Micro/extended-nano sampling interface from a living single cell.

Single-cell analysis is of increasing importance in many fields, but is challenging due to the ultra-small volumes (picoliters) of single cells. Indeed, analysis of a specific analyte might require the analysis of a single molecule or several molecules. Analytical processes usually include sampling, chemical processing, and detection. Although several papers have reported chemical processing and detection methods for single cells, a sampling method compatible with maintaining the viability of a single cell during sampling has yet to be developed. Here, we propose a femtoliter sampling method from a living single cell using micro/nanofluidic device technology. The sampling of 39 fL of cytoplasm from a single human aortic endothelial cell was demonstrated and its viability after sampling was confirmed.

Detection of zeptomole quantities of nonfluorescent molecules in a 10(1) nm nanochannel by thermal lens microscopy.

Nanofluidics in 10(1) nm space, whose scale is comparable to the electric double layer (EDL) and the size of biomolecules, promises novel functional analytical devices. However, the detection, which is indispensable to the integrated chemical system, is still challenging in such an ultra-small space. Previously, we reported a differential interference contrast thermal lens microscope (DIC-TLM) based on the photothermal interferometry principle and succeeded in detection of nonfluorescent molecules in 10(2) nm spaces. However, the thermal diffusion into substrates becomes a problem for detection in 10(1) nm spaces. The DIC-TLM signals are significantly cancelled out in spaces much smaller than the confocal length (∼10(2) nm), which makes DIC-TLM detection in 10(1) nm space quite difficult. To overcome this problem, we propose a new channel structure that benefits the thermal diffusion and sensitivity enhancement in DIC-TLM by employing TiO2 as a substrate material for compensating the signal cancellation effect. As a result, DIC-TLM detection of nonfluorescent molecules (800 molecules) was successfully demonstrated in a nanochannel with a depth of 50 nm. The developed detection method will contribute to the functional nanofluidic devices utilizing 10(1) nm spaces.

Integration of a wet analysis system on a glass chip: determination of Co(ii) as 2-nitroso-1-naphthol chelates by solvent extraction and thermal lens microscopy.

The integration of a wet analysis system on a glass chip was demonstrated and determination of Co(II) was performed using this system. The Co(II) was extracted into m-xylene from aqueous solution as 2-nitroso-1-naphthol chelates, and colorimetric determination of the m-xylene phase was applied by a thermal lens microscope. The integration of the chemical operation procedures shown here leads to a considerable reduction in analyzing time. The time for extraction in the integrated system, 10 min, was about tenfold shorter than a conventional system using a separatory funnel and mechanical shaker. Moreover, troublesome operations such as phase separation necessary for the conventional system could be omitted. The determination of Co(II) in the range 2 x 10(-7)-1 x 10(-8) M, which was estimated to be 0.072-1.44 zmol, was achieved.

Integration of a microextraction system on a glass chip: ion-pair solvent extraction of Fe(II) with 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid and tri-n-octylmethylammonium chloride

An ion-pair solvent extraction was performed in a microchannel fabricated in a quartz glass chip. the aqueous solution of Fe-bathophenanthrolinedisulfonic acid complex and the chloroform solution of tri-n-octylmethylammonium chloride were introduced into the microchannel, and a parallel two-phase laminar flow was formed. The ion-pair product extracted in chloroform was monitored by the thermal lens microscope. The ion-pair product was gradually extracted from aqueous solution into chloroform when the flow was very slow or stopped, while nothing was extracted into chloroform when the flow was fast. The time for extraction in the present 250 microns microchannel, 45 s, roughly coincided with the molecular diffusion time, and the extraction time was at least 1 order shorter compared with the ordinary extraction time using a separatory funnel and mechanical shaking. The microspace in the microchannel was characterized by the large specific interface area and short diffusion distance, and these characteristics may contribute to highly efficient extraction without mechanical shaking. The success of this molecular transport may lead to the integration of more complicated separation and chemical operations on a microchip and more applications.

Quantitation of IgE and carcinoembryonic antigen (CEA) by optical beam deflection (OBD) measurement of dot-immunobinding assay patterns visualized by an ELISA technique.

