Browsing by Author "Wu, Vivian C. H."

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A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles
(2012-10-01)
Guo, Xiao
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Lin, Chih-Sheng
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Chen, Sz-Hau
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Ye, Rebecca
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Wu, Vivian C. H.
A sensitive bacteria enrichment and detection system for viable Escherichia coil O157:H7 was developed using a piezoelectric biosensor-quartz crystal microbalance (QCM) with antibody-functionalized gold nanoparticles (AuNPs) used as detection verifiers and amplifiers. In the circulating-flow QCM system, capture antibodies for E. coil 0157:H7 were first immobilized onto the QCM chip. The sample containing E. coli 0157:H7 was circulated through the system in the presence of 10 ml of brain heart infusion (BHI) broth for 18 h. The cells of E. coli 0157:H7 specifically captured and enriched on the chip surface of the QCM were identified by QCM frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. After bacterial enrichment, detection antibody-functionalized AuNPs were added to enhance the changes in detection signal. The use of BHI enrichment further enhanced the sensitivity of the developed system, achieving a detection limit of 0-1 log CFU/ml or g. The real-time monitoring method for viable E. coli 0157:H7 developed in this study can be used to enrich and detect viable cells simultaneously within 24 h. The unique advantages of the system developed offer great potential in the microbial analysis of food samples in routine settings. (C) 2012 Elsevier B.V. All rights reserved.
Application of cranberry concentrate (Vaccinium macrocarpon) to control Escherichia coli O157:H7 in ground beef and its antimicrobial mechanism related to the downregulated slp, hdeA and cfa
(2009-02-01)
Wu, Vivian C. H.
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Qiu, Xujian
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de los Reyes, Benildo G.
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Lin, Chih-Sheng
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Pan, Yingjie
The possible use of cranberry concentrate (CC) as a natural food preservative was studied by examining its antimicrobial effect on the growth of Escherichia coli O157:H7 inoculated in ground beef, its organoleptical effect oil beef patties, and its antimicrobial mechanism on the gene regulation level. Inoculated ground beef was added with CC and stored at 4 degrees C for 5 days. Bacteria were detected on day 0, 1, 3, and 5. Cranberry concentrate (2.5%, 5%, and 7.5% w/w) reduced total aerobic bacteria 1.5 log, 2.1 log, and 2.7 log CFU/g and E coli O157:H7 0.4 log, 0.7 log, and 2.4 log CFU/g, respectively, when compared to the control on day 5. Fifty panelists evaluated the burgers supplemented with CC. No differences in appearance, flavor, and taste were found among burgers with 0%, 2.5%, and 5% CC. The expression of E coli O157:H7 cyclopropane fatty acyl phospholipid synthase (cfa), hypothetical protein (hdeA), outer membrane porin protein C (ompC), hyperosmotically inducible periplasmic protein (osmY), and outer membrane protein induced after carbon starvation (sip) genes with or without CC (2.5% v/v) treatment was investigated by quantitative real-time PCR. Compared to the control, sip, hdeA, and cfa were markedly downregulated, ompC was slightly downregulated, while osmY was slightly affected. (C) 2008 Elsevier Ltd. All rights reserved.
Real-time detection of Escherichia coli O157 : H7 sequences using a circulating-flow system of quartz crystal microbalance
(2007-06-15)
Wu, Vivian C. H.
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Chen, Sz-Hau
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Lin, Chih-Sheng
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生物科技學系
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Department of Biological Science and Technology
A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5'-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5'-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis. (c) 2006 Elsevier B.V. All rights reserved.
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.
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de los Reyes, Benildo G.
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Lin, Chih-Sheng
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Wu, Vivian C. H.
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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.
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de los Reyes, Benildo G.
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Lin, Chih-Sheng
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Wu, Vivian C. H.
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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.
Using oligonucleotide-functionalized Au nanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor
(2008-04-01)
Chen, Sz-Hau
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Wu, Vivian C. H.
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Chuang, Yao-Chen
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Lin, Chih-Sheng
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生物科技學系
A circulating-flow piezoelectric biosensor, based on an Au nanoparticle amplification and verification method, was used for real-time detection of a foodbome pathogen, Escherichia coli O157:H7. A synthesized thiolated probe (Probe 1; 30-mer) specific to E. coli O157:H7 eaeA gene was immobilized onto the piezoelectric biosensor surface. Hybridization was induced by exposing the immobilized probe to the E. coli O157:H7 eaeA gene fragment (104-bp) amplified by PCR, resulting in a mass change and a consequent frequency shift of the piezoelectric biosensor. A second thiolated probe (Probe 2), complementary to the target sequence, was conjugated to the An nanoparticles and used as a "mass enhancer" and "sequence verifier" to amplify the frequency change of the piezoelectric biosensor. The PCR products amplified from concentrations of 1.2 x 10(2) CFU/ml of E. coli O157:H7 were detectable by the piezoelectric biosensor. A linear correlation was found when the E. coli O157:H7 detected from 10(2) to 10(6) CFU/ml. The piezoelectric biosensor was able to detect targets from real food samples. (C) 2008 Elsevier B.V. All rights reserved.