Mitochondria in a live HepG2 cell were visualized with a fluorescent probe to specify their location and state in a living cell. Then, mitochondria were selectively captured with a nanospray tip under fluorescence microscope, and thousands of small molecular peaks were revealed and unique steroids specific to mitochondria were also found. This fluorescence imaging combined with live single-cell mass spectrometry opens the door to the analysis of site- and state-specific molecular detection to elucidate precise molecular mechanisms at the single-cell and organelle level.

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Direct trapping of a single floating cell, i.e. a white blood cell from a drop of blood, within a nanospray tip was followed by super-sonication after the addition of ionization solvent. Molecular detection of an increased number of peaks with a higher intensity and a wider m/z range, which extends from metabolites to lipids, was acquired than of that without sonication. This method was applied to a few separated circulating tumor cells (CTC) from a neuroblastoma patient’s blood to obtain their lipido-metabolomic molecular profile at the single cell level. In addition to vital molecules such as amino acids, catechol amine metabolites, which are specific to neuroblastoma, and drugs included in the patient’s course of therapy were detected. This established “direct single-cell lipido-metabolomic method” seems to be useful for direct and wide range molecular detection not only for many live single-cells, but also for rare cells, such as CTCs, for future molecular diagnosis.

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This paper provides two approaches to estimate the standard deviation of measurements from baseline noise in instrumental output when (i) in theory, the noise can be approximated by a well-established random process in statistics and mathematics, referred to as a stationary process and (ii) in practice, the baseline noise is the predominant source of measurement error. For the first approach proposed, a general evaluation equation for measurement precision, when the baseline noise can be treated as a stationary process, is derived as a function of the process autocorrelations and process variance of the noise. In particular, for the second approach, when the baseline noise is a mixed random process of white noise and a first order autoregressive (AR(1)) process, the corresponding equation for the precision is also derived. The equations derived in the present paper include some results published elsewhere as special cases. For illustration, an example is presented.

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A polyadenine film (PAE) modified glassy carbon electrode (GCE) was employed for the simultaneous determination of dopamine (DA), uric acid (UA) and guanine (GA). Experimental results showed that this modified electrode had good electrocatalytic properties for the oxidation of DA, UA and GA. Under optimal conditions, DA, UA and GA reflected their electrochemical response into three separated and well-defined oxidation peaks, whose currents increased 47-, 12-, and 7-fold, respectively, compared with those at bare electrode. Moreover, their oxidation peak currents were linear proportional to their concentrations within the ranges of 2.5 – 75, 10 – 750, and 7.5 – 75 μmol L⁻¹, respectively, and the limits of detection (LOD) (S/N = 3) were 0.075, 0.35, and 0.025 μmol L⁻¹, respectively. Compared with a variety of modified electrodes, this designed sensor had a wider linear range and lower LOD. Furthermore, the sensor exhibited good stability and reproducibility.

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The aggregation formation of monoclonal antibodies as biopharmaceuticals induced by heat stress was evaluated by size-exclusion chromatography, and the formation rate was correlated with several physicochemical parameters of the antibodies to clarify the factors to govern the aggregate formation. The parameters we studied were: the melting temperature (T_m) and the standard enthalpy of the melting point (Δ_mH°) evaluated by differential scanning calorimetry under given and common conditions; the wavelength (λ_max) and the intensity (F_int) of the maximum fluorescence peak of 1-anilinonaphthalene-8-sulfonate as a probe dye; the z-average diameter (D) evaluated by dynamic light scattering; and the isoelectric point (pI) and the hydrophobic index (H_pho) of the complementarity determining region calculated from the amino acid sequence. Multivariate statistical analysis with these explanatory variables based on Akaike’s information criterion indicates that the soluble aggregate formation is negatively correlated with T_m and pI, while the insoluble aggregate formation is positively correlated with F_int and pI. Based on these results, the mechanisms of the aggregate formation and methods to prevent the formation are discussed.

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In this work, an electroactive porous Mb-CA’s composite film was fabricated by incorporating myoglobin (Mb) in a three-dimension (3D) porous calcium alginate (CA) film with polyvinyl alcohol, glycerol, and gelatin. The porous Mb-CA’s film modified electrodes exhibited a pair of well-defined, quasi-reversible cyclic voltammetric (CV) peaks at about –0.37 V vs. SCE in pH 7.0 buffers, characteristic of Mb heme Fe^(III)/Fe^(II) redox couples. The electrochemical parameters, such as formal potentials (E^o′) and apparent heterogeneous electron-transfer rate constants (k_s), were estimated by square-wave voltammetry with nonlinear regression analysis. The porous CA’s composite film could form hydrogel in aqueous solution. The positions of the Soret absorbance band suggest that Mb in the CA’s composite film kept its native states in the medium pH range. Hydrogen peroxide, oxygen, and nitrite were electrochemically catalyzed by the Mb-CA’s composite film with significant lowering of the reduction overpotential.

