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 sources often has drastic and adverse effects on communities without access to sufficiently filtered water, and food contamination has led to a great number of illnesses and product recall efforts.^5,6

In order to reduce the occurrence of illnesses caused by bacterial contamination, there have been a number of efforts to develop methods by which water and food can be efficiently scanned prior to sale or consumption.³ Techniques such as PCR,^1,7,8,9,10 ELISA,^11,12 loop-mediated isothermal amplification (LAMP),^13,14 among others,^{15,16,17,18,19,20,21,22,23,24} have recently been used for detection of various pathogens. Compared to traditional bacterial culturing methods, these techniques are far more efficient with regards to specificity and time. However, these techniques still struggle with false positives and negatives, complex procedures, and cost.^1,3,25 It is for this very reason that multiparametric magneto-fluorescent nanosensors (MFnS) are proposed as an alternative method for bacterial detection.

These nanosensors uniquely pair together magnetic relaxation and fluorescent modalities, allowing for a dual-detection platform that is both rapid and accurate. Using E. coli O157:H7 as a sample contaminant, the ability of MFnS to detect as little as 1 CFU within minutes is demonstrated. Pathogen-specific antibodies are used to increase specificity, and the combination of both magnetic and fluorescent modalities allows for the detection and quantification of bacterial contaminants in both low- and high-contamination ranges.¹⁶ In the case of bacterial contamination, the nanosensors will swarm around the bacteria due to the targeting abilities of the pathogen-specific antibodies. The binding between the magnetic nanosensors and bacteria limits the interaction between the magnetic iron core and the surrounding water protons. This causes an increase in the T2 relaxation times, as recorded by a magnetic relaxometer. As the concentration of bacteria in solution rises, the nanosensors disperse with the increased number of bacteria, resulting in lower T2 values. Conversely, fluorescence emission will increase in proportion with the concentration of bacteria, due to the increased number of nanosensors directly bound to pathogen. Centrifugation of the samples, and isolation of the bacterial pellet, will only conserve the nanoparticles directly attached to the bacteria, removing any free-floating nanosensors, and directly correlating the fluorescence emission with the number of bacteria present in solution. A schematic representation of this mechanism is represented in Figure 1.

This MFnS platform has been designed with point-of-care screening in mind, resulting in low-cost and portable characteristics. MFnS are stable at room temperature, and are only required in very low concentrations for accurate detection of bacterial contaminants. Furthermore, after synthesis, use of the MFnS is simple and does not require the use of trained professionals in the field. Lastly, this diagnostic platform allows for highly customizable targeting, providing a means by which this one platform may be used to detect pathogens of all kinds, in many different settings.

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.
   3. Centrifuge for 20 min at 1,620 x g. Centrifuge the supernatant for 20 min at 2,880 x g.
   4. Purify the final supernatant via dialysis. Add the supernatant to a dialysis bag (MWCO 6−8 K) and place it in a beaker containing phosphate buffered saline (PBS) (pH = 7.4) and spin bar. Let it soak for 12 h, replacing with fresh buffer every 2-3 h.
2. Conjugation of target-specific antibodies to surface of IONPs
   1. Prepare the following four solutions: Solution A: 5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in 250 µL MES [2-(N-morpholino)ethanesulfonic acid] buffer (0.1 M, pH = 6.8), Solution B: 3 mg of N-Hydroxysuccinimide (NHS) in 250 µL of MES buffer (0.1 M, pH = 6.8), Solution C: 5 µg of IgG1, the E. coli mAb, in 225 µL of phosphate-buffered saline (PBS) (pH 7.4), and Solution D: 4 mL of IONP (5.0 mmol) in 1 mL of PBS (pH 7.4).
   2. Add 2-(N-Morpholino)ethanesulfonic acid (MES) buffer to Solution A, then immediately add Solution A to Solution D in small increments within 15 s, inverting the solution after each addition for mixing.
   3. Immediately begin to add Solution B to Solution D in small increments over a three-minute period. Mix by inverting the solution after each addition.
   4. Add Solution C to solution in increments of 5 µL, inverting the solution after each addition for mixing.
   5. Allow reaction to continue for 3 h at room temperature and then continue the incubation at 4 °C overnight.
   6. Purify the resulting Ab-conjugated IONPs via magnetic column using PBS (pH = 7.4, final [Fe] = 3.5 mmol) to remove any unconjugated antibodies.
      1. Wash the magnetic column with PBS (1 mL). Attach the column to the magnetic plate, and add IONP solution.
      2. Wash the magnetic column with PBS (1 mL) again. Remove the magnetic column from magnetic plate. Add PBS to the column and collect the solution. Store at 4 °C
3. Encapsulation of fluorescent 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiI) dye into the polyacrylic acid (PAA) coating of the antibody-conjugated IONPs using a solvent diffusion method.
   1. To 4 mL of the IONPs, add dropwise 2.0 µL of DiI dye (2 mmol) in 100 µL of DMSO with continuous mixing at 1100 rpm.
   2. Dialyze the resulting solution (MWCO 6-8 KDa, 12 h) against PBS, creating a final MFnS solution with a final iron concentration of [Fe] = 2 mmol.
4. Characterization of MFnS
   1. For spectrophotometric analysis, use a plate reader to detect encapsulated DiI dye with fluorescence emission at 595 nM.
   2. Dynamic Light Scattering
      1. Place a sample of the MFnS solution into a zetasizer to determine the average size and surface charge of the MFnS.
         Note: Here, the average size and surface charge of functional MFnS were found to be 77.09 nm and -22.3 mV, respectively.

