2system in alkaline solution compared with other common catalysts. In this system, luminol with very low concentration can get excellent experimental result with higher selectivity than other luminol system. We have developed a novel CL sensor array to determine Triacetone Triperoxid (TATP) in the suspected items. We have developed a novel CL method to determine the removal rate constant of H2O2by human erythrocytes. We have developed a novel CL approch to determine trace quantities of H2O2in exhaled breath condensate (EBC) where it is a mediator of oxidative stress and a promising biomarker for diagnosing. We also have developed bifunctional CL probe arrays and sensors to determine a variety of biochemical substances, which were based on mesoporous nanocomposite material.
The application of metal oxide nanomaterials in analysis is summarized in Chapter
1. The research work of the dissertation is made up of two sections:
(I) The study of metal oxide nanomaterials used as catalyter in luminol-H2O2CL system and its application
Metal oxide nanomaterials (Co3O4, Cr2O3, CeO2, NiO, a-Fe2O3,CuO, Y2O3, Nd2O3, possess more excellent catalysis in H2O2CL system than previously reported (such as Co2+, Cu2+, Cr3+, HRP and so on). The selectivity CL reaction of luminol-H2O2metal oxide nanomaterials has been proposed.
(1) New CL techniques based on metal oxide nanomaterials catalysis for high-throughput determination of the explosive Triacetone Triperoxide at the scene
TATP is a new organic peroxide based explosive, which decomposes via an entropically favorable mechanism to form4gas phase molecules (a10g sample gave250cm3expansion in the trauzl test as compared to300cm3for TNT). TATP is a highly unstable compound. Explosions may occur by friction, impact or temperature changes, especially in solid phase samples. TATP can be dissolved in a variety of solvents to enhance the stability, such as using an ordinary hair gel, shampoo, shower gel, or other liquids and gels. Metal detector, X-ray baggage scanner, and many detection devices that readily detect tradition explosives made of organic nitro and nitrate compounds fail to detect TATP.
Here we report on the finding that CeO2nanoparticles are capable of strongly enhancing the CL of the luminol-H2O2system. CeO2nanoparticles can be firmly adsorbed on the96-well plates made of polystyrene to form a very solid sensing membrane. The vapor pressure of TATP is8×10-2Torr at25℃. Its vapor can be absorbed onto suitcases and rapidly hydrolyze into H2O2in wet air. Soaked swabs were used to sample the suspected items (pen, plastic bag, banknotes, paper, card), which may be possible carried on an airplane. Immediately after collection, samples were transferred to10mL centrifuge tubes with5mL ultrapure water, which were then sealed for storage.50μL sample solution and50μL (5×10-5M) luminol were injected into the CL sensor array and readings were acquired. The integrated kinetic curve for each sensor can be obtained within5s. The process is easy to perform. It has been successfully applied to the detection of TATP in the suspected items, which may be possible carried on an airplane.
In this paper, we also synthesized Cr2O3nanoparticles (about20nm). Cr2O3nanoparticles were found to greatly enhance the CL intensity of luminol-H2O2system. A reaction mechanism was discussed. The novel CL method also has been successfully applied to the detection of TATP at the scene. Especially, some organic hydroperoxides such as carbamide peroxide, tert-butyl hydroperoxide and cumene hydroperoxide do not interfere with the determination.
(2) CL assay for hydrogen peroxide in exhaled breath condensate using Co3O4 nanoparticle-based catalysis
We systematically studied the nanoparticles-luminol-H2O2CL system. The catalytic activity of three nanoparticles (Co3O4, NiO, a-Fe2O3,) were investigated using microarray method. The CL method based on the use of the Co3O4nanoparticles is ultrasensitive and particularly selective. Under the optimum conditions described above, the linear calibration range prolonged over3orders of magnitude from1.0nm to1000nM. The limit of detection (LOD,3σ) for hydrogen peroxide was0.3n M. A new CL method for the determination of H2O2in exhaled breath condensate (EBC) was built by combining with flow injection technology. The method was successfully applied to the determination of trace quantities of H2O2in EBC where it is a mediator of oxidative stress and a promising biomarker for diagnosing (healthy subjects, rheum subjects, and feverish subjects). Our data suggested that the average H2O2concentration of EBC from feverish subjects was significantly higher than healthy subjects and rheumatic subjects.