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A novel flow-injection chemiluminescence (FI-CL) method was described for the determination of sinomenine hydrochloride (SIN). The method was based on the inhibitory effect of SIN on the CL reaction of luminol and K₃Fe(CN)₆ in an alkaline solution, which was sensitized by CdTe/CdS quantum dots (QDs). Under the optimized conditions, the linear range for the determination of SIN was 1.0 × 10⁻⁸ to 1.4 × 10⁻⁶ mol/L. The detection limit was 7.5 × 10⁻⁹ mol/L, and the relative standard deviation was 2.47% (n = 11). The current CL method was applied to determine SIN in pharmaceutical formulations and biological fluids with satisfactory results. The possible CL reaction mechanism was discussed briefly.

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In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.

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Microfluidics is a rapidly growing field in which small volumes of liquid are moved through channels in a large variety of applications. Fabricating such channels can be expensive. Here, we describe an inexpensive method for making 3D channels in fluidic chips by using a sacrificial template made of coated metal wire or metal tubes. A 3D template is embedded in polymer or glass and then dissolved, leaving channels in the chip, without the need for expensive instruments. By changing the mold, chips of various shapes can be made.

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We developed a capillary chromatography system using a phase-separated solvent mixture as a carrier solution—i.e., a water-hydrophilic/hydrophobic organic solvent mixture—which we call “tube radial distribution chromatography” (TRDC). Here, we attempted to apply the TRDC system to a microchip incorporating microchannels with a double T-junction for injection of analyte solution and a three-to-one, narrow-to-wide channel confluence point for tube radial distribution phenomenon (TRDP) at room temperature. A ternary mixed solvent of water, acetonitrile and ethyl acetate was used as a carrier solution. TRDP in the wide microchannel was examined using various flow rates, temperatures, and component solvent ratios. Successful observation was carried out using a fluorescence microscope-CCD camera. Model analytes perylene (hydrophobic) and Eosin Y (hydrophilic) were separated by flowing through the microchannel, without any treatment such as packed columns or coating, at room temperature (25°C).

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A separation method for a biopolymer, ε-poly-L-lysine (εPL), with its colorimetric determination using a yellow anionic dye, dipicrylamine anion (DPA⁻), is presented. The εPL-producing culture broth was mixed with a NaDPA solution to precipitate the εPL in polycationic form with the DPA⁻ anion. The precipitate was dissolved into acetonitrile (AN). The AN solution was yellowish, and gave an absorption maximum at around 420 nm. Thus, εPL in the culture broth can be assayed colorimetrically a with multiwell microtiter plate. By the addition of bis(triphenylphosphoranylidene)ammonium chloride, the polycationic εPL was precipitated from the AN solution as the hydrochloride salt. On the other hand, the DPA⁻ anion remained in the AN solution as the quaternary ammonium salt. Thus, εPL larger than tetramer can be separated and purified after the high-throughput screening of the synthetic enzyme.

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A pyridinium benzocrown ether conjugated compound, 1, and its analogue with a non-crown ether unit, 2, have been prepared. Both compounds showed similar absorption spectra with two absorption bands at around 260 and 330 nm in acetonitrile. The bands at the longer wavelength side are associated with intramolecular charge transfer (ICT) absorption, in which the dialkoxyphenyl unit in benzocrown ether and the pyridinium unit act as the donor and acceptor, respectively. The addition of a guest, such as Li⁺ or Mg²⁺, caused a blue shift in the ICT absorption band for 1, but not for 2. This is explained by the formation of a 1:1 host-guest inclusion complex of 1 with the guest. The guest-induced absorption variation of 1 can be used for alkali and alkaline metal ion sensing. Compound 1 could detect divalent cations, especially for Mg²⁺, rather than univalent ones (Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺), although Li⁺ was detected with high sensitivity among the alkali metal ions. Compound 3, which has a pyridyl unit at the para position on the pyridinium of 1, showed a similar trend to that of 1 with lower sensitivity than that of 1. The fact that the Mg²⁺/Li⁺ sensitivity ratio of 1 and 3 was estimated to be 8.63 and 5.08, respectively, suggests a higher Mg²⁺-preference of 1 rather than 3, while the Ca²⁺/Na⁺ ones were 4.98 and 4.85, respectively, when compared ions with similar ionic radii. The sensitivity values of 1 were roughly proportional to their binding constants, as shown by the binding constants with Li⁺, Na⁺, Mg²⁺, and Ca²⁺ with values of 2100, 910, 11500, and 2000 M⁻¹ for 1, respectively. The binding constants of 3 were estimated to be 1710, 650, 3000, and 1400 M⁻¹ for Li⁺, Na⁺, Mg²⁺, and Ca²⁺, respectively, but could not be obtained for alkaline metal ions. The limit concentration for the detection of 1 for Mg²⁺ was estimated to be 0.0156 mM, which was the smallest value in this system.

