Molecular oxygen-sensitive fluorescent lipobeads for intracellular oxygen measurements in murine macrophages

Intracellular oxygen concentration is of primary importance in determining numerous physiological and pathological processes in biological systems. This paper describes the development and application of micrometer-sized oxygen-sensitive fluorescence lipobeads for intracellular measurements of molecular oxygen in J774 murine macrophages. A ruthenium diimine complex [Ru(bpy-pyr)(bpy)2]C12 (bpy = 2,2'-bipyridine, bpy-pyr = 4-(1"-pyrenyl)-2,2'-bipyridine) is used as the oxygen indicator. The indicator exhibits high chemical and photostability and high sensitivity to oxygen. The indicator molecules are immobilized in a phospholipid membrane that coats polystyrene microparticles. The fluorescence of the lipobeads is effectively quenched by molecular oxygen. The fluorescence intensity of the oxygen-sensitive lipobeads is 3 times higher in a nitrogenated solution than in an oxygenated solution. The lipobeads are internalized by murine macrophages through phagocytosis. They maintain their spectral properties for 24 h in living cells when the cells are stored in phosphate-buffered saline at pH 7.4. The photostability, reversibility, and effect of hypoxia, hyperoxia, and oxidative stress on the intracellular level of oxygen in J774 murine macrophages are described.     

Development and critical evaluation of fluorescent chloride nanosensors

In this study, we describe the preparation and evaluation of new fluorescent sensor nanoparticles for the ratiometric measurement of chloride concentrations. Both a chloride-sensitive dye (lucigenin) and a reference dye (sulforhodamine derivative) were incorporated into polyacrylamide nanoparticles via inverse microemulsion polymerization and investigated for their response to chloride ions in buffered suspension as well as in living cells. The fluorescence intensity of lucigenin reversibly decreased in the presence of chloride ions due to a collisional quenching process, which can be described with the Stern-Volmer equation. The determined Stern-Volmer constant K SV for the quenching of lucigenin incorporated into particles was found to be 53 M (-1) and is considerably smaller than the Stern-Volmer constant for quenching of free lucigenin ( K SV = 250 M (-1)) under the same conditions. To test the nanosensors in living cells, we incorporated them into Chinese hamster ovary cells and mouse fibroblasts by using the conventional lipofectamin technique and monitored the response to changing chloride concentrations in the cell.     

Copper ion-selective fluorescent sensor based on the inner filter effect using a spiropyran derivative

A highly selective copper(II) ion fluorescent sensor has been designed based on the UV-visible absorption of a spiropyran derivative coupled with the use of a metal porphyrin operative on the fluorescence inner filter effect. Spiropyrans, which combine the characteristics of metal binding and signal transduction, have been widely utilized in cationic ion recognition by UV-visible spectroscopy. In the present work, the viability of converting the absorption signal of the spiropyran molecule into a fluorescence signal was explored. On account of overlap of the absorption band of the spiropyran (lambda(abs) = 547 nm) in the presence of copper ion with the Q-band of an added fluorophore, zinc meso-tetraphenylporphyrin (lambda(abs) = 556 nm), the effective light absorbed by the porphyrin and concomitantly the emitted light intensity vary as a result of varying absorption of the spiropyran via fluorescence inner filter effect. The metal binding characteristic of the spiropyran presents an excellent selectivity for copper ion in comparison with several other heavy or transition metal ions. Since the changes in the absorbance of the absorber translate into exponential changes in fluorescence of the fluorophore, the novelty of the present device is that the analytical signal is more sensitive over that of the absorptiometry or that of the fluorometry using one single dye. To realize a practical fluorescent sensor, both the absorber and fluorophore were immobilized in a plasticized poly(vinyl chloride) membrane, and the sensing characteristics of the membrane for copper ion were investigated. The sensor is useful for measuring Cu2+ at concentrations ranging from 7.5 x 10(-7) to 3.6 x 10(-5) M with a detection limit of 1.5 x 10(-7) M. The sensor is chemically reversible, the fluorescence was switched off by immersing the membrane in copper ion solution and switched on by washing it with EDTA solution.     

