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We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.

Forensic human identification (HID) laboratories occasionally encounter non-specific peaks generated by non-human DNA. Casework samples for human short tandem repeat (STR) profiling may be contaminated by animal DNA because of the specific environment or situation from which they were obtained. Validation studies for HID kits have reported that non-specific peaks generated from some animals are observed near the human amelogenin peak. In this study, we first revealed that DNA sequences associated with the non-specific peaks generated from animal DNA differ from one animal family to the other. However, non-specific peaks cannot be analyzed using the remainder of polymerase chain reaction (PCR) products left over from conventional HID kits when human and animal DNA are mixed. To overcome this issue, we have developed a novel analysis method of using non-specific peaks generated from animal DNA in human STR profiling to identify the source of contaminating animal DNA at the family level. The method applied here is termed as blocking PCR, which involves selective animal DNA re-amplification by blocking nontarget human amelogenin DNA amplification using an oligonucleotide probe that specifically binds to human amelogenin using the remaining PCR product from the HID kit. Our data demonstrated that HID and family discrimination among animals that are often encountered in forensic contexts could be performed simultaneously. This study enabled recovery of more information from limited quantities of casework samples contaminated with animal DNA, which would be useful for forensic HID scientists.

We investigated whether bloodstain examination and DNA typing can be performed on washed bloodstains on clothes. Blood was dropped onto T-shirts made from 100% cotton or 100% polyester. After drying, the T-shirts were hand-washed with handwashing soap, dishwashing detergent, laundry detergent, soap, or just water until the bloodstains could not be seen. After drying the T-shirts, DNA and RNA were extracted simultaneously from the bloodstained areas using commercial kits. RNA was reverse-transcribed to DNA, and then the detection of the mRNAs for HBB, ACTB, and 18S rRNA was examined. DNA was quantified via real-time PCR, and then STR typing was performed with a commercial kit. The luminol and leucomalachite green tests were used as preliminary bloodstain tests, and an immuno-chromatography kit was used to identify human bloodstains. DNA could be extracted from all washed bloodstains, but more DNA was extracted from cotton T-shirts than from polyester T-shirts. STR typing was successful for all bloodstains without issues such as PCR inhibition. In the human bloodstain identification test using mRNA, almost all bloodstains produced a Ct value for HBB and all bloodstains produced a Ct value for 18S rRNA, whereas few bloodstains produced a Ct value for ACTB. All bloodstains reacted positively to luminol, but some were negative for leucomalachite green. Most of the bloodstains did not react positively in the human bloodstain identification test using the immuno-chromatography kit. The results suggest that human bloodstain identification and DNA typing can still be performed after clothes with bloodstains are washed.

A new radiolabeling method using a microreactor was developed for the rapid synthesis of [(11)C]raclopride. A chip bearing a Y-shaped mixing junction with a 200 µm (width)×20 µm (depth)×250 mm (length) flow channel was designed, and the efficiency of O-[11C]methylation was evaluated. Dimethyl sulfoxide solutions containing the O-desmethyl precursor or [11C]CH3I were introduced into separate injection ports by infusion syringes, and the radiochemical yields were measured under various conditions. The decay-corrected radiochemical yield of microreactor-derived [11C]raclopride reached 12% in 20 s at 25 °C, which was observed to increase with increasing temperature. In contrast, batch synthesis at 25 °C produced a yield of 5%: this indicates that this device could effectively achieve O-[11C]methylation in a shorter period of time. The microreactor technique may facilitate simple and efficient routine production of 11C-labeled compounds via O-[11C]methylation with [11C]CH3I.

