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.     

Synthesis and application of submicrometer fluorescence sensing particles for lysosomal pH measurements in murine macrophages

Phagocytosis of bioparticles such as bacteria and viruses by macrophages is a critical component of the immune response against infections. In this paper we describe the synthesis of submicrometer fluorescent particles with pH sensing capability. The particles are used to measure the pH and to monitor the effect of chloroquine, an antimalarial drug, on the pH in the lysosome, the cellular organelle involved in the phagocytosis process. The synthesis of the pH sensing particles is realized by the covalent attachment of amine reactive forms of Oregon Green (pH sensitive dye) and Texas Red (pH insensitive dye) to the surface of amino-modified submicrometer polystyrene particles. The particles are absorbed by J774 Murine Macrophages through phagocytosis and directed to lysosomes. Despite the high lysosomal levels of digestive enzymes and acidity, the absorbed particles remain stable for 12 h in the cells when they are stored in a PBS buffer solution at pH 7.4. The pH dynamic range of the sensing particles is between pH 4.5 and 7 with a sensitivity of 0.1 pH units. Exposure of the cells to chloroquine increases the lysosomal pH from 4.8 to 6.5. The effect is concentration-dependent.     

Real-time measurements of dissolved oxygen inside live cells by organically modified silicate fluorescent nanosensors

Optical PEBBLE (probes encapsulated by biologically localized embedding) nanosensors have been developed for dissolved oxygen using organically modified silicate (ormosil) nanoparticles as a matrix. The ormosil nanoparticles are prepared via a sol-gel-based process, which includes the formation of core particles with phenyltrimethoxysilane as a precursor followed by the formation of a coating layer with methyltrimethoxysilane as a precursor. The average diameter of the resultant particles is 120 nm. These sensors incorporate the oxygen-sensitive platinum porphyrin dye as an indicator and an oxygen-insensitive dye as a reference for ratiometric intensity measurement. Two pairs of indicator dye and reference dye, respectively, platinum(II) octaethylporphine and 3,3'-dioctadecyloxacarbocyanine perchlorate, and platinum(II) octaethylporphine ketone and octaethylporphine, were used. The sensors have excellent sensitivity with an overall quenching response of 97%, as well as excellent linearity of the Stern-Volmer plot (r(2) = 0.999) over the whole range of dissolved oxygen concentrations (0-43 ppm). In vitro intracellular changes of dissolved oxygen due to cell respiration were monitored, with gene gun injected PEBBLEs, in rat C6 glioma cells. A significant change was observed with a fluorescence ratio increase of up to 500% after 1 h, for nine different sets of cells, which corresponds to a 90% reduction in terms of dissolved oxygen concentration. These results clearly show the validity of the delivery method for intracellular studies of PEBBLE sensors, as well as the high sensitivity, which is needed to achieve real-time measurements of intracellular dissolved oxygen concentration.     

Characterization of glucose microsensors for intracellular measurements.

Ultrasmall glucose sensors have been constructed by using platinum-deposited carbon ring microelectrodes with glucose oxidase. Response times as low as 270 ms have been obtained with these sensors. Moreover, there is a linear relationship between sensor tip diameter and response times. The use of these sensors has been demonstrated in the detection of glucose in single-cell cytoplasm of the large dopamine cell of the pond snail Planorbis corneus. Current responses obtained at these sensors implanted into a cell increase following injection of 2 pL of glucose solution (3 M) into the cell. Results obtained from these experiments show that these sensors are suitable for glucose monitoring in ultrasmall environments. In addition, characterizations of these sensors have been investigated under different O2 concentrations. At atmospheric oxygen concentrations, glucose levels in the submillimolar range can be measured without oxygen interference; however, oxygen interference can be substantial at low oxygen concentrations.     

