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.

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.

Expression of two kinds of recombinant glutamate dehydrogenase from Aeropyrum pernix with different N-terminal sequence length in Escherichia coli.

Two recombinant Aeropyrum pernix glutamate dehydrogenase (GDH) enzymes with different length N-termini were cloned and expressed in Escherichia coli: sGDH begins with the amino acid sequence of the extracted native enzyme (M-Q-P-T-D-P-L-E-E), whereas lGDH begins with the sequence of the predicted ORF (M-E-V-L-A-L-Q-P-T-D) and is longer than sGDH by five amino acids (M-E-V-L-A). Purified recombinant lGDH was more stable than sGDH, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant lGDH. This stabilising effect of extending the N-terminal sequence on an oligomeric enzyme such as GDH is novel; factors affecting stabilisation have previously only been discussed in the context of the contribution of internal amino acids.

On-chip capillary electrophoresis for alkaline phosphatase testing.

We fabricated an on-chip capillary electrophoresis device for blood analysis. An on-chip capillary electrophoresis device was photolithographically fabricated on a glass chip. Alkaline phosphatase (ALP) was employed as a sample enzyme. Small amounts of enzyme in the mixture of other proteins were detected with the electrophoretically mediated microanalysis (EMMA) method. Fluorescein diphosphate was used as fluorogenic substrate. The detection of ALP activity was achieved with laser-induced fluorescence monitoring fluorescein that was produced in enzyme reaction in capillary. Several methods to reduce the adhesion of protein are also discussed.

High-throughput PCR in silicon based microchamber array.

Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 degrees C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).

Yeast-immobilized SPV device for koji quality control in sake brewing process.

The malted rice, koji, is an indispensable material for the brewing of sake. It saccharifies rice starch and supplies vitamins for the yeast in sake brewing. Since the quality of sake depends strongly on the quality of koji, quality control of koji is very important in the brewing. There are some methods to measure the activity of enzymes and the quantity of vitamins with the quality of koji. None of these methods, however, directly relate to the yeast metabolism. We constructed a sensor system to monitor the yeast metabolism in sake brewing by use of immobilized Saccharomyces cerevisiae and a Surface PhotoVoltage device (SPV). In this system, S. cerevisiae K701 and K9, designed for use in sake brewing by the Brewing Society of Japan, were employed as immobilized microbe. The pH change due to the production of organic acids in sake brewing is measured using the SPV. A linear relationship was observed between decrease in the photocurrent (the metabolism response) and the concentration to less than 60 mM of glucose (r=0.990). Then we measured the koji extract and observed the difference of response between K701 and K9 which corresponded to the productivity of acidic substances by batch test.

Characterization of native glutamate dehydrogenase from an aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

Glutamate dehydrogenase (GDH) was purified and characterized from an aerobic hyperthermophilic archaeon Aeropyrum pernix (A. pernix) K1. The enzyme has a hexameric structure with a native molecular mass of about 285 +/- 15 kDa. It was specific for NADP and thermostable (74% activity was remained after 5 h incubation at 100 degrees C). The activity of the enzyme increased in the presence of polar water-miscible organic solvents such as acetonitrile, methanol, and ethanol. The N-terminal sequence of GDH is Met-Gln-Pro-Thr-Asp-Pro-Leu-Glu-Glu-Ala. This sequence, except for the methionine, corresponds to amino acids 7-15 of the open reading frame (ORF) encoding the predicted GDH (ORF APE 1386). In the ORF nucleotide sequence, the codon TTG appears at the position of the methionine, suggesting that the leucine codon might be recognized as an initiation codon and translated to methionine in A. pernix GDH.

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.

Development of a microchamber array for picoliter PCR.

A microchamber array for PCR was developed by semiconductor microfabrication technology. The microchambers were designed to be of picoliter quantity. To optimize fluid retention, the surface states of the substrate and the inner walls were examine for four different types of microchamber. The substrate was silicon, while silicon dioxide was selected for the inner walls. PCR was performed in the microchamber array, and the amplification of DNA was detected using a technique based on the energy transfer of fluorescent dyes. The lower volume limit for PCR was investigated using various sizes of microchambers. Microchambers with volume greater than 86 pL gave successful PCR. In addition, the system was improved in order to take up the PCR product. To prevent mixing of the samples, the samples were dried after PCR using a membrane that permeates only vapor.

