This article presents an overview of the recent progress made by our group in the development of bioelectrochemical imaging devices and systems with micro/nanoelectrodes. The topics include bioimaging of enzymes and live cells by scanning electrochemical microscopy (SECM), high-resolution bioimaging by SECM equipped with a nanoprobe, comprehensive measurements and bioimaging with local-redox cycling-based electrochemical (LRC-EC) devices, and rapid and sensitive bioimaging with BioLSI.

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We describe an implantable patch sensor for monitoring L-glutamate in hippocampal slices in submerged and interface preparations. This patch sensor is prepared by excising the cell membrane in the CA3 region of a hippocampal slice in a submerged preparation, and then implanted in the target neuronal region (CA1) of mouse hippocampal slices. The lifetime of the sensor was 3 – 8 min for the interface slice and 40 – 50 min for the submerged slice. The calibration of an implanted sensor can be achieved by adding an L-glutamate solution to a bath (ACSF) solution. The monitoring of L-glutamate release in the CA1 region of mouse hippocampal slices under chemical stimulation with γ-aminobutyric acid (GABA) and potassium chloride (KCl) was demonstrated.

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The purpose of this study is to apply optical waveguide (OWG) spectroscopy to characterize the encapsulation behavior of enzymes modified with polyethylene glycol (PEG), i.e. pegylation, in a hydrophobic mesoporous silica film. For that purpose, pegylated myoglobin (PEG-Mb) was introduced into the silica mesopores modified with octadecylsilyl (ODS) groups and studied by OWG spectroscopy. OWG spectroscopy confirmed that the hydrophobic interaction between the PEG group and the surface ODS group promoted the encapsulation of PEG-Mb into the hydrophobic silica mesopores. The surface density of ODS affected the adsorbed amount of PEG-Mb and the higher surface density of the ODS group resulted in the suppression of adsorption and diffusion of PEG-Mb inside the pore. Since the desorption rate of PEG-Mb was found to be much slower than the adsorption rate, the pegylation of an enzyme could be effective for the enzyme encapsulation into the hydrophobic mesoporous silica host.

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Uracil-DNA glycosylase (UDG) plays a crucial role in DNA lesion repair because it is one of the most important base excision repair (BER) enzymes. Quantitative analysis of UDG activity is of fundamental importance in bioanalysis. Here, an electrochemical sensing platform combined with enzymatic amplification was developed for simple and sensitive assay of UDG activity and its inhibition. This strategy relies on the release of a biotinylated signal probe from the electrode surface, due to the lowered melting temperature of the duplex UDG substrate after UDG treatment. A biotin modification was used as a tracer in the signal probe and streptavidin-alkaline phosphatase (SA-ALP) was taken as a reporter molecule. Upon reacting with UDG, the loss of biotin label led to a decrease in the amount of bound SA-ALP on the electrode surface, resulting in a weaker electrochemical signal. This strategy allowed for a simple, cost-effective, sensitive and selective assay for UDG with a wide linear response range from 0.01 to 10 U/mL and a low detection limit of 0.0079 U/mL. In addition, the effects of drugs on UDG activity have also been investigated. The proposed strategy not only provides a universal platform for the assay of BER enzymes, but also shows potential application for drug screening.

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The calcium ion concentration in cells was measured by a phase-modulation fluorescence lifetime method with compensation for proteins. A high-accuracy measurement of the calcium ion concentration is best realized by fluorescence lifetime measurements, because the fluorescence lifetime is independent of the fluorescence intensity. The fluorescence intensity is easily varied by the scattering of excitation and emission light in cells, photobleaching, the concentration of fluorochromes, and wavelength dispersion of optical elements. A phase-modulation fluorescence lifetime measurement, however, provides high accuracy and precision, and can measure not only the calcium ion concentration, but also other ion concentrations, such as that of magnesium, sodium, and potassium. We have examined the phase-modulation fluorescence lifetime shift using protein compensation in cells, and have measured the calcium ion concentration in cells stimulated with bradykinin.

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A low-conversion poly(butyl methacrylate-co-ethylene dimethacrylate)-based polymer monolithic column was prepared by ultraviolet (UV) irradiation for a short time at a low temperature (–15°C). By UV irradiation for 2 min, the monolithic column exhibited a high permeability of 5.6 × 10⁻¹³ m² and a high column efficiency of over 100000 plates m⁻¹. At this polymerization time, the conversions of butyl methacrylate and ethylene dimethacrylate were only 10 and 21%, respectively, as determined by pyrolysis gas chromatography. The low conversion led to high porosity, which in turn resulted in high permeability. The reduction in conversion also contributed to improve the compositional homogeneity of the prepared polymer monolith, which would promote high column efficiency. Using the prepared low-conversion column in conjunction with a vacuum-driven low-pressure HPLC without a conventional high-pressure pump, the separation of alkylbenzenes was successfully achieved using a low pressure of only −0.045 MPa to generate the mobile phase stream.

