Design and Application of Sensors for Chemical Cytometry

Abstract

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.

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

“…The dynamic range required for chemical cytometry spans 3-5 orders of magnitude to match the range of the total amount of reporter loaded into a cell for peptide kinase reporters (10 -21 to 10 -19 mols) and for lipid kinase reporters (10 -19 to 10 -17 mols). 3,21 The SiPM in this configuration has a dynamic range that spans five orders of magnitude while detecting as little as 10 -12 M or 10 -21 moles of fluorescein. Thus, the SiPM detector possesses comparable performance characteristics as a PMT and is suitable for chemical cytometry assays by CE.…”

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

“…Component substitutions can be made depending on experimental requirements and have been discussed in previous work (Dickinson et al, 2013;Hellman, Rau, Yoon, & Venugopalan, 2008;Jiang, Sims, & Allbritton, 2010;Lai et al, 2008;Mainz, Serafin, et al, 2016;Mainz, Wang, et al, 2016;McNamara et al, 2017;Phillips, Bair, Lawrence, Sims, & Allbritton, 2013;Proctor & Allbritton, 2018;Proctor et al, 2014Proctor et al, , 2017Proctor et al, , 2016Rau, Quinto-Su, Hellman, & Venugopalan, 2006;A. H. Turner et al, 2016;Vickerman, Anttila, Petersen, Allbritton, & Lawrence, 2018). In subsequent sections, we describe each of the major instrumentation components and their operation.…”

Selection and optimization of enzyme reporters for chemical cytometry

“…(Dovichi, 2010;Dovichi and Hu, 2003) Microelectrophoretic chemical cytometry is well-suited to address many of the challenges of single-cell analysis by virtue of its low sample volume requirements (pL to nL), superb resolving power (100s of analytes), and extremely low detection limits (10 −21 moles), enabling separation of a large number of analytes from single cells with sub-pM detection limits. (Vickerman et al, 2018) These attributes, in combination with the absence of the need for cell genetic engineering, make this technique ideal for analyzing single cells from small mixed populations such as that from a primary clinical sample. Furthermore, chemical cytometry can provide a direct readout of enzyme activity, irrespective of the DNA, RNA, or protein levels in the cells, yielding valuable information about active cellular processes that cannot be obtained from genetic or expression information alone.…”