Dot-immunobinding assays of IgE and CEA were performed by a conventional dot-ELISA technique with diaminobenzidine staining, and the quantitative results were compared by densitometry and a new, spectroscopic, optical beam deflection (OBD) method using the same membrane. It was possible with the OBD method to detect quantities of these substances at least ten times smaller than with densitometry. Better intra-assay reproducibility for IgE and CEA measurements was obtained by the OBD method. The measurable ranges of the OBD method was broader than that of densitometry, because dark bands caused OBD in proportion to their color densities. When the dot-immunobinding assay with OBD measurement for CEA was also compared with a microtube ELISA using biotin-avidin conjugates, the sensitivities and reproducibilities of the two methods were found to be similar, with a correlation coefficient of 0.991.

Antiaggregatory, antithrombotic effects of MS-180, a novel platelet glycoprotein IIb/IIIa receptor antagonist.

The antiaggregatory and antithrombotic effects of (S)-(-)-ethyl[6-[4-(morpholinoformimidoyl)benzamido]-3,4-dihydro-2 H-1-benzo-pyran-3-yl]acetate hydrochloride (MS-180), a novel platelet glycoprotein IIb/IIIa receptor antagonist, were investigated. Ma-HCl, (S)-(-)-[6-[4-(Morpholinoformimidoyl)benzamido]-3,4-dihydro-2H-1-b enzopyran-3-yl]acetic acid hydrochloride, the hydrochloride salt of Ma (active metabolite), inhibited the binding of fibrinogen to immobilized human glycoprotein IIb/III receptor with an IC50 value of 0.12+/-0.03 nM without affecting binding to either fibronectin or vitronectin receptors. In anesthetized guinea pigs, intraduodenal administration of MS-180 caused dose-dependent inhibition of both ADP- and collagen-induced ex vivo platelet aggregation. At the same dosages, occluded thrombus formation and platelet release reactions were also markedly suppressed. In anesthetized dogs, the bleeding time was prolonged slightly even when submaximal inhibition (< 90%) of ex vivo platelet aggregation was achieved following i.v. administration of Ma-HCl. Aspirin (100 mg/kg) prolonged the bleeding time to the same extent as MS-180 (1 mg/kg), although it suppressed only collagen-induced platelet aggregation. Therefore, MS-180 may be clinically useful for the treatment of thrombotic diseases.

Integration of a microextraction system solvent extraction of a Co-2-nitroso-5-dimethylaminophenol complex on a microchip.

A newly designed microchannel for solvent extraction was fabricated in a quartz glass chip and applied to solvent extraction of a Co-2-nitroso-5-dimethylaminophenol complex. The aqueous solution of Co complex and toluene were introduced into the microchannel, and the Co complex extracted in toluene was detected by thermal lens microscopy (TLM). The Co complex was quickly extracted into toluene when the flow was stopped. The observed extraction time, ca. 50 s, was almost equivalent to the value calculated using the diffusion distance and diffusion coefficient. The dependence of the TLM signal on the concentration of the Co complex showed good linearity in the range of 1 x 10(-7) - 1 x 10(-6) M.

Non-contact photothermal control of enzyme reactions on a microchip by using a compact diode laser.

Photothermal temperature control of an enzyme-catalyzed reaction in a microchip using a diode laser was demonstrated. A laser beam with energy of 10 mW was used to irradiate an absorbing target placed on top of the microchip cover plate. Theoretical calculations have shown that temperature in the microchannel can be locally increased by 5-7 degrees C during short time intervals, due to heat released by the target. The rate of the enzyme reaction, which was initially inhibited due to cooling of the chip to low temperature, was increased when the target was irradiated. The products were detected by a thermal lens microscope. The product concentration was shown to depend on irradiation time, laser intensity and substrate concentration. Reaction characteristics (rate constant of the reaction) were then derived from these dependencies. The reaction volume and absolute quantity of the reaction product were estimated as 10 nl and 100 fmol, respectively. It was also demonstrated that a direct solvent heating method using infrared radiation could control the reaction in the microchannel.

Highly sensitive and direct detection of DNA fragments using a laser-induced capillary vibration effect.

