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ABSTRACT: 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 hanging drop culture method. Fluidigm Biomark dynamic array was used for comprehensive and qRT-PCR analysis on the samples whose respiratory activity had been measured. Genes involved in cellular senescence, glucose metabolism indicated significantly higher for PDMS microwell culture method than for hanging drop culture method (P < 0.05). Interestingly, samples formed with PDMS microwell culture method showed stronger responses for glycolysis than those formed with hanging drop method. These results illustrate the power of multi-parameter analysis to characterize multicellular spheroids cultured in different microenvironment even if they have the same morphology.
Analytical Biochemistry 04/2013; · 3.00 Impact Factor
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ABSTRACT: 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.
Biosensors & bioelectronics 04/2013; 48C:12-18. · 5.43 Impact Factor
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ABSTRACT: 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. Real-time monitoring of the EB ALP activity for 3.5 h indicated decrease in the ALP activity after 2 h in a glucose-free buffer solution. 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 3 h. The chip is expected to be useful for the evaluation of 3D tissues.
Biosensors and Bioelectronics 03/2013; · 5.60 Impact Factor
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ABSTRACT: 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.
Analytical Chemistry 03/2013; · 5.86 Impact Factor
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ABSTRACT: In this study, tubular hydrogel structures were constructed via electrodeposition using alginate gels. Electrolysis of water in alginate solutions with calcium carbonate particles induced gel aggregation around Pt wire electrodes, forming tubular alginate gel structures. The simple method is a promising approach for construction of multi-layer tubular hydrogel structures for tissue engineering.
Journal of Bioscience and Bioengineering 12/2012; · 1.79 Impact Factor
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ABSTRACT: Mercury is considered the most important heavy metal pollutant because of the likelihood of bioaccumulation and toxicity. Monitoring widespread ionic mercury (Hg2+) contamination requires high-throughput and cost-effective methods to screen large numbers of environmental samples. In this study, we developed a simple and sensitive analysis for Hg2+ in environmental aqueous samples by combining a microfluidic immunoassay and solid-phase extraction (SPE). By using a microfluidic platform, an ultrasensitive Hg2+ immunoassay, which yields results in only 10 min and with a lower detection limit (LOD) of 0.13 µg/L, was developed. To allow application of the developed immunoassay to actual environmental aqueous samples, we developed an ion exchange resin (IER) based SPE for selective Hg2+ extraction from among an ion mixture. When using optimized SPE conditions, followed by the microfluidic immunoassay, the LOD of the assay was 0.83 μg/L, which satisfied the guideline values for drinking water suggested by the US EPA (2 µg/L; total mercury), and the WHO (6 µg/L; inorganic mercury). Actual water samples, including tap water, mineral water, and river water, which had been spiked with trace levels of Hg2+, were well analyzed by SPE followed by microfluidic Hg2+ immunoassay, and the results were agreed with reduction vaporizing-atomic adsorption spectroscopy.
Analytical Chemistry 11/2012; · 5.86 Impact Factor
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ABSTRACT: Engineered skeletal muscle tissues are ideal candidates for applications in drug screening systems, bio-actuators, and as implantable constructs in tissue engineering. Electrical field stimulation considerably improves the differentiation of muscle cells to muscle myofibers. Currently used electrical stimulators often use direct contact of electrodes with tissue constructs or their culture medium, which may cause hydrolysis of the culture medium, joule heating of the medium, contamination of the culture medium due to products of electrodes corrosion, and surface fouling of electrodes. Here, we used an interdigitated array of electrodes combined with an isolator coverslip as a contactless platform to electrically stimulate engineered muscle tissue, which eliminates the aforementioned problems. The effective stimulation of muscle myofibers using this device was demonstrated in terms of contractile activity and higher maturation as compared to muscle tissues without applying the electrical field. Due to the wide array of potential applications of electrical stimulation to two- and three-dimensional (2D and 3D) cell and tissue constructs, this device could be of great interest for a variety of biological applications as a tool to create noninvasive, safe, and highly reproducible electric fields.
Biomedical Microdevices 09/2012; · 3.03 Impact Factor
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ABSTRACT: A lab-on-a-chip device is described for the electrochemical detection of alkaline phosphatase (ALP) secreted by transformed single HeLa cells. Detection on the chip device is based on local redox cycling at 256 individually addressable sensor points. Ring-disk electrodes (generator/collector) are arranged at individual sensor points to amplify the signal due to redox-cycling with only 32 connector pads. The surface of each sensor point is modified with antibodies for secreted alkaline phosphatase (SEAP) immobilization, which facilitates separation and detection of SEAP. Separation of SEAP from HeLa cells enables elimination of endogenous ALP and prevents HeLa cells from damage due to exposure to high level pH used during electrochemical detection. The large number of sensor points enables the simultaneous analysis of a large amount of single cells using the chip. The system is useful for gene reporter assays and for the detection of several types of secreted proteins.
