A poly(dimethylsiloxane) microfluidic sheet reversibly adhered on a glass plate for creation of emulsion droplets for droplet digital PCR.

A PDMS microfluidic chip with T-junction channel geometry, two inlet reservoirs, and one outlet reservoir was reversibly adhered on a glass plate through the viscoelastic properties of PDMS. This formed a detachable microfluidic device for creation of water-in-oil emulsion droplets that were used as discrete reaction compartments for the droplet digital PCR. The PDMS/glass device could continuously produce monodisperse droplets without leakage of fluids using a vacuum-driven autonomous micropumping method. This droplet preparation technique only required evacuation of air dissolved in the PDMS before loading of oil and aqueous phases into separate inlet reservoirs. Degassing of the PDMS chip at approximately 300 Pa for 1.5 h in a vacuum desiccator gave 40 000 droplets in 80 min, which corresponded to a generation frequency of up to nine droplets per second. Over multiple runs the droplet creation was very reproducible, and the size reproducibility of generated droplets (polydispersity of up to 4.1%) was comparable to that acquired using other microfluidic droplet preparation techniques. Because the PDMS chip can be peeled off the glass plate, blocked channels can easily be fixed when they arise, and this extends the lifetime of the chip. Single DNA molecules partitioned into the droplets were successfully amplified by PCR. In addition, the droplet digital PCR platform allowed absolute quantification of low copy numbers of target DNA, and was robust against instrumental variance.

Hands-off preparation of monodisperse emulsion droplets using a poly(dimethylsiloxane) microfluidic chip for droplet digital PCR.

A fully autonomous method of creating highly monodispersed emulsion droplets with a low sample dead volume was realized using a degassed poly(dimethylsiloxane) (PDMS) microfluidic chip possessing a simple T-junction channel geometry with two inlet reservoirs for oil and water to be loaded and one outlet reservoir for the collection of generated droplets. Autonomous transport of oil and water phases in the channel was executed by permeation of air confined inside the outlet reservoir into the degassed PDMS. The only operation required for droplet creation was simple pipetting of oil and aqueous solutions into the inlet reservoirs. Long-lasting fluid transport in the current system enabled us to create ca. 51,000 monodispersed droplets (with a coefficient of variation of <3% for the droplet diameter) in 80 min with a maximum droplet generation rate of ca. 12 Hz using a PDMS chip that had been degassed overnight. With multiple time-course measurements, the reproducibility in the current method of droplet preparation was confirmed, with tunable droplet sizes achieved simply by changing the cross-sectional dimensions of the microchannel. Furthermore, it was verified that the resultant droplets could serve as microreactors for digital polymerase chain reactions. This hands-free technique for preparing monodispersed droplets in a very facile and inexpensive fashion is intended for, but not limited to, bioanalytical applications and is also applicable to material syntheses.

Microbead-based ligase detection reaction assay using a molecular beacon probe for the detection of low-abundance point mutations.

A microbead-based ligase detection reaction (LDR) assay using a molecular beacon probe was developed for the facile and rapid detection of point mutations present in low copy numbers in a mixed population of wild-type DNA. Biotin-tagged ligation products generated in the LDR were captured on the surface of streptavidin-modified magnetic beads for purification and concentration. The resulting product-tethered microbeads were combined with a molecular beacon probe solution, and the suspension was directly flowed into a capillary. The microbeads were accumulated in a confined space within the capillary using a bar magnet. The packed bead sample was then scanned by a fluorescence scanning imager to detect the presence of any mutations. With the developed methodology, we were able to successfully detect one cancer mutation in a mixture of 400 wild-type templates (t test at 95% confidence level). Furthermore, the post-LDR processing, typically the most laborious and time-consuming step in LDR-based mutation detection assays, could be carried out much more rapidly (approximately 20 min). This was enabled by the simple bead and fluid manipulations involved in the present assay.

Discriminative detection of low-abundance point mutations using a PCR/ligase detection reaction/capillary gel electrophoresis method and fluorescence dual-channel monitoring.

