Talanta 94 (2012) 30–35 Contents lists available at SciVerse ScienceDirect Talanta jo u r n al hom epage: www.elsevier.com/locate/talanta A Pt layer/Pt disk electrode configuration to evaluate respiration and alkaline phosphatase activities of mouse embryoid bodies Raquel Obregon a , Yoshiko Horiguchi a , Toshiharu Arai a , Shihomi Abe a , Yuanshu Zhou a , RyosukeTakahashi b , Akiko Hisada b , Kosuke Ino a , Hitoshi Shiku a,∗ , Tomokazu Matsue a,c,∗∗ a Graduate School of Environmental Studies, Tohoku University, 6-6-11, Aramaki, Aoba, Sendai 980-8579, Japan b Advanced Research Laboratory, Hitachi Ltd., Hatoyama, Saitama 350-0395, Japan c WPI Advanced Institute of Materials Research, Tohoku University, 2-1-1 Katahira, Aoba, Sendai 980-8577, Japan a r t i c l e i n f o Article history: Received 21 November 2011 Received in revised form 27 January 2012 Accepted 30 January 2012 Available online 3 March 2012 Keywords: Embryonic stem cell Embryoid body Respiration Alkaline phosphatase Scanning electrochemical microscopy Pseudo reference electrode a b s t r a c t A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E 1/2 ) for [Fe(CN) 6 ] 4- oxidation and O 2 reduction measured versus the internal Pt pseudo reference was shifted by about -0.2 V, compared with the E 1/2 measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxy- gen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at -0.7 V versus the Pt pseudo reference and compared with those monitored at -0.5 V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cul- tivation dates. We have plotted the oxygen consumption rate (F(O 2 )) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determina- tion R 2 = 0.90). The slope of the least squares method was 1. In a 1.0 mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E 1/2 for PAP oxidation mea- sured versus the Pt layer was not shifted, compared with the E 1/2 versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3 V versus the Pt layer and compared with those monitored at +0.3 V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very use- ful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Scanning electrochemical microscopy (SECM) can be used with different types of probe (disk, ring, disk–ring, dual disk) and mate- rials with sizes ranging from several micrometers to just a few ∗ Corresponding author. Tel.: +81 22 795 7209; fax: +81 22 795 7209. ∗∗ Corresponding author at: Graduate School of Environmental Studies, Tohoku University, 6-6-11, Aramaki, Aoba, Sendai 980-8579, Japan. Tel.: +81 22 795 7209; fax: +81 22 795 7209. E-mail addresses: shiku@bioinfo.che.tohoku.ac.jp (H. Shiku), matsue@bioinfo.che.tohoku.ac.jp (T. Matsue). nanometers. Microelectrodes have been fabricated from small diameter metal wires or carbon fibers sealed into tapered tips of pulled glass micropipettes and/or insulating polymers [1–11]. In the present study, the outer surface of the glass insulation of a disk- shaped microelectrode was sputtered with a Pt layer [12,13]. This is particularly useful because one does not then need to insert a reference electrode into a measuring solution in order to obtain an electrical connection, and electrochemical measurement in a small volume becomes much easier. Miniaturization and microfabrication of reference electrodes play an important role in the development of integrated microsen- sors. Until now, 3-electrode configuration has generally been performed in a commercial 96-well plate, with a sample volume 0039-9140/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2012.01.059