荧光纳米粒子探针的制备及其应用研究
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摘要
纳米技术和生物技术是21世纪的两大领先技术,在这两者之间存在着许多技术交叉。其中,纳米探针技术和传感技术就是纳米技术、生物技术与探针技术和传感技术相结合的产物,已经引起了人们的广泛关注。纳米材料是指在三维空间中至少有一维空间的尺度在1nm-100 nm之间的物质。具有着独特的化学性质和物理性质,如表面效应、微尺寸效应、量子效应和宏观量子隧道效应等,呈现出常规材料不具备的优越性能。将纳米材料引入生物探针和传感领域后,如何利用纳米材料构建纳米粒子探针和传感器、提高分析测定的灵敏度、稳定性和其它性能,建立不同分析对象的分析方法,使其在实际应用中真正得到更好和更广泛的应用,仍然是存在的一个问题。
本研究建立了一种环境友好、制备方法简单、成本低廉的改良St(o|¨)ber合成法和一种制备方法简单、成本低廉的沉淀聚合法,并利用其成功的制各了功能化的荧光纳米粒子和新型聚合物荧光纳米粒子,通过特定的生物修饰,作成荧光纳米粒子探针,实现了癌细胞的识别和检测;以反相乳液聚合法制备的荧光纳米粒子为标记物建立了高灵敏度测定临床疾病标志物IL-6、TNF-α和毒素SEC_1的免疫分析新方法;制备了电活性物质Ru(bpy)_3Cl_2掺杂的SiO_2纳米粒子/Nafion、壳聚糖复合膜和电活性物质Ru(bpy)_3Cl_2掺杂的TiO_2纳米粒子/Nafion复合膜修饰电极,据此构建了测定甲氧氯普胺、伊托必利和三丙胺的新型高灵敏度的电化学发光传感器。
第一部分荧光纳米粒子标记癌细胞成像
第1章荧光纳米粒子标记癌细胞成像研究进展
就纳米粒子作为标记物在癌细胞成像中的研究进展进行了论述,内容主要包括量子点的性质及其在癌细胞成像中的研究;二氧化硅荧光纳米粒子的性质及其在癌细胞成像中的研究和多功能二氧化硅荧光纳米粒子在癌细胞成像中的研究。
第2章叶酸修饰的荧光纳米粒子探针在宫颈癌细胞成像中的研究
利用改良St(o|¨)ber合成方法制备了易于标记的二氧化硅荧光纳米粒子。该方法具有制备简单、成本低和环境友好等特点。并将叶酸对所制备的Ru(bpy)_3~(2+)掺杂的二氧化硅荧光纳米粒子进行了修饰(叶酸修饰的Ru(bpy)_3~(2+)掺杂的荧光二氧化硅纳米粒子,FCFNs),利用叶酸与宫颈癌细胞表面叶酸受体的特异性识别,结合荧光显微成像技术实现了宫颈癌细胞的识别和检测。实验结果表明,FCFNs具有光稳定性好,并能够有效、灵敏的识别宫颈癌细胞。这将使其在宫颈癌的临床诊断和疗效评价和叶酸受体的检测中得以应用。
第3章抗表皮生长因子抗体修饰的荧光纳米粒子探针在乳腺癌细胞成像中的研究
在世界范围内,尽管肺癌是妇女死亡的最主要的疾病,但是在恶性肿瘤的临床诊断中,乳腺癌的诊断率仍然仅次于皮肤癌,位居第二位。本研究利用抗表皮生长因子(anti-epidermal growthfactor,anti-EGF)抗体对由改良St(o|¨)ber合成方法制备的荧光纳米粒子进行了修饰,并通过荧光显微成像技术方便的实现了对乳腺癌的检测和识别。该方法也可以适用于其它表达anti-EGFR的细胞系和组织的识别和检测。
第4章新型聚合物荧光纳米粒子探针的制备及其在卵巢癌细胞成像中的研究
本研究以甲基丙烯酸为单体,三羟甲基丙烷三甲基丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,丁基罗丹明B为荧光染料,利用沉淀聚合法非常容易的制备了一种新型聚合物荧光纳米粒子(PFNPs)。该PFNPs具有光稳定性好、泄漏率低和粒度均一的特点。随后将PFNPs与抗Her-2单克隆抗体进行交联,制备得到抗Her-2单克隆抗体修饰的PFNPs探针,并利用其对卵巢癌细胞进行了识别和荧光显微成像检测。
第二部分纳米粒子标记荧光免疫分析
第1章纳米粒子标记荧光免疫分析的研究进展
就荧光免疫分析中常见的荧光纳米标记物:半导体量子点、二氧化硅荧光纳米粒子、高分子荧光纳米微球、稀土元素氧化物纳米粒子、上转换荧光纳米粒子和脂质体荧光纳米粒子在荧光免疫分析中的研究进行了评述。
第2章Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子标记荧光免疫分析法测定白介素-6
生物分子修饰的纳米粒子在许多领域有着广泛的应用。本文利用Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子为标记物建立了一种高灵敏度测定白介素(IL-6)的荧光免疫新方法。基于IL-6抗原与抗IL-6单克隆抗体交联的Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子之间的特异性捕获作用和“三明治”免疫夹心法实现了IL-6的测定。在最佳实验条件下,荧光强度和IL-6浓度在20.0～1250.0 pg/mL范围内成线性,检出限为7 pg/mL。回归方程为I_F=7.66+32.50[IL-6](ng/mL)(R=0.9980)。对78.0pg/mL的IL-6进行11次测定的RSD为3.2%。此外,还利用荧光显微成像技术对纳米粒子标记荧光免疫法检测IL-6进行了研究。结果表明,本方法在IL-6检测中具有灵敏度高,操作简便和精密度高的特点,并成功的用于人血清中IL-6含量的测定。
第3章Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子标记荧光免疫分析法测定金葡萄球菌肠毒素
本研究以功能化荧光纳米粒子为标记物,利用双抗体夹心免疫分析反应原理,建立了一种高灵敏度检测金黄色葡萄球菌肠毒素(SEC_1)的荧光免疫分析新方法。首先,以Ru(bpy)_3Cl_2(Rubpy)为荧光染料,正硅酸乙脂(TEOS)和(3-氨基丙基)三乙氧基硅烷(APS)为硅源进行共水解,利用反相微乳液聚合法制备了功能化荧光纳米粒子。再利用共水解在纳米粒子表面产生的氨基实现了功能化荧光纳米粒子与抗SEC_1单克隆抗体的交联,然后利用双抗体夹心法对SEC_1进行了测定。在最佳实验条件下,荧光强度与SEC_1浓度在1.0～75.0 ng/mL范围内呈良好线性关系,检出限为0.3 ng/mL(3σ)。回归方程为I_F=24.58+0.64[SEC_1](ng/mL)(R=0.9991)。对25.0ng/mL SEC_1测定11次的RSD为2.5%。此外,还探索了应用荧光显微镜成像技术以功能化荧光纳米粒子为标记物,利用双抗体夹心法对SEC_1的测定。实验结果表明,该检测SEC_1的方法具有抗体易于标记,方法简单,测定灵敏高和精密度好的优点。并将其成功的应用于检测实际样品中的SEC_1含量。
第4章Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子标记荧光免疫分析法测定肿瘤坏死因子
