Alexander S. Groombridge

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.

Microcapsule Buckling Triggered by Compression-Induced Interfacial Phase Change

There is an emerging trend toward the fabrication of microcapsules at liquid interfaces. In order to control the parameters of such capsules, the interfacial processes governing their formation must be understood. Here, poly(vinyl alcohol) films are assembled at the interface of water-in-oil microfluidic droplets. The polymer is cross-linked using cucurbit[8]uril ternary supramolecular complexes. It is shown that compression-induced phase change causes the onset of buckling in the interfacial film. On evaporative compression, the interfacial film both increases in density and thickens, until it reaches a critical density and a phase change occurs. We show that this increase in density can be simply related to the film Poisson ratio and area compression. This description captures fundamentals of many compressive interfacial phase changes and can also explain the observation of a fixed thickness-to-radius ratio at buckling, [Formula: see text].

Modified high performance concentric nebulizer for inductively coupled plasma optical emission spectrometry

The high performance triple-tube concentric nebulizer (HPCN) was modified and evaluated for sample introduction into inductively coupled plasma optical emission spectrometry (ICP-OES) at liquid flow rates of 0.25 to 0.8 mL min−1. The acceptable liquid flow rate for the use of HPCN was extended by replacing the tapered center capillary tube of the nebulizer (i.d./o.d.: 50 μm/150 μm) with a large bore tube (i.d./o.d.: 110 μm/170 μm). The nebulization efficiency was much improved by reducing the inner diameter of the nebulizer nozzle from 250 μm to 200 μm. At a liquid flow rate of 0.8 mL min−1 and a nebulizer gas flow rate of 1 L min−1, the Sauter mean diameter (D3,2) of the primary aerosol generated by the modified HPCN was 3.4 μm, and over 90% (v/v) of the aerosol droplets were below 10 μm in diameter. The D3,2 value was smaller than those generated by conventional nebulizers, Meinhard nebulizer type C (8.2 μm), Glass Expansion Conikal nebulizer (15.8 μm), SeaSpray (14.8 μm), and MiraMist (5.6 μm). The amount of the tertiary aerosol of the modified HPCN generated through a non-baffled cyclone chamber was approximately 1.8 to 3.1 times higher than those of the other nebulizers, with similar size distributions. The sensitivity in ICP-OES with the modified HPCN was 1.5- to 3.2-fold higher than those with the other nebulizers when the liquid flow rates were the same. The plasma robustness estimated from the commonly used ratio Mg(II)/Mg(I) was the same or slightly better than that of the conventional nebulizers. The HPCN also showed a good tolerance to high total dissolved solids (TDS) using 20% NaCl solution. Validation of the modified HPCN was performed by a recovery test of spiked seawater and analyzing the NMIJ CRM 7502-a white rice flour. The observed values for the ten elements Na, Mg, P, K, Ca, Mn, Fe, Cu, Zn and Cd were in good agreement with their certified values. We concluded the modified HPCN is a very useful nebulizer for ICP-OES with good performance at a large range of sample flow rates.

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.

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.

Research data supporting "Microcapsule Buckling Triggered by Compression-Induced Interfacial Phase Change"

Numerical data from each graph are provided in tab-delimited text format. Uncropped versions of each image used are provided in png format. AFM data are enclosed in a zip folder containing mi format files. mi files can be opened with Gwyddion software which is freely available online. The files are labelled according to the relevant figure number. Refer to the manuscript and ESI for a description of the data.