The effects of different storage methods on both DNA degradation and the results of preliminary blood tests were evaluated. Bloodstains stored at room temperature, 4°C, –20°C, and –80°C for 20 years; whole blood samples stored at –20°C and –80°C for 20 years; and fresh whole blood samples were analyzed. DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41bp fragments. Preliminary testing included leuco-malachite-green staining, anti-human hemoglobin (Hb) testing by immunochromatography, and the detection of the hemoglobin-beta (HBB) mRNA. Bloodstains stored at room temperature and 4°C were more highly degraded than fresh blood, as indicated by the ratio of 129:41bp DNA fragments and 305:41bp DNA fragments. All samples tested positive using leuco-malachite-green staining and anti-human Hb assays. HBB was not detected in any whole blood samples stored at –20°C or –80°C, although HBB was detected in all bloodstain samples. Therefore, to prevent DNA degradation during long-term storage, it is recommended that blood samples be stored at below –20°C. In addition, stored bloodstains are suitable for preliminary tests of blood, rather than storing blood.

The relationships between DNA degradation ratios and the number of detected loci were explored in extremely old seminal stains evaluated using three short tandem repeat (STR) kits: the AmpFlSTR® Identifiler™ PCR Amplification Kit (Identifiler), the AmpFlSTR® Yfiler™ PCR Amplification Kit (Yfiler), and the AmpFlSTR® MiniFiler™ PCR Amplification Kit (MiniFiler). DNA degradation ratios based on 41, 129, and 305bp DNA fragments were calculated (129:41 and 305:41), and the relationships between the ratios and storage duration were also explored. Using the Identifiler kit, the number of loci detected was strongly correlated with the 129:41 ratio (r=0.887), whereas the correlation with the 305:41 ratio was moderate (r=0.656). Using the Yfiler kit, the DYS385 amplicon was detected in all samples, suggesting that DYS385 may be resistant to degradation. The number of detected loci was strongly correlated with the 129:41 ratio (r=0.768), and moderately so with the 305:41 ratio (r=0.515). MiniFiler detected at least seven loci in all samples. In samples that did not yield full profiles, the undetected loci were D7S820 and D21S11, or D21S11 only, suggesting that these loci might be easily degraded. The number of loci detected using STR kits correlated with the DNA degradation ratios. In particular, the 129:41 ratio was particularly useful for estimating the number of loci detectable by STR kits. On the other hand, we suggest that storage duration cannot be accurately estimated using DNA degradation ratios; these ratios were not strongly correlated with storage duration (129:41; r=-0.698, 305:41; r=-0.550). However, the ratios may allow the identification of samples that have been stored for more than 40years.

The Planar electrostatic ion trap expands the trapping space of the linear electrostatic ion trap, giving rise to higher tolerance to space charge. A rotational symmetrical design was made, which has a trapping field between two layers of concentric circular electrodes, and the ions are trapped to oscillate around the center plane between the electrodes. The oscillatory motions of the ions were simulated and the field distribution was optimized to achieve isochronous motion against energy spread in R, z, and φ directions. The image charge signal can be picked up by more than one circular electrode and using FFT the mass resolution for the optimized trap can reach 80,000 FWHM. While Fourier transform of the image charge signal generates many high harmonic peaks, the unwanted harmonic peaks can be eliminated by linear combination of image charge signals from multiple pick-up electrodes to give satisfactory results.

Glucose fluctuation (GF) is a risk factor for coronary artery disease (CAD). However, it remains unknown whether specific indices of GF are risk factors for CAD. Therefore, we evaluated the relationship between GF, as determined by a continuous glucose monitoring system (CGMS) or the glucose level at 2h after a 75-g oral glucose tolerance test (75g OGTT 120), and the severity of CAD in prediabetic patients. We also evaluated whether nitrotyrosine (NT) and glyceraldehyde-derived advanced glycation end-products (Glycer-AGE) were induced by GF.Twenty-eight prediabetic patients underwent coronary angiography (CAG), and the Gensini score and the SYNTAX score were evaluated as the severity of CAD, while the mean amplitude of glycemic excursions (MAGE) by CGMS and 75g OGTT 120 were evaluated. Serum NT and Glycer-AGE were measured.The MAGE was closely associated with the Gensini score (r=0.742, p0.001) and the SYNTAX score (r=0.776, p0.001), respectively. The 75g OGTT 120 was not associated with the Gensini score (r=0.36, p=0.06), but it was significantly associated with the SYNTAX score (r=0.413, p=0.036). Multiple linear regression analysis showed that the MAGE was the only independent determinant for the severity of CAD. The levels of NT and Glycer-AGE were significantly higher in the high MAGE group than in the low MAGE group.Diurnal GF is associated with the severity of CAD, even in prediabetic patients. GF, NT, and Glycer-AGE may play a pathological role in the progression of CAD.

