Shin-ichi Miyashita

Highly efficient single-cell analysis of microbial cells by time-resolved inductively coupled plasma mass spectrometry

To realise highly efficient single-cell analysis of microbial cells by time-resolved inductively coupled plasma mass spectrometry (ICP-MS), we developed a modified high efficiency cell introduction system (HECIS), consisting of a large-bore high performance concentric nebulizer (LB-HPCN) with a centre capillary tube of 150 μm inner diameter and a custom-made small-volume (15 cm3) on-axis spray chamber that uses a sheath gas flow near the chamber exit to suppress cell deposition. We also assembled an external ion pulse counting unit to directly read the ion pulse current from the electron multiplier of the ICP-MS via a function generator with no dead time, in order to obtain data with sufficiently high time resolution (i.e., 0.05–1 ms). As compared to a conventional ICP-MS working at its minimum integration time (10 ms), this assembly led to more than ca. 13-fold higher signal-to-background ratios for 31P, and made higher throughput of cells to the plasma more feasible. By using the modified HECIS and the external ion pulse counting unit for determination of the cell introduction efficiencies of different-sized unicellular microbes, including yeast (Saccharomyces cerevisiae), cyanobacterium (Synechocystis sp. PCC 6803), red algae (Cyanidioschyzon merolae 10D and Galdieria sulphuraria), and green alga (Chlamydomonas reinhardtii CC-125), it was revealed that their cell introduction efficiencies ranged from 86% (for C. reinhardtii CC-125 with a mean cell diameter of 6.4 μm) to ca. 100% (for other microbes with mean cell diameters of 2.0–3.0 μm), implying that by use of the ICP-MS system, the cell introduction efficiencies are able to reach approximately 100% and tend to decrease with increasing cell sizes (at least more than 3.1 μm in mean diameter). A wide range of biologically important elements, such as C, Mg, Al, P, S, K, Ca, Cr, Mn, Fe, and Zn, were tested for reasonable detection using the ICP-MS system. Results likely corresponding to separate cell events were obtained for some elements present in each microbe.

Effective and selective recovery of gold and palladium ions from metal wastewater using a sulfothermophilic red alga, Galdieria sulphuraria.

The demand for precious metals has increased in recent years. However, low concentrations of precious metals dissolved in wastewater are yet to be recovered because of high operation costs and technical problems. The unicellular red alga, Galdieria sulphuraria, efficiently absorbs precious metals through biosorption. In this study, over 90% of gold and palladium could be selectively recovered from aqua regia-based metal wastewater by using G. sulphuraria. These metals were eluted from the cells into ammonium solutions containing 0.2M ammonium salts without other contaminating metals. The use of G. sulphuraria is an eco-friendly and cost-effective way of recovering low concentrations of gold and palladium discarded in metal wastewater.

A coupling system of capillary gel electrophoresis with inductively coupled plasma-mass spectrometry for the determination of double stranded DNA fragments

The coupling system of capillary gel electrophoresis (CGE) and inductively coupled plasma-mass spectrometry (ICP-MS) was newly developed and successfully applied to the double-stranded (ds) DNA quantification. The developed system combines the separation technique for large biomolecules and element selective detection of ICP-MS. This coupling was achieved by using the modified high performance concentric nebulizer (HPCN) with the PTFE tube (HPCN-PT), which can produce the liquid jet by the flow focusing effect. The HPCN-PT effectively nebulizes the highly viscous solution containing gel buffer even at a low flow rate. At a liquid flow rate of 0.010 mL min(-1) and a nebulizer gas flow rate of 1 L min(-1), the Sauter mean diameter (D3,2) of primary aerosols generated by the HPCN-PT was 3.4 μm, and over 90% (v/v) of the aerosol droplets were less than 10 μm in diameter. The electrophoresis capillary filled with gel buffer was connected to the HPCN-PT via the interface. This interface has two connectors and an electrode that can connect CE and ICP-MS. After the electrophoretic separation at atmospheric pressure, the samples were transferred to the ICP-MS through the interface by applying additional pressure. Fragments of dsDNA, which were commercially available as a ladder marker solution, were successfully separated and analyzed by measuring (31)P(+) with CGE-ICP-MS, and a linear calibration curve of the phosphorus standard solution (R(2) = 0.999) was obtained from 2.7 to 27 mg kg(-1). The detection limit (LOD) and absolute detection limit of P were 3.7 μg kg(-1) and 0.6 pg (equivalent to 6 pg of DNA), respectively. This absolute detection limit value was equal to the conventional fluorescence determination of DNA.

