Toemsak Srikhirin

Immobilization of glucose oxidase onto Langmuir–Blodgett films of poly-3-hexylthiophene

Abstract Monolayers of poly(3-hexyl thiophene) (P3HT) were obtained by dispensing a solution of P3HT with stearic acid (SA) in chloroform at air-water interface using Joyce–Loebl LB trough. These films were transferred onto indium-tin-oxide coated glass plate by vertical dipping method. Enzyme glucose oxidase (GOX) was immobilized on the films using LB technique. These films were characterized by X-ray diffraction and atomic force microscopic techniques. Photometric response of these P3HT/SA/GOX films was obtained as a function of glucose concentration. These P3HT/SA/GOX LB films show a linearity from 100 to 500 mg/dl of glucose concentration.

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.

ABO blood-typing using an antibody array technique based on surface plasmon resonance imaging.

In this study, readily available antibodies that are used in standard agglutination tests were evaluated for their use in ABO blood typing by a surface plasmon resonance imaging (SPR imaging) technique. Five groups of antibodies, including mixed clones of anti-A, anti-B, and anti-AB, and single clones of anti-A and anti-B, were used to construct the five-line detection arrays using a multichannel flow cell in the SPR imager. The red blood cell (RBC) samples were applied to a multichannel flow cell that was orthogonal to the detection line arrays for blood group typing. We found that the blood samples were correctly grouped in less than 12 min by the SPR imaging technique, and the results were consistent with those of the standard agglutination technique for all 60 samples. We found that mixed clones of antibodies provided 33%–68% greater change in the SPR signal than the single-clone antibodies. Applying the SPR imaging technique using readily available antibodies may reduce the costs of the antibodies, shorten the measurement time, and increase the throughput.

SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification

In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA.

Fiber optic electric field sensors using polymer-dispersed liquid crystal coatings and evanescent field interactions

Abstract We report a simple evanescent field fiber optic electric field sensor constructed by coating the exposed fiber optic core with a polymer-dispersed liquid crystal (PDLC). It is well known that the effective refractive index of the liquid-crystal polymers has a large dependence on the direction and the magnitude of an applied electric field. This dependence was large enough in our devices to enable simple transmission measurements to detect the presence of an applied electric field across the fiber optic. The sensors showed good sensitivity and reproducibility with its response dominated by the RC time constant of the structure rather than the response of the PDLC. Its response time was approximately 3 min with 15–20 min relaxation time. Using an electric circuit model of the device, we also discuss how the sensors response time can be improved by many orders of magnitude.

Antimicrobial effects of silver zeolite, silver zirconium phosphate silicate and silver zirconium phosphate against oral microorganisms

OBJECTIVE To evaluate the antimicrobial activities of silver inorganic materials, including silver zeolite (AgZ), silver zirconium phosphate silicate (AgZrPSi) and silver zirconium phosphate (AgZrP), against oral microorganisms. In line with this objective, the morphology and structure of each type of silver based powders were also investigated. METHODS The antimicrobial activities of AgZ, AgZrPSi and AgZrP were tested against Streptococcus mutans, Lactobacillus casei, Candida albicans and Staphylococcus aureus using disk diffusion assay as a screening test. The minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) were determined using the modified membrane method. Scanning electron microscope and X-ray diffraction were used to investigate the morphology and structure of these silver materials. RESULTS All forms of silver inorganic materials could inhibit the growth of all test microorganisms. The MIC of AgZ, AgZrPSi and AgZrP was 10.0 g/L whereas MLC ranged between 10.0-60.0 g/L. In terms of morphology and structure, AgZrPSi and AgZrP had smaller sized particles (1.5-3.0 µm) and more uniformly shaped than AgZ. CONCLUSIONS Silver inorganic materials in the form of AgZ, AgZrPSi and AgZrP had antimicrobial effects against all test oral microorganisms and those activities may be influenced by the crystal structure of carriers. These results suggest that these silver materials may be useful metals applied to oral hygiene products to provide antimicrobial activity against oral infection.

