Abstract
Cellular heterogeneity occurs, and should be probed, at multiple levels of cellular structure and physiology from the genome to enzyme activity. In particular, single-cell measures of protein levels are complemented by single-cell measurements of the activity of these proteins. Microfluidic assays of enzyme activity at the single-cell level combine moderate to high throughput with low dead volumes and the potential for automation. Herein, we describe the steps required to fabricate and operate a microfluidic device for chemical cytometry of fluorescent or fluorogenic reporters of enzyme activity in individual cells.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kovarik ML, Allbritton NL (2011) Measuring enzyme activity in single cells. Trends Biotechnol 29:222–230
Ubersax JA, Ferrell JE Jr (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8:530–541
Phillips KS, Lai HH, Johnson E, Sims CE, Allbritton NL (2011) Continuous analysis of dye-loaded, single cells on a microfluidic chip. Lab Chip 11:1333–1341
Kovarik ML, Shah PK, Armistead PM, Allbritton NL (2013) Microfluidic chemical cytometry of peptide degradation in single drug-treated acute myeloid leukemia cells. Anal Chem 85:4991–4997
Kovarik ML, Dickinson AJ, Roy P, Poonnen RA, Fine JP, Allbritton NL (2014) Response of single leukemic cells to peptidase inhibitor therapy across time and dose using a microfluidic device. Integr Biol. doi:10.1039/C3IB40249E
Shackman JG, Watson CJ, Kennedy RT (2004) High-throughput automated post-processing of separation data. J Chromatogr A 1040:273–282
Lai HH, Quinto-Su PA, Sims CE, Bachman M, Li GP, Venugopalan V, Allbritton NL (2008) Characterization and use of laser-based lysis for cell analysis on-chip. J R Soc Interface 5:S113–S121
OZ Optics collimators and focusers – receptacle style. http://www.ozoptics.com/ALLNEW_PDF/DTS0094.pdf. Accessed 17 Jan 2014
McClain MA, Culbertson CT, Jacobson SC, Allbritton NL, Sims CE, Ramsey JM (2003) Microfluidic devices for the high-throughput chemical analysis of cells. Anal Chem 75:5646–5655
Metto EC, Evans K, Barney P, Culbertson AH, Gunasekara DB, Caruso G, Hulvey MK, Fracassi da Silva JA, Lunte SM, Culbertson CT (2013) An integrated microfluidic device for monitoring changes in nitric oxide production in single T-lymphocyte (Jurkat) cells. Anal Chem 85:10188–10195
Sun Y, Yin X (2006) Novel multi-depth microfluidic chip for single cell analysis. J Chromatogr A 1117:228–233
Wang HY, Lu C (2006) Microfluidic chemical cytometry based on modulation of local field strength. Chem Commun 33:3528–3530
Greif D, Galla L, Ros A, Anselmetti D (2008) Single cell analysis in full body quartz glass chips with native UV laser-induced fluorescence detection. J Chromatogr A 1206:83–88
Mellors JS, Jorabchi K, Smith LM, Ramsey JM (2010) Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry. Anal Chem 82:967–973
Xia F, Jin W, Yin X, Fang Z (2005) Single-cell analysis by electrochemical detection with a microfluidic device. J Chromatogr A 1063:227–233
Ye F, Huang Y, Xu Q, Shi M, Zhao S (2010) Quantification of taurine and amino acids in mice single fibrosarcoma cell by microchip electrophoresis coupled with chemiluminescence detection. Electrophoresis 31:1630–1636
Xu C, Wang M, Yin X (2011) Three-dimensional (3D) hydrodynamic focusing for continuous sampling and analysis of adherent cells. Analyst 136:3877–3883
Phillips KS, Kottegoda S, Kang KM, Sims CE, Allbritton NL (2008) Separations in poly(dimethylsiloxane) microchips coated with supported bilayer membranes. Anal Chem 80:9756–9762
Kovarik ML, Lai HH, Xiong JC, Allbritton NL (2011) Sample transport and electrokinetic injection in a microchip device for chemical cytometry. Electrophoresis 32:3180–3187
OSHA Directorate of Training and Education. How electrical current affects the human body. https://www.osha.gov/SLTC/etools/construction/electrical_incidents/eleccurrent.html. Accessed 6 Jan 2014
Dickinson AJ, Armistead PM, Allbritton NL (2013) Automated capillary electrophoresis system for fast single-cell analysis. Anal Chem 85:4797–4804
Jiang D, Sims CE, Allbritton NL (2010) Microelectrophoresis platform for fast serial analysis of single cells. Electrophoresis 31:2558–2565
Okada CY, Rechsteiner M (1982) Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell 29:33–41
Tsong TY (1991) Electroporation of cell membranes. Biophys J 60:297–306
Nelson AR, Borland L, Allbritton NL, Sims CE (2007) Myristoyl-based transport of peptides into living cells. Biochemistry 46:14771–14781
Madani F, Lindberg S, Langel U, Futaki S, Graslund A (2011) Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys 2011:414729
Acknowledgment
The authors thank Trinity College for financial support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Shehaj, L., de la Vega, L.L., Kovarik, M.L. (2015). Microfluidic Chemical Cytometry for Enzyme Assays of Single Cells. In: Singh, A., Chandrasekaran, A. (eds) Single Cell Protein Analysis. Methods in Molecular Biology, vol 1346. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2987-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2987-0_15
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2986-3
Online ISBN: 978-1-4939-2987-0
eBook Packages: Springer Protocols