A Pt layer/Pt disk electrode configuration to evaluate respiration and alkaline phosphatase activities of mouse embryoid bodies

Raquel Obregon, Yoshiko Horiguchi, Toshiharu Arai, Shihomi Abe, Yuanshu Zhou, Ryosuketakahashi, Akiko Hisada, Kosuke Ino, Hitoshi Shiku, Tomokazu Matsue

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

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 (E1/2) for [Fe(CN)6]4- oxidation and O2 reduction measured versus the internal Pt pseudo reference was shifted by about -0.2 V, compared with the E1/2 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 oxygen 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 cultivation dates. We have plotted the oxygen consumption rate (F(O2)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R2 = 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 E1/2 for PAP oxidation measured versus the Pt layer was not shifted, compared with the E1/2 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 useful 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.

Original languageEnglish
Pages (from-to)30-35
Number of pages6
JournalTalanta
Volume94
DOIs
Publication statusPublished - 2012 May 30

Keywords

  • Alkaline phosphatase
  • Embryoid body
  • Embryonic stem cell
  • Pseudo reference electrode
  • Respiration
  • Scanning electrochemical microscopy

ASJC Scopus subject areas

  • Analytical Chemistry

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