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A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen-enzyme-conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10-1000 ng mL(-1) linear range, is reported.

We report an effective and a quantitative analysis method for one of pharmaceuticals, sulpiride, in river water by online solid phase extraction (SPE) connected with liquid chromatography–mass spectrometry (LC–MS) using a molecularly imprinted polymer as a preconcentration medium. The polymer prepared with a pseudo template molecule showed the selective retention ability based on the interval recognition of functional groups in sulpiride. Also, the imprinted polymer provided an effective concentration of a trace level of sulpiride in offline SPE with dual washing processes using water and acetonitrile, although another imprinted polymer prepared by an authentic method using sulpiride and methacrylic acid as a template and a functional monomer, respectively, showed the selective adsorption only in organic solvents. Furthermore, we employed the imprinted polymer as the preconcentration column of online SPE-LC–MS and the results supposed that the proposed system allowed the quantitative analysis of sulpiride with high sensitivity and recovery (10 ng/L at 96%). Additionally, the determination of sulpiride in real river water without an additional spiking was effectively achieved by the system.

This short communication describes a newly developed open-tubular capillary which was coated with C60-fullerene by a covalent bonding via a photo/thermal active agent. We utilized perfluorophenyl azide (PFPA) as an active agent, which can be used for the "photo click" coupling of the carbon materials. The inner wall of a fused silica capillary was treated with silane conjugated PFPA, and then C60-fullerene was chemically modified by a photoreaction or a thermal reaction. Through evaluations of the capillaries by liquid chromatography, the separation characteristics of three polycyclic aromatic hydrocarbons (PAHs) were confirmed in both capillaries. With comparison of the retention behavior to a commonly used C18 column, the prepared capillaries showed the specific separation ability based on the π-π stacking by C60-fullerene. The capillary prepared by the thermal reaction provided the base line separation of phenanthrene, triphenylene, and benz[a]pyrene within 3min at 18.8cm capillary length.

This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1–2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

Here, we developed a highly sensitive label-free plasmonic crystal (PC). The PC is composed of two types of nanoperiodic metal structures, nanodiscs and nanohole arrays, fabricated simultaneously by nanoimprint lithography using a nanostructured polymer mold. The PC absorbed light at specific wavelengths based on localized surface plasmon resonance (LSPR). The strongly enhanced electric field was excited by the combined structures of nanodiscs and nanohole arrays; thus, highly sensitive biosensing was possible. The LSPR-based optical characteristics of the PC were analyzed by finite-difference time-domain simulation; the structure (metal layer thickness) was optimized to respond to changes in the surrounding refractive index with high sensitivity. PC-based biosensor chips were prepared by immobilizing anti-human immunoglobulin G, which was successfully detected in the 200 pg/mL to 200 ng/mL range. Our approach introduces an easy and rapid process allowing large-area fabrication of PCs, resulting in a highly sensitive label-free biosensor device.

A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow pump (LVSEP) and field-amplified sample injection (FASI), was investigated in CE to achieve highly sensitive oligosaccharide analysis. In CE with LVSEP-FASI, analytes injected throughout the capillary were concentrated on the basis of LVSEP, followed by an electrokinetic introduction of concentrated analytes from the inlet vial by the FASI mechanism. After switching the inlet vial solution from the sample to running buffer, the concentrated analytes were then separated by CZE. In the present LVSEP-FASI-CZE, pressure was applied to the capillary inlet until the inlet vial solution was exchanged. The applied pressure generated a counterflow against the EOF. It kept the stacked sample zone within the capillary, minimizing loss of concentrated analytes. Fluorescein was first analyzed by LVSEP-FASI-CZE to optimize preconcentration condition. Up to 110 000-fold sensitivity increase was obtained with 200 μL of sample, compared to normal CZE with sample injection of 0.3 psi for 3 s (ca. 1.7 nL). From the results, the pressure application improved the efficiency of the FASI-mode concentration significantly at total concentration time longer than 10 min. In the analysis of maltoheptaose, a 10 000-fold sensitivity increase was achieved, which is the highest concentration efficiency ever reported in CE of oligosaccharides. The relative standard deviations of the detection time and peak height were 2.4 and 11%, respectively. In the analysis of glucose oligomer, up to 8600-fold sensitivity increases were achieved without reducing the separation performance of conventional CZE.

This review highlights recent developments and applications of on-line sample preconcentration techniques to enhance the detection sensitivity in microchip electrophoresis (MCE); references are mainly from 2008 and later. Among various developed techniques, we focus on the sample preconcentration based on the changes in the migration velocity of analytes in two or three discontinuous solutions system, since they can provide the sensitivity enhancement with relatively easy experimental procedures and short analysis times. The characteristic features of the on-line sample preconcentration applied to microchip electrophoresis (MCE) are presented, categorized on the basis of "field strength-" or "chemically" induced changes in the migration velocity. The preconcentration techniques utilizing field strength-induced changes in the velocity include field-amplified sample stacking, isotachophoresis and transient-isotachophoresis, whereas those based on chemically induced changes in the velocity are sweeping, transient-trapping and dynamic pH junction.

