Fluorescence ratiometric pH sensor prepared from covalently immobilized porphyrin and benzothioxanthene

A fluorescence ratiometric sensor for pH determination is described in this paper. The sensor incorporated the pH-sensitive dye meso-5,10,15,20-tetra-(4-allyloxyphenyl)porphyrin (TAPP) as an indicator and a pH-insensitive dye N-(2-methacryloxyethyl)benzo[k,l]thioxanthene-3,4-dicarboximide (MBTD), a benzothioxanthene derivative, as a reference for fluorescence ratiometric measurement. To prevent leakage of the dyes, both were photocopolymerized with acrylamide, hydroxyethyl methacrylate, and triethylene glycol dimethacrylate on the silanized glass surface. The reproducibility and response time of the prepared sensor were sufficient. Most common coexisting inorganic ions and organic compounds did not interfere with pH sensing. In the acidic pH range from 1.5 to 5.0 the fluorescence intensity ratio of the two dyes varied linearly as a function of pH. The sensing membrane was found to have a lifetime of at least one month. The sensor was applied to the analysis of waste water and artificial samples.     

Dual‐fluorophore Raspberry‐like Nanohybrids for Ratiometric pH Sensing

We report on the development of raspberry‐like silica structures formed by the adsorption of 8‐hydroxypyrene‐1,3,6‐trisulfonate (HPTS)@silica nanoparticles (NPs) on rhodamine B isothiocyanate (RBTIC)@silica NPs for ratiometric fluorescence‐based pH sensing. To overcome the well‐known problem of dye leaching which occurs during encapsulation of anionic HPTS dye in silica NPs, we utilized a polyelectrolyte‐assisted incorporation of the anionic HPTS. The morphological and optical characterization of the as‐synthesized dye‐doped NPs and the resulting nanohybrids were carried out. The pH‐sensitive dye, HPTS, incorporated in the HPTS‐doped silica NPs provided a pH‐dependent fluorescence response while the RBITC‐doped silica provided the reference signal for ratiometric sensing. We evaluated the effectiveness of the nanohybrids for pH sensing; the ratio of the fluorescence emission intensity at 510 nm and 583 nm at excitation wavelengths of 454 nm and 555 nm, respectively. The results showed a dynamic response in the acidic pH range. With this approach, nanohybrids containing different dyes or receptors could be developed for multifunctioning and multiplexing applications.     

Magnetic core-shell fluorescent pH ratiometric nanosensor using a Stöber coating method

We describe the use of a modified St?ber method for coating maghemite (γ-Fe(2)O(3)) nanocrystals with silica shells in order to built magnetic fluorescent sensor nanoparticles in the 50-70nm diameter range. In detail, the magnetic cores were coated by two successive silica shells embedding two fluorophores (two different silylated dye derivatives), which allows for ratiometric pH-measurements in the pH range 5-8. Silica coated magnetic nanoparticles were prepared using maghemite nanocrystals as cores (5-10nm in diameter) coated by tetraethoxyorthosilicate via hydrolysis/condensation in ethanol, catalyzed by ammonia. In the inner shell was covalently attached a sulforhodamine B, which was used as a reference dye; while a pH-sensitive fluorescein was incorporated into the outer shell. Once synthesized, the particles were characterized in terms of morphology, size, composition and magnetization, using dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD) and vibrating sample magnetometry (VSM). TEM analysis showed the nanoparticles to be very uniform in size. Wide-angle X-ray diffractograms showed, for uncoated as well as coated nanoparticles, typical peaks for the spinel structure of maghemite at the same diffraction angle, with no structural changes after coating. When using VSM, we obtained the magnetization curves of the resulting nanoparticles and the typical magnetization parameters as saturation magnetization (M(s)), coercivity (H(c)), and remanent magnetization (M(r)). The dual-dye doped magnetic-silica nanoparticles showed a satisfactory magnetization that could be suitable for nanoparticle separation and localized concentration of them. Changes in fluorescence intensity of the pH indicator in the different pH buffered solutions were observed within few seconds indicating an easy accessibility of the embedded dye by protons through the pores of the silica shell. The relationship between the ratio in fluorescence (sensor/reference dyes) and pH was adjusted to a sigmoidal fit using a Boltzmann type equation. Finally, the proposed method was statistically validated against a reference procedure using samples of water and physiological buffer with 2% (w/v) of horse serum added, indicating that there are no significant statistical differences at a 95% confidence level.     

