The possibility to conduct complete cell assays under a precisely controlled environment while consuming minor amounts of chemicals and precious drugs have made microfluidics an interesting candidate for quantitative single-cell studies. Here, we present an application-specific microfluidic device, cellcomb, capable of conducting high-throughput single-cell experiments. The system employs pure hydrodynamic forces for easy cell trapping and is readily fabricated in polydimethylsiloxane (PDMS) using soft lithography techniques. The cell-trapping array consists of V-shaped pockets designed to accommodate up to six Saccharomyces cerevisiae (yeast cells) with the average diameter of 4 μm. We used this platform to monitor the impact of flow rate modulation on the arsenite (As(III)) uptake in yeast. Redistribution of a green fluorescent protein (GFP)-tagged version of the heat shock protein Hsp104 was followed over time as read out. Results showed a clear reverse correlation between the arsenite uptake and three different adjusted low = 25 nL min ?1, moderate = 50 nL min ?1, and high = 100 nL min ?1 flow rates. We consider the presented device as the first building block of a future integrated application-specific cell-trapping array that can be used to conduct complete single cell experiments on different cell types.
References
[1]
Figeys, D.; Pinto, D. Lab-on-a-chip: A revolution in biological and medical sciences. Anal. Chem. 2000, 72, 330A–335A.
[2]
Haeberle, S.; Zengerle, R. Microfluidic platforms for lab-on-a-chip applications. Lab Chip 2007, 7, 1094–1110, doi:10.1039/b706364b.
[3]
Squires, T.M.; Quake, S.R. Microfluidics: Fluid physics at the nanoliter scale. Rev. Mod. Phys. 2005, 77, 977–1026, doi:10.1103/RevModPhys.77.977.
[4]
Elowitz, M.B.; Levine, A.J.; Siggia, E.D.; Swain, P.S. Stochastic gene expression in a single cell. Sci. Signal. 2002, 297, 1183–1186.
[5]
K?rn, M.; Elston, T.C.; Blake, W.J.; Collins, J.J. Stochasticity in gene expression: From theories to phenotypes. Nat. Rev. Genet. 2005, 6, 451–464, doi:10.1038/nrg1615.
[6]
Burnette, W.N. “Western blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein a. Anal. Biochem. 1981, 112, 195–203, doi:10.1016/0003-2697(81)90281-5.
[7]
Dorak, M.T. Real-Time PCR; Taylor & Francis: New York, NY, USA, 2006.
[8]
Svahn, H.A.; van den Berg, A. Single cells or large populations? Lab Chip 2007, 7, 544–546, doi:10.1039/b704632b.
Muzzey, D.; van Oudenaarden, A. Quantitative time-lapse fluorescence microscopy in single cells. Ann. Rev. Cell Dev. Biol. 2009, 25, 301–327, doi:10.1146/annurev.cellbio.042308.113408.
[11]
Jen, C.-P.; Hsiao, J.-H.; Maslov, N.A. Single-cell chemical lysis on microfluidic chips with arrays of microwells. Sensors 2011, 12, 347–358, doi:10.3390/s120100347.
[12]
Rettig, J.R.; Folch, A. Large-scale single-cell trapping and imaging using microwell arrays. Anal. Chem. 2005, 77, 5628–5634, doi:10.1021/ac0505977.
[13]
Yamamura, S.; Kishi, H.; Tokimitsu, Y.; Kondo, S.; Honda, R.; Rao, S.R.; Omori, M.; Tamiya, E.; Muraguchi, A. Single-cell microarray for analyzing cellular response. Anal. Chem. 2005, 77, 8050–8056, doi:10.1021/ac0515632.
[14]
Irimia, D.; Toner, M. Cell handling using microstructured membranes. Lab Chip 2006, 6, 345–352, doi:10.1039/b515983k.
Ashkin, A.; Dziedzic, J.; Yamane, T. Optical trapping and manipulation of single cells using infrared laser beams. Nature 1987, 330, 769–771, doi:10.1038/330769a0.
[17]
Eriksson, E.; Scrimgeour, J.; Graneli, A.; Ramser, K.; Wellander, R.; Enger, J.; Hanstorp, D.; Goks?r, M. Optical manipulation and microfluidics for studies of single cell dynamics. J. Opt. A Pure Appl. Opt. 2007, 9, S113, doi:10.1088/1464-4258/9/8/S02.
[18]
Ramser, K.; Hanstorp, D. Optical manipulation for single-cell studies. J. Biophotonics 2010, 3, 187–206, doi:10.1002/jbio.200910050.
[19]
Taff, B.M.; Voldman, J. A scalable addressable positive-dielectrophoretic cell-sorting array. Anal. Chem. 2005, 77, 7976–7983, doi:10.1021/ac0513616.
[20]
Gascoyne, P.; Mahidol, C.; Ruchirawat, M.; Satayavivad, J.; Watcharasit, P.; Becker, F.F. Microsample preparation by dielectrophoresis: Isolation of malaria. Lab Chip 2002, 2, 70–75, doi:10.1039/b110990c.
[21]
Gagnon, Z.R. Cellular dielectrophoresis: Applications to the characterization, manipulation, separation and patterning of cells. Electrophoresis 2011, 32, 2466–2487, doi:10.1002/elps.201100060.