2. Bacterial Culturing and Stock Solution Preparation

1. Bacterial Culturing
   1. Hydrate a freeze-dried pellet (E. coli O157:H7) in 1 mL of nutrient broth and then add an additional 5 mL of broth.
   2. Apply 100 µL of this solution to an agar plate and streak using a sterile inoculation loop, followed by incubation at 37 °C for 24 h.
   3. After 24 h, select an isolated colony from the agar plate and add it to a 15 mL of culture broth. Incubate at 37 °C for 4-6 h, while monitoring the optical density value (absorbance at 600 nm).
   4. Once an optical density value of 0.1 is obtained, stop culturing, and perform serial dilutions.
2. Serial dilutions
   1. Add 900 µL of nutrient broth to eight sterile microcentrifuge tubes.
   2. Add 100 µL of the bacterial suspension to the first tube (dilution 10^-1).
   3. Take 100 µL from the first tube and add it to the second, to obtain a dilution of 10^-2. Continue this pattern for the remaining tubes, ending with a final dilution of 10^-8.
   4. Transfer 100 µL from each tube to separate agar plates, and incubate for 24 h at 37 °C.
   5. The next day, count the colony forming units (CFUs) on each plate. Select the plate with ~100 CFUs. Use the corresponding dilutions for further experiments (100 µL = ~100 CFUs).
      Note: Here it was the 10^-6 dilution that resulted in ~100 CFUs.

3. Rapid Detection of E. coli O157:H7 using MFnS

1. Spike various PBS solutions (1X, pH 7.4, 300 µL) with increasing amounts of the 10^-6 bacterial stock, resulting in CFU ranges from 1-100. Add a consistent amount of MFnS (100 µL) to these solutions.
2. Create one baseline solution that contains only PBS (1X, pH 7.4, 300 µL) and MFnS (100 µL).
3. Incubate solutions for 30 min at 37 °C and then allow them to cool to room temperature.
4. Transfer individual solutions to the magnetic relaxometer (0.47 Tesla) and record changes in relaxation times (T2) relative to the CFUs in each solution.
   1. To record changes in T2 values, begin by measuring the T2 value of the baseline solution that contains only PBS and MFnS.
      1. Place the solution in the magnetic relaxometer. Open the respective software, select the "T2 relaxation" setting and press "measure."
   2. Following the collection of the baseline T2, measure the T2 values of the additional solutions that were spiked with various concentrations of bacteria.
      Note: The change in T2 is equivalent to baseline T2 subtracted from the spiked T2.
5. Remove samples from the magnetic relaxometer and centrifuge the tubes at 2880 x g for 10 min.
6. Decant the supernatant and resuspend bacterial pellets in 100 µL of PBS (1X, pH 7.4).
7. Add 80 µL of each resuspension to a 96-well plate and record fluorescence intensities at 595 nM.
   Note: Testing can be repeated using different solvents, including lake water, milk and others as described in the results section

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