Identification of possible technical problems in determination of the major inorganic constituents of brown-rice flour by evaluating proficiency test results

AbstractTo support skill upgrading in analysis of inorganic constituents of environmental and food samples, the National Metrology Institute of Japan (NMIJ) and the National Food Research Institute (NFRI) have organized a proficiency test (PT) of determination of Mn, Fe, Cu, Zn, As, and Cd in brown-rice flour based on the international standard (ISO/IEC 17043:2010). One hundred and thirty-three sets of reports were assessed by use of the En-number and z-score approaches in accordance with ISO/IEC 17043 and the international harmonized protocol for PT. The PT results and analytical procedures, reported in detail, were reviewed, and possible technical reasons for questionable or unsatisfactory results are discussed. Distribution of reported values for cadmium in the test material according to the measurement methods used. The mean values reported with standard deviations (error bars) are given in the order of lower to higher values. Solid lines indicate the expanded uncertainty (coverage factor k = 2) of assigned value.

Separation and quantification of RNA molecules using size-exclusion chromatography hyphenated with inductively coupled plasma-mass spectrometry.

The hyphenation of SEC with ICP‐MS was successfully applied to RNA quantification. The developed method combines the separation technique for large biomolecules and element selective detection of ICP‐MS. The separation of RNA molecules was performed under the SEC condition without additive reagents such as salts to prevent the adhesion of RNA molecules on the column resin. Fragments of RNA, which were commercially available as a ladder marker solution and certified reference materials, were successfully separated and analyzed by measuring 31P+ with this method. RNA was quantified with good repeatability (RSD of peak area; 2.7%, n = 3) and linearity (R2 = 0.999) using a P standard solution as a calibrant. LOD and absolute detection limit of RNA were 6.7 μg/kg and 67 pg, respectively, which were equal to the values obtained by the analysis of a P standard solution. The accuracy of the proposed measurement was evaluated by measuring certified reference materials of RNA solutions for quantitative analysis (NMIJ CRM 6204‐a). The results obtained by this method agreed with the certified values within uncertainty. The proposed analysis method, which demonstrates good accuracy and high precision and is free from interference by nucleotide analogues, qualifies as a method of quality control for the RNA synthesis and extraction process.

Arsenic metabolism in cyanobacteria

Environmental context Cyanobacteria are ecologically important, photosynthetic organisms that are widely distributed throughout the environment. They play a central role in arsenic transformations in terms of both mineralisation and formation of organoarsenic species as the primary producers in aquatic ecosystems. In this review, arsenic resistance, transport and biotransformation in cyanobacteria are reviewed and compared with those in other organisms. Abstract Arsenic is a toxic element that is widely distributed in the lithosphere, hydrosphere and biosphere. Some species of cyanobacteria can grow in high concentrations of arsenate (pentavalent inorganic arsenic compound) (100mM) and in low-millimolar concentrations of arsenite (trivalent inorganic arsenic compound). Arsenate, which is a molecular analogue of phosphate, is taken up by cells through phosphate transporters, and inhibits oxidative phosphorylation and photophosphorylation. Arsenite, which enters the cell through a concentration gradient, shows higher toxicity than arsenate by binding to sulfhydryl groups and impairing the functions of many proteins. Detoxification mechanisms for arsenic in cyanobacterial cells include efflux of intracellular inorganic arsenic compounds, and biosynthesis of methylarsonic acid and dimethylarsinic acid through methylation of intracellular inorganic arsenic compounds. In some cyanobacteria, ars genes coding for an arsenate reductase (arsC), a membrane-bound protein involved in arsenic efflux (arsB) and an arsenite S-adenosylmethionine methyltransferase (arsM) have been found. Furthermore, cyanobacteria can produce more complex arsenic species such as arsenosugars. In this review, arsenic metabolism in cyanobacteria is reviewed, compared with that in other organisms. Knowledge gaps remain regarding both arsenic transport (e.g. uptake of methylated arsenicals and excretion of arsenate) and biotransformation (especially production of lipid-soluble arsenicals). Further studies in these areas are required, not only for a better understanding of the role of cyanobacteria in the circulation of arsenic in aquatic environments, but also for their application to arsenic bioremediation.

