An optical pickup enzyme-linked immunosorbent assay (ELISA) with a microfluidic disk.

We evaluated optical pickup ELISA with an original microfluidic disk that contains eight radially arranged channels, which enable semi-automatic sample loading and washing. This disk-shaped chip composed of acrylic plates was fabricated by CO2 laser machining and capture antibodies were immobilized in the channels. After the immunoreaction with antigens and enzyme-linked secondary antibodies, an enzyme-catalyzed nanoaggregation of o-phenylenediamine was detected by measuring the reflectivity change of a laser beam focused in the channel. The assay of C-reactive protein (CRP) was successfully performed in a short amount of time (approximately 20 min from CRP loading). The limit of detection was determined to be 2 ng mL-1, which is more sensitive as compared with conventional ELISA using microplates.

Electrical stimulation of hybridoma cells producing monoclonal antibody to cAMP.

Electrical stimulation was applied to hybridoma cells in order to activate metabolic activities and increase the monoclonal antibody production. Hybridoma cells that produce monoclonal antibody to adenosine 3':5'-cyclic monophosphate were placed on a transparent glass electrode immersed in medium and subjected to electric pulses (pulse shape, alternating rectangular; field strength, 4 X 10(3) V X m-1; frequency, 5 kHz; pulse mode, 0.5 min application and 4.5 min pause). After 48 h of incubation, the concentration of lactic acid in the medium reached 8.4 mM, approx. 30% higher than that obtained without electric stimulation. Similarly, cell growth rate was promoted by the electric stimulation, reaching a maximum stimulation after 40 h. When the hybridoma was cultured for 48 h with electrical stimulation, the antibody concentration in the medium reached 22.3 microgram X ml-1, approx. 10% higher than the control, with a concomitant 16% increase in cell concentration. Longer periods of electric pulse application, however, caused an inhibitory effect on the hybridoma growth. The most probable cause of the inhibition are reactive oxygen species such as superoxide and hydrogen peroxide, which are inevitably generated by electrolysis. The presence of superoxide dismutase (EC 1.15.1.1) reduced the inhibitory effects. In conclusion, metabolic activities including monoclonal antibody production were activated by the electrical stimulation.

Centrifugal microfluidic platform for single-cell level cardiomyocyte-based drug profiling and screening.

Drug screening and profiling is an important phase in drug discovery, development, and marketing. However, some profiling tests are not routinely done because of the needed additional technical skills and costly maintenance, which leads to cases of unexpected side effects or adverse drug reactions (ADRs). This study presents the design and operation of a microfluidic chip for single-cell level drug screening and profiling as an alternative platform for this purpose. Centrifugation was utilized to trap isolated single and groups of primary cultured neonatal rat cardiomyocytes in the same chip. In the off-spin operation of the chip, the cells can be observed under a microscope and movies of the beat motion can be recorded. The beat profiles of the cells were generated by image correlation analysis of the recorded video to study the contractile characteristics (beating rate, beating strength, and inter-beat duration). By utilizing this non-invasive tool, long term continuous monitoring, right after trapping, was made possible and cell growth and dynamics were successfully observed in the chip. Media and liquid replacement does not require further centrifugation but instead utilizes capillary flow only. The effect of carbachol (100 µM) and isoproterenol (4 µg mL(-1)) on single cells and groups of cells was demonstrated and the feature for immunostaining (ß-actin) applicability of the chip was revealed. Furthermore, these findings can be helpful for the headway of non-invasive profiling of cardiomyocytes and for future chip design and operation of high-throughput lab-on-a-chip devices.

New cell fusion method using polymer membrane.

A porous polymer membrane of nitrocellulose or tetrafluoroethylene (TFE) was employed for fusion of Saccharomyces cerevisiae (AH22 and D13-1A) protoplasts. Protoplasts were adsorbed on the membrane with slight suction. Some part of the protoplasts was trapped in pores of the membrane as observed by electron microscopy. The membrane retaining protoplasts was placed on a selective medium. Several colonies appeared on the medium after 5-7 days incubation at 30 degrees C. The fusion of the two strains was ascertained by DNA content and genetic markers. Fusion frequency was 1.2 X 10(-6) in the case of the TFE membrane.

DNA cleavage based on high voltage electric pulse.

A high voltage electric pulse was applied to DNA cleavage. The DNA cleavage reaction was dependent on the voltage amplitude, pulse number and pulse width. Radical scavengers and ESR data indicated the possibility that active species such as OH radical were strongly related to DNA cleavage.

Purification and characterization of cold-active L-glutamate dehydrogenase independent of NAD(P) and oxygen.

L-Glutamate dehydrogenase (GLDH) independent of NAD(P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp. L101, originally isolated from the organs of salmon (Oncorhynchus keta). GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5.7. Maximum activity was obtained at 55 degrees C and pH 8.5. The activity of GLDH at 4 and 20 degrees C was 38 and 50%, respectively, of that at 50 degrees C. GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo[alpha]phenoxazin-7-ium chloride (meldola's blue), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4, 4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H tetrazolium chloride] (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H tetrazolium (INT). The presence of NAD(P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.

