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Recently, high-throughput analysis with minimal reagent consumption has been desired to assess interactions between drug candidates and disease-related oligonucleotides. To realize an ideal assay for drug screening, a rapid assay based on affinity capillary electrophoresis was generated to reduce the consumption of samples/reagents by a partial-filling technique under nonequilibrium conditions. In the proposed method, the first sample, oligonucleotide as a ligand, and second sample zones were injected into a capillary with spacers of background solution between the samples and oligonucleotide zones. After applying voltages, only the second sample zone passed through the partially filled oligonucleotide zone, resulting in variations in the peak parameters owing to this interaction. The electropherograms obtained were analyzed using equilibrium, reaction kinetics, and moment theories. In the interaction analyses between small molecules and DNA aptamers, only the small molecules binding to the aptamer showed significant changes in their peak heights and shapes. The estimated kinetic parameters were in good agreement with the reported values, indicating the applicability of the proposed method for drug screening. When interactions between drug candidates and disease-related RNAs were analyzed, one of the candidates showed remarkable variation in the peak parameters upon the addition of potassium ions. Consequently, the proposed method could be one of the ideal assays for drug screening.

Here, we used a fluorophore-bound, porphyrin-targeted DNA aptamer to increase the sensitivity of capillary electrophoresis for the detection of a model porphyrin. The complex of the DNA aptamer wit...

Aptamers, single-stranded DNAs/RNAs with a strong and specific interaction towards a target molecule, have wide applications in the fields of medicine and biosensors. In conventional aptamer selection methods, it is difficult to obtain "preorganized" and/or "induced-fit" type of aptamers selectively. In this study, separation and fractionation of single-stranded DNAs with/without stable preorganized structures were carried out using capillary sieving electrophoresis. The fractionated DNAs showed different mobilities and thermodynamic stabilities of their secondary structures; this outcome is deemed to be necessary for the synthesis of novel aptasensors with a desirable sensing mechanism.

A novel electrokinetic filtration device using a plugged hydrogel was developed to directly measure the initial rate of enzyme reactions. In the proposed method, the enzyme reaction proceeded only for a short time when the substrate was passed through a thin layer of enzyme trapped by the hydrogel without any lag times for mixing and detection. In experimental conditions, alkaline phosphatase (enzyme) was filtrated at a cathodic-side interface of the plugged hydrogel by molecular sieving effect, providing the thin enzyme zone whose thickness was approximately 100 μm. When 4-methylumberiferyl phosphate (substrate) was electrokinetically introduced into the device after trapping the enzyme, 4-methylumberiferone (product) was generated by the enzyme reaction for only 1.26 s as the substrate passed through the trapped enzyme zone. As a result, the initial rate of the enzyme reaction could be directly calculated to 31.0 μM/s by simply dividing the concentration of the product by the tunable reaction time. Compared to the initial rate obtained by mixing the enzyme and substrate solutions, the value of the maximum velocity of the enzyme reaction was 30-fold larger than that in the mixing method due to the preconcentration of the enzyme by trapping. The Michaelis-Menten constant in the proposed method was 2.7-fold larger than that in the mixing method, suggesting the variation of changes in the equilibrium of complex formation under the experimental conditions.

Optical sensors based on solvent polymeric membranes have the potential to measure analytes present in an aqueous solution through the development of a tailored method for a specific target. However, limits in the concentrations of the component dyes have prevented improvements in sensitivity. We propose a Forster resonance energy transfer (FRET)-based fluorescence amplification system for ion-selective optical sensors using a highly fluorescent liquid material composed of a lipophilic phosphonium cation and a pyrene modifying sulfonate anion ([P66614][HP-SO3]), as both the plasticizer and donor, in addition to a combination of the lipophilic phosphonium cation and the fluorescein dodecyl ester anion ([P66614][12-FL]) as the fluorescent sensing dye acceptor. For ion extraction-based sensing, the donor and acceptor were retained in the plasticized PVC membrane with negligible leaching upon exposure to acidic and basic aqueous solutions. Systematic investigation of the donor and acceptor ratios clarified the effect of the amplification factor and the sensitivity of the sensor. At an acceptor doping level of 0.5 mol % (vs donor), an approximately 22-fold higher sensitivity was obtained compared to that of a conventional PVC membrane optical sensor. During ion measurement based on the coextraction of protons and anions, selectivity following the Hofmeister order was observed, which was controlled by the addition of ionophores. The proposed FRET system based on a lipophilic fluorescent liquid material has the potential to significantly improve the sensitivities of optical sensors using solvent polymeric membranes with high selectivities for various target analytes.

