Toward million-fold sensitivity enhancement by sweeping in capillary electrophoresis combined with thermal lens microscopic detection using an interface chip.

This paper reports a thermal lens microscope (TLM) detection coupled with capillary electrophoresis (CE) by using an interface chip (IFChip) to achieve highly sensitive detection with high reproducibility. Fused silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel on the IFChip. In comparison with an on-capillary detection method in CE-TLM, ca. 10-fold improvements in the reproducibility for peak height were obtained by using IFChips. The detection limit of an azo dye was estimated to be 3.6 x 10(-7)M (100 ppb), which was above 100-times lower than that of conventional absorbance detection. Toward further improvement of the detectability for nonfluorescent compounds, on-line sample preconcentration by sweeping was applied to the CE-TLM using the IFChip. Due to the sweeping effect, 3900000-fold increase in the sensitivity was successfully achieved.

[Clinical evaluation of ECG at the onset subjective symptoms using real-time analysis electrocardiograph (event recorder)].

Examination of patient complaining of palpitation, chest pain and chest discomfort is usually performed by 12-lead electrocardiograph. However, the recording time is short and there are few opportunities to capture an ECG demonstrating conditions during subjective symptoms. To investigate the cause, we need to obtain an ECG during subjective symptoms. Thus, we frequently use a Holter ECG, which can be recorded for 24 hours. However, some patients have a low frequency of subjective symptoms, which may not appear during a 24-hour examination. We used a real-time electrocardiograph (Event Recorder CG-6106 made by Card Guard Scientific Survival Limited) as a monitor during subjective symptoms. Thereafter, ECG findings at the onset of subjective symptoms could be analyzed in 30 patients who did not have a clear cardiac disease. In this examination, arrhythmia was recorded in 25 of 30 cases. Although in these cases ECG during subjective symptoms could not be captured even when Holter examination was performed several times ECG during subjective symptoms was captured using an Event Recorder. This method using an Event Recorder is simple and convenient, moreover, is considered very useful for investigation of subjective symptoms. In the future, the use of an Event Recorder for heart-health-care in the daily life of healthy people and/or cardiac disease patient is highly anticipated.

[The assessment of arteriolosclerosis by serum lipids data, and carotid ultrasonography and pulse wave velocity, and its evaluation as a tool for preventive medicine: II. Carotid ultrasonography and pulse wave velocity for hyperlipemia and diabetes mellitus].

We studied the test results of carotid ultrasonography and pulse wave velocity against a sample of hyperlipemia and diabetes mellitus. Sixty four hyperlipemia samples (HL), 85 diabetes mellitus samples (DS), and 27 complicated samples (CS) were compared with 56 healthy samples (HS). Hyperlipemia samples were selected from cholesterol under 300 mg/dl, and neutral fat under 300 mg/dl. Diabetes mellitus samples were selected from fasting plasma glucose (FBS) under 200 mg/dl. Samples from severe conditions with various disease were excluded. Ratio over 1.1 mm intima-media thickness (IMT) was 0% in HS, 48% in HL, 40% in DS and 33% in CS. PWV value was max 1896cm/s in CS. There was no significant correlation within IMT, serum lipid(Total Cholesterol, Neutral Fat, HDL-Cholesterol, LDL-Cholesterol) and FBS. For early treatment or accurate diagnosis of arteriosclerosis in hyperlipemia or diabetes mellitus patients, who are at high risk of developing arteriosclerosis, to vital function tests (carotid ultrasonography and pulse wave velocity) should be performed, in addition to normal blood tests.

Optimization of an interface chip for coupling capillary electrophoresis with thermal lens microscopic detection.

This paper presents a capillary-to-microchip connection, which can be used as an interface for coupling capillary electrophoresis (CE) with a thermal lens microscope (TLM). It is difficult to directly apply TLM to samples in a capillary with a curved surface, and such an interface chip at the end of a CE separation column is needed for reliable TLM measurements. The dependence of the TLM signal intensity on the TLM detection point in the interface chip and the dependence of the theoretical plate number of CE separation on the channel dimensions of the interface chip were investigated and optimized with a mixture of 4-dimethylaminoazobenze-4'-sulfonyl (DABSYL)-derivatized amino acids (glycine, alanine, methionine, and proline) as a model sample. By using an optimized interface chip, theoretical plate numbers of DABSYL-glycine, -methionine, -alanine, and -proline were obtained to be 104000, 95000, 104000, and 95000, respectively.

An interface chip connection between capillary electrophoresis and thermal lens microscope.

A thermal lens microscope (TLM) detection of capillary electrophoresis (CE) utilizing microchip technology was developed. Fused-silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel in a microchip. The detection limit by TLM was estimated as 2.8 x 10(-7) absorbance by measuring pure water. The detection limit of derivatized amino acids determined by CE-TLM was estimated as 2.4 x 10(-8) M, which was 100 times lower than that of conventional absorbance detection.

Chemicofunctional membrane for integrated chemical processes on a microchip.

Here we report a design and synthesis of a chemically functional polymer membrane by an interfacial polycondensation reaction and multilayer flow inside a microchannel. Single and parallel dual-membrane structures are successfully prepared by using organic/aqueous two-layer flow and organic/aqueous/organic three-layer flow inside the microchannel followed by an interfacial polycondensation reaction. By using the inner-channel membrane, permeation of ammonia species through the inner-channel membrane is successfully achieved. Furthermore, horseradish peroxidase is immobilized on one side of the membrane surface to integrate the chemical transform function onto the inner-channel membrane. Here substrate permeation through the membrane and subsequent chemical transformation at the membrane surface are realized. The polymer membrane prepared inside the microchannel has an important role in ensuring stable contact of different phases such as gas/liquid or liquid/ liquid and the permeation of chemical species through the membrane. Furthermore, membrane surface modification chemistry allows chemical transformation of permeated chemical species. These methods are expected to lead to development of complicated and sophisticated chemical systems involving membrane permeation and chemical reactions.

