Review—Egg Cells on a Semiconductor: Potentials in Drug Screening and Clinical Diagnostics

In this review paper

With the rapid increase of knowledge in the fields of medicine and biology, micro-and nano-technologies in electronics have been applied to these fields for parallel processing of information, miniaturization of analysis system and exploring molecular mechanism of life. 1,2 A biosensor is one of typical examples of fusion between biotechnology and microelectronics, which consists of transducers and membranes on which biologically active substances are immobilized. 3 Physical and chemical changes at the membrane as a result of biochemical reaction are transduced to the electrical signals in the transducer. Electrochemical electrodes such as ion-selective electrodes and oxygen electrodes are commonly used as the transducer in the conventional biosensors. 4 Most biological phenomena in vivo are closely related to ionic behaviors such as ion channel activities at the cell membrane or behaviors of various ions in the blood. The direct detection of ions enables the straightforward analysis of various biomolecular recognition events. The principle of semiconductor-based biosensor on the basis of the field effect, a field-effect transistor (FET)-based biosensor (bio-FET), can be utilized to directly detect ionic behavior in a solution. 5,6 In a few decades, some methods for the electrical measurement of cellular functions using the bio FETs have been reported. [7][8][9][10][11][12][13][14] The principle of the bio-FET is based on the potentiometric detection of changes in charge density caused by specific biomolecular reactions at a gate insulator/solution interface. Ionic charges induced at the gate insulator electrostatically interact with electrons in a silicon substrate across a thin gate insulator, resulting in a change in the threshold voltage. In 1970, Bergverd et al. showed electrical detection method of pH variation based on change of positive charges of hydrogen ions using a FET on the basis of semiconductor principle. 5,6 This is called ionsensitive FET (ISFET), as shown in Fig. 1a. Semiconductor material is separated with solution across gate insulator, the thickness of which is not more than a few hundred nm. The gate insulator is usually composed of oxide such as SiO 2 , Ta 2 O 5 , Al 2 O 3 and so on or nitride such as Si 3 N 4 . 15 The hydroxyl groups are formed at the surface of gate insulator in a solution and are so sensitive to hydrogen ions (Fig.  1b). These positive charges at the gate surface interact electrostatically with electrons at the channel in silicon crystal. The field effect caused by charge density changes at the gate induces the change of threshold z E-mail: sakata@biofet.t.u-tokyo.ac.jp voltage ( V T ) at a constant drain-source current (I D ). This electrical response to hydrogen ions of ISFET shows Nernstian response, about 59.1 mV/pH at room temperature (25 • C). A platform based on a bio-FET device has some good abilities in terms of label-free, noninvasive and real-time measurements, ease of downsizing, and integration with conventional semiconductor micro fabrication processes.
Here, a novel potential of bio-FET with egg cells is reviewed in this article, which will come in useful for pharmaceutical discovery and clinical diagnostics.

Transporter-Substrate Interaction at Oocyte Membrane for Drug Screening 9
The liver plays a primary role in the excretion of drugs and drug metabolites. The clearance process involves membrane transport systems that mediate the hepatocellular uptake of bile acids, organic anions, and organic cations. [16][17][18] In particular, the organic anion transport pathway has been shown to mediate the elimination of various drugs. Xenopus laevis oocytes efficiently express the membrane-bound transporters and can be used as a convenient model system in pharmaceutical lead discovery to predict drug disposition, drug clearance, and drug-drug interactions. 19,20 Oocyte expression systems have been used extensively to study the function of membrane proteins such as transporters, ion channels, and pumps because of their low background and high expression levels. In case of the uptake measurement of substrates mediated by transporters, radioisotope (RI)-labeled compounds are usually used and fundamental characteristics such as substrate selectivity and uptake rates are investigated. In this method, oocytes need to be solved and fractured before detecting specific radioactivity. On the other hand, the patch clamp technique has been accepted as a label-free standard method for studies of ion channel proteins by use of oocyte expression systems. 21,22 The patch clamp technique is basically an invasive method. A fine glass pipet tip has to be pressed against the cell membrane carefully and a tiny area of the cell membrane is sucked into it, although the chip-based planar patch clamp technique has been developed. 23 In the whole cell patch clamp mode, the cell membrane is broken by suction to exchange cytoplasm. It is therefore not possible to obtain information on cell activity noninvasively. Moreover, high skill is required to achieve a good contact between the cell membrane and the pipet tip and to measure the ionic currents resulting from openings of ion channels  without any leakage. It would be preferable to monitor the interaction between membrane proteins/transporters and ligands at the cell membrane noninvasively while the cells are cultured on the materials.
