嵌合异硫氰酸罗丹明B核壳荧光纳米颗粒制备的新方法研究

随着纳米技术的发展,结合了纳米技术与材料制备技术而发展起来的荧光染料嵌合的核壳荧光纳米颗粒的制备为生物医学领域的研究提供了新的材料、技术和方法。何晓晓等以联钉吡啶配合物为核材料,制备了嵌合无机金属配合物的核壳荧光纳米颗粒,段菁华等用异硫氰酸荧光素FITC与蛋白质IgG相结合,     

吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒的制备及其性质研究

以蛋白质或多肽修饰的吲哚类菁染料Cy3为内核, 采用实验条件简单的油包水反相微乳液方法成核, 通过正硅酸乙酯水解形成的网状二氧化硅包壳的方法制备吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒. 考察了以不同等电点的蛋白质和多肽修饰的Cy3为内核材料对吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒制备的影响. 结果表明, 分别采用人免疫球蛋白(IgG)或多聚赖氨酸修饰的Cy3为内核材料, 都能制备荧光强度高、荧光稳定性强和染料泄漏极少的Cy3嵌入的核壳荧光纳米颗粒. 进一步对Cy3嵌入的核壳荧光纳米颗粒进行了表征, 并将基于这一新型的荧光纳米颗粒建立起来的生物标记方法初步应用于流感病毒DNA的检测, 其检测线性范围为3.18×10-10～1.27×10-9 mol/L, 检测下限为3.51×10-10 mol/L, 相关系数r为0.986 5.     

基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.     

异硫氰酸荧光纳米复合粒子的研制与应用

采用改进的St ber法制备了二氧化硅外壳的纳米复合荧光粒子。利用异硫氰酸荧光素(FITC)与3-氨基丙基三乙氧基硅烷(APTEOS)反应制备前驱体,再用正硅酸乙酯(TEOS)在一定的条件下水解与缩合,制备有机-无机纳米复合荧光颗粒,利用透射电子显微镜(TEM)测试表明,此纳米复合颗粒呈球形、大小均一,直径约为70 nm。制备的纳米复合荧光粒子经过多次水洗后,仍有较强的荧光特性,有效地防止FITC泄露。用激光共聚焦显微镜观测纳米复合荧光粒子标记的牛血清白蛋白(BSA),可以明显看出BSA上的绿色荧光。     

新型FITC掺杂的二氧化硅荧光纳米粒子的合成及其应用于pH传感的研究

将异硫氰酸荧光素(FITC)与3-氨基丙基三甲氧基硅烷(APTMS)反应制得前驱体FITC-APTMS,采用油包水微乳液法,利用APTMS与正硅酸乙酯(TEOS)的共水解与聚合作用,制备了FITC掺杂的二氧化硅核壳型荧光纳米粒子。经TEM与荧光光谱表征及光稳定性实验与染料泄露实验等,表明所制得纳米颗粒呈规则球形,粒径为(150±15)nm,具有良好的单分散性与光稳定性,不易发生染料泄露。这种纳米颗粒对pH值敏感,在pH3.6～9.7范围内,荧光强度与溶液酸度有良好的响应,其中在pH6.0～9.0之间呈良好的线性关系,此纳米颗粒能被单个小鼠神经干细胞吞噬,可应用于细胞的pH值实时监测。     

新型双荧光二氧化硅纳米颗粒的制备

本文采用改良的反相微乳液法制备得到双染料掺杂的二氧化硅荧光纳米颗粒。扫描电镜结果显示,荧光纳米颗粒呈球形,平均粒径约为90nm。利用荧光光谱及共聚焦荧光显微镜对该纳米颗粒的光学性能进行表征,结果发现其可同时发出红、绿双色荧光,这为进一步制备具有更多种荧光信号的硅纳米颗粒提供基础。同时,由于硅纳米颗粒生物相容性好,易功能化的特点,使得该新型硅纳米荧光颗粒在生物标记及生物成像的多组分分析中具有潜在的应用。     

温敏性荧光纳米材料的合成

合成了以荧光材料罗丹明B/SiO2为核,交联聚N-异丙基丙烯酰胺(PNIPAM)为壳的具有核/壳结构的纳米颗粒。用氢氟酸除去二氧化硅模板核后,形成了核壳结构的温敏荧光微球。28～36℃范围内的温敏性实验表明,该粒子的低临界溶解温度(LCST)为33℃,具有温敏性,用SEM、TEM、XRD、FT-IR等手段对温敏荧光微球的组成和结构进行了表征和分析,探讨了磁性温敏纳米颗粒的制备机理。     