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     

Nanoparticles as scaffolds for FRET-based ratiometric detection of mercury ions in water with QDs as donors

The quenching of quantum dots' emission by some analytes (Hg(2+), Pb(2+), etc.) has long been hindering the fabrication of QD-based 'turn-on' or ratiometric fluorescent sensors for these analytes. In this study, we demonstrate a facile solution for constructing a robust FRET-based ratiometric sensor for Hg(2+) detection in water with CdTe QDs as the donor. By using the reverse microemulsion approach, CdTe QDs were first embedded into nanosized silica particles, forming the QDs/silica cores, a positively charged ultrathin spacer layer was then deposited on each QDs/silica core, followed by the coating of a mercury ion probe on the particle surfaces. The resultant multilayered QDs/silica composite nanoparticles are dispersible in HEPES buffered water; and in the presence of mercury ions, the QDs inside the nanoparticles will not be quenched by mercury ions due to the existence of the positively charged spacer layer, but can transfer their excited energy to the acceptors (probe/Hg(2+) complex), thus achieving the FRET-based ratiometric sensing for mercury ions in totally aqueous media. With its detection limit of 260 nM, this QD-based sensor exhibits high selectivity toward mercury ion and can be used in a wide pH range. This strategy may be used to construct QDs-based ratiometric assays for other ions which quench the emission of QDs.     

Ratiometric sensing of mercury(II) based on a FRET process on silica core-shell nanoparticles acting as vehicles

We report on a fluorescence resonance energy transfer (FRET)-based ratiometric sensor for the detection of Hg(II) ion. First, silica nanoparticles were labeled with a hydrophobic fluorescent nitrobenzoxadiazolyl dye which acts as a FRET donor. A spirolactam rhodamine was then covalently linked to the surface of the silica particles. Exposure of the nanoparticles to Hg(II) in water induced a ring-opening reaction of the spirolactam rhodamine moieties, leading to the formation of a fluorescent derivative that can serve as the FRET acceptor. Ratiometric sensing of Hg(II) was accomplished by ratioing the fluorescence intensities at 520 nm and 578 nm. The average decay time for the donor decreases from 9.09 ns to 7.37 ns upon addition of Hg(II), which proves the occurrence of a FRET process. The detection limit of the assay is 100 nM (ca. 20 ppb). The sensor also exhibits a large Stokes shift (>150 nm) which can eliminate backscattering effects of excitation light.

Effects of surfactant/water ratio and dye amount on the fluorescent silica nanoparticles

Effects of surfactant/water volume ratios and dye amounts on the properties of micelles and fluorescence silica nanoparticles were studied in microemulsions containing nonionic surfactant Triton X-100, hexanol as co-surfactant, cyclohexane as organic solvent, and metal organic dye (tris(2,2′-bipyridyl)dichlororuthenium) via fluorescence probe technique, TEM, and XPS. Fluorescence probe measurements show that the micelle microenvironment becomes stable at the surfactant/water volume ratio higher than 3.5 and the incubation time longer than 10 h. The data suggest that the silica shell, which is formed at the surfactant/water ratio of 3.5, yields an efficient protection of dye molecules against the e-beam irradiation and result in high photostability of fluorescent silica. We pioneered the localization of dye molecules on the surface of dye-doped silica and found that an increase of dye amounts, beyond a threshold, in the microemulsion cannot enhance the fluorescence intensity of dye-doped nanoparticles. These results are of significant importance for optimizing the synthesis of fluorescent nanoparticles with high photostability and low cost.     

新型有机荧光染料嵌合的核壳荧光纳米材料的研制

采用油包水的反相微乳液方法,首次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,成功地制备了FITC的核壳荧光纳米颗粒,克服了采用传统方法制备核壳荧光纳米颗粒中存在的荧光染料泄露的问题.制备的这种核壳荧光纳米颗粒比细胞小很多,且具有生物亲和性,可为纳米生物传感器件提供新型材料.基于该核壳荧光纳米颗粒的标记方法也为生物医学提供了一种新型的非同位素分析方法.     

基于聚合物核壳纳米粒子的Hg~(2+)比率型荧光检测传感器

采用一步聚合的方法,制备了以疏水的聚甲基丙烯酸甲酯(PMMA)为核、亲水性的聚电解质支化聚乙烯亚胺(PEI)为壳的纳米粒子分散液.将供体荧光团4-胺基-7-硝基-N-辛基苯并[1,2,5]噁二唑(NBD)以包埋的方式在聚合过程中直接引入PMMA核内部,而受体荧光团罗丹明衍生物SRHB通过吸附作用进入PEI-PMMA核壳界面,构成了含有两种不同荧光分子且可对Hg2+进行荧光比率检测的传感器.考察了含荧光分子的聚合物粒子光谱学性质,证明两种荧光分子均被引入了聚合物粒子体系.在汞离子的荧光检测试验中,加入Hg2+后,体系中的NBD荧光强度下降,而罗丹明的特征发射峰在579 nm处出现,并随着Hg2+浓度的增加,受体/供体的荧光强度比值呈现增长趋势.研究还发现,聚合物粒子基荧光探针对于Hg2+具有较好的选择性,且最佳使用范围是体系pH值在5～8之间,其检测Hg2+的最低浓度可达到1μmol/L.     