Considering the backgrounds, we introduce an oocyte-based field effect transistor (oocyte-based FET) as a noninvasive and easy method for drug transport analysis, in which target transporters are expressed at the cell membrane of the oocyte. In particular, noninvasive monitoring of the uptake kinetics of substrates mediated by membrane-bound transporters is shown in this Transporter-substrate interaction at oocyte membrane for drug screening 9 section.
Noninvasive monitoring of the uptake kinetics of substrates mediated by membrane-bound transporters.-The schematic illustration of the oocyte-based FET is shown in Fig. 2. A Xenopus laevis oocyte is placed on the surface of the gate insulator of the FET. The n-channel depletion type FET was used and the width (W) and length (L) of the gate channel were 340 μm and 10 μm, respectively. The oocytebased FET is immersed in a measurement solution together with an Ag/AgCl reference electrode with a saturated KCl solution. The potential of a measurement solution is controlled and fixed by the gate voltage (V G ) through the reference electrode. The extracellular potential changes are induced at the interface between the gate insulator and cell membrane by the uptake of substrates. The change in the electrical characteristics caused by interaction between transporters and substrates at the cell membrane is measured and monitored using the oocyte-based FET system. The electrical characteristics of FET such as the V G -I D characteristic and the surface potential at the gate surface are measured in a solution using a semiconductor parameter analyzer (B1500A, Agilent). As the basic electrical characteristic, V T is defined as the difference of V G -I D characteristics at a constant I D . The time course of the surface potential at the gate surface is monitored using a source follower circuit 24 with which the potential change at the interface between an aqueous solution and the gate insulator can be read out directly at a constant I D . The FET chip was mounted on a polyimide printed circuit board and encapsulated with a polymer cover with holes in which cells are cultured. The details of the FET device and the fabrication process have been reported previously. 24 The changes in the surface potentials at the gate surface of the oocyte-based FETs were monitored after adding a substrate (Figs. 3a-3c). Here, two types of oocyte-based FETs were prepared; one was the FET with oocyte in which transporters were expressed, and the other was the FET with oocyte in which transporters were not expressed. With the use of these oocyte-based FETs, differential measurements were performed in order to eliminate the common background noises such as temperature change, change in ion concentration, and so on. The oocyte-based FET chip was treated as a single use tool for monitoring transporting function at cell membrane for the present. The reference FET without the oocyte itself was used in order to take the effect of oocyte placed at the gate surface into account. The estrone-3-sulfate (E3S) was used as a substrate in the uptake measurement for a human organic anion transporting peptide C (hOATP-C). When the E3S was introduced into two kinds of oocytebased FETs and the reference FET, the surface potential of the oocyte FET with hOATP-C increased drastically during the uptake of E3S, while the oocyte FET without hOATP-C expression and the reference FET showed little surface potential changes. This result was similar to those obtained with RI measurement (Fig. 3d), in which [3H]-labeled E3S was used as a substrate. Monotonous increase of the E3S uptake during incubation time of 2 h was found. However, the time course of the uptake signal obtained with the oocyte-based FET was different from those of the conventional RI measurements. The surface potential of the oocyte-based FET reached steady-state in about 30 min after introduction of E3S, while the intensity of radioactivity increased over 2 h in the RI measurement. This means that the observed phenomena based on the oocyte-based FET would be disparate from the uptake amount of [3H]-labeled E3S. Although the molecular mechanism during the uptake of E3S through hOATP-C has not yet been elucidated, the uptakes of substrates mediated by some transporters are known to be associated with a substrate-dependent current under voltageclamped conditions. 25 In the case of OAT1, one of the family of organic anion transporters, entry of organic anion is reported to be accompanied by efflux of dicarboxylate by a 1:1 ratio. 26 This exchange of singly charged organic anion with doubly charged dicarboxylates leads to a net loss of one negative charge per transporter cycle and change of the membrane potential in the positive direction. Since the positive change of the surface potential was obtained for the oocyte-based FET (Fig. 3), similar exchange of charged species is considered to take place during the uptake of E3S in the case of hOATP-C.  flux of charged species continues during the uptake of E3S, which results in steady-state of the surface potential of the oocyte-based FET. However, the change in pH at the oocyte/gate interface may be caused by the stimulation of E3S to hOATP-C at the oocyte membrane, because the ISFET devices basically responds to the change in pH. Further investigation is underway to elucidate the response mechanism of the oocyte-based FET.

