Foodborne pathogens detection with nanoporous anodic aluminum oxide membrane based biosensor

Abstract

This research investigated nanoporous anodic aluminum oxide membrane based immnuosensor for foodborne pathogenic bacteria detection by impedance spectrum. Polydimethylsiloxane (PDMS) based immnuobiosensors integrated with nanoporous alumina membrane were developed for detection of E. coli O157:H7 and Staphylococcus aureus respectively and simultaneously. Firstly, antibodies to the targeted bacteria were covalently immobilized on the nanoporous alumina membrane via self assembled (3-glycidoxypropyl)trimethoxysilane (GPMS) silane. The successful covalent immobilization of silane monolayer on nanoporous membrane surface was characterized by X-ray photoelectron spectroscopy (XPS). The antibody immobilization on silane modified membrane was also confirmed by XPS and Atomic Force Microscopy (AFM). Then, single type bacteria detection system and simultaneous detection system for multiple type bacteria detection were developed for impedance measurement. For single type bacteria detection, impedance spectrum was first recorded for target bacteria samples using specific antibody immobilized nanoporous membrane with a frequency range from 1 Hz to 10,000 Hz. Impedance amplitude changes induced by target bacteria capturing with specific antibody modified membrane were found to increase with the bacteria concentrations. The sensing limit was around 103 CFU/ml. Cross experiments between antibody immobilized membranes and non-target bacteria were also performed to test the specificity of this device. No obvious impedance amplitude change was found for non-target bacteria samples. These results were also confirmed by sandwich type fluorescence immunoassay. For the simultaneous detection system for multiple type bacteria detection, mixed bacteria samples with E. coli O157:H7 and Staphylococcus aureus, and samples with only one type bacteria were tested. The impedance changes on E. coli O157:H7 detection chamber and Staphylococcus aureus detection chamber showed the increase with related bacteria concentrations in the mixture samples, respectively. However, when only one kind of bacteria such as E. coli O157:H7 or Staphylococcus aureus added into the system, only the specific antibody modified membrane side showed an increase of impedance amplitude change. For response time detection experiment, the detection assay for both E. coli O157:H7 and Staphylococcus aureus could be completed in around 3 hours.