Kooranee 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.

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.

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.

Validation of a Rapid Visual Screening of Campylobacter jejuni in Chicken Using Antibody-Conjugated Fluorescent Dye-Doped Silica Nanoparticle Reporters

The objective of this work was to validate the antibody-conjugated fluorescent dye-doped silica nanoparticle- (FDS-NP-) based assay for the rapid detection of Campylobacter jejuni in chicken samples using ISO16140:2003 single-laboratory validation guidelines. This assay reversed the paradigm of microbial testing. The noncultured FDS-NPs increased the fluorescent light signal of the test, not the cell numbers, and significantly reduced the detection time. Validation results showed that relative detection level (LOD) of the assay was 103 cfu/ml. The antibody-conjugated FDS-NPs were evaluated for the detection of C. jejuni in 140 chicken samples collected from slaughterhouses (50 carcass rinse water, 60 rectal swabs, and 30 viscera contents) against ISO 10272 reference method with duplex PCR confirmation. The relative accuracy (AC), relative specificity (SP), and relative sensitivity (SE) were 95.67%, 100%, and 94.87%, respectively. The inclusivity test of C. jejuni strains was 100% positive. The exclusivity test demonstrated no cross-reactivity with 32 strains of non-Campylobacter strains. The FDS-NP assay was very fast and specific, with time to result of 30 min compared with the 2–5 days required by the reference method. The noncultured FDS-NPs demonstrated comparable performance to the cultured reference method to detect C. jejuni in poultry samples. It is applicable for effective screening of poultry product category.

Antibody-conjugated ferromagnetic nanoparticles with lateral flow test strip assay for rapid detection of Campylobacter jejuni in poultry samples

The aim of this study was to develop a nanoparticle-based cell capture system combined with a lateral flow test strip (LFT) assay for rapid detection of Campylobacter jejuni from poultry samples. The developed assay was bench-marked against the standard modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) method according to ISO16140:2003 procedures. The synthesized ferromagnetic nanoparticles (FMNs) were modified with glutaraldehyde, then functionalized with polyclonal antibodies for specific C. jejuni capture and concentration from poultry samples. After lysing captured cells, DNA from C. jejuni was amplified by PCR using the primers designed to target the hipO gene, and the PCR amplicons were detected with the lateral flow test strip assay. Following the ISO16140:2003 guidelines, the relative detection limit, and the inclusivity and exclusivity tests were determined. The results showed that the limit of detection (LOD) of the assay was 100 or 1 cfu/ml with C. jejuni in pure culture and 101-102 cfu/ml with target cells spiked in poultry sample. In addition, the inclusivity and exclusivity tests were found to be 100%. Using field chicken samples (n = 60), the assay showed relative accuracy, relative specificity, and relative sensitivity of 96.67%, 100% and 93.33%, respectively. The positive predictive values (PPV) and negative predictive values (NPV), and the kappa index of concordance (k) were calculated as 100% and 93.75%, and 0.93, respectively. The developed assay required approximately 3 h to complete and gave results comparable to those analyzed by the standard culture method, which required 5-7 days. The assay is rapid, easy-to-use, and has potential to be directly applied to C. jejuni detection in various categories of poultry samples.

Fluorescent Dye-Doped Silica Nanoparticles with Polyclonal Antibodies for the Rapid Detection of Salmonella spp.

This study investigated a novel detection method of Salmonella spp. based on immunofluorescence microscopy using fluorescent dye-doped silica nanoparticles (FDS-NPs). Tetraethyl orthosilicate (TEOS) was used as a precursor. The FDS-NPs produced with microemulsion combined with sol-gel techniques were spherical in shape and 40-70 nm in diameter. The observation of the particles under fluorescence microscope showed high intensity orange color luminescent (Rubpy dye) with high photostability. Surface modifications for bioconjugation with polyclonal antibodies were performed by two methods, amine and carboxyl group modifications. The FDS-NPs were coated with polyclonal antibodies (purified IgGs) against Salmonella spp. Results of bacterial detection by FDSNPs indicated that nanoparticles with amine modified surface could attach onto the cells of the test organisms, Salmonella weltevreden and S. enteritidis, producing a distinctively bright color under fluorescence microscope. However, the nanoparticles with carboxyl group modification alone did not bind well with the test organisms. The FDS-NPs developed here show a high potential for use in the rapid detection of Salmonella and other bio-molecules.