Browsing by Author "Thidarat Wangkam"

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    Adsorption of bovine serum albumin (BSA) on polystyrene (PS) and its acid copolymer
    (2012-01-01) Thidarat Wangkam; Sirasa Yodmongkol; Jarupat Disrattakit; Boonsong Sutapun; Rathasart Amarit; Armote Somboonkaew; Toemsak Srikhirin; Mahidol University; Materials Science and Engineering Programme; Thailand National Electronics and Computer Technology Center
    The effect of surface polarity on the adsorption of bovine serum albumin (BSA) on polystyrene (PS), 7% polystyrene-co-maleic anhydride (7%PSMAn) and 50% polystyrene-co-maleic acid (50%PSMA), at pH 7.4, was investigated. Polystyrene represented the non-polar surface while 7%PSMAn and 50%PSMA represented a low and high acid content copolymer. The amount of the adsorbed BSA depended on the amount of the acid content in the copolymer. BSA formed a monolayer with a side-on orientation on the low polarity PS surface, a mixed side-on and end-on orientation on 7%PSMAn and a predominantly side-on orientation on 50%PSMA. The thickness of adsorbed BSA, measured with an atomic force microscope (AFM), varied from 3 nm to 5 nm for the side-on orientation and from 10 nm to 15 nm for the end-on orientation. The average area occupied per BSA molecule was consistent with the proposed orientation, and was 34.8 nm 2 , 27.8 nm 2 and 18.0 nm 2 for PS, 7%PSMAn and 50%PSMA, respectively. The adsorption showed a concentration dependency following the Freundlich isotherm, which indicated the interactions among adsorbed BSA molecules on the polymer surface. The adsorption took place as an island-like morphology and started to fuse into a patch-like morphology at higher concentrations before achieving a complete monolayer formation. A non-uniform surface coverage and defects were observed in all cases. It is recommended that for an effective blocking of PS, 7%PSMAn and 50%PSMA, the BSA concentration should be higher than 3 mg/mL. © 2011 Elsevier B.V. All rights reserved.
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    Design sensor chip for repelling the non-specific binding for its application in biomedical sensor
    (2014-01-01) Thidarat Wangkam; Toemsak Srikhirin; King Mongkut's University of Technology North Bangkok; Mahidol University
    © Springer International Publishing Switzerland 2014. The important parameter for the application in biomedical sensor is specificity on the specimen. However, there is the limitation in a low detection signal in real sample applications. The measurement cannot avoid contaminant from some biomolecules, components in patient samples such as blood or serum, which are effect on low detection or error diagnostic. Thus, to decrease this non-specific binding from some contaminants, we designed the sensor chips which are capability on repelling the non-specific binding in order to increase the efficiency of specificity. The ultra thin film and polymeric film were chosen for design. The atomic force microscope (AFM) was used for monitoring behavior of the adsorption of the non-specific binding on the designed chip. The results were found that the ultra thin film can repel some components of samples better than polymeric chip by observation the topography of the sensor chip via AFM. It was found that the area of the covered biomolecules was shown the inhomogeneous and higher roughness in the polymeric thin film than the ultra thin film. It means that there is more residual contaminant from serum on the polymeric chip and less efficiency repelling the contaminant than the results from the ultra thin film. This result was also correlated to measurement the non-specific binding of pateints’ serum via surface Plasmon resonance technique.
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    Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody
    (2011-01-15) Chokchai Puttharugsa; Thidarat Wangkam; Nongluck Huangkamhang; Oraprapai Gajanandana; Orawan Himananto; Boonsong Sutapun; Ratthasart Amarit; Armote Somboonkaew; Toemsak Srikhirin; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Thailand National Electronics and Computer Technology Center
    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. © 2010 Elsevier B.V.
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    Effect of pH on the formation of a bovine serum albumin layer on a poly(stryren-co-maleic acid) surface
    (2010-02-05) Tippavan Hongkachern; Verawat Champreda; Toemsak Srikhirin; Thidarat Wangkam; Tanakorn Osotchan; Mahidol University; Thailand National Science and Technology Development Agency
    The layer formation of bovine serum albumin (BSA) on a poly(styrene-co- maleic acid) (PSMA) surface was investigated by using quartz crystal microbalance (QCM) technique at various pH values. The formation of a BSA surface was examined by atomic force microscopy (AFM). To study the effect on the layer formation, the pH of solution was varied from 2 to 7.4 while the concentration of BSA was in the range of 0.01 to 5 mg/ml during the layer absorption. It was found that the BSA adsorption strongly depends on the pH of solution, and the concentration of BSA. The absorption layer occurred maximum at the pH value of 3.5 which resulted from the charge of PSMA and BSA molecules. The layer formation reached the saturate value at the concentration higher than 3 mg/ml. The molecular packing of the BSA layer at different pH values was determined by AFM and total mass change of QCM. © (2010) Trans Tech Publications.
