Toshiharu Arai

Novel electrochemical methodology for activity estimation of alkaline phosphatase based on solubility difference.

We propose a novel electrochemical detection system for alkaline phosphatase (ALP) activity using the difference in water and oil solubilities between the substrate, ferrocene ethyl phosphate ester (FcEtOPO(3)(2-)), and the enzymatic product, ferroceneethanol (FcEtOH). In this system, water droplets containing ALP and FcEtOPO(3)(2-) were placed on a Pt disk microelectrode and surrounded by a mineral oil. By the ALP-catalyzed reaction, FcEtOPO(3)(2-) was converted to FcEtOH, which was then transferred to the mineral oil from the water droplets with FcEtOPO(3)(2-) remaining in the water droplets. After partitioning FcEtOH from the water droplets, FcEtOPO(3)(2-) was detected at the Pt disk microelectrode to estimate the ALP activity. Using this novel system, the ALP activity of embryoid bodies was successfully detected. We believe that the present system will be widely applicable to ALP-based bioassays.

LSI-based amperometric sensor for real-time monitoring of embryoid bodies

A large scale integration (LSI)-based amperometric sensor is used for electrochemical evaluation and real-time monitoring of the alkaline phosphatase (ALP) activity of mouse embryoid bodies (EBs). EBs were prepared by the hanging drop culture of embryonic stem (ES) cells. The ALP activity of EBs with various sizes was electrochemically detected at 400 measurement points on a Bio-LSI chip. The electrochemical measurements revealed that the relative ALP activity was low for large EBs and decreased with progress of the differentiation level of the ES cells. The ALP activity of the EBs was successfully monitored in real time for 3.5h, and their ALP activity in a glucose-free buffer decreased after 2h. To the best of our knowledge, this is the first report on the application of an LSI-based amperometric sensor for real-time cell monitoring over 3h. The chip is expected to be useful for the evaluation of cell activities.

Multiparameter analyses of three-dimensionally cultured tumor spheroids based on respiratory activity and comprehensive gene expression profiles

Multicellular spheroids of human breast cancer cells (MCF-7) formed with two different three-dimensional (3D) culture methods were evaluated in detail on the basis of respiratory activity and high-throughput gene expression analysis. The spheroids formed with poly(dimethylsiloxane) (PDMS) microwell arrays indicated significant restriction of the spheroid size, whereas their respiratory activity was 2-fold greater than that formed with the hanging drop culture method. Fluidigm BioMark dynamic array was used for comprehensive and quantitative real-time polymerase chain reaction (qRT-PCR) analysis on the samples whose respiratory activity had been measured. Genes involved in cellular senescence and glucose metabolism indicated significantly higher values for the PDMS microwell culture method than for the hanging drop culture method (P<0.05). Interestingly, samples formed with the PDMS microwell culture method showed stronger responses for glycolysis than those formed with the hanging drop method. These results illustrate the power of multiparameter analysis to characterize multicellular spheroids cultured in different microenvironments even if they have the same morphology.

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

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 -0.2V, 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 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(O(2))) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R(2)=0.90). The slope of the least squares method was 1. In a 1.0mM 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 measured 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 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.

Carbon-Ag/AgCl Probes for Detection of Cell Activity in Droplets

In this study, we fabricated a probe consisting of a carbon nanoelectrode and an Ag/AgCl reference electrode for detecting the activity of cells in single droplets. HeLa cells were confined into a single droplet, and the alkaline phosphatase (ALP) activity of the cells was electrochemically measured using the probe inserted into the droplet. The ALP of the confined cells catalyzed the hydrolysis of p-aminophenyl phosphate (PAPP) to yield p-aminophenol (PAP) that gave electrochemical responses. Since the tip of the carbon-Ag/AgCl probe is very small, it is useful for electrochemical analysis of cells using droplets.

Noninvasive Measurement of Alkaline Phosphatase Activity in Embryoid Bodies and Coculture Spheroids with Scanning Electrochemical Microscopy

Alkaline phosphatase (ALP) is an enzyme commonly used as an undifferentiated marker of embryonic stem cells (ESCs). Although noninvasive ALP detection has long been desired for stem cell research and in cell transplantation therapy, little progress has been made in developing such techniques. In this study, we propose a noninvasive evaluation method for detecting ALP activity in mouse embryoid bodies (mEBs) using scanning electrochemical microscopy (SECM). SECM has several advantages, including being noninvasive, nonlabeled, quantitative, and highly sensitive. First, we found that SECM-based ALP evaluation permits the comparison of ALP activity among mEBs of different sizes by monitoring the p-aminophenol (PAP) production rate in aqueous solution containing p-aminophenylphosphate (PAPP) normal to the surface area of each sample. Second, coculture spheroids, consisting of mEB and MCF-7 cells for the core and the concentric outer layer, respectively, were prepared as model samples showing heterogeneous ALP activities. The overall PAP production rate dramatically declined in the presence of the MCF-7 cell outer layer, which blocked the mass transfer of PAPP to inner mEB. This result indicated that the SECM response mainly originated from ALP located at the surface of the cellular aggregate, including mEBs and coculture spheroids. Third, taking advantage of the noninvasive nature of SECM, we examined the relevance of ALP activity and cardiomyocyte differentiation. Collectively, these results suggested that noninvasive SECM-based ALP activity normalized by the sample surface enables the selection of EBs with a higher potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research.