结合免疫分析的特异性和荧光纳米粒子标记技术的高灵敏度优点,建立了一种测定肿瘤坏死因子(TNF-α)的荧光免疫分析新方法。首先,利用反相乳液聚合法制备了Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子(RuDFSNs),然后将抗TNF-α单克隆抗体标记在RuDFSNs上,并用荧光免疫分析法,将抗TNF-α单克隆抗体标记的RuDFSNs用于人血清样品中TNF-α的测定。在最佳实验条件下,荧光强度和TNF-α浓度在1.0～250.0 pg/mL范围内成线性,检出限为0.1 pg/mL。对浓度为2.0、20.0、200.0 pg/mL的TNF-α样品5次平行测定的日内和日间精密度分别为4.9%、4.4%、4.6%、6.1%、5.9%、5.3%。采用标准加入法对方法进行了评价,对人血清样品测定回收率为95.5-105.0%。此外,还利用荧光显微成像技术对纳米粒子标记荧光免疫法检测TNF-α进行了研究。结果表明,本方法在TNF-α检测中具有灵敏度高,操作简便和精密度高的特点,并成功的用于人血清中TNF-α含量的测定。
第三部分纳米粒子修饰电极电化学发光传感器
第1章纳米粒子修饰电极电化学发光传感器的研究进展
重点就纳米粒子修饰电极电化学发光的研究进展进行了论述,主要包括碳纳米管、纳米金、纳米二氧化硅和纳米二氧化钛这几类纳米物质在修饰电极电化学发光中的研究及Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子在电化学发光中的研究进行了综述。
第2章Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子/Nafion复合膜修饰电极电化学发光传感器测定甲氧氯普胺
本文利用Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子/Nafion复合膜对玻碳电极进行了修饰,基于盐酸甲氧氯普胺(MCP)对固定化的Ru(bpy)_3~(2+)的电化学发光信号的增强作用,构建了一种高灵敏度测定MCP的电化学发光传感器。对该电化学发光传感器的特性和测定MCP的分析特性进行了详细的研究。在最佳实验条件下,MCP的浓度在2.0×10~(-8)～1.0×10~(-5) mol/L(R=0.9989)范围内与电化学发光强度有良好的线性关系,检出限为7×10~(-9)mol/L,对1.2×10~(-6)mol/L的MCP测定的RSD为3.2%(n=11)。该电化学发光传感器已经成功地用于药物制剂和尿样中MCP的测定。加标回收实验结果表明该传感器测定MCP的回收率为97.0-104.4%。并利用统计检验对电化学发光传感器测定结果与标准方法测定结果进行了分析,结果表明两者之间无明显的差异。
第3章Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子/壳聚糖复合膜修饰电极电化学发光传感器测定伊托必利
利用Ru(bpy)_3~(2+)搀杂的SiO_2(RuDS)纳米粒子/壳聚糖复合膜修饰电极建立了一种测定伊托必利的电化学发光传感器。首先利用改良的St(o|¨)ber合成法制备了RuDS纳米粒子,并利用透射电镜、电化学和荧光方法对Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子进行了表征。结果表明,Ru(bpy)_3~(2+)掺杂在纳米粒子中仍然保持着其电化学活性,并且由于纳米粒子的保护作用降低了Ru(bpy)_3~(2+)分子的泄漏。并对Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子/壳聚糖复合膜修饰电极电化学发光传感器测定伊托必利的分析特性进行了详细的研究。在最佳实验条件下,伊托必利的浓度在1.0×10~(-8)～2.0×10~(-5)g/mL(R=0.9978)范围内与电化学发光强度有良好的线性关系,检出限为3×10~(-9) g/mL,对浓度为8.0×10~(-8) g/mL的伊托必利测定的RSD为2.3%(n=11)。该电化学发光传感器已经成功地用于药物制剂和尿样中伊托必利的测定。结果表明,该电化学发光传感器具有高的灵敏度和好的稳定性。
第4章新型Ru(bpy)_3~(2+)搀杂的TiO_2纳米粒子/Nafion复合膜修饰电极电化学发光传感器的研究
利用反相乳液聚合法制备了电活性成份Ru(bpy)_3~(2+)掺杂的二氧化钛纳米粒子,然后将Ru(bpy)_3~(2+)掺杂的二氧化钛纳米粒子/Nafion复合膜对玻碳电极进行了修饰,建立了Ru(bpy)_3~(2+)掺杂的二氧化钛纳米粒子/Nafion复合膜修饰电极化学发光传感器。利用透射电镜、扫描电镜和电化学方法对纳米粒子和复合膜进行了表征。结果表明,Ru(bpy)_3~(2+)掺杂的二氧化钛纳米粒子呈球形,粒径大小38±3 nm;Ru(bpy)_3~(2+)掺杂在纳米粒子中仍然保持着其电化学活性,并且由于纳米粒子的保护作用降低了Ru(bpy)_3~(2+)的泄漏;纳米粒子均一的分布在复合膜中。由于大量的Ru(bpy)_3~(2+)固定在电极上,使得电化学发光信号明显增强,从而可以提高测定的灵敏度。详细研究了这种电化学发光传感器检测三丙胺(TPA)的电化学发光行为。实验结果表明,该传感器具有高的度灵敏度,对TPA测定的检出限为1 nmol/L。此外,该电化学发光传感器还表现出了良好的稳定性。
Nanotechnology and biotechnology are two key technologies of the 21 st century.Herein, nanoprobe technology and nanobiosensing technology are one of the intersectant research areas of nanotechnology,biotechnology,probe technology and sensing technology and becomes an emerging area and has attracted world-wide attention and research.Nowadays,nanomaterials,or matrices with at least one of their dimensions ranging in scale from 1 to 100 nm,display unique physical and chemical features because of the quantum size effect,mini size effect,surface effect and macro-quantum tunnel effect.Nanomaterials are revolutionizing the development of bioprobes and biosensors.How to construct and improve the sensitivity,stability and other attributes of nanoparticles probes and sensors for better application and a wider use by using nanomaterials remains to be further studied.
