Interface of an Array of Five Capillaries with an Array of One-Nanoliter Wells for High-Resolution Electrophoretic Analysis as an Approach to High-Throughput Chemical Cytometry

Abstract

We report a system that allows the simultaneous aspiration of one or more cells into each of five capillaries for electrophoresis analysis. A glass wafer was etched to create an array of 1 nL wells. The glass was treated with poly(2-hydroxyethyl methacrylate) to control cell adherence. A suspension of formalin-fixed cells was placed on the surface, and cells were allowed to settle. The concentration of cells and the settling time were chosen so that there was, on average, one cell per well. Next, an array of five capillaries was placed so that the tip of each capillary was in contact with a single well. A pulse of vacuum was applied to the distal end of the capillaries to aspirate the content of each well into a capillary. Next, the tips of the capillaries were placed in running buffer and potential was applied. The cells lysed upon contact with the running buffer, and fluorescent components were detected at the distal end of the capillaries by laser-induced fluorescence. The electrophoretic separation efficiency was outstanding, generating over 750,000 theoretical plates (1,800,000 plates/meter). In this example, AtT-20 cells were used that had been treated with TMR-G M1 . The cells were allowed to metabolize this substrate into a series of products before the cells were fixed. The number of cells found in each well was estimated visually under the microscope and was described by a Poisson distribution with mean of 0.95 cells/well. This system provides an approach to high-throughput chemical cytometry.Cytometry is the analysis of individual cells. Flow cytometry and image cytometry are powerful tools that characterize large numbers of cells. 1 However, these measurements can characterize only a handful of specific target molecules. In contrast, chemical cytometry employs powerful analytical tools to characterize the composition of single cells. 2-24 While classic cytometry is capable of analyzing a few components from a very large number of cells, most instrumentation for chemical cytometry is capable of analyzing a large number of components but at a much lower rate. Cells were mixed with surfactant to effect lysis. The lysate was mixed with a fluorogenic substrate that was enzymatically converted to fluorescent product, which was detected downstream from the point of lysis. This system produced an analysis rate of perhaps 10 cells/ minute. However, this system did not employ a separation step and was able to resolve only one component. Wang reported a similar system with a slightly different geometric design that allowed analysis rates approaching 1 cell per second, but again measuring only one component. 26Wu reported a microfluidic system that can capture a cell, lyse the cell, derivatize the lysate, and separate the labeled components with fluorescence detection. 27 In this example, amino acids were determined from single cells. The separation efficiency of this system was limited by the interface between the reaction and separation portions of the device and separation efficiency appeared to be le...

“Fix and assay”: separating in-cellulo sphingolipid reactions from analytical assay in time and space using an aldehyde-based fixative

“…Dovichi and colleagues used formalin-based fixation of cells loaded with fluorescent glycosphingolipid to stop the chemical reactions of these lipids within cells and to measure their intracellular metabolism. 25,26,[29][30][31] Building on this strategy, Proctor and colleagues described a method for analyzing phosphatidylinositol lipids within fixed cells, demonstrating that glutaraldehyde was the best fixative to reliably preserve the phosphatidylinositol metabolites within the framework of a cross-linked cellular environment. 24 However, unlike the glycosphingolipids and phosphatidylinositol lipids, sphingolipids possess a free amine group that might be irreversibly cross-linked to cellular proteins by the fixative, making these analytes unavailable for downstream analysis.…”

Chemical Analysis of Single Cells

“…They reported a microfluidic system that can capture a cell, lyse the cell, derivatize the lysate, and separate the labeled components with fluorescence detection for amino acids from single cells. 21 This system is applied to the analysis of ganglioside metabolism in AtT-20 cells. Cells are incubated with a fluorescent substrate, tetramethylrhodamine (TMR)-GM1.…”

Sampling techniques for single-cell electrophoresis

“…Automated mechanical manipulations not only minimize user handling, they reduce the time it takes to isolate and introduce cells into a separation system. In one strategy demonstrated by Boardman and co-workers, 71 parallel separation of the ganglioside metabolites of AtT-20 cells was accomplished. An array of five capillaries was used to inject single cells from etched nanolitre wells onto a poly(2-hydroxyethyl methacrylate)-coated glass substrate with a minimal amount of manual manipulation.…”