Skip to main content

Advertisement

SpringerLink
Log in

Quantification of kinase activity in cell lysates via photopatterned macroporous poly(ethylene glycol) hydrogel arrays in microfluidic channels

  • Published:
Biomedical Microdevices Aims and scope Submit manuscript

Abstract

The efficacy of tyrosine kinase inhibitors (TKIs) as cancer therapeutics varies amongst individual patients as a result of patient-specific differences in molecular regulation of cancer development and progression, and acquisition of resistance to TKIs during therapy. A sensitive assay that can quantify kinase activity and predict inhibition of that activity from minimally invasive patient tissue samples may aid design of efficacious individualized TKI treatments. A microfluidic format can be useful in reducing limitations in standard protein kinase assays, including sensitivity required and low sample volume available. We present photopatterned macroporous poly(ethylene glycol) diacrylate hydrogel pillars functionalized with kinase substrates within microchannels for quantifying kinase activity in complex cellular lysates. We determined the effect of using a porogen to induce macroporosity in hydrogel pillars and showed that hydrogel poration enhanced the sensitivity of detecting Bcr-Abl activity in cell lysates by an order of magnitude. Bcr-Abl tyrosine kinase activity in K562 cell lysates could be detected from 0.01 μg/μL of cell lysate, corresponding to approximately 500 cells, using GST-Crkl immobilized in macroporous hydrogels. This device was also capable of quantifying inhibition of Bcr-Abl activity by imatinib mesylate, which demonstrates the potential to predict the biochemical response to drug inhibitors. These results indicate that microfluidic devices containing macroporous hydrogels functionalized with kinase substrates provide a promising platform for sensitive and specific quantification of kinase activity and efficacy of kinase inhibitors in cancer cell lysates.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • J.K. Armstrong, R.B. Wenby, H.J. Meiselman, T.C. Fisher, Biophys. J. 87, 4259 (2004)

    Article  Google Scholar 

  • M.V. Badiger, M.E. Mcneill, N.B. Graham, Biomaterials 14, 1059 (1993)

    Article  Google Scholar 

  • D.J. Beebe, J.S. Moore, Q. Yu, R.H. Liu, M.L. Kraft, B.H. Jo, C. Devadoss, Proc. Natl. Acad. Sci. USA 97, 13488 (2000)

    Article  Google Scholar 

  • S.B. Brueggemeier, S.J. Kron, S.P. Palecek, Anal. Biochem. 329, 180 (2004)

    Article  Google Scholar 

  • S.B. Brueggemeier, D. Wu, S.J. Kron, S.P. Palecek, Biomacromolecules 6, 2765 (2005)

    Article  Google Scholar 

  • T. Caykara, M. Bulut, N. Dilsiz, Y. Akyuz, J. Macromol, Sci. Part A-Pure Appl. Chem. 43, 889 (2006)

    Google Scholar 

  • A.S. Corbin, P. La Rosee, E.P. Stoffregen, B.J. Druker, M.W. Deininger, Blood 101, 4611 (2003)

    Article  Google Scholar 

  • J. Deere, R.F. De Oliveira, B. Tomaszewski, S. Millar, A. Lalaouni, L.F. Solares, S.L. Flitsch, P.J. Halling, Langmuir 24, 11762 (2008)

    Article  Google Scholar 

  • B.J. Druker, F. Guilhot, S.G. O’Brien, I. Gathmann, H. Kantarjian, N. Gattermann, M.W. Deininger, R.T. Silver, J.M. Goldman, R.M. Stone, F. Cervantes, A. Hochhaus, B.L. Powell, J.L. Gabrilove, P. Rousselot, J. Reiffers, J.J. Cornelissen, T. Hughes, H. Agis, T. Fischer, G. Verhoef, J. Shepherd, G. Saglio, A. Gratwohl, J.L. Nielsen, J.P. Radich, B. Simonsson, K. Taylor, M. Baccarani, C. So, L. Letvak, R.A. Larson, N. Engl. J. Med. 355, 2408 (2006)

    Article  Google Scholar 

  • C. Fang, Y.J. Wang, N.T. Vu, W.Y. Lin, Y.T. Hsieh, L. Rubbi, M.E. Phelps, M. Muschen, Y.M. Kim, A.F. Chatziioannou, H.R. Tseng, T.G. Graeber, Cancer Res. 70, 8299 (2010)

    Article  Google Scholar 

  • P.B. Gaspers, C.R. Robertson, A.P. Gast, Langmuir 10, 2699 (1994)

    Article  Google Scholar 

  • P.B. Gaspers, A.P. Gast, C.R. Robertson, J. Colloid Interface Sci. 172, 518 (1995)

    Article  Google Scholar 

  • G. Ghosh, A.G. Lee, S.P. Palecek, Anal. Biochem. 393, 205 (2009)

    Article  Google Scholar 

  • G. Ghosh, X. Yan, A.G. Lee, S.J. Kron, S.P. Palecek, Biosens. Bioelectron. 26, 424 (2010)

    Article  Google Scholar 

  • R. Hart, R. Lec, H. Noh, Sens. Actuator B-Chem. 147, 366 (2010)

    Article  Google Scholar 

  • J. Heo, R.M. Crooks, Anal. Chem. 77, 6843 (2005)