A simple,cost-effective and environmentally-friendly modified St(o|¨)ber synthesis method and a simple and cost-effective precipitation polymerization method were proposed in this dissertation, Fluorescent silica nanoparticles and a novel kind of polymer fluorescent nanoparticles were synthesized with these methods.The new nanoparticles peobes prepared with the synthesized nanoparticles through modification were used to detect cancer cells with fluorescence microscopy imaging technology.Novel fluoroimmunoassay methods with the fluorescent silica nanoparticles which prepared with inverse microemulsion polymerisation method labeling technique for the determination of staphylococcal enterotoxin C_1(SEC_1),recombinant human interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were proposed.Electrogenerated chemiluminescence sensors for metoclopramide,itopride and tripropylamine with Ru(bpy)_3~(2+)-doped silica nanoparticles/chitosan,perfluoinated ion-exchange resin and Ru(bpy)_3~(2+)-doped titania nanoparticles/perfluoinated ion-exchange resin composite films modified electrode were developed.
This dissertation consists of three parts and very part contains four chapters.The major contents in this dissertation are described as follows:
Part One Fluorescent Nanoparticle Probe for Cancer Imaging
Chapter One The Development of Fluorescent Nanoparticle Probe for Cancer Imaging
In this chapter,the development and the tendency of the cancer imaging based on the fluorescent nanoparticle probes are reviewed.It covers characteristics of the quantum dots(QDs),fluorescent silica nanoparticle and multimodal fluorescent silica nanoparticle and their applications in the cancer imaging.
Chapter Two Folate Conjugated Fluorescent Nanoparticle Probe for Cervical Cancer Imaging
Fluorescent nanoparticles with excellent character such as non-toxicity and photostability were first synthesized with a simple,cost-effective and environmentally-friendly modified St(o|¨)ber synthesis method,and then successfully modified with folate.This kind of fluorescence probe based on the folate conjugated fluorescent nanoparticles(FCFNs) has been used to detect cervical cancer cells with fluorescence microscopy imaging technology.The experimental results demonstrate that the folate conjugated fluorescent nanoparticles can effectively recognize cervical cancer cells and exhibited good sensitivity and exceptional photostability,which would provide a novel way for the diagnosis and curative effect observation of cervical cancer cells and offer a new method in detecting folate receptors.
Chapter Three Anti-Epidermal Growth Factor Receptor Antibody Conjugated Fluorescent Nanoparticle Probe for Breast Cancer Imaging
Although lung cancer is now the leading cause of cancer death among women,breast cancer still constitutes the most commonly diagnosed malignancy in women after skin cancer.In this study, anti-EGFR(epidermal growth factor receptor) antibody conjugated fluorescent nanoparticles anti-EGFR antibody conjugated fluorescent nanoparticles probe prepared with modified St(o|¨)ber synthesis method was used to detect breast cancer cells with fluorescence microscopy imaging technology.This method may be used in the detecting and recognizing of breast cancer cells and tissue which express epidermal growth factor receptor.
Chapter Four The Preparation of a Novel Kind of Polymer Fluorescent Nanoparticle Probe and its Application in Ovarian Cancer Imaging
A novel kind of polymer fluorescent nanoparticles(PFNPs) was synthesized with precipitation polymerization by using methaerylic acid as monomer,trimethylolpropane trimethacrylate as cross-linker,azobisisobutyronitrile as radical initiator and butyl rhodamine B as fluorescent dye.With this method the PFNPs can be prepared easily.And then the PFNPs were successfully modified with anti-Her-2 monoclonal antibody.The fluorescence probe based on anti-Her-2 monoclonal antibody conjugated PFNPs has been used to detect ovarian cancer cells with fluorescence microscopy imaging technology.The experimental results demonstrate that the PFNPs can effectively recognize ovarian cancer cells and exhibited good sensitivity and exceptional photostability.
Part Two Fluorescent Nanoparticle Labeled Fluoroimmunoassay
Chapter One The Development of Fluoroimmunoassay Using Nanoparticles as Labels
In this chapter,the developments and applications of fluoroimmunoassay using nanoparticles as labels were reviewed.The labels including quantum dots,silica fluorescent nanopaticals,polymer fluorescent nanopaticals,europium oxide nanoparticles,up-converting nanoparticles and liposome fluorescent nanoparticles are all reviewed.
Chapter Two Fluorescent Nanoparticles Used as a Fluorescent Labels in Fluoroimmunoassay for IL-6
Nanoparticle labels conjugated with biomolecules are used in a variety of different assay applications.In this paper,a sensitive fluoroimmunoassay for recombinant human interleukin-6(IL-6) with the Rubpy-encapsulated fluorescent silica nanoparticles labeling technique has been proposed. IL-6 was measured based on the specific interaction between captured IL-6 antigen and fluorescent nanoparticles-labeled anti-IL-6 monoclonal antibody.The calibration graph for IL-6 was linear over the range 20.0-1250.0 pg/mL with a detection limit of 7 pg/mL(3σ).The regression equation of the working curve is I_F=7.66+32.50[IL-6](ng/mL)(R=0.9980).The RSD for eleven parallel measurements of 78.0 pg/mL IL-6 was 3.2%.Furthermore,the application of fluorescence microscopy imaging in the study of the antibody labeling and sandwich fluoroimmunoassay with the fluorescent nanoparticles was also explored.This proposed method has the advantage of showing the specificity of immunoassay and sensitivity of fluorescent nanoparticle labels technology.The results demonstrate that the method offers potential advantages of sensitivity,simplicity and reproducibility for the determination of IL-6,and is applicable to the determination of IL-6 in serum samples and enabling fluorescence microscopy imaging for the determination of IL-6.
Chapter Three A Novel Sensitive Staphylococcal Enterotoxin C_1 Fluoroimmunoassay Based on Fluorescent Nanoparticle Labels
A highly sensitive fluoroimmunoassay for the determination of staphylococcal enterotoxin C_1 (SEC_1) is proposed.It is based on the fluorescent nanoparticles as the label coated with anti-SEC_1 monoclonal antibodies in "sandwich" fluoroimmunoassay.With the simple inverse microemulsion polymerisation method the fluorescent nanoparticles were prepared easily.The preparation process produces a silica shell on the surface of the Ru(bpy)_3Cl_2(Rubpy) dye with one step cohydrolysis of tetraethylorthosilicate(TEOS) and the coupling agent(3-aminopropyl)triethoxysilane(APS) provided the amine groups that can be used for biological conjugation.The nanoparticles were then labeled with the anti-SEC_1 monoclonal antibodies and the antibody-labeled nanoparticles were successfully used for the determination of SEC_1.The calibration graph for SEC_1 was linear over the range 1.0～75.0 ng/mL with a detection limit of 0.3 ng/mL.The regression equation of the working curve was I_F=24.58+ 0.64[SEC_1](ng/mL)(R=0.9991).The RSD for eleven parallel measurements of 25.0 ng/mL SEC_1 was 2.5%.Furthermore,the application of fluorescence microscopy imaging in the study of the antibody labeling and sandwich fluoroimmunoassay with the fluorescent nanoparticles was also explored.The results demonstrate that the method offers potential advantages of easily labeling to the antibody,sensitivity,simplicity and reproducibility for the determination of SEC_1 and is applicable to the determination of SEC_1 in real samples and enabling fluorescence microscopy imaging for the determination of SEC_1.