Discrimination of transporting ability among genotypes of the transporters using the oocyte-based FET.-hOATP-C is a
liver-specific transporter involved in the hepatocellular uptake of a variety of endogenous chemicals, such as taurocholate, 27 estrone sulfate, 28 estradiol 17β-D-glucuronide, 19 leukotriene C 4 , 19 prostaglandin E 2 , 19 and thyroid hormone. 19 Genetic polymorphisms in hOATP-C have been investigated because of pharmacologic, toxicologic, and pathologic significances. [29][30][31] We attempted to detect the difference of the transporting ability between the wild-type and mutant-type transporters using the oocyte-based FETs. The estradiol 17β-D-glucuronide (E 2 17βG) was used as a substrate mediated by the wild-type hOATP-C * 1a (the same transporter as hOATP-C) and the mutant-type hOATP-C * 15. The uptake of the RI-labeled substrate was first measured for both the wild type and the mutant type transporters as a control experiment. The [3H]-labeled E 2 17βG was detected in both hOATPC * 1a-and hOATP-C * 15-expressed oocytes (Figs. 4a and 4b). The uptake amount of [3H]-labeled E 2 17βG for the wild-type transporter was 2 times larger than that of the mutant-type transporter. The difference of the uptake amount among the genotypes in the hOATP-C transporter has been reported in the previous work. 31 The oocyte with the wild type hOATP-C * 1a or the mutant-type hOATP-C * 15 transporter was placed on the gate surface of the oocyte-based FET. Figure 4c shows the potential behaviors of the oocyte-based FETs during the uptake of E 2 17βG without the RI label. The surface potentials of both oocyte-based FETs increased after the introduction of E 2 17βG while the control oocyte FET showed a little change in the surface potential. The amount of the surface potential change of the oocyte-based FET with the wild-type transporter was approximately twice as large as that of the oocyte-based FET with the mutant-type transporter. The difference of the surface potential change between the wild type and the mutant type transporters was in good agreement with that obtained in the RI measurement (Figs. 4a and 4b). Thus, the transporting kinetics of the substrate mediated by the wild-type and the mutant-type transporters were distinguished by use of the oocyte-based FETs.
It is possible to integrate multiple oocyte-based FETs and signal processing circuits in a single chip using advanced semiconductor technology. Simultaneous analyses of different transporters and membrane proteins can therefore be realized based on the integrated oocyte-based FETs. Because of the output of the oocyte-based FET is an electrical signal, it would be easy to quantify the transporting ability of various transporters in the future. The platform based on the oocyte-based FETs is suitable for a simple, accurate, and inexpensive system for high-throughput screening in pharmaceutical lead discovery.

Real-Time Electrical Detection of Molecular Charges at Ovum Membrane 10
Understanding molecular behavior at cell membrane in the integrated field of biology and electronics is very important for life science. Glycoproteins or glycolipids exist in the plasma membrane and have important roles in interactions of the cell with its surroundings. 32,33 Some of them have charges, exposing their sialic acid residues or sulfated saccharides at the cell surface. Information on molecular charges at the surface of cell membrane is therefore useful not only for understanding the cell functions but also for practical application.