A compactly integrated flow cell with a chemiluminescent FIA system for determining lactate concentration in serum.

We have fabricated an integrated flow cell as a total microanalysis system (microTAS). This flow cell (size, 15 x 20 mm; total inner volume, 12.2 microL) was designed for a rational analyzing system of lactate determination for serum. This cell was made by micromachining techniques and consisted of two hollows of a lactate oxidase (LOD) reactor and a mixing cell, a spiral groove, and three penetrated holes. To form the reactor and capillary, these patterns, etched on a silicon wafer, were attached to a glass plate by the anodic bonding method. A photodiode was put under part of the spiral capillary. The compactly accumulated devices were integrated into a flow injection analysis (FIA) system. In the flow cell, lactate was catalyzed to pyruvate and hydrogen peroxide at the LOD reactor; subsequently, hydrogen peroxide reacted with the luminol-ferricyanic reagent at the mixing cell. The resulting chemiluminescent light was detected by the photodiode. Using the miniaturized flow cell, the sample volume for one measurement was greatly reduced to 0.2 microL. The response to lactate was obtained within 30 s and was linear between 0.5 and 5.0 mM (4.5 and 45 mg/dL) lactate with excellent correlative variances of 3.2% (average of three measurements at 5.0 mM). For practical application, the lactate concentration in control human serum was determined using this system. The results showed a good correlation coefficient (r = 0.979) with the results obtained by the spectrophotometric reference method. No difference in sera (normal or pathological) was found. Consequently, this integrated flow cell shows potential as a clinical device for lactate determination in serum. In this article, the effect of the design on the chemiluminescent FIA system is also described.

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.

Are Metallic Markers Necessary for Coronary Artery Bypass Grafts? A Study Using X-ray Computed Tomography and Selective Graft Angiography.

Using X-ray computed tomography (CT) and selective graft angiography, the authors studied the necessity of metallic markers in coronary artery bypass grafts on 45 patients (mean age 57.2 years) with 87 saphenous vein grafts. Eight patients had 17 markers. X-ray CT was performed after surgery using an apparatus with a 1-second scanning time. Noncontrast X-ray CT was performed on horizontal sections, at 5-mm intervals, from the lower margin of the aortic arch to the lower left ventricle. A contrast medium was then injected into the antecubital vein (3 ml/second, total 30 ml) in one cross-section at the level of bifurcation of the pulmonary artery. Aortography (60 degrees in the left anterior and oblique positions, 20 ml/second, total 40 ml) was performed concurrently. Selective graft angiography was taken in the same direction, using 4 cm right of the Judkins with reference to the aortographic image and position of five clips on the sternum. Aortography revealed 79 patent and 8 occluded grafts. Selective graft angiography was easily performed even in grafts without markers. A cross-section of the occluded graft could not be seen with X-ray CT. Grafts with markers were often masked by artifacts produced by markers on X-ray CT. The number of observed graft slices (marker-positive grafts) was only 1.2 +/- 1.1 slices, significantly (p < 0.01) lower than marker-negative grafts (4.1 +/- 3.1 slices). In particular, the number of marker-positive right coronary artery grafts was 0.4 +/- 0.9 slices. Four of five right coronary artery grafts were unobservable due to artifacts. In grafts without markers, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of X-ray CT to graft patency were 100%, 85.7%, 98.4%, 100%, and 98.6%, respectively. This study suggests that metallic markers may not be necessary for coronary artery bypass grafts.

[Department of microbiosensing system using micromachine techniques].