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,     

Dimerization of Organic Dyes on Luminescent Gold Nanoparticles for Ratiometric pH Sensing

Synergistic effects arising from the conjugation of organic dyes onto non‐luminescent metal nanoparticles (NPs) have greatly broadened their applications in both imaging and sensing. Herein, we report that conjugation of a well‐known pH‐insensitive dye, tetramethyl‐rhodamine (TAMRA), to pH‐insensitive luminescent gold nanoparticles (AuNPs) can lead to an ultrasmall nanoindicator that can fluorescently report local pH in a ratiometric way. Such synergy originated from the dimerization of TAMRA on AuNPs, of which geometry was very sensitive to surface charges of the AuNPs and can be reversely modulated through protonation of surrounding glutathione ligands. Not limited to pH‐insensitive dyes, this pH‐dependent dimerization can also enhance the pH sensitivity of fluorescein, a well‐known pH‐sensitive dye, within a larger pH range, opening up a new pathway to design ultrasmall fluorescent ratiometric nanoindicators with tunable wavelengths and pH response ranges.     

Synthesis and evaluation of self-calibrating ratiometric viscosity sensors

We describe the design, synthesis and fluorescent profile of a family of self-calibrating dyes that provide ratiometric measurements of fluid viscosity. The design is based on covalently linking a primary fluorophore (reference) that displays a viscosity-independent fluorescence emission with a secondary fluorophore (sensor) that exhibits a viscosity-sensitive fluorescence emission. Characterization of fluorescent properties was made with separate excitation of the units and through Resonance Energy Transfer from the reference to the sensor dye. The chemical structures of both fluorophores and the linker length have been evaluated in order to optimize the overall brightness and sensitivity of the viscosity measurements. We also present an application of such ratiometric dyes for the detection of membrane viscosity changes in a liposome model.     

pH响应的介孔二氧化硅纳米颗粒的制备及可控释放

选择带负电荷且溶解度和分子结构对pH值非常敏感的聚丙烯酸作为封堵分子, 采用静电吸附的修饰方法, 制备了pH响应的MCM-41型介孔二氧化硅纳米颗粒. 利用高倍透射电子显微镜(TEM)、 X射线衍射(XRD)、 傅里叶变换红外光谱(FTIR)及比表面积分析等手段表征了介孔二氧化硅纳米颗粒的物理化学性质. 以联钌吡啶染料分子作为模式客体分子, 研究了pH调控下的模式客体分子在介孔二氧化硅纳米颗粒中的包裹及释放行为. 结果表明, 该介孔二氧化硅纳米颗粒对pH具有很好的响应性; 在近中性条件下, 带正电的二氧化硅纳米颗粒通过静电吸附作用吸附带负电的聚丙烯酸, 导致介孔封堵, 使包载的染料分子几乎无释放; 客体分子的释放率随着pH值的降低而升高, 当pH≤5时, 染料分子显著释放, pH=1时客体分子的释放率高达98％, 可以实现对包载客体分子的控制释放. 该pH响应的介孔二氧化硅纳米颗粒载体具有制备简便、 价格低廉和包载量大等优点, 有望应用于药物的控制释放.     

Uniform silica nanoparticles encapsulating two-photon absorbing fluorescent dye

We have prepared uniform silica nanoparticles (NPs) doped with a two-photon absorbing zwitterionic hemicyanine dye by reverse microemulsion method. Obvious solvatochromism on the absorption spectra of dye-doped NPs indicates that solvents can partly penetrate into the silica matrix and then affect the ground and excited state of dye molecules. For dye-doped NP suspensions, both one-photon and two-photon excited fluorescence are much stronger and recorded at shorter wavelength compared to those of free dye solutions with comparative overall dye concentration. This behavior is possibly attributed to the restricted twisted intramolecular charge transfer (TICT), which reduces fluorescence quenching when dye molecules are trapped in the silica matrix. Images from two-photon laser scanning fluorescence microscopy demonstrate that the dye-doped silica NPs can be actively uptaken by Hela cells with low cytotoxicity.     