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Chemosensors provide an efficient and low-cost approach to the detection of various metal ions and small molecules. In the present work, we present a detailed investigation of a simple, easily-prepared, yet efficient, coumarin-based sensor for Hg²⁺. The sensor is based on a fluorescent group of coumarin. When exposed to Hg²⁺, the sensor solution shows a significant color change from yellow to red, and a fluorescence change from green to orange. Further study revealed that the sensor responds solely to Hg²⁺, and shows excellent selectivity even in the presence of other potential competition metal ions, including Na⁺, K⁺, Co²⁺, Fe²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Ag⁺, Zn²⁺, Mg²⁺, Mn²⁺, Pb²⁺, Fe³⁺. Furthermore, the sensor has been successfully applied to detect Hg²⁺ in a form of test paper, which may promote easy and effective application of the sensor.

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A colorimetric method for monosaccharide determination (Anal. Sci., 2013, 29, 1021) was optimized for the high-throughput screening of α-glucosidase, which hydrolyzes an α-1,4-glycosidic bond of starch and related oligo- and polysaccharides, followed by the release of D-glucose from the non-reducing ends. In a microplate, 40 μL of a sample solution was mixed with 160 μL of a 50 mM Na₂SiO₃, 600 mM Na₂MoO₄, 1.5 M CH₃COOH, and 20% (v/v) dimethyl sulfoxide solution, which was yellowish due to the formation of a yellow molybdosilicate. The mixture was kept at 80°C for 60 min. In the mixture, glucose reduced the Mo(VI) species directly to form a blue heteropolymolybdate(V/VI). Thus, 0.1 mM level glucose can be determined by the color change from yellow to blue. Since maltose cannot render the mixture blue as strongly as glucose, the present method has been successfully applied to a microtiter plate assay of α-glucosidase with the disaccharide. Also, the method has been applied to an assay of α-glucosidase inhibitors, acarbose and quercetin.

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Honokiol is a potential candidate for the treatment of intervertebral disc (IVD) degeneration. In this study, we develop in vitro and in vivo methods to detect the distribution of honokiol in intervertebral discs using high-performance liquid chromatography. A rat tail disc was used for both experimental models. For the in vivo animal experiment, blood samples and tail discs were collected at 15, 30, 60, 120 and 240 min after honokiol administration (30 mg/kg, i.v.). The analyte was separated by a mobile phase of methanol and 10 mM NaH₂PO₄ buffer at pH 2.8 (78:22, v/v) and pumped through a reversed-phase analytical column (250 × 4.6 mm, particle size 5 μm) at room temperature. The in vitro experimental results demonstrated that honokiol diffused into the intervertebral disc and was concentration-dependent. The active concentration is obtained for the therapeutic level at 15 and 30 min after honokiol administration in the in vivo model.

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A speciation method for Cr(III) and Cr(VI) by on-line solid-phase extraction using a flow-injection system with FAAS has been proposed. The Cr(III) was selectively retained at pH 5 on the AXAD16-XO chelating resin and was eluted using 0.5 mol L⁻¹ HNO₃. The preconcentration factor and the detection limit (3σ) of 73 and 0.11 μg L⁻¹, respectively, were obtained for a 120-s preconcentration time. The precision (%RSD) at the 100 μg L⁻¹ level was 1.08%. The linearity for Cr(III) measurements was up to 600 μg L⁻¹. The total chromium was determined by reducing Cr(VI) to Cr(III) using hydroxylamine hydrochloride, and the Cr(VI) concentration was obtained by subtraction from the total chromium concentration. The effect of interfering ions was examined towards the selectivity of the proposed system for Cr(III). Spike recovery studies in industrial water samples were carried out using standard Cr(III) and Cr(VI) solutions traceable to NIST. The proposed method was validated using SRM 1643e (Trace Elements in Water) supplied by NIST, USA.