Ovum of sea urchin is surrounded by gelatinous layer called the jelly coat that is made up of a mixture of sulfated fucose-rich polysaccharides and sialic acid-rich glycoproteins. 34 Sulfated fucose-rich polysaccharide is believed to be a major component for acrosome reaction at the jelly coat of sea urchin ovum surface (Fig. 5), although this reason is not still clarified as to whether the pure sulfated polysaccharide activates the acrosome reaction or not. Thus the ova surface of sea urchin have negative charges of molecules such as sialic acids and sulfated saccharides at glycolipids and glycoproteins in an aqueous solution so that intrinsic molecular charges at the ovum surface related to the interaction between ovum and sperm can be detected in principle by measuring the change in the electrical characteristics of FET devices. Since sialic acids at cell membrane are related to various recognition processes for cells, quantitative analysis of molecular charges of sialic acids at cell membrane would be a good measure to analyze cell functions. In particular, molecular charges at the ovum surface of sea urchin might be significant for fertilization, because sulfated oligosialic acid units at the ovum surface of sea urchin functionate as receptor for sperm. 33 In this Real-time electrical detection of molecular charges at ovum membrane 10 section, we show the real-time electrical detection of intrinsic molecular charges of sialic acids and sulfated saccharides, which play an important role on fertilization, at the ovum surface of sea urchin by use of the FET devices.  was immersed in a measurement solution together with an Ag/AgCl reference electrode with a saturated KCl solution. The molecular charges at the ovum surface on the gate interact electrostatically with electrons in silicon crystal through the thin gate insulator and induce electrical signals by the field effect, and are monitored as the change in the surface potential on the gate at a constant I D using the FET measurement system.

Real-time electrical detection of intrinsic molecular charges at
The change in the surface potential at the gate surface of the ISFET was monitored after adding sea urchin ova (Fig. 6). Here, two types of ISFETs were utilized; one was the ISFET on which sea urchin ova were seeded, and the other was the ISFET on which nothing is introduced. Using these ISFETs, differential measurements were performed in order to eliminate the common background noises such as temperature change and so on. In case of the reference ISFET, the surface potential have never shifted because no ova was introduced (Fig. 6a). When the sea urchin ova were introduced into the ISFET, the surface potential decreased during the adhesion of sea urchin ova on the gate surface (Fig. 6b). As shown in Fig. 7, two or three sea urchin ova were observed on the gate surface of ISFET. The change in surface potential at the gate of FET device depends on the change in charge per unit area of the gate. Since the change of surface potential of ISFET was about 40-50 mV after introducing the sea urchin eggs on the gate surface as shown in Fig. 6, the surface potential shift of about 10-20 mV would be obtained for one egg by use of the ISFET. The negative shift of surface potential by use of the FET system indicates the increase of negative charges on the gate surface. The negative charges are based on mainly sialic acids and sulfated saccharides at the ovum surface of sea urchin. In the other work, sulfated polysialic acid chains at carbohydrate-rich vitelline envelope (under the jelly coat shown in Fig. 1), which is believed to be important for fertilization and has been found as a component of the receptor protein for sperm, inhibited fertilization, while the nonsulfated form of this polysialic acid chains has little inhibitory activity. 35 Therefore, the ovum with low ability for acrosome reaction and subsequent fertilization may be distinguished from the normal ovum by comparing the surface potentials based on the charge amount at the ovum surface using the FET devices.

Single Embryo-Coupled Gate Field Effect Transistor for Elective
Single Embryo Transfer 13,14 Recently, assisted reproductive technology (ART) has been expected to be one of therapeutic methods of sterility. Engineers other than obstetricians have been required for assured success of ART programs. For in vitro fertilization (IVF) of one of ART programs, how to evaluate embryo quality and select an embryo in good condition are significant. Morphological evaluation has been widely used to rank embryo quality because microscopic analysis is noninvasive and useful in predicting pregnancy rates. 36,37 However, the standard of classification for embryo quality seems to be ambiguous among  operators because it is a subjective method. Moreover, elective single embryo transfer (eSET) will be recommended in the future in order to prevent a multiple pregnancy. 38 Therefore, a novel principle to evaluate the quality of a single embryo quantitatively and noninvasively in a real-time manner is required for practical use in ART. The detection principle of ISFET is based on the potentiometric detection of charge density changes at the gate insulator and is applied for various biosensing. Since the gate insulator usually consists of Si 3 N 4 or Ta 2 O 5 with hydroxyl group at the surface in solutions, furthermore, the ISFET is sensitive to the concentration of hydrogen ion with positive charge and should be utilized as the pH sensor. Therefore, pH variation based on respiration activity of the embryo will be monitored quantitatively and noninvasively in a real-time manner using a single embryo-coupled gate FET for eSET (eSET-FET), because pH at the interface between the embryo and gate membrane of FET will change sensitively according to dissolution of carbon dioxide into medium generated by metabolism and respiration activity in an embryo. Thus, the platform based on the eSET-FET sensor will be valuable for the development of an evaluation system to select a single embryo with good quality for eSET in the future.