Differential absorption sensor applied for liquid oxygen measurements

Differential laser absorption was investigated for its merit in liquid oxygen (LOX) sensing. Whereas previous researchers have used differential absorption to detect trace concentrations of a substance, we use differential absorption to monitor small changes in large amounts of a substance. Two lasers of different wavelengths were intensity modulated 180 deg out of phase from each other and multiplexed into a single beam. After probing the LOX, the total transmitted signal was demodulated by a lock-in amplifier. Our experiment simulated rapid changes in LOX number density by varying the length of an approximately 73 mm path through pure LOX. In this experiment, we demonstrated the ability to monitor LOX number density with an uncertainty of approximately 1% with a time constant of 3 micros. The uncertainty could be halved by doubling the path length, and this improvement could be repeated as long as the relative intensity noise of the lasers is the dominating factor. We discuss the benefits of differential absorption for problems requiring an extended dynamic range.     

Molecular engineering of fluorescent penicillins for molecularly imprinted polymer assays

The interaction of seven novel fluorescent labeled beta-lactams with a library of six polymer materials molecularly imprinted (MI) with penicillin G (PenG) has been evaluated using both radioactive and fluorescence competitive assays. The highly fluorescent competitors (emission quantum yields of 0.4-0.95) have been molecularly engineered to contain pyrene or dansyl labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers. Pyrenemethylacetamidopenicillanic acid (PAAP) is the tagged antibiotic that provides the highest selectivity when competing with PenG for the specific binding sites in a MI polymer prepared with methacrylic acid and trimethylolpropane trimethacrylate (10:15 molar ratio) in acetonitrile in the presence of PenG. Molecular modeling shows that recognition of the fluorescent analogues of PenG by the MI material is due to a combination of size and shape selectivity and demonstrates how critical the choice of label and tether chain is. PAAP has been applied to the development of a fluorescence competitive assay for PenG analysis with a dynamic range of 3-890 muM in 99:1 acetonitrile-water solution. Competitive binding studies demonstrate various degrees of cross-reactivity for some antibiotics derived from 6-aminopenicillanic acid, particularly amoxicillin, ampicillin, and penicillin V (but not oxacillin, cloxacillin, dicloxacillin, or nafcillin). Other antibiotics, such as chloramphenicol, tetracycline, or cephapirin, do not compete with PAAP for binding to the imprinted polymer. The MI assay has successfully been tested for PenG analysis in a pharmaceutical formulation.     

Single color fluorescent indicators of protein phosphorylation for multicolor imaging of intracellular signal flow dynamics

Existing monitoring methods for protein phosphorylation involved in intracellular signal transduction in vivo are exclusively based on fluorescence resonance energy transfer, which needs the measurement of the change in fluorescence intensities at two wavelengths. Therefore, it is difficult to monitor protein phosphorylation together with other related signaling processes, such as second messengers and protein translocation. To overcome this problem, we developed novel fluorescent indicators, each containing a differently colored (cyan and green) single fluorophore. The present indicator is a tandem fusion protein containing a kinase substrate domain, a circularly permuted fluorescent protein (cpFP), and a phosphorylation recognition domain. The cpFP is obtained by dividing a green fluorescent protein mutant (GFP) at residue 144-145 and linking the carboxy and amino portions thereof with a peptide linker. The substrate domain used in this study is a peptide sequence that is phosphorylated by insulin receptor. Phosphorylation of the substrate domain induces its interaction with the phosphorylation recognition domain, which causes a conformational change in the cpFP and a change in its fluorescence. The cyan and green indicators exhibited 10% decrease and 15% increase, respectively, in their fluorescence intensities upon phosphorylation. Using this cyan indicator and GFP-tagged mitogen-activated protein kinase (MAPK), we found that insulin-induced protein phosphorylation occurred immediately upon the addition of insulin, whereas nuclear translocation of MAPK occurred 7 min later. By tailoring the substrate domains and the phosphorylation recognition domains in these cyan and green indicators, the present approach should be applicable to the in vivo analysis of a broad range of protein phosphorylation processes, together with other intracellular signaling processes.     