Synthesis and characterization of surface-enhanced Raman scattering tags with Ag/SiO2 core-shell nanostructures using reverse micelle technology

A novel simplified method for synthesis of surface-enhanced Raman scattering tags has been reported. This synthesis method is based on reverse micelle technique using Igepal CO-520 as a surfactant and the mixed solution of silver nitrate and rhodamine dyes with isothiocyanate group as water pool followed by hydrazine hydrate reduction and TEOS polymerization leading to the formation of silica layer surrounding the silver core. Compared to the method reported in literature, the proposed methodology eliminates the necessity of vitrophilic pretreatment and makes it possible to complete all different processes including the preparation of silver nanoparticles, the conjugation of dye molecules and the formation of silica shell in the microreactor. The nanoparticle-based surface-enhanced Raman tags obtained are composed of silver core conjugated with rhodamine dyes and an encasing silica shell. Both the dyes themselves and the Ag/SiO2 core-shell nanoparticles without the encapsulation of dyes exhibit no Raman signals. However, the Ag/SiO2 core-shell nanoparticles exhibit strong Raman signals when encapsulated with these dyes. This is due to the appearance of fluorescence quenching and surface-enhanced Raman scattering effect resulting from the conjugation of dyes and silver core. The Raman tags were characterized using transmission electron microscopy (TEM), UV-visible absorption spectrometry, and Raman spectrometry.     

Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging

Polyacrylamide-based, ratiometric, spherical, optical nanosensors, or polyacrylamide PEBBLEs (Probes Encapsulated By Biologically Localized Embedding), have been fabricated, aimed at real-time glucose imaging in intact biological systems, i.e. living cells. These nanosensors are prepared using a microemulsion polymerization process, and their average size is about 45 nm in diameter. The sensors incorporate glucose oxidase (GOx), an oxygen sensitive fluorescent indicator (Ru[dpp(SO3Na)2]3)Cl2, and an oxygen insensitive fluorescent dye, Oregon Green 488-dextran or Texas Red-dextran, as a reference for the purpose of ratiometric intensity measurements. The enzymatic oxidation of glucose to gluconic acid results in the local depletion of oxygen, which is measured by the oxygen sensitive ruthenium dye. The small size and inert matrix of these sensors allows them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. The PEBBLE matrix protects the enzyme and fluorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis. Conversely, the matrix also significantly reduces the toxicity of the indicator and reference dyes to the cells, so that a larger variety of dyes can be used in optimal fashion. Furthermore, the PEBBLE matrix enables the synergistic approach in which there is a steady state of local oxygen consumption, and this cannot be achieved by separately introducing free enzyme and dyes into a cell. The work presented here describes the production and characterization of glucose sensitive PEBBLEs, and their potential for intracellular glucose measurements. The sensor response is determined in terms of the linear range, ratiometric operation, response time, sensor stability, reversibility and immunity to interferences.     

双重对象同时检测的新型荧光纳米粒子传感器的制备

报道了一种基于荧光共振能量转移理论合成的新型纳米粒子. 新型纳米粒子A中掺杂了由亲和素-生物素桥联并可发生荧光共振能量转移(FRET)的供体染料和受体染料; 新型纳米粒子B中只掺杂了供体染料. 在使用供体染料的特征激发波长同时激发纳米粒子A和B时, 纳米粒子A由于荧光共振能量转移而发射出受体染料的特征波长, 而纳米粒子B则只发射供体染料的特征波长. 这样, 在单一波长激发下, 可很容易地实现双重对象同时检测.     

Ratiometric pH sensor based on mesoporous silica nanoparticles and Förster resonance energy transfer

Incorporation of a dual-FRET dye pair into mesoporous silica nanoparticles yields sensitive and sensing-range tunable nanosensors with good reversibility that can be used for ratiometric pH measurements under a single-wavelength excitation.     

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.     

Dextran-coated silica nanoparticles for calcium-sensing

This article describes the synthesis and characterisation of fluorescent composite nanoparticles consisting of a silica core and a dextran shell. The silica core contains a rhodamine-based reference dye, which allows ratiometric measurements and the dextran shell is labelled with the Ca(2+)-sensitive dye Fluo-4. The nanoparticles have an average hydrodynamic diameter of 95 nm, good colloidal stability and show a 2.9-fold increase in fluorescence intensity upon binding to Ca(2+) ions. The apparent dissociation constant of K'(d) ≈ 520 nM is well suited for measurements in the physiological range.