We applied a facile LIF dual-channel monitoring system recently developed and reported by our group to the polymerase chain reaction/ligase detection reaction/CGE method for detecting low-abundance point mutations present in a wild-type sequence-dominated population. Mutation discrimination limits and signaling fidelity of the analytical system were evaluated using three mutant variations in codon 12 of the K-ras oncogene that have high diagnostic value for colorectal cancer. We demonstrated the high sensitivity of the present method by detecting rare mutations present among an excess of wild-type alleles (one mutation among ~100 normal sequences). This method also simultaneously interrogated the allelic compositions of the test samples with high specificity through spectral discrimination of the dye-tagged ligase detection reaction products using the dual-channel monitoring system.

Phase-separation multiphase flow: preliminary application to analytical chemistry.

A two-phase separation mixed solution can undergo phase separation from one phase to two phases (i.e., upper and lower phases) in a batch vessel in response to changes in temperature and/or pressure. This phase separation is reversible. When the mixed solution undergoes a phase change while being fed into a microspace region, a dynamic liquid-liquid interface is formed, leading to a multiphase structure. This flow is called a phase-separation multiphase flow. Annular flow in a microspace, which is one such phase-separation multiphase flow, is interesting and has been applied to chromatography, extraction, reaction fields, and mixing. Here, research papers related to phase-separation multiphase flows-ranging from the discovery of the phenomenon to basic and technical research from the viewpoint of analytical science-are reviewed. In addition, the development of a new separation mode in a high-performance liquid chromatography system based on phase-separation multiphase flow is introduced.

Development of an HPLC device with water/acetonitrile with NaCl mixed solution that induces a phase-separation multiphase flow as the eluent in the separation column.

We developed a novel HPLC device where the phase-separation multiphase flow worked as the eluent in the separation column by using a water/acetonitrile/ethyl acetate triple mixed solution as a dual-phase-separation solution. Dual-phase-separation solutions form a phase-separation multiphase flow in a microscopic space. The new separation mechanism in the HPLC is called phase-separation mode. In this study, we used water and acetonitrile with NaCl mixed solution as a dual-phase-separation solution instead of the triple mixed solution. Octadecylsilyl (ODS)-modified particle- and porous silica particle-packed separation columns were united with the HPLC device for phase-separation mode caused by phase-separation multiphase flow. NA (1-naphthol) and NDS (2,6-naphthalenedisulfonic acid) were analyzed by the device as model sample. Using the water and acetonitrile with NaCl mixed solution at the solvent volume ratio of 5:5, NA and NDS were not separated on either column at 25 °C. On the other hand, they were separated with the order NDS and NA on the ODS column and separated with the order NA and NDS on the silica column in phase-separation mode at 0 °C. We discuss the separation mechanism of phase-separation mode using the water and acetonitrile with NaCl mixed solution at 0 °C.

Development of a ligase detection reaction/CGE method using a LIF dual-channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild-type DNA.

We developed an inexpensive LIF dual-channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low-abundance point mutations in a large excess of wild-type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K-ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K-ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild-type sequence-dominated population, and to disclose their allelic composition, was thus demonstrated successfully.

Michrochip chromatography using an open-tubular microchannel and a ternary water-ACN-ethyl acetate mixture carrier solution.

A capillary chromatography system has been developed using a ternary mixed-solvents solution, i.e. water-hydrophilic/hydrophobic organic solvent mixture as a carrier solution. Here, we tried to carry out the chromatographic system on a microchip incorporating the open-tubular microchannels. A model analyte solution of isoluminol isothiocyanate (ILITC) and ILITC-labeled biomolecule was injected to the double T-junction part on the microchip. The analyte solution was delivered in the separation microchannel (40 µm deep, 100 µm wide, and 22 cm long) with the ternary water-ACN-ethyl acetate mixture carrier solution (3:8:4 volume ratio, the organic solvent rich or 15:3:2 volume ratio, the water-rich). The analyte, free-ILITC and labeled BSA mixture, was separated through the microchannel, where the carrier solvents were radially distributed in the separation channel generating inner and outer phases. The outer phase acts as a pseudo-stationary phase under laminar flow conditions in the system. The ILITC and the labeled BSA were eluted and detected with chemiluminescence reaction.