Lab on a Chip 08/2012; 12(21):4328-35. · 5.67 Impact Factor
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ABSTRACT: 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.
Analytical Chemistry 08/2012; 84(18):7593-8. · 5.86 Impact Factor
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ABSTRACT: A lab-on-a-chip device is described for the electrochemical detection of alkaline
phosphatase (ALP) secreted by transformed single HeLa cells. Detection on the chip
device is based on local redox cycling at 256 individually addressable sensor points.
Ring-disk electrodes (generator/collector) are arranged at individual sensor points to
amplify the signal due to redox-cycling with only 32 connector pads. The surface of each
sensor point is modified with antibodies for secreted alkaline phosphatase (SEAP)
immobilization, which facilitates separation and detection of SEAP. Separation of SEAP
from HeLa cells enables elimination of endogenous ALP and prevents HeLa cells from
damage due to exposure to high level pH used during electrochemical detection. The
large number of sensor points enables the simultaneous analysis of a large amount of
single cells using the chip. The system is useful for gene reporter assays and for the
detection of several types of secreted proteins.
Lab on a Chip 08/2012; · 5.67 Impact Factor
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ABSTRACT: Engineered skeletal muscle tissues could be useful for applications in tissue engineering, drug screening, and bio-robotics. It is well-known that skeletal muscle cells are able to differentiate under electrical stimulation (ES), with an increase in myosin production, along with the formation of myofibers and contractile proteins. In this study, we describe the use of an interdigitated array of electrodes as a novel platform to electrically stimulate engineered muscle tissues. The resulting muscle myofibers were analyzed and quantified in terms of their myotube characteristics and gene expression. The engineered muscle tissues stimulated through the interdigitated array of electrodes demonstrated superior performance and maturation compared to the corresponding tissues stimulated through a conventional setup (i.e., through Pt wires in close proximity to the muscle tissue). In particular, the ES of muscle tissue (voltage 6 V, frequency 1 Hz and duration 10 ms for 1 day) through the interdigitated array of electrodes resulted in a higher degree of C2C12 myotube alignment (∼80%) as compared to ES using Pt wires (∼65%). In addition, higher amounts of C2C12 myotube coverage area, myotube length, muscle transcription factors and protein biomarkers were found for myotubes stimulated through the interdigitated array of electrodes compared to those stimulated using the Pt wires. Due to the wide array of potential applications of ES for two- and three-dimensional (2D and 3D) engineered tissues, the suggested platform could be employed for a variety of cell and tissue structures to more efficiently investigate their response to electrical fields.
Lab on a Chip 07/2012; 12(18):3491-503. · 5.67 Impact Factor
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Kumi Y Inoue,
Masahki Matsudaira,
Reyushi Kubo,
Masanori Nakano,
Shinya Yoshida,
Sakae Matsuzaki,
Atsushi Suda,
Ryota Kunikata,
Tatsuo Kimura,
Ryota Tsurumi,
Toshihito Shioya,
Kosuke Ino,
Hitoshi Shiku,
Shiro Satoh,
Masayoshi Esashi, Tomokazu Matsue
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ABSTRACT: We have developed an LSI-based amperometric sensor called "Bio-LSI" with 400 measurement points as a platform for electrochemical bio-imaging and multi-point biosensing. The system is comprised of a 10.4 mm × 10.4 mm CMOS sensor chip with 20 × 20 unit cells, an external circuit box, a control unit for data acquisition, and a DC power box. Each unit cell of the chip contains an operational amplifier with a switched-capacitor type I-V converter for in-pixel signal amplification. We successfully realized a wide dynamic range from ±1 pA to ±100 nA with a well-organized circuit design and operating software. In particular, in-pixel signal amplification and an original program to control the signal read-out contribute to the lower detection limit and wide detection range of Bio-LSI. The spacial resolution is 250 μm and the temporal resolution is 18-125 ms/400 points, which depends on the desired current detection range. The coefficient of variance of the current for 400 points is within 5%. We also demonstrated the real-time imaging of a biological molecule using Bio-LSI. The LSI coated with an Os-HRP film was successfully applied to the monitoring of the changes of hydrogen peroxide concentration in a flow. The Os-HRP-coated LSI was spotted with glucose oxidase and used for bioelectrochemical imaging of the glucose oxidase (GOx)-catalyzed oxidation of glucose. Bio-LSI is a promising platform for a wide range of analytical fields, including diagnostics, environmental measurements and basic biochemistry.
Lab on a Chip 07/2012; 12(18):3481-90. · 5.67 Impact Factor
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ABSTRACT: This report describes the electrochemical detection of a redox component in droplets using a local redox cycling-based electrochemical (LRC-EC) chip device consisting of 256 sensors. The time-course analyses showed that the redox compound in the droplet was dynamically changed during droplet evaporation or mass transfer through a water/oil interface.
Chemical Communications 07/2012; 48(68):8505-7. · 6.17 Impact Factor
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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 (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.