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    Heterologous expression of polyhydroxyalkanoate depolymerase from Thermobifida sp. in Pichia pastoris and catalytic analysis by surface plasmon resonance
    (2009-02-01) Chitwadee Phithakrotchanakoon; Ratama Daduang; Arinthip Thamchaipenet; Thidarat Wangkam; Toemsak Srikhirin; Lily Eurwilaichitr; Verawat Champreda; Thailand National Center for Genetic Engineering and Biotechnology; Kasetsart University; Mahidol University
    A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His6-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline-serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with Vmaxand Kmof 3.63∈±∈0.16 μmol min-1mg-1and 0.79∈±∈0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50-55°C and pH 7-8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm-2h-1for poly[(R)-3-hydroxybutyrate-co-3- hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation. © 2008 Springer-Verlag.
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    Investigation of enzyme reaction by surface plasmon resonance (SPR) technique
    (2009-06-04) Thidarat Wangkam; Toemsak Srikhirin; Phenphichar Wanachantararak; Vipul Baxi; Boonsong Sutapun; Rathasart Amarit; Mahidol University; Chiang Mai University; Thailand National Science and Technology Development Agency
    An investigation into the surface plasmon resonance (SPR) sensor surface for the detection of enzyme reaction is reported. The thin polymeric film is prepared by spin casting poly (styrene-co-maleic acid) [PSMA] on a chromium/gold-coated SPR substrate. Bovine serum albumin (BSA) is adsorbed onto this surface with a mix of side-on and end-on orientation. The adsorbed BSA was tested against the protease where the cleavage reaction was followed by the change in the resonance angle of the SPR. The kinetic of the enzyme reaction follows the Michaelis-Menten equation where the reaction depends on the concentration of protease. The protease concentration is in the range of 2.5 μg/ml to 1.25 mg/ml. The different topology of protein surface before and after enzyme cleavage was observed by atomic force microscope (AFM). The AFM result shows uncleaved BSA on the surface even at highest protease concentrations owing to the steric hindrance of the adsorbed protease on the BSA surface. The sensor surface can be cleaned by cleaning solution and the surface can be reused, which avoids repetition of the complicated preparing of the sensor surface. © 2009 Elsevier B.V. All rights reserved.
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    Kinetic study of protein formation and digestion by quartz crystal microbalance
    (2010-05-05) Tippavan Hongkacharn; Verawat Champreda; Toemsak Srikhirin; Thidarat Wangkam; Tanakorn Osotchan; Mahidol University; Thailand National Science and Technology Development Agency
    Quartz crystal microbalance technique was used to study kinetic of formation and digestion of bovine serum albumin (BSA) protein on polystyrene-co-maleic acids (PSMA) surface. The kinetic of formation and digestion of BSA was investigated by measuring the frequency shift and resistance shift as a function of time. In order to study pH and concentration effect to layer formation kinetic, the pH of solution was varied from 2.0 to 7.4 while the concentration of BSA was varied in the range of 0.001 to 10 mg/ml. The kinetic of formation appears to be sensitive to the pH of solution and concentration. The formation layer at about pH 3.0 to 3.5 gives the different characteristic from the others. The layer formation increases as the concentration increase then reached the saturate value at the concentration of over 3 mg/ml. The kinetic of digestion was evaluate by applying proteinase enzyme on varies densities of BSA layer. It found that the rate of digestion depends on the density of the molecular BSA packing, modified by varying pH and concentration of BSA. ©2010 IEEE.