Chapter Four Fluoroimmunoassay for Tumor Necrosis Factor-αin Human Serum Using Fluorescent Nanoparticles as Labels
A novel fluoroimmunoassay method was developed for the determination of tumor necrosis factor-α(TNF-α) in this study.The proposed method has the advantage of showing the specificity of immunoassays and sensitivity of fluorescent nanoparticles label technology.With the well-established inverse microemulsion polymerisation process,the tris(2′2-bipyridyl)dichlororuthenium(Ⅱ) hexahydrate(Rubpy)-doped fluorescent silica nanoparticles(RuDFSNs) were prepared.Then a RuDFSNs-labeled anti-TNF-αmonoclonal antibody was prepared and used for fluoroimmunoassay of TNF-αin human serum samples with a sandwich fluoroimmunoassay by using the low-fluorescent ninety-six well transparent microtiter plates.The assay response was linear from 1.0 to about 250.0 pg/mL with a detection limit of 0.1 pg/mL for TNF-α.The intra- and inter-assay precision are 4.9%, 4.4%,4.6%;6.1%,5.9%,5.3%for five parallel measurements of 2.0,20.0,200.0 pg/mL TNF-αrespectively,and the recoveries are in the range of 95.5～105.0%for human serum sample measurements by standard-addition method.We also explored the application of fluorescence microscopy imaging in the study of the fluoroimmunoassay for TNF-αwith the fluorescent nanoparticales labels.The results demonstrate that the method offers potential advantages of sensitivity,simplicity and good reproducibility for the determination of TNF-α,and is applicable to the determination of TNF-αin serum samples and being capable of fluorescence microscopy imaging for the determination of TNF-α.
Part Three Electrogenerated Chemiluminescence Sensor Based on Nanoparticles Modified Electrode
Chapter One The Progress of Electrogenerated Chemiluminescence Sensor Based on Nanoparticles Modified Electrode
The review gives emphasis on the development of electrogenerated chemiluminescence(ECL) with modified electrodes using nanomaterials including carbon nanotubes,gold nanoparticles,silica nanoparticles and titania nanoparticles.Furthermore,the development of Ru(bpy)_3~(2+)-doped silica nanoparticles in ECL was also disscused.
Chapter Two Electrogenerated Chemiluminescence Sensor for Metoclopramide Determination Based on Ru(bpy)_3~(2+)-doped Silica Nanoparticles Dispersed in Nafion on Glassy Carbon Electrode
The aim of the study presented here is to develop and validate a novel method for the determination of metoclopramide(MCP) with the electrogenerated chemiluminescence(ECL) by using tris(2,2′-bipyridyl)dichlororuthenium(Ⅱ)(Ru(bpy)_3~(2+))-doped silica(RuDS) nanoparticles/perfluoinated ion-exchange resin(Nafion) nanocomposites membrane modified glassy carbon electrode(GCE).The immobilization of Ru(bpy)_3~(2+) in a RuDS nanoparticles/Nafion nanocomposites membrane modified GCE was achieved in this study.The Ru(bpy)_3~(2+) encapsulation interior of the silica nanoparticle maintains its electrochemical activities and also reduces Ru(bpy)_3~(2+) leaching from the silica matrix when immersed in water due to the electrostatic interaction.The ECL analytical performances of this ECL sensor for MCP were investigated in detail.Under the optimum experimental conditions,it showed good linearity in the concentration range from 2.0×10~(-8) to 1.0×10~(-5) mol/L(R=0.9989) with a detection 7×10~(-9) mol/L.The RSD(n=11) was 3.2%for detecting 1.2×10~(-6) mol/L MCP.And the recoveries are in the range of 97.0～104.4%for sample measurements by standard-addition method. This method has been applied successfully to determine MCP in pharmaceutical preparations and in human urine.Statistical analysis(Student's t-test and variance ratio F-test) of the obtained results showed no significant difference between the proposed method and the reference method.
Chapter Three Electrogenerated Chemiluminescence Sensor for Itopride with Ru(bpy)_3~(2+)-doped Silica Nanoparticles/Chitosan Composite Films Modified Electrode
A electrogenerated chemiluminescence(ECL) sensor for itopride was developed based on tris(2,2′-bipyridyl)dichlororuthenium(Ⅱ)(mu(bpy)_3~(2+))-doped silica(RuDS) nanoparticles/biopolymer chitosan composites membrane modified glassy carbon electrode(GCE).The RuDS nanoparticles(52±5nm) were prepared by a modified St(o|¨)ber synthesis method and were characterized by electrochemical,fluorometric and transmission electron microscopy technology.The Ru(bpy)_3~(2+) encapsulation interior of the silica nanoparticle maintains its electrochemical activities and also reduces Ru(bpy)_3~(2+) leaching from the silica matrix when immersed in water due to the electrostatic interaction. The ECL analytical performances of this ECL sensor for itopride based on its enhancement ECL emission of Ru(bpy)_3~(2+) were investigated in details.Under the optimum condition,the enhanced ECL intensity was linear with the itopride concentration in the range of 1.0×10~(-8) to 2.0×10~(-5) g/mL(R= 0.9978).The detection limit was 3×10~(-9) g/mL,and the RSD was 2.3%for 8.0×10~(-8) g/mL itopride(n= 11).The method was successfully applied to the determination of itopride in pharmaceutical and human serum samples with satisfactory results.The as-prepared ECL sensor for the determination of itopride displayed good sensitivity and stability.
Chapter Four A Novel Electrogenerated Chemiluminescence Sensor Based on Ru(bpy)_3~(2+)-doped Titania Nanoparticles Dispersed in Nafion on Glassy Carbon Electrode
A novel electrogenerated chemiluminescence(ECL) sensor based on Ru(bpy)_3~(2+)-doped titania (RuDT) nanoparticles dispersed in a perfluorosulfonated ionomer(Nafion) on a glassy carbon electrode (GCE) was developed in this paper.The electroactive component-Ru(bpy)_3~(2+) was entrapped within the titania nanoparticles by the inverse microemulsion polymerization process that produced spherical sensors in the size region of 38±3nm.The RuDT nanoparticles were characterized by electrochemical,transmission electron and scanning microscopy technology.The Ru(bpy)_3~(2+) encapsulation interior of the titania nanoparticles maintains its ECL efficiency and also reduces Ru(bpy)_3~(2+) leaching from the titania matrix when immersed in water due to the electrostatic interaction. This is the first attempt to prepare the RuDT nanoparticles and extend the application of electroactive component-doped nanoparticles into the field of ECL.Since a large amount of Ru(bpy)_3~(2+) was immobilized three-dimensionally on the electrode,the Ru(bpy)_3~(2+) ECL signal could be enhanced greatly,which finally resulted in the increased sensitivity.The ECL analytical performance of this ECL sensor for tripropylamine(TPA) was investigated in detail.This sensor shows a detection limit of 1 nmol/L for TPA.Furthermore,the present ECL sensor displays outstanding long-term stability.
