Biochip for cellomics and proteomics

我们已经开发了生物芯片，用于保留单个生物细胞，用于长期监测细胞变化和反应。特别是，我们对酵母、心肌细胞和癌细胞在不同试剂刺激下的细胞内钙进行了测量。我们还组装了聚合物芯片，以微阵列的形式进行DNA探针固定和样品杂交。这是使用所有微流控操作而不使用任何点样程序来实现的。这项拟议研究的目标是将这些方法扩展到细胞运输过程和蛋白质-小分子相互作用的基础研究。首先，生物芯片可以实现单细胞保留，用于长期细胞监测，使我们能够研究各种必要的生化过程。例如，对单个癌细胞外排药物的机制的研究将有助于阐明化疗药物多药耐药的动力学。到目前为止，传统的流式细胞术方法只允许测量一批细胞来研究药物的排泄和积累。生物芯片方法可以用来监测药物进入癌细胞，然后在排泄时离开的动力学过程。这种由细胞膜排泄蛋白1介导的过程将在单细胞形式下进行详细研究。其次，利用已开发的用于DNA微阵列的生物芯片，构建用于细胞-药物相互作用研究的细胞微阵列和用于酶结合研究的小分子阵列。此外，还将利用微流控芯片上的复杂通道网络进行蛋白质分离。例如，将在生物芯片上构建一个径向和多个螺旋通道，对细胞蛋白质进行二维分离。这种生物芯片蛋白质分离将一端连接从单个细胞中提取蛋白质，另一端连接分离蛋白质的后续分析。我们预计，我们对总体蛋白质，特别是细胞蛋白质的芯片研究方法将在蛋白质组学和细胞组学领域具有非常重要的意义。

We have developed biochips for retaining a single biological cell for long-term monitoring of cellular changes and reactions. In particular, we have carryied out measurements of intracellular calcium in a yeast, a heart muscle cell and a cancerous cell as they were stimulated by various reagents. We have also assembled polymeric chips to conduct DNA probe immobilization and sample hybridization in a microarray format. This is achieved using all microfluidic operations without using any spotting procedures. The objectives of this proposed research is to extend these methods for the basic studies of cellular transport processes and protein-small molecule interactions. First, the biochip that achieves single-cell retention for long-term cellular monitoring allows us to study various essential biochemical processes. For instance, the mechanistic study of drug efflux from a single cancerous cell will benefit the elucidiation of the kinetics of multi-drug resistance of chemotherapeutic drugs. To date, the conventional method of flow cytometry only allows the measurements of a batch of cells for the study of excretion and accumulation of drugs. The biochip method can be used to monitor the kinetic process of a drug when it enters a cancerous cell, and then leaves it upon excretion. Such a process as mediated by a cellular membrane excretory protein  will be studied in detail in the single-cell format. Second, the biochip that has been developed for DNA microarray will be exploited to construct cell microarray for cell-drug interaction studies and small molecule array for enzyme binding studies. In addition, the complex channel network on the microfluidic chip will be employed to carry out protein separation. For instance, one radial and numerous spiral channels will be constructed on the biochip to conduct 2-dimensional separation for cellular proteins. This biochip protein separation will link the extraction of proteins from a single cell on the one end, and the subsequent analysis of separated proteins on the other.We envision that our approach of on-chip study of proteins in general and cellular proteins in particular will be highly significant in the field of proteomics and cellomics.