A simple and robust method to immobilize cells onto a glass substrate is presented. The method employs a photochemical reaction of benzophenone, which is modified on the substrate using a standard silane coupling agent, with cells. Cells were immobilized to an area irradiated with UV light from a standard light source under an inverted microscope. The dependence of immobilization on the light power intensity and irradiation time was investigated. In situ DNA analysis within the immobilized cells was demonstrated using target-primed rolling circle amplification and fluorescent detection.
The use of a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium chloride (C4mimCl), as a salting-out phase separation agent to a water–tetrahydrofuran homogeneous system was studied for possible applications to novel aqueous biphasic extraction separation. The IL showed a salting-out phase-separation ability. Also, differences in the polarity between the formed two phases were smaller than that when using NaCl as a salting-out agent. This result suggested that C4mimCl remaining in water-rich phase acts not only as a salting-out agent, but also a component of a mixed-solvent. Possible uses of C4mimCl/NaCl mixed salting-out agent system were also discussed.
The purpose of this review article is to outline recent progress in near-infrared (NIR) spectroscopy. Some particular emphasis is put on the delineation of its versatility in analytical chemistry. NIR spectroscopy is versatile in many aspects. For example, it is electronic spectroscopy as well as vibrational spectroscopy. It is also all-round in applications from basic to practical applications. NIR spectroscopy can be applied to various kinds of materials, bulk materials, thin or thick polymers, tablets, human bodies, and so on. It is particularly powerful in non-invasive, non-destructive, and in situ analysis. In this review, the principles and advantages of NIR spectroscopy are described first, and then its applications to various fields, including polymer science, on-line monitoring, inorganic material research, medical diagnosis, and NIR imaging are introduced.
The potential response of an ion-exchanger type PVC ion-selective electrode (ISE) for a drug ion, desipramine+ (DES+), was analyzed by a mixed-potential (MP) theory proposed previously. The transfer of DES+ and its analogous ions, imipramine+ (IMP+) and neostigmine+ (NEO+), at a micro ο-nitrophenyl octyl ether/water interface was studied by ion-transfer voltammetry; also, the standard ion-transfer potentials (ΔOWφjo) of the ions were then determined. The application of MP theory with the ΔOWφjo values successfully explained the under-Nernstian response of DES+-ISE due to interference from IMP+ or NEO+. In this study, a universal method based on numerical calculations was developed for evaluating MP associated with plural interfering ions, which was impossible in the previous method based on analytical equations. Using the universal method, we could well predict the detection limit of DES+-ISE theoretically. The MP theory is promising for the sophisticated design of ISEs, which is not due to conventional “trial-and-error” procedures.
In this research, it was found that a composite film could be formed by mixing Nafion with chitosan on the graphite electrode surface. Then, based on the strong absorption of both chitosan for AuCl4− and Nafion for Ru(bpy)32+, respectively, the gold nanoparticles and electrochemiluminescence (ECL) active molecules, Ru(bpy)32+, were effectively incorporated into the composite film of chitosan with Nafion. Lastly, based on the interaction of Ru(bpy)32+ with Nafion inside the Nafion-chitosan/gold nanoparticles film, Ru(bpy)32+ was successfully immobilized within this composite film. In this case, a new composite film for fabricating Ru(bpy)32+-based ECL sensors was developed. The performances of this composite film were characterized by transmission electron microscopy (TEM), electrochemistry and electrochemiluminescence methods. Our results showed that: firstly, the gold nanoparticles in the resulting composite film could act as conducting pathways to connect Ru(bpy)32+ sites; the electrode surface accelerated the charge transport through the composite film, and the diffusion coefficient of Ru(bpy)32+ within this composite film modified electrode was 65 times higher than that of the pure Nafion film modified electrode. Secondly, due to its unique polymeric cationic character and better film-forming properties, chitosan could improve the compact structure of pure Nafion and greatly enhance the mass-transfer speed of Ru(bpy)32+. Then, the co-reactant tripropylamine (TPA) inside the composite film could offer better ECL performances such as more rapid ECL response speed, longer-term stability and higher sensitivity compared with the performances of pure Nafion film.
