Evaluation of osteoblastic cell behavior upon culture on titanium substrates photo-functionalized by vacuum ultra-violet treatment.

Photo-functionalization of titanium orthopedic/prosthetic implants using ultraviolet illumination is known to improve osteogenesis. Therefore, in this study, we aimed to examine the influence of vacuum ultraviolet (VUV)-treated titanium surfaces on osteoblast cell adhesion, activity, and differentiation. Osteoblastic cells were cultured on titanium substrates treated with various VUV treatment conditions (0, 6.2, 18.7, and 37.4 J/cm2) and their behavior was evaluated. The results revealed that cell adhesion was increased whereas cell activity and differentiation ability were decreased upon cell culture on VUV-treated substrates. In particular, cell activity and differentiation ability were dramatically suppressed with 18.7 J/cm2 VUV irradiation. Within the limitations of this cell-based experiment, we clarified the VUV treatment conditions in which cell adhesion was improved but cell activity and differentiation ability were suppressed. These results indicate that VUV-treatment can be used to influence cell growth properties and can be used to accelerate or suppress cell differentiation on implant substrates.

Preliminary system of rapid analysis of blood retinol level in cattle.

Control of blood retinol levels in cattle during fattening is important in the production of marbled beef. However, it is difficult to easily measure the blood retinol concentration in the field. In this study, we attempted to develop an analysis method that does not require blood cell separation and uses a compact fluorescence analyzer that can be carried around as a preliminary system for measuring blood retinol concentration in the field. This system was used to monitor blood retinol levels in 12 fattening cattle (14 to 27 months old) and demonstrated a strong correlation (r = 0.78) with the results obtained by the standard high-performance liquid chromatography (HPLC) method. Stronger correlations (r = 0.87) were obtained until the cattle were 24 months of age. These results suggest that higher correlations can be expected to be obtained by improving the robustness of the extraction system. Refinements for practical use need to be considered, but whole blood extraction and the vitamin A analyzer that was developed show potential to be used for on-farm monitoring of retinol levels.

An Improved Reflection Colorimeter Integrated with a Coaxial Optical-fiber Cable for Highly Sensitive Solid-phase Colorimetry Using a Membrane Filter.

Highly sensitive solid-phase colorimetry for nickel ion was demonstrated using an improved reflection colorimeter equipped with a coaxial optical-fiber cable. The nickel complex with α-furil dioxime was collected on a small-size membrane filter embedded in a disposable syringe filter unit. The leading edge of the optical-fiber cable was connected to the syringe filter unit via a Luer-lock fitting, and the color intensity of the sample on the filter was evaluated accurately. The detection limit was 0.8 ng in 2.5 mL of the complex solution (0.3 µg L-1). This improved configuration is applicable to highly sensitive on-site analysis without expensive instruments nor high laboratory skills.

Enzyme-linked immunosorbent assay based on light absorption of enzymatically generated aniline oligomer: Flow injection analysis for 3-phenoxybenzoic acid with anti-3-phenoxybenzoic acid monoclonal antibody.

A flow enzyme-linked immunosorbent assay (ELISA) method based on light absorption by enzymatically generated aniline oligomer in the presence of horseradish peroxidase (HRP), H2O2, and aniline is proposed. Aniline oligomer is rapidly formed through the polymerization reaction via the enzymatic reaction, and its fast reaction rate is beneficial for flow ELISA. An anti-3-phenoxybenzoic acid monoclonal antibody (mAb) was produced by mice, and was used for the flow competitive ELISA for the determination of 3-phenoxybenzoic acid (3PBA), which was performed on an acrylic plate having a Y-shaped channel. ABS resin beads (d = 1 mm) were filled in the channel to increase the surface area for the adsorption of the mAb. A clank-type detection chamber (optical length: 1 cm) made of polydimethylsiloxane (PDMS) containing carbon black, which can significantly decrease light scattering, was fabricated with a 3D printer. The PDMS detection chamber was connected to the outlet of the acrylic flow chip with a tube. A blue LED was used as a light source for the flow ELISA. The inhabitation concentration at 50% and the detection range (absorbance change from 90 to 10%) for the proposed flow competitive ELISA were 0.5 ppm and 0.05-5 ppm, respectively. We also performed the flow competitive ELISA in an artificial and real urine, and no significant matrix effect of the urine samples on the ELISA was found.