Development of HPLC system that uses phase-separation multiphase flow as an eluent.

We developed a new type of HPLC system that uses phase-separation multiphase flow as an eluent. A commercially available HPLC system with a packed separation column filled with octadecyl-modified silica (ODS) particles was used. First, as preliminary experiments, 25 kinds of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were supplied to the system to act as eluents at 20 °C. 2,6-Naphthalenedisulfonic acid (NDS) and 1-naphthol (NA) mixture was used as a model and mixed analyte was injected into the system. Roughly speaking, they were not separated in organic solvent-rich eluents and well separated in water-rich eluents, in which NDS eluted faster than NA. This means that HPLC worked under a reverse-phase mode for separation at 20 °C. Next, the separation of the mixed analyte was examined on HPLC at 5 °C, and then after judging the results, four kinds of ternary mixed solutions were in detail as eluents on HPLC at 20 °C and 5 °C. Based on their volume ratio, the ternary mixed solutions acted as a two-phase separation mixed solution, leading to a phase-separation multiphase flow. Consequently, the solutions flowed homogeneously and heterogeneously in the column at 20 °C and 5 °C, respectively. For example, the ternary mixed solutions containing water/acetonitrile/ethyl acetate at volume ratios of 20:60:20 (organic solvent-rich) and 70:23:7 (water-rich) were delivered into the system as eluents at 20 °C and 5 °C. In the organic solvent-rich eluent, the mixture of NDS and NA was not separated at 20 °C but was separated at 5 °C, the elution of NA being faster than the one of NDS (phase-separation mode). In the water-rich eluent, the mixture of analytes was separated at both 20 °C and 5 °C, the elution of NDS being faster than the one of NA. The separation at 5 °C was more effective than at 20 °C (reverse-phase mode and phase-separation mode). This separation performance and elution order can be attributed to the phase-separation multiphase flow at 5 °C.

Observation of the phase-separation multiphase flow using a polyethylene glycol/phosphate mixed solutions and the aqueous two-phase distribution of red blood cells in the flow system.

Phase-separation multiphase flow at a liquid-liquid interface was successfully formed in an aqueous two-phase system of polyethylene glycol/phosphate mixed solutions when fed into a microchannel (100 µm wide and 40 µm deep) on a microchip and a fused-silica capillary tube (100 µm ID). As one example, tube radial distribution flow (annular flow) was observed when 10.0 wt% polyethylene glycol 6000 and 8.5 wt% dipotassium hydrogen phosphate aqueous solution containing 1.0 mM Rhodamine B was fed at 40 â„ƒ, recorded by bright field microscopy. It exhibited a dipotassium hydrogen phosphate-rich inner phase and polyethylene glycol-rich outer phase. Effects of conditions including composition, flow rate, viscosity, and contact angle on tube radial distribution flow were analyzed. It was found out that although the viscosity of PEG-rich solution was much higher than that of phosphate-rich one, the phase configuration in tube radial distribution flow did not necessarily obey the viscous dissipation law in untreated microchannel and capillary tube, as well as for all the types of PEG/phosphate mixed solution the PEG-rich solution occupied the outer phase near the ODS-treated inner wall of both microchannel and capillary tube against the law. To assess the use of microfluidic flow in applications, we examined the distribution of red blood cells in the inner and outer phases fed into double capillary tubes with different inner diameters. Cell distribution was found to concentrate in the inner (dipotassium hydrogen phosphate-rich) phase compared to the outer (polyethylene glycol-rich) phase at a ratio of 1.8.

Development of a HPLC system using a phase-separation multiphase flow as an eluent coupled to a silica-particle packed column.

