Fabrication of Ca2+-K+ Image Sensor Using an Inkjet Method and Its Application to Living Cells

Abstract

Introduction It is extremely important to know the change in the ion concentration in order to understand the dynamics of living cells. Nowadays, considerable attention has been paid to the method which can display ion concentration intuitively. In our laboratory, we have developed several ion image sensors by modifying a CCD-type pH image sensor with plasticized poly(vinyl chloride) (PVC) membranes. Recently, Na+-K+ image sensor [1] was fabricated using a inkjet method. In this paper, we developed Ca2+-K+ image sensor. Ca2+ and K+are very important ions in cells. Cell membranes have each ion channel and each ion pump. Both of ions exist on lower concentration in extracellular fluid than in cells. Materials and methods The half of a CCD-type pH image sensor was modified with Ca2+ selective membrane using a inkjet method. Similarly, the other half was modified with K+ selective membrane. The K+ selective membrane was prepared in our previous paper [1].The inkjet condition of the Ca2+ selective membrane was optimized, and the membrane with Nernstian response was prepared. The K+ selective membrane consisted of 30.0 % of PVC (MW = 82000), 30.0 % of 1,3,5,7,9,11,13,15-octa(propylmethacryl)pentacyclo[9.5.1.13,9.15,15.17,13]octasiloxane (POSS), 36.0 % of dioctyl sebacate (DOS), 2.7 % of valinomycin, and 1.3 % of potassium tetrakis (4-chlorophenyl)borate (K-TCPB). Ca2+ selective membrane consisted of 30.0 % of PVC, 10.0 % of POSS, 55.0 % of 2-nitrophenyl octyl ether (NPOE), 3.6 % of 10,19-bis[(octadecylcarbamoyl)methoxyacetyl]-1,4,7,13,16-pentaoxa-10,19-diazacycloheneicosane (K23E1) and 1.4 % of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (Na-TFPB). Real-time bio-images for PC12 cells (rat pheochromocytoma cell line) and Hela cells (human cervical cancer cell line) were captured by the Ca2+-K+image sensor. Results and Discussion After several days culture of PC12 cells on the collagen sheet, the cells on the Ca2+-K+ image sensor were stimulated by acetylcholine (Ach). As a result, the Ca2+ concentration decreased after the stimulation. On the other hand, there was no significant change in K+ concentration. The previous study [2] reported that the stimulation brought about the decrease in the Ca2+ concentration. This study confirmed that the decrease of Ca2+ wasn’t accompanied by the change in the K+concentration. After a day culture of HeLa cells, the cells were stimulated by Amphotericin B. As a result, the K+ concentration increased after the stimulation. Meanwhile, there was no significant change in Ca2+ concentration. A related result that showed the Amphotericin B-induced decreasing of K+ concentration in HeLa cells due to the K+ efflux was reported by Ohtsuka et al. [3] through the fluorometric imaging. The present study indicated that the change can be recorded only in the K+concentration with a non-invasive and easy method. In conclusion, the Ca2+-K+image sensor demonstrated successfully the real-time monitoring of living cells stimulated. [1]T. Hattori, H. Sato, K. Tokunaga, R. Kato, K. Sawada, Sensors and Materials, 27, (2015) ,1023-1034 [2] H. Taki, T. Sakurai, Y. Masaki, T. Hattori, K. Takahashi, S. Terakawa, M. Ishida, K. Okumura, K. Sawada, 2012 International Symposium on Chemical-Environmental-Biomedhical Technology, P_BE1_09, September 2-5, Tainan, Taiwan (2012) [3]K. Ohtsuka, S. Sato, Y. Sato, K. Sota, S. Ohzawa, T. Matsuda, K. Takemoto , N. Takamune, B. Juskowiak, T. Nagai, S. Takenaka, Chem. Commun., 48, (2012), 4740-4742