(FITC/SiO2-CdTe QDs)-SiO2复合结构编码荧光纳米颗粒的制备

制备了一种(FITC/SiO2-CdTe QDs)-SiO2复合结构编码荧光纳米颗粒。荧光光谱、扫描隧道显微镜和透射电镜对FITC/SiO2和(FITC/SiO2-CdTe QDs)-SiO2等荧光纳米颗粒进行了表征。结果表明,FITC/SiO2NPs和(FITC/SiO2-CdTe QDs)-SiO2均呈球形,粒径分别约为200和300 nm,分布均匀,且具有可分辨的双重荧光信号。并用Cu2+对(FITC/SiO2-CdTe QDs)-SiO2编码荧光纳米颗粒进行了后编码。     

硅壳包被的核壳型量子点荧光纳米颗粒的制备及其细菌计数的应用

以氧化镉和硬脂酸锌为前驱体,合成了CdSe/ZnS核壳型量子点(QDs).采用反相微乳液技术,在温和条件下实现了硅壳包被的CdSe/ZnS荧光纳米颗粒的成功制备.在戊二醛的交联作用下,以金黄色葡萄球菌(S.aureus)为目标细菌、荧光纳米颗粒为荧光探针,建立了一种高灵敏的、简单快速的细菌计数的方法,并借助荧光显微镜成功地进行成像探测研究.通过考察荧光纳米颗粒与细菌的孵育时间、包入硅壳的核壳量子点质量等多种因素的影响.在最优化条件下,本方法的线性范围为5×102～5×107 CFU/mL;检出限为500 CFU/mL;线性回归方程为Y=494.96749X- 1194.25738(R=0.9960).本方法操作简单,检测时间短,有效克服了传统平板计数方法和基于有机染料的荧光检测方法存在的缺陷,提高了灵敏度.将此法用于6种实际样品的细菌数量测定,检测结果与平板计数方法基本一致,相对标准偏差在3.1％～8.2％之间,结果令人满意.     

纳米粒子标记激光诱导荧光免疫分析测定癌胚抗原

建立了一种核壳型SiO₂荧光纳米粒子标记的激光诱导荧光免疫测定的新方法. 该方法用 掺杂的核壳型SiO₂荧光纳米粒子为标记物, 采用双抗体夹心法, 在载玻片上固定鼠抗人癌胚抗原(CEA)单克隆抗体, 加入1～2 μL样品, 再加入纳米粒子标记的另一种鼠抗人癌胚抗原(CEA)单克隆抗体. 识别后在光导纤维激光诱导荧光毫米阵列检测平台上测量荧光信号, 进行定量. 在优化条件下, 荧光强度和癌胚抗原的量在1～80 pg的范围内具有良好的线性关系, 相关系数r＝0.9933, 方法的检出限为0.3 pg (20 amol). 对40 pg的CEA平行测定5次, 相对标准偏差为5.5%.     

Preparation of luminescent Cy5 doped core-shell SFNPs and its application as a near-infrared fluorescent marker

Cy5 dye is widely used as a biomarker in the research fields of life science because of its excitation at wavelengths above 600 nm where autofluorescence of bio-matter is much reduced. However, Cy5 dye could not be encapsulate into silica directly to form stable nanoparticles by using of the traditional methods. In this paper, an improved method had been developed to prepare Cy5 dye doped core-shell silica fluorescent nanoparticles (SFNPs), employing biomolecules conjugated Cy5 as the core material and silica coating produced from the hydrolysis TEOS (tetraethyl orthosilicate) in the water-in-oil microemulsion. To obtain stable Cy5 dye doped SFNPs with core-shell structure, five kinds of biomolecules with different iso-electric point (pI) have been selected to conjugate Cy5 for preparation of core-shell SFNPs. Results demonstrated that very bright and photostable Cy5 doped core-shell SFNPs could be both prepared by use of positive polysine conjugated Cy5 or IgG conjugated Cy5 as the core material, respectively. IgG conjugated Cy5 doped core-shell SFNPs was selected as a demonstration to be characterized and applied as a near-infrared fluorescent marker in cell recognition. The results showed that Cy5 doped core-shell SFNPs prepared by conjugating with a positive biomolecules IgG as the core material were luminescent and stable. About 110 Cy5 dye molecules could be doped in one nanoparticle with size of 42 ± 5 nm. The breast cancer cells had been selectively recognized by use of the near-infrared fluorescent marker based on the Cy5-IgG doped core-shell SFNPs. And the results demonstrated that this Cy5 doped core-shell SFNPs fluorescence marker was superior to the pure Cy5 dye marker for cell recognition in photostability and detection sensitivity.     

