Search

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.

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.

We describe the fabrication and simple packaging of a mass-producible analytical microdevice, the third-generation capillary-assembled microchip (CAs-CHIP-3G), capable of single-step and multiplexed bioassay. The packaged CAs-CHIP format is an assembly of a laminated-dry reagent-release capillary (dRRC) array (CAs-CHIP-3G), paper pad, and vinyl tape (electrical tape) that acts as biosensor array, absorbent of excess sample solution, and adhesive for forming sample introduction microchannel, respectively. Sample introduction is carried out by simply piercing the vinyl tape with syringe and subsequent manual injection. No external pump is necessary since the fluid actuation for sample introduction into the dRRC array and subsequent removal of excess sample solution by paper pad are both spontaneously occurred by capillary action. Evaporation of sample solution is prevented by sealing both ends of dRRC array with oil allowing long time incubation for enzyme reaction. To demonstrate the potential application for simple and multiplexed bioassay, multiplexed glycosidase enzymes (α- and β-galactosidase, α-glucosidase, α-mannosidase) assay are carried out as an example.

Metal nanostructures have great potential for optical label-free biosensors based on localized surface plasmon resonance (LSPR). The sensitivity of a metal nanostructure-based label-free biosensor (i.e., plasmonic sensor) depends on its plasmonic properties, which suffer a decrease in sensitivity by energy losses in the metal material. Here, we demonstrate an approach to improve the plasmonic properties of metal nanostructures by controlling the carrier density in the base polymer material using titanium nitride (TiN). It is expected that the light energy absorbed by TiN is converted into excitons, and it will assist LSPs in the metal nanostructure; thus, the losses of the metal material are compensated by the excitons excited in TiN. We designed a TiN-contained polymer-metal/core-shell structured nanocone array (NCA), comprising TiN nanoparticles (NPs) in a polymer core and metal shell (Au or Ag), and realized improvement of the refractive index (RI) sensitivity of a label-free biosensor by optimizing the TiN-contained polymer composition. As a result, the TiN-contained polymer-metal NCA, with a TiN NP concentration of 10 wt% in the polymer core, had a 1.5-fold higher RI sensitivity than that of the same NCA without TiN NPs. The results of the resistance measurement of the metal surface with the TiN NP-contained polymer (10 wt%) under light exposure suggest the conversion of exposed light into LSPs of metal via TiN. It is suggested that plasmonic properties and sensor performances can be improved by the presented approach. Moreover, in DNA hybridization detection, an extremely low limit of detection of 117.5 fM was achieved.

In this study, a highly sensitive capillary-based enzyme-linked immunosorbent assay (ELISA) has been developed for the analysis of picomolar levels of thrombin-cleaved osteopontin (trOPN), a potential biomarker for ischemic stroke, in human plasma. Using a square capillary coated with 8.5 μg/ml anti-human trOPN capture antibody for ELISA, the linear range obtained was 2 to 16 pM trOPN antigen. This concentration range was in the detection window of trOPN antigen in plasma samples. Compared with the conventional microplate-based ELISA, the current capillary technique significantly reduced the amounts of reagent from milliliter to microliter, reduced the analysis time from 8 to 3 h, and had a better sensitivity and detection limit performance from approximately 50 pM down to 2 pM of trOPN antigen. These results indicate that this capillary-based immunoassay is a potential tool for biomarker detection and may be useful in clinical trials and medical diagnostic applications.

An on-line sample preconcentration based on transient trapping was applied to cyclodextrin electrokinetic chromatography (CDEKC) to realize a highly sensitive chiral analysis in capillary electrophoresis. The partial introduction of a micellar solution before the electrokinetic injection of a sample solution provided the effective preconcentration and enantioseparation of chiral compounds, resulting in the up to 240-fold enhancements of peak intensity and 100-fold improvements of limit of detection of (R),(S)-1-aminoindan as the model analyte. The demonstrated method could be applied to the other pharmaceutical compounds, which allowed five chiral analytes to be resolved with 40- to 160-fold sensitivity enhancements at once. The enantioseparation efficiency of the proposed method was slightly lower as compared to the conventional CDEKC, while the acceptable baseline separations of enantiomers were obtained in transient trapping-CDEKC relative to the undesirable resolution in the CDEKC with other preconcentration techniques. Additionally, it was clarified that transient trapping-CDEKC was also applicable to the analysis of enantiomeric excess, providing the sensitive detection of 43 ppb of (R)-chlorpheniramine in 5 ppm (S)-chlorpheniramine solution commercially available.

