"Fix and Click" for Assay of Sphingolipid Signaling in Single Primary Human Intestinal Epithelial Cells.

Capillary electrophoresis with fluorescence detection (CE-F) is a powerful method to measure enzyme activation in single cells. However, cellular enzymatic assays used in CE-F routinely utilize reporter substrates that possess a bulky fluorophore that may impact enzyme kinetics. To address these challenges, we describe a "fix and click" method utilizing an alkyne-terminated enzyme activation reporter, aldehyde-based fixation, and a click chemistry reaction to attach a fluorophore prior to analysis by single-cell CE-F. The "fix and click" strategy was utilized to investigate sphingolipid signaling in both immortalized cell lines and primary human colonic epithelial cells. When the sphingosine alkyne reporter was loaded into cells, this reporter was metabolized to ceramide (31.6 ± 3.3% peak area) without the production of sphingosine-1-phosphate. In contrast, when the reporter sphingosine fluorescein was introduced into cells, sphingosine fluorescein was converted to sphingosine-1-phosphate and downstream products (32.8 ± 5.7% peak area) without the formation of ceramide. Sphingolipid metabolism was measured in single cells from both differentiated and stem/proliferative human colonic epithelium using "fix and click" paired with CE-F to highlight the diversity of sphingosine metabolism in single cells from primary human colonic epithelium. This novel method will find widespread utility for the performance of single-cell enzyme assays by virtue of its ability to temporally and spatially separate cellular reactions with alkyne-terminated reporters, followed by the assay of enzyme activation at a later time and place.

Silicon Photomultipliers as a Low-Cost Fluorescence Detector for Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation method capable of handling small sample volumes (∼pL) and low (∼yoctomole) detection limits and, as such, is ideal for applications that require high sensitivity, such as single-cell analysis (Chen et al. Anal. Chem. 1996, 68 (4), 690-696; Cohen et al. Annu. Rev. Anal. Chem. 2008, 1 (1), 165-190; Vickerman et al. ACS Chem. Biol. 2018, 13 (7), 1741-1751). Low-cost CE instrumentation is quickly expanding, but low-cost, open-source fluorescence detectors with ultrasensitive detection limits are lacking (Vickerman et al. ACS Chem. Biol. 2018, 13 (7), 1741-1751; Fang et al. Electrophoresis 2016, 37 (17-18), 2376-2383; Casto et al. Anal. Chem. 2019, 40 (1), 65-78). Silicon photomultipliers (SiPM) are inexpensive, low-footprint detectors with the potential to fill the role as a detector when cost, size, and customization are important. In this work, we demonstrate the use of a SiPM in CE with zeptomolar detection limits and a dynamic range spanning 5 orders of magnitude, comparable to photomultiplier detectors. The performance of these detectors was measured using a continuous wave excitation laser in an epifluorescence detection configuration. We characterize the performance of the SiPM as a highly sensitive detector by measuring enzyme activity in single cells. This simple, small footprint, and low-cost (<$130) light detection circuit will be beneficial for open-source, portable, and budget-friendly instrumentation requiring high sensitivity.

Design of an automated capillary electrophoresis platform for single-cell analysis.

Single-cell analysis of cellular contents by highly sensitive analytical instruments is known as chemical cytometry. A chemical cytometer typically samples one cell at a time, quantifies the cellular contents of interest, and then processes and reports that data. Automation adds the potential to perform this entire sequence of events with minimal intervention, increasing throughput and repeatability. In this chapter, we discuss the design considerations for an automated capillary electrophoresis-based instrument for assay of enzymatic activity within single cells. We describe the key requirements of the microscope base and capillary electrophoresis platforms. We also provide detailed protocols and schematic designs of our cell isolation, lysis, sampling, and detection strategies. Additionally, we describe our signal processing and instrument automation workflows. The described automated system has demonstrated single-cell throughput at rates above 100cells/h and analyte limits of detection as low as 10-20mol.

Design and Application of Sensors for Chemical Cytometry.

The bulk cell population response to a stimulus, be it a growth factor or a cytotoxic agent, neglects the cell-to-cell variability that can serve as a friend or as a foe in human biology. Biochemical variations among closely related cells furnish the basis for the adaptability of the immune system but also act as the root cause of resistance to chemotherapy by tumors. Consequently, the ability to probe for the presence of key biochemical variables at the single-cell level is now recognized to be of significant biological and biomedical impact. Chemical cytometry has emerged as an ultrasensitive single-cell platform with the flexibility to measure an array of cellular components, ranging from metabolite concentrations to enzyme activities. We briefly review the various chemical cytometry strategies, including recent advances in reporter design, probe and metabolite separation, and detection instrumentation. We also describe strategies for improving intracellular delivery, biochemical specificity, metabolic stability, and detection sensitivity of probes. Recent applications of these strategies to small molecules, lipids, proteins, and other analytes are discussed. Finally, we assess the current scope and limitations of chemical cytometry and discuss areas for future development to meet the needs of single-cell research.

Simultaneous chromatography and electrophoresis: two-dimensional planar separations.

Single-dimension separations are routinely coupled in series to achieve two-dimensional separations, yet little has been done to simultaneously exploit multiple dimensions during separation. In this work, simultaneous chromatography and electrophoresis is introduced and evaluated for its potential to achieve two-dimensional separations. In simultaneous chromatography and electrophoresis, chromatography occurs via capillary action while an orthogonal electric field concurrently promotes electrophoresis in a second dimension. A novel apparatus with a dual solvent reservoir was designed to apply the concurrent electric field. Various compounds were used to characterize the apparatus and technique, i.e., vitamins, amino acids, and dyes. Improved separation is reported with equivalent analysis times in comparison to planar chromatography alone. The feasibility of simultaneously employing chromatography and electrophoresis in two dimensions is discussed.