This paper reviews devices for Point Of Care (POC) testing, especially focusing on current and future biosensors and biosensing fields. POC testing involves on-site diagnosis such as bedside diagnosis and near-patient testing. Biosensors and biosensing devices are a key technology to measure all clinically important parameters. For many reasons such as historical, simplicity, the high stability and activity of the enzyme required, and large demand from diabetics, only glucose sensors are commercially available. Various biosensors are waiting for commercialization. Not only simple biosensors having a biofunctional membrane on a transducer, but more complicated biosensing systems are fabricated on a microchip. Micro-Total Analysis System (mu TAS) is a research field to fabricate miniaturized sensing devices or systems. In these devices or systems, biochemical reactions for detection can be well controlled by running buffers, even though the consumption of reagents and sample is very small. Microarray chip technology, such as the DNA chip, is now receiving much attention in not only genetic research, but other biochemical research and application fields as well. Detection apparatus for the DNA chip is currently bench-top size. According to recent research, however, the size will be reduced as to a portable-size that is suitable for POC testing. Research and development of POC devices are advanced not alternative methods of conventional laboratory test, but as complement technology with advantages such as mobility, speed, and low-cost.

Multifunctional biocompatible membrane and its application to fabricate a miniaturized glucose sensor with potential for use in vivo.

A multifunctional membrane with biocompatibility, diffusion-limiting effect, and the ability to curtail the responses of an H(2)O(2) electrode to ascorbate and urate was prepared. It was composed of MB, AB, and CTA, where MB is the copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butylmethacrylate (BMA), AB is the copolymer of acrylamide-2-methylpropane sulfonic acid (AMPS) and BMA, CTA is cellulose triacetate. Investigation of the biocompatibility of this membrane showed that, compared with CTA, relatively few platelets bound to it. The membrane was coated onto the working electrode of a needle-type glucose sensor on which immobilized glucose oxidase membrane has been coated. The sensor did not respond to ascorbate and urate at their concentration normally encountered in blood. Its response was not inhibited by metal ions in blood at usual concentration. The sensor exhibited superior thermostability in addition to a rapid response (<90 seconds in batch operation), good reproducibility (RE<5%), good stability (more than 36 hours continuously in heparinized whole blood), and a wide dynamic range (5-650 mg/dl glucose). The sensor was used to determine glucose in serum. The data obtained from the sensor showed good agreement with that from a clinical autoanalyzer (R=0.973).

Effects of Postoperative Pericardial Effusion on Coronary Artery Bypass Grafts: A Study with X-ray Computed Tomography.

The aim of this study was to evaluate the effects of pericardial effusion on coronary artery bypass grafts and their patency using X-ray computed tomography (CT). Uncontrasted CT of horizontal sections from the lower margin of the aortic arch to the left ventricle was done at 5-mm intervals. In one cross-section of the pulmonary bifurcation level, 30 ml of a contrast media (lohexol 350) was injected at a rate of 3 ml/second into the antecubital vein. All slices of uncontrasted CT were analyzed for the presence or absence of effusion. The severity was expressed as the maximum value of the thickness of effusion. CT was repeated about every 6 months postoperatively under the same conditions. Selective angiography was also performed 7.1 +/- 3.9 months postoperatively. A total of 46 patients (mean age 57 years) underwent CT and angiography. A total of 95 grafts were implanted: 90 saphenous veins and 5 internal thoracic arteries. Selective angiography revealed that 79 grafts were patent and 16 were occluded. The first postoperative CT (at 2.6 +/- 2.1 months) showed the retention of effusion in all patients. The mean maximum value was 1.0 +/- 0.5 cm; there were no significant differences between patent grafts (1.0 +/- 0.5 cm) and occluded grafts (1.0 +/- 0.5 cm). Occlusion was found in 10 grafts by the first CT (2.9 +/- 2.7 months postoperatively) and another 6 grafts by the second CT (11.3 +/- 4.2 months). Thereafter, all grafts were patent. Previously occluded grafts showed no cross-section images on uncontrasted or contrasted CT. Except for two grafts, all patent grafts could be observed even without contrast enhancement. The remaining two grafts were masked with effusion, but patency was confirmed by a contrast media. In conclusion, retention of effusion does not affect the patency of grafts. Occlusion occurs early after surgery, and grafts cannot be imaged on CT. Patent grafts can be observed by uncontrasted CT, as well as contrasted CT, except where a large amount of effusion is present.