Cerebral vasoreactivity is an important indicator of the reserve capacity of the cerebral circulation. To make a quantitative analysis of cerebral vasoreactivity in individual major arterial territories, we evaluated the response to acetazolamide using three-dimensional time-of-flight magnetic resonance angiography. We examined 10 healthy volunteers and 6 patients with unilateral stenosis of the middle cerebral artery by a 1.5-T superconducting magnetic resonance imaging system. After a baseline vascular image was obtained, each subject received 17 mg/kg IV of acetazolamide; a second scan was performed 20 minutes later. Using a generally available personal computer and image analysis software, we measured the areas of the individual major arteries on collapsed axial vascular images and then calculated the vasoreactivity. The average vasoreactivity of individual major cerebral arterial territories in the healthy volunteers was as follows: anterior cerebral artery complex, 33%; right middle cerebral artery, 71%; left middle cerebral artery, 74%; right posterior cerebral artery, 68%; and left posterior cerebral artery, 68%. In the patient group, the vasoreactivity of the stenotic middle cerebral arteries was significantly smaller than that of the nonstenotic arteries (P < .05). In addition, the nonstenotic middle cerebral arteries showed significantly less vasoreactivity than the right arteries of the healthy volunteers (P < .01). Three-dimensional time-of-flight magnetic resonance angiography can be used to quantitatively evaluate acetazolamide-induced vasoreactivity in individual major cerebral arterial territories.

To obtain high sensitivity in capillary electrophoresis of oligosaccharide without reducing the high resolution with an easy experimental procedure, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was investigated. As a fundamental study, effect of the conductivity of a sample solution in LVSEP was examined. It was revealed that LVSEP was successfully carried out even in using a sample solution with the ionic strength of 150 μM and the conductivity ratio of 20, indicating a good applicability of LVSEP to the analysis of real samples containing salts. When glucose oligomer was analyzed as a model sample in LVSEP-capillary zone electrophoresis (CZE), all peaks were well resolved with decreasing only 5% of the peak-to-peak distance, which suggested 95% of the whole capillary could be used for the effective separation. In the analysis of maltoheptaose, a good calibration line with correlation coefficient of 0.9995 was obtained. The limit of detection was estimated as 2 pM, which was 500-fold lower than that in the conventional CZE. N-linked glycans released from three glycoproteins, bovine ribonuclease B, bovine fetuin, and human α(1)-acid glycoprotein were also analyzed by LVSEP-CZE. By the sample purification with a gel filtration column, further sample dilution to reduce the sample conductivity for LVSEP was not needed. All glycan samples were well concentrated and separated with up to a 770-fold sensitivity increase. The run-to-run repeatabilities of the migration time, peak height, and peak area were good with relative standard deviations of 0.1-1.3%, 1.2-1.7%, and 2.8-4.9%, respectively.

Silent brain infarction is fairly common in the elderly, but predictive factors have not been definitively established. This study focuses attention on ischemic heart disease and cerebrovascular risk factors about the frequency of silent brain infarction.The existence of silent brain infarction, the extent of coronary artery stenosis, and cerebrovascular risk factors of consecutive 92 case series with suspected ischemic heart disease were surveyed.A hospital for patients with ischemic heart disease.Ninety-two consecutive Japanese patients with suspected ischemic heart disease were recruited.All subjects were evaluated for coronary atherosclerosis (number of coronary arteries with significant stenosis and Gensini score), the number of silent brain infarctions detected by computed tomography, the extent of carotid atherosclerosis as determined by B-mode ultrasonography, and cerebrovascular risk factors.Patients with silent cerebral infarctions were older (66.2 +/- 10.4 years) than those without such events (60.1 +/- 8.8 years) (P.01). The extent of coronary atherosclerosis in patients with silent cerebral infarctions was significantly greater than in those without such events after adjustment for the effect of age (P.001). The extent of carotid atherosclerosis and the percentages of individuals with hypertension, diabetes mellitus, a smoking habit, hypercholesterolemia, hypertriglyceridemia, and a low serum high-density lipoprotein cholesterol level did not differ between the groups with and without silent brain infarction. The frequency of silent brain infarction increased with the severity of coronary stenosis.Coronary atherosclerosis and age were important risk factors for silent brain infarction.