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    A polymer surface for antibody detection by using surface plasmon resonance via immobilized antigen
    (2013-08-01) Chokchai Puttharugsa; Thidarat Wangkam; Nongluck Houngkamhang; Sirisa Yodmongkol; Oraprapai Gajanandana; Orawan Himananto; Boonsong Sutapun; Ratthasart Amarit; Armote Somboonkaew; Toemsak Srikhirin; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Suranaree University of Technology; Thailand National Electronics and Computer Technology Center
    A polymer substrate based surface plasmon resonance (SPR) technique was developed for detection of specific monoclonal antibody 10B2 (MAb 10B2) against bacterium Acidovorax avenae subsp. citrulli (Aac). The monolayer of Aac antigen was physically immobilized on 95:5 polystryrene-copoly acrylic acid (95PSMA) for detection of antibody. The amount of antigen-antibody binding was found to depend on the surface density of immobilized Aac on the sensor surface and the antibody concentration. The detection limit was 5 μg/ml which was lower than the required concentration during the normal production of the antibody at 10-100 μg/ml. This suggests a possible use of surface for the antibody screening. Moreover, an application in antibody screening was explored by combination of surface plasmon resonance imaging (SPR imaging) and antibody detection assay on the 95PSMA surface. Two antigens of bovine serum albumin (BSA) and Aac were used as a model system for antibody screening. The result shows that both antibodies can be distinguished using the immobilized antigens on the 95PSMA surface based SPR imaging technique. © 2013 Elsevier B.V. All rights reserved.
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    Polymethyl methacrylate (PMMA) point of care for ABO-Rh(D) blood typing
    (2018-11-10) Chinnawut Pipatpanukul; Chanwit Kataphiniharn; Thidarat Wangkam; Boonsong Sutapun; Pimpun Kitpoka; Mongkol Kunakorn; Toemsak Srikhirin; King Mongkut's University of Technology North Bangkok; Suranaree University of Technology; Faculty of Medicine, Ramathibodi Hospital, Mahidol University; Mahidol University; Burapha University
    © 2018 Elsevier B.V. Polymethyl methacrylate (PMMA) based point of care (POC) for ABO-Rh(D) blood grouping by the naked eye was investigated. PMMA substrate was covalently linked with carboxyl methyl dextran (CMD) and coupled to the blood group specific antibodies anti A, anti B, and anti D, which were patterned into “A”, “B”, and “+”. The red read out color originated from the color of hemoglobin in the RBCs. The major human blood group, ABO-Rh(D), was successfully sorted using these patterns. PMMA strip-based assays for blood grouping by the naked eye resulted in correct blood group identification of all 74 samples in comparison to slide agglutination test. The determination of captured RBC Rh(D) required a longer reaction time with immobilized anti D antibody on the PMMA substrate than other blood groups to produce sufficient color for unambiguous visual identification. The proper RBC concentration was found to be higher than 1% v/v for naked eye detection. The dilution of blood sample is playing a crucial role in reducing interference from blood proteins during detection. A used of undiluted blood sample for the testing should be avoided. The proper blood dilution was found to be 10% or 1:10 dilution. The proposed technique presents a quick, cheap, simple, and almost instantaneous assay for blood grouping.
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    Recombinant expression of BTA hydrolase in Streptomyces rimosus and catalytic analysis on polyesters by surface plasmon resonance
    (2010-05-01) Nitat Sinsereekul; Thidarat Wangkam; Arinthip Thamchaipenet; Toemsak Srikhirin; Lily Eurwilaichitr; Verawat Champreda; Kasetsart University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology
    A recombinant polyester-degrading hydrolase from Thermobifida sp. BCC23166 targeting on aliphatic-aromatic copolyester (rTfH) was produced in Streptomyces rimosus R7. rTfH was expressed by induction with thiostrepton as a C-terminal His6fusion from the native gene sequence under the control of tipA promoter and purified from the culture supernatant to high homogeneity by a single step affinity purification on Ni-Sepharose matrix. The enzyme worked optimally at 50-55°C and showed esterase activity on C3-C16 p-nitrophenyl alkanoates with a specific activity of 76.5 U/mg on p-nitrophenyl palmitate. Study of rTfH catalysis on surface degradation of polyester films using surface plasmon resonance analysis revealed that the degradation rates were in the order of poly-ε-caprolactone >∈Ecoflex®∈>∈ polyhydroxybutyrate. Efficient hydrolysis of Ecoflex®by rTfH was observed in mild alkaline conditions, with the highest activity at pH 8.0 and ionic strength at 250 mM sodium chloride, with the maximal specific activity of 0.79 mg-1min-1mg-1protein. Under the optimal conditions, rTfH showed a remarkable 110-time higher specific activity on Ecoflex®in comparison to a lipase from Thermomyces lanuginosus, while less difference in degradation efficiency of the two enzymes was observed on the aliphatic polyesters, suggesting greater specificities of rTfH to the aliphatic-aromatic copolyester. This study demonstrated the use of streptomycetes as an alternative expression system for production of the multi-polyester-degrading enzyme of actinomycete origin and provided insights on its catalytic properties on surface degradation contributing to further biotechnological application of this enzyme. © 2010 Springer-Verlag.