The effects of silane-SiO2 nanocomposite films on Candida albicans adhesion and the surface and physical properties of acrylic resin denture base material

STATEMENT OF PROBLEM Polysiloxane has been used as a coupling material in restorative dental materials for several decades. However, few studies are available on the application of polysiloxane in other dental prosthesis functions. PURPOSE The purpose of this study was to investigate the effects of silane-SiO2 nanocomposite films on Candida albicans adhesion and the surface and physical properties of acrylic resin denture base materials. MATERIAL AND METHODS Specimens were separated into 2 groups, uncoated and coated. They were coated with a film by using the dip-coating method. Specimens were incubated with Candida albicans 10(7) cells/mL for 1 hour, and the adherent cells were counted under an optical microscope. The following surface properties were measured: surface chemical composition with Fourier-transform infrared spectrometry, surface roughness with a surface profiler, surface energy with the sessile drop method, and surface hardness with a microhardness tester. The physical properties, including water sorption, water solubility, ultimate flexural strength, and flexural modulus, were evaluated according to International Organization for Standardization 20795-1 requirements. The adhesion of Candida albicans and the surface properties of the specimens were investigated after cleaning with effervescent tablets and brushing. An MTT assay was used to evaluate the coated specimens. The results were statistically analyzed with the Mann-Whitney U test (α=.05). RESULTS A significant reduction in Candida albicans adhesion (P=.002) was observed before cleaning. In addition, the surface energy was comparable (P=.100), the surface hardness increased significantly (P=.008), and the surface roughness remained unchanged (P=.310). After cleaning with effervescent tablets, a significant decrease in Candida albicans adhesion (P=.002) and in surface roughness (P=.008) was observed; however, similar surface energies were measured (P=.100). After cleaning with a toothbrush, the adhesion of Candida albicans was significantly higher on the coated specimen than on the uncoated specimen (P=.004). The surface roughness values were significantly different (P=.008), and the surface energies could not be determined. The coated specimen had a silicon-oxygen-silicon peak instead of an ester bond in the polymethyl methacrylate structure. The coating film reduced the water sorption (P=.008) and water solubility (P=.032), and increased the ultimate flexural strength (P=.008) and flexural modulus (P=.032) of the specimen. The coated specimen also had satisfactory toxicity results. CONCLUSIONS Reduced Candida albicans adhesion was observed on the coated specimens. The polymeric film did not change the surface roughness of the acrylic resin specimen; however, it did slightly reduce the surface energy. The physical properties of the acrylic resin did not decrease after it was coated with the film.

Amino acid residues in the N-terminal region of the BinB subunit of Lysinibacillus sphaericus binary toxin play a critical role during receptor binding and membrane insertion

The binary toxin produced by Lysinibacillus sphaericus is composed of BinA and BinB subunits that work together in governing toxicity against mosquito larvae. BinA is proposed to be important for toxicity, whereas BinB has been shown to act as a specific receptor-binding component. The precise function of both subunits, however, is not well established. Here, we investigated the function of the N-terminal region of BinB subunit initially by introducing triple alanine substitutions at positions 35PEI37 and 41FYN43. Both block mutations abolished the larvicidal activity. Single point mutations (P35A, E36A, I37A, F41A, Y42A, N43A) were generated in order to identify amino acids that are critical for the toxin activity. Mosquito-larvicidal activity was significantly reduced in P35A, E36A, F41A and Y42A mutants. However, these mutants retained ability to form in vitro interaction with the BinA counterpart. Immunohistochemistry analysis revealed that P35A, F41A and N43A bind to the larval midgut membrane at comparable levels to that of the wild type BinB. In contrast, greatly reduced binding activity was observed in the Y42A, suggesting an important role of this residue in receptor binding. Alanine substitution at P35 resulted in a marked decrease in membrane penetration, indicating its functional importance for the membrane insertion. These results suggest the important roles of the N-terminal region of BinB in both the receptor recognition and the membrane interaction.

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

Bacillus sphaericus produces mosquito-larvicidal binary toxin composed of BinA and BinB. While BinB is expected to bind to a specific receptor on the cell membrane, BinA interacts to BinB or BinB receptor complex and translocates into the cytosol to exert its activity via unknown mechanism. To investigate functional roles of aromatic cluster in BinA, amino acids at positions Y213, Y214, Y215, W222 and W226 were substituted by leucine. All mutant proteins were highly produced and their secondary structures were not affected by these substitutions. All mutants are able to insert into lipid monolayers as observed by Langmuir-Blodgett trough and could permeabilize the liposomes in a similar manner as the wild type. However, mosquito-larvicidal activity was abolished for W222L and W226L mutants suggesting that tryptophan residues at both positions play an important role in the toxicity of BinA, possibly involved in the cytopathological process after toxin entry into the cells.

Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population.

To develop reliable and convenient methods for Miltenberger (Mia) blood group typing.