本研究建立了一种环境友好、制备方法简单、成本低廉的改良St(o|¨)ber合成法和一种制备方法简单、成本低廉的沉淀聚合法,并利用其成功的制各了功能化的荧光纳米粒子和新型聚合物荧光纳米粒子,通过特定的生物修饰,作成荧光纳米粒子探针,实现了癌细胞的识别和检测;以反相乳液聚合法制备的荧光纳米粒子为标记物建立了高灵敏度测定临床疾病标志物IL-6、TNF-α和毒素SEC_1的免疫分析新方法;制备了电活性物质Ru(bpy)_3Cl_2掺杂的SiO_2纳米粒子/Nafion、壳聚糖复合膜和电活性物质Ru(bpy)_3Cl_2掺杂的TiO_2纳米粒子/Nafion复合膜修饰电极,据此构建了测定甲氧氯普胺、伊托必利和三丙胺的新型高灵敏度的电化学发光传感器。
第一部分荧光纳米粒子标记癌细胞成像
第1章荧光纳米粒子标记癌细胞成像研究进展
就纳米粒子作为标记物在癌细胞成像中的研究进展进行了论述,内容主要包括量子点的性质及其在癌细胞成像中的研究;二氧化硅荧光纳米粒子的性质及其在癌细胞成像中的研究和多功能二氧化硅荧光纳米粒子在癌细胞成像中的研究。
第2章叶酸修饰的荧光纳米粒子探针在宫颈癌细胞成像中的研究
利用改良St(o|¨)ber合成方法制备了易于标记的二氧化硅荧光纳米粒子。该方法具有制备简单、成本低和环境友好等特点。并将叶酸对所制备的Ru(bpy)_3~(2+)掺杂的二氧化硅荧光纳米粒子进行了修饰(叶酸修饰的Ru(bpy)_3~(2+)掺杂的荧光二氧化硅纳米粒子,FCFNs),利用叶酸与宫颈癌细胞表面叶酸受体的特异性识别,结合荧光显微成像技术实现了宫颈癌细胞的识别和检测。实验结果表明,FCFNs具有光稳定性好,并能够有效、灵敏的识别宫颈癌细胞。这将使其在宫颈癌的临床诊断和疗效评价和叶酸受体的检测中得以应用。
第3章抗表皮生长因子抗体修饰的荧光纳米粒子探针在乳腺癌细胞成像中的研究
在世界范围内,尽管肺癌是妇女死亡的最主要的疾病,但是在恶性肿瘤的临床诊断中,乳腺癌的诊断率仍然仅次于皮肤癌,位居第二位。本研究利用抗表皮生长因子(anti-epidermal growthfactor,anti-EGF)抗体对由改良St(o|¨)ber合成方法制备的荧光纳米粒子进行了修饰,并通过荧光显微成像技术方便的实现了对乳腺癌的检测和识别。该方法也可以适用于其它表达anti-EGFR的细胞系和组织的识别和检测。
第4章新型聚合物荧光纳米粒子探针的制备及其在卵巢癌细胞成像中的研究
本研究以甲基丙烯酸为单体,三羟甲基丙烷三甲基丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,丁基罗丹明B为荧光染料,利用沉淀聚合法非常容易的制备了一种新型聚合物荧光纳米粒子(PFNPs)。该PFNPs具有光稳定性好、泄漏率低和粒度均一的特点。随后将PFNPs与抗Her-2单克隆抗体进行交联,制备得到抗Her-2单克隆抗体修饰的PFNPs探针,并利用其对卵巢癌细胞进行了识别和荧光显微成像检测。
第二部分纳米粒子标记荧光免疫分析
第1章纳米粒子标记荧光免疫分析的研究进展
就荧光免疫分析中常见的荧光纳米标记物:半导体量子点、二氧化硅荧光纳米粒子、高分子荧光纳米微球、稀土元素氧化物纳米粒子、上转换荧光纳米粒子和脂质体荧光纳米粒子在荧光免疫分析中的研究进行了评述。
第2章Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子标记荧光免疫分析法测定白介素-6
生物分子修饰的纳米粒子在许多领域有着广泛的应用。本文利用Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子为标记物建立了一种高灵敏度测定白介素(IL-6)的荧光免疫新方法。基于IL-6抗原与抗IL-6单克隆抗体交联的Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子之间的特异性捕获作用和“三明治”免疫夹心法实现了IL-6的测定。在最佳实验条件下,荧光强度和IL-6浓度在20.0～1250.0 pg/mL范围内成线性,检出限为7 pg/mL。回归方程为I_F=7.66+32.50[IL-6](ng/mL)(R=0.9980)。对78.0pg/mL的IL-6进行11次测定的RSD为3.2%。此外,还利用荧光显微成像技术对纳米粒子标记荧光免疫法检测IL-6进行了研究。结果表明,本方法在IL-6检测中具有灵敏度高,操作简便和精密度高的特点,并成功的用于人血清中IL-6含量的测定。
第3章Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子标记荧光免疫分析法测定金葡萄球菌肠毒素
本研究以功能化荧光纳米粒子为标记物,利用双抗体夹心免疫分析反应原理,建立了一种高灵敏度检测金黄色葡萄球菌肠毒素(SEC_1)的荧光免疫分析新方法。首先,以Ru(bpy)_3Cl_2(Rubpy)为荧光染料,正硅酸乙脂(TEOS)和(3-氨基丙基)三乙氧基硅烷(APS)为硅源进行共水解,利用反相微乳液聚合法制备了功能化荧光纳米粒子。再利用共水解在纳米粒子表面产生的氨基实现了功能化荧光纳米粒子与抗SEC_1单克隆抗体的交联,然后利用双抗体夹心法对SEC_1进行了测定。在最佳实验条件下,荧光强度与SEC_1浓度在1.0～75.0 ng/mL范围内呈良好线性关系,检出限为0.3 ng/mL(3σ)。回归方程为I_F=24.58+0.64[SEC_1](ng/mL)(R=0.9991)。对25.0ng/mL SEC_1测定11次的RSD为2.5%。此外,还探索了应用荧光显微镜成像技术以功能化荧光纳米粒子为标记物,利用双抗体夹心法对SEC_1的测定。实验结果表明,该检测SEC_1的方法具有抗体易于标记,方法简单,测定灵敏高和精密度好的优点。并将其成功的应用于检测实际样品中的SEC_1含量。
第4章Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子标记荧光免疫分析法测定肿瘤坏死因子
结合免疫分析的特异性和荧光纳米粒子标记技术的高灵敏度优点,建立了一种测定肿瘤坏死因子(TNF-α)的荧光免疫分析新方法。首先,利用反相乳液聚合法制备了Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子(RuDFSNs),然后将抗TNF-α单克隆抗体标记在RuDFSNs上,并用荧光免疫分析法,将抗TNF-α单克隆抗体标记的RuDFSNs用于人血清样品中TNF-α的测定。在最佳实验条件下,荧光强度和TNF-α浓度在1.0～250.0 pg/mL范围内成线性,检出限为0.1 pg/mL。对浓度为2.0、20.0、200.0 pg/mL的TNF-α样品5次平行测定的日内和日间精密度分别为4.9%、4.4%、4.6%、6.1%、5.9%、5.3%。采用标准加入法对方法进行了评价,对人血清样品测定回收率为95.5-105.0%。此外,还利用荧光显微成像技术对纳米粒子标记荧光免疫法检测TNF-α进行了研究。结果表明,本方法在TNF-α检测中具有灵敏度高,操作简便和精密度高的特点,并成功的用于人血清中TNF-α含量的测定。
第三部分纳米粒子修饰电极电化学发光传感器
第1章纳米粒子修饰电极电化学发光传感器的研究进展
重点就纳米粒子修饰电极电化学发光的研究进展进行了论述,主要包括碳纳米管、纳米金、纳米二氧化硅和纳米二氧化钛这几类纳米物质在修饰电极电化学发光中的研究及Ru(bpy)_3Cl_2掺杂SiO_2纳米粒子在电化学发光中的研究进行了综述。
第2章Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子/Nafion复合膜修饰电极电化学发光传感器测定甲氧氯普胺