Talanta 05/2012; 94:30-5. · 3.79 Impact Factor
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ABSTRACT: Monitoring cellular activity: A local redox-cycling-based electrochemical chip device has been used to entrap three-dimensional culture cells and evaluate their activity. Deep microwells were incorporated into the chip device for the trapping of embryoid bodies. This chip device is useful for the evaluation of 3D organ tissues.
Angewandte Chemie International Edition 05/2012; 51(27):6648-52. · 13.45 Impact Factor
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Yasufumi Takahashi,
Andrew I Shevchuk,
Pavel Novak,
Babak Babakinejad,
Julie Macpherson,
Patrick R Unwin,
Hitoshi Shiku,
Julia Gorelik,
David Klenerman,
Yuri E Korchev, Tomokazu Matsue
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ABSTRACT: We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5-100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5-3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.
Proceedings of the National Academy of Sciences 05/2012; 109(29):11540-5. · 9.68 Impact Factor
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ABSTRACT: A simple and rapid flow-based multioperation immunoassay for heavy metals using a microfluidic device was developed. The antigen-immobilized microparticles in a sub-channel were introduced as the solid phase into a main channel structures through a channel flow mechanism and packed into a detection area enclosed by dam-like structures in the microfluidic device. A mixture of a heavy metal and a gold nanoparticle-labeled antibody was made to flow toward the corresponding metal through the main channel and make brief contact with the solid phase. A small portion of the free antibody was captured and accumulated on the packed solid phase. The measured absorbance of the gold label was proportional to the free antibody portion and, thus, to the metal concentration. Each of the monoclonal antibodies specific for cadmium-EDTA, chromium-EDTA, or lead-DTPA was applied to the single-channel microfluidic device. Under optimized conditions of flow rate, volume, and antibody concentration, the theoretical (antibody K(d)-limited) detection levels of the three heavy metal species were achieved within only 7 min. The dynamic range for cadmium, chromium, and lead was 0.57-60.06 ppb, 0.03-0.97 ppb, and 0.04-5.28 ppb, respectively. An integrated microchannel device for simultaneous multiflow was also successfully developed and evaluated. The multiplex cadmium immunoassay of four samples was completed within 8 min for a dynamic range of 0.42-37.48 ppb. Present microfluidic heavy metal immunoassays satisfied the Japanese environmental standard for cadmium, chromium and, lead, which provided in the soil contamination countermeasures act.
Biosensors & bioelectronics 03/2012; 33(1):106-12. · 5.43 Impact Factor
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ABSTRACT: A new electrochemical assay for the detection of secreted alkaline phosphatase (SEAP) from transfectant HeLa cells is proposed using a microarray device and scanning electrochemical microscopy (SECM). The assay consists of two steps: the first is the incubation of a transfected cell in a microarray culture device covered with a substrate modified with anti-SEAP under physiological conditions without any additives. The array device consists of a 4 × 4 array of microwells having a size of 100 µm × 100 µm (diameter × depth). The second step is SECM measurement of secreted SEAP at the antibody-immobilized substrate. This assay ensures accuracy and intactness because the undesired influence of endogeneous ALP is eliminated and the transfected cells are incubated in a culture device under suitable conditions. We successfully detected the expression of SEAP from intact cells at the single-cell level using this assay. The system is useful as a cell-based gene-expression assay.
Biotechnology and Bioengineering 02/2012; 109(8):2163-7. · 3.95 Impact Factor
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ABSTRACT: A microfluidic device with analytical chambers for electrochemical measurements has been employed to detect photosynthetic activity at single cell level. The flowing cells (Microcystis viridis) in a main channel are individually guided to the chamber with microelectrodes by an electrophoretic manipulation. The reduction current of oxygen was continuously monitored to determine the photosynthetic activity upon light irradiation. The average rates for oxygen generation were estimated and found to be 10(-18) mol/s level.
Analytical Sciences 01/2012; 28(1):69. · 1.25 Impact Factor
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ABSTRACT: An electrochemical platform for parallel monitoring of secreted alkaline phosphatase (SEAP) has been microfabricated on a device with a mammalian-cell array chip. A 4 × 4 ring-ring electrode array was designed at the rim of the round cellular pattern with a diameter of 270 μm. Electrochemical characterization was carried out, and it was found that the collection efficiency was about 50% in dual mode when the inner-ring and the outer-ring electrodes were selected as the collector and generator electrodes, respectively. The current amplification ratio for the dual mode normal to single mode was 2.84. SEAP expressing from the cells was parallelly monitored by using a multiplexer switching system at the 16 round cellular spots. The reduction current for HeLa cells transfected with plasmid encoding SEAP observed at the collector outer ring electrode was found to be significantly higher than that for wild-type HeLa. Finally, the top of the microwell with the round cellular pattern was covered with a poly(dimethylsiloxane) block for 5 min to accumulate the secreted enzyme and the product of the enzyme reaction so that further signal enhancement could be observed.
The Analyst 12/2011; 136(23):4991-6. · 4.23 Impact Factor