A pulsed laser-induced stationary wave capillary vibration detection method was applied to the sensitive detection of capillary gel electrophoresis, and the direct detection of non-labeled nucleic acids, such as DNA sequencing products, was demonstrated. An excimer laser operating at 248 nm was used as a CVL excitation source, and polynucleotides were sensitively detected without derivatization. From an investigation on the endurance of several matrixes to pulsed laser irradiation, a polyacrylamide without a cross-linker (0%C) was found to have adequate endurance, and it exhibited no serious damage during an analysis. A cytosine-terminated sequence reaction product was detected with a sensitivity close to that of laser-induced fluorometry (LIF). These results suggest the feasibility of the highly sensitive detection of ultramicro amounts of biological materials without a pre- or post-column derivatization, which has usually been required in sensitive detection procedures, such as LIF. Furthermore, the feasibility of a novel DNA sequencing method is also suggested.

Integrated multilayer flow system on a microchip.

We utilized microchip technology and found that the multilayer flow of liquids can be formed in microchannels. Liquid/liquid interfaces were formed parallel to the side wall of the microchannels, because the surface tension and friction force are stronger than the force of gravity. A water/ethylacetate/water interface was formed in a 70-microm-wide and 30-microm-deep channel. The interface was observed to be quite stable and to be maintained for a distance of more than 18 cm. As an example of a multilayer flow application, we demonstrated the liquid/liquid extraction of Co-dimethylaminophenol complex in a microchannel. The solvent-extraction process of the complex into m-xylene in the multilayer flow was found to reach equilibrium in 4 s, while it took 60 s in a simple two-phase extraction.

[Two cases of rhabdomyosarcoma of the bladder in children].

A 1-year-old boy was admitted to our hospital with chief complaint of urinary retention and a lower abdominal large mass. The mass was shown in the bladder by computerized tomography (CT) associated with paraaortic lymph node swelling. Tumor biopsy revealed rhabdomyosarcoma, embryonal type. Complete remission (CR) was obtained by chemotherapy based on STS' 88. He has been healthy 40 months without recurrence. A similar case of rhabdomyosarcoma, embryonal type, in the bladder in a 3-year-old girl with a chief complaint of macrohematuria was verified by CT and tumor biopsy. The same chemotherapy was performed, resulting in partial remission (PR). A complete resection of the tumor was achieved by partial cystectomy. She is alive without recurrence 18 months after the cystectomy.

[Influential factors on the therapeutic response in the conditioning treatment of enuresis with an original therapeutic machine].

The conditioning treatment of enuresis with our original therapeutic machine, that is to awake the patient before enuresis may occur, has been performed since 1987. Influential factors on the therapeutic response were investigated. Twenty two patients with enuresis Type I were admitted and were treated for 5 nights with the therapeutic machine. Seven patients were cured (the cured group) and a certain effectiveness (decrease of the frequency of enuresis of more than 50%) was observed in 8 patients (the effective group). No effectiveness was obtained in 7 patients (the no change group). The average age of the cured group was higher than that of the no change group, and the difference was significant. No significant differences were found among the three groups in sex, the frequency of enuresis or the past experience of awakening before enuresis. Significant differences among the three groups were found in the average awakening score (how easily the patient awoke when a nurse called the patient after the machine alarmed) and the change of awakening score during treatment. The average awakening score of the cured group was the highest and that of the no change group was the lowest. The change of awakening score during treatment of the no change group was significantly lower than that of the cured group or that of the effective group. The desire to cure, scored 0-2 points at the time of discharge, was significantly stronger in the cured group than in the no change group. No significant differences were noticed among the three groups in the sleeping condition and the remembrance of awakening at the next morning.

[Ofloxacin for the treatment of chlamydial urethritis].

The effect of Ofloxacin administered orally 600 mg everyday for 14 days was evaluated in 20 male patients with Chlamydial urethritis, in whom Chlamydia was identified from urethral secretion by immunohistochemical technique using monoclonal primary antibody specific for all known serotypes of Chlamydia trachomatis and psittaci. 19 out of the 20 patients showed an improvement of subjective and/or objective symptoms after the treatment. The Chlamydia reaction turned to negative in 13 patients and to (+/-) in another patient. The overall clinical effect was excellent in 7, good in 6 and poor in 7, showing a effective rate of 65%. No side effects were observed.