In the field of positron emission tomography (PET) radiochemistry, compact microreactors provide reliable and reproducible synthesis methods that reduce the use of expensive precursors for radiolabeling and make effective use of the limited space in a hot cell. To develop more compact microreactors for radiosynthesis of 18F-labeled compounds required for the multistep procedure, we attempted radiosynthesis of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) via a three-step procedure using a microreactor. We examined individual steps for [18F]SFB using a batch reactor and microreactor and developed a new continuous-flow synthetic method with a single microfluidic chip to achieve rapid and efficient radiosynthesis of [18F]SFB. In the synthesis of [18F]SFB using this continuous-flow method, the three-step reaction was successfully completed within 6.5 min and the radiochemical yield was 64 ± 2% (n = 5). In addition, it was shown that the quality of [18F]SFB synthesized on this method was equal to that synthesized by conventional methods using a batch reactor in the radiolabeling of bovine serum albumin with [18F]SFB.

We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice.

The various potential factors affecting the performance of nanopillar chips on DNA separation were investigated from the viewpoints of both numerical calculations and actual experiments. To yield higher performance and replace the conventional DNA separation techniques such as microchip electrophoresis, the phenomenon specific to the nanopillar chips should be deeply understood. In this paper, although various factors affecting the performance of the nanopillar chips are considered, we focused on the effect of electroosmotic flow, which is particularly noticeable in quartz-made nanopillar chips. High-resolution separation of DNA was realized when an electroosmotic flow was suppressed by simply using a higher concentration of buffer, but DNA separation failed in the presence of an electroosmotic flow. It was confirmed from the numerical simulations and the direct observations that the deformation of DNA band during the injection process was induced by electroosmotic flow and consequently led to a poor resolution.

To achieve a rapid assay for ABO blood grouping using a latex reagent, two latex reagents were produced, one of which combined with mouse monoclonal immunoglobulin M (IgM) isolated from commercial ABO blood grouping reagent, and the other of which combined with its F(ab')2 fragment prepared by cold pepsin digestion. The latex reagent adsorbing the F(ab')2 fragment was able to detect the 1000-fold diluted saliva and provided much better sensitivity than that of IgM. This suggests that the difference in sensitivity between the two latex reagents is responsible for adsorption orientation of the antigen site on the latex particles. The new assay successfully completed the ABO blood grouping of cigarette ends within 30 min.

We successfully developed a measurement system for real-time analysis of cellular function using a newly designed microchip. This microchip was equipped with a micro cell incubation chamber (240 nl) and was stimulated by a very small amount of stimuli (as small as 24 nl). Using the microchip system, cultivation of mast cells was successfully carried out. Monitoring of the cellular events after stimulation with an extremely small amount of fluid on a microchip was performed. This system could be applicable for various types of cellular analysis including real-time monitoring of cellular response by stimulation.

This paper presents a sheath flow multiple sample injector that realizes concentration-conservative sample injection. Stepwise flow is used to reduce the dilution of the sample reagent. Based on the CFD simulation results, it is estimated that sample reagent is diluted to 94% in a conventional sample injector. With stepwise flow, however, the level of dilution is reduced and the average concentration is estimated to be near 100%. For the experiment evaluation, a sample injector is fabricated and the sample injection using stepwise flow is visualized using a fluorescent measuring system. The accuracy of the injected sample volume and the feasibility of multiple sample injection are evaluated based on the experiment results.

We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1-38 kbp) and large DNA fragments (lambda DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of lambda DNA and T4 DNA was successfully separated by a 380-microm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 microm long) was 1000-3000 (0.7 x 10(6)-2.1 x 10(6) plates/m). A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas lambda DNA kept a rather spherical conformation. We demonstrated that, even under a dc electric field, the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time.