On the other hand, oxygen consumption has been considered to be the parameter that provides the best indication of overall metabolic activity of a single embryo, [39][40][41][42][43] although embryo metabolism has previously been assessed by measurement of nutrient consumption, such as glucose, pyruvate, and amino acids. [44][45][46][47] As one of the detection methods for the evaluation of embryo quality, the electrochemical system is being developed. Shiku et al. reported previously the detection concept of oxygen consumption based on the respiration activity of an embryo. 48 In this method, the oxygen reduction current was detected near the surface of a single embryo using the cyclic voltammetry technique. However, this method is unsuitable for real-time measurement for a long-term such as cleavage of mammalian embryo. Therefore, we propose a semiconductor-based embryo sensing device for eSET (eSET-FET) to monitor not only sea urchin embryo but also single mouse embryo activities based on cellular respiration in a real-time, quantitative, and noninvasive manner. Additionally, we report to have developed the simultaneous analysis system composed of microscopic observation and electrical measurement of eSET-FET under the adequate embryo culture condition in an incubator. In this section, sea urchin embryos and a single mouse embryo were treated for evaluations. 13 .- Figure 8 shows the conceptual structure of the bio-ISFET intended to detect embryo activity after fertilization. Sea urchin ova were seeded on the surface of the gate insulator of the ISFET. Then, sperms were added to ova, resulting in the formation of fertilization membrane. The gate surface of the ISFET was immersed in a measurement solution together with an Ag/AgCl reference elec- trode with a saturated KCl solution. The charge density changes based on hydrogen ions could be detected as the shift of surface potential of the bio-ISFET, as mentioned in the above sections. Basically, the ion or molecular charges at the gate interact electrostatically with electrons in silicon crystal through the thin gate insulator and induce electrical signals by the field effect, and are monitored using the bio-ISFET system. Furthermore, two types of bio-ISFETs were prepared for differential measurements in this study; one was the bio-ISFET on which sea urchin ova were kept and fertilization was accomplished by induction of sperms, and the other was the control bio-ISFET on which there were ova, but sperms were not introduced. Using these bio-ISFETs, differential measurements were performed in order to eliminate the common background noise such as temperature change, non-specific adsorption and change in ion concentration. The change in the surface potential at the gate surface of the bio-ISFET was monitored after adding sea urchin ova and sperms in turn (Fig. 9a). First of all, the surface potential showed a negative shift after introducing ova at t 1 on the gate because of negative charges of the ovum membrane in contact with the gate surface. The negative charges of the ovum membrane derived from mainly sialic acids and sulfated saccharides of its jelly coat, as shown in the Real-time electrical detection of molecular charges at ovum membrane 10 section. After ova were kept on the gate area, sperms were added there at t 2 but little surface potential change of bio-ISFET could be detected, although some noise based on temperature and ion concentration changes could be found. The size of a sperm is smaller than that of an ovum (about 100 μm) and the diameter of the head is about 5 μm. They were running The surface potential of the bio-ISFET increased gradually to the 8-cell stage, then decreased after that. The increase of surface potential at the beginning of cell division indicates an increase of hydrogen ions induced by dissolved carbon dioxide created by respiration. Since little surface potential change was measured before fertilization, when virginal ova were kept on the gate surface, the shift of surface potential based on the increase of hydrogen ion might reveal the change of function of the respiration system due to fertilization. On the other hand, the control bio-ISFET showed little the electrical response due to non-fertilization (Fig. 9b).

