The main goal of this protocol is to design and synthesize dual-modal nanosensors capable of detecting bacterial contaminants such as E.coli O157:H7. Magneto-fluorescent nanosensors are synthesized by functionalizing iron oxide nanoparticles via a two-step procedure. First, targeting antibodies are conjugated to the surface of the nanoparticle.
Then fluorescent dye is loaded into its coding. The paring of these modalities allows for rapid and sensitive detection of bacterial contaminants in both low and highly concentrated solutions. In the presence of a small amount of bacteria, the nanosensors will swarm around the bacteria due to the targeting of the conjugated antibodies.
This swarming alters the interactions of the nanosensors magnetic core with the surrounding water protons, allowing for sensitive detection of changes in magnetic relaxation values. As the concentration of bacterial contaminants in solutions increases, swarming decreases and the magnetic modality's ability to quantify contamination is reduced. However, as the bacterial concentration increases, so does the fluorescent submission of the bio nanosensors.
It is for this reason that the pairing of magnetic and fluorescent modalities is important. The combination has allowed for the detection of as little as one colony forming unit of E.coli O157:H7 within minutes. The first step in the systhesis of magneto-fluorescent nanosensors is the preparation of a iron salt solution consisting of both ferous and ferric chloride.
The iron will provide the nanosensors with a magnetic core which allows for its adaptability to magnetic relaxation platforms. Additional solutions needed are polyacrylic acid and ammonium hydroxide. Hydrochloric acid is introduced to the iron salt solution, which is then added to the ammonium hydroxide solution while vortexing.
Finally the polyacrylic acid solution is added, and the resulting mixture is vortexed for an additional hour while the reaction continues. The resulting solution is centrifuged in order to precipitate the larger molecules and isolate nanoparticles that are smaller than 100 nanometers in size. The solution is then dialyzed overnight for purification.
The next step is the conjugation of targeting antibodies to the polyacrylic acid coding of the newly synthesized iron acid nanoparticles. Materials needed include MES buffer, EDC, NHS and the selected antibodies. EDC is first added to the nanoparticle solution, followed by NHS and finally the antibody.
The mixture is then placed on a tabletop mixer for three hours while the reaction continues. The solution is purified using a magnetic column. The magnetized column captures the nanoparticles, allowing only free floating antibodies to exit.
Afterwards the column is washed with PBS buffer, and the conjugated nanosensors are collected. The nanoparticles are then ready for the addition of fluorescent dye, providing the second key modality used for detection. One of the most key features of our nanosensor is the combination of the magnetic and fluorescent modalities, which is important for a number of reasons.
Firstly, the combination of the modalities allows each one to sort of cross check the other one, which greatly reduces the risk of false positives and false negatives, which is one of the greatest hurdles faced by different diagnostic platforms used today. And secondly, the combination of the modalities greatly widens the range within which we can not only detect but also quantify bacterial contamination. The final step in the preparation of the nanosensors is the encapsulation of fluorescent dye in the coding of the nanoparticle.
This is simply achieved by adding dye to the solution while vortexing, and then allowing further time for the dye to diffuse into the coding of the nanosensor. The fully functionalized nanosensors are then dialyzed one final time for purification. In order to characterize the nanosensors, the size and fluorescent submission are recorded using a zetasizer and a fluorescence plate reader.
A small sample of the nanosensor is added to a cuvette water solution and placed in the zetasizer for the recording of size. For the fluorescence analysis a sample of the nanosensor is placed on a 96 well plate and inserted into the fluorescence plate reader. Ideally the nanosensors will be around 70 nanometers in diameter, and have a fluorescent submission of 575 nanometers.
In addition to the synthesis of the nanosensors, the culturing of bacteria is necessary to provide the bacterial targets in the lab. A glycerol stock of bacteria is used to prepare a culture in nutrient broth. This solution is then allowed to incubate.
Following the initial incubatory period, serial dilutions are performed in order to achieve a wide range of bacterial concentrations. One hundred microliters of each concentration are plated to an nutrient auger plate and then incubated for 24 hours. Following this incubation the colonies on each plate are counted in order to determine the colony forming unit, or CFU count per mil of each diluted stock.
Now that the bacterial stock solutions have been prepared, they are able to be used in conjunction with the prepared nanosensors. And one important aspect of this particular serotype of the bacteria that distinguishes it from other serotypes is like suppose if you get infected with the other serotypes, you need to have many colony forming units to get an infection. But in this particular E.coli 10 to 100 CFUs or colony forming units are good enough to give you an infection.
Our technology is established in such a way that you don't miss even a single colony bacterial contamination is there. In order to prepare solutions for reading in the magnetic relaxometer, 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 and 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 reading are less quantifiable, which is why the use of fluorescent subsmission 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 supernatent is discarded, and the bacterial pellet is resuspended in PBS. Finally the sample may be analyzed via fluorescent submission. The strength of the emission will be relative to the amount of nanosensors remaining in solution, and therefore also the amount of bacterial present.
As can be seen, fluorescent submission strengthens as the bacterial concentration increases, and it becomes more sensitive as well. This is why the pairing of both magnetic and fluorescent modalities allows for the detection and quantification of bacterial contamination in both early and late developmental stages. In addition to the detection of bacteria in simple solutions such as PBS, these nanosensors also possess the capability to function in more complex media, such as lake water or milk.
Furthermore, these nanosensors have been tested for the specificity in the presence of non-target bacteria and heat inactivated target bacteria. As shown magneto-fluorescent nanosensors have little to no reaction with non-targeted or non-living bacteria due to the specificity of the conjugated antibodies. This demonstrates their effectiveness for the detection of specific bacteria while in the presence of other species.
Finally, it is also important to note that these nanosensors may be easily customized for the detection of other pathogens as well. You can synthesize or you can formulate these nanosensors that we did in our lab within one month now. You can do it within one month, and that one month of product can give you like 10 years of application.