Improvement in fuel consumption rate requires a reduction in vehicle weight. Research and development for materials substitution in the brake rotor, from the conventional cast iron to aluminum, has been undertaken. In this study, we developed aluminum metal matrix composites brake rotor and pads, which have equivalent braking effects and wear resistance to those of the conventional cast iron rotor, by optimization of the quantities and the particle diameter ratio of hard particles used for the rotor and the pad.

Using a convergent growth approach, a series of novel 5-aminolevulinic acid (ALA)-containing dendrimers have been synthesized. In these molecules, ALA residues are attached to the periphery by ester linkages, with amide bonds connecting the dendrons. Three first-generation dendrimers, bearing either 6 or 9 ALA residues, were synthesized by attachment of a tris(Boc-protected ALA)-containing wedge (1) to a di- or tripodent aromatic, or tripodent aliphatic core. Two second generation 18-ALA-containing dendrimers were also synthesized using a 3,3'-iminodipropionic acid spacer unit between wedge 1 and the aromatic core. These compounds differed only in the distance between the core and the linker unit. The Boc-protected dendrimers were deprotected using trifluoroacetic acid and isolated as their TFA salts. The potential of these ALA ester dendrimers as macromolecular prodrugs for photodynamic therapy has been demonstrated in the tumorigenic keratinocyte PAM 212 cell line.

Microchip electrophoresis for the short-time analysis of amino sugars is described. D-Glucosamine, D-galactosamine and their reduced forms were labeled with 4-nitro-2,1,3-benzoxadiazole 7-fluoride (NBD-F) at pH 6.0 and the fluorescent derivatives were purified on an octadecyl silica (ODS) gel plate. The derivatives were analyzed by electrophoresis on a microfabricated chip with a 33 mm long separation channel with argon laser-induced fluorescence detection. Under the established conditions, these amino sugar derivatives were well separated from each other within 60 s. Amino sugars of as small an amount as 0.5 fmol could be detected with a signal-to-noise (S/N) ratio of 3, and peak response showed good linearity between at least 0.8 and 8 fmol of samples with a relative standard deviation (RSD) of ca. 4%. This method was also applied to the analysis of amino sugar composition of O-linked glycans released from bovine submaxillary mucin with alkali in the presence of borohydride. The result of amino sugar composition analysis for individual O-glycans fractionated by high-performance liquid chromatography was quite useful for their identification.

To evaluate organic pollution in water, we did preliminarily studies on high-throughput characterization of organic pollution in water using microchip-based capillary electrophoresis (CE) with laseer-induced fluorescence (LIF) detection. The applied voltage was investigated to control the gated valve injection and CE separation for conventional cross type microchips using a self-made personal computer (PC)-based controller as the voltage supply. We obtained high-throughput data for the reproducible separation of fluorescein isothiocyanate (FITC)-labeled river-water samples using a zwitter-ion based buffer solution to avoid adsorption of the labeled sample onto the channel of a microchip made from quartz glass. We used real samples from the Hino River that flows into Lake Biwa, from ten sampling points and obtained several reproducible peaks in different separation patterns for each sample within 2 min. We successfully demonstrated high-throughput characterization of dissolved organic carbon (DOC) in environmental water using the microchip.

In order to improve detection sensitivity of UV absorption detection on a chip for capillary electrophoresis (CE), we have developped the microfabricated CE chips which have silicon shade at bonding interface. Although the CE chips are fabricated with a simple process, the Si shade is ideal structure as an optical slit. It was confirmed, using the CE chips, that the detection sensitivity of UV absorption detection was improves about ten times, and a DNA sample analysis with UV absorption detection was perfomed.