    Article  Google Scholar 

  • A. Hochhaus, S. Kreil, A.S. Corbin, P. La Rosee, M.C. Muller, T. Lahaye, B. Hanfstein, C. Schoch, N.C. Cross, U. Berger, H. Gschaidmeier, B.J. Druker, R. Hehlmann, Leukemia 16, 2190 (2002)

    Article  Google Scholar 

  • J.H. Kim, S. Roy, J.T. Kellis, A.J. Poulose, A.P. Gast, C.R. Robertson, Langmuir 18, 6312 (2002)

    Article  Google Scholar 

  • D.N. Kim, W. Lee, W.G. Koh, Anal. Chim. Acta 609, 59 (2008)

    Article  Google Scholar 

  • W. Kusnezow, Y.V. Syagailo, I. Goychuk, J.D. Hoheisel, D.G. Wild, Expert. Rev. Mol. Diagn. 6, 111 (2006)

    Article  Google Scholar 

  • H.J. Lee, A.W. Wark, T.T. Goodrich, S.P. Fang, R.M. Corn, Langmuir 21, 4050 (2005)

    Article  Google Scholar 

  • J.H. Lee, B.D. Cosgrove, D.A. Lauffenburger, J. Han, J. Am. Chem. Soc. 131, 10340 (2009)

    Article  Google Scholar 

  • A.G. Lee, C.P. Arena, D.J. Beebe, S.P. Palecek, Biomacromolecules 11, 3316 (2010)

    Article  Google Scholar 

  • K. Lewandowski, K. Warzocha, A. Hellmann, A. Skotnicki, W. Prejzner, K. Foryciarz, T. Sacha, M. Gniot, M. Majewski, I. Solarska, G. Nowak, B. Wasag, M. Kobelski, C. Scibiorski, M. Siemiatkowski, M. Lewandowska, M. Komarnicki, Pol. Arch. Med. Wewn. 119, 789 (2009)

    Google Scholar 

  • L.A. Liotta, V. Espina, A.I. Mehta, V. Calvert, K. Rosenblatt, D. Geho, P.J. Munson, L. Young, J. Wulfkuhle, E.F. Petricoin, Cancer Cell 3, 317 (2003)

    Article  Google Scholar 

  • V.I. Lozinsky, I.Y. Galaev, F.M. Plieva, I.N. Savinal, H. Jungvid, B. Mattiasson, Trends Biotechnol. 21, 445 (2003)

    Article  Google Scholar 

  • O. Okay, Prog. Polym. Sci. 25, 711 (2000)

    Article  Google Scholar 

  • J.S. Ross, J.A. Fletcher, Oncologist 3, 237 (1998)

    Google Scholar 

  • A.Y. Rubina, E.I. Dementieva, A.A. Stomakhin, E.L. Darii, S.V. Pan’kov, V.E. Barsky, S.M. Ivanov, E.V. Konovalova, A.D. Mirzabekov, Biotechniques 34, 1008 (2003)

    Google Scholar 

  • R.L. Schnaar, Y.C. Lee, Biochemistry 14, 1535 (1975)

    Article  Google Scholar 

  • M. Sigurdson, D. Wang, C.D. Meinhart, Lab Chip 5, 1366 (2005)

    Article  Google Scholar 

  • W.C. Sung, H.H. Chen, H. Makamba, S.H. Chen, Anal. Chem. 81, 7967 (2009)

    Article  Google Scholar 

  • G.M. Walker, D.J. Beebe, Lab Chip 2, 131 (2002)

    Article  Google Scholar 

  • E. Weisberg, P.W. Manley, S.W. Cowan-Jacob, A. Hochhaus, J.D. Griffin, Nat. Rev. Cancer 7, 345 (2007)

    Article  Google Scholar 

  • D. White, V. Saunders, A.B. Lyons, S. Branford, A. Grigg, L.B. To, T. Hughes, Blood 106, 2520 (2005)

    Article  Google Scholar 

  • Y.-H. Wu, H.B. Park, T. Kai, B.D. Freeman, D.S. Kalika, J. Membr. Sci. 347, 197 (2010)

    Article  Google Scholar 

  • V.K. Yadavalli, W.G. Koh, G.J. Lazur, M.V. Pishko, Sens. Actuator B-Chem. 97, 290 (2004)

    Article  Google Scholar 

  • D.A. Zubtsov, E.N. Savvateeva, A.Y. Rubina, S.V. Pan’kov, E.V. Konovalova, O.V. Moiseeva, V.R. Chechetkin, A.S. Zasedatelev, Anal. Biochem. 368, 205 (2007)

    Article  Google Scholar 

Download references

Acknowledgements

We thank Alicia Powers for helpful discussion and revisions in preparing this manuscript. This work was supported by the NIH/NIGMS Grant R01GM074691.

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, University of Wisconsin—Madison, 1415 Engineering Drive, Madison, WI, 53706, USA

    Andrew G. Lee & Sean P. Palecek

  2. Department of Biomedical Engineering, University of Wisconsin—Madison, 1550 Engineering Drive, Madison, WI, 53706, USA

    David J. Beebe & Sean P. Palecek

Authors
  1. Andrew G. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David J. Beebe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sean P. Palecek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sean P. Palecek.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, A.G., Beebe, D.J. & Palecek, S.P. Quantification of kinase activity in cell lysates via photopatterned macroporous poly(ethylene glycol) hydrogel arrays in microfluidic channels. Biomed Microdevices 14, 247–257 (2012). https://doi.org/10.1007/s10544-011-9602-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10544-011-9602-y

Keywords

Search

Navigation