Synthesis, characterization, and application of fluorescence sensing lipobeads for intracellular pH measurements

This paper describes the synthesis and characterization of micrometric phospholipid-coated polystyrene particles, named lipobeads, with pH-sensing capability and their application for intracellular pH measurements in murine macrophages. The phospholipids used to coat the particles are labeled with fluorescein (a pH-sensitive dye) and tetramethylrhodamine (a pH-insensitive dye), which serves as a referencing fluorophore for increased accuracy of the pH measurements. The synthesis of the pH-sensing lipobeads is realized by the covalent attachment of the fluorescent phospholipids to the surface of carboxylated polystyrene particles. The pH dynamic range of the sensing particles is between 5.5 and 7.0 with a sensitivity of 0.1 pH unit. The excitation light intensity is reduced to minimize photobleaching of the fluorescein-phospholipid conjugates. The fluorescent lipobeads are used to measure the pH in single macrophages. The lipobeads are ingested by the macrophages and directed to lysosomes, which are the cellular organelles involved in the phagocytosis process. Despite the high lysosomal levels of digestive enzymes and acidity, the absorbed particles remain stable for over 6 h in the cells when they are stored in a phosphate-buffered saline solution at pH 7.4.     

3,3',5,5'-tetramethyl-N-(9-anthrylmethyl)benzidine: a dual-signaling fluorescent reagent for optical sensing of aliphatic aldehydes

A new optical chemical sensor for continuous monitoring of aliphatic aldehydes has been proposed based on the reversible chemical reaction between a new sensing reagent, 3,3',5,5'-tetramethyl-N-(9-anthrylmethyl)benzidine (TMAB), and the analytes. TMAB, containing two receptors and two fluorescent reporters, can perform dual fluorescence responses corresponding to the reactions of hydrogen ion and carbonyl compound. When immobilized in a plasticized poly(vinyl chloride) membrane, TMAB extracts aliphatic aldehydes from aqueous solution into the bulk membrane phase and reacts with the analyte by forming a Schiff base. Since the extraction equilibrium and chemical reaction are accompanied by fluorescence increase of the sensing membrane, the chemical recognition process could be directly translated into an optical signal. At pH 3.20, the sensor exhibits a dynamic detection range from 0.017 to 4.2 mM n-butyraldehyde with a limit of detection of 0.003 mM. The forward response time (t95) of the sensor is 3-5 min, and the reverse response time is 5-7 min. The responses of the sensor toward different kinds of aldehydes and ketones depend on the lipophilicity and the reactivity of the analytes. Since the fluorescence enhancement of the sensing membrane at 296 nm/410 nm is only related to the formation of Schiff base, the measurement of aldehydes is independent of pH.     

Capillary-assembled microchip for universal integration of various chemical functions onto a single microfluidic device

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon.     

Immobilized fluorescent cyclodextrin on a cellulose membrane as a chemosensor for molecule detection

Dansylglycine-modified cyclodextrin (DnsC4-beta-CD) was prepared as a fluorescent host that is capable of being immobilized on a cellulose membrane (DnsC4-beta-CD membrane). DnsC4-beta-CD immobilized on the cellulose membrane decreased its fluorescence intensity with increasing concentration of guest molecules, indicating that the host changes the location of the dansyl group from inside to outside the cyclodextrin cavity upon guest accommodation, which is similar to DnsC4-beta-CD in solution; thereby, the DnsC4-beta-CD membrane is useful as a novel chemosensor for detecting molecules. This result demonstrates that the cellulose membrane is useful as a practical supporting material for various chromophore-modified cyclodextrins.     