An enzyme-responsive fluorescent nanoemulsion (NE) based on lipophilic dye liquid (LDL) was developed for alkaline phosphatase (ALP). The response mechanism of the NE involved enzymatic reactions and simultaneous extraction of anions. The LDL-based NE exhibited 3.8 times higher sensitivity than plasticizer-based conventional NE. Detection limit and response range were 2.7 (U L-1) and 5-50 (U L-1), respectively. The response time was reduced to less than half that of the LDL-based membrane.

An on-line sample preconcentration technique based on transient trapping (tr-trapping) in micellar electrokinetic chromatography (MEKC) was applied for steroid detection with UV (tr-trapping-UV) and electrospray ionization mass spectrometry detection (tr-trapping-ESI-MS). ESI-MS was used to improve the sensitivity in MEKC. The MEKC separation was carried out using volatile ammonium formate as a background solution to facilitate the coupling with ESI-MS. The partial introduction of a sodium dodecyl sulfate (SDS) micellar solution before the introduction of a sample solution to the capillary provided the effective preconcentration of analytes. At the same time, the SDS micelle would not enter the ESI-MS system, so its interference in ESI-MS detection was suppressed under the optimal condition, then five steroids can be separated by the developed method. In tr-trapping-ESI-MS, an acidic condition of pH 3.5 was employed to suppress the electroosmotic flow, which can avoid micellar solution migrating to the MS instrument. The developed method showed that the micellar solution requires a twofold slower time than the sample to migrate along the column, which can prohibit the cause of the problem with the MS instrument and interference signal of SDS in the steroid's detection. The tr-trapping-ESI-MS protocol showed up to 540-fold enhancements of the peak intensity and 50-fold improvement of the limit of detection compared with capillary zone electrophoresis using androsterone as a model sample.

Ionophore-based dye liquid nanoemulsion sensors exhibiting rapid response, high selectivity, and high sensitivity to chloride were developed. Since nanoemulsions contain extremely high concentrations of dyes and have large surface areas, rapid and highly sensitive measurements were possible.

Metal nanostructures exhibit specific optical characteristics owing to their localized surface plasmon resonance (LSPR) and have been studied for applications in various optical devices. The LSPR property strongly depends on the size and shape of metal nanostructures, thus, plasmonic devices must be designed and fabricated according to their uses. Nanoimprint lithography (NIL) is an effective process for repeatedly fabricating metal nanostructures with controlled sizes and shapes and require optical properties. NIL is a powerful method for mass-producible, low-cost, and large-area fabrication. However, the process lacks flexibility in adjusting the size and shape according to the desirable optical characteristics because the size and shape of metal nanostructures are determined by a single corresponding mold. Here, we conducted a re-shaping process through the air-plasma etching of a polymer’s secondary mold (two-dimensional nanopillar array made of cyclo-olefin polymer (COP)) to modulate the sizes and shapes of nanopillars, then, we controlled the spectral characteristics of the imprinted plasmonic devices. The relationship between the structural change of the mold, which was based on etching time, and the optical characteristics of the corresponding plasmonic device was evaluated through experiments and simulations. According to evaluation results, the diameter of the nanopillar was controlled from 248 to 139 nm due to the etching time and formation of a pit structure. Consequently, the spectral properties changed, and responsivity to the surrounding dielectric environment was improved. Therefore, plasmonic devices based on the re-shaped COP mold exhibited a high responsivity to a refractive index of 906 nm/RIU at a wavelength of 625 nm.

The refractive index sensitivity of a localized surface plasmon resonance (LSPR) sensor is correlated to an enhanced local electromagnetic (EM) field originating from noble metal nanostructures. He...

