Browsing by Author "Quintela, Irwin A."

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Simultaneous Colorimetric Detection of a Variety of Salmonella spp. in Food and Environmental Samples by Optical Biosensing Using Oligonucleotide-Gold Nanoparticles
(2019-05-31)
Quintela, Irwin A.
;
de los Reyes, Benildo G.
;
Lin, Chih-Sheng
;
Wu, Vivian C. H.
;
生物科技學系
Optical biosensors for rapid detection of significant foodborne pathogens are steadily gaining popularity due to its simplicity and sensitivity. While nanomaterials such as gold nanoparticles (AuNPs) are commonly used as signal amplifiers for optical biosensors, AuNPs can also be utilized as a robust biosensing platform. Many reported optical biosensors were designed for individual pathogen detection in a single assay and have high detection limit (DL). Salmonella spp. is one of the major causative agents of foodborne sickness, hospitalization and deaths. Unfortunately, there are around 2,000 serotypes of Salmonella worldwide, and rapid and simultaneous detection of multiple strains in a single assay is lacking. In this study, a comprehensive and highly sensitive simultaneous colorimetric detection of nineteen (19) environmental and outbreak Salmonella spp. strains was achieved by a novel optical biosensing platform using oligonucleotide-functionalized AuNPs. A pair of newly designed single stranded oligonucleotides (30-mer) was displayed onto the surface of AuNPs (13 nm) as detection probes to hybridize with a conserved genomic region (192-bases) of ttrRSBCA found on a broad range of Salmonella spp. strains. The sandwich hybridization (30 min, 55 degrees C) resulted in a structural formation of highly stable oligonucleotide/AuNPs-DNA complexes which remained undisturbed even after subjecting to an increased salt concentration (2 M, final), thus allowing a direct discrimination via color change of target (red color) from non-target (purplish-blue color) reaction mixtures by direct observation using the naked eye. In food matrices (blueberries and chicken meat), nineteen different Salmonella spp. strains were concentrated using immunomagnetic separation and then simultaneously detected in a 96-well microplate by oligonucleotide-functionalized AuNPs after DNA preparation. Successful oligonucleotide/AuNPs-DNA hybridization was confirmed by gel electrophoresis while AuNPs aggregation in non-target and control reaction mixtures was verified by both spectrophotometric analysis and TEM images. Results showed that the optical AuNP biosensing platform can simultaneously screen nineteen (19) viable Salmonella spp. strains tested with 100% specificity and a superior detection limit of <10 CFU/mL or g for both pure culture and complex matrices setups. The highly sensitive colorimetric detection system can significantly improve the screening and detection of viable Salmonella spp. strains present in complex food and environmental matrices, therefore reducing the risks of contamination and incidence of foodborne diseases.
Simultaneous direct detection of Shiga-toxin producing Escherichia coli (STEC) strains by optical biosensing with oligonucleotide-functionalized gold nanoparticles
(2015-01-01)
Quintela, Irwin A.
;
de los Reyes, Benildo G.
;
Lin, Chih-Sheng
;
Wu, Vivian C. H.
;
生物科技學系
A simultaneous direct detection of Shiga-toxin producing strains of E. coli (STEC; "Big Six"-O26, O45, O103, O111, O121, and O145) as well as O157 strains by optical biosensing with oligonucleotide-functionalized gold nanoparticles (AuNPs) was developed. Initially, conserved regions of stx genes were amplified by asymmetric polymerase chain reaction (asPCR). Pairs of single stranded thiol-modified oligonucleotides (30-mer) were immobilized onto AuNPs and used as probes to capture regions of stx1 (119-bp) and/or stx2 (104-bp) genes from STEC strains. DNA samples from pure cultures and food samples were sandwich hybridized with AuNP-oligo probes at optimal conditions (50 degrees C, 30 min). A complex was formed from the hybridization of AuNP-probes and target DNA fragments that retained the initial red color of the reaction solutions. For non-target DNA, a color change from red to purplish-blue was observed following an increase in salt concentration, thus providing the basis of simultaneous direct colorimetric detection of target DNA in the samples. Enrichment and pooling systems were incorporated to efficiently process a large number of food samples (ground beef and blueberries) and detection of live targets. The detection limit was <1 log CFU g(-1), requiring less than 1 h to complete after DNA sample preparation with 100% specificity. Gel electrophoresis verified AuNP-DNA hybridization while spectrophotometric data and transmission electron microscope (TEM) images supported color discrimination based on the occurrence of molecular aggregation. In conclusion, the significant features of this approach took advantage of the unique colorimetric properties of AuNPs as a low-cost and simple approach yet with high specificity for simultaneous detection of STEC strains.