本文利用Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子/Nafion复合膜对玻碳电极进行了修饰,基于盐酸甲氧氯普胺(MCP)对固定化的Ru(bpy)_3~(2+)的电化学发光信号的增强作用,构建了一种高灵敏度测定MCP的电化学发光传感器。对该电化学发光传感器的特性和测定MCP的分析特性进行了详细的研究。在最佳实验条件下,MCP的浓度在2.0×10~(-8)～1.0×10~(-5) mol/L(R=0.9989)范围内与电化学发光强度有良好的线性关系,检出限为7×10~(-9)mol/L,对1.2×10~(-6)mol/L的MCP测定的RSD为3.2%(n=11)。该电化学发光传感器已经成功地用于药物制剂和尿样中MCP的测定。加标回收实验结果表明该传感器测定MCP的回收率为97.0-104.4%。并利用统计检验对电化学发光传感器测定结果与标准方法测定结果进行了分析,结果表明两者之间无明显的差异。
第3章Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子/壳聚糖复合膜修饰电极电化学发光传感器测定伊托必利
利用Ru(bpy)_3~(2+)搀杂的SiO_2(RuDS)纳米粒子/壳聚糖复合膜修饰电极建立了一种测定伊托必利的电化学发光传感器。首先利用改良的St(o|¨)ber合成法制备了RuDS纳米粒子,并利用透射电镜、电化学和荧光方法对Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子进行了表征。结果表明,Ru(bpy)_3~(2+)掺杂在纳米粒子中仍然保持着其电化学活性,并且由于纳米粒子的保护作用降低了Ru(bpy)_3~(2+)分子的泄漏。并对Ru(bpy)_3~(2+)搀杂的SiO_2纳米粒子/壳聚糖复合膜修饰电极电化学发光传感器测定伊托必利的分析特性进行了详细的研究。在最佳实验条件下,伊托必利的浓度在1.0×10~(-8)～2.0×10~(-5)g/mL(R=0.9978)范围内与电化学发光强度有良好的线性关系,检出限为3×10~(-9) g/mL,对浓度为8.0×10~(-8) g/mL的伊托必利测定的RSD为2.3%(n=11)。该电化学发光传感器已经成功地用于药物制剂和尿样中伊托必利的测定。结果表明,该电化学发光传感器具有高的灵敏度和好的稳定性。
第4章新型Ru(bpy)_3~(2+)搀杂的TiO_2纳米粒子/Nafion复合膜修饰电极电化学发光传感器的研究
利用反相乳液聚合法制备了电活性成份Ru(bpy)_3~(2+)掺杂的二氧化钛纳米粒子,然后将Ru(bpy)_3~(2+)掺杂的二氧化钛纳米粒子/Nafion复合膜对玻碳电极进行了修饰,建立了Ru(bpy)_3~(2+)掺杂的二氧化钛纳米粒子/Nafion复合膜修饰电极化学发光传感器。利用透射电镜、扫描电镜和电化学方法对纳米粒子和复合膜进行了表征。结果表明,Ru(bpy)_3~(2+)掺杂的二氧化钛纳米粒子呈球形,粒径大小38±3 nm;Ru(bpy)_3~(2+)掺杂在纳米粒子中仍然保持着其电化学活性,并且由于纳米粒子的保护作用降低了Ru(bpy)_3~(2+)的泄漏;纳米粒子均一的分布在复合膜中。由于大量的Ru(bpy)_3~(2+)固定在电极上,使得电化学发光信号明显增强,从而可以提高测定的灵敏度。详细研究了这种电化学发光传感器检测三丙胺(TPA)的电化学发光行为。实验结果表明,该传感器具有高的度灵敏度,对TPA测定的检出限为1 nmol/L。此外,该电化学发光传感器还表现出了良好的稳定性。
Nanotechnology and biotechnology are two key technologies of the 21 st century.Herein, nanoprobe technology and nanobiosensing technology are one of the intersectant research areas of nanotechnology,biotechnology,probe technology and sensing technology and becomes an emerging area and has attracted world-wide attention and research.Nowadays,nanomaterials,or matrices with at least one of their dimensions ranging in scale from 1 to 100 nm,display unique physical and chemical features because of the quantum size effect,mini size effect,surface effect and macro-quantum tunnel effect.Nanomaterials are revolutionizing the development of bioprobes and biosensors.How to construct and improve the sensitivity,stability and other attributes of nanoparticles probes and sensors for better application and a wider use by using nanomaterials remains to be further studied.
A simple,cost-effective and environmentally-friendly modified St(o|¨)ber synthesis method and a simple and cost-effective precipitation polymerization method were proposed in this dissertation, Fluorescent silica nanoparticles and a novel kind of polymer fluorescent nanoparticles were synthesized with these methods.The new nanoparticles peobes prepared with the synthesized nanoparticles through modification were used to detect cancer cells with fluorescence microscopy imaging technology.Novel fluoroimmunoassay methods with the fluorescent silica nanoparticles which prepared with inverse microemulsion polymerisation method labeling technique for the determination of staphylococcal enterotoxin C_1(SEC_1),recombinant human interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were proposed.Electrogenerated chemiluminescence sensors for metoclopramide,itopride and tripropylamine with Ru(bpy)_3~(2+)-doped silica nanoparticles/chitosan,perfluoinated ion-exchange resin and Ru(bpy)_3~(2+)-doped titania nanoparticles/perfluoinated ion-exchange resin composite films modified electrode were developed.