In this paper, we report a single-step trypsin inhibitor assay on a microchannel array device immobilizing enzymes and substrates by inkjet printing. The microdevice is composed of a poly(dimethylsiloxane) (PDMS) microchannel array that immobilizes trypsin and fluorescent substrates as reactive reagents at the two bottom corners of a microchannel. Inkjet printers allow simple, accurate, and position-selective immobilization of reagents as nanoliter spots. Therefore, plural reactive reagents, such as enzymes and substrates, can be separately immobilized at different positions in the same microchannel without mixing, and thus allowing for single-step operation by simply introducing a sample solution through capillary action. Furthermore, reproducible fabrication and mass production of the device could be expected. In this study, the efficiency of an acidic solution as a spotting agent for protease immobilization to prevent decrease in the fluorescence intensity was confirmed. Additionally, single-step trypsin inhibitor screening was performed using three inhibitors. Finally, we investigated the storage stability of the device and confirmed that it remained stable for at least 10 days.

Background and Purpose: High blood pressure increases bleeding risk during treatment with antithrombotic medication. The association between blood pressure levels and the risk of recurrent stroke during long-term secondary stroke prevention with thienopyridines (particularly prasugrel) has not been well studied. Methods: This was a post hoc analysis of the randomized, double-blind, multicenter PRASTRO-I trial (Comparison of Prasugrel and Clopidogrel in Japanese Patients With Ischemic Stroke-I). Patients with noncardioembolic stroke were randomly assigned (1:1) to receive prasugrel 3.75 mg/day or clopidogrel 75 mg/day for 96 to 104 weeks. Risks of any ischemic or hemorrhagic stroke, combined ischemic events, and combined bleeding events were determined based on the mean level and visit-to-visit variability, including successive variation, of systolic blood pressure (SBP) throughout the observational period. These risks were also compared between quartiles of mean SBP level and successive variation of SBP. Results: A total of 3747 patients (age 62.1±8.5 years, 797 women), with a median average SBP level during the observational period of 132.5 mm Hg, were studied. All the risks of any stroke (146 events; hazard ratio, 1.318 [95% CI, 1.094–1.583] per 10-mm Hg increase), ischemic stroke (133 events, 1.219 [1.010–1.466]), hemorrhagic stroke (13 events, 3.247 [1.660–6.296]), ischemic events (142 events, 1.219 [1.020–1.466]), and bleeding events (47 events, 1.629 [1.172–2.261]) correlated with increasing mean SBP overall. Similarly, an increased risk of these events correlated with increasing successive variation of SBP (hazard ratio, 3.078 [95% CI, 2.220–4.225] per 10-mm Hg increase; 3.051 [2.179–4.262]; 3.276 [1.172–9.092]; 2.865 [2.042–4.011]; 2.764 [1.524–5.016], respectively). Event rates did not differ between the clopidogrel and prasugrel groups within each quartile of SBP or successive variation of SBP. Conclusions: Both high mean SBP level and high visit-to-visit variability in SBP were significantly associated with the risk of recurrent stroke during long-term medication with either prasugrel or clopidogrel after stroke. Control of hypertension would be important regardless of the type of antiplatelet drugs. Registration: URL: https://www.clinicaltrials.jp ; Unique identifier: JapicCTI-111582.

An ionic liquid-based thin (~ 1 µm) colorimetric membrane (CM) is a key nano-tool for optical ion sensing, and a two-dimensional photonic crystal slab (PCS) is an important nano-platform for ultimate light control. For highly sensitive optical ion sensing, this report proposes a hybrid of these two optical nano-elements, namely, a CM/PCS hybrid. This structure was successfully fabricated by a simple and rapid process using nanoimprinting and spin-coating, which enabled control of the CM thickness. Optical characterization of the hybrid structure was conducted by optical measurement and simulation of the reflection spectrum, indicating that the light confined in the holes of the PCS was drastically absorbed by the CM when the spectrum overlapped with the absorption spectrum of the CM. This optical property obtained by the hybridization of CM and PCS enabled drastic improvement in the absorption sensitivity in Ca ion sensing, by ca. 78 times compared to that without PCS. Experimental and simulated investigation of the relation between the CM thickness and absorption sensitivity enhancement suggested that the controlled light in the PCS enhanced the absorption cross-section of the dye molecules within the CM based on the enhanced local density of states. This highly sensitive optical ion sensor is expected to be applied for micro-scale bio-analysis like cell-dynamics based on reflectometric Ca ion detection.