双原子氧气液界面特性的分子动力学模拟

采用分子动力学模拟方法,考虑氧分子中原子间键的谐振势作用,研究了双原子氧的平衡态气液相变特性以及不均匀相界面区的表面张力等性质。根据双原子氧气液相饱和密度的实验数据,确定Lennard-Jones势能模型中氧原子的参数,模拟得到了不同温度下气液相的密度和饱和压强,与实验数据符合良好,验证了确定的氧原子参数的正确性。通过模拟得到了界面压力张量的法向和切向分量的分布曲线以及界面表面张力随温度的变化曲线,在气液界面区,压力张量的法向与切向分量存在较大的势阱和势垒,界面的表面张力随温度线性降低,当达到临界温度时,表面张力减小到0。     

Simultaneous electrochemical measurements of oxygen and dopamine in vivo.

Fast-scan cyclic voltammetry, a demonstrated analytical method for the in vivo detection of catecholamine neurotransmitters, is extended to the simultaneous determination of molecular oxygen (O2). Cyclic voltammograms were recorded at a scan rate of 400 V/s at carbon-fiber disk electrodes coated with a perfluorinated ion-exchange material. The peak current for O2 occurs near -1.2 V under these conditions. In flow-injection experiments, these electrodes respond to step changes in dopamine and O2 with a half-rise time of less than 200 ms. The voltammetric peak current is independent of flow rate, indicating a diffusion-limited response unaffected by convection. Several compounds present in the in vivo matrix (adenosine, glutathione, and NAD and glutamic, lactic, and uric acids) were tested and shown not to interfere with the voltammetric signal for O2. These electrodes maintain a stable response in vivo for at least 6 h. They have been used to measure transient increases in both dopamine and O2 in the extracellular fluid of the caudate nucleus of an anesthetized rat in response to an electrical stimulus.     

Polymeric optodes based on upconverting nanorods for fluorescent measurements of pH and metal ions in blood samples

Optical thin films incorporating NaYF(4):Er,Yb upconverting nanorods and chromoionophore ETH 5418 in hydrophobic polymer matrixes have been developed for the first time to measure pH and metal ions based on the ion-exchange mechanism. The absorption spectra of protonated and unprotonated ETH 5418 overlap the two emission peaks of upconverting material, respectively, which makes the inert nanorods ion-sensitive. Optodes for pH and metal ions (Na(+), K(+), Ca(2+), and Cu(2+)) were investigated and exhibited excellent sensitivity, selectivity, and reproducibility. Because of excitation by the 980 nm laser source, detection in the near-infrared region at 656 nm, and high quantum yield of the nanorods in hydrophobic membrane, the proposed sensors have been successfully used in whole blood measurements with minimized background absorption and sample autofluorescence.     

Conductivity measurements on cobalt and nickel oxides in highly-enriched oxygen atmospheres

The pressure dependencies of the conductivities of nickel and cobalt monoxides have been investigated in the range 10^–2 to 7.5 × 10² atmospheres at temperatures in the region of 1000° C. Conductivity saturation occurs in CoO due to the formation of Co₃O₄, and the experimental data correlates well with a model for the separation of the higher phase. NiO exhibits no saturation effect in the pressure range investigated and the p^1/5 dependence of the conductivity at 950° C with this oxide above 0.1 atm is interpreted in terms of the formation of singly ionised vacancies.     

"Molecular beacon"-based fluorescent assay for selective detection of glutathione and cysteine

We report on the development of a fluorescence turn-on "molecular beacon" probe for the detection of glutathione (GSH) and cysteine (Cys). The method is based on a competitive ligation of Hg(2+) ions by GSH/Cys and thymine-thymine (T-T) mismatches in a DNA strand of the self-hybridizing beacon strand. The assay relies on the distance-dependent optical properties of the fluorophore/quencher pair attached to the ends of the molecular beacon DNA strand. In a very selective coordination of Hg(2+) to GSH/Cys, the fluorophore/quencher distance increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg(2+)-T due to the extraction of Hg(2+) ions. This process results in switching the molecular beacon to the "on" state. The concentration range of the probe is 4-200 nM with the limit of detection (LOD) of 4.1 nM for GSH and 4.2 nM Cys. The probe tested satisfactorily against interference for a range of amino acids including sulfur-containing methionine.     