(3) A CL microarray based on catalysis by CeO2nanoparticles, and its application to determine the rate of removal of hydrogen peroxide by human erythrocytes
It is important that nano-oxide surface defects in the catalytic reactions. CeO2nanoparticles vacancies present on the surface. It has excellent oxygen storage capacity. Hydrophilic CeO2nanoparticles were also shown highly active and selective for the luminol-H2O2system. H2O2is a normal metabolite in living cells. It is important for cells to remove hydrogen peroxide due to superfluous H2O2may oxidize cellular components. Tight control of cellular H2O2levels is important to avoid oxidative damage of cellular components and to maintain the regulation of various cell responses. The microarray CL method based on the use of the CeO2nanoparticles is ultrasensitive and particularly selective. It has been successfully applied to the measurement of removal rate constant of hydrogen peroxide by human erythrocytes.
(II) The study of metal oxide nanomaterials used as bifunctional CL probe arrays and sensors and its application in analysis a variety of biochemical substances.
Mesoporous material is a material containing pores with diameters between2and50nm. Mesoporous metal oxide nanoparticles have good monodisperse pore rules, adjustable aperture, large surface area, good thermal stability and other characteristics. Mesoporous metal material with over1000m2.g-1surface area and pore structures can provide the target-receptor a lot sites and sufficient interface to help adsorbed target and enhance the concentration of the target. Mesoporous material is suitable for immobilization of various substances, such as drugs, enzymes and antibodies. Our research found that mesoporous metal oxide nanomaterial itself has an excellent catalytic effect on the CL reactions. The novel bifunctional CL probe array has been applied to the high-throughput determination of a variety of biochemical substances.
(1) A novel bifunctional CL probe array for the determination of glucose in human serum.
A new CL probe array assay approach was first developed. The new CL probe array was based on Co3O4-SiO2mesoporous nanocomposite material, which not only has an excellent catalytic effect on the luminol-H2O2CL reaction in alkaline medium but also can be used for the immobilization of enzymes. As a model, the novel bifunctional CL probe array has been applied to the high-throughput determination of glucose in human. The linear range of the glucose concentration was3-90μM and the detection limit was0.36μM.
(2) A novel bifunctional CL probe array for the determination of lactose in milk was established.
In our work, we further explored the mesoporous Co3O4-SiO2bifunctional CL probe array. The new type CL probe array was also based on enzyme mimics of Co3O4-SiO2mesoporous nanocomposite material, which not only have an excellent catalytic effect on the luminol-H2O2CL reaction in alkaline medium but also can be used for the immobilization of enzymes.β-galactosidase and glucose oxidase were selected as a model for enzyme assays to demonstrate the applicability of Co3O4-SiO2mesoporous nanocomposite material in multienzyme immobilization. It is different from the traditional probe. The linear range of the lactose concentration was3.0×10-7to1.0×10-5g·mL-1and the detection limit was6.9×10-8g·mL-1. It has been successfully applied to determine lactose in milk.
(3)A novel CL sensor array combined with mesoporous enzyme reactor for high-throughput determination of glucose in human serum
A novel enzyme reactor was prepared by calcium alginate fiber and amino modified nanosized mesoporous silica (CAF-AMNMS) as support. Combination the adsorption of enzyme on AMNMS with the cage effect of the polymer greatly increases catalytic activity and stability of immobilized enzyme. It was showed that the lifetime, stability and catalytic activity of enzyme reactor greatly improved by incorporating AMS into CAF to efficiently encapsulate enzyme. Glucose oxidase ((pI=4.0)) was chosen as a model enzyme to explore the possibility of CAF-AMNMS as a matrix for enzyme immobilization in the design of a CL microreactor. The unique sensor array combined with the enzyme reactor has been successfully applied to the high-throughput determination of glucose in human
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