A fluorescence ratiometric nano-pH sensor based on dual-fluorophore-doped silica nanoparticles

We have synthesized dual-fluorophore-doped core-shell silica nanoparticles used as ratiometric pH sensor. The nanoparticles were prepared with a reverse microemulsion technique by simultaneously encapsulating two different fluorophores, the pH-sensitive dye fluorescein as a pH indicator and the pH-insensitive dye phenosafranine as an internal reference for fluorescence ratiometric measurement, into silica shell. The nanoparticles prevent the fluorescence dyes leaching from the silica matrix when immersed inside water. The hydrophilic silica shells were made by hydrolysing and polymerizing tetraethoxysilane (TEOS) in water-in-oil microemulsion. The fluorescence intensity ratio of the two dyes varied linearly as a function of pH in the range from 4.0 to 8.0. The sensor was also applied to measure pH of real water samples. The results are in good agreements with that using the conventional glass electrode method. The as-prepared fluorescent nanoparticles showed rapid response, excellent stability and high reproducibility as pH sensors.     

Core‐shell type dually fluorescent polymer nanoparticles for ratiometric pH‐sensing

The synthesis and pH‐sensing properties of fluorescent polymer nanoparticles (NPs) in the 20 nm diameter range with a sensitive dye covalently attached to the particle surface and a reference dye entrapped within the particle core are presented. Fluorescein‐functionalized NPs were readily obtained by conjugation of fluorescein isothiocyanate (FITC) to amine‐coated crosslinked polystyrene‐based nanoparticles prepared by microemulsion polymerization followed by postfunctionalization. This all water‐based method gave access to stable aqueous suspensions of pH‐sensing fluorescent NPs. The encapsulation of the insensitive reference fluorescent dye (1,9‐diphenylanthracene, DPA) was then conveniently achieved by soaking leading to dual fluorescent NPs containing about 20 DPA and 55 fluorescein, as deduced from spectroscopic analyses. This core‐shell type architecture maximizes the interactions of the sensing dye with the medium while protecting the reference dye. The variations of the ratio of the fluorescence emission intensities of the sensitive dye (fluorescein) to the reference dye (DPA) with pH show that the dual fluorescent NPs act as a ratiometric pH sensor with a measuring range between pH 4 and pH 8. This pH nanosensor was found to be fast, fully reversible, and robust without any leaching of dye over a long period of time. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6206–6213, 2008     

Fluorescence Enhancement of IgG-FITC Based on Surface Plasmon Resonance of Ag@SiO2 Nanoparticles as Application of Biomarker

Ag@SiO₂ nanoparticles with the core-shell structure have been prepared, of which the silver core was about 50 nm and the thickness of silica shell was approximately 10 nm. In slightly alkaline aqueous solution (pH = 8), through electrostatic force between cationic polymer PDDA (i.e., poly-diallyldimethylammonium chloride) and the obtained Ag@SiO₂ nanoparticles, PDDA molecules were fixed on the surface of Ag@SiO₂ nanoparticles. The prepared Ag@SiO₂/PDDA nanoparticles have both rich positive surface charges and rich micro-holes of silica shell. Based on micro-hole adsorption, the small molecule FITC (i.e., fluorescein isothiocyanate) marking on IgG (i.e., immunoglobulin) was adsorbed into the rich microholes of silica shell; at the same time, the negatively charge macromolecule IgG marked by FITC was firmly fixed on the rich positive charges surface of Ag@SiO₂/PDDA nanoparticles by electrostatic interaction. And then, Ag@SiO₂/PDDA/IgG-FITC fluorescent nanoparticles with the SPR fluorescence enhancement were prepared. The shell-type SiO₂/PDDA/IgG-FITC nanoparticles were obtained by dissolving the silver core in the prepared core-shell Ag@SiO₂/PDDA/IgG-FITC nanoparticles by using H₂O₂. Compared with the shell-type nanoparticles, the fluorescence intensity of Ag@SiO₂/PDDA/IgG-FITC was enhanced 1.7 times. The prepared Ag@SiO₂/PDDA/IgG-FITC nanoparticles have both SPR-based fluorescence enhancement ability and the surface distributing IgG–based obvious advantages including good biocompatibility and easy marking with other biomolecules.     