[Ofloxacin concentration in prostatic tissue].

The concentration of ofloxacin in prostatic tissue and serum was determined in order to evaluate the permeability of ofloxacin into prostatic tissue in patients with benign prostatic hypertrophy. Ofloxacin was administered orally at a dose of 200 mg several hours before subcapsular prostatectomy. The determination was performed in the surgically removed adenoma and in the serum taken one hour before and at the removal of the adenoma. The peak level in the prostatic tissue was 1.46 microgram/g at 4.5 hour after the administration. The ratio of prostatic tissue level to the serum level was 1.00. In conclusion, ofloxacin was thought to be very useful for the treatment of acute and chronic prostatitis.

[Long-term results of systematic treatment for nocturnal enuresis based on overnight simultaneous monitoring of electroencephalography and cystometry].

BACKGROUND: The long-term results of systematic treatment based on overnight simultaneous monitoring of electroencephalography (EEG) and cystometry in the patients with enuresis were evaluated. METHODS: From January to December a total of 213 patients were classified into 3 types. Enuresis Type I: such cases show a normal cystometrogram (CMG) with an awakening response on the EEG before enuresis, but they do not awake. Enuresis Type IIa; such cases show a normal CMG without an awakening response on the EEG. Enuresis Type IIb: such cases show an abnormal CMG with no awakening response on the EEG. When such cases enter the deep sleep stage, continuous uninhibited contractions of the bladder are observed on the CMG. 136 cases were of Type I, 20 cases of Type IIa, and 57 cases of Type IIb. RESULTS: Out of 213 patients who were followed up for 2 years, cured cases were 94 (44%), effective cases were 81 (38%) and unchanged cases were 38 (18%). In 136 patients with Type I, cured cases were 71 (52%), effective cases were 50 (37%) and unchanged cases were 15 (11%). In 20 patients with Type IIa, cured cases were 8 (40%), effective cases were 9 (45%) and unchanged cases were 3 (15%). In 57 patients with Type IIb, cured cases were 15 (26%), effective cases were 22 (39%) and unchanged cases were 20 (35%). The age of the effective group, which included cured cases and effective cases, was significantly higher than that of the unchanged group. In enuresis Type I, the percentage of the patients with incontinence in daytime were significantly in the unchanged group than in the effective group. No significant differences in the frequency of enuresis and the percentage of the patients who had awakened spontaneously by urinary sensation were found between the two groups. CONCLUSION: The therapeutic response was best in enuresis Type 1 and worst in enuresis Type IIb. The patients, in whom not only frequency of enuresis but also type of enuresis was unchanged by systematic treatment for two years, was approximately 10% across the types. Accordingly they were thought to be non-responders to this systematic treatment.

Individual detection of single-nanometer-sized particles in liquid by photothermal microscope.

We have developed a thermal lens microscope for liquid-phase and surface microanalyses. By applying the thermal lens microscope to particle detection, we succeeded in detecting a pulsed photothermal signal from single-nanometer-sized particles in liquid and counting them individually. The samples were polystyrene latex particles (190 and 80 nm in diameter) and colloidal Ag particles (10 nm in diameter). To verify that the detected pulsed signals corresponded to the single-particle photothermal effects, we confirmed the items as described below using 190-nm polystyrene particles. First, no pulsed signal was generated under irradiation by either the excitation beam or the probe beam. Second, the pulse counts were proportional to the expectation value of the particles in the detection volume and zero for ultrapure water blank. Third, the pulse counts' distribution in a series of unit times had a Poisson distribution when the expectation value of the sample was much less than 1. Then, we demonstrated counting 80-nm polystyrene particles and 10-nm Ag particles in water. The pulsed signals were clearly distinguished from noise, and the signal-to-noise ratio was as large as 5. Finally, we discussed differences between the conventional thermal lens effect and the single-particle photothermal effect. Individual nanometer-sized particle detection by photothermal effect was the first demonstration.