Electrochemical Genosensor To Detect Pathogenic Bacteria (Escherichia coli O157:H7) As Applied in Real Food Samples (Fresh Beef) To Improve Food Safety and Quality Control.

The electrochemical genosensor is one of the most promising methods for the rapid and reliable detection of pathogenic bacteria. In a previous work, we performed an efficient electrochemical genosensor detection of Staphylococcus aureus by using lead sulfide nanoparticles (PbSNPs). As a continuation of this study, in the present work, the electrochemical genosensor was used to detect Escherichia coli O157:H7. The primer and probes were designed using NCBI database and Sigma-Aldrich primer and probe software. The capture and signalizing probes were modified by thiol (SH) and amine (NH2), respectively. Then, the signalizing probe was connected using cadmium sulfide nanoparticles (CdSNPs), which showed well-defined peaks after electrochemical detection. The genosensor was prepared by immobilization of complementary DNA on the gold electrode surface, which hybridizes with a specific fragment gene from pathogenic to make a sandwich structure. The conductivity and sensitivity of the sensor were increased by using multiwalled carbon nanotubes (MWCNT) that had been modified using chitosan deposited as a thin layer on the glass carbon electrode (GCE) surface, followed by a deposit of bismuth. The peak currents of E. coli O157:H7 correlated in a linear fashion with the concentration of tDNA. The detection limit was 1.97 × 10(-14) M, and the correlation coefficient was 0.989. A poorly defined current response was observed as the negative control and baseline. Our results showed high sensitivity and selectivity of the electrochemical DNA biosensor to the pathogenic bacteria E. coli O157:H7. The biosensor was also used to evaluate the detection of pathogen in real beef samples contaminated artificially. Compared with other electrochemical DNA biosensors, we conclude that this genosensor provides for very efficient detection of pathogenic bacteria. Therefore, this method may have potential application in food safety and related fields.

[1]  M. Ligaj,et al.  Electrochemical DNA biosensor for the detection of pathogenic bacteria Aeromonas hydrophila , 2014 .

[2]  E. Alocilja,et al.  A multiplex nanoparticle-based bio-barcoded DNA sensor for the simultaneous detection of multiple pathogens. , 2010, Biosensors & bioelectronics.

[3]  Feng Gao,et al.  Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR , 2013, European Food Research and Technology.

[4]  J. Odumeru,et al.  Detection and enumeration of E. coli O157:H7 in water samples by culture and molecular methods. , 2013, Journal of microbiological methods.

[5]  Hui-fang Huang,et al.  An electrochemical immunosensor for sensitive detection of Escherichia coli O157:H7 using C60 based biocompatible platform and enzyme functionalized Pt nanochains tracing tag. , 2013, Biosensors & bioelectronics.

[6]  Hongyuan Chen,et al.  CdS nanocrystal-based electrochemiluminescence biosensor for the detection of low-density lipoprotein by increasing sensitivity with gold nanoparticle amplification. , 2007, Analytical chemistry.

[7]  S. Chand,et al.  A green approach for direct growth of CdS nanoparticles network in poly(3-hexylthiophene-2,5-diyl) polymer film for hybrid photovoltaic , 2012 .

[8]  B. Nigović,et al.  Multi-walled carbon nanotubes/Nafion composite film modified electrode as a sensor for simultaneous determination of ondansetron and morphine. , 2014, Talanta.

[9]  E. Malinowska,et al.  Electrochemical uranyl cation biosensor with DNA oligonucleotides as receptor layer. , 2014, Bioelectrochemistry.

[10]  Cha-Mei Tang,et al.  Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay. , 2011, Biosensors & bioelectronics.

[11]  Yanfei Liu,et al.  Hydroxylation of multi-walled carbon nanotubes reduces their cytotoxicity by limiting the activation of mitochondrial mediated apoptotic pathway , 2014, Journal of Materials Science: Materials in Medicine.

[12]  Mehmet Lütfi Yola,et al.  Molecular imprinted nanosensor based on surface plasmon resonance: Application to the sensitive determination of amoxicillin , 2014 .

[13]  Zong Dai,et al.  A label-free and PCR-free electrochemical assay for multiplexed microRNA profiles by ligase chain reaction coupling with quantum dots barcodes. , 2014, Biosensors & bioelectronics.

[14]  Yuzhong Zhang,et al.  A sensitive electrochemical DNA biosensor based on gold nanomaterial and graphene amplified signal , 2014 .

[15]  Jian Ji,et al.  Rapid and sensitive detection of foodborne pathogenic bacteria (Staphylococcus aureus) using an electrochemical DNA genomic biosensor and its application in fresh beef. , 2014, Journal of agricultural and food chemistry.

[16]  Ningning Zhu,et al.  Electrochemical detection of DNA hybridization using methylene blue and electro-deposited zirconia thin films on gold electrodes , 2004 .

[17]  Regina Célia Santos Mendonça,et al.  Prediction of Escherichia coli O157:H7 adhesion and potential to form biofilm under experimental conditions , 2012 .

[18]  Hongmei Xu,et al.  Electrochemical DNA nano-biosensor for the detection of genotoxins in water samples , 2014 .

[19]  M. Nishibuchi,et al.  Simultaneous detection of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in sliced fruits using multiplex PCR , 2011 .

[20]  F. Gao,et al.  Development of a novel electrochemical DNA biosensor based on elongated hexagonal-pyramid CdS and poly-isonicotinic acid composite film. , 2014, Biosensors & bioelectronics.

[21]  F. Reynaldi,et al.  Simultaneous detection of bee viruses by multiplex PCR. , 2013, Journal of virological methods.

[22]  Wei Sun,et al.  Application of cadmium sulfide nanoparticles as oligonucleotide labels for the electrochemical detection of NOS terminator gene sequences , 2007, Analytical and bioanalytical chemistry.

[23]  Hai-Bo Wang,et al.  A sensitive and label-free electrochemical impedance biosensor for protein detection based on terminal protection of small molecule-linked DNA , 2014 .

[24]  M. Hassan,et al.  Synthesis of nanostructured cadmium and zinc sulfides in aqueous solutions of hyperbranched polyethyleneimine , 2008 .

[25]  Wei Sun,et al.  Electrochemical DNA biosensor with chitosan-Co(3)O(4) nanorod-graphene composite for the sensitive detection of Staphylococcus aureus nuc gene sequence. , 2012, Bioelectrochemistry.

[26]  R. McCormick,et al.  c-di-GMP signaling regulates E. coli O157:H7 adhesion to colonic epithelium. , 2013, Veterinary microbiology.

[27]  Y. Chai,et al.  In situ hybridization chain reaction amplification for universal and highly sensitive electrochemiluminescent detection of DNA. , 2012, Analytical chemistry.

[28]  X. Mao,et al.  A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7. , 2006, Biosensors & bioelectronics.