Controlled synthesis of fluorescent silica nanoparticles inside microfluidic droplets

We study the droplet-based synthesis of fluorescent silica nanoparticles (50-350 nm size) in a microfluidic chip. Fluorescein-isothiocyanate (FITC) dye is first chemically linked to aminopropyl triethoxysilane (APTES) in ethanol and this reaction product is subsequently mixed with tetraethyl orthosilicate (TEOS) to yield a fluorescent silicon alkoxide precursor solution. The latter reacts with an aqueous ethanol-ammonia hydrolysing mixture inside droplets, forming fluorescent silica nanoparticles. The droplets are obtained by pinching-off side-by-side flowing streams of alkoxide solution/hydrolysing mixture on a microfluidic chip using a Fluorinert oil continuous phase flow. Synthesis in droplets leads to a faster reaction and allows drastically improved nanoparticle size uniformity (down to 3% relative standard deviation for 350 nm size particles) when compared to conventional bulk synthesis methods, thanks to the precise control of reagent concentrations and reaction times offered by the microfluidic format. Incorporating FITC inside silica nanoparticles using our method leads to reduced dye leakage and increases the dye's stability, as evidenced by a reduced photochemical bleaching compared to a pure FITC solution.     

Preparation and Characterization of PEGylated Thiophilic Nanoparticles for Rapid Antibody Separation

Magnetic nanoparticles with novel core-shell structure were prepared for immunoglobulin (IgG) separation, in which thiophilic property of sulfone groups and protein resistance of poly(ethylene glycol) (PEG) moieties were integrated. The step-wise surface reactions on the nanoparticles were characterized by ¹H nuclear magnetic resonance (NMR) and surface zeta potential measurements. With human IgG and bovine serum albumin (BSA) as model proteins, the effects of PEG chain length, conjugation group, solution pH and salt concentration on IgG selectivity were investigated using static adsorption experiments. The experiment results showed that mPEG2000-NH₂ modified magnetic nanoparticles had an adsorption capacity of 132.8 mg g^?1 and selectivity of 32.5 towards IgG under the condition of pH 7.45 and 0.15 M NaCl. In complex biological fluids, the PEG modified magnetic nanoparticles could separate IgG from fetal calf serum and Omalizumab from cell culture supernatant with purities of 96% and 99%, respectively. Moreover, the binding affinities of the proposed core-shell structure towards IgG from four animal species (human, bovine, rabbit and goat) were quantified by bio-layer interferometer (BLI). The results showed that the selectivity of this structure towards IgG varied from traditional Protein A method, suggesting its potentials in rapid separation and purification of IgG with low affinity towards Protein A.     

Synthesis and characterization of new fluorescent nanoparticles

A novel kind of fluorescent nanoparticles（FNPs）has been prepared using a precipitation polymerization method.Methacrylic acid,trimethylolpropane trimethacrylate and azobisisobutyronitrile were used as functional-monomer,cross-linker and initiator, respectively.Compared with other fluorescent nanoparticles,the FNPs have the characteristics including low dye leakage and good photostability.The fluorescence microscopy imaging indicates that the FNPs can be used as fluorescent labels in bioanalysis.     

A core-shell nanoparticle approach to photoreversible fluorescence modulation of a hydrophobic dye in aqueous media

Amphiphilic core-shell nanoparticles containing spiropyran moieties have been prepared in aqueous media. The nanoparticles consist of hydrophilic and biocompatible poly(ethyleneimine) (PEI) chain segments, which serve as the shell, and a hydrophobic copolymer of methyl methacrylate (MMA), a spiropyran-linked methacrylate, and a cross-linker, which forms the core of the nanoparticles. A hydrophobic fluorescent dye based on the nitrobenzoxadiazolyl (NBD) group was introduced into the nanoparticles to form NBD-nanoparticle complexes in water. The nanoparticles not only greatly enhance the fluorescence emission of the hydrophobic dye NBD in aqueous media, probably by accommodating the dye molecules in the interface between the hydrophilic shells and the hydrophobic cores, but also modulate the fluorescence of the dye through intraparticle energy transfer. This biocompatible and photoresponsive nanoparticle complex may find applications in biological areas such as biological diagnosis, imaging, and detection. In addition, this nanoparticle approach will open up possibilities for the fluorescence modulation of other hydrophobic fluorophores in aqueous media.