Ultrasensitive Single Cell Metabolomics by Capillary Electrophoresis-Mass Spectrometry with a Thin-Walled Tapered Emitter and Large-Volume Dual Sample Preconcentration.

Single cell metabolome analysis is essential for studying microscale life phenomena such as neuronal networks and tumor microenvironments. Capillary electrophoresis-mass spectrometry (CE-MS) is one of the most sensitive technologies; however, its sensitivity is still not enough for single cell analysis on general human cells such as HeLa. To address these issues, we first developed an efficient ionization emitter, named as a "nanoCESI" emitter, that had a thin-walled (∼10 µm) and tapered (5-10 µm) end. The thin conductive wall enabled sheathless ionization and minimized the flow rate of ionizing sample, and the tapered end efficiently ionized analytes via an electrospray ionization mechanism, providing up to 3.5-fold increase in sensitivity compared with a conventional sheathless emitter. Fifty repetitive analyses on 20 amino acids were successfully achieved with a nanoCESI emitter. Relative standard deviations of 50 analyses were 1.5%, 4.4%, and 6.8% for migration time, peak height, and peak area, respectively, where a limit of detection (LOD) of 170 pM (850 zmol) was achieved. Second, a sample enrichment method, large-volume dual preconcentration by isotachophoresis and stacking (LDIS), was applied to a newly designed protocol of nanoCESI-MS. This approach achieved up to 380-fold enhanced sensitivity and LOD of 450 fM. Compared with normal sheathless CE-MS, coupling of nanoCESI and LDIS provided up to 800-fold increase of sensitivity in total. Finally, metabolome analyses of single HeLa cells were performed, where 20 amino acids were successfully quantified with triple-quadrupole MS and 40 metabolites were identified with quadrupole-time-of-flight MS, as a promising analytical platform for microscale bioanalysis for the next generation.

Micro/nanoparticle separation via curved nano-gap device with enhanced size resolution.

Micro/nanoparticles are widely found in industry and biological field to play important roles and particle size distribution is an important factor to evaluate these particles. Nano-gap device has advantages in size determination for particles in diverse size and/or shape, but it has difficulty in practical use due to severe requirement on instrumental alignment to reproduce the gap profile and non-quantitative sample injection based on capillary action. To solve these problems, curved nano-gap device (CGD) was fabricated from two flat glass plates via a simple microfabrication process to gain enhanced size resolution, and pressure-driven liquid delivery system was coupled to CGD. The gap was precisely controlled by wet etching with hydrofluoric acid on a glass plate to obtain the depth of 35.5±15.0nm on average. CGD utilized glass deflection with 18.1nm elevation/µm lateral distance that achieved practical size resolutions of 14.5nm, which was 15.7% smaller than that of conventional linear nano-gap device. Using CGD, particles from 0.5 to 10µm diameter were trapped and separated. The estimated sizes of the trapped particles matched the suggested values well. Cell sizes were also measured by CGD and the measured values matched with the values found by microscope observation. CGD acquired reproducible instrumental setup that resulted in robust analysis on size of micro/nanoparticles.