Preparation And Application Of Nanoparticles Probes

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 C₁(SEC₁),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)₃²⁺-doped silica nanoparticles/chitosan,perfluoinated ion-exchange resin and Ru(bpy)₃²⁺-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 ImagingChapter One The Development of Fluorescent Nanoparticle Probe for Cancer ImagingIn 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 ImagingAlthough 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 ImagingA 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 FluoroimmunoassayChapter One The Development of Fluoroimmunoassay Using Nanoparticles as LabelsIn 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-6Nanoparticle 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 C₁ Fluoroimmunoassay Based on Fluorescent Nanoparticle LabelsA highly sensitive fluoroimmunoassay for the determination of staphylococcal enterotoxin C₁ (SEC₁) is proposed.It is based on the fluorescent nanoparticles as the label coated with anti-SEC₁ 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)₃Cl₂(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-SEC₁ monoclonal antibodies and the antibody-labeled nanoparticles were successfully used for the determination of SEC₁.The calibration graph for SEC₁ 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[SEC₁](ng/mL)(R=0.9991).The RSD for eleven parallel measurements of 25.0 ng/mL SEC₁ 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 SEC₁ and is applicable to the determination of SEC₁ in real samples and enabling fluorescence microscopy imaging for the determination of SEC₁.Chapter Four Fluoroimmunoassay for Tumor Necrosis Factor-αin Human Serum Using Fluorescent Nanoparticles as LabelsA 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(â…¡) 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 ElectrodeChapter One The Progress of Electrogenerated Chemiluminescence Sensor Based on Nanoparticles Modified ElectrodeThe 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)₃²⁺-doped silica nanoparticles in ECL was also disscused.Chapter Two Electrogenerated Chemiluminescence Sensor for Metoclopramide Determination Based on Ru(bpy)₃²⁺-doped Silica Nanoparticles Dispersed in Nafion on Glassy Carbon ElectrodeThe 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(â…¡)(Ru(bpy)₃²⁺)-doped silica(RuDS) nanoparticles/perfluoinated ion-exchange resin(Nafion) nanocomposites membrane modified glassy carbon electrode(GCE).The immobilization of Ru(bpy)₃²⁺ in a RuDS nanoparticles/Nafion nanocomposites membrane modified GCE was achieved in this study.The Ru(bpy)₃²⁺ encapsulation interior of the silica nanoparticle maintains its electrochemical activities and also reduces Ru(bpy)₃²⁺ 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)₃²⁺-doped Silica Nanoparticles/Chitosan Composite Films Modified ElectrodeA electrogenerated chemiluminescence(ECL) sensor for itopride was developed based on tris(2,2′-bipyridyl)dichlororuthenium(â…¡)(mu(bpy)₃²⁺)-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)₃²⁺ encapsulation interior of the silica nanoparticle maintains its electrochemical activities and also reduces Ru(bpy)₃²⁺ 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)₃²⁺ 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)₃²⁺-doped Titania Nanoparticles Dispersed in Nafion on Glassy Carbon ElectrodeA novel electrogenerated chemiluminescence(ECL) sensor based on Ru(bpy)₃²⁺-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)₃²⁺ 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)₃²⁺ encapsulation interior of the titania nanoparticles maintains its ECL efficiency and also reduces Ru(bpy)₃²⁺ 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)₃²⁺ was immobilized three-dimensionally on the electrode,the Ru(bpy)₃²⁺ 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.