The photodynamic inactivation of the membrane functions of bacteria was analyzed in situ, using K+ and tetraphenylphosphonium (TPP+) electrodes, as well as an oxygen electrode. Tetrakis(4-N-trimethylaminophenyl)porphine (TTMAPP) and rose bengal were used, since both dyes act strongly on bacteria, such as Staphylococcus aureus. After a short time lag, they inhibited the respiration of bacteria and increased the permeability of the cytoplasmic membrane to K+, while dissipating the membrane potential. This combination of sensors is quite useful for visualizing the actions of photosensitizers on the bacterial membrane. TTMAPP and rose bengal impaired the bacterial function by reducing the membrane potential within minutes of photo-irradiation.
This paper reviews chemically selective imaging at the single-atom/molecule level by molecular STM tips. The molecular tips enable the recognition of a particular chemical species on the basis of chemical interactions with a sample molecule, including hydrogen-bond, metal-coordination, and charge-transfer interactions. The chemical selectivity can be tailored by designing functional groups of the tip molecules. Moreover, the rational design of the molecular tip allows sophisticated chemical recognition. The discrimination of DNA bases and chiral recognition on a single molecule basis are thereby achieved. The molecular tips also revealed rectified electron transmission within an electron donor-acceptor molecular pair. Self-assembled monolayers, carboxylated carbon nanotube, and conducting polymers can be utilized for the preparation of molecular tips. This technique may be coined “intermolecular tunneling microscopy” as its principle goes, and is of general significance for novel molecular imaging of chemical identities at the membrane and solid surfaces.
Amniotic fluid-derived stem cells (AFSCs) are becoming an important source of cells for regenerative medicine given with apparent advantages of accessibility, renewal capacity and multipotentiality. In this study, the mechanical properties of human amniotic fluid-derived stem cells (hAFSCs), such as the average Young’s modulus, were determined by atomic force microscopy (3.97 ± 0.53 kPa for hAFSCs vs. 1.52 ± 0.63 kPa for fully differentiated osteoblasts). These differences in cell elasticity result primarily from differential actin cytoskeleton organization in these two cell types. Furthermore, ultrastructures, nanostructural details on the surface of cell, were visualized by atomic force microscopy (AFM). It was clearly shown that surface of osteoblasts were covered by mineralized particles, and the histogram of particles size showed that most of the particles on the surface of osteoblasts distributed from 200 to 400 nm in diameter, while the diameter of hAFSCs particles ranged from 100 to 200 nm. In contrast, there were some dips on the surface of hAFSCs, and particles were smaller than that of osteoblasts. Additionally, as osteogenic differentiation of hAFSCs progressed, more and more stress fibers were replaced by a thinner actin network which is characteristic of mature osteoblasts. These results can improve our understanding of the mechanical properties of hAFSCs during osteogenic differentiation. AFM can be used as a powerful tool for detecting ultrastructures and mechanical properties.
Two-dimensional real-time observation of potassium ion distributions was achieved using an ion imaging device based on charge-coupled device (CCD) and metal-oxide semiconductor technologies, and an ion selective membrane. The CCD potassium ion image sensor was equipped with an array of 32 × 32 pixels (1024 pixels). It could record five frames per second with an area of 4.16 × 4.16 mm2. Potassium ion images were produced instantly. The leaching of potassium ion from a 3.3 M KCl Ag/AgCl reference electrode was dynamically monitored in aqueous solution. The potassium ion selective membrane on the semiconductor consisted of plasticized poly(vinyl chloride) (PVC) with bis(benzo-15-crown-5). The addition of a polyhedral oligomeric silsesquioxane to the plasticized PVC membrane greatly improved adhesion of the membrane onto Si3N4 of the semiconductor surface, and the potential response was stabilized. The potential response was linear from 10−2 to 10−5 M logarithmic concentration of potassium ion. The selectivity coefficients were KK+,Li+pot = 10−2.85, KK+,Na+pot = 10−2.30, KK+,Rb+pot =10−1.16, and KK+,Cs+pot = 10−2.05.