Sea urchin embryo
Mitochondria play an important role to respiration inside cells. Pyruvate molecules produced by glycolysis are transported into the mitochondrion matrix through the inner membrane, where they are oxidized and combined with coenzyme A to form carbon dioxide, acetyl-CoA, and NADH. The fertilized ova would become activated, accompanied by cell division, resulting in changes of shape and amount of mitochondria, 49 which produce ATP and are closely related to the metabolism of the embryo. Cell division progressed to the 8-cell phase in a relatively short time, so the surface potential shifted increasingly due to the increase of hydrogen ions. On the other hand, the surface potential decreased gradually after reaching the 8-cell phase. The cell division of each blastomere occurred at random, and each step of cell division took longer than the previous step. For example, the transition from 8-cell to 16-cell took longer than that from 2-cell to 4-cell, resulting in the degeneration of respiration for one fertilized ovum in a constant period. This explains why the concentration of hydrogen ions would decrease based on the diffusion from the interface between the embryo and the gate surface to the bulk solution after the 8-cell phase. This means the effect of diffusion of hydrogen ions on the electrical signal was stronger than that of soluble carbon dioxide due to respiration. Thus, the embryo activity at the beginning of development can be easily detected in a real-time and noninvasive manner by use of the bio-ISFET.
Single mouse embryo 14 .-A single mouse embryo was put on one gate sensing area of the eSET-FET (Fig. 10a). The embryologist put a single mouse embryo on a gate area by use of a micropipet. The mammalian embryo is very sensitive to external environments such as ion strength of culture medium, temperature, and so on. This is why the total system for evaluation of embryo based on IVF should be prepared appropriately. Figure 10b shows the simultaneous analysis system of embryo activity by microscopic observation and electrical culture is useful to control the position of the single embryo on the gate and detect ion concentration change due to low volume. The change of surface potential at the gate surface of eSET-FET sensor was monitored after IVF (Fig. 11). The surface potential of the eSET-FET sensor with a single embryo to blastocyst increased gradually as shown in Fig. 11a. The increase of surface potential at the beginning of cleavage indicates the increase of hydrogen ions with positive charges based on dissolution of carbon dioxide generated by cellular respiration. Since little surface potential change was measured before fertilization, when virginal ova were kept on the gate surface, the shift of surface potential based on the increase of hydrogen ion would reveal the change of metabolism due to fertilization, as discussed in the above Sea urchin embryo 13 section. On the other hand, the surface potential change of the eSET-FET sensor with a single embryo accompanied by a 2-cell block decreased gradually, which was similar with that of a control sensor without embryo, although it increased drastically only for several hours before a 2-cell stage. This electrical signal indicates an abnormal behavior of an embryo before a 2-cell block and that hydrogen ions with positive charges based on respiration activity increased drastically at the interface between embryo and sensor surface. This may be because metabolism in a single embryo was activated accompanied by autophagy before cell death, 50 because proteins would be degraded to amino acids in autolysosome, which would be utilized for cellular respiration. Lastly, the control sensor without embryo showed actually the pH variation of culture medium for 90 h under the embryo culture condition. The surface potential change of the control sensor reached about −20 mV at around 40 h, which could be calculated as pH 7.4 to 7.7 because the semiconductor sensor had the detection ability of about 60 mV/pH. Moreover, the differential electrical signals were calculated as shown in Fig. 11b. Basically, the electrical signal of sample sensor against control sensor should be evaluated considering unexpected signals such as temperature change, pH variation of culture medium, and so on. The electrical signal of the eSET-FET sensor with normal embryo against the control FET (normal eSET-FET) was clearly different from that of the eSET-FET sensor with abnormal embryo against the control FET (abnormal eSET-FET) after around the 2-cell stage. Interestingly, the surface potential for normal eSET-FET increased gradually after IVF, but its inclination changed significantly after around the 2-cell stage of 10−15 h. This might show the change of metabolism during cleavage at around a 2-cell stage. Figure 11c shows the differential signal at 40 h after IVF in Fig. 11b, which corresponds to around an 8-cell stage for normal eSET-FET particularly. All the signals were The size of gate sensing area was 10 μm × 340 μm. The control sensor was placed at the distance of about 1 mm from the embryo sensor under the same condition. The photograph was taken at the 2-cell stage by upright microscope system in the incubator. (b) Simultaneous embryo analysis system. Electrical measurement using the eSET-FET sensor can be continuously performed in various intervals of second or minute to ∼ a few weeks or more in the incubator controlled at 37 • C and 5% CO 2 . The upright microscopy was set up in the incubator. Therefore, the simultaneous analysis of embryo quality can be realized by microscopic observation and noninvasive electrical measurement under the culture condition in the incubator system. (c) Sensing structure. Droplet of 20 μl was covered by mineral oil so that embryo culture can be conducted for a long time. Mineral oil is non-toxicity and has permeability of gases such as CO 2 and O 2 . Since embryo culture needs to be performed in a safe environment, polydimethylsiloxane (PDMS) was utilized for molding of glass ring and sensor electrodes. Reproduced with permission. 14 Copyright 2013, American Chemical Society. calculated on the basis of the differential measurements between a sample sensor with embryo and a control sensor without embryo. Twenty-one embryos were cultured at 37 • C on the gate in monitoring surface potential. Eighteen of them were normally cultured to the blastocyst-hatching stage, but 3 of them stopped cleavage around the 2-cell stage. The difference of surface potential changes at 40 h between normal eSET-FET and abnormal eSET-FET were distinguished as a significant difference (p < 0.01). Thus, which embryo reaches the blastocyst normally or not can be predicted at the early stage of cell division by evaluating the surface potential change of the eSET-FET sensor.