Transient trapping (tr-trapping) was developed as one of the on-line sample preconcentration techniques to improve a low concentration-sensitivity in microchip electrophoresis (MCE), providing highly effective preconcentration and separation based on the trap-and-release mechanism. However, a poor performance to hydrophilic analytes limited the applicability of tr-trapping. To overcome this drawback, tr-trapping was combined with a sample labeling using a hydrophobic reagent in CE. Three commercially available fluorescent dyes, fluorescein isothiocyanate, succinimidyl esters of Alexa Fluor 488 and BODIPY FL-X, were tested as derivatization reagents to increase the hydrophobicity of amino acids (AAs) that were undetectable due to no fluorescence/UV-absorbance. As a result, it was confirmed that BODIPY labeling allowed various AAs to be analyzed in tr-trapping-micellar electrokinetic chromatography (tr-trapping-MEKC) by the increase in the hydrophobicity. In tr-trapping-MEKC, both the improvement of the resolution and 106-125-fold enhancements of the detectability of labeled AAs were achieved relative to the conventional capillary zone electrophoresis. The limit of detection of labeled phenylalanine was improved from 800 to 5 pM by applying tr-trapping-MEKC. In tr-trapping-microchip MEKC, furthermore, an 80-160-fold enhancement of the peak intensity and a baseline separation was also achieved within 30 s. These results clearly demonstrate that the tr-trapping technique with hydrophobic labeling will make CE/MCE more sensitive for various analytes.

The applicability of an online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP), to microchip zone electrophoresis (MCZE) for the analysis of oligosaccharides was investigated. Since the sample stacking and separation proceeded continuously without polarity switching in LVSEP, a single "straight" channel microchip could be employed. In the MCZE analysis of oligosaccharides, sample adsorption onto the channel surface should be suppressed, so the straight microchannel was modified with poly(vinyl alcohol) (PVA). So far, the mechanism of LVSEP in the polymer-coated capillary or microchannel has not been reported, and thus, the LVSEP process in the PVA-coated channel was investigated by fluorescence imaging. Although it is well-known that the PVA coating can suppress the electroosmotic flow (EOF), an enhanced EOF with a mobility of 4.4 x 10(-4) cm(2)/(V x s) was observed in a low ionic strength sample solution. It was revealed that such temporarily enhanced EOF in the sample zone worked as the driving force to remove the sample matrix in LVSEP. To evaluate the analytical performance of LVSEP-MCZE, oligosaccharides were analyzed in the PVA-coated straight channel. As a result, both the glucose ladder and oligosaccharides obtained from bovine ribonuclease B were well enriched and separated with up to 2200-2900-fold sensitivity enhancement compared to those in a conventional MCZE analysis. The run-to-run repeatabilities of the migration time and peak height were good with relative standard deviations of 1.1% and 7.2%, respectively, which were better than those of normal MCZE. By applying the LVSEP technique to MCZE, a complicated voltage program for fluidic control could be simplified from four channels for two steps to two channels for one step.

A simple method for a chemical surface modification of poly(methyl methacrylate) (PMMA) microchips with amino-poly(ethyleneglycol) (PEG–NH 2 ) by nucleophilic addition–elimination reaction was developed to improve the separation efficiency and analytical reproducibility in a microchip electrophoresis (MCE) analysis of biomolecules such as proteins and enantiomers. In our procedure, the PEG chains were robustly immobilized only by introducing an aqueous solution of PEG–NH 2 into the PMMA microchannel. The electroosmotic mobilities on the modified chips remained almost constant during 35 days with 37 runs without any recoating. The PEG–NH 2 modified chip provided a fast, reproducible, efficient MCE separation of proteins with a wide variety of isoelectric points within 15 s. Furthermore, the application of the modified chip to affinity electrophoresis using bovine serum albumin gave a good chiral separation of amino acids.

In order to provide an insight into the basic nature of ischemic brain injury, we sequentially studied cerebral blood flow with [99mTc]hexamethylpropyleneamine oxime single photon emission computed tomography (CBF imaging) in a patient with diffuse brain ischemia due'to prolonged cardiac arrest. On the 10th postarrest day, concentrated blood flow over superior-medial portion of the occipital lobe was demonstrated. On the 18th postarrest day, the same region became high density on a CT scan, while the concentrated flow on the CBF imaging had diminished. Thus, an abnormal cerebral blood flow (CBF) pattern preceded the density change on CT scan. On the 23rd postarrest day, remarkably concentrated flow over the brainstem was demonstrated. This might have illustrated the reduced metabolic demand of the damaged tissue over the cerebral and cerebellar hemispheres with relative preservation of brainstem perfusion. In patients with diffuse brain ischemia, a CBF imaging may be a useful tool for clarifying pathological process and prognosis.

