Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Summary

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Abstract

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Introduction

The persistent occurrence of bacterial contamination in both commercially produced food and water sources has created a need for increasingly rapid and specific diagnostic platforms.^1,2 Some of the more common bacterial contaminants responsible for food and water contamination are from the Salmonella, Staphylococcus, Listeria, Vibrio, Shigella, Bacillus, and Escherichia genera.^3,4 Bacterial contamination by these pathogens often results in symptoms such as fever, cholera, gastroenteritis, and diarrhea.⁴ Contamination of water so....

Protocol

1.Synthesis and Functionalization of Multi-Parametric Magneto-Fluorescent Nanosensors (MFnS).

1. Synthesis of superparamagnetic iron oxide nanoparticles (IONPs)
   1. To prepare for IONP synthesis, prepare the following 3 solutions: Solution 1: FeCl₃ (0.70 g) and FeCl₂ in H₂O (2 mL), Solution 2: NH₄OH (2.0 mL, 13.4 M) in H₂O (15 mL), and Solution 3: polyacrylic acid (0.855 g) in H₂O (5 mL).
   2. Add 90 µL of 2 M Hydrochloric acid (HCl) to Solution 1 and then immediately mix with Solution 2 while vortexing at 875 rpm. Then add Solution 3. Continue vortexing for 60 min.

Results

The mechanism of MFnS action is represented in Figure 1. The clustering of MFnS around the surface of bacterial contaminants interferes with the interactions between the magnetic cores of the MFnS and the surrounding hydrogen nuclei. As a result of this clustering, magnetic relaxation values increase. As the concentration of bacterial contaminants increases, clustering reduces, and the change in T2 values decreases. Therefore, the addition of a fluorescent modality is crucial. As the bacteri.......

Discussion

This protocol has been designed to produce fully functional MFnS as simply as possible. However, there are many key points at which alteration of the protocol may be useful, depending on the user's end goal. For example, the use of different antibodies would allow for targeting of many other pathogens. In addition, this protocol is not limited to the use of antibodies as targeting molecules. Any molecule which has specific binding affinity for target pathogens, such as host cell receptors.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ferrous Chloride Tetrahydrate	Fisher Scientific	I90-500
Ferric Chloride Hexahydrate	Fisher Scientific	I88-500
Ammonium Hydroxide	Fisher Scientific	A669S-500
Hydrochloric Acid	Fisher Scientific	A144S-500
Polyacryllic Acid	Sigma-Aldrich	323667-100G
EDC	Thermofisher Scientific	22980
NHS	Fisher Scientific	AC157270250
Anti-E. coli O111 antibody	sera care	5310-0352
Anti-E. coli O157:H7 antibody [P3C6]	Abcam	ab75244
DiI Stain	Fisher Scientific	D282
Nutrient Broth	Difco	233000
Freeze-dried E. coli O157:H7 pellet	ATCC	700728
Magnetic Relaxomteter	Bruker	mq20
Zetasizer	Malvern	NANO-ZS90
Plate Reader	Tecan	Infinite M200 PRO
Magnetic Column	QuadroMACS	130-090-976
Centrifuge	Eppendorf	5804 Series
Centrifuge (accuSpin Micro 17)	Fisher Scientific	13-100-676
Floor Model Shaking Incubator	SHEL LAB	SSI5
Analytical Balance	Metler Toledo	ME104E
Digital Vortex Mixer	Fisher Scientific	02-215-370
Open-Air Rocking Shaker	Fisher Scientific	02-217-765