The study reports the development of a high-performance liquid chromatography (HPLC) system in which a phase-separation multiphase flow as the eluent and a silica-particle based packed column as the separation column were combined to form the phase separation mode. Twenty-four types of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were applied as eluents to the system at 20 °C. 2,6-Naphthalenedisulfonic acid (NDS) and 1-naphthol (NA) were injected as model analytes into the system. They showed separation tendency in organic solvent-rich eluents in normal-phase mode and NA was detected earlier than NDS. Subsequently, seven types of the ternary mixed solutions were examined as eluents in the HPLC system at 20 °C and 0 °C. These mixed solutions worked as a two-phase separation mixed solution, providing a phase-separation multiphase flow at 0 °C in the separation column. In the organic solvent-rich eluent, the mixture of analytes was separated at both 20 °C (normal-phase mode) and 0 °C (phase-separation mode), with NA being detected earlier than NDS. The separation at 0 °C was more efficient than at 20 °C. In the water-rich eluent, the mixture of NDS and NA was not separated at 20 °C but was separated at 0 °C (phase-separation mode), with NDS being detected earlier than NA. We also discussed the separation mechanism of phase-separation mode in HPLC together with the computer simulation for the multiphase flow in the cylindrical tube having sub-µm inner diameter.

Novel separation mode of HPLC based on phase-separation multiphase flow.

A novel separation mode for high-performance liquid chromatography (HPLC) is proposed based on phase-separation multiphase flow. A commercially available HPLC system was used with a packed-separation column of octadecyl-silica (ODS)-modified particles. Water/acetonitrile/ethyl acetate ternary mixed solutions, (a) 1:8:1, (b) 1:3:1, and (c) 16:3:1 (v/v/v), were delivered into the system as an eluent at 20 and 5 °C. The ternary mixed solution acted as a two-phase separation solution leading to phase-separation multiphase flow. The solution flowed in the column homogeneously and heterogeneously at 20 and 5 °C, respectively. 1-Naphthol (NA) and 2,6-naphthalenedisulfonic acid (NDS) were injected into the system as model analytes. At 20 °C, the analyte mixture did not separate in solutions (a) and (b) while it separated in solution (c) with the elution order of NDS followed by NA. At 5 °C, it did not separate in solution (a), while it separated in solution (b) with elution order of NA followed by NDS and solution (c) with elution order of NDS followed by NA more effectively than 20 °C. The separation behavior and elution order are possibly caused by the phase-separation multiphase flow.

Microfluidic behavior of ternary mixed solutions of water/acetonitrile/ethyl acetate through experiments and computer simulations.

When ternary mixed solutions of water/acetonitrile/ethyl acetate are delivered into a microspace under laminar flow conditions, the solvent molecules show specific microfluidic flows, such as microfluidic inverted flow and tube radial distribution flow, which have been applied to novel analytical methods. In this paper, inverted flow was examined using various Y-type microchannels that had mixing angles of 0°, 90°, 180°, and 270°. Inverted flow was experimentally observed and the trigger phenomenon was also successfully expressed through computer simulations. Tube radial distribution flow, that is, annular flow, in a capillary tube is reported to cause exchange of the inner and outer phases based on the solvent composition of the ternary mixed solution. Tube radial distribution flow for an organic solvent-rich inner and a water-rich outer phases, as well as for a water-rich inner and an organic solvent-rich outer phases, could be well recreated by computer simulations for a ternary mixed solution. This highlights the effectiveness of computer simulations for such flow scenarios and will allow optimization of the operating conditions and design of microfluidic analytical devices.

Fluorescence observation supporting capillary chromatography based on tube radial distribution of carrier solvents under laminar flow conditions.

We developed a capillary chromatography system by using an open capillary tube made of fused-silica, polyethylene, or polytetrafluoroethylene, and a water-hydrophilic/hydrophobic organic mixture carrier solution, called tube radial distribution chromatography (TRDC) system. By comparing with chromatograms obtained via the TRDC system, fluorescence photographs and profiles of the fluorescent dyes dissolved in the carrier solvents in capillary tubes were observed under laminar flow conditions. The chromatograms were obtained for a model mixture analyte consisting of 1-naphthol and 2,6-naphthalenedisulfonic acid with the TRDC system, by using a fused-silica capillary tube and a water-acetonitrile-ethyl acetate carrier solution. By altering the carrier flow rates, we examined the fluorescence photographs and profiles of the dyes, perylene and Eosin Y, dissolved in the carrier solvents in the capillary tube by using a fluorescence microscope equipped with a CCD camera. As confirmed by fluorescence observations, the major inner and minor outer phases generated in the capillary tube were based on the tube's radial distribution of the carrier solvents. We designed and manufactured a microreactor incorporating microchannels in which three narrow channels combined to form one wide channel. When the carrier solvents containing the dyes were fed into the channels, the inner and outer phase generations were also observed in the narrow and wide channels, strongly supporting the conclusions concerning the tube radial distribution phenomenon of the solvents.

