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.