Development of Electrochemical Biolithograpy and application to Cellular Chips

微流控系统在生物领域的应用已经得到了广泛的研究，在诊断分析和生物反应器的发展方面具有很大的前景。表面图图化技术与微流体系统的结合为多功能、高通量和高成本效益的分析铺平了道路，其中微通道内生物元素的“实时”和“按需”微图图化将是必不可少的，因为这种微妙的材料对干燥、氧化和加热不稳定。然而，大多数基于光刻的技术都无法在密封的微通道内应用。在这项研究中，我们开发了一种新的技术“电化学生物光刻”，可以在微流控通道等三维微结构中实现蛋白质和细胞的局部固定。该技术的原理是基于我们的发现，白蛋白或肝素涂层的表面，最初是抗生物污垢的，在暴露于活性氧化剂（如次溴酸）后迅速成为蛋白质和细胞粘合剂，次溴酸可以通过生物缓冲溶液中的溴离子的电化学氧化产生。由于这种光刻技术可以在典型的生理条件下进行，因此可以对细胞在基质上的粘附和生长进行时空控制；它为多型蛋白阵列和多表型细胞阵列的逐步固定化提供了便利。重要的是，该技术足够简单，可以集成到微型和半封闭系统中，如微流控装置，这表明在使用微流控生物装置之前，可能“按需”固定蛋白质和活细胞。此外，我们还将电化学生物光刻与负电介质电泳（DEP）相结合，在半封闭的微流控通道中实现了细胞的快速排列。

Microfluidic systems have been widely investigated for biological applications and hold great promise in the development of diagnostic assays and bioreactors. The combination of the surface patterning techniques with the microfluidic systems paves the way to multi-functional, high-throughput, and cost-effective analysis, in which the "real-time" and "on-demand" micropatterning of bioelements within the microchannels would be essentially required since such delicate materials are unstable to desiccation, oxidation, and heat. However, most of the photolithography-based techniques are unable to be applied within the sealed microchannels.In this research, we have developed a novel technique "electrochemical bio-lithography", which enables the localized immobilization of proteins and cells within 3D microstructures such as microfluidic channels. The principle of the technique is based on our finding that the albumin- or heparin-coated surfaces, initially anti biofouling, rapidly becomes protein- and cell-adhesive upon exposure to the reactive oxidizing agent such as hypobromous acid, which can be produced by the electrochemical oxidation of bromide ion in a biological buffer solution. Since this lithography can be conducted under typical physiological conditions, it enables the spatiotemporal control of cell adhesion and growth on substrates; it facilities the stepwise immobilization of multitype protein arrays and multiphenotype cell arrays. And importantly, this technique is simple enough to be integrated into the miniaturized and semi-closed systems such as microfluidic devices, indicating the possible "on-demand" immobilization of proteins and living cells just prior to use of the microfluidic biodevices. In addition, we have achieved rapid cellular arrangement in the semi-closed microfluidic channel by combining electrochemical bio-lithography with negative dielectrophoresis (DEP).