Use of tube radial distribution of ternary mixed carrier solvents for introduction of absorption reagent for metal ion separation and online detection into capillary.

When ternary mixed solvents consisting of water-hydrophilic/hydrophobic organic solvents are fed into a micro-space under laminar flow conditions, the solvent molecules are radially distributed in the micro-space. The specific fluidic behavior of the solvents is called the "tube radial distribution phenomenon (TRDP)". A novel capillary chromatography method was developed based on the TRDP that creates the inner major and outer minor phases in a tube, where the outer phase acts as a pseudo-stationary phase. This is called "tube radial distribution chromatography (TRDC)". In this study, Chrome Azurol S as an absorption reagent was introduced into the TRDC system for metal ion separation and online detection. The fused-silica capillary tube (75 µm id and 110 cm length) and water-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio) including 20 mM Chrome Azurol S as a carrier solution were used. Metal ions, i.e. Co(II), Cu(II), Ni(II), Al(III), and Fe(III), as models were injected into the present TRDC system. Characteristic individual absorption characteristics and elution times were obtained as the result of complex formation between the metal ions and Chrome Azurol S in the water-acetonitrile-ethyl acetate mixture solution. The elution times of the metal ions were examined based on their absorption behavior; Co(II), Ni(II), Al(III), Fe(III), and Cu(II) were eluted in this order over the elution times of 4.7-6.8 min. The elution orders were determined from the molar ratios of metal ion to Chrome Azurol S and Irving-Williams series for bivalent metal ions.

Consecutive Sample Injection Analysis in Tube Radial Distribution Chromatography.

Tube radial distribution chromatography based on the tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported, where the annular flow is created through phase-separation multiphase flow. We have proposed the first-ever procedure for consecutive sample injection analysis using chromatography. In basic terms, a commercially available HPLC system could be used with a sample injector (0.2 µL volume) and a fused-silica capillary tube (250 cm long) as a separation column instead of a normal packed one, while the built-in detection cell was replaced by improved on-capillary detection. A ternary mixed solution of water/acetonitrile/ethyl acetate (3:8:4 volume ratio) was delivered into the capillary tube as an eluent at a flow rate of 2.0 µL min-1. Model sample solutions of 1-naphthol and 2,6-naphthalenedisulfonic acid were consecutively analyzed by the present chromatography with a processing rate of 6 samples per hour. Simple and rapid consecutive analysis could be performed because washing and initialization of the separation tube was no longer necessary. The obtained results provide clues to developing new methodologies which combine features of both chromatography (separation) and the flow injection method (consecutive analysis).

Metal ion analysis using microchip CE with chemiluminescence detection based on 1,10-phenanthroline-hydrogen peroxide reaction.

We developed a microchip CE method with chemiluminescence (CL) detection using the reaction of 1,10-phenanthroline and hydrogen peroxide for separation and determination of metal ions, where the metal ions acted as catalysts for the CL reaction. The microchip consisted of two microchannels that crossed at the intersection and four reservoirs that accessed the ends of the channels. The metal ions in the sample solution migrated in the channel along with 1,10-phenanthroline included in a running solution, and then mixed with hydrogen peroxide in one of the reservoirs to emit CL. The light was detected with a photomultiplier tube located just above the reservoir. Two metal ion groups, the platinum metal group (Ru(III), Rh(III), Pd(II), Os(VIII), Ir(III), and Pt(IV)) and the fourth periodic transition metal group (Cu(II), Fe(II), Co(II), and Ni(II)) were examined using the present system. The lowest detection limit was observed for Os(VIII); Os(VIII) responded over the range of 7.5x10(-12)-1.0x10(-8 )M with the detection limit of 7.5x10(-12 )M (about 38 zmol) (S/N = 3). The mixed solution of Ru(III), Rh(III), Pd(II), Os(VIII), Ir(III), and Pt(IV) could be analyzed using this system within about 2.5 min. In addition, the system was applied to the determination of Cu(II) concentration in a city water supply.