Apoptosis induction and attenuation of inflammatory gene expression in murine macrophages via multitherapeutic nanomembranes

The realization of optimized therapeutic delivery is impaired by the challenge of localized drug activity and by the dangers of systemic cytotoxicity which often contribute to patient treatment complications. Here we demonstrate the block copolymer-mediated deposition and release of multiple therapeutics which include an LXRα/β agonist 3-((4-methoxyphenyl)amino)-4-phenyl-1-(phenylmethyl)-1H-pyrrole-2,5-dione (LXRa) and doxorubicin hydrochloride (Dox) at the air-water interface via Langmuir-Blodgett deposition, as well as copolymer-mediated potent drug elution toward the Raw 264.7 murine macrophage cell line. The resultant copolymer-therapeutic hybrid serves as a localized platform that can be functionalized with virtually any drug due to the integrated hydrophilic and hydrophobic components of the polymer structure. In addition, the sequestering function of the copolymer to anchor the drugs to implant surfaces can enhance delivery specificity when compared to systemic drug administration. Confirmation of drug functionality was confirmed via suppression of the interleukin 6 (Il-6) and tumor necrosis factor alpha (TNFα) inflammatory cytokines (LXRa), as well as DNA fragmentation analysis (Dox). Furthermore, the fragmentation assays and gene expression analysis demonstrated the innate biocompatibility of the copolymeric material at the gene expression level via the confirmed absence of material-induced apoptosis and a lack of inflammatory gene expression. This modality enables layer-by-layer control of agonist and chemotherapeutic functionalization at the nanoscale for the localization of drug dosage, while simultaneously utilizing the copolymer platform as an anchoring mechanism for drug sequestering, all with an innate material thickness of 4?nm per layer, which is orders of magnitude thinner than existing commercial technologies. Furthermore, these studies comprehensively confirmed the potential translational applicability of copolymeric nanomaterials as localized multitherapeutic thin film platforms.     

Spot measurements of intermediate frequency electric fields in the vicinity of compact fluorescent lamps

Starting in 2009, certain types of incandescent light bulbs will be withdrawn from the market in the European Union and elsewhere. However, compact fluorescent lamps that are among the candidates to replace them produce intermediate frequency electric fields (EFs) much higher than any other device or appliance previously available to the general public. Measurement results of these EFs showed that the maximum recorded EF strength in the 1.2-100 kHz frequency range in close proximity to the lamps was > 42 V m(-1) for all tested lamps. In nine cases, the field strength exceeded 87 V m(-1) and the highest measured value was 216 V m(-1).     

Polyelectrolyte microshells as carriers for fluorescent sensors: loading and sensing properties of a ruthenium-based oxygen indicator

A strategy for the design and fabrication of microcapsule-based fluorescent biosensors containing indicators and internal references is described. The rationale for this work is the physical immobilization and chemical separation of assay chemistry for use in biological environments. Using the general approach of depositing oppositely charged species on colloidal micro/nanotemplates, a sensor system employing polyelectrolyte microshells for uptake of functional molecules is proposed, and experiments to demonstrate the feasibility of nanoengineering the sensor properties are described in the context of an oxygen sensor. Methods for immobilization and entrapment of fluorescent indicator and reference dyes are shown, along with the pH dependence of this process. Embedded dyes are shown to be stable and retain their function, as demonstrated with oxygen-sensitivity experiments of loaded microcapsules. Although oxygen sensitivity is presented as an example of a specific application, the overall strategy is likely more generally useful. The work suggests that polyelectrolyte microshells may be used as a platform to develop novel sensors by entrapment of functional materials.     

Desorption kinetics of oxygen in plasma treated SWNTs by in situ thermoelectric power measurements

The functionalization and defect formation of SWNTs caused by isotropic plasma treatments were studied using oxygen desorption/adsorption kinetics by measuring the time dependence of the in situ thermoelectric power (TEP). It is shown that the plasma treatments result in the formation of low binding energy sites for oxygen adsorption. Raman and x-ray photoelectron spectroscopy (XPS) data are in good agreement with the results.