Quantification of Kinase Activity in Cell Lysates Via Photopatterned Macroporous Poly(ethylene Glycol) Hydrogel Arrays in Microfluidic Channels

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.