Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

Magnetic nanoparticles are ideal tools for analytical science and nano medicine due to their strong magnetic properties and the easy functionalization of their surface. We report here the first instance of a conjugate between amino functionalized silica magnetic particles and the siderophore feroxamine along with evaluation for the capture of photogenic material. Siderophores are small, organic molecules which play a key role in the iron partake mechanism, and they are recognized by specific outer membrane receptors of bacteria.

This video displays, step-by-step, an efficient protocol to prepare magnetic iron nanoparticles. Then, they were coated with silica to protect dispersion and to protect the magnetic nucleus. The resulting reactor surface was functionalized with amine groups.

Synthesis of magnetic nanoparticles conjugated with feroxamine. Add 0.5 gram of Fe(acac)3 in a 20 mL glass vial. Then, mix with a 10 mL of benzyl alcohol.

Sonicate this mixture for 2 minutes. Then, transfer to a heating block and heat at 180 degrees Centigrades for 72 hours. Rinse the nanoparticles with 96%ethanol.

And centrifuge for 30 minutes. Separate the nanoparticles from the supernatant by magnetic attraction, using a neodymium magnet. Discard the residual solvent.

Discard the supernatant, alternating with sonication, until the solvent looks clear. Prepare a suspension of 2 grams of MNP in 80 milliliters of isopropanol, add 4 milliliters of 21%ammonia, 7.5 milliliters of distilled water, and 0.56 milliliters of TEOS. Heat the mixture at 40 degrees Centigrades for 2 hours, with continuous stirring.

Then, sonicate for 1 hour. Separate the MNP with a magnet, discard the supernatant, disperse it in 30 milliliters of isopropanol, add ammonia, distilled water, and TEOS, in the same order as described before. Heat the mixture at 40 degrees Centigrades for two hours with continuous stirring.

Then, sonicate for one hour. Remove and wash conveniently the magnetic stir bar, to recover all the material. Discard the supernatant and rinse the nanoparticles with 96%ethanol three times, alternating with sonication.

Dry the nanoparticles under vacuum at room temperature for 12 hours. Rinse 500 milligrams of the MNP@SIO2 obtained from previous step with dimethylformamide. Sonicate them and discard.

Re-suspend the particles in a round-bottom flask, stir with a magnetic bar and add 9 milliliters of APTES. Stir the mixture at 60 degrees Centigrades for 12 hours. Discard the supernatant and rinse the nanoparticles with 96%ethanol three times, alternating with sonication.

Dissolve a 100 mg of deferoxamine, mesylate salt and 53.0 milligrams of iron acetylacetonate in 5 milliliters of distilled water. Stir the mixture overnight at room temperature. Wash the resulting product three times with 20 milliliters of ethyl acetate in a separation funnel.

Remove the organic solvent under vacuum. Freeze-dry the aqueous phase to afford feroxamine and a red solid. Add 350 milligrams of succinic anhydride to a solution of 100 mg of feroxamine in 5 milliliters of pyridine in a 50 milliliters round-bottom flask.

Stir the resulting mixture, at room temperature, for 16 hours. Remove the excess of pyridine under reduced pressure. Dissolve the reaction crude in 3 milliliters of methanol.

Transfer the methanolic solution into a LH-20 column and elute at 0.5 mL/minute. Collect the red fraction and remove the methanol under vacuum. Rinse 30 mg of dry amine functionalized nanoparticles twice with DMF and sonicate the nanoparticles in a 100 milliliter Erlenmeyer flask for 30 minutes.

Prepare a solution of 200 milligrams N-succinylferoxamine, 173 milligrams Benzotriazole 1-yl-oxy-tris-phosphonium hexafluorophosphate BOP, 46 mg 1-hydroxybenzotriazole HOBt and 128.8 milligrams N, N-diisopropylethylamine DIPEA, in 10 mL of DMF, in a 50 milliliter round-bottom flask. Suspend the previously rinsed MNP@SiO2@NH2 in a 3 milliliter of DMF, under sonication in dry and oxygen free conditions. Use argon atmosphere.

Add, dropwise, mix A to mix B.Shake the final mixture, using an orbital shaker, at room temperature overnight. Separate the resulting conjugate from the suspension using a magnet. Bacterial assay with Y.enterocolitica strains to quantify the isolation and capture of pathogenic bacteria with nanoparticles.

Prepare a suspension of all the intermediate nanoparticles and the final conjugate in PBS at 1 mg/mL in sterile tubes. Prepare a culture of Y.entorocolitica in 5 mL of Luria Bertani, LB, broth overnight. Prepare a 5 mL of iron deficient Trypcase Soy Broth, TSB, by the addition of 50 microliters of 10 mM 2, 2-bipyridyl solution.

Inoculate the 5 milliliter of iron deficient TSB broth with 50 microliters of the overnight culture of Y.enterocolitica. Incubate at 37 degrees Centigrades with agitation until an OD600 of 0.5 to 0.8 is reached. Take a 100 microliters of the overnight culture and dilute it in a tube containing 900 microliters of PBS to obtain the first 1/10 dilution.

Then, prepare a 1/100 dilution from the first dilution using the same procedure to get a concentration of bacterial cells at 10 to the power of 6 Colony Forming Units per milliliter approximately. Add a 100 microliters of nanoparticles suspension and 1 mg/mL to 1 milliliter of the 1/100 bacteria dilution. Separate the MNP bacteria aggregates, by using a magnet, discard, carefully, the supernatant.

Rinse the separated nanoparticles twice with 1 millimeter PBS using a vortex. Prepare four successive 1/10 dilutions from the former suspension, until a 10 to the power of minus 4 dilution. Plate 10 microliters of each dilution onto TS agar plates.

Photograph the plate with a gel digitalizer in epi white mode. Process the image, using the convenient software, to amplify a spot to count the number of individual colonies. To confirm the formation of the covalent bond between the nanoparticles and the siderophore, we employ FT-IR Raman spectroscopy to monitoring the synthesis, observing how new bands appear in agreement with a new functional groups in each step.

TEM and SEM microscopy were used to observe the morphology and particle size. Thermogravimetric analysis to measure the weight loss due to the addition of organic material. And XPS allow us to analyze the different oxidation states of the atoms in the surface and confirming the formation of covalent bonds.

Finally, we used all the intermediates on the final conjugate to test its ability to capture bacteria, in order to establish their properties as biosensor. This protocol was designed to take advantage of the high biocompatibility of magnetite nanoparticles. The coating with silica improves the dispersion and protect the magnetic nucleus from degradation in aqueous media.

And also give a reactive surface to functionalize with amine groups which are necessary to covalently bind an acid modified siderophore by carbodiimide chemistry. In this case, N-succynilferoxamine. The conjugate bacteria capture capability was tested and as a result, a low number of colonies was found, without significant difference with the intermediates due to a specific interactions on bulky effects presented in the PBS bacterial suspension.