Herein, a fully lipophilic ionic liquid (IL) comprising a lipophilic fluorescein anion and a trihexyltetradecylphosphonium cation was synthesized and used as the plasticizer for a plasticized poly(vinyl chloride) (PVC) membrane optode. Systematic investigation of the alkyl chain length of the fluorescein anion proved the significance of lipophilicity for obtaining the reversible absorbance measurements. A PVC membrane fabricated with the synthesized lipophilic IL was observed to comprise an unusually high dye concentration (915 mmol kg−1) and exhibited good sensitivity as well as response time in its sensor performance. The sensitivity of the presented PVC membrane was 26-fold higher than that of a conventional optode membrane with the same membrane thickness and the same lipophilic dye of typical dye content (1 wt%). The response time was observed to be >120-fold faster by using a significantly thinner PVC membrane (approx. 140 nm). Heparin is known to be a polyanionic anticoagulant, and the presented PVC membrane exhibited an extremely fast response (20–150 seconds) to the heparin in diluted serum within the required concentration region. Thus, the lipophilic IL-based dye could significantly improve the sensor performance in conventional optodes, especially for an analyte showing slow diffusion, such as macromolecular heparin.

A highly lipophilic ionic liquid (IL) consisting of stoichiometrically equal amounts of two purely functional chemical sensing molecules, an anionic calcium ionophore and a cationic dye, was synthesized for the first time, and used as a component for a poly(vinyl chloride)-based or neat liquid membrane optical sensor. This is the first report of an IL consisting of purely functional chemical sensing molecules, which is completely different from the previously reported ionic liquids typically consisting of imidazolium or other lipophilic cations. Since the present IL contained an extremely high concentration of dyes, the IL-based sensor showed dramatically enhanced sensitivity (13 times higher compared to that of a conventional sensor), and fully reversible and selective response to Ca2+. Preliminary investigation on the unique response characteristics of the present liquid membrane IL-based sensor was performed with the responses to Ca2+ and Na+.

In this study, we developed a novel particle-type material comprised of graphene and polyethylene glycol (PEG) to solve the slow response problem of the previously reported microdevice for single-step immunoassay. Characterization of these graphene/PEG hybrids (GPH) was carried out using a transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, and fluorescence analysis. We found that PEG attached to graphene surfaces behave like molecular sieves to separate free fluorescently-labeled antibodies from antigen-bound antibody immunocomplexes. The free antibody is PEG-permeable and directly adsorbs onto the graphene surface, where fluorescence is quenched by fluorescence resonance energy transfer. On the contrary, immunocomplexes are PEG-impermeable; thus, as the concentration of the antigen increases, the concentration of PEG-impermeable immunocomplexes also increases; consequently, increasing the fluorescence intensity of the solution. After characterizing GPH, single-step immunoassay microdevices were prepared by separately immobilizing the GPH and fluorescently-labeled antibodies on two independent microchannel arrays and combining them. Finally, we tested the proposed immunoassay microdevices with various known concentrations of C-reactive protein (CRP), an inflammatory marker protein, and successfully measured CRP concentration within an approximate time of 2 min by monitoring the fluorescence intensity change.

Exosomes, a type of extracellular vesicle with a diameter of 30&ndash, 150 nm, perform key biological functions such as intercellular communication. Recently, size sorting of exosomes has received increasing attention in order to clarify the correlation between their size and components. However, such sorting remains extremely difficult. Here, we propose to sort their size by controlling their electrokinetic migration in nanochannels in a micro-nanofluidic device, which is achieved by tuning the thickness of the electric double layers in the nanochannels. This approach was demonstrated experimentally for exosomes smaller than 250 nm. Using different running buffer concentrations (1 &times, 10&minus, 3, 1 &times, 4, and 1 &times, 5 M), most of the exosomes larger than 140, 110, and 80 nm were successfully cut off at the downstream of the nanochannels, respectively. Therefore, it is clarified that the proposed method is applicable for the size sorting of exosomes.