To determine the factors that affect the partitioning of solutes in micellar systems, we investigated the partitioning of several β-diketones and their tris-complexes with iron(III) between the bulk aqueous phase and micelles of various polyoxyethylene (POE)-type nonionic surfactants (C12POE8, Brij 35, Brij 58, and Triton X-100). The trends of the partition constants in the micellar systems differed from those in typical liquid–liquid systems; these differences may have been due to the effects of the substituent groups on the extractants, and to the effects of the inner-sphere chemistry of the micelles. The bulkiness and the low wettability of the extractants and the complexes hindered their extraction into the micellar phase. The interaction between the polyoxyethylene moiety of the surfactants and water molecules dissolved in the micellar mantle may have hampered the penetration of such solutes with bulky or low-wettability substituents into the mantle. The locus of the solutes in the micelles seemed to play an important role in the partitioning behavior.
A selective dopamine (DA) sensor was developed using gold nanoparticles (Au-NPs) dispersed overoxidized-polyaniline (PANIox) based on a boron-doped diamond (BDD) thin-film electrode. The concentration of the DA was determined using voltammetry as a non-enzymatic sensor. BDD thin film has a high signal-to-noise ratio, a long-term stability, a high sensitivity, and a good reproducibility. PANI nanocomposites were directly synthesized on the BDD electrode and overoxidized using 0.5 M H2SO4 solution. The overoxidized PANI film enhances selectivity and sensitivity toward DA. The Au-NPs were dispersed on the PANI nanocomposite by electrochemical deposition. The nanometer-sized Au-NPs favor the sensing of DA in the presence of ascorbic acid (AA). The combination of the PANI with the Au-NPs and the BDD electrode can create synergetic effects for the performance of the biosensor, such as a fast response time, a lower detection limit, a wider linear range, enhanced selectivity, and higher sensitivity for the determination of DA.
A new direct method for log P determination by dispersive liquid/liquid microextraction (DLLME) coupled with derivatized magnetic nanoparticles (DMNPs) predispersed in 1-octanol phase is discussed. First, the aim of DMNPs predispersed into 1-octanol phase was to provide the magnetic force when an ultrastrong magnet was used to separate the two phases. Second, the interaction of 1-octanol with inner DMNPs nuclei prevented emulsion formation in the DLLME process. Moreover, interruption of absorption of DMNPs due to the partition equilibrium of the model compound was negligible. The equilibrium of model compound between the two phases was reached in less than 3 min. The two phases were separated quickly by a super magnet because model compounds in the two phases did not interfere with each other. Fourteen model compounds of varied log P values were measured using this method. The log P values fall in the range of 0.6 to 4.8, which are in agreement with the published results. This method is a rapid, efficient and facile method for direct measurement of log P values.
Three kinds of lake plankton were cultivated, and the properties of protein-like fluorophores released from the plankton were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results were compared with those by gel chromatography with a fluorescence detector and three-dimensional excitation-emission matrix (3-DEEM). The concentrated protein-like fluorophores of algal dissolved organic matter (DOM) were successfully separated from the fulvic-like fluorophores, and analyzed using SDS-PAGE. SDS-PAGE analysis revealed that the protein-like fluorescence DOM released from Microcystis aeruginosa consisted of proteins with molecular weights of 17, 37, 50, 75, 150 kDa, and greater than 250 kDa. The results of SDS-PAGE were consistent with those of gel chromatography. Those substances with molecular weights greater than 250 kDa may be a polysaccharide-peptide complex, called peptidoglycan, which is a component of bacterial cell walls. The molecular weights of protein-like fluorescence DOM from Staurastrum dorsidentiferum were determined to be 37 and 50 kDa. For Cryptomonas ovata, its DOM was found to be composed of substances with molecular weights of between 10 and 150 kDa. The results by high-performance size exclusion chromatography with chemiluminescent nitrogen detection (HPSEC/CLND) analysis suggest that the protein-like fluorophores from the plankton might be composed of substances containing organic nitrogen.
