Pravate Tuitemwong

Recent Trends in Rapid Environmental Monitoring of Pathogens and Toxicants: Potential of Nanoparticle-Based Biosensor and Applications

Of global concern, environmental pollution adversely affects human health and socioeconomic development. The presence of environmental contaminants, especially bacterial, viral, and parasitic pathogens and their toxins as well as chemical substances, poses serious public health concerns. Nanoparticle-based biosensors are considered as potential tools for rapid, specific, and highly sensitive detection of the analyte of interest (both biotic and abiotic contaminants). In particular, there are several limitations of conventional detection methods for water-borne pathogens due to low concentrations and interference with various enzymatic inhibitors in the environmental samples. The increase of cells to detection levels requires long incubation time. This review describes current state of biosensor nanotechnology, the advantage over conventional detection methods, and the challenges due to testing of environmental samples. The major approach is to use nanoparticles as signal reporter to increase output rather than spending time to increase cell concentrations. Trends in future development of novel detection devices and their advantages over other environmental monitoring methodologies are also discussed.

Synthesis of antibodies-conjugated fluorescent dye-doped silica nanoparticles for a rapid single step detection of campylobacter jejuni in live poultry

The preparation of antibodies-conjugated fluorescent dye-doped silica nanoparticles (FDS-NPs) was developed to detect Campylobacter jejuni cells under a fluorescence microscope. The particles prepared by sol-gel microemulsion techniques have a round shape with an average size of 43±4 nm. They were highly photo stable and could emit strong orange fluorescent for 60min. Both amine- and carboxyl-functionalized properties were evident from FTIR and FT Raman spectra. The FDS-NPs conjugated with antibodies against C. jejuni were well dispersed in PBS solution at 20mM of NaCl. The conjugation with monoclonal antibodies against C. jejuni was successful. The direct observation of the antibodies-conjugated FDS-NPs- that bounds C. jejuni with Petroff Hausser counting chamber at 40x was clear. The different focus lengths clearly separated bound and unbound FDSNPs under the microscope. We successfully synthesis the bio-conjugated dye doped silica nanoparticles for C. jejuni that are easy to use and giving clear detection in due time.

Exposure assessment and process sensitivity analysis of the contamination of Campylobacter in poultry products

Studies were conducted in a Thai poultry plant to identify the factors that affected numbers of Campylobacter jejuni in chicken carcasses. The concentrations of Campylobacter were determined using the SimPlate most probable number and modified charcoal cefoperazone deoxycholate plating methods. Results indicated that the mean concentrations of C. jejuni in carcasses after scalding, plucking, and chilling were 2.93 ± 0.31, 2.98 ± 0.38, 2.88 ± 0.31, and 0.85 ± 0.95 log cfu, whereas the concentrations of C. jejuni in the scalding tank water, plucked feathers, and chicken breast portion were 1.39 ± 0.70, 3.28 ± 0.52, and 0.50 ± 1.22 log cfu, respectively. Sensitivity analysis using tornado order correlation analysis showed that risk parameters affecting the contamination of C. jejuni in the chicken slaughter and processing plant could be ranked as chilling water pH, number of pathogens in the scald tank water, scalding water temperature, number of C. jejuni on plucked feathers, and residual chlorine in the chill water, respectively. The exposure assessment and analysis of process parameters indicated that some of the current critical control points were not effective. The suggested interventions included preventing fecal contamination during transportation; increasing the scalding temperature, giving the scalding water a higher countercurrent flow rate; reducing contamination of feathers in the scalding tank to decrease C. jejuni in the scalding water; spraying water to reduce contamination at the plucking step; monitoring and maintaining the chill water pH at 6.0 to 6.5; and increasing the residual chlorine in the chill water. These interventions were recommended for inclusion in the hazard analysis and critical control point plan of the plant.

A Novel Type of Formaldehyde-Oxidizing Enzyme from the Membrane of Acetobacter sp. SKU 14

Membrane-bound NAD(P)-independent formaldehyde-oxidizing enzyme was purified to homogeneity from the membrane of Acetobacter sp. SKU 14 isolated in Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Tween 20 at pH 2.85, and purified to homogeneity through the steps of column chromatographies on DEAE-Sephadex A-50 and Q-Sepharose in the presence of 0.1% Tween 20 and 0.1% Triton X-100. The enzyme purified together with a cytochrome c showed a single protein band on native-PAGE, and was dissociated into three different subunits upon SDS–PAGE with molecular masses of 78 kDa, 55 kDa, and 18 kDa. The purified enzyme was finally characterized as a quinoprotein alcohol dehydrogenase (QADH), and this is the first indication that QADH highly oxidizes formaldehyde. The substrate specificity of the enzyme was found to be broad toward aldehydes and alcohols, and alcohols, especially n-butanol, n-propanol, and ethanol, were oxidized more rapidly than formaldehyde.

Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand

Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels.

