Toyohiro Naito

Effective determination of a pharmaceutical, sulpiride, in river water by online SPE-LC-MS using a molecularly imprinted polymer as a preconcentration medium.

We report an effective and a quantitative analysis method for one of pharmaceuticals, sulpiride, in river water by online solid phase extraction (SPE) connected with liquid chromatography-mass spectrometry (LC-MS) using a molecularly imprinted polymer as a preconcentration medium. The polymer prepared with a pseudo template molecule showed the selective retention ability based on the interval recognition of functional groups in sulpiride. Also, the imprinted polymer provided an effective concentration of a trace level of sulpiride in offline SPE with dual washing processes using water and acetonitrile, although another imprinted polymer prepared by an authentic method using sulpiride and methacrylic acid as a template and a functional monomer, respectively, showed the selective adsorption only in organic solvents. Furthermore, we employed the imprinted polymer as the preconcentration column of online SPE-LC-MS and the results supposed that the proposed system allowed the quantitative analysis of sulpiride with high sensitivity and recovery (10ng/L at 96%). Additionally, the determination of sulpiride in real river water without an additional spiking was effectively achieved by the system.

Inkjet Injection of DNA Droplets for Microchannel Array Electrophoresis

We demonstrated DNA droplets could be injected with an inkjet injector for microchannel array electrophoresis and attained high throughput analysis of biomolecules. This injection method greatly reduced both analysis time and sample amount, compared with a conventional microchip electrophoresis method, and allowed high parallelization of a microchannel array on a small substrate. Since we do not need to use complicated electric programs or microchannel design, our injection method should facilitate omics analyses and contribute to high performance clinical assays.

Three-Dimensional Fabrication for Microfluidics by Conventional Techniques and Equipment Used in Mass Production

This paper presents a simple three-dimensional (3D) fabrication method based on soft lithography techniques and laminated object manufacturing. The method can create 3D structures that have undercuts with general machines for mass production and laboratory scale prototyping. The minimum layer thickness of the method is at least 4 µm and bonding strength between layers is over 330 kPa. The performance reaches conventional fabrication techniques used for two-dimensionally (2D)-designed microfluidic devices. We fabricated some 3D structures, i.e., fractal structures, spiral structures, and a channel-in-channel structure, in microfluidic channels and demonstrated 3D microfluidics. The fabrication method can be achieved with a simple black light for bio-molecule detection; thus, it is useful for not only lab-scale rapid prototyping, but also for commercial manufacturing.

Unique Separation Behavior of a C60 Fullerene-Bonded Silica Monolith Prepared by an Effective Thermal Coupling Agent.

Herein, we report a newly developed C60 fullerene-bonded silica monolith in a capillary with unique retention behavior due to the structure of C60 fullerene. N-Hydroxysuccinimide (NHS)-conjugated C60 fullerene was successfully synthesized by a thermal coupling agent, perfluorophenyl azide (PFPA), and assigned by spectroscopic analyses. Then, NHS-PFPA-C60 fullerene was attached onto the surface of a silica monolith in a capillary. The capillary provided specific separation ability for polycyclic aromatic hydrocarbons in liquid chromatography by an effective π-π interaction. Furthermore, corannulene, which has a hemispherical structure, was selectively retained in the capillary based on the specific structural recognition due to the spherical C60 fullerene. This is the first report revealing the spherical recognition ability by C60 fullerene in liquid chromatographic separation.

Tunable separations based on a molecular size effect for biomolecules by poly(ethylene glycol) gel-based capillary electrophoresis

We report novel capillary gel electrophoresis (CGE) with poly(ethylene glycol) (PEG)-based hydrogels for the effective separations of biomolecules containing sugars and DNAs based on a molecular size effect. The gel capillaries were prepared in a fused silica capillary modified with 3-(trimethoxysilyl)propylmethacrylate using a variety of the PEG-based hydrogels. After the fundamental evaluations in CGE regarding the separation based on the molecular size effect depending on the crosslinking density, the optimized capillary provided the efficient separation of glucose ladder (G1 to G20). In addition, another capillary showed the successful separation of DNA ladder in the range of 10-1100 base pair, which is superior to an authentic acrylamide-based gel capillary. For both glucose and DNA ladders, the separation ranges against the molecular size were simply controllable by alteration of the concentration and/or units of ethylene oxide in the PEG-based crosslinker. Finally, we demonstrated the separations of real samples, which included sugars carved out from monoclonal antibodies, mAbs, and then the efficient separations based on the molecular size effect were achieved.

