Related Experiment Video
Updated: May 27, 2026
Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
Published on: October 23, 2019
Quantification of kinase activity in cell lysates via photopatterned macroporous poly(ethylene glycol) hydrogel
Andrew G Lee1, David J Beebe, Sean P Palecek
1Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
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 microg/microL 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.
Insights
This study introduces a new microfluidic device using porous hydrogels to precisely measure cancer-driving kinase activity. This assay enhances sensitivity for detecting Bcr-Abl activity and predicting drug responses in tyrosine kinase inhibitor (TKI) treatments.
Area of Science:
- Biochemistry
- Materials Science
- Oncology
Background:
- Tyrosine kinase inhibitors (TKIs) show variable efficacy in cancer treatment due to patient-specific factors and drug resistance.
- Sensitive assays are needed to quantify kinase activity and predict TKI inhibition from minimal patient samples for personalized therapy.
- Microfluidic platforms offer advantages for protein kinase assays, addressing limitations in sensitivity and sample volume.
Purpose of the Study:
- To develop a sensitive microfluidic assay for quantifying kinase activity in complex cell lysates.
- To evaluate the use of macroporous hydrogels for enhanced kinase detection sensitivity.
- To demonstrate the potential for predicting drug inhibitor efficacy using the developed assay.
Main Methods:
- Fabrication of photopatterned macroporous poly(ethylene glycol) diacrylate hydrogel pillars within microchannels.
- Functionalization of hydrogel pillars with kinase substrates for activity detection.
- Quantification of Bcr-Abl tyrosine kinase activity in K562 cell lysates and its inhibition by imatinib mesylate.
Main Results:
- Macroporous hydrogel pillars significantly enhanced the sensitivity of detecting Bcr-Abl activity by an order of magnitude.
- The assay detected Bcr-Abl activity from as little as 0.01 µg/µL of cell lysate (approx. 500 cells).
- The device successfully quantified the inhibition of Bcr-Abl activity by imatinib mesylate, demonstrating predictive potential.
Conclusions:
- Microfluidic devices with macroporous hydrogels offer a sensitive and specific platform for kinase activity quantification.
- This technology holds promise for personalized cancer therapy by predicting patient response to TKIs.
- The assay facilitates the evaluation of kinase inhibitor efficacy in complex biological samples.
More Related Videos
09:24Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing
Published on: June 9, 2016
08:07Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions
Published on: August 2, 2015