A possible formation mechanism of the ionic aggregate of poly(oxyethylene) dodecyl ether nonionic surfactants with a ferric-thiocyanate chromophore is presented, and the dependence of their colorimetric sensitivity on the degree of ethoxylation can be clearly explained through both experimental and theoretical approaches. The stoichiometry of ferric thiocyanate was confirmed to be a monovalent anion comprised of the 1:4 complex, [Fe(SCN)4]−, both by the extractive mole-ratio method and the continuous variation method. The poly(oxyethylene) moiety can form a stable helical conformation by including an alkali cation inside the helix, leading such nonionic surfactants into cationic complexes that can produce ionic aggregates with a bulky anionic complex, such as ferric thiocyanate. The colorimetric sensitivity of the ionic aggregates can depend on the inter-charge distance between the cation and the counter anion. The newly introduced conformational ideas based on experimental results are theoretically supported by CHARMM29 molecular mechanics/dynamics simulations. Molecular dynamics simulations successfully provided the most stable conformation for the K+-included heptaoxyethylene dodecyl ether aggregated with the bulky counter anion.
In this paper, we describe DNA detection experiments using our two original technologies, power-free microchip and laminar flow-assisted dendritic amplification (LFDA), which were previously applied to immunoassays. A microchip was fabricated by combining a poly(dimethylsiloxane) (PDMS) part having microchannel patterns and a glass plate modified with probe DNA. We carried out two kinds of experiments: the detection of 21-base biotinylated target DNA and the detection of single-nucleotide polymorphism (SNP) in 56-base unlabeled target DNA by sandwich hybridization with biotinylated probe DNA. For both of the experiments, the necessary solutions were injected into microchannels not by an external power source, but by air dissolution into the PDMS part. After a hybridization reaction, the LFDA was started by injecting FITC-labeled streptavidin and biotinylated anti-streptavidin antibody onto the reaction site. With a detection time of 20 min, the limit of detection (LOD) for the biotinylated target was 2.2 pM, and the LOD for the SNP was 10 – 30 pM, depending on the SNP type.
Voltammetric detection of biological molecules was carried out using chopped carbon fibers produced from carbon fiber reinforced plastics that are biocompatible and inexpensive. Because chopped carbon fibers normally are covered with a sizing agent, they are difficult to use as an electrode. However, when the surface of a chopped carbon fiber was treated with ethanol and hydrochloric acid, it became conductive. To evaluate the functioning of chopped carbon fibers, voltammetric measurements of [Fe(CN)6]3− were carried out. Redoxes of FAD, ascorbic acid and NADH as biomolecules were recorded using cyclic voltammetry. The sizing agents used to bundle the fibers were epoxy, polyamide and polyurethane resins. The peak currents were the greatest when using the chopped carbon fibers that were created with epoxy resins. When the electrode response of the chopped carbon fibers was compared with that of a glassy carbon electrode, the peak currents and the reversibility of the electrode reaction were sufficient. Therefore, the chopped carbon fibers will be useful as disposable electrodes for the sensing of biomolecules.
The interaction of 4-nonylphenol (NP) with deoxyribonucleic acid (DNA) was explored electrochemically by using differential pulse voltammetry (DPV) in combination with unmodified and modified pencil graphite electrodes (PGE) with single walled carbon nanotubes (SWCNT). The differentiation of the two oxidation signals coming from NP and DNA base, guanine was studied before and after the interaction process. In addition, the effect of NP concentration was investigated in order to determine the optimum experimental conditions. The detection limit and the reproducibility were determined by using CNT-modified electrodes.