We have developed quartz microchips for electrophoresis and a linear imaging UV detector along with the microchip. The microchips have an optical slit, which cut off the stray light in order to improve the sensitivity of UV absorption detection on the chip, at the bonding interface. They have been successfully fabricated on synthesized quartz glass substrates using the hydrofluoric acid (HF) solution bonding method. The signal level of UV absorption detection was effectively improved by applying microchips with the "on-chip" optical slit. It is also possible to improve the signal-to-noise ratio by repetitive scanning of linear photodiode array located along the separation channel, and signal averaging during elimination of the potential. Furthermore, the analysis may be performed until the separation of the target component is complete, because the real-time migration pattern of each component in the sample can be seen just as in a slab-gel electrophoresis, thus enabling a shorter analysis time.

We investigated the microfabrication of PCR reaction vessel, capillary array electrophoresis (CAE), and laser-induced fluorescence detection cell using synchrotron radiation in polymethylmethacrylate (PMMA) substrates towards the development of novel technology for ultra-fast DNA analysis. Laser-induced fluorescence detection system for the chip has been developed and successfully applied to the imaging of a band for DNA fragment labeled by fluorescent dye during migrating in the array of microchannels to optimize the separation conditions for DNA analysis. Based on the imaging data, we can estimate that 10-s separation of some DNA fragments will be achievable using 10-mm separation channel on a chip.

In order to apply SiO2–SiO2 bonding with hydrofluoric acid (HF bonding) for micro-electro-mechanical systems (MEMS) fabrication, the optimal bonding conditions were examined under different temperature, HF concentration and bonding time. The necessary HF concentration and the necessary time for bonding are reduced by elevating the bonding temperature. The time for bonding was reduced from 24 h at room temperature to 30 min at 80°C, 60 min at 60°C under the conditions of 0.5 wt.% HF concentration and 1.3 MPa applied pressure. The bonding time is comparable to that of anodic bonding. Reliability of the HF bonding was confirmed by the results of temperature cyclic tests and thermal shock tests. A long term stability of the bonded sample was also evaluated by helium (He) leak detection. The measured He leak rate was less than 2.0×10−9 atm cm3/s which is much smaller than that calculated value through component materials of the sample. A novel quartz UV detection micro flow cell for chemical analysis having Si shade structure was fabricated by the HF bonding. The absorbance unit for UV absorption detection of the cell was improved remarkably.

Studies on SiO 2 –SiO 2 bonding with hydrofluoric acid (HF) are described. This method has a remarkable feature that bonding can be obtained at room temperature. Advantages of this method are low thermal damage, low residual stress and simplicity of the bonding process, which are expected for the packaging and assembly of micro-electro-mechanical systems (MEMS). The bond characteristics were measured under different bonding conditions of HF concentration, applied pressure, another chemicals for bonding and so on. The bond strength depends on the applied pressure during bonding. To achieve reliable bonding, HF concentration of higher than 0.5 wt.% and a large applied pressure of 1.3 MPa are required. The bonding is also observed using KOH solution in stead of HF. Transmission electron microscopy (TEM), secondary ion mass spectrometry (SIMS), radioactive isotope (RI) analysis and electron probe micro analysis (EPMA) were applied to evaluate the bonded interface. The results of these analysis indicated that an interlayer of a silicon oxide complex including hydrogen and fluorine atoms is formed between bonded SiO 2 to SiO 2 . The thickness of the interlayer depends strongly on the applied pressure during bonding. Large bond strength is obtained when the interlayer is thin. The bonding mechanism is expected when the SiO 2 at both surfaces is dissolved in HF solution, and that the interlayer, which is a binding layer, is formed between substrates by resolidification of dissolved silicon dioxide. Formation of the interlayer plays very important roles for the characteristics of HF-bonding.

A laser-induced fluorescence detection system coupled with a highly sensitive silicon-intensified target (SIT) camera is successfully applied to the imaging of a band for DNA fragment labeling by fluorescence dye in a microchannel, and to the visualizing of the separation process on a microfabricated chip. We demonstrated that an only 6 mm separation channel is sufficient for the separation of triplet repeat DNA fragment and DNA molecular marker within only 12 s. The separation using the microfabricated capillary electrophoresis device is confirmed to be at least 18 times faster than the same separation carried out by conventional capillary electrophoresis with 24.5 cm effective length. The use of a short capillary with 8.5 cm effective length is also efficient for fast separation of DNA; however, the microchip technology is even faster than capillary electrophoresis using a short capillary.

