Development of Electrochemical Biolithograpy and application to Cellular Chips

微流控系统在生物应用方面得到了广泛的研究，并在诊断分析和生物反应器的发展方面有着巨大的前景。表面图案化技术与微流控系统的结合为多功能、高通量和成本效益分析铺平了道路，在这种分析中，由于这种微细材料对干燥、氧化和加热不稳定，因此基本上需要在微通道内对生物元素进行“实时”和“按需”的微图案化。然而，大多数基于光刻的技术无法应用于封闭的微通道中。在本研究中，我们开发了一种新的技术--电化学生物光刻，该技术能够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。这项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抗生物污垢，当暴露在次溴酸等反应性氧化剂中时，迅速成为蛋白质和细胞粘附剂，次溴酸可以通过生物缓冲溶液中的溴离子电化学氧化产生。由于这种光刻可以在典型的生理条件下进行，因此它能够在时空上控制细胞在基质上的黏附和生长；它便于多类型蛋白质阵列和多表型细胞阵列的逐步固定化。重要的是，这项技术非常简单，可以集成到微流控设备等小型化和半封闭式系统中，这表明在使用微流控生物设备之前，可以按需固定蛋白质和活细胞。此外，我们还将电化学生物光刻技术与负介电泳法(DEP)相结合，实现了细胞在半封闭微流控通道中的快速排列。

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).