[Left ventricular wall motion dynamics of asymmetric septal hypertrophy: assessment by intramyocardial pulsed Doppler echocardiography].

Apical pulsed Doppler tissue imaging can be used to assess the function of regional myocardium. We hypothesized that septal dysfunction might be clarified in the hypertrophic cardiomyopathy (asymmetric septal hypertrophy) by this method. Twenty-one patients with asymmetric septal hypertrophy (mean age 54.8 +/- 11 years) and age-matched 24 normal subjects (52.4 +/- 8 years) were studied. The E/A ratio measured by mitral inflow Doppler was not different between the groups (1.1 vs 1.2). E wave velocities of the septum were significantly decreased in the hypertrophy group compared to the control group (4.0 +/- 1.5 vs 8.1 +/- 2.2 cm/sec), and A wave velocities were increased in the hypertrophic septum, resulting in a significantly lower E/A ratio (0.5 +/- 0.3) compared to the E/A ratio (0.9 +/- 0.3) of the normal septum. Deceleration time of the E wave and isovolumic relaxation time were significantly prolonged in the thick septum compared to the normal septum (136 +/- 51 vs 107 +/- 28 msec, 91 +/- 36 vs 63 +/- 19 msec, respectively). In conclusion, asymmetric septal hypertrophy was characterized by diastolic dysfunction of the thickened septum. Intramyocardial pulsed Doppler echocardiography can detect regional myocardial dysfunction earlier than the mitral inflow Doppler method.

[Ebstein's anomaly associated with left ventricular dysfunction: a case report].

A 66-year-old man presented with Ebstein's anomaly associated with left ventricular dysfunction. He had been followed since 40 years of age for cardiomegaly and arrhythmia, and experienced episodes of orthopnea at the age of 64. He was referred to our hospital in April 1997 because of lower extremity edema. Physical examination revealed dilated external jugular vein, tenderness of the right hypocondorium, and lower extremity edema. Electrocardiography confirmed atrial fibrillation. Transthoracic echocardiography revealed bilateral atrial and ventricular dilation, and paradoxical septal movement. The apical four-chamber view demonstrated 15 mm apical displacement of the septal leaflet. Color Doppler echocardiography revealed moderate tricuspid regurgitation. Transesophageal echocardiography revealed low echoic and hypoplastic tricuspid valve. Left ventriculography showed diffuse hypokinesis, and the ejection fraction was 49%. The coronary artery was normal. Atrial septal defect was not detected. Diffuse fibrosis, which may be found in the hearts of patients with Ebstein's anomaly at autopsy may have been responsible for the left ventricular depressed systolic function in this patient.

Properties of a cold-active protease from psychrotrophic Flavobacterium balustinum P104.

Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 degrees C, which was isolated from salmon (Oncorhynchus keta) intestine. The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographyies. The molecular mass of the protease was 70 kDa, and its isoelectric point was close to 3.5. Maximal activity toward azocasein was observed at 40 degrees C and from pH 7.0 to 9.0. The activity was strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease. The N-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu- Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Gly-Asn. A search through the database for sequence homology yielded no significant match. The initial cleavage sites for oxidized insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate.

Simultaneous topographic and fluorescence imagings of recombinant bacterial cells containing a green fluorescent protein gene detected by a scanning near-field optical/atomic force microscope.

A scanning near-field optical/atomic force microscope (SNOAM) system was applied for simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever and as a scanning near-field optical microscopy probe. Optical resolution of this system was about 50-100 nm in fluorescence mode for fluorescent latex beads on a quartz glass plate. Green fluorescent protein (GFP) is a convenient indicator of transformation and should allow cells to be separated by fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant Escherichia coli. The SNOAM system used 458- or 488-nm irradiation from a multiline Ar ion laser for excitation of GFP, since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. A nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, it can be seen that individual E. coli cells expressed different fluorescence intensities. Fluorescence obtained by SNOAM indicated that GFP oxidation possibly occurred near the cell surface. A SNOAM system also indicated the possibility of precise imaging of native cells in liquid.