Takanori Ichiki

Immunoelectrophoresis of red blood cells performed on microcapillary chips

Immunoanalysis of blood cells on a microcapillary electrophoresis (νCE) chip has been studied using sheep erythrocytes (ShE) as an example. Two different buffer solutions, the phosphate‐buffered saline (PBS) and the gelatin veronal buffer (GVB) were examined in regard to the electrokinetic transport behavior of ShE suspended in these solutions inside the rectangular channel engraved on a quartz chip. This clarified two advantages of the use of GVB for on‐chip cell electrophoresis: gelatin coatings prevent (i) nonspecific sticking of ShE on the channel wall, and cause (ii) an appreciable reduction in the zeta potential of the wall suppressing the electroosmotic flow of the buffer solution. As a result ShE suspended in the GVB can smoothly migrate from the cathode to the anode, which is the opposite flow direction of immunoglobulin G (IgG) antibodies under the physiological pH condition of 7.4. Based on these results, on‐chip capillary cell immunoelectrophoresis of ShE and rabbit anti ShE antibodies (IgG) have been proposed and successfully accomplished using the GVB. It is demonstrated that the variation of the cell migration velocity originating from the change in the surface charge after binding antibodies is applicable to the fast detection of immune reactions and also to single‐cell typing.

Solid-Phase Cell-Free Protein Synthesis to Improve Protein Foldability

Proteins are the most abundant molecules in biology which control virtually every biological process on which our lives depend. Therefore, understanding how newly synthesized proteins folds into the correct native structure and achieve their biologically functional states inside the cell is of paramount importance. Most of what is currently known about the process of protein folding has been studied by analyzing proteins outside the cells in a ‘dilute solution’ under in vitro conditions. The pioneering work on the creation of cell-free (in vitro) protein synthesis (CFPS) reported by Nirenberg and Matthaei in 1961 has been a powerful and ever expanding tool for large-scale analysis of proteins [1]. In general, these systems are derived from the crude extract of cells engaged in a high rate of protein synthesis and are consist of all the macromolecular components required for translation of exogenous mRNA which are added separately in the system. The cell-free system offer several advantages over traditional cell-based (in vivo) systems which are specially not good at making exogenous proteins and those which are toxic to the host cell, undergoes rapid proteolytic degradation or forms inclusion bodies. Cell-free system provides the ability to easily manipulate the reaction components and conditions to favor protein synthesis, decreased sensitivity to product toxicity and suitability for miniaturization and high-throughput applications. With these advantages, there is continuous increasing interest in CFPS system among biotechnologists, molecular biologists and medical or pharmacologists. However, CFPS systems rely on the correct folding of the expressed polypeptide chain into a fully functional three-dimensional protein. Thus ‘foldability’ of expressed protein in a cell-free system is one of the most challenging conundrums of CFPS science.