Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors

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.