Name: synthesis of functionalized magnetic nanoparticles, their conjugation with the siderophore feroxamine and its evaluation for bacteria detection
Uploaded: 2020-06-16T14:30:00
Description: Watch this Scientific Journal Video about Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection at JoVE.com

The authors gratefully acknowledge Professor Klaus Hantke (University of T&#252;bingen, Germany) for kindly supply the Yersinia enterocolitica strains used in this work. This work was supported by grants AGL2015-63740-C2-1/2-R and RTI2018-093634-B-C21/C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union. Work in University of Santiago de Compostela and University of A Coru&#241;a was also supported by grants GRC2018/018, GRC2018/039, and ED431E 2018/03 (CICA-INIBIC strategic group) from Xunta de Galicia. Finally, we want to thank to Nuria Calvo for her great collaboration doing the voice-off this video protocol.

NOTE: For the reactions performed under inert atmosphere conditions, all the glassware was previously dried in an oven at 65 &#176;C, sealed with a rubber septum and purged with argon three times.
1. Synthesis of magnetic nanoparticles conjugated with feroxamine
<ol>
	<li>Synthesis of Fe3O4 magnetic nanoparticles (MNPs)
	<ol>
		<li>Add 0.5 g of Fe(acac)3 in a 20 mL glass vial and then mix with 10 mL of benzyl alcohol.</li>
		<li>Sonicate this mixture for 2 mi...

<ol>
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This protocol describes the synthesis of a conjugate between magnetic nanoparticles and the siderophore feroxamine by covalent bonding. The synthesis of magnetite was carried out using the protocol reported by Pinna et al.5 followed by silica coating to protect the magnetic core of corrosion in aqueous systems, to minimize the aggregation and to provide a suitable surface for functionalization6. The silica coating process was modified. Instead of carrying out three coatings...

The bacteria detection methods using MNP are based on the molecular recognition of antibodies, aptamers, bioprotein, carbohydrates conjugated to MNP by the pathogenic bacteria1. Taking into account that siderophores are recognized by specific receptors on the outer membrane of bacteria, they could also linked to MNP to increase their specificity2. Siderophores are small organic molecules involved in the Fe3+ uptake by bacteria3,4. The preparation of conjugates between siderophores and MNP along with their evaluation for the capture and isolation of bacteria has not yet been reported.
One of the crucial steps in the synthesis of conjugates of magnetic nanoparticles with small molecules is the selection of the type of bond or interaction between them to ensure that the small molecule is attached to the surface of the MNP. For this reason, the procedure to prepare the conjugate between magnetic nanoparticles and feroxamine&#8212;the siderophore recognized by Yersinia enterocolitica&#8212;was focused at the generation of a modifiable surface of the MNP to allow linking it covalently to the siderophore by carbodiimide chemistry. In order to get an uniform magnetite nanoparticles (MNP) and to improve nucleation and size control, a solvolysis reaction with benzyl alcohol was carried in a thermal block without shaking5. Then, a silica coating was generated by St&#246;ber method to confer protection and improve the stability of the nanoparticles suspension in aqueous media6. Taking into account the structure of the feroxamine, the introduction of amine groups is necessary to produce suitable nanoparticles (MNP@SiO2@NH2) to be conjugated with the siderophore. This was achieved by condensation of (3-aminopropyl)triethoxysilane (APTES) with the alcohol groups present on the surface of the silica modified nanoparticles (MNP@SiO2) using a sol-gel method7.
In parallel, the feroxamine iron(III) complex was prepared by complexation of the commercially available deferoxamine with iron acetyl acetonate in aqueous solution. N-succinylferoxamine, bearing succinyl groups that will act as linkers, was obtained by the reaction of feroxamine with succinic anhydride.
The conjugation between MNP@SiO2@NH2 and N-succinylferoxamine to give MNP@SiO2@NH@Fa was carried out through carbodiimide chemistry using as coupling reagents benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) and 1-hydroxybenzotriazole (HOBt) in a soft basic media to activate the terminal acid group in N-succinylferoxamine8.
Once the MNPs were characterized, we evaluated the capabilities of bare and functionalized magnetic nanoparticles to capture wild type (WC-A) and a mutant of Y. enterocolitica lacking feroxamine receptor FoxA (FoxA WC-A 12-8). Plain MNPs, functionalized MNPs and the conjugate MNP@SiO2@NH@Fa were allowed to interact with each Y. enterocolitica strain. The bacteria-conjugate aggregates were separated from the bacteria suspension by the application of a magnetic field. The separated aggregates were rinsed twice with phosphate buffered saline (PBS), re-suspended in PBS to prepare serial dilutions and then, they were plated for colony counting. This protocol demonstrates each step of the synthesis of MNP@SiO2@NH@Fa, the structural characterization of all the intermediates and the conjugate, and a bacterium capture assay as an easy way to evaluate the specificity of the conjugate in relation to the intermediates.9

<table>
	<tbody>
		<tr>
			<td>1-Hydroxybenzotriazole hydrate 
			HOBT</td>
			<td>Acros</td>
			<td>300561000</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2&#8242;-Bipyridyl</td>
			<td>Sigma Aldrich</td>
			<td>D216305</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Aminopropyltriethoxysilane 99%</td>
			<td>Acros</td>
			<td>151081000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution 28% NH3</td>
			<td>Sigma Aldrich</td>
			<td>338818</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate BOP Reagent</td>
			<td>Acros</td>
			<td>209800050</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>822259</td>
			<td></td>
		</tr>
		<tr>
			<td>Deferoxamine mesylate salt &#62;92,5% (TLC)</td>
			<td>Sigma Aldrich</td>
			<td>D9533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, anhydrous, 96%</td>
			<td>Panreac</td>
			<td>131085</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate, Extra Pure, SLR, Fisher Chemical</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) acetylacetonate 97%</td>
			<td>Sigma Aldrich</td>
			<td>F300</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth (Lennox)</td>
			<td>Sigma Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine, 99.5+%, AcroSeal</td>
			<td>Acros</td>
			<td>459591000</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide, 99.8%, Extra Dry, AcroSeal</td>
			<td>Acros</td>
			<td>326871000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridine, 99.5%, Extra Dry, AcroSeal</td>
			<td>Acros</td>
			<td>339421000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex LH-20</td>
			<td>Sigma Aldrich</td>
			<td>LH20100</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinic anhydride &#62;99%</td>
			<td>Sigma Aldrich</td>
			<td>239690</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetraethyl orthosolicate &#62;99,0%</td>
			<td>Sigma Aldrich</td>
			<td>86578</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of functionalized magnetic nanoparticles, their conjugation with the siderophore feroxamine and its evaluation for bacteria detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

An exhaustive structural characterization is carried out in order to determine the morphology and the properties of each intermediate and the final conjugate. For this purpose, the techniques XRD, FT-IR, Raman spectroscopy, TGA, TEM, EDX mapping and XPS are used in order to demonstrate the formation of the conjugate. The oxidation states of the atoms at the surface of the nanoparticles acquired by X-ray photoelectron spectroscopy (XPS) are the most relevant data to confirm the formation of covalent bonds between the nano...

Watch this Scientific Journal Video about Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection at JoVE.com

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

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

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.

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