This paper reports on the development of a micro-potentiometric sensor based on external microelectrodes introduced into a microchip. We miniaturized reference and ion-selective electrodes (ISEs) and embedded them into a plastic (PDMS) microchip; the miniaturization of ISE was attained by using a monolithic capillary-based membrane. This sensor was applied to the detection of alkali ions (Na+, K+ and NH4+) in a microflow on the microg/L level.

Micromachined pyrolyzed polymer electrode is evaluated as 18F− deposition and recovery electrode toward positron emission tomography (PET) application for the first time. Electrical separation of 18F− from H 2 18O water has been studied for a preparation of a PET probe. A typical deposition and recovery cell of 18F− is illustrated in Fig. 1. We propose pyrolyzed polymer electrode instead of conventional bulk glassy carbon (GC) electrode in order to open the way for an integrated cell system with benefit of MEMS technology in replacement of bulk GC. It will be reported that pyrolyzed polymer electrode can deposit and recover 18F− (77% and 38% yield, respectively). Furthermore, we propose a composite of pyrolyzed polymer and GC particles to take advantage of both materials. This paper evaluates developed electrode for PET application and discuss its potential possibilities.

A new chemical approach to the preparation of [1-11C]fatty acids from [11C]HCN has been developed. Four straight chain [1-11C]fatty acids (C15C18) and one branched chain [1-11C]fatty acid (3-methylheptadecanoic acid) were produced by the reaction of alkyl bromides with 11CN− and subsequent hydrolysis. The synthesis time was 47–67 min after the end of bombardment, with radiochemical yields of 16–20 mCi (70–83%) for a series of straight chain [1-11C]fatty acids and 4–8 mCi (33–42%) for 3-methyl [1-11C]heptadecanoic acid (from 120 mCi of trapped [11C]HCN). This method has some advantages, such as no requirement of absolutely anhydrous solvent, easy 11C-labeling using an extremely small amount of substrate, and good reproducibility.

This article presents a real-time monitoring system for cellular analysis using micro total analysis systems technology. Time-resolved luminescence anisotropy analysis was adopted for real-time detection of small amounts of a target protein produced by a small number of cells. The system was tested by real-time monitoring of the antibody secretion by hybridomas. The cells were successfully cultivated in a micro-incubation chamber (240 nl) fabricated on a microchip. The quantification of the antibody was achieved using the Ru(II) complex-labeled Staphylococcus aureus protein A probe, which can bind specifically to the Fc region of the antibody. Using this system, we detected as little as 24 fmol of immunoglobulin G under physiological conditions without the bound/free separation protocol. We successfully achieved real-time and quantitative monitoring of small amounts of antibody production by approximately 200 hybridoma cells. This method could be applied to various cellular analyses using small numbers of cells.

In this paper, we describe a new method of local hydrophilic treatment of polymer micro-channel using surface discharge generated by a micro-electrode array. The method uses a pair of interdigitating electrodes embedded beneath a hydrophobic polymer (e.g. PDMS) micro-channel. When RF voltage is applied to the electrodes, surface discharge occurs inside the channel, and its inside surface is modified to be hydrophilic. Because the diffusion length of the generated plasma is only a few tens of micrometers, the treatment is local and selective. By using such a hydrophobic/hydrophilic patterning method, a droplet dispenser is developed. A straight PDMS channel having a side branch at the middle is fabricated, and the hydrophilic treatment is applied to the straight part. Due to capillary force, water solution fed from one end of the channel fills the entire straight channel, and when air is fed from the side, the liquid is divided into two parts, one ejected from the outlet. The ejection releases the pressure, and the channel is refilled, ready for the next ejection. The process produces a series of droplets, whose volume is precisely determined by the volume of the hydrophilic channel.

