We report the lithographic microfabrication of a movable thin film microelectrode array (MEA) probe consisting of 16 platinum band electrodes placed on top of a supporting borosilicate glass substrate. These 1.2 μm wide electrodes were tightly packed and positioned parallel in two opposite rows within a 20 μm × 25 μm square area and with a distance less than 10 μm from the edge of the glass substrate. We demonstrate the ability to control and place the probe in close proximity to the surface of adherent bovine chromaffin cells and to amperometrically record single exocytosis release events with high spatiotemporal resolution. The two-dimensional position of single exocytotic events occurring in the center gap area separating the two rows of MEA band electrodes and that were codetected by electrodes in both rows was determined by analysis of the fractional detection of catecholamine released between electrodes and exploiting random walk simulations. Hence, two-dimensional electrochemical imaging recording of exocytosis release between the electrodes within this area was achieved. Similarly, by modeling the current spikes codetected by parallel adjacent band electrodes positioned in the same electrode row, a one-dimensional imaging of exocytosis with submicrometer resolution was accomplished within the area. The one- and two-dimensional electrochemical imaging using the MEA probe allowed for high spatial resolution of exocytosis activity and revealed heterogeneous release of catecholamine at the chromaffin cell surface.

We report the fabrication and characterization of microwell-based individually addressable microelectrode arrays (MEAs) and their application to spatially and temporally resolved detection of neurotransmitter release across a single pheochromocytoma (PC12) cell. The microwell-based MEAs consist of 16 4-μm-width square ultramicroelectrodes, 25 3-μm-width square ultramicroelectrodes, or 36 2-μm-width square ultramicroelectrodes, all inside a 40 × 40 μm square SU-8 microwell. MEAs were fabricated on glass substrates by photolithography, thin film deposition, and reactive ion etching. The ultramicroelectrodes in each MEA are tightly defined in a 30 × 30 μm square area, which is further encased inside the SU-8 microwell. With this method, we demonstrate that these microelectrodes are stable, reproducible, and demonstrate good electrochemical properties using cyclic voltammetry. Effective targeting and culture of a single cell is achieved by combining cell-sized microwell trapping and cell-picking micropipet techniques. The surface of the microelectrodes in the MEA was coated with collagen IV to promote cell adhesion and further single-cell culture, as good adhesion between the cell membrane and the electrode surface is critical for the quality of the measurements. Imaging the spatial distribution of exocytosis at the surface of a single PC12 cell has also been demonstrated with this system. Exocytotic signals have been successfully recorded from eight independent 2-μm-wide ultramicroelectrodes from a single PC12 cell showing that the subcellular heterogeneity in single-cell exocytosis can be precisely analyzed with these microwell-based MEAs.

We report the application of individually addressable microelectrode arrays (MEAs) to study the heterogeneity of cell exocytosis at the subcellular level. Multiple subcellular-size electrodes are covered by a single PC12 cell for the investigation of subcellular exocytosis. PC12 cells have been seeded and cultured on top of three kinds of MEAs containing 16, 25, or 36 square microelectrodes (4 μm width in a 4 by 4 MEA, 3 μm width in a 5 by 5 MEA, 2 μm width in a 6 by 6 MEA). After collagen coating, single cells were found to cover several electrodes and these were selected for the study of subcellular exocytosis. Amperometric results show that single cell and subcellular heterogeneity in single cell exocytosis can be electrochemically detected with these MEAs. The results also show that these MEAs are suitable for detecting fast chemical events at single cells, as well as for developing multifunctional electrochemical sensors.

An organic lateral resolution test device has been developed to measure the performance of imaging mass spectrometry (IMS) systems. The device contains periodic gratings of polyethylene glycol (PEG) and lipid bars covering a wide range of spatial frequencies. Microfabrication technologies were employed to produce well-defined chemical interfaces, which allow lateral resolution to be assessed using the edge-spread function (ESF). In addition, the design of the device allows for the direct measurement of the modulation transfer function (MTF) to assess image quality. Scanning electron microscopy (SEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used to characterize the device. TOF-SIMS imaging was used to measure the chemical displacement of biomolecules in matrix-assisted laser desorption/ionization (MALDI) matrix crystals. In a proof-of-concept experiment, the platform was also used to evaluate MALDI matrix application methods, specifically aerosol spray and sublimation methods.

Thin-film platinum ultramicroelectrode arrays (MEAs) with subcellular microelectrodes were developed for the spatial measurement of neurotransmitter release across single cells or clusters of single cells. MEAs consisting of 16, 25, and 36 square ultramicroelectrodes with respective widths of 4, 3, and 2 μm were fabricated on glass substrates by photolithography, thin-film deposition, and reactive ion etching. The electrodes in each MEA are tightly defined in a 30 μm × 30 μm square, which is potentially useful to measure exocytosis across a single cell or clusters of single cells. These MEAs have been characterized with scanning electron microscopy and cyclic voltammetry and show excellent stability and reproducibility. Culturing PC12 cells on top of the MEAs has been achieved by modifying the array with a poly(dimethylsiloxane) chamber and coating a thin layer of collagen IV on top of the electrode surface. The electrochemical response to dopamine has been characterized after coating the surface with the cell-adhering molecules and then with cells attached. Amperometric detection demonstrates that individual exocytotic events can be recorded at these arrays with spatial resolution for dynamic electrochemical measurements near 2 μm. In contrast to previous single-cell experiments, the effect of dopaminergic drugs on imaging single vesicle exocytotic release from PC12 cell clusters is presented at cell clusters incubated with the dopamine precursor and Parkinson's therapy agent, L-3,4-dihydroxyphenylalanine, and at cell clusters incubated with the vesicular monoamine transport inhibitor, reserpine. The results of electrochemical imaging demonstrate that the drug effect on PC12 cell clusters is consistent with previous single-cell experiments.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTChemical Analysis of Single CellsRaphaël Trouillon†, Melissa K. Passarelli†, Jun Wang†, Michael E. Kurczy‡, and Andrew G. Ewing*†‡View Author Information† University of Gothenburg, Department of Chemistry and Molecular Biology, 41296 Gothenburg, Sweden‡ Chalmers University, Department of Chemistry and Biological Engineering, 41296 Gothenburg, Sweden*E-mail: [email protected]Cite this: Anal. Chem. 2013, 85, 2, 522–542Publication Date (Web):November 14, 2012Publication History Published online30 November 2012Published inissue 15 January 2013https://pubs.acs.org/doi/10.1021/ac303290shttps://doi.org/10.1021/ac303290sreview-articleACS PublicationsCopyright © 2012 American Chemical SocietyRequest reuse permissionsArticle Views7590Altmetric-Citations156LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Cells,Electrochemical cells,Electrodes,Fluorescence,Mass spectrometry Get e-Alerts

Dry film resist SU-8 was used to make a thick mold for soft lithography of a poly(dimethylsiloxane) (PDMS) microfluidic chip with deep channels. The stacking of the SU-8 film enabled an ultra-thick (up to 500 mu m) resist process on Si wafer. This process was fast and highly reproducible compared with the conventional liquid SU-8 process. The deep channel in the PDMS chip was utilized as a micro-flow cell for sensitive absorbance measurement. Sunset Yellow FCF dye was used to demonstrate absorption spectroscopy in the deep channel. Since the channel depth was proportional to the optical path length, which was proportional to the absorbance value, the PDMS chip achieved a detection limit (15.9 mu M) comparable to U- or Z-shaped microfabricated absorbance detection cells in glass. Calibration curves for different solution concentrations were obtained with good R-2 values (similar to 1).