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The analytical performance of 265 nm femtosecond laser ablation (fs-LA) and 213 nm nanosecond laser ablation (ns-LA) systems coupled with multi-collector inductively coupled plasma mass spectrometry (MC-ICPMS) for Pb isotope ratio measurements of solder were compared. Although the time-resolved signals of Pb measured by fs-LA-MC-ICPMS showed smoother signals compared to those obtained by ns-LA-MC-ICPMS, similar precisions on Pb isotope ratio measurements were obtained between them, even though their operating conditions were slightly different. The mass bias correction of the Pb isotope ratio measurement was carried out by a comparison method using a Pb standard solution prepared from NIST SRM 981 Pb metal isotopic standard, which was introduced into the ICP by a desolvation nebulizer (DSN) via a dual-sample introduction system, and it was successfully demonstrated for Pb isotope ratio measurements for either NIST 981 metal isotopic standard or solder by fs-LA-MC-ICPMS since the analytical results agreed well with the certified value as well as the determined value within their standard deviations obtained and the expanded uncertainty of the certified or determined value. The Pb isotope ratios of solder obtained by ns-LA-MC-ICPMS also showed agreement with respect to the determined value within their standard deviations and expanded uncertainty. From these results, it was evaluated that the mass bias correction applied in the present study was useful and both LA-MC-ICPMS could show similar analytical performance for the Pb isotope ratio microanalysis of metallic samples such as solder.

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The identification of ancient silk is of great importance in both archaeology and academia. In the present work, a specific antibody having the characteristics of low cost, easy operation and extensive applicability was developed directly through immunizing rabbits with complete antigen (silk fibroin, SF). Then, antibody-based immunoassays, i.e. enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM), were established and conducted in tandem to identify the corresponding protein in ancient silks. The anti-SF antibody exhibits high sensitivity and specificity for the identification of modern and ancient silks. The detection limit of the ELISA method is about 0.1 ng/mL, and no cross-reactions with other possible interference antigens have been noted. IFM makes it possible to localize target proteins in archaeological samples, and also ensure the reliability of the ELISA results. Based on these advantages, immunological techniques have the potential to become powerful analytical tools at archaeological sites and conservation science laboratories.

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We describe a unique UV-visible absorption spectral property of 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (THPP) in the presence of organophosphorus (OP) pesticides. Upon titrating each 16 among total 40 different OP pesticides, the Soret band was significantly red-shifted, and a very intense Q band appeared. They were attributed to the diprotonation of THPP. A suitable solvent for this reaction was determined to be methanol. THPP would become a potential sensor molecule used to detect OP pesticides with high sensitivity in the concentration range of 10⁻⁶ – 10⁻⁴ M.

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There is considerable evidence that the antioxidant property of ascorbic acid (AH) is effective for reducing oxidative stress of phospholipids. Herein, a liquid crystals (LCs)-based method was developed for the optical amplification of resistance to phospholipid oxidation by AH. Phospholipid peroxidation initiated by free radicals was monitored from a homeotropic-to-planar anchoring transition of LCs via polarized optical microscopy. Alternatively, consistent homeotropic anchoring of LCs was observed when the oxidation caused by free radicals was blocked by AH.

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Fimasartan is a novel angiotensin II receptor blocker with strong anti-hypertensive activity. In this study, a more rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method to determine fimasartan in human plasma was developed and fully validated. The quantification of analytes was conducted by MS/MS in a multiple reaction monitoring mode at m/z 502.2 → 207.1 for fimasartan and m/z 526.3 → 207.1 for the internal standard (IS, BR-A-563). The method showed a linear response from 3 to 1000 ng/mL (r > 0.9950). The intra- and inter-day accuracy values were 86.9 – 98.2 and 93.3 – 100.1%, respectively. The intra- and inter-day precision values were 2.0 – 3.1 and 0.8 – 8.0%, respectively. This UPLC-MS/MS method was validated for specificity, linearity, precision, accuracy, recovery, system suitability, and stability, and was found to be acceptable for bioanalytical applications. Finally, this fully validated method was successfully applied to a pharmacokinetic study of fimasartan in healthy volunteers following oral administration.

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A novel amphipathic phenolic compound, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), that can be isolated from the Pacific oyster (Crassostrea gigas) has been found to protect human hepatocytes against oxidative stress. This study aims to establish a method for the measurement of DHMBA for industrial application. Liquid chromatography–tandem mass spectrometry using deuterated DHMBA as an internal standard and a polar end-capped ODS (Hypersil GOLD aQ) as the solid phase was validated. The limit of detection was 0.04 pmol (S/N = 5), and the limit of quantitation was 0.1 pmol (S/N = 10). The calibration curve was linear throughout the range of 0.1 – 16 pmol (r² = 0.9995). This method successfully quantified DHMBA in oysters from 11 sea areas in Japan. The results showed that the yield of DHMBA was variable from 9.8 to 58.8 μg g⁻¹ whole oyster meat wet weight but not affected by the seawater temperature. The proposed LC-MS/MS method is useful in quantitative studies for DHMBA and potentially for other amphipathic substances.

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