This dissertation consists of three parts and very part contains four chapters.The major contents in this dissertation are described as follows:
Part One Fluorescent Nanoparticle Probe for Cancer Imaging
Chapter One The Development of Fluorescent Nanoparticle Probe for Cancer Imaging
In this chapter,the development and the tendency of the cancer imaging based on the fluorescent nanoparticle probes are reviewed.It covers characteristics of the quantum dots(QDs),fluorescent silica nanoparticle and multimodal fluorescent silica nanoparticle and their applications in the cancer imaging.
Chapter Two Folate Conjugated Fluorescent Nanoparticle Probe for Cervical Cancer Imaging
Fluorescent nanoparticles with excellent character such as non-toxicity and photostability were first synthesized with a simple,cost-effective and environmentally-friendly modified St(o|¨)ber synthesis method,and then successfully modified with folate.This kind of fluorescence probe based on the folate conjugated fluorescent nanoparticles(FCFNs) has been used to detect cervical cancer cells with fluorescence microscopy imaging technology.The experimental results demonstrate that the folate conjugated fluorescent nanoparticles can effectively recognize cervical cancer cells and exhibited good sensitivity and exceptional photostability,which would provide a novel way for the diagnosis and curative effect observation of cervical cancer cells and offer a new method in detecting folate receptors.
Chapter Three Anti-Epidermal Growth Factor Receptor Antibody Conjugated Fluorescent Nanoparticle Probe for Breast Cancer Imaging
Although lung cancer is now the leading cause of cancer death among women,breast cancer still constitutes the most commonly diagnosed malignancy in women after skin cancer.In this study, anti-EGFR(epidermal growth factor receptor) antibody conjugated fluorescent nanoparticles anti-EGFR antibody conjugated fluorescent nanoparticles probe prepared with modified St(o|¨)ber synthesis method was used to detect breast cancer cells with fluorescence microscopy imaging technology.This method may be used in the detecting and recognizing of breast cancer cells and tissue which express epidermal growth factor receptor.
Chapter Four The Preparation of a Novel Kind of Polymer Fluorescent Nanoparticle Probe and its Application in Ovarian Cancer Imaging
A novel kind of polymer fluorescent nanoparticles(PFNPs) was synthesized with precipitation polymerization by using methaerylic acid as monomer,trimethylolpropane trimethacrylate as cross-linker,azobisisobutyronitrile as radical initiator and butyl rhodamine B as fluorescent dye.With this method the PFNPs can be prepared easily.And then the PFNPs were successfully modified with anti-Her-2 monoclonal antibody.The fluorescence probe based on anti-Her-2 monoclonal antibody conjugated PFNPs has been used to detect ovarian cancer cells with fluorescence microscopy imaging technology.The experimental results demonstrate that the PFNPs can effectively recognize ovarian cancer cells and exhibited good sensitivity and exceptional photostability.
Part Two Fluorescent Nanoparticle Labeled Fluoroimmunoassay
Chapter One The Development of Fluoroimmunoassay Using Nanoparticles as Labels
In this chapter,the developments and applications of fluoroimmunoassay using nanoparticles as labels were reviewed.The labels including quantum dots,silica fluorescent nanopaticals,polymer fluorescent nanopaticals,europium oxide nanoparticles,up-converting nanoparticles and liposome fluorescent nanoparticles are all reviewed.
Chapter Two Fluorescent Nanoparticles Used as a Fluorescent Labels in Fluoroimmunoassay for IL-6
Nanoparticle labels conjugated with biomolecules are used in a variety of different assay applications.In this paper,a sensitive fluoroimmunoassay for recombinant human interleukin-6(IL-6) with the Rubpy-encapsulated fluorescent silica nanoparticles labeling technique has been proposed. IL-6 was measured based on the specific interaction between captured IL-6 antigen and fluorescent nanoparticles-labeled anti-IL-6 monoclonal antibody.The calibration graph for IL-6 was linear over the range 20.0-1250.0 pg/mL with a detection limit of 7 pg/mL(3σ).The regression equation of the working curve is I_F=7.66+32.50[IL-6](ng/mL)(R=0.9980).The RSD for eleven parallel measurements of 78.0 pg/mL IL-6 was 3.2%.Furthermore,the application of fluorescence microscopy imaging in the study of the antibody labeling and sandwich fluoroimmunoassay with the fluorescent nanoparticles was also explored.This proposed method has the advantage of showing the specificity of immunoassay and sensitivity of fluorescent nanoparticle labels technology.The results demonstrate that the method offers potential advantages of sensitivity,simplicity and reproducibility for the determination of IL-6,and is applicable to the determination of IL-6 in serum samples and enabling fluorescence microscopy imaging for the determination of IL-6.
Chapter Three A Novel Sensitive Staphylococcal Enterotoxin C_1 Fluoroimmunoassay Based on Fluorescent Nanoparticle Labels
A highly sensitive fluoroimmunoassay for the determination of staphylococcal enterotoxin C_1 (SEC_1) is proposed.It is based on the fluorescent nanoparticles as the label coated with anti-SEC_1 monoclonal antibodies in "sandwich" fluoroimmunoassay.With the simple inverse microemulsion polymerisation method the fluorescent nanoparticles were prepared easily.The preparation process produces a silica shell on the surface of the Ru(bpy)_3Cl_2(Rubpy) dye with one step cohydrolysis of tetraethylorthosilicate(TEOS) and the coupling agent(3-aminopropyl)triethoxysilane(APS) provided the amine groups that can be used for biological conjugation.The nanoparticles were then labeled with the anti-SEC_1 monoclonal antibodies and the antibody-labeled nanoparticles were successfully used for the determination of SEC_1.The calibration graph for SEC_1 was linear over the range 1.0～75.0 ng/mL with a detection limit of 0.3 ng/mL.The regression equation of the working curve was I_F=24.58+ 0.64[SEC_1](ng/mL)(R=0.9991).The RSD for eleven parallel measurements of 25.0 ng/mL SEC_1 was 2.5%.Furthermore,the application of fluorescence microscopy imaging in the study of the antibody labeling and sandwich fluoroimmunoassay with the fluorescent nanoparticles was also explored.The results demonstrate that the method offers potential advantages of easily labeling to the antibody,sensitivity,simplicity and reproducibility for the determination of SEC_1 and is applicable to the determination of SEC_1 in real samples and enabling fluorescence microscopy imaging for the determination of SEC_1.
