Development of a Single Assay Comprising Nano-immunomagnetic Separation of Escherichia coli O157:H7 from Apple Juice Followed by Surface Enhanced Raman Scattering-based Detection

Introduction: The successful detection of foodborne pathogens is dependent on efficient separation and concentration of target organism from the food matrices. Immunomagnetic separation (IMS) is one of the commonly used methods for isolation of pathogens from food and beverages. However, traditional IMS is time consuming and requires additional steps to confirm the identity of the captured microbes. Therefore, the need to develop a rapid IMS procedure enabling both microbial separation and identification in a single assay is highly desirable.

Purpose: The purpose of this study was to develop a method combining gold-coated iron nanoparticle-based IMS and surface enhanced Raman spectroscopy (SERS)-based detection in a single assay for screening of Escherichia coli O157:H7 from model liquid food.

Methods: Magnetite-gold (Fe₃O₄/Au) nanoparticles (MNP) were synthesized using Polyethyleneimine (PEI).  The capture antibody (cAb) was immobilized on gold coated MNP.  These cAb-MNP were used in the separation and concentration of the E coli O157:H7 cells from spiked apple juice samples.  The capture efficiency was obtained in different E. coli O157:H7 concentrations (10²–10⁸ CFU /ml). The specificity of the assay was tested by using a group of different gram positive and gram negative bacteria. For detection of target pathogen, SERS labels were prepared by conjugating gold nanoparticles with Raman reporter molecules and the detector antibody (dAb).  Two different Raman reporter molecules were examined.  Gold-Raman label-dAb was interacted with gold coated MNP-cAb-E. coli O157:H7 complex. Following IMS and washing, the nano-aggregates were directly analyzed using a Raman spectrometer.

Results: The percentage of captured bacteria (expressed as capture efficiency, CE) for E. coli O157:H7 was found to be about 85-94%.  No cross reactivity was observed with background non-target organisms at a concentration of 10⁶ CFU /ml. There was a significant difference in the mean CE of bacteria captured by MNP and commercially sourced immunomagnetic microbeads (P < 0.05). The limit of detection of bacterial cell in apple juice using nanoparticles was 10² CFU/ml.

Significance: This newly developed IMS method, employing Fe₃O₄/Au nanoclusters, presents a convenient way for isolation as well as detection of target pathogen (E. coli O157:H7) in food sample.  The method is rapid and sensitive to target organisms with a total assay time of less than 60 minutes.