公开(公告)号：US09448178B2

公开(公告)日：2016-09-20

申请号：US13783283

申请日：2013-03-02

Applicant: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Inventor： Thomas G. Graeber ,  Hsian-Rong Tseng ,  Arion F. Hadjioannou (Chatziioannou) ,  Cong Fang ,  Yanju Wang ,  Nam T. Vu

IPC: B01L3/00 ,  G01N21/75 ,  C12Q1/48 ,  G01N33/543 ,  G01N33/573

CPC classification number: G01N21/75 ,  B01L3/502707 ,  B01L3/502761 ,  B01L2200/0668 ,  B01L2300/0816 ,  B01L2300/088 ,  B01L2400/0481 ,  B01L2400/0487 ,  B01L2400/086 ,  C12Q1/485 ,  G01N33/54366 ,  G01N33/573

Abstract: An immunocapture-based in vitro kinase assay on an integrated polydimethylsiloxane (PDMS) microfluidics platform that can reproducibly measure kinase activity from as few as 3,000 cells is described. For this platform, the standard radiometric 32P-ATP labeled phosphate transfer assay was adopted. Implementation on a microfluidic device required the development of methods for repeated trapping and mixing of solid-phase affinity micro beads. A solid state beta-particle camera imbedded directly below the microfluidic device was used to provide real-time quantitative detection of the signal from this and other microfluidic radio bioassays. The integrated device can measure ABL protein kinase activity from BCR-ABL positive leukemia patient samples, and can measure the small molecule phosphorylation such as phosphorylation of the deoxycytidine analog 18F-FAC by deoxycytidine kinase (dCK) isolated from cell lysates.

Abstract translation: 描述了可复制地测量从3,000个细胞的激酶活性的整合聚二甲基硅氧烷（PDMS）微流体平台的基于免疫捕获的体外激酶测定。 对于该平台，采用标准辐射32P-ATP标记的磷酸盐转移测定法。 在微流体装置上的实施需要开发用于重复捕获和混合固相亲和微珠的方法。 使用嵌入微流体装置正下方的固态β粒子相机来提供来自该和其他微流体放射生物测定的信号的实时定量检测。 一体化装置可以测量BCR-ABL阳性白血病患者样品中的ABL蛋白激酶活性，并可以通过从细胞裂解液中分离的脱氧胞苷激酶（dCK）测量小分子磷酸化如脱氧胞苷类似物18F-FAC的磷酸化。