Herein, we describe the development of a novel material, "enzyme-responsive fluorescent ionic liquid", which enabled a highly sensitive detection of alkaline phosphatase (ALP). We prepared a plasticized poly(vinyl chloride) (PVC) membrane using this new material as a plasticizer and quantified ALP in aqueous solutions. Preliminary results suggested that the PVC membrane responded to ALP at an interface between the membrane and the sample solution with anion extraction to maintain electroneutrality in the membrane phase. The developed PVC membrane showed an approximately six-times higher sensitivity than the conventional membrane, thereby demonstrating highly sensitive ALP detection. These results suggested the potential applicability of the proposed membrane for highly sensitive protein detection by using ALP-labeled antibodies.

In this study, we developed a polymer/TiO2 hybrid two-dimensional photonic crystal (2D-PhC) optical ion sensor that shows fluorescence enhancement based on the matching of the fluorescence wavelength and the confinable wavelength of a 2D-PhC. The polymer/TiO2 hybrid PhC was fabricated from a TiO2 pillar array, where the interspace was filled with a polymer containing a fluorescent dye. We developed a method of fabricating the TiO2 pillar array based on nanoimprint lithography using liquid phase deposition. The polymer/TiO2 hybrid PhC was fabricated on a poly(ethylene terephthalate) (PET) film. Thus, it can be easily cut because of its high flexibility and used for ion sensing applications. By using a fluorescent dye-containing polymer (poly(methyl methacrylate), PMMA) for the fabrication of the polymer/TiO2 hybrid PhC, the fluorescence intensity was drastically (25 times) increased. Subsequently, we used an ion-sensing polymer composed of plasticized poly(vinyl chloride) (PVC), a fluorescent dye, and ion recognition elements in the polymer/TiO2 hybrid PhC to enable its use as a highly sensitive ion sensor. As a result, the ion sensor based on the plasticized-PVC/TiO2 hybrid PhC exhibited four times greater fluorescence intensity compared with that based on the plasticized PVC without the PhC structure.

An ionic liquid-based dye was synthesized and directly used as a plasticizer in a plasticized poly(vinyl chloride) (PVC) membrane-based anion optode. In contrast to conventional PVC-based optode membranes, the optode membrane presented can contain a very high concentration (∼296.1 mmol/kg) of dye molecules (for conventional membranes, the concentration is typically 5–50 mmol/kg), which allowed the preparation of a very thin membrane (∼0.8 μm) exhibiting a rapid response time (6.5 s), with sufficient sensitivity (1.2 Absorbance unit (AU)) and excellent response reversibility. We prepared the ionic liquid-based dye using trihexyltetradecylphosphonium (P66614) as a lipophilic monocation and naphtholphthalein (NP) as a dianion. The prepared ionic liquid-based dye contained approximately 40% dye relative to the formula [P66614]2[NP], as confirmed by proton NMR measurements and elemental analysis. Optimized PVC content of the plasticized PVC membrane, without leaching of dye, was 10 wt%. Under these conditions, surprisingly, the membrane exhibited fully reversible responses to acidic and basic solutions, even though the dye was not lipophilized using long alkyl chains, something that has been considered an indispensable chemical modification for dyes used in typical ion-selective optodes. A comparison with conventional plasticized PVC membranes, using nitrophenyl octyl ether (NPOE) as a typical plasticizer and a lipophilic derivative of NP as a lipophilic dye, was carried out in terms of the response time and sensitivity, the results of which demonstrated the advantages of the present approach. Finally, as the simplest application, a plasticized PVC membrane based on an ionic liquid-based dye was successfully applied to coextraction-based anion sensing.

In conventional microfluidic bioassays, short optical path length and extremely small quantities of analytes sometimes can significantly reduce detectability. Various sample preconcentration techniques have been reported for improving the detectability of bioassays. In the present study, we developed a novel preconcentration technique, "double sweeping", utilizing cationic and anionic micelles simultaneously. Microscopic observations demonstrated that double sweeping enabled a sample solution, which initially filled a capillary, to be focused into an extremely narrow band. Initially, the sample molecules are swept from cathode to anode, or anode to cathode, based on conventional sweeping with an anionic or cationic micellar solution, respectively. Then, the fronts of the swept bands collide with each other in the capillary and halt at the interface between the bands. The sample molecules in the micellar solutions continue to move toward the interface because of the electrophoretic migration of the micelles, which results in further focusing and suppression of the band broadening due to molecular diffusion. We demonstrated that higher preconcentration efficiency was achieved using double sweeping than using conventional sweeping. In addition, double sweeping was successfully incorporated into a multiple enzyme activity assay using an arrayed reagent-release capillary, resulting in simple, rapid, simultaneous, and highly sensitive assays of caspase-3, alkaline phosphatase, and trypsin.