Along with the recent changes in lifestyle in Japan, the incidence of coronary artery disease has increased while the incidence of stroke appears to be decreasing. We investigated the relation between the progression of carotid atherosclerosis and the severity of coronary artery disease in the Japanese population. The 2-year change in extracranial carotid atherosclerosis in 50 Japanese patients who underwent coronary angiography was evaluated using carotid echotomography. To quantify the extent of carotid atherosclerosis, the maximal thickness measurements of all plaques were summed for an individual plaque score, except for new plaques found on reexamination. Carotid disease progression was evaluated by the sum of plaque score change and the thickness of the new plaque found on reexamination. The plaque score changed by -3.2 to 10.1 mm (mean +/- SD, 1.06 +/- 2.42 mm). The extent of coronary atherosclerosis (p less than 0.02) and serum total cholesterol level (p less than 0.01) were different between the progressing (n = 17) and the nonprogressing (n = 30) groups of carotid atherosclerosis when the progressing group included the patients with a delta plaque score of greater than or equal to 1.0 mm. Neither age, serum triglyceride level, serum high-density lipoprotein cholesterol level, pack-years of smoking, percentage of smokers, percentage of hypertensive patients, nor percentage of diabetic patients was different between the two groups. Carotid disease progression was significantly higher in patients with three-vessel coronary disease than in patients without significant coronary artery disease (p less than 0.005). There was a significant positive linear correlation between carotid disease progression and Gensini's coronary artery disease score (R = 0.411, p less than 0.005). Our data showed that severe coronary artery disease and a high serum total cholesterol level were strong predictors for carotid disease progression in Japanese patients with high rates of coronary artery disease.

A forty-four-year-old woman with Takayasu's arteritis and involvement of the aortic arch and its main branches complained of precordial pain on effort. Exercise electrocardiograms revealed significant ST segment depression in leads II, III, aVF, and V4-6. Coronary arteriograms demonstrated no stenosis. However, the right coronary arteriogram revealed collateral circulation arising from the sinus node artery to the bilateral vertebral arteries and the left internal carotid artery. The collateral circulation was considered to be an important route of blood flow supply to the brain and, at the same time, a cause of coro nary steal syndrome and, consequently, of angina pectoris.

In this study, the authors examined relations between coronary and carotid atherosclerosis and between coronary atherosclerosis and silent cerebral infarc tion. They ascertained the risk factors for carotid atherosclerosis and silent cerebral infarction complicating coronary heart disease (CHD) in 77 Japanese subjects. As coronary atherosclerosis progressed, the carotid ultrasonographic score and the brain computed tomographic score increased. Multivariate analy ses showed that the significant and independent risk factors for carotid athero sclerosis in patients with CHD were age (p < 0.01) and apolipoprotein (apo) B (p < 0.05) and the factors for silent cerebral infarction were age (p < 0.05) and hypertension (p < 0.05). Their study confirms a positive relation between coronary atherosclerosis and carotid atherosclerosis and between coronary atherosclerosis and silent ce rebral infarction in patients with CHD. Their data suggest that carotid athero sclerosis should be looked for in patients with CHD who are old and have a high value of apo B, and silent cerebral infarction should be looked for in those who are old and have hypertension, to prevent complicating symptomatic cerebral vascular disease (CVD). If severe carotid atherosclerosis or silent cerebral in farction are detected, antithrombotic medication should be given.

健常例, 高血圧症, 高脂血症, 糖尿病, 一過性脳虚血発作, 脳梗塞からなる183例を対象としてリニヤ走査電子スキャナー, 5MHz探触子を用いて頚動脈超音波断層法を施行した.本法による血管狭窄度と血管写によるそれとの間には有意正相関が成立し, 本法の血管写を対照とした診断正答率は閉塞67%, 健常82%, 狭窄100%であった.本法による頚動脈病変検出率は健常例7%, 高血圧症45%, 高脂血症45%, 糖尿病50%, 一過性脳虚血発作73%, 脳梗塞67%であった.atheromatous plaqueの形態を8型に分類し各疾患での出現頻度を検討すると健常例, 高血圧症では微小型, 糖尿病では広基底性表面平滑型, 高脂血症では半円形表面平滑型が多くこれら非脳血管障害例では微小型, 表面平滑型が主体を占めるのに対し脳血管障害例の病因側頚動脈では閉塞型が最も多く表面不整型および結節型の増加がみられた.非病因側頚動脈では表面平滑型が主体を占めた.

CO2変化時の脳血管反応性を経頭蓋超音波ドプラ法による中大脳動脈血流速 (MCAV) 測定ならびにアルゴン法による脳血流量測定の両者で計測しその比較検討をおこなった.過呼吸, 3%, 5%, 7%CO2吸入による25.0mmHgから51.7mmHgまでのPaCO2変動に対しMCAVは34.5cm/sec.から96.8cm/sec., CBFは27.3ml/100g/min.から86.2ml/100g/min.まで変化した.MCAV測定による脳血管反応性は0.0391±0.0062 (Mean±S.D.) でありCBF測定から算出した脳血管反応性は0.0406±0.0060であり両者間には有意の正相関が成立した (r=0.890, p<