微量血液蛋白组自动化检测设备用于胃癌靶向药物疗效标志物的筛选

Blood that circulates to all major organs is an informative window to both health and disease. All organs secrete proteins into the blood. The levels of each protein in a particular blood fingerprint will report the status of that organ and thus distinguish health from disease - and if disease, which disease. However, only a handful of plasma proteins are utilized in routine clinical tests for cancer. This is due to a host of reasons such as the complexity of the plasma proteome, the heterogeneity of cancer and the fast kinetics of blood protein degradation. Automatic and cheap technologies that allow systems examination of large numbers of plasma proteins, from small amounts of blood, and within minutes of sample measurement, would assist in solving these problems and be useful in clinic. We plan to develop an automatic blood proteomics platform, including the microfluidic chips and the operating system, allowing rapid separation of plasma from blood cells and simultaneous measurement of a panel of plasma proteins from a finger prick of whole blood within a few minutes to avoid the protein degradation that can occur when plasma is stored. The secreting protein blood fingerprint is captured within 10 minutes which avoids the protein degradation, and the total assay completes within 30 minutes. This blood proteomics chip will be capable of rapid, large-scale measurement of tens to one hundred of plasma proteins to identify and stratify disease, to monitor progression of disease and the response of disease to therapy. It holds potential for inexpensive, non-invasive, and point-of-care clinical diagnosis. Our hypothesis is that at least some of these molecules are secreted into the blood at detectable levels and hence constitute a molecular fingerprint for each organ whose protein components change individually in their levels of expression as one shifts from the normal to a diseased state and as one progresses through the disease state. Hence, we plan to test the hypothesis that these blood fingerprints become a multiparameter panel of proteins capable of predicting efficiency of molecularly targeted therapy (Bevacizumab) for gastric cancer patients - and will do so by using blood proteomics techniques. The measurement should be carried out rapidly, quantitatively, cheaply, on very small blood samples, and fully automatically.

流经全身各器官的血液是反映人体肿瘤状况的窗口。肿瘤细胞向血液中分泌蛋白，同时肿瘤细胞内蛋白与细胞表面受体由于肿瘤细胞的死亡被释放到血液中，这些蛋白的组成谱是特定条件下肿瘤基因表达、蛋白质相互作用以及代谢网络运行的忠实而精细的分子水平的反映。采集血液并对其中的蛋白标志物进行检测是一种低侵袭的检测方法。但是，血液是一个极其复杂的体系，其中的蛋白的浓度范围可相差11个数量级，而且大量蛋白，包括潜在的生物标志物都以很低的浓度存在（pg/ml量级）。此外，血液中部分蛋白在离开人体后会很快降解，存储的血清和血浆样品可能难以准确地反映真实信息。本课题将一种新型抗体微阵列技术与微流控芯片相结合而发展出一种高灵敏、低成本、自动化的定量蛋白组检测设备，并用于临床上对胃癌患者的微量血液（约20微升）中的40种蛋白进行定量检测以筛选蛋白标志物组合来预测贝伐单抗联合化疗治疗晚期胃癌的临床预后。

采集血液并对其中的蛋白标志物进行检测是一种低侵袭的检测方法。本课题发展了基于新型高密度抗体微阵列与微流控芯片的自动化微量血液蛋白组定量检测设备及基于智能手机的远程控制App，能够通过智能手机控制芯片上的自动进样以及整个运行过程。同时，手机App具有手动控制与自动控制两种模式，分别用于芯片的测试与实际样本的检测。本课题还发展了两种基于核酸的蛋白检测信号放大方法用于提高低丰度蛋白检测的灵敏度，其中一种是基于杂交链式反应（hybridization chain reaction, HCR）的信号放大机制，另一种是基于树枝状DNA（branched DNA）的信号放大机制，两种方法均经过了优化和评估，能够提高将灵敏度提升一个数量级以上，大大增加了低丰度蛋白的检测成功率与可靠性。基于芯片上的双抗体夹心法的免疫检测原理以及上述荧光信号的放大手段，能够在微流控芯片上实现30分钟以内的检测，且灵敏度达到ELISA的水平。本课题验证了32种蛋白的检测抗体并根据其相互交叉反应性质对其在芯片上分组检测，以降低交叉反应，提高检测特异性，并最终将上述设备、方法学用于临床血液样本的检测，尤其是接受贝伐单抗联合化疗治疗的晚期胃癌患者的血液样本。