A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field-amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed-polarity voltages on a cross-channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP-FASI. The applied pressure generated a counter-flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520-fold sensitivity increase was obtained in the LVSEP-FASI analysis of a standard dye, which was 33-times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100-fold sensitivity increase was achieved with the 75-μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d-leucine.

SUMMARY We fabricated 1 STEP biomarker protein analysis device using silver nanoparticles (AgNPs)-contained hydrogel and reagent-immobilized cartridge. Specific color change of AgNPs based on localized surface plasmon resonance (LSPR) was applied to develop the novel colorimetric protein analysis device. In this report, we constructed this device by four components, AgNPs-contained hydrogel, glucose oxidase (GOx) antibody release pad, glucose release pad, and antibody-immobilized membrane. AgNPs-contained hydrogel detected 0.001 to 10 μg/mL GOx-antibody. AgNPs-contained hydrogel absorbance became altered 90.0% GOx release pad as compared to GOx-antibody solution under the reaction time 60 min. AgNPs-contained hydrogel absorbance became altered 98.4% glucose release pad as compared to glucose solution under the reaction time 60 min. We applied AgNPs-contained hydrogel and antibody-immobilized membrane to detect human immunoglobulin G (IgG). As a result, we found these could detect 1 μg/mL or lower concentration of human IgG.

Herein, we describe the development of a novel material, "enzyme-responsive fluorescent ionic liquid", which enabled a highly sensitive detection of alkaline phosphatase (ALP). We prepared a plasticized poly(vinyl chloride) (PVC) membrane using this new material as a plasticizer and quantified ALP in aqueous solutions. Preliminary results suggested that the PVC membrane responded to ALP at an interface between the membrane and the sample solution with anion extraction to maintain electroneutrality in the membrane phase. The developed PVC membrane showed an approximately six-times higher sensitivity than the conventional membrane, thereby demonstrating highly sensitive ALP detection. These results suggested the potential applicability of the proposed membrane for highly sensitive protein detection by using ALP-labeled antibodies.

A novel method for the intact immobilization of a very-thin and soft PVC membrane on a convex-shaped poly(dimethylsiloxane) (PDMS) surface is described. The present method using PVA film as a sacrificial layer allowed successful immobilization of an intact PVC membrane using an ionic liquid-based dye on only the convex-shaped PDMS surface without any deformation or increase of the inhomogeneity. In addition, two different kinds of PVC membranes were successfully immobilized simultaneously toward multiplexed detection.

To achieve an on-line coupling of the sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) with microchip gel electrophoresis (MCGE), a sample solution, a background solution for LVSEP and a sieving solution for MCGE were loaded in a T-form channel and three reservoirs on PDMS microchips. By utilizing the difference in the flow resistance of the two channels, a low-viscosity sample and a viscous polymer solution were easily introduced into the LVSEP and MCGE channels, respectively. Fluorescence imaging of the sequential LVSEP-MCGE processes clearly demonstrated that a faster stacking of anionic fluorescein and successive introduction into the MCGE channel can be carried out on the T-channel chip. To evaluate the preconcentration performance, a conventional MCZE analysis of fluorescein on the cross-channel chip was compared with LVSEP-MCGE on the short T-channel chip, and as a result that the value of sensitive enhancement factor (SEF) was estimated to be 370. The repeatability of the peak height was good with the RSD value of 3.2%, indicating the robustness of the enrichment performance. In the successive LVSEP-MCGE analysis of φX174/HaeIII digest, the DNA fragments were well enriched to a sharp peak in the LVSEP channel, and they were separated in the MCGE channel, whose electropherogram was well-resembled with that in the conventional MCGE. The values of SEF for the DNA fragments were calculated to be ranging from 74 to 108. Thus, the successive LVSEP-MCGE analysis was effective for both preconcentrating and separating DNA fragments.