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A superheated water chromatography (SWC) method for the separation of alkyl esters of 4-hydroxybenzoic acid (parabens) using a zirconia-based stationary phase was developed and applied to real sample analysis. First, the SWC system was optimized in terms of the proper length of the preheating coil for establishing thermal equilibration of the mobile phase entering the column at the oven temperature. Next, the effect of the column temperature on the retention was investigated at 100 – 180°C. The elution time for all parabens decreased with increasing column temperature, and linear relationships between ln k and 1/T were obtained. At higher column temperatures, the elution time was further shortened because of the increased mobile-phase flow rate. Nevertheless, the loss of column efficiency at the higher flow rates was not significant. The application of the present method to the analysis of commercial lotions was then demonstrated. The quantification results obtained from SWC showed good agreement with those from a conventional HPLC method.

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Gas chromatographic (GC) method has been developed for the determination of the guanidino compounds: guanidine (G), methylguanidine (MG), guanidinoacetic acid (GAA), guanidinopropionic acid (GPA), guanidinobutyric acid (GBA) and guanidinosuccinic acid (GSA) was carried out after precolumn derivatization with glyoxal and ethyl chloroformate from the column HP-5 (30 m × 0.32 mm i.d.) at 90°C for 3 min, followed by a heating rate 25°C/min up to 260°C with a nitrogen flow rate of 2 ml/min. Detection was by FID. The linear calibrations were obtained within 0.1 – 20.0 μmol/L, with limits of detection (LODs) within 0.014 – 0.024 μmol/L. The separation and derivatization was repeatable (n = 6) with relative standard deviations (RSD) within 0.8 – 1.9% in retention time and 0.5 – 1.8% in peak height/peak area. A number of additives and amino acids did not affect the determination. The method was applied for the determination of guanidino compounds from the serum and urine of 9 healthy volunteers and 8 uremic patients and the amounts found were in the range 0.08 – 0.48 and below the limit of detection (LOD) – 345 μmol/L and 1.82 – 13.88 and 0.77 – 432.0 μmol/L with RSDs within 4.2%, respectively.

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A sensitive, novel and rapid chemiluminescence (CL) method combined with high-performance liquid chromatography (HPLC) separation for the determination of salicylic acid (SA) is described in this work. The method was based on the fact that SA could significantly enhance the CL of the reaction of cerium sulfate and the tris(2,2-bipyridyl) ruthenium(II) CL system in the presence of acid. Under the optimal conditions, the CL intensity was linear over concentrations of SA in the range of 0.02 – 10 × 10⁻⁶ g/mL, with a detection limit of 8 × 10⁻⁹ g/mL (S/N = 3). Also, the relative standard detection was 2.2% for 1.0 × 10⁻⁷ g/mL (n = 11). The proposed method has been successfully applied to the analysis of SA in human serum samples and urine samples with satisfactory results.

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The reagent-free mineralization of dissolved organic matter (DOM) in river water was achieved within 1 min using a lamp-pass-through photoreactor containing a narrow reaction tube (2 mm i.d.) passing through a 40 W mercury lamp. The structure efficiently irradiated the sample solution in the tube with vacuum ultraviolet (VUV; 185 nm) light from the lamp, which rapidly decomposed the DOM with hydroxyl radicals generated efficiently from the water and oxygen that are naturally present in the solution. The photoreactor was also applicable to oxidizing reagent-free online toatal organic carbon (TOC) analysis of DOM in river-water samples using a non-dispersive infrared radiation detector after acidification of the sample using 20 mmol L⁻¹ phosphoric acid. The detection limit for phthalate at the injection of 390 μL was 6.2 μg of carbon L⁻¹. The repeatability, as expressed by the relative standard deviation, was 2.5% for thrice-repeated analyses of a river sample with 1.85 mg of carbon L⁻¹.