The average of the surface potential change for normal eSET-FETs, in which cleavage to the blastocyst-hatching stage occurred, was 36.7 mV with standard error ± 4.9 mV (n = 18) at the 8-cell stage of 40 h after IVF in Fig. 11c, considering the differential measurements. Basically, the eSET-FET sensor utilized in this measurement showed the average change of gate voltage 55.6 mV/pH for pH variation. Therefore, the respiration activity triggered by fertilization caused the change of about pH = 0.7 at the interface between embryo and gate surface. Strictly, the shift of pH from 7.4 to 6.7 was detected at the interface by the eSET-FET sensor. The eliminated carbon dioxide was calculated as about 1.6 × 10 −7 M based on the concentration change of hydrogen ion corresponding to pH variation, according to the equilibrium of carbon dioxide in solutions (CO 2 + H 2 O H + + HCO 3− ). In the calculation, the oxygen consumption, the ATP synthesis, and so on in the citric acid cycle inside the mitochondrion could be estimated from the amount of eliminated carbon dioxide, which is worked out from the electrical signal of the eSET-FET sensor. However, the diffusion of carbon dioxide at the interface between embryo and gate surface or from embryo surface apart from the gate has to be considered in order to estimate accurately its generation.
The volume of culture medium might actually affect the signals of pH variation based on embryos. In this case, however, the volume of 20 μL used in this study was large enough for the size of the embryo (about 100 μm), and actually, we could not find electrical signals when the embryo did not have contact with the gate area and was placed far from the gate in the same volume. Therefore, we need to directly put the embryo on the gate surface for measurement. Thus, the electrical signal of eSET-FET sensor for single mouse embryo activity should become an effective indication to evaluate objectively embryo activity as its morphology is observed subjectively after IVF. The platform based on the eSET-FET sensor will contribute to promote eSET in human ART.

Conclusions
In this review, some egg cells were targeted for the electrical monitoring with the semiconductor devices. Generally, cells can be directly and noninvasively observed by microscopy, which enables the imaging of fluorescent-labeled biomolecules as well as the observation of cellular morphologies. A quality check of an embryo obtained by IVF is performed before transplantation according to the criterion of the embryo grade, 36,37 as introduced in this paper, and the differential behaviors of stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are also investigated using green fluorescent protein (GFP)-induced gene transfection. [51][52][53] Microscopic observation in vitro provides considerable information on cellular functions to researchers and doctors. In particular, observation by inverted microscopy enables the high-resolution imaging of cells on a conventional cell culture dish owing to its transparency. However, the quantitative analysis of invisible cellular activities such as ion behaviors through cell membrane proteins is difficult to per- form at the same time as microscopic observation. It is very important to simultaneously analyze a number of functions of the same cell as rapidly as possible under the same conditions, because the evaluation of living cells for transplantation into a human body should be performed concurrently for identical cells in a noninvasive manner. However, silicon-based semiconductor devices focused in this paper are not suitable for microscopic observation because of their nontransparency. By developing transparent devices for cell sensing, 54 therefore, a simultaneous measurement system that allows visible and invisible information to be obtained simultaneously will be realized in the medical fields of clinical diagnosis and tissue engineering in the future.