A method for micrometer resolution patterning of primary culture neurons for SPM analysis.

In this work we present a method for ultra-fine patterning of primary culture neuron cell growth, which is compatible for scanning near-field optical atomic force microscopy (SNOAM) analysis. SNOAM uses near-field optics to break the fundamental diffraction limit imposed on normal microscopy. SNOAM can achieve sub-100 nm optical resolutions, but requires transparent, open substrates. The ability to do physiological measurements on patterns of neurons, combined with ultra high resolution optical and fluorescent analysis, is useful in the study of long-term potentiation. The patterning method consists of chemical guidance with an element of physical confinement and allows for ultra-fine patterning of neural growth on transparent glass substrates. Substrates consist of microfabricated perfluoropolymer barrier structures on glass. Poly-L-lysine was selectively deposited using a silicone-based microfluidic stencil aligned to the perfluoropolymer/glass substrate. Primary culture neurons were extracted from 8-day-old chicks and grown for 3 days to form good networks. This patterning system shows very specific growth with patterning separations down to the level of individual neurites. Fluorescent imaging was carried out on both cell viability during growth and immuno-tagged microtubule-associated proteins on the neurites. Neurons inside the patterned structures were imaged and analyzed with a tapping mode SNOAM.

Ultramicrobiosensors for monitoring of neurotransmitters.

Carbon fiber electrodes are used to construct ultramicrobiosensors with 7-15 microns diameter. Electrochemical operations for preelectrolysis and measuring were examined for sensitive determination of hydrogen peroxide. Determination limit was 0.1 microM of hydrogen peroxide. Reproducible determination of hydrogen peroxide is possible even in samples containing albumin protein. A micro-acetylcholine sensor was fabricated by immobilizing acetylcholine esterase and choline oxidase on the carbon fiber by entrapment with PVA-SbQ. This sensor gave a linear calibration plot for the range from 0.1 to 1.0 mM with a linear correlation coefficient of 0.9842. A micro-glutamate sensor consisted of a platinized carbon fiber disk electrode modified with immobilized glutamate oxidase membrane. This sensor gave a linear calibration for the range 2 microM to 1.2 mM. Release of glutamate in the cerebellar cortex was detected after potassium stimulation.

Fluorescence polarization immunoassay employing immobilized antibody.

The use of an antibody immobilized on latex or silver colloid in fluorescence polarization immunoassay (FPI) is assessed. In FPI it is possible to detect antigens of high molecular weight because the molecular weight of the antibody is effectively increased. In the assay for rabbit immunoglobulin G a limit of detection lower by two orders of magnitude and an assay range wider by one order of magnitude can be obtained in comparison with conventional FPI. The detection limit is 10(-10) mol l-1 and the total assay time for one sample is 8 min. This assay combines a low detection limit with a short assay time.

Microbiosensors for acetylcholine and glucose.

Microbiosensors based on carbon and and platinum fibers are described. Carbon fibers were used to construct microelectrodes of 7 microm diameter. Electrochemical operations for pre-electrolysis and measuring were examined for the highly sensitive determination of hydrogen peroxide. A triangular potential (-2 to +2V vs Ag/AgCl) was applied before measuring each pair of double pulses (first pulse: 750 mV; second pulse: 1100 mV). The determination limit was 0.1 microM of hydrogen peroxide. The reproducible determination of hydrogen peroxide is possible even in samples containing albumin protein. The separation of hydrogen peroxide from ascorbic acid is also possible because the oxidation potential of ascorbic acid is different from that of hydrogen peroxide. An acetylcholine microsensor was fabricated by immobilizing acetylcholine esterase and choline oxidase on the carbon fiber by entrapment with poly(vinyl alcohol)-quarternized stilbazole (PVA-SbQ). This sensor gave a linear calibration plot for the range 0.1-1.0 mM with a linear correlation coefficient of 0.9842. Glucose oxidase (GOD) and glucose dehydrogenase (GDH) immobilized cylindrical platinum microelectrodes were fabricated, and their characteristics were evaluated, respectively, by using 1,4-benzoquinone (BQ) and ferricyanide as electron mediators. Each enzyme was immobilized by using PVA-SbQ on a cylindrical microelectrode of 2 microm diameter. A linear range in the calibration curve of the GOD-based glucose microsensor was observed to be wider than that obtained using a disk electrode of 1 mm diameter. The mediated response of the 2 microm glucose sensor was compared with the response resulting from hydrogen peroxide detection. This result showed that a higher response and a wider linear range were observed with highly concentrated mediator. A much higher response of the GDH immobilized 2 microm microelectrode was obtained when not only ferricyanide but also diaphorase was employed to reoxidize the NADH produced by the enzyme reaction of GDH. The GHD-based glucose microsensor was found to be unaffected by the concentration of dissolved oxygen.