Single Cell Analysis by Using ICP-MS

Single cell analysis by using inductively coupled plasma mass spectrometry (ICP-MS) has been attracting attention for investigating a cell-to-cell variation of content of elements and biomolecules. ICP-MS has a unique feature that can be applied to a highly sensitive elemental analysis for evaluating a cell-to-cell variance or cellular uptake of elements, drugs, and nanoparticles and a multiparametric analysis for characterizing a cell population based on multiparameters, like a flow cytometry (so-called mass cytometry). In addition, subcellular localization analysis including multiparametric imaging can be performed with the coupling of a laser ablation system. In this chapter, we describe an overview of single cell analysis by using ICP-MS and its future prospects, focusing on cytometric analysis and imaging analysis.

Report of the key comparison CCQM-K108 determination of arsenic species, total arsenic and cadmium in brown rice flour

The CCQM-K108 key comparison was organised by the Inorganic Analysis Working Group (IAWG) of CCQM to test the abilities of national metrology institutes (NMIs) or designated institutes (DIs) to measure the mass fractions of arsenic species, total arsenic and cadmium in brown rice flour. The National Metrology Institute of Japan (NMIJ) acted as the coordinating laboratory. The participants used different measurement methods, though most of them used inductively coupled plasma mass spectrometry (ICP-MS) or isotope-dilution inductively coupled plasma mass spectrometry (ID-ICP-MS) for Cd and ICP-MS for total arsenic. Regarding arsenic speciation, all participants used ICP-MS coupled with liquid chromatography (LC). Accounting for relative expanded uncertainty, comparability of measurement results for each of total arsenic and cadmium was successfully demonstrated by the participating NMIs or DIs for the measurement of the measurand at the level of less than 0.5 mg/kg. Regarding arsenic species (inorganic arsenic and dimethylarsinic acid (DMAA)), there was, however, a measurement problem still to be solved and that part of CCQM-K108 will be repeated. It is expected that arsenic, cadmium and other metals at mass fractions greater than approximately 0.1 mg/kg in rice flour can be determined by each participant using the same technique(s) employed for this key comparison to achieve similar uncertainties mentioned in the present report. Furthermore, the results of this key comparison can be utilised along with the IAWG core capability approach. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

A novel concentric grid nebulizer for inductively coupled plasma optical emission spectrometry

