Detection of low levels of Escherichia coli in fresh spinach by surface plasmon resonance spectroscopy with a TMB-based enzymatic signal enhancement method
ABSTRACT The development of effective Escherichia coli (E. coli) sensors has been of great importance and urgent need to public health and safety in the wake of the spinach outbreak in California in 2006. We report here an enzymatic signal enhancement method for highly sensitive and fast detection of E. coli based on the generation of a mass-enhancing product at the sensing interface that is quantified by surface plasmon resonance (SPR) spectroscopy. The insoluble product is formed through the reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) with a horse radish peroxidase (HRP) conjugated antibody that attaches to the bacteria captured on a self-assembled monolayer (SAM) surface. Results indicate a signal enhancement of E. coli cells on the order of 250% as compared to the control samples. In addition, this signal enhancement was consistent for over six orders of concentration range. The limit of detection for E. coli is determined to be 103 cfu/mL by this method, offering excellent detection sensitivity for probing actual E. coli levels in produce and other real world samples. The method was further demonstrated for E. coli measurement in the complex matrix of spinach leaves where a dynamic range over three orders of magnitude of concentrations was obtained with a limit of detection of 104 cfu/mL. This approach is simple, fast and sensitive; given the relative simple procedure of adding an HRP tag to an antibody, it opens new route for detection of low levels of biomolecules, in particular bacteria and viruses, in a complex matrix.
Article: Subtractive inhibition assay for the detection of E. coli O157:H7 using surface plasmon resonance.[show abstract] [hide abstract]
ABSTRACT: A surface plasmon resonance (SPR) immunosensor was developed for the detection of E. coli O157:H7 by means of a new subtractive inhibition assay. In the subtractive inhibition assay, E. coli O157:H7 cells and goat polyclonal antibodies for E. coli O157:H7 were incubated for a short of time, and then the E. coli O157:H7 cells which bound antibodies were removed by a stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using a BIAcore 3000 biosensor. The results showed that the signal was inversely correlated with the concentration of E. coli O157:H7 cells in a range from 3.0 × 10(4) to 3.0 × 10(8) cfu/mL with a detection limit of 3.0 × 10(4) cfu/mL. Compared with direct SPR by immobilizing antibodies on the chip surface to capture the bacterial cells and ELISA for E. coli O157:H7 (detection limit: both 3.0 × 10(5) cfu/mL in this paper), the detection limit of subtractive inhibition assay method was reduced by one order of magnitude. The method simplifies bacterial cell detection to protein-protein interaction, which has the potential for providing a practical alternative for the monitoring of E. coli O157:H7 and other pathogens.Sensors 01/2011; 11(3):2728-39. · 1.74 Impact Factor