[22]
Falconnet, D.; Csucs, G.; Michelle Grandin, H.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044–3063, doi:10.1016/j.biomaterials.2005.12.024.
[23]
Lim, J.Y.; Donahue, H.J. Cell sensing and response to micro-and nanostructured surfaces produced by chemical and topographic patterning. Tissue Eng. 2007, 13, 1879–1891, doi:10.1089/ten.2006.0154.
[24]
Di Carlo, D.; Wu, L.Y.; Lee, L.P. Dynamic single cell culture array. Lab Chip 2006, 6, 1445–1449, doi:10.1039/b605937f.
[25]
Zhu, Z.; Frey, O.; Ottoz, D.S.; Rudolf, F.; Hierlemann, A. Microfluidic single-cell cultivation chip with controllable immobilization and selective release of yeast cells. Lab Chip 2012, 12, 906–915, doi:10.1039/c2lc20911j.
[26]
Van den Brink, F.T.; Gool, E.; Frimat, J.P.; Bomer, J.; van den Berg, A.; le Gac, S. Parallel single-cell analysis microfluidic platform. Electrophoresis 2011, 32, 3094–3100, doi:10.1002/elps.201100413.
[27]
Tan, W.-H.; Takeuchi, S. A trap-and-release integrated microfluidic system for dynamic microarray applications. Proc. Natl. Acad. Sci. USA 2007, 104, 1146–1151, doi:10.1073/pnas.0606625104.
[28]
Kobel, S.; Valero, A.; Latt, J.; Renaud, P.; Lutolf, M. Optimization of microfluidic single cell trapping for long-term on-chip culture. Lab Chip 2010, 10, 857–863, doi:10.1039/b918055a.
[29]
Chung, K.; Rivet, C.A.; Kemp, M.L.; Lu, H. Imaging single-cell signaling dynamics with a deterministic high-density single-cell trap array. Anal. Chem. 2011, 83, 7044–7052, doi:10.1021/ac2011153.
[30]
Eriksson, E.; Enger, J.; Nordlander, B.; Erjavec, N.; Ramser, K.; Goks?r, M.; Hohmann, S.; Nystr?m, T.; Hanstorp, D. A microfluidic system in combination with optical tweezers for analyzing rapid and reversible cytological alterations in single cells upon environmental changes. Lab Chip 2006, 7, 71–76.
[31]
Eriksson, E.; Sott, K.; Lundqvist, F.; Sveningsson, M.; Scrimgeour, J.; Hanstorp, D.; Goks?r, M.; Granéli, A. A microfluidic device for reversible environmental changes around single cells using optical tweezers for cell selection and positioning. Lab Chip 2010, 10, 617–625, doi:10.1039/b913587a.
[32]
Jacobson, T.; Navarrete, C.; Sharma, S.K.; Sideri, T.C.; Ibstedt, S.; Priya, S.; Grant, C.M.; Christen, P.; Goloubinoff, P.; Tamás, M.J. Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast. J. Cell Sci. 2012, 125, 5073–5083, doi:10.1242/jcs.107029.
[33]
Tan, S.H.; Nguyen, N.-T.; Chua, Y.C.; Kang, T.G. Oxygen plasma treatment for reducing hydrophobicity of a sealed polydimethylsiloxane microchannel. Biomicrofluidics 2010, 4, 32204, doi:10.1063/1.3466882.
[34]
Sott, K.; Eriksson, E.; Goks?r, M. Acquisition of Single Cell Data in an Optical Microscope. In Lab on a Chip Technology: Biomolecular Separation and Analysis; Caister Academic Press: Norfolk, UK, 2009; pp. 151–166.
[35]
Smedh, M.; Beck, C.; Sott, K.; Goks?r, M. Cellstress-Open Source Image Analysis Program for Single-Cell Analysis. In Proceedings of SPIE 7762, Optical Trapping and Optical Micromanipulation VII, 77622N, San Diego, CA, USA, 27 August 2010; International Society for Optics and Photonics: Bellingham, WA, USA, 2010.
[36]
Wysocki, R.; Chéry, C.C.; Wawrzycka, D.; van Hulle, M.; Cornelis, R.; Thevelein, J.M.; Tamás, M.J. The glycerol channel fps1p mediates the uptake of arsenite and antimonite in Saccharomyces cerevisiae. Mol. Microbiol. 2001, 40, 1391–1401, doi:10.1046/j.1365-2958.2001.02485.x.
[37]
Tamás, M.J.; Karlgren, S.; Bill, R.M.; Hedfalk, K.; Allegri, L.; Ferreira, M.; Thevelein, J.M.; Rydstr?m, J.; Mullins, J.G.; Hohmann, S. A short regulatory domain restricts glycerol transport through yeast fps1p. J. Biol. Chem. 2003, 278, 6337–6345, doi:10.1074/jbc.M209792200.
[38]
Maciaszczyk-Dziubinska, E.; Migdal, I.; Migocka, M.; Bocer, T.; Wysocki, R. The yeast aquaglyceroporin Fps1p is a bidirectional arsenite channel. FEBS Lett. 2010, 584, 726–732, doi:10.1016/j.febslet.2009.12.027.