Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors

Published: March 29, 2024

Abstract

Source: Shelby, T., Sulthana, S., McAfee, J., Banerjee, T., Santra, S. Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7. J. Vis. Exp. (2017).

This video demonstrates a technique to detect bacterial contaminants in food and water samples using nanosensors. The target pathogenic bacteria are detected through a combination of magnetic relaxation and fluorescence emission modalities.

Protocol

1. 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.

開示

The authors have nothing to disclose.

Materials

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

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記事を引用
Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors. J. Vis. Exp. (Pending Publication), e22063, doi: (2024).

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