Stimulus-Response Biosensor for Determining Bacteria Viability Using Lectin-Glycoenzyme Nanobrushes

E.S. McLamore, I. Khondaker, C. Gomes, D. Alves De Oliveira
University of Florida,
United States

Keywords: pathogen, viability, food safety, nanobrush, biogenic amine


Contamination of food with pathogens not only sickens, but may lead to hospitalization and even death in people with compromised immune systems. Public demand for organic, non-pasteurized food products is inducing pressure on the food industry to provide high quality/safe products, which requires rapid sensors to test food products in situ. While many detection strategies exist, there are few approaches for determining pathogen viability. The objective of this work was to develop a nanobrush material with two distinct features: 1) the ability to selectively capture pathogens based on stimulus-response capture, and 2) determination of viability based on the decarboxylation of exogenous amino acids based on a cascade reaction. A mannose binding lectin (concanavalin A, Con A) and the glycoenzyme diamine oxidase (DOx) were assembled in a layer-by-layer approach for developing a multilayer pH-sensitive nanobrush. The outermost layer of the brush was terminated with ConA or a 64mer aptamer to facilitate capture of Escherichia coli O157:H7. Lectin-mediated cell capture was interrogated using electrochemical impedance spectroscopy and cyclic voltammetry. After cell capture, exogenous amino acids were added and metabolism by viable E. coli produced biogenic amines (BA). DC potential amperometry was used to detect microbe-produced BA at +400mV. The optimal lectin and enzyme concentrations for creating the nanobrush layers were determined to be 0.8 mg/mL and 1.0 mg/mL, respectively; with an optimum time of 20 min at room temperature using PBS as a binding buffer. The total assay time for capture/viability was 40 minutes: 10 min for cell capture and 30 min for viability. Ongoing testing aims to optimize the amino acid concentration/type and examine the effect of stimulus response. This new sensor approach can be expanded to target specific foodborne pathogens by altering the outermost capture probe of the nanobrush assembly (e.g., use of aptamer-decorated polymer nanobrushes), enabling rapid determination of food pathogen presence and viability without addition of expensive reagents.