Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, University of California, Los Angeles, California 90095, USA.
Cancer Res. 2010 Nov 1;70(21):8299-308. doi: 10.1158/0008-5472.CAN-10-0851. Epub 2010 Sep 13.
Oncogenic kinase activity and the resulting aberrant growth and survival signaling are a common driving force of cancer. Accordingly, many successful molecularly targeted anticancer therapeutics are directed at inhibiting kinase activity. To assess kinase activity in minute patient samples, we have developed an immunocapture-based in vitro kinase assay on an integrated polydimethylsiloxane microfluidics platform that can reproducibly measure kinase activity from as few as 3,000 cells. For this platform, we adopted the standard radiometric (32)P-ATP-labeled phosphate transfer assay. Implementation on a microfluidic device required us to develop methods for repeated trapping and mixing of solid-phase affinity microbeads. We also developed a solid-state beta-particle camera imbedded directly below the microfluidic device for real-time quantitative detection of the signal from this and other microfluidic radiobioassays. We show that the resulting integrated device can measure ABL kinase activity from BCR-ABL-positive leukemia patient samples. The low sample input requirement of the device creates new potential for direct kinase activity experimentation and diagnostics on patient blood, bone marrow, and needle biopsy samples.
致癌激酶活性以及由此导致的异常生长和存活信号是癌症的常见驱动因素。因此，许多成功的分子靶向抗癌疗法旨在抑制激酶活性。为了评估微小患者样本中的激酶活性，我们在集成的聚二甲基硅氧烷微流控平台上开发了一种基于免疫捕获的体外激酶测定法，该方法可从少至 3000 个细胞中重现性地测量激酶活性。对于该平台，我们采用了标准的放射性 (32)P-ATP 标记的磷酸转移测定法。在微流控设备上的实施要求我们开发出用于重复捕获和混合固相亲和微珠的方法。我们还开发了直接嵌入在微流控设备下方的固态β-粒子相机，用于实时定量检测来自该微流控放射性生物测定法和其他微流控放射性生物测定法的信号。我们表明，所得的集成设备可以测量来自 BCR-ABL 阳性白血病患者样本的 ABL 激酶活性。该设备的低样本输入要求为直接在患者血液、骨髓和针吸活检样本上进行激酶活性实验和诊断创造了新的潜力。
