Local Redox-Cycling-Based Electrochemical Chip Device with Deep Microwells for Evaluation of Embryoid Bodies

摘要

Three-dimensional (3D) cell cultures are important for applications in modern cell biology and tissue engineering, because these cell cultures offer architectural microenviron-ments very close to those of natural tissues, compared to conventional two-dimensional cultures. The environment of 3D cultures has an important effect on cell biology, including differentiation of stem cells. For example, embryonic stem (ES) cells, which can differentiate into any body organ tissues and are expected to have significant potential as cell sources for regenerative medicine, can develop into cardiomyocytes by forming 3D organ tissues, such as embryoid bodies (EBs).Several biomarkers, such as alkaline phosphatase (ALP) that express undifferentiated ES cells, are used to check the differentiation level of ES cells. Several types of cell arrays have recently been developed for high-throughput cell assays on cell activity and differentiation. Although fluorescence detection has been performed with cell arrays, because its sensitivity is high, the fluorescent detection has some disadvantages as described in the Supporting Information. As an alternative method, electrochemical detection has been incorporated into biosensor devices. Since electrochemical techniques have many advantages, several kinds of microelectrode arrays have been proposed and applied to chemical and biological analyses including cell arrays. However, it is difficult to collect electrochemical responses at many individual measurement points using conventional electrochemical devices, because sufficient space for the bond pads is not available on the chip border. To solve this problem, we have proposed a novel method to realize individually addressable electrochemical measurements using a device consisting of two sets of microelectrode arrays to induce local redox cycling, and we have designated the novel methodology as local redox cycling-based electrochemical (LRC-EC) detection. In the system, two arrays of band microelectrodes are orthogonally set to form an n x n array of crossing points with only 2n bonding pads for external connection. By setting the potentials at the row and column electrodes to appropriate values and inducing local redox cycling at a particular crossing point, the local redox cycling-based signals can be acquired from an individual crossing point. The detection system has been applied for the expression of reporter proteins in a cell culture array. In addition, we have developed the LRC-EC chip device with a comb-type interdigitated array (IDA) electrode that has an open space on the sensor to introduce and collect samples with ease.