Multianalyte detection of food pathogens/toxins through enzymatic bio-nanotransduction on an electrochemical biosensor array

Enzymatic Bio-nanotransduction is a biological detection system based on the use of an enzyme (biological transducer) to change a specific biological recognition event into nucleic acid (RNA) nano-signal molecules. We used biological recognition elements (antibodies, DNA sequences) linked to DNA templates with T7 promoter regions for detection of specific target molecules. In vitro transcription of DNA templates bound to target molecules produced RNA nano-signals specific for every target in the sample. Discrimination of RNA nano-signals using a standard enzyme linked oligonucleotide fluorescence assay (ELOFA) provided a correlation between nano-signal profiles and target concentrations.;The system was capable of detecting and distinguishing three species of specific IgG molecules at a level of 0.2 ng. The system was also shown to be capable of detecting mixed protein and DNA targets. The flexibility of this system was substantiated through the simultaneous detection of Escherichia coli O157 microorganisms and Staphylococcal enterotoxin B (SEB) using a labeled secondary antibody approach.;By using primary antibodies to functionalize magnetic beads and to produce biological recognition elements (antibodies) conjugated to nano-signal producing DNA templates we were able to simultaneously detect Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and Staphylococcal enterotoxin B. Using an optimized protocol we were able to estimate our limit of detection as 2.4 x 103 cfu/ml for E. coli O157:H7, 1.9 x 104 cfu/ml for Salmonella enterica serovar Typhimurium and 0.11 ng/ml for SEB with multi-analyte detection in buffer. We also examined the detection of targets in a model food product (milk) and provided evidence for the improvement of detection in milk using heat treatment.;We were able to extend these results to the development of a nucleic acid array electrochemical biosensor for multiplex detection of food pathogens/toxins. Estimated limits of detection were 1.2 x 104 cfu/ml for Escherichia coli O157:H7, 6 x 104 for Salmonella enterica serovar Typhimurium and 0.32 ng/ml for SEB. A threshold cut-off approach was established to show the effective detection of all three targets in mixed samples.