Ion sensing and inhibition studies using the transmembrane ion channel peptide gramicidin A entrapped in sol-gel-derived silica

The development of new, targeted drugs relies heavily on innovative technologies that allow for high-throughput screening of drug libraries against biologically relevant targets, particularly membrane-associated receptors. Therefore, immobilization of natural receptors is of the utmost importance to allow for screening of small molecule libraries. Herein, we describe the immobilization of liposomes containing the transmembrane peptide ion-channel gramicidin A into sol-gel-derived silicate materials. Steady-state fluorescence measurements of the intrinsic tryptophan residues of reconstituted gramicidin A in phospholipid vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were obtained in solution and following entrapment in diglyceryl silane (DGS)-derived silicate to examine the effects of entrapment on the conformation of the ion channel. Only minor deviations were observed in the fluorescence properties of gramicidin following entrapment in DGS-derived silicate. DOPC vesicles containing a 50 microM internal solution of the potential sensitive fluorescent dye safranine O were used to study ion flux through the membrane ion channel. The dependence of ion flux on both ion concentration and amount of gramicidin embedded in the membrane were examined before and after entrapment in sol-gel-derived silicate. It was found that ion channel activity upon entrapment in DGS-derived silicate mirrored very closely that observed in solution. Moreover, the ability to inhibit ion flux through gramicidin A due to blockage by calcium ions was retained after the immobilization procedure. The implications for development of drug-screening and -sensing platforms are discussed.     

苯磺酸甜菜碱表面改性阳离子交换膜

采用等离子体辉光放电技术,在以聚笨乙烯为基膜的阳离子交换膜表面接枝苯磺酸甜菜碱(SBMA)单体,制备出具有高选择透过性的阳离子交换膜.对改性前后的膜进行扫描电镜(SEM)、傅里叶红外光谱分析(FTIR)及氯离子泄漏率测试,表征膜的改性效果.结果表明:在SBMA浓度为60 g/L,等离子照射强度为0.7 W/cm2,照射时间为7 min,照射气体氛围为氩气的条件下,改性效果最佳.改性膜含有的活性基团明显增多,表面致密均匀,氯离子泄漏率由原膜的14％降低至2％以下.     

Color-tuned FRET polystyrene microspheres by single wavelength excitation

Fluorescent monodisperse polystyrene microspheres were prepared by two-stage dispersion polymerization, which successfully covalently labeled microspheres with two dyes without disturbing the final particle size and size distribution. By varying the dye concentrations, microspheres show tuned colors with different fluorescent intensity under a single wavelength excitation. Fluorescence resonance energy transfer (FRET) between two labeled dyes was proved to contribute to the emission of the longer-wavelength dye at a shorter-wavelength excitation. There is no dye leakage for microspheres because of the covalent incorporation of dye molecules. The microsphere matrix provides good protection of dye molecules and blocks the influence of media outside on the fluorescence of microspheres. Single microsphere shows intense fluorescence due to a large number of encapsulated dye molecules. These uniform barcoding fluorescent microspheres have potential application in multiplexed bioanalysis.     

苯磺酸甜菜碱表面改性阳离子交换膜

采用等离子体辉光放电技术,在以聚苯乙烯为基膜的阳离子交换膜表面接枝苯磺酸甜菜碱(SBMA)单体,制备出具有高选择透过性的阳离子交换膜。对改性前后的膜进行扫描电镜(SEM)、傅里叶红外光谱分析(FTIR)及氯离子泄漏率测试,表征膜的改性效果。结果表明:在SBMA浓度为60g/L,等离子照射强度为0.7 W/cm2,照射时间为7min,照射气体氛围为氩气的条件下,改性效果最佳。改性膜含有的活性基团明显增多,表面致密均匀,氯离子泄漏率由原膜的14%降低至2%以下。     

Molecular boxes on a molecular printboard: encapsulation of anionic dyes in immobilized dendrimers

Fifth-generation poly(propylene imine) dendrimers, modified with 64 apolar adamantyl groups, have been immobilized on cyclodextrin host monolayers ("molecular printboards") on glass by supramolecular microcontact printing. The immobilized dendrimers retain their guest-binding properties and function as "molecular boxes" that can be filled with fluorescent dye molecules from solution. Alternatively, part of the immobilized dendrimers were filled with dye molecules by cross-microcontact printing while the remaining, empty dendrimers were filled with a different dye from solution, resulting in alternating patterns of dye molecules. In addition, we demonstrate that encapsulation of dyes in immobilized dendrimers is reversible: immobilized molecular boxes can be filled with a dye, emptied, and subsequently refilled with a different dye.     