Studies on SiO/sub 2/-SiO/sub 2/ bonding with hydrofluoric acid (HF) are described. This method has a remarkable feature that bonding can be obtained at room temperature. Advantages of this method are low thermal damage, low residual stress and simplicity of the bonding process, which are expected for the packaging and assembly of MEMS. The bond characteristics were measured under different bonding conditions of HF concentration, pressure, chemicals and so on. The bond strength depends on the applied pressure during bonding. HF concentration can be reduced to 0.1%. The bonding is also observed using KOH solution instead of HF. TEM, SIMS, RI and EPMA were applied to evaluate the bonded interface. From the TEM results, an interlayer is formed between SiO/sub 2/-SiO/sub 2/. The thickness of the interlayer depends strongly on the applied pressure during bonding. The SIMS results showed that hydrogen and fluorine partially exist in the interlayer. Considering the result of the RI analysis, surplus HF solution is squeezed out from the interface as the bonding progress. From these results, both surfaces of the SiO/sub 2/ are solved by HF and an interlayer, which is a binding layer, is formed. Formation of the interlayer plays a very important role for the characteristics of HF-bonding.

Capillary electrophoresis (CE) devices have been fabricated in a small size on quartz or glass substrates. To fabricate a quartz device, we developed a novel bonding method with HF solution. HF-bonding was carried out with 1% HF solution with a pressure of /spl sim/40 gf/cm/sup 2/. With an evaluation of the new bonding method, we confirm that the method ensures enough strength, air tightness and optical property. An advantage of this method is simplicity of bonding process in comparison with a conventional thermal bonding. Using the glass /spl mu/CE devices, we obtained the original device design and process procedure through our experiments. It was proved that /spl mu/CE chip permits to apply higher electric power in comparison with the conventional CE system. We show that our design permits to perform quantitative sample injection (several 10 pl or less), effective separation (the number of theoretical plates is over 2500) and high speed analysis (over 10 times as high as the conventional CE system). These features promise an advantage of /spl mu/CE devices over conventional analytical equipments.

Electrophoresis features of large DNA in dense pillar array structures were studied. The sieving structures were all-quartz-made, 200 – 500nm in diameter, 5 jim in height, and fabricated by dry etching process employing high selective Ni mask. Electrophoresis velocities of DNA in the structure were measured by a fluorescence microscope. The separation ability of T4 and lambda in the pillar region was dependent on the pillar size. The ability was significantly enhanced at the boundary where the DNA entered into the dense pillar region from free solution region. These results shows the importance of pillar pattern design for the DNA separation, and the possibility of high performance separation by optimizing the pillar size and the boundary effect.

We have proposed a field-inversion electrophoresis on a microchip for a shorter effective length, and investigated the external frequency for the DNA analysis based on the field-inversion electrophoresis device. By using the optimized frequency, we demonstrated that the field-inversion electrophoresis has great potentials for the separation of DNA fragments with shorter effective length.

The effects of heat treatment during fabrication process and NaOH treatment after fabrication on surface silanol (Si-OH) concentration profile are discussed. FTIR-ATR (Fourier transform Infrared Spectroscopy - Attenuated Total Reflection) method is applied to measure the surface OH concentration of materials. The result revealed that FTIR-ATR can be a simple solution for estimation of OH concentration on the glass surface.

We have developed novel μ-CE chips with “on chip” optical slit which cut off the stray light in order to improve the sensitivity of the optical absorption detection on μ-CE chips. Our chips which have an optical slit at the bonding interface have been successfully fabricated on synthesized quartz glass substrates using HF (hydrofluoric acid solution) bonding method [1],[2]. The signal level of UV absorption detection was improved about ten times by applying the “on chip” optical slit compared with that of conventional µ-CE chips.

Fabrication method and characteristics of micro capillary electrophoresis (μ-CE) chips fabricated on synthetic fused silica (quartz glass) substrates are described. A novel room temperature bonding method with hydrofluoric acid solution is applied [1], [2]. Advantages of this method are low thermal damage, low residual stress due to the room temperature bonding and simplicity of the bonding process which are expected for the assembly of microfluidic devices. The migration time variations are investigated to confirm the reproducibility of the fabricated μ-CE chips.