A novel concentric type grid nebulizer (CGrid) was developed for sample introduction into an inductively coupled plasma optical emission spectrometer (ICP-OES). The CGrid has a concentric structure and a grid screen (over 350 meshes per inch) that is set inside the nozzle. The grid screen acts as both an effective gas–liquid mixing filter and a gas flow damper, and then the liquid breaks up into small droplets by passing through the grid with low velocity. By this unique nebulizing process, the CGrid showed excellent nebulizer performances on comparing with commercially available nebulizers, such as Meinhard nebulizer type C (MHN), modified high performance concentric nebulizer (m-HPCN), and OneNeb. The primary aerosols generated with the CGrid were finer and their velocities were lower than those with the other nebulizers. This nebulization feature gave a high transport efficiency of aerosols into the plasma, resulting in high sensitivity in ICP-OES. In the range of the liquid flow rate of 0.25 mL min−1 to 2.0 mL min−1 with the optimized nebulizer gas flow rate for obtaining the highest Mg(II)/Mg(I) signal intensity ratio, the maximum loading amount of aerosols into the plasma obtained with the CGrid was higher than those with the MHN (2.1-fold) and m-HPCN (1.4-fold), and almost the same as that with the OneNeb. The maximum sensitivity in ICP-OES obtained with the CGrid was 1.8- to 3.7-fold, 1.5- to 1.9-fold, and 1.1- to 1.2-fold higher than those with the MHN, m-HPCN, and OneNeb, respectively. The CGrid also showed a good tolerance for high total dissolved solid (TDS) concentrations. No clogging was observed when saturated NaCl solution was continuously nebulized for 5 hours. The limits of detection (LODs) obtained with the CGrid were better than those of the MHN, 1.6- to 5.3-fold improved, except for Cd I 228.802 nm, and similar to those of the m-HPCN and OneNeb. The plasma robustness estimated from the Mg(II)/Mg(I) signal intensity ratio obtained with the CGrid (10.6) was also better than those of the MHN (9.6), and similar to those of the m-HPCN (10.2) and OneNeb (10.4). The short-term stability on measuring spiked seawater (45 min) was within 3% of the relative standard deviation, and the recoveries of the spiked elements were in the range of 99% to 106%. The validation of the CGrid was performed by analyzing the NMIJ CRM 7531-a brown rice flour. The observed values for the five elements Mn, Fe, Cu, Zn, and Cd were in good agreement with their certified values. It was concluded that the CGrid is very useful for ICP-OES with good performance on sensitivity and high TDS solution analysis.

Final report on APMP.QM-S5: Essential and toxic elements in seafood

The supplementary comparison APMP.QM-S5 was undertaken to demonstrate the capability of participating national metrology institutes (NMIs) and designated institutes (DIs) in measuring the contents of the incurred essential elements (iron and zinc) and toxic elements (total arsenic and cadmium) at ?g/g levels in a test sample of dried shrimp by various analytical techniques. At the APMP TCQM Meeting held in Pattaya, Thailand in November 2010, Government Laboratory of the Government of the Hong Kong Special Administrative Region (GLHK) proposed this APMP supplementary comparison. The proposal was further discussed and agreed upon at the CCQM Inorganic Analysis Working Group Meeting held in Paris in April 2011. GLHK was the coordinating laboratory for the supplementary comparison. For enhancing the collaboration amongst specialized regional bodies in Asia-Pacific and to help build the laboratory capacity of NMIs/DIs from developing economies, the reference values of the supplementary comparison are used for evaluation of performance of participants of an APMP proficiency testing programme (APMP PT 11-01), an Asia-Pacific Laboratory Accreditation Cooperation proficiency testing programme (APLAC T082) and an Asia-Pacific Economic Co-operation proficiency testing programme (APEC PT), which were concurrently run using the same testing material as in APMP.QM-S5. The supplementary comparison serves to facilitate claims by participants on the Calibration and Measurement Capabilities (CMCs) as listed in Appendix C of the Key Comparison Database (KCDB) under the Mutual Recognition Arrangement of the International Committee for Weights and Measures (CIPM MRA). A total of 18 institutes registered for the supplementary comparison and all of them submitted their results. Most of the participants used microwave acid digestion methods for sample dissolution. For the instrumental determination, a variety of techniques like ICP-MS, ICP-OES, INAA and AAS were employed by the participants. For this supplementary comparison, inorganic core capabilities have been demonstrated by concerned participants with respect to methods including ICP-MS (without isotope dilution), ID-ICP-MS, ICP-OES, INAA and AAS on the determination of total arsenic, cadmium, iron and zinc in a matrix of seafood. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the APMP, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).