A multi-wall carbon nanotubes (MWCNTs) composite with Nafion was modified on a glass carbon electrode. The modified electrode was then used as a voltammetric sensor in detecting 2,4,6-triaminopyrimidine (TAP). The surface morphology of the Nafion/MWCNTs composite film was characterized by atomic force microscopy (AFM), and the electrochemical behavior of TAP at this sensor was investigated in detail. The results indicated that the Nafion/MWCNTs modified electrode exhibited efficient electrocatalytic oxidation for TAP with relatively high sensitivity, stability and lifetime. Under the optimized condition using linear sweep voltammetry (LSV), the Nafion/MWCNTs modified electrode exhibited a linear voltammetric response for TAP in the concentration range of 2.0 × 10−7 to 3.6 × 10−5 mol L−1, with a detection limit of 5.0 × 10−8 mol L−1. The electrode was applied to detect TAP added to human blood serum, with an average recovery value of 101.3%.
In this paper, a highly selective and sensitive probe for fluoride ions (F−), containing a phenylpyridylvinylene derivative reporter and a Si–O bond receptor, was designed and characterized. The reaction mechanism is based on the intramolecular charge transfer (ICT) mechanism. Upon addition of F−, probe 1 showed a remarkable red-shift (183 nm) in the absorption spectra accompanying with the color changes from colorless to purple, so probe 1 could serve as a “naked-eye” probe for F−. The absorbance of probe 1 at 545 nm increased linearly with the concentration of F− from 20 to 150 μM. The detection limit was calculated to be 0.1 μM. Besides, “off-on-off” fluorescence intensity changes were also observed in the fluorescence spectra. The present results may provide a useful approach for the development of highly selective dual-channel probes for F−.
The degradation products of Cefepime dihydrochloride that emerged throughout stress stability studies have been determined, identified and characterized. The two new impurities were detected by gradient reverse-phase high performance liquid chromatography (HPLC), and Impurity-I was formed in the range from 0.2 to 11.0% and Impurity-II range from 0.2 to 3.5%. These impurities have been identified by LC/MS, and were not reported in the literature. These impurities were synthesized, isolated and characterized. Based on the spectral data, the impurities were named (6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-3-[(1-methyl-1-pyrrolidinium)methyl]-3-cephem-4-carboxylate-1-oxide (Impurity-I); (2RS)-2[[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-methyl]-1,2,5,7-tetrahydro-7-oxo-4H-furo[3,4-d][1,3]thiazine (Impurity-II). The structures were established unambiguously by independent synthesis and co-injection in HPLC to confirm the retention times and relative retention times. The structural elucidation of these impurities by spectral data (1H NMR, 13C NMR, 2D-NMR (COSY, HSQC and HMBC), LC/MS, TOF-MS, elemental analysis and IR), synthesis, isolation and the formation of these impurities are discussed in detail.
A capillary modified by assembling a molecular film was presented for the chiral separation of sertraline by microemulsion electrokinetic chromatography. The assembling molecular film was constructed with poly(diallyldimethylammonium-chloride) and β-cyclodextrin via inclusion complexation. The separation efficiency of cis-trans isomers and enantiomers of sertraline was improved with a running microemulsion that contained the acetonitrile, sodium dodecyl sulfate, n-butanol and n-hexane buffered with sodium tetraborate. The baseline separation of four sertraline cis-trans isomers and enantiomers was achieved under the optimum conditions. The detection limit for isomers and enantiomers of sertraline (1S,4S, 1R,4R, 1S,4R, 1R,4S) was 0.15, 0.15, 0.30, 0.30 mg/L, respectively. The mechanism of chiral separation was studied and it could be applied for the determination of commercial Zoloft tablet samples satisfactorily.