Antibody-conjugated rubpy dye-doped silica nanoparticles as signal amplification for microscopic detection of vibrio cholerae O1

This study demonstrated the potential application of antibody-conjugated Rubpy dye-doped silica nanoparticles for immunofluorescence microscopic detection of Vibrio cholerae O1. The particle synthesis of 20X of the original ratio was accomplished yielding spherical nanoparticles with an average size of 45 ± 3 nm. The nanoparticles were carboxyl functionalized and then conjugated with either monoclonal antibody or polyclonal antibody against V. cholerae O1. The antibody-conjugated nanoparticles were tested with two target bacteria and three challenge strains. The result showed that monoclonal antibody-conjugated Rubpy dye-doped silica nanoparticles could be effectively used as signal amplification to detect V. cholerae O1 under a fluorescence microscope. Their extremely strong fluorescence signal also enables the detection of a single cell bacterium.

Formaldehyde elimination with formaldehyde and formate oxidase in membrane of acetic acid bacteria

Formaldehyde elimination was successfully carried out with Acetobacter sp. SKU 14, having strong formaldehyde-oxidizing activity in the cytoplasmic membrane. Formaldehyde was decomposed via formate to carbon dioxide by formaldehyde- and formate-oxidizing activities. A resting-cell suspension of the organism was more convenient for practical purposes than the isolated membrane fraction. In Gluconobacter suboxydans IFO 12528, formaldehyde elimination was not so prominent when compared with that in Acetobacter sp. SKU 14.

Single Step Synthesis of Amino-Functionalized Magnetic Nanoparticles with Polyol Technique at Low Temperature

The synthesis and characterization of amino-functionalized magnetic nanoparticles (amino-MNPs) were carried out. Amino-MNPs were prepared using polyol technique in an autoclave (121°C) without inert gas. The characterization of particles employed conventional SEM-EDS, TEM, XRD, FTIR, and VSM techniques. Results showed that amount of NaOH affected to the speed of MNP formation. The desirable uniform cubic shape of amino-MNPs was obtained from the addition of 2.50% w/v NaOH for 3 reaction cycles (2 hours/cycle). The amino-MNPs obtained from this condition have cubic shapes with the average size of 43 ± 9 nm. Results from elemental and structure analyses confirmed that the product was pure magnetite. The magnetic properties were ferromagnetism and were very close to a complete superparamagnetism. Fourier transform infrared (FT-IR) spectrum showed that the amino group existed on particle surface. The amino-MNPs of cubic shape were synthesized with facile single step at low temperature, and they have potential to be used for rapid microbial detection and many biological applications.

Rubpy dye-doped silica nanoparticles as signal reporter in a dot fluorescence immunoassay strip

This paper describes an application of Rubpy dye-doped silica nanoparticles (RSNPs) as signal reporter in a dot fluorescence immunoassay strip for rapid screening of Vibrio cholera O1 (VCO1). These nanoparticles have a spherical shape with an average diameter of 45 nm. They appear luminescent orange when excited with a 312nm UV lamp. Based on the sandwich immunoassay principle, a test strip was made of a nitrocellulose membrane dotted with monoclonal antibodies against VCO1 as analyte capture molecules. After introducing a test sample, followed by polyclonal rabbit anti-VCO1 antibody conjugated RSNPs as detection reporters and one washing step, the presence or absence of the target bacteria could be identified under UV light by naked eyes. A positive sample would signal a bright orange dot on the strip. The proposed assay had a detection limit of 4.3 × 103 cfu/mL and was successfully applied as a rapid screening test for VCO1 in food samples with high sensitivity, specificity, and accuracy.

Facile and sensitive epifluorescent silica nanoparticles for the rapid screening of EHEC

This study was to develop antibodies conjugated fluorescent dye-doped silica nanoparticles (FDS-NPs) aiming to increase signals for the rapid detection of Escherichia coli O157:H7 with glass slide method. The FDS-NPs were produced with microemulsion/solgel techniques resulting in spherical in shape with 47 ± 6 nm in diameter. The particles showed high intensity and stable orange color Rubpy luminescent dye. The XRDspectrumshowed a broad diffraction peak in the range of 18-30° (centered at 22°) indicating an amorphous structure. Surface modifications for bioconjugation with affinity chromatography purified (IgGs) antibodies were successful. The properties were evident from FTIR spectra at 1631.7 cm-1. Results indicated that nanoparticles could attach onto cells of E. coli O157:H7 coated on a glass slide, and give distinctively bright color under epifluorescence microscope (400x). It was shown that FDS-NPs could detect a very low amount of cells of E. coli O157:H7 (16 CFU in 10ml) in 60 min. The phosphate buffered saline (PBS)with ionic strength of 1.70 gave zeta potential of good particle dispersion (-40mV). This work demonstrated that highly sensitive bioconjugated E. coli O157:H7 FDS-NPs were successfully developed with a potential to be used for the rapid detection of E. coli O157:H7 in foods.