Fluorescent cyclodextrin immobilized on a cellulose membrane as a chemosensor system for detecting molecules

Dansyl glutamate-modified cyclodextrin (DnsGlu-beta-CD) was prepared as a fluorescent host, which is capable of being immobilized on a cellulose membrane (DnsGlu-beta-CD-membrane). The fluorescence intensity of DnsGlu-beta-CD decreased with increasing concentration of guest molecules, indicating that the host changes the location of the dansyl group from inside to outside the cyclodextrin cavity upon guest accommodation. Similar guest-induced decrease in the fluorescence intensity was observed for DnsGlu-beta-CD immobilized to a cellulose membrane. This result demonstrates that the cellulose membrane may be used as a practical supporting material of various chromophore-modified cyclodextrins and that DnsGlu-beta-CD-membrane is useful as a novel disposable chemosensor for molecules.     

Application of ganglioside-sensitized liposomes in a flow injection immunoanalytical system for the determination of cholera toxin

Cholera, an acute infectious disease associated with water and seafood contamination, is caused by the bacterium Vibrio cholerae, which lives and colonizes in the small intestine and secretes cholera toxin (CT), a causative agent for diarrhea in humans. Based on earlier lateral flow assays, a flow injection liposome immunoanalysis (FILIA) system with excellent sensitivity was developed in this study for the determination of CT at zeptomole levels. Ganglioside (GM1), found to have specific affinity toward CT, was inserted into the phospholipid bilayer during the liposome synthesis. These GM1-sensitized, sulforhodamine B (SRB) dye-entrapping liposomes were used as probes in the FILIA system. Anti-CT antibodies were immobilized in its microcapillary. CT was detected by the formation of a sandwich complex between the immobilized antibody and GM1 liposomes. During the assay, the sample was introduced first into the column, and then liposomes were injected to bind to all CT captured by the antibody in the microcapillary. Subsequently, the SRB dye molecules were released from the bound liposomes via the addition of the detergent octyl glucopyranoside. The released dye molecules were transported to a flow-through fluorescence detector for quantification. The FILIA system was optimized with respect to flow rate, antibody concentration, liposome concentration, and injected sample volume. The calibration curve for CT had a linear range of 10-16 to 10-14 g mL-1. The detection limit of this immunosensor was 6.6 x 10(-17) g mL-1 in 200-microL samples (equivalent to 13 ag or 1.1 zmol).     

均匀设计法优选低温等离子体改性阳离子交换膜耐氯氧化性能

利用离子交换膜构建双膜三室电积生产金属钴是一种新的绿色湿法冶金工艺。目前市场上普通的非均相阳离子交换膜应用于该工艺时易被阳极生产的次氯酸氧化而造成膜性能下降,影响工艺效果,因此提高离子交换膜的耐氯性能是该工艺的核心技术。通过均匀试验设计,对阳离子交换膜进行低温等离子体改性,选用聚偏氟乙烯(PVDF)为接枝溶液,以期在膜表面产生高键能的含氟键官能团保护层,改善离子交换膜的耐氯氧化性能。利用活性氯浸泡法模拟实际应用中的强氧化环境,以氯离子泄漏率为表征指标,利用软件SPSS 23对均匀试验数据进行回归分析,得到实验数据显著性回归方程(R2=0.972);通过对方程系数进行t检验,挑选出影响最为显著的四个因素——等离子体放电电流强度、放电时长、接枝浓度、接枝时长;最后采用反演结合等值线图方法对方程进行寻优,预测得到的优化改性参数为:放电电流6A,放电时长5min,接枝液浓度8%,接枝时长60min。最终的验证试验表明,改性后离子交换膜的氯离子泄漏率较原膜降低了57.56%,膜的耐氯氧化性能大幅提高。     

Dual-color polystyrene microspheres by two-stage dispersion copolymerization

Dual-color monodisperse polystyrene microspheres have been prepared by two-stage dispersion polymerization, whose addition of the dye-comonomer was deferred until the nucleation stage was complete. Two dyes were covalently incorporated into microspheres by copolymerization without disturbing the final particle size and size distribution. No dye leakage occurs for the microsphere suspension because of covalent bonding of dye molecules. By varying the dye concentrations in microspheres, we obtained fluorescent microspheres with two distinguishing parts of fluorescence and varied fluorescent intensity ratios under a single-wavelength excitation. The microspheres present intense fluorescence for the large amount of encapsulated dye molecules.     