A simple method using solid-phase extraction combined with metal furnace atomic absorption spectrometry was developed for the determination of Cr(III) and Cr(VI) at sub-ppb levels in water. A 500-ml water sample was adjusted to pH 3 with nitric acid and then passed through an iminodiacetate extraction disk placed on a cation-exchange extraction disk at a flow rate of 20 – 40 ml min−1 for concentrating Cr(III). The filtrate was adjusted to pH 10 with aqueous ammonia and then passed through an anion-exchange extraction disk at a flow rate of 2 ml min−1 for concentrating Cr(VI). The Cr(III) and Cr(VI) collected were eluted with 40 ml of 3 mol l−1 nitric acid for Cr(III) and 40 ml of 1 g l−1 diphenylcarbazide solution for Cr(VI). Each eluate was diluted to 50 ml with deionized water and injected into a U-type tungsten board on the metal furnace. The calibration curves of Cr(III) and Cr(VI) showed good linearity in the range of 0.1 – 0.5 ng. The detection limits corresponding to three times the standard deviation (n = 5) of blank values were 8.1 pg for both Cr(III) and Cr(VI). The analytical value of total Cr (Cr(III) + Cr(VI)) in certified reference material of river water (JSAC 0302-3) was in good agreement with the reference value. The recovery test for 0.50 μg (1.00 μg l−1) of Cr(III) and Cr(VI) added to 500 ml of the water samples showed sufficient values (98.1 – 106%), except for river water sampled downstream due to relatively higher CODMn value. The relative standard deviations (n = 5) were less than 5% for both Cr(III) and Cr(VI).
A rapid and sensitive method for analysis of blonanserin in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry is presented. After pretreatment of a plasma sample by solid-phase extraction, blonanserin was analyzed by the system with a C18 column. This method gave satisfactory recovery rates, reproducibility, and good linearity of calibration curve in the range of 0.01 – 10.0 ng/mL for quality control samples spiked with blonanserin. The detection limit was as low as 1 pg/mL. This method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.
The concentration level of extracellular L-glutamate released from region CA3 of mouse hippocampal slices under tetraethylammonium (TEA) chloride and KCl stimulation was measured with independent methods, i.e., a capillary-based enzyme sensor, a patch sensor, and an enzyme-based imaging method. The L-glutamate level was compared with those at regions CA1 and DG. It was found that the enhanced concentration level at CA3 by TEA stimulation is very similar to that at CA1, but it is much lower than that at DG. The order of the regional distribution of L-glutamate, i.e., DG > CA1 ≈ CA3, was the same as that obtained by K+ stimulation. However, in the presence of an uptake inhibitor, DL-TBOA, KCl stimulation showed the strongest L-glutamate flux at CA1, while TEA stimulation exhibited the strongest flux at CA3. The usefulness of the present approach for knowing the extracellular L-glutamate level in acute hippocampal slices is discussed.
WaterLOGSY and STD experiments are widely used as NMR-based screening techniques in drug research. In the present study, an improved STD pulse sequence was developed, and its efficiency and applicability of observing the ligand signals were evaluated compared with the WaterLOGSY experiment. A combination of presaturation, a WET sequence and subsequent repeated Z-filters can provide the most effective water suppression, which is incorporated into the STD pulse sequence. In a sample solution of tryptophan and glucose in the presence of human serum albumin, the improved STD experiment only succeeded in selective detections of the bound ligand signals, even resonating close to water.
A simple and rapid high-performance liquid chromatography method was developed for the determination of eleven nucleosides and bases in beer, herring sperm DNA and RNA soft capsules. The separation was carried out on an Agilent extend-C18 column with a simple gradient elution of acetonitrile and water as the mobile phase. Good linear relationships between the peak areas and the concentrations of the analytes were obtained. The detection limits for eleven analytes were in the range of 0.007 – 0.037 mg/L by UV detection at 260 nm. The relative standard deviations (RSDs) of the retention times were in the range of 0.78 – 1.85% for intra-day and 0.87 – 1.94% for inter-day, respectively. The RSDs of the peak areas were in the range of 2.71 – 3.22% for intra-day and 3.03 – 3.39% for inter-day, respectively. This method has been successfully applied to simultaneous determination of eleven nucleosides and bases in beer, herring sperm DNA and RNA soft capsules with the recoveries in the range of 93.7 – 108.3%.