Continuous-flow chemical processing on a microchip by combining microunit operations and a multiphase flow network.

A new design and construction methodology for integration of complicated chemical processing on a microchip was proposed. This methodology, continuous-flow chemical processing (CFCP), is based on a combination of microunit operations (MUOs) and a multiphase flow network. Chemical operations in microchannels, such as mixing, reaction, and extraction, were classified into several MUOs. The complete procedure for Co(II) wet analysis, including a chelating reaction, solvent extraction, and purification was decomposed into MUOs and reconstructed as CFCP on a microchip. Chemical reaction and molecular transport were realized in and between continuous liquid flows in a multiphase flow network, such as aqueous/aqueous, aqueous/organic, and aqueous/organic/aqueous flows. When the determination of Co(II) in an admixture of Cu(II) was carried out using this methodology, the determination limit (2sigma) was obtained as 18 nM, and the absolute amount of Co chelates detected was 0.13 zmol, that is, 78 chelates. The sample analysis time was faster than that of a conventional processing system. Moreover, troublesome operations such as phase separation and acid and alkali washing, all necessary for the conventional system, were simplified. The CFCP methodology proposed here can be applied to various on-chip applications.

Stabilization of liquid interface and control of two-phase confluence and separation in glass microchips by utilizing octadecylsilane modification of microchannels.

We demonstrated a liquid/liquid and a gas/liquid two-phase crossing flow in glass microchips. A 250-microm-wide microchannel for aqueous-phase flow was fabricated on a top glass plate. Then, as a way to utilize the surface energy difference for stable phase confluence and separation, a 250-microm-wide microchannel for organic-phase (or gas-phase) flow was fabricated on a bottom glass plate and the wall was chemically modified by octadecylsilane (ODS) group. The top and bottom plates were sealed only by pressure. A microchannel pattern was designed so that the two phases made contact at the crossing point of the straight microchannels. The crossing point was observed with an optical microscope. Results showed that the ODS modification of the microchannel wall clearly improved stability of the interface between the two fluids. Pressure difference between fluids was measured and the interface of water and nitrobenzene was stable for the pressure difference from +300 Pa to -200 Pa. The pressure drop in a countercurrent flow configuration was also estimated, and the pressure difference required to realize the counter current flow was less than the allowable pressure range. Finally, we discussed the advantages of utilizing this approach.

Three-layer flow membrane system on a microchip for investigation of molecular transport.

A stable three-layer flow system, water/organic solvent/water, has been successfully applied for the first time in a microchannel to get rapid transport through an organic liquid membrane. In the continuous laminar flow region, the analyte (methyl red) was rapidly extracted across the microchannel from the donor to the acceptor phase through the organic solvent phase (cyclohexane). Thermal lens microscopy was used to monitor the process. The thickness of the organic phase, sandwiched by the two aqueous phases, was approximately 64 microm, and it was considered as a thin liquid organic membrane. Permeability studies showed the effects of molecular diffusion, layer thickness, and organic solvent-water partition coefficient on the molecular transport. In the microchip, complete equilibration was achieved in several seconds, in contrast to a conventionally used apparatus, where it takes tens of minutes. The thickness of the organic and aqueous boundary layers was defined as equal to the microchannel dimensions, and the organic solvent-water partition coefficient was determined on a microchip using the liquid/liquid extraction system. Experimental data on molecular transport across the organic membrane were in agreement with the calculated permeability based on the three-compartment water/organic solvent/water model. This kind of experiment can be performed only in a microspace, and the system can be considered as a potential biological membrane for future in vitro study of drug transport.

Glass microchip with three-dimensional microchannel network for 2 x 2 parallel synthesis.

An integrated multireactor system for 2 x 2 parallel organic synthesis has been developed on a single glass microchip. Three-dimensional channel circuits in the chip were fabricated by laminating three glass plate layers. The fabrication method is a straightforward extension of the conventional one, and topological equivalence for any three-dimensional circuits can be constructed easily with it. 2 x 2 phase-transfer amide formation reactions, which constitute a simple model for combinatorial synthesis, were successfully carried out on the microchip, and the integrity of the three-dimensional circuits was confirmed. Combinatorial chemistry with multi-microreactors, in conjunction with a high-throughput screening method based on micro-TAS technologies, is expected to provide an efficient tool for drug discovery.

Pile-up glass microreactor.

We made a 'pile-up' microreactor in which ten levels of microchannel circuits were integrated to form a single glass entity. Solutions were distributed to each layer via cylindrical holes with a diameter much larger than that of the microchannel. Fabrication of the pile-up reactor was completed using only conventional photolithography, wet etching and thermal bonding techniques, and no special facilities or instruments were required. An amide formation reaction between amine in aqueous solution and acid chloride in organic solution was carried out using the pile-up reactor. The yield of the amide formation reaction is dependent on the size of the specific surface area between the two solutions, and the small space inside the microchannels is good for acquiring a large specific surface area without any stirring processes. The maximum throughput for the ten-layered pile-up reactor was ten times larger than that of a single-layered one, yet the reaction yield was still high. Productivity of the pile-up reactor for the reaction was as high as on a gram per hour scale. This value suggests that many conventional plants producing fine chemicals can be replaced by microreactors through the numbering-up technology.