Highly sensitive detection of clenbuterol in urine sample by using surface plasmon resonance immunosensor.

This study proposed the filtration method for removal of inhibitors from real urine samples for immunoassay without centrifuge. Although the inhibitors could not be removed by the physical filtration, the carboxyl group terminated silica effectively removed the inhibitors. In a low pH, antibody formed aggregation due to the protonation. We propose to adjust pH of the sample solution by adding a phosphate buffer solution (pH 7.5). As a result of pretreatment, the SPR immunosensing achieved the SPR signal of 45 mdeg and a low limit of detection with 100 ppq (100 fg mL-1).

Mechanism of surface plasmon resonance sensing by indirect competitive inhibition immunoassay using Au nanoparticle labeled antibody.

We investigated the use of a surface plasmon resonance (SPR) biosensor using an antibody (Ab) labeled with Au-nanoparticle (Ab-AuNP conjugate). As clenbuterol is a small molecule, an indirect competitive inhibition immunoassay was used. The SPR immunoassay using Ab-AuNP conjugate had an extremely low limit of the detection (LOD) with a magnitude of 0.05 ppt (0.05pgmL-1), which was 40 times lower than that of unlabeled Ab. To identify the key factor in determining the LOD of the indirect competitive inhibition immunoassay, affinity constants of the surface immunoreaction (K1) and of the premixed solution (αK2) were evaluated. We found that the dielectric constant change due to AuNP labeling of Ab did not affect on the affinity constants, because all the amplification magnitude terms canceled out in the equations. Thus, the K1 and αK2 values were determined to 3.0×1011M-1 and 2.9×1012M-1, respectively, which were three and four orders of magnitude higher, respectively, than those of unlabeled Ab. The simulation plot of LOD with respect to K1 and αK2 showed that a K1 one order of magnitude lower than αK2 produced a ppt level LOD. Because the affinity constants are determined by the molar concentrations of reactant and product, the molar mass of the Ab or Ab-AuNP conjugate in the sample solution containing 1ppm (1µgmL-1) highly affects the constants. Consequently, molar mass adjustment can be used to adjust the LOD in an indirect competitive inhibition immunoassay as needed for a practical application.

Carbon-polydimethylsiloxane-based integratable optical technology for spectroscopic analysis.

A polydimethylsiloxane (PDMS)-based optical system has been demonstrated. To suppress intense background radiation due to multiple internal scatting in a transparent material, a composite structure of a carbon-PDMS compound and PDMS was proposed. The index matching of the real part of the refractive index can suppress internal scattering, and an absorption of 99-99.7% was attained by using carbon micro particles and carbon nano tubes. The black-PDMS light channel functions as a light filter for straight pass, and an optical density of 5 was obtained by bending the filter.

Highly selective and sensitive detection of ß-agonists using a surface plasmon resonance sensor based on an alkanethiol monolayer functionalized on a Au surface.

Immunosensor surfaces for surface plasmon resonance (SPR) have been constructed using a functionalized succinimidyl propanethiol monolayer as a linker to immobilize ß-agonist protein conjugates on a Au surface. Because ß-agonist is a small molecule, an indirect competitive inhibition immunoassay was used for detection. The lowest detection limits for ractopamine and salbutamol were 10 ppt (10 pg mL(-1)) and 5 ppt (5 pg mL(-1)), respectively. The fabricated immunosensor surface can be used again for detection after regeneration in 0.1 M sodium hydroxide. It was found that the same sensor surface could be reused for performing over 100 rapid immunoreactions. Moreover, one immunosensing-regeneration cycle requires only 600 s. The fabricated immunosensor surfaces were characterized using SPR and scanning tunneling microscopy observation. In the kinetic study of the indirect competitive immunosensing inhibition, the affinity constant (K1) of salbutamol antibody was smaller than the K1 of ractopamine antibody. Compared to a previous study of clenbuterol detection, it was concluded that the high K1 was coupled with low sensitivity. In the selectivity study, both immunosensor surfaces provided >90% of confidence level for the specific detection of ß-agonist compounds. The fabrication of highly selective and sensitive sensor surfaces for detecting ß-agonist compounds was confirmed.