Capillary chromatography based on tube radial distribution of aqueous-organic mixture carrier solvents: Introduction of inner-wall-modified capillary tubes.

We developed a novel capillary chromatography using an open capillary tube and a water-hydrophilic/hydrophobic organic solvent mixture as a carrier solution. The capillary chromatography was called a tube radial distribution chromatography (TRDC) system. In this study we tried to introduce inner-wall-modified (e.g. phenylboronic-acid- and iminodiacetic-acid-modified) fused-silica capillary tubes to the TRDC system to separate model mixture analytes. The phenylboronic-acid-modified capillary tube was combined with absorption detection to analyze a mixture of adenosine and deoxyadenosine. The iminodiacetic-acid-modified capillary tube was combined with chemiluminescence detection using a luminol reaction to analyze a mixture of copper(II) and hematin. A water (carbonate buffer)/ACN/ethyl acetate (2:7:4 v/v/v) and a water (carbonate buffer)/ACN/ethyl acetate (15:3:2 v/v/v) mixture solution were used as carrier solutions in the TRDC system, and typical carbonate buffer solutions not containing any organic solvents were also used as carrier solutions as reference solutions. In both modified capillary tubes, the organic-solvent-rich carrier solution successfully improved the separation of the mixture analytes in the system, and the water-rich carrier solution greatly depressed their separation, when compared with chromatography using carbonate buffer carrier solutions containing no organic solvents. Such observed phenomena were discussed considering the separation mechanism of the TRDC system.

Phase Separation and Collection of Annular Flow by Phase Transformation.

A polyethylene glycol/citrate mixed solution was fed into a single channel of a Y-type micro-channel on a microchip as an aqueous two-phase system. A phase separation multi-phase flow with a liquid-liquid interface was generated due to a phase transformation. An annular flow, one of the flow types in the phase separation multi-phase flow, was observed through bright-field microscopy. The flow consisted of citrate-rich inner and polyethylene glycol-rich outer phases. We attempted to separate and collect the two phases in the single channel into two separate Y-type channels. When the pressure losses for the separated channels were not very different, we observed symmetric flow in the Y-type channel. When the pressure losses were quite different, the polyethylene glycol-rich phase with higher viscosity was selectively distributed to the separated channel with lower pressure loss. Thus, the polyethylene glycol-rich phase was successfully and intentionally collected from the chosen Y-type channel via the creation of annular flow in the single channel.

Microfluidic Inverted Flow of Ternary Water/Hydrophilic/Hydrophobic Organic Solvent Solution in a Y-Type Microchannel and a Proposal of the Response Microfluidic Analysis through the Experiment.

Two solutions that are individually fed at the same flow rate into two separate microchannels of a microchip, combine to form a single channel (a Y-type microchannel). This flow is either parallel for immiscible solutions or initially parallel, but then becomes homogeneous through diffusion, for miscible solutions. However, a new type of microfluidic behavior in a Y-type microchannel that was neither parallel nor homogeneous flow has been observed using, for example, water/acetonitrile (3:4.5, v/v) and acetonitrile/ethyl acetate (3.5:4, v/v) mixed solutions. Each mixed solution was marked with distinctive dyes and delivered at the same flow rate into a Y-type microchannel under laminar flow conditions. In the single channel, the two phases were initially observed to flow in parallel, but then apparently swapped to flow on the opposite wall while retaining parallel flow with a slight change in the components of the two phases. We have named this type of laminar flow "microfluidic inverted flow" for ternary water/hydrophilic/hydrophobic organic solvent mixed solutions. The inverted flow of a ternary water/acetonitrile/ethyl acetate system was examined in detail under various flow conditions. We also proposed a concept of response microfluidic analysis based on such microfluidic inverted flow.