Lipophilic polymer membrane optical sensor with a synthetic receptor for saccharide detection

An optical sensing approach for the detection of saccharides based on reversible boronic acid-diol complexation in a lipophilic polymer membrane is presented. The complexation of saccharides with phenyl boronic acids that are immobilized in a hydrophobic polymer matrix produces a stable boronate anion and liberates a mobile hydrogen ion. The change in fluorescence intensity of the sensing films resulting from the increase in hydrogen ion concentration is thus directly related to the ambient saccharide concentration. By monitoring the pH change in a bulk optical film, we were able to detect D-glucose, D-fructose, D-galactose, and D-sorbitol with a concentration range from 0.1 to 100 mM at physiological pH 7.4.     

Dual lifetime referencing as applied to a chloride optical sensor

A membrane with an optical response to chloride has been developed that contains two luminophores that display two largely different decay times. The first luminophore (the "reference") is a chloride-insensitive ruthenium metal-ligand complex possessing a decay time in the microsecond range. The second luminophore is the short-lived chloride-quenchable fluorescent probe lucigenin. Both are contained in a hydrogel matrix and are excited by a blue LED emitting sinusoidally modulated light. Under these conditions, the chloride-dependent fluorescence intensity of lucigenin can be converted in an analyte-dependent fluorescence phase shift that depends on the ratio of the two luminescence intensities and can be measured at modulation frequencies of typically 45 kHz. The dynamic range of this sensor can be adjusted by either varying the ratio of the two luminophores or selecting a particular optical filter combination.     

Coupling fiber optics to a permeation liquid membrane for heavy metal sensor development

We present the first sensing system for metal ions based on the combination of separation/preconcentration by a permeation liquid membrane (PLM) and fluorescence detection with an optical fiber. As a model, a system for the detection of Cu(II) ions was developed. The wall of a polypropylene hollow fiber serves as support for the permeable liquid membrane. The lumen of the fiber contains the strip solution in which Cu(II) is accumulated. Calcein, a fluorochromic dye, acts as stripping agent and at the same time as metal indicator. The quenching of the calcein fluorescence upon metal accumulation in the strip phase is detected with a multimode optical fiber, which is incorporated into the lumen. Fluorescence is excited with a blue LED and detected with a photon counter. Taking advantage of the high selectivity and sensitivity of PLM preconcentration, a detection limit for Cu(II) of approximately 50 nM was achieved. Among five tested heavy metal ions, Pb(II) was the only major interfering species. The incorporation of small silica optical fibers into the polypropylene capillary allows for real-time monitoring of the Cu(II) accumulation process.     

Quantitative fluorescence microscopy to determine molecular occupancy of phospholipid vesicles

Encapsulation of molecules in phospholipid vesicles provides unique opportunities to study chemical reactions in small volumes as well as the behavior of individual proteins, enzymes, and ribozymes in a confined region without requiring a tether to immobilize the molecule to a surface. These experiments generally depend on generating a predictable loading of vesicles with small numbers of target molecules and thus raise a significant measurement challenge, namely, to quantify molecular occupancy of vesicles at the single-molecule level. In this work, we describe an imaging experiment to measure the time-dependent fluorescence from individual dye molecules encapsulated in ~130 nm vesicles that are adhered to a glass surface. For determining a fit of the molecular occupancy data to a Poisson model, it is critical to count empty vesicles in the population since these dominate the sample when the mean occupancy is small, λ ≤ ~1. Counting empty vesicles was accomplished by subsequently labeling all the vesicles with a lipophilic dye and reimaging the sample. By counting both the empty vesicles and those containing fluors, and quantifying the number of fluors present, we demonstrate a self-consistent Poisson distribution of molecular occupancy for well-solvated molecules, as well as anomalies due to aggregation of dye, which can arise even at very low solution concentrations. By observation of many vesicles in parallel in an image, this approach provides quantitative information about the distribution of molecular occupancy in a population of vesicles.