This study presents a method for quantifying bacterial concentrations using magneto-fluorescent nanosensors. The approach leverages changes in magnetic properties and fluorescence emission to detect bacteria effectively.
Begin by adding magneto-fluorescent nanosensors to the bacterial samples. These nanoparticles contain bacteria-specific antibodies and a fluorescent dye.
Incubate to allow interaction with bacteria, causing detectable changes in the magnetic properties of the solution.
Include a baseline nanosensor solution without bacteria as a control.
Transfer each solution to a magnetic relaxometer, where a constant magnetic field aligns the magnetic particles within the sample.
Apply a brief pulse to disrupt alignment temporarily.
The relaxometer measures how quickly the particles return to their original state, known as the T2 relaxation time.
Nanosensors clustering around bacteria increases T2 compared to the control, enabling quantification even at low bacterial concentrations.
Conversely, higher bacterial concentrations decrease nanosensor clustering, affecting T2 values.
For accurate quantification, perform fluorescence measurement.
Accordingly, centrifuge to isolate bacteria and bound nanosensors.
Resuspend the pellet for fluorescence measurement.
Upon excitation, the fluorescent nanosensors emit light, aiding in bacterial measurement.
In order to prepare solutions for reading in the magnetic relaxometry, 300 microliters of PBS is first pipetted into an eppendorf tube. Then, a sample of bacterial stock is added, followed by the addition of nanosensors. The solution is then transferred to a glass tube, and a piece of parafilm is placed on top in order to prevent evaporation.
The glass tube is then placed inside a larger NMR tube and inserted into the magnetic relaxometer. A baseline solution containing no bacteria and only nanosensor in PBS is used to get a baseline T2 reading as shown. Then, solutions containing various concentrations of bacteria are inserted into the magnetic relaxometer for analysis, and the changes in T2 values are caused by the binding between the nanosensors and the bacteria.
As shown, the presence of as few as one bacterial CFU can be detected within minutes using this modality. However, as the bacterial concentration increases, the MR readings are less quantifiable, which is why the use of fluorescence emission data is also tantamount to an accurate bacterial quantification. Before fluorescence data can be collected, the sample must first be centrifuged.
The solution is transferred from the glass tube to an eppendorf tube, and then centrifuged. This separates the bacteria and the nanosensors bound to it from free-floating nanosensors in solution. The supernatant is discarded and the bacterial pellet is resuspended in PBS. Finally, the sample may be analyzed via fluorescence emission. The strength of the emission will be relative to the amount of nanosensors remaining in solution, and therefore, also to the amount of bacteria present.
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