Highly Sensitive Electrochemical Endotoxin Sensor Based on Redox Cycling Using an Interdigitated Array Electrode Device.

The Limulus amebocyte lysate (LAL) reaction-based assay, the most commonly used endotoxin detection method, requires a skilled technician. In this study, to develop an easy-to-use and highly sensitive endotoxin sensor, we created an electrochemical endotoxin sensor by using an interdigitated array electrode (IDAE) device with advantages of amplifiable signals via redox cycling and portability. We added Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as an electrochemical substrate for an LAL reaction and detected p-aminophenol (pAP) released from LGR-pAP as a product of an endotoxin-induced LAL reaction via an IDAE device. The IDAE device showed a great redox cycling efficiency of 79.8%, and a 4.79-fold signal amplification rate. Then, we confirmed that pAP was detectable in the presence of LGR-pAP through chronoamperometry with the potential of the anode stepped from -0.3 to 0.5 V vs. Ag/AgCl while the cathode was biased at -0.3 V vs. Ag/AgCl. Then, we performed an endotoxin assay by using the IDAE device. Our endotoxin sensor detected as low as 0.7 and 1.0 endotoxin unit/L after the LAL reaction for 1 h and 45 min, respectively, and these data were within the cut-off value for ultrapure dialysis fluid. Therefore, our highly sensitive endotoxin sensor is useful for ensuring medical safety.

Developmental assessment of human vitrified-warmed blastocysts based on oxygen consumption.

BACKGROUND: In this study, we aimed to develop a model for embryo selection based on oxygen consumption following cryopreservation, the relationship between the developmental competence of blastocysts and their oxygen consumption was assessed. METHODS: Oxygen consumption of vitrified-warmed human blastocysts was measured at 0, 1.5, 3, 4.5, 6, 7.5, 9 and 24 h after warming using scanning electrochemical microscopy. On the basis of their developmental stage at 24 h, blastocysts were classified into four groups (hatched, hatching, arrested and degenerated). Moreover, cytochrome c oxidase (CCO) activity in vitrified-warmed blastocysts was assessed at 0 and 24 h. RESULTS: The oxygen consumption rate of blastocysts just after warming was significantly lower than that of non-vitrified blastocysts (P< 0.05). The oxygen consumption rate of blastocysts was significantly higher in the hatched group than in the arrested and the degenerated groups after 1.5 h (P< 0.05) and than in the hatching group (P< 0.05) at 7.5 and 9 h. Moreover, CCO activity was absent in vitrified-warmed blastocysts at 0 h, but was detected at 24 h. CONCLUSIONS: The respiratory rate of vitrified blastocysts after warming was significantly lower than non-cryopreserved blastocysts. Oxygen consumption of blastocysts with high developmental potential was restored earlier than that of blastocysts with low developmental potential. The results of this study suggest that it is possible to select vitrified-warmed blastocysts with high developmental potential based on their respiratory activity.

Monitoring oxygen consumption of single mouse embryos using an integrated electrochemical microdevice.

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36±0.33×10(-15) mol s(-1), 1.38±0.58×10(-15) mol s(-1), and 3.44±2.07×10(-15) mol s(-1), respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM).

A microfluidic dual capillary probe to collect messenger RNA from adherent cells and spheroids.

Collection of bioanalytes from single cells is still a challenging technology despite the recent progress in many integrated microfluidic devices. A microfluidic dual capillary probe was prepared from a theta (theta)-shaped glass capillary to analyze messenger RNA (mRNA) from adherent cells and spheroids. The cell lysis buffer solution was introduced from the injection aperture, and the cell-lysed solution from the aspiration aperture was collected for further mRNA analysis based on reverse transcription real-time PCR. The cell lysis buffer can be introduced at any targeted cells and never spilled out of the targeted area by using the microfluidic dual capillary probe because laminar flow was locally formed near the probe under the optimized injection/aspiration flow rates. This method realizes the sensitivity of mRNA at the single cell level and the identification of the cell types on the basis of the relative gene expression profiles.

Measurement of gene expression from single adherent cells and spheroids collected using fast electrical lysis.

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 microm above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10 micros between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 microL of the lysed solution was collected using a micro-injector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although single-cell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the single-cell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroids-the aggregated cellular masses formed during the three-dimensional culture -- as a model system to isolate small cellular clusters from tissues and organs.