Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors

Overview

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.

Materials

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