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The PVC (polyvinyl chloride) and the PP (polypropylene) resin pellet certified reference materials (CRMs, NMIJ CRM 8123-a and 8133-a) with respect to the RoHS (restriction of the use of hazardous substances in electrical and electronics equipment) directive were developed in the present study. Both the sample pretreatment and the measurement procedures for the determination of Cd, Cr, Hg and Pb in the PVC and the PP resin pellets were examined, and several analytical methods were developed. Microwave acid digestion and dry-ashing followed by acid digestion procedures were examined as sample pretreatment procedures. In the case of measurement procedures, an inductively coupled plasma mass spectrometry (ICPMS) including an isotope dilution mass spectrometry (IDMS) and an ICP optical emission spectrometry (ICPOES) were used. Since the analytical results obtained by different analytical methods showed good agreement within their uncertainties estimated, it was concluded that the analytical methods established in this study were reliable with high accuracy. The homogeneity and the stability of Cd, Cr, Hg and Pb in these plastic CRMs were also evaluated, and the results revealed sufficient homogeneity of 0.3 – 0.7% and stability for more than 5 years. From these results, it is considered that these CRMs are useful CRM for the determination of Cd, Cr, Hg and Pb in plastics with respect to the RoHS directive.

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A certified reference material (CRM), NMIJ CRM 7512-a, was developed for the determination of trace elements in milk powder. At least three independent analytical methods were applied to characterize the certified value of each element; all of these analytical methods were based on microwave acid digestions and carried out using different analytical instruments. The certified value was given on a dry-mass basis, where the dry-mass correction factor was obtained by drying the sample at 65°C for 15 to 25 h. The certified values in the units of mass fractions for 13 elements were as follows: Ca, 8.65 (0.38) g kg⁻¹; Fe, 0.104 (0.007) g kg⁻¹; K, 8.41 (0.33) g kg⁻¹; Mg, 0.819 (0.024) g kg⁻¹; Na, 1.87 (0.09) g kg⁻¹; P, 5.62 (0.23) g kg⁻¹; Ba, 0.449 (0.013) mg kg⁻¹; Cu, 4.66 (0.23) mg kg⁻¹; Mn, 0.931 (0.032) mg kg⁻¹; Mo, 0.223 (0.012) mg kg⁻¹; Rb, 8.93 (0.31) mg kg⁻¹; Sr, 5.88 (0.20) mg kg⁻¹; and Zn, 41.3 (1.4) mg kg⁻¹, where the numbers in the parentheses are the expanded uncertainties with a coverage factor of 2. The expanded uncertainties were estimated considering the contribution of the analytical methods, the method-to-method variance, the sample homogeneity, the dry-mass correction factor, and the concentrations of the standard solutions for calibration. The concentrations of As (2.1 μg kg⁻¹), Cd (0.2 μg kg⁻¹), Cr (1.3 μg kg⁻¹), Pb (0.3 μg kg⁻¹), and Y (64 μg kg⁻¹) were given as information values for the present CRM.

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This study describes the optimum conditions at the staining time and the signal intensity for using 8-anilino-1-naphthalenesulfonate (ANS) as a fluorescent probe to detect proteins in SDS-PAGE. Using the optimized protocol, protein can be easily detected by short time fixing (20 min) and washing (2 × 5 min), followed by 10 min of staining. As low as 1 – 2 ng of the protein band can be detected, approximately thirty-fold higher than that of the original protocol. Furthermore, the compatibility of the staining method with MS was also explored by comparing the peptide mass fingerprinting results data of serial dilutions of BSA and ovalbumin stained by ANS with SYPRO Ruby.

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We report a simplified micropatterning method for the straight-line extension of the neurites of cultured neurons. We prepared a poly-D-lysine (PDL)-patterned surface using a polydimethylsiloxane microfluidic stamp. Hippocampal neurons were cultured on the PDL-bound substrate with the stamp removed, allowing for conventional cell seeding and detailed optical observation without fluorescent label. Cultured neurons elongated neurites along straight lines at the single-cell level and displayed spontaneous firing as detected by time-lapse imaging and Ca²⁺ imaging.

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The aim of this study was the preparation and characterization of a bioluminescence bacteria sheet in view of the fabrication of an oxygen marker inside a biofilm. Photobacterium kishitanii sheets were prepared using sodium alginate, and a biofilm of Pseudomonas aeruginosa was dropped over it. The relationship between the dissolved oxygen (DO) concentration in the biofilm and luminescence from the sheet was examined. As a conclusion, the alginate-gel film method was effective in fabricating a DO-monitoring thin film.

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A method was developed for the determination of ¹²⁹I in soil samples that uses an ICP-MS equipped with an octopole reaction system. Oxygen was used as the reaction gas for reducing the background intensity of m/z 129, principally by ¹²⁹Xe⁺. The contribution of polyatomic ions, such as ¹²⁷IH₂⁺, could be effectively corrected for by assuming a production ratio (¹²⁷IH₂⁺/¹²⁷I⁺ = 3 × 10⁻⁸). The measured ¹²⁹I/¹²⁷I ratios in a NIST standard solution and Fukushima soil samples are consistent with the expected value within the analytical error. This method provides a powerful tool for investigating radioiodine contamination in Fukushima and elsewhere.

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