An iron(II) solution is often used as a reducing agent in titrimetry and standardized with cerium(IV) or potassium dichromate. Such an iron(II) standard solution is needed for not only titrimetric analyses, but also instrumental ones. Iron(II) is unstable even in a highly acidic solution, mainly due to air-oxidation; therefore, its standardization is required before use. In the present study, the concentration of an iron(II) solution was accurately determined by coulometric titration with electrogenerated cerium(IV), and also by gravimetric titration with a standard potassium dichromate; new useful information concerning the stability of iron(II) solutions in aqueous sulfuric acid was obtained. The current efficiency of the coulometric titration with electrogenerated cerium(IV) was not very high; however, it was found that the titration efficiency was sufficient to assay an iron(II) solution.
Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.
In this study, we describe a novel application for light scattering, a method widely used for separation of molecules in solution based on their size. We demonstrate that light scattering analysis can monitor the change in particle size of protein 4.1R prior to and after binding to red blood cell inside-out-vesicles in solution. Light scattering constitutes therefore a novel tool to analyze protein-binding association constants.
An open-tubular capillary chromatography was developed based on the tube radial distribution of the ternary mixed carrier solvents that generated the inner and outer phases under laminar flow conditions. This is called “tube radial distribution chromatography” (TRDC). In this report, the elution behavior of lambda-DNA (48502 bp) as a biopolymer was examined by the TRDC system. The ternary mixture of water–acetonitrile–ethyl acetate, 15:3:2 or 3:8:4 volume ratio, as a carrier solution was fed into the capillary tube made of polytetrafluoroethylene (PTFE) or fused-silica. The mixture of hydrophobic 1-naphthol and hydrophilic lambda-DNA was subjected to the TRDC system using the water-rich carrier solution. Lambda-DNA and 1-naphthol were distributed between the inner and outer phases due to their hydrophilic and hydrophobic nature, and then eluted in this order, undergoing chromatographic separation. The mixture of hydrophilic 2,6-naphthalenedisulfonic acid and hydrophobic lambda-DNA that was treated with surfactants was also examined with the organic solvent-rich carrier solution. The modified hydrophobic DNA and 2,6-naphthalenedisulfonic acid were distributed and eluted in this order due to their nature.
Polyamines such as spermidine (Spd) and spermine (Spm), produced by aminopropyltransferase (Apt), play roles in cell growth and differentiation. A sensitive and simple fluorometric high-performance liquid chromatographic determination for Apt activity of spermidine synthase (Spdsyn) and spermine synthase (Spmsyn) was developed in order to examine cellular functions of polyamine synthesis. The derivatization procedure for methylthioadenosine (MTA) produced from decarboxylated S-adenosylmethionine by Apt was the reaction with 2-chloroacetaldehyde to give fluorescent 1, N6-etheno methylthioadenosine. The reaction conditions for derivatization were optimized. A calibration curve was established, ranging from 0.01 to 25 pmol. Quantification of derivatized MTA was confirmed to be identical to Spd or Spm production. The developed method determined Spdsyn and Spmsyn activities in HepG2 cells treated with oleic acid as a cellular lipid accumulation model.
The utility of a cationic polymer polyethylenimines (PEI) for coating some commercially available silica-based stationary phases packed in capillaries of 0.32 mm i.d. and 10 cm long was studied for the separation and direct UV detection of inorganic anions (iodate, bromate, nitrite, bromide, and nitrate ions). With a super-endcapped octadecylated silica stationary phase, which yielded the best separation in terms of resolution and retention time of individual anions, the effect of the pH and composition of the eluent on the elution behavior of individual anions was studied. The relative standard deviations of the retention time, peak area and peak height for 60 successive injections (running time of more than 10 h) were not more than 1.32, 2.30 and 1.94, respectively, with the exceptions of the peak area and peak height of the bromate ion. The detection limits at 210 nm ranged from 1 to 7 ppm. This method was successfully applied to determine concentrations of nitrate ions present in pills and beverage samples.