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

Summary

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.

Abstract

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 Stöber 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.

Introduction

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 bacteria^3,4. The preparation of conjugates between siderophores and MNP along with their evaluation for the capture and isolation of bac....

Protocol

NOTE: For the reactions performed under inert atmosphere conditions, all the glassware was previously dried in an oven at 65 °C, sealed with a rubber septum and purged with argon three times.

1. Synthesis of magnetic nanoparticles conjugated with feroxamine

1. Synthesis of Fe₃O₄ magnetic nanoparticles (MNPs)
   1. Add 0.5 g of Fe(acac)₃ in a 20 mL glass vial and then mix with 10 mL of benzyl alcohol.
   2. Sonicate this mixture for 2 min, then transfer to a heating block and heat at 180 °C for 72 h.
   3. After the reaction is completed, allow the vials to cool down, rinse ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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