Simple and Effective Label-Free Capillary Electrophoretic Analysis of Sugars by Complexation Using Quinoline Boronic Acids

An effective separation and detection procedure for sugars by capillary electrophoresis (CE) using a complexation between quinolineboronic acid (QBA) and multiple hydroxyl structure of sugar alcohol is reported. We investigated the variation of fluorescence spectra of a variety of QBAs with sorbitol at a wide range of pH conditions and then found that 5-isoQBA strongly enhanced the fluorescence intensity by the complexation at basic pH conditions. The other sugar alcohols having multiple hydroxyls also revealed the enhancement of the fluorescence intensity with 5-isoQBA, whereas the alternation of the intensity was not found in the sugars such as glucose. After optimization of the 5-isoQBA concentration and pH of the buffered solution in CE analysis, 6 sugar alcohols were successfully separated in the order based on the formation constants with 5-isoQBA, which were calculated from the variation of the fluorescence intensity with each sugar alcohol and 5-isoQBA. Furthermore, the limits of detection for sorbitol and xylitol by the CE method were estimated at 15 and 27 μM, respectively.

Quantitative Ligand Immobilization Using Alginate Hydrogel Formed in a Capillary: Application for Online Affinity Concentration

To simplify a quantitative immobilization procedure of ligands with maintaining their activities, we developed an automated preparation method using an alginate hydrogel partially formed in a capillary. After a sodium alginate solution containing a ligand was injected into the capillary, a background solution containing Ca(2+) was then introduced into the sodium alginate solution zone by applying an appropriate voltage for the hydrogelation, resulting in encapsulation of the ligand by the formed alginate hydrogel. According to the estimated binding capacity for biotin by the encapsulated avidin, the injected avidin was immobilized quantitatively by the formed hydrogel with keeping its affinity. When avidin (3.5-35.2 ng) was immobilized by the proposed method, the immobilization efficiency was estimated to be almost 100%. Furthermore, by using the prepared capillary, biotinylated fluorescein was specifically trapped and separated due to the affinity of the encapsulated avidin. By the change of pH of the background solution, the concentrated analytes could be easily eluted, resulting in 90% recovery with high reproducibility by using 1.18 fmol biotinylated sample.

New platform for simple and rapid protein-based affinity reactions

We developed a spongy-like porous polymer (spongy monolith) consisting of poly(ethylene-co-glycidyl methacrylate) with continuous macropores that allowed efficient in situ reaction between the epoxy groups and proteins of interest. Immobilization of protein A on the spongy monolith enabled high-yield collection of immunoglobulin G (IgG) from cell culture supernatant even at a high flow rate. In addition, immobilization of pepsin on the spongy monolith enabled efficient online digestion at a high flow rate.

Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A detected by sodium dodecyl sulfate-capillary gel electrophoresis

&NA; This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal antibody‐A (mAb‐A), detected by sodium dodecyl sulfate‐capillary gel electrophoresis (SDS‐cGE). The fragment migrated close to the internal standard (10 kDa marker) of SDS‐cGE and increased about 0.5% under a 25 °C condition for 6 months. Generally, identification of fragments observed in SDS‐cGE is challenging to carry out due to the difficulty of collecting analytical amounts of fractionations from the capillary. In this study, in‐gel digestion peptide mapping and reversed phase liquid chromatography‐mass spectrometry (RPLC–MS) were employed to elucidate the structure of the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain HC1‐104. The fragment contained complementarity determining regions (CDRs), which are significant to antigen binding, and thus would affect the efficacy of mAb‐A. In addition, SDS‐cGE without the 10 kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment revealed that the fragment increases 0.2% per year in storage at 5 °C. The combination of the three analytical methodologies successfully identified the impurity peak detected by SDS‐cGE, providing information critical to assuring the quality and stability of the biotherapeutics. Graphical abstract Figure. No caption available. HighlightsA fragment of monoclonal antibody was detected close to the 10 kDa marker in SDS‐capillary gel electrophoresis and increased about 0.5% under 25 °C for 6 months.The fragment was identified and characterized by using in‐gel digestion peptide mapping, RPLC–MS, and a Gelfree 8100 fractionation system.Three analytical methods showed comparable results proving the fragment was a fraction of heavy chain HC1‐104.

Selective adsorption of trypsin using molecularlyimprinted polymers prepared with PEG-basedhydrogels containing anionic functionalmonomers

Abstract Molecularly imprinting (MI) hydrogels for selective adsorption of trypsin are reported. The trypsin imprinted hydrogels were prepared using a polyethylene glycol (PEG)-based dimethacrylate as a crosslinker and anionic functional monomers. The hydrogel prepared without any functional monomers showed significantly low ability to adsorb a variety of proteins. We optimized the concentration and the length of PEG units of the crosslinkers to achieve the complete removal of the template molecule and suitable selective adsorption. Additionally, the functional monomers chosen were anionic since the template, trypsin, is a basic protein. The adsorption tests for proteins, done on the prepared MI gels, indicated that the MI gel prepared with sodium allyl sulfonate (AS) as a functional monomer showed much higher selective adsorption for trypsin, even though a mixture of trypsin and cytochrome c was used as the protein solution. The selective adsorption was more effective in a NaCl solution in which the non-specific adsorption by a sulfonate is suppressed, similarly to our findings in a previous study. The MI gel prepared with acrylic acid also showed the selectivity, although the adsorption strength was lower than that of the MI gel containing AS. We believe that the present study constitutes the first approach for the selective adsorption of trypsin using PEG-based hydrogels.