To realize the high-performance and simple-operation analysis of cationic compounds in capillary electrophoresis, we investigated large-volume sample stacking with an electroosmotic flow pump (LVSEP) using capillaries with hydrophilic and weakly cationic inner surface. Three capillary modification methods were employed: thermally passivated physical coating with polymer mixture of poly(vinyl alcohol) and poly(allylamine); covalent modification with random copolymer of acryl amide and 3-(methacryloylamino)propyltrimethylammonium chloride; easily preparable physical coating with dimethyldioctadecylammonium bromide and polyoxyethylene stearate. In these capillaries, the electroosmotic flow (EOF) was well suppressed in the high ionic strength (I) electrolyte under the acidic and basic pH, whereas the EOF was enhanced in the low I electrolyte, indicating a suitable EOF property for the rapid LVSEP and following separation. In the LVSEP-capillary zone electrophoresis (CZE) analyses of benzylamine and 1-naphthylethylamine, up to 550-fold sensitivity increases were successfully obtained in the three capillaries without significantly reducing the repeatability and resolution. LVSEP-cyclodextrin-modified CZE of chlorpheniramine and brompheniramine was also carried out, resulting in up to 380-fold sensitivity enhancement with keeping the baseline separation for the enantiomers. Finally, we performed the LVSEP-CZE analysis of basic proteins, where up to 100-fold sensitivity increases were achieved, but a peak broadening was observed due to the sample adsorption in the low I sample matrix.

To improve the sensitivity in chiral analysis by capillary electrophoresis without loss of optical resolution, application of large-volume sample stacking with an electroosmotic flow pump (LVSEP) was investigated. Effects of the addition of cyclodextrin (CD) into a running solution on the LVSEP preconcentration was theoretically studied, where the preconcentration efficiency and effective separation length would be slightly increased if the effective electrophoretic velocity (v(ep, eff, BGS)) of the analytes was decreased by interacting with CD. In LVSEP-CD-modified capillary zone electrophoresis (CDCZE) and LVSEP-CD electrokinetic chromatography with reduced v(ep, eff, BGS), up to 1000-fold sensitivity increases were achieved with almost no loss of resolution. In LVSEP-CD-modified micellar electrokinetic chromatography of amino acids with increased v(ep, eff, BGS), a 1300-fold sensitivity increase was achieved without much loss of resolution, indicating the versatile applicability of LVSEP to many separation modes. An enantio-excess (EE) assay was also carried out in LVSEP-CDCZE, resulting in successful analyses of up to 99.6% EE. Finally, we analyzed ibuprofen in urine by desalting with a C(18) solid-phase extraction column. As a typical result, 250ppb ibuprofen was well concentrated and optically resolved with 84.0-86.6% recovery in LVSEP-CDCZE, indicating the applicability of LVSEP to real samples containing a large amount of unnecessary background salts.

In order to provide an insight into the basic nature of ischemic brain injury, we sequentially studied cerebral blood flow with [99mTc]hexamethylpropyleneamine oxime single photon emission computed tomography (CBF imaging) in a patient with diffuse brain ischemia due'to prolonged cardiac arrest. On the 10th postarrest day, concentrated blood flow over superior-medial portion of the occipital lobe was demonstrated. On the 18th postarrest day, the same region became high density on a CT scan, while the concentrated flow on the CBF imaging had diminished. Thus, an abnormal cerebral blood flow (CBF) pattern preceded the density change on CT scan. On the 23rd postarrest day, remarkably concentrated flow over the brainstem was demonstrated. This might have illustrated the reduced metabolic demand of the damaged tissue over the cerebral and cerebellar hemispheres with relative preservation of brainstem perfusion. In patients with diffuse brain ischemia, a CBF imaging may be a useful tool for clarifying pathological process and prognosis.