Communication to the Editor
A new electrochemical assay method for gene expression using hela cells with a secreted alkaline phosphatase (SEAP) reporter system
Article first published online: 22 FEB 2012
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 109, Issue 8, pages 2163–2167, August 2012
How to Cite
Şen, M., Ino, K., Shiku, H. and Matsue, T. (2012), A new electrochemical assay method for gene expression using hela cells with a secreted alkaline phosphatase (SEAP) reporter system. Biotechnol. Bioeng., 109: 2163–2167. doi: 10.1002/bit.24461
- Issue published online: 18 JUN 2012
- Article first published online: 22 FEB 2012
- Accepted manuscript online: 13 FEB 2012 08:36AM EST
- Manuscript Accepted: 23 JAN 2012
- Manuscript Revised: 11 JAN 2012
- Manuscript Received: 7 NOV 2011
- Japan Society for the Promotion of Science. Grant Numbers: 22245011, 23760745
- Asahi Glass Foundation
- Mitsui Sumitomo Insurance Welfare Foundation
- Formation of Innovation Center for Fusion of Advanced Technologies, Sepecial Coordination Funds for Promoting Science and Technology, Ministry of Education, Culture, Sports, Science and Technology
Additional Supporting Information may be found in the online version of this article.
|bit_24461_sm_SupplFigS1.tif||94K||Supplementary Figure S1. Illustration of the differences between the present and a previous study. (a) In the previous method, SEAPs were detected in the presence of cells at pH 9.5. (b) In the present study, a special substrate covered by anti-SEAP substrate was used. After incubation of cells at pH 7.0 in a closed microenvironment, the substrate capturing the SEAP and the cells were separated from each other and the substrate with the captured SEAP was subsequently scanned at pH 9.5.|
|bit_24461_sm_SupplFigS2.tif||351K||Supplementary Figure S2. Measurement of the viability of individual cells in a closed microenvironment. The transformed HeLa cells were stained with a double staining kit (PI, calcein-AM) before entrapment inside the microwells to investigate cell viability during culture in the microwell array. The cells were introduced into the microwell array, and the microwell array was covered with the substrate. A phase microscope image (a) and fluorescent images (b, c) were acquired before the culture, and a fluorescent image (d) was acquired after 4 h of culture. The live cells were stained with calcein-AM (green), and the dead cells were stained with PI (red). (e) The cell viability on trapping 1-3 cells in a microwell was calculated. The scale bars are 150 µm.|
|bit_24461_sm_SupplFigS3.tif||96K||Supplementary Figure S3. Dependence of the electrochemical signals on the ALP concentration. A PDMS layer having microwells (diameter, 200 µm) was fabricated and placed on the antibody-immobilized substrate. The ALP solution was introduced into the microwells and incubated at 37° C for 1 h. The PDMS layer was peeled off and 4.7 mM PAPP solution was introduced onto the substrate for measuring the ALP activity from a single enzyme spot by using the SECM. The current outside the enzyme spot were subtracted from the current on the enzyme spot and the value was plotted onto a graph. Although it is a supplementary result, the result shows that the electrochemical signals depended on the ALP concentration.|
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