The LIGA (Lithographie Galvanoformung Abformung) process using synchrotron radiation lithography is applied to the microfabrication of capillary array electrophoresis (CAE) device. Laser-induced fluorescence detection system for the CAE device has been constructed by the modification of laser confocal fluorescence microscopy. DNA molecules were detected during migrating in the microchannels filled with polymer separation matrices under electric field to optimize the separation conditions for DNA analysis. Based on this observation, we demonstrated that microfabricated CAE device is realized the fast separation of DNA.

This paper describes the cultivation of primary cells in a microchamber and the real-time monitoring of small amounts of antibody secretion after the introduction of a minute amount of stimulus by a microinjector using time-resolved fluorescence anisotropy analysis.

A 4-vane-type Radio-Frequency Quadrupole (RFQ) accelerator system for MeV ion implantation has been constructed and ion beams of boron and nitrogen have been accelerated successfully up to an energy of 1.01 and 1.22 MeV, respectively. The acceleration of phosphorus is now ongoing. The design was performed with two computer codes called SUPERFISH and PARMTEQ. The energy of the accelerated ions was measured by Rutherford backscattering spectroscopy. The obtained values agreed well with the designed ones. Thus we have confirmed the validity of our design and have found the possibility that the present RFQ will break through the production-use difficulty of MeV ion implantation.

The presence of 2-deoxy-2-[ 18 F]fluoro- d -mannose ([ 18 F]FDM) in 2-deoxy-2-[ 18 F]fluoro- d -glucose ([ 18 F]FDG) prepared by the reaction of 3,4,6-tri- O -acetyl- d -glucal (TAG) with [ 18 F]acetyl hypofluorite ([ 18 F]CH 3 COOF) or [ 18 F]F 2 was quantified by radio HPLC analysis of reacetylated [ 18 F]FDG. The solvent effects on the stereoselectivity of the reaction of TAG with [ 18 F]CH 3 COOF were examined in six kinds of solvent. Reaction of TAG with [ 18 F]CH 3 COOF in Freon-11 results in the least contamination of [ 18 F]FDM (4–5%). The presence of [ 18 F]FDM in the [ 18 F]FDG prepared from TAG with [ 18 F]F 2 was also indicated, but by careful chromatographic separation of hexopyranosyl difluorides the amount was reduced to nearly that resulting from the [ 18 F]CH 3 COOF method.

The optimal conditions for the catalytic production of [11C]HCN from [11C]CO2 were investigated. [11C]CO2 was reduced to [11C]CH4 with H2 on Ni and then converted to [11C]HCN by reaction with NH3 on Pt in a radiochemical yield of more than 95% under the optimized conditions of an NH3 concentration of 5 vol%, a Pt furnace temperature of 920 degrees C, and a reaction gas flow rate of over 200 mL/min. Absorbers were used to remove O2 and H2O from the reaction gas. The synthesis of no-carrier-added [1-11C]amino acids from [11C]HCN via [11C]aminonitriles was successfully carried out. This method is suitable for automation of [1-11C]amino acid production.

To accommodate the increasing need for [1- 11 C]fatty acids for use in routine medical studies and the large amounts of radioactivity required, a more advanced automated synthesis system for the preparation of [1- 11 C]fatty acids has been constructed. The synthesis of [1- 11 C]fatty acids has been completely automated using a new separation unit (isobaric Sepaltor) for the extraction procedure, which had caused some difficulties with automation. The yield of [1- 11 C]palmitic acid was 20–30 mCi, and that of 3-methyl [1- 11 C]heptadecanoic acid was 2–7 mCi. The time required for the synthesis was less than 30 min from the start of the 11 CO 2 trapping. This system has been used for more than 20 preparations of [1- 11 C]palmitic acid and more than 20 preparations of 3-methyl [1- 11 C]heptadecanoic acid.