Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Biomed Microdevices. 2012 Apr;14(2):247-57. doi: 10.1007/s10544-011-9602-y.
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.
酪氨酸激酶抑制剂 (TKIs) 作为癌症治疗药物的疗效因患者个体在癌症发展和进展的分子调控以及在治疗过程中对 TKI 的耐药性获得方面的差异而有所不同。一种能够量化激酶活性并预测从微创患者组织样本中抑制该活性的灵敏测定法可能有助于设计有效的个体化 TKI 治疗方法。微流控格式在减少标准蛋白激酶测定法的局限性方面非常有用，包括所需的灵敏度和可用的低样品量。我们提出了一种在微通道内用激酶底物功能化的光图案化大孔聚乙二醇二丙烯酸酯水凝胶柱，用于量化复杂细胞裂解物中的激酶活性。我们确定了使用致孔剂在水凝胶柱中诱导大孔的效果，并表明凝胶穿孔使检测细胞裂解物中 Bcr-Abl 活性的灵敏度提高了一个数量级。使用 GST-Crkl 固定在大孔水凝胶中，可以从 0.01μg/μL 的细胞裂解物中检测到 K562 细胞裂解物中的 Bcr-Abl 酪氨酸激酶活性，相当于大约 500 个细胞。该设备还能够定量测量甲磺酸伊马替尼对 Bcr-Abl 活性的抑制作用，这表明有可能预测对药物抑制剂的生化反应。这些结果表明，含有用激酶底物功能化的大孔水凝胶的微流控设备为在癌细胞裂解物中灵敏和特异性地定量激酶活性和激酶抑制剂的功效提供了一个有前途的平台。
