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.

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.

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.

Relation between population density and body size in stream communities.

The existence of a general relation between population density and body size in animal assemblages has been debated because of known biases and ambiguities in the published data and data handling. Using new comprehensive data sets from two geographically separated stream communities that encompass 448 and 260 invertebrate taxa with a wide range of body sizes, we show that an inverse proportionality between density and body size is a consistent feature in these communities. The scaling across taxa is not statistically different between the two systems, indicating a convergent pattern of communities. Variation in the regression slope among different taxonomic groups indicates that these communities are not governed universally by a single ecological or energetic rule.

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.

Development of a tunable UV laser system synchronizing precisely with synchrotron radiation pulses from UVSOR.

A mode-locked Ti:sapphire laser is made to oscillate at the frequency of the UVSOR storage ring, 90.115 MHz, in a multi-bunch operation mode. The third harmonic of the laser is available in the wavelength range 243-280 nm. Synchrotron radiation from an undulator is monochromated by a grazing-incidence monochromator and introduced coaxially with the laser. The temporal profile of the photon pulses is monitored in situ by a luminescing substance/photomultiplier combination. The delay timing between the laser and synchrotron radiation can be changed from 0 to 11 ns by adjusting an electronic module that provides phase-locked loop stabilization of the laser pulse. The reliability and feasibility of this laser-synchrotron radiation combination technique are demonstrated by applying pump-probe experiments to two physical systems. The first system is photodissociation of iodomethane (CHA) with a laser photon, followed by photoionization of I and CH3 fragments with synchrotron radiation. The second, two-photon ionization of He atoms, is studied as the prototype of a time-resolved experiment. The He+ signal counts as a function of the laser-synchrotron radiation delay are found to be enhanced in a narrow time window, which can be interpreted in terms of a short lifetime of the resonant state, He*(1s2p 1P), produced by primary synchrotron radiation excitation.