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.

Use of a thermal lens microscope in integrated catecholamine determination on a microchip.

A new method for determination for catecholamines (CA) utilizing microchip technology and a thermal lens microscope has been developed. Microchannels with a 250 microm x 10 microm cross section were used for mixing, reaction, and detection. Epinephrine (EP), nor-epinephrine (NE), dopamine (DA), and L-dopa (LD) were determined by using coloring oxidization to aminochromes by sodium metaperiodate. A thermal lens microscope (TLM) was used for detection of the product. The sensitivity of the system was comparable for the four CA and required only 15 s for mixing of sample and reagent. The calibration lines indicated excellent linearity for concentrations of 5-20 microg mL(-1). The relative standard deviations for 10 microg mL(-1) solution were 1.08, 2.18, 2.2, and 2.5% for EP, NE, DA, and LD, respectively. CA in pharmaceutical injections were also determined by use of the system and the results correlated very well with nominal values. Results obtained by use of the integrated system suggested there was a sufficient possibility to realize high-throughput medical diagnosis systems.

Photothermal temperature control of a chemical reaction on a microchip using an infrared diode laser.

We have demonstrated that a miniaturized device with IR laser heating of the solvent, based on a photothermal effect, is capable of fast and localized control of an enzymatic reaction on a microchip under flow conditions. Using noncontact spectroscopic temperature-sensing techniques, we measured temperature dynamics and spatial distribution and compared the measurements with results of numerical simulation analysis. The device was operated at ultrafast heating and cooling rates of 67 and 53 degrees C/s, respectively, which is 30 times faster than conventional systems and 3-6 times faster than electrothermal miniaturized thermocyclers. The IR laser-mediated heater is characterized by a significantly reduced heated volume of only 5 nL, compared to existing chip-based systems with electrothermal heating. Direct heating of a sample with extremely small heat capacity led us to a fast heating rate, and efficient heat removal through heat transfer to the glass substrate resulted in a fast cooling rate. Reproducible temperature levels with dwell times shorter than 0.5 s were achieved. The enzyme reaction on a chip was successfully controlled with 0.6-s time resolution, using periodic photothermal heating by IR laser. The IR diode laser is compact and thus suits well the miniaturized system design. Our work gives the basis for integration in a chip format of a variety of chemical processes that require fast temperature control.

Assay of spherical cell surface molecules by thermal lens microscopy and its application to blood cell substances.

To detect and quantitate uneven cell surface molecules, such as blood group antigens on a blood cell and immunoglobulin molecules on a mast cell, an improved method of thermal lens microscopy was employed. The antigen molecules were immunologically stained with their antibodies, which were labeled with colloidal gold. Since the surface of the biological cells was not flat but spherical, the focal point of the probe laser beam inevitably deviated from the sample surface on the moving stage. Therefore, the deviation of the focal point of the probe beam was corrected by adjusting the phase of the signal. Using this technique, a three-dimensional antigen distribution on each cell surface was imaged. Despite the convex surface of cells, labeled colloidal gold was correctly quantified. In the measurement of erythrocyte antigens, a small quantity of Lewis antigens was successfully detected on the umbilical cord erythrocytes. Immunoglobulin E on a mast cell, derived from the allergic human mucosa fungus, was also observed by this method, and the distribution of IgE molecules on the cell surface was quantitatively imaged. A thermal lens microscope, which measures spherical samples correcting the deviation, made it possible for us to observe and assay the substances on biological specimens that have complicated forms, such as living cells in vivo or in situ.

Determination of subyoctomole amounts of nonfluorescent molecules using a thermal lens microscope: subsingle-molecule determination.

The photothermal effect of an ultratrace amount of nonfluorescent molecules in liquid was determined by optimizing the optical arrangement for a thermal lens microscope. The optimized experimental setup could be determined from the evaluation of probing volume and the concentration of the sample solutions even when the expectation of the molecule number in the probing region was less than a single molecule. The minimum expectation, which is explained as being the time average, was 0.4 molecule of Pb(II) octaethylporphyrin (OEP) in benzene. The concentrations in the 9.7 x 10(-11)-7.8 x 10(-10) M region used in this work corresponded to the expected number of 0.4-3.4 molecules, and the calibration curve in this region showed good linearity. Taking into account the enhancement factor of solvent, the molar absorption coefficient of solute, and the optimization of the optical arrangement, the present result, which was the determination limit of 0.34, was consistent with that previously reported. The relation between molecular behavior in the probing volume and the signal was discussed. The average temperature rise in the probing volume by the photothermal effect for the single OEP molecule was estimated as 3.1 muK, and this value was detectable, based on conventional thermal lens measurements for bulk scale sample.

Determination of carcinoembryonic antigen in human sera by integrated bead-bed immunoassay in a microchip for cancer diagnosis.

A bead-bed immunoassay system was structured on a microchip and applied to determine carcinoembryonic antigen (CEA), which is a commonly used marker of colon cancer. Polystyrene beads precoated with anti-CEA antibody were introduced into a microchannel, and then a serum sample containing CEA, the first antibody, and the second antibody conjugated with colloidal gold were reacted successively. The resulting antigen-antibodies complex, fixed on the bead surface, was detected using a thermal lens microscope (TLM). A highly selective and sensitive determination of an ultratrace amount of CEA in human sera was made possible by a sandwich immunoassay system that needs three antibodies for an assay. A detection limit dozens of times lower than the conventional ELISA was achieved. Moreover, when serum samples for 13 patients were assayed with this system, there was a high correlation (r = 0.917) with the conventional ELISA. The integration reduced the time necessary for the antigen-antibody reaction to approximately 1%, thus shortening the overall analysis time from 45 h to 35 min. Moreover, troublesome operations required for conventional heterogeneous immunoassays could be much simplified. This microchip-based diagnosis system is the first microchip-based system that is practically useful for clinical diagnoses with short analysis time, high sensitivity, and easy procedures.