Chapter Four Fluoroimmunoassay for Tumor Necrosis Factor-αin Human Serum Using Fluorescent Nanoparticles as Labels
A novel fluoroimmunoassay method was developed for the determination of tumor necrosis factor-α(TNF-α) in this study.The proposed method has the advantage of showing the specificity of immunoassays and sensitivity of fluorescent nanoparticles label technology.With the well-established inverse microemulsion polymerisation process,the tris(2′2-bipyridyl)dichlororuthenium(Ⅱ) hexahydrate(Rubpy)-doped fluorescent silica nanoparticles(RuDFSNs) were prepared.Then a RuDFSNs-labeled anti-TNF-αmonoclonal antibody was prepared and used for fluoroimmunoassay of TNF-αin human serum samples with a sandwich fluoroimmunoassay by using the low-fluorescent ninety-six well transparent microtiter plates.The assay response was linear from 1.0 to about 250.0 pg/mL with a detection limit of 0.1 pg/mL for TNF-α.The intra- and inter-assay precision are 4.9%, 4.4%,4.6%;6.1%,5.9%,5.3%for five parallel measurements of 2.0,20.0,200.0 pg/mL TNF-αrespectively,and the recoveries are in the range of 95.5～105.0%for human serum sample measurements by standard-addition method.We also explored the application of fluorescence microscopy imaging in the study of the fluoroimmunoassay for TNF-αwith the fluorescent nanoparticales labels.The results demonstrate that the method offers potential advantages of sensitivity,simplicity and good reproducibility for the determination of TNF-α,and is applicable to the determination of TNF-αin serum samples and being capable of fluorescence microscopy imaging for the determination of TNF-α.
Part Three Electrogenerated Chemiluminescence Sensor Based on Nanoparticles Modified Electrode
Chapter One The Progress of Electrogenerated Chemiluminescence Sensor Based on Nanoparticles Modified Electrode
The review gives emphasis on the development of electrogenerated chemiluminescence(ECL) with modified electrodes using nanomaterials including carbon nanotubes,gold nanoparticles,silica nanoparticles and titania nanoparticles.Furthermore,the development of Ru(bpy)_3~(2+)-doped silica nanoparticles in ECL was also disscused.
Chapter Two Electrogenerated Chemiluminescence Sensor for Metoclopramide Determination Based on Ru(bpy)_3~(2+)-doped Silica Nanoparticles Dispersed in Nafion on Glassy Carbon Electrode
The aim of the study presented here is to develop and validate a novel method for the determination of metoclopramide(MCP) with the electrogenerated chemiluminescence(ECL) by using tris(2,2′-bipyridyl)dichlororuthenium(Ⅱ)(Ru(bpy)_3~(2+))-doped silica(RuDS) nanoparticles/perfluoinated ion-exchange resin(Nafion) nanocomposites membrane modified glassy carbon electrode(GCE).The immobilization of Ru(bpy)_3~(2+) in a RuDS nanoparticles/Nafion nanocomposites membrane modified GCE was achieved in this study.The Ru(bpy)_3~(2+) encapsulation interior of the silica nanoparticle maintains its electrochemical activities and also reduces Ru(bpy)_3~(2+) leaching from the silica matrix when immersed in water due to the electrostatic interaction.The ECL analytical performances of this ECL sensor for MCP were investigated in detail.Under the optimum experimental conditions,it showed good linearity in the concentration range from 2.0×10~(-8) to 1.0×10~(-5) mol/L(R=0.9989) with a detection 7×10~(-9) mol/L.The RSD(n=11) was 3.2%for detecting 1.2×10~(-6) mol/L MCP.And the recoveries are in the range of 97.0～104.4%for sample measurements by standard-addition method. This method has been applied successfully to determine MCP in pharmaceutical preparations and in human urine.Statistical analysis(Student's t-test and variance ratio F-test) of the obtained results showed no significant difference between the proposed method and the reference method.
Chapter Three Electrogenerated Chemiluminescence Sensor for Itopride with Ru(bpy)_3~(2+)-doped Silica Nanoparticles/Chitosan Composite Films Modified Electrode
A electrogenerated chemiluminescence(ECL) sensor for itopride was developed based on tris(2,2′-bipyridyl)dichlororuthenium(Ⅱ)(mu(bpy)_3~(2+))-doped silica(RuDS) nanoparticles/biopolymer chitosan composites membrane modified glassy carbon electrode(GCE).The RuDS nanoparticles(52±5nm) were prepared by a modified St(o|¨)ber synthesis method and were characterized by electrochemical,fluorometric and transmission electron microscopy technology.The Ru(bpy)_3~(2+) encapsulation interior of the silica nanoparticle maintains its electrochemical activities and also reduces Ru(bpy)_3~(2+) leaching from the silica matrix when immersed in water due to the electrostatic interaction. The ECL analytical performances of this ECL sensor for itopride based on its enhancement ECL emission of Ru(bpy)_3~(2+) were investigated in details.Under the optimum condition,the enhanced ECL intensity was linear with the itopride concentration in the range of 1.0×10~(-8) to 2.0×10~(-5) g/mL(R= 0.9978).The detection limit was 3×10~(-9) g/mL,and the RSD was 2.3%for 8.0×10~(-8) g/mL itopride(n= 11).The method was successfully applied to the determination of itopride in pharmaceutical and human serum samples with satisfactory results.The as-prepared ECL sensor for the determination of itopride displayed good sensitivity and stability.
Chapter Four A Novel Electrogenerated Chemiluminescence Sensor Based on Ru(bpy)_3~(2+)-doped Titania Nanoparticles Dispersed in Nafion on Glassy Carbon Electrode
A novel electrogenerated chemiluminescence(ECL) sensor based on Ru(bpy)_3~(2+)-doped titania (RuDT) nanoparticles dispersed in a perfluorosulfonated ionomer(Nafion) on a glassy carbon electrode (GCE) was developed in this paper.The electroactive component-Ru(bpy)_3~(2+) was entrapped within the titania nanoparticles by the inverse microemulsion polymerization process that produced spherical sensors in the size region of 38±3nm.The RuDT nanoparticles were characterized by electrochemical,transmission electron and scanning microscopy technology.The Ru(bpy)_3~(2+) encapsulation interior of the titania nanoparticles maintains its ECL efficiency and also reduces Ru(bpy)_3~(2+) leaching from the titania matrix when immersed in water due to the electrostatic interaction. This is the first attempt to prepare the RuDT nanoparticles and extend the application of electroactive component-doped nanoparticles into the field of ECL.Since a large amount of Ru(bpy)_3~(2+) was immobilized three-dimensionally on the electrode,the Ru(bpy)_3~(2+) ECL signal could be enhanced greatly,which finally resulted in the increased sensitivity.The ECL analytical performance of this ECL sensor for tripropylamine(TPA) was investigated in detail.This sensor shows a detection limit of 1 nmol/L for TPA.Furthermore,the present ECL sensor displays outstanding long-term stability.
引文
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