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.

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.

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.

On-chip integration of sequential ion-sensing system based on intermittent reagent pumping and formation of two-layer flow.

A sequential ion-sensing system using a single microchip was successfully realized. The system developed here involves intermittent pumping of plural organic phases into a microchannel, followed by contact with a single aqueous phase to form a stable organic-aqueous two-layer flow inside the microchannel. Because the plural organic phases created by intermittent flow contain the same lipophilic pH indicator dye but different ion-selective neutral ionophores, different ions can be sequentially and selectively extracted into the different organic phases, where they can be determined by thermal lens microscopy (TLM). We used KD-A3 as the lipophilic pH indicator dye and valinomycin and DD16C5 as neutral ionophores to demonstrate sequential ion sensing of potassium and sodium ions by measuring the deprotonated dye caused by the ion extraction. The integrated microfluidic system proposed here allows multi-ion sensing, which is not easily demonstrated by conventional ion sensor technology using a solvent polymeric membrane. The minimum volume of single organic phase needed to obtain an equilibrium response without dilution by cross dispersion of two organic phases was ca. 500 nL in our system, indicating that the required amounts of expensive reagents in one measurement could be reduced to 1.7 ng and 2.8 ng for the dye and ionophore molecules, respectively.

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.

Detection and measurement of a single blood cell surface antigen by thermal lens microscopy.

A highly sensitive method for detection of antigens on the surface of a single blood cell using thermal lens microscopy is described. Colloidal gold, coated with antibody, was used to stain membrane antigens of leukocytes. Human leukocyte antigens on the lymphocytes and mononuclear leukocytes were observed by new thermal lens microscopy, which involves spectrometry using a laser-induced thermal-lens effect. Antigens of HLA-A, -B, and -C loci on the lymphocytes were identified and quantitated using a single cell. The image of HLA-A, -B, and -C antigen distribution on a mononuclear leukocyte was obtained. Our laser microscope, newly devised for measuring convex surface cells, is a powerful analytical tool for detecting and quantitating localized antigens in a single cell and/or cell-surface-associated molecules.

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.

Integration of an immunosorbent assay system: analysis of secretory human immunoglobulin A on polystyrene beads in a microchip.

An immunosorbent assay system was integrated into a glass microchip. Polystyrene beads were introduced into a microchannel, and then human secretory immunoglobulin A (s-IgA) adsorbed on the bead surface was reacted with colloidal gold conjugated anti-s-IgA antibody and detected by a thermal lens microscope. The scale merits of liquid microspace on the molecular behavior remarkably contributed to reduced assay time. The integration cut the time necessary for the antigen-antibody reaction by 1/90, thus shortening the overall analysis time from 24 h to less than 1 h. Moreover, troublesome operations required for conventional immunosorbent assays could be replaced by simple operations.

Miniaturized ultrathin slab gel electrophoresis with thermal lens microscope detection and its application to fast genetic diagnosis.

A miniaturized ultrathin slab gel electrophoresis (MUSGE) apparatus was developed, and fast separation of DNA fragments was obtained using it. To obtain sufficient separation efficiency in a limited space, a discontinuous buffer system was used. In general, it is difficult to cast a discontinuous ultrathin slab gel of adequate quality. However, the miniaturized resolving gel could be cast by taking advantage of the "capillary phenomenon" of the ultrathin channel. A gradient plate was used to control the height of the resolving gel and to form a clear interface between the concentrating gel and the resolving gel. This method was used to cast multiple gels simultaneously and reproducibly. The gradient plate also facilitated sample introduction, which was carried out by using a micropipet. A 25-mm-long and 80-micron thick-resolving gel was used to separate the 100-base pair ladder DNA within 10 min. Bandwidth was reduced to 100-200 microns, thus improving the number of theoretical plates to 22,000, which was comparable to that in conventional slab gel electrophoresis even though the migration distance was reduced to 1/10. Satisfactory lane-to-lane reproducibility (RSD < 1.0%, n = 6) and gel-to-gel reproducibility (RSD < 2.7%, n = 4) were obtained. Finally, the MUSGE apparatus was successfully applied to get a rapid genetic diagnosis.

Critical increment of Lewis blood group antigen in serum by cancer found by photothermal immunoassay.

Lewis blood group antigen levels in human sera were assayed with a highly sensitive photothermal immunoassay which is based on laser-induced photothermal detection. Comparison of 32 colon cancer patients' sera and 34 healthy persons' sera showed that cancer patients' sera contained more Lewis antigens than healthy persons' sera. Le(a) antigen level in Le(a-) type persons and Le(b) level in Le(b-) type persons differentiated healthy persons and colon cancer patients. Furthermore, it was found that in Lewis blood phenotype (a-) several cancer patients' sera specimens changed to (a+). Many reports demonstrated that Lewis phenotype of erythrocytes changed with various conditions, including carcinomas, but they dealt mostly with erythrocytes and salivas or showed immunohistochemical evidence, and there are no reports on the quantitative analysis of ordinary (noncancerated) Lewis antigen levels in human sera. This is because Lewis antigens in sera, unlike those found in saliva, are too small to quantify with conventional immunoassay and there has been no highly selective method to measure Lewis antigens in sera. The increase of Lewis antigen in cancer patients' sera is presumed to antecede the blood type change. Our assay presented here, a highly sensitive assay of Lewis antigens, will greatly contribute to an early detection or diagnosis of cancers.