Multifunctional magnetite and silica–magnetite nanoparticles: Synthesis, surface activation and applications in life sciences

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Abstract

A method for the introduction of amine groups onto the surface of magnetite and silica-coated magnetite nanoparticles has been established based on the condensation of aminopropyltriethoxysilane. Amine-modified particles were grafted with an oligonucleotide and used in the capture of a complimentary sequence. The particles’ efficiency at capture was observed to correlate directly with amine group surface density.

Introduction

Magnetic separation was introduced in the context of biotechnology in the early seventies by Robinson et al. [1] and since then it has become a routine technique for the quick, easy, sensitive and reliable separation of biomolecules [2], [3]. The basic procedure in magnetic bioseparations comprises three steps: the selective binding of the biomaterial of interest to a paramagnetic solid phase support, the separation of the support from the surrounding matrix using a magnetic field, and the recovery of the bound molecular species by elution. The paramagnetic materials used in such processes are superparamagnetic, meaning that they respond strongly to magnetic fields, but retain no residual magnetism after the field is removed. The morphology, surface area and the magnetic susceptibility of the support contribute in a major fashion to the efficiency of the separation processes. Additionally, the chemical nature of the support surface can be used to specify the separation process: the adsorption of the molecular species can be driven by the interactions at the molecular level between the surface groups of the paramagnetic particles and those of the target molecules (eg. antigens/antibodies or complementarity in the case of nucleic acids).

Magnetite nanoparticles (pure Fe3O4) have been synthesised and used as a basis for production of such supports [4], [5], [6] and magnetite nanoparticles in the size range of 5–10 nm [7], [8] with various morphologies [9], [10] have been reported. We have recently developed a scaled-up process for the easy, reproducible and inexpensive bulk synthesis of magnetite nanoparticles (30–100 nm), as well as a method for their surface coating with silica [7]. The surface of these materials can be also modified with polymers [11], [12], [13], [14], [15] or other molecules, eg. phospholipids [16], to improve the affinity of the particles for specific target species. In this context, the use of organosilanes has recently been described by Ma et al. [17] and Liu et al. [18], but no detailed study on their surface modification via silanisation in terms of optimisation has been performed so far.

Organosilanes are bifunctional molecules containing a trialkoxy or trichlorosilane group (which can bind covalently to the free –OH groups at the surface of the particles) and an organic head-group functionality that determines the final chemical character of the modified surface (e.g. –NH2, –OH, –SH etc.). Working with alkoxysilanes involves several difficulties. The moisture sensitivity and highly chemically reactive nature of the alkoxysilane moiety can lead to uncontrollable, inhomogeneous and non-reproducible surface coverage if the surface activation reaction is performed under inappropriate experimental conditions. For these reasons, a systematic study of the parameters involved, and their influence on the nature of the surface of the final material is necessary to optimise the efficiency of the method. We report a simple and effective procedure for the surface modification of pure magnetite and silica-coated magnetite with an –NH2 linker using aminopropyltriethoxysilane (APTS) as the surface modification agent. The amine activated surface can be used to covalently link specific biomolecules and thereby generate “bioactive” nanoparticles [19], [20], [21], [22]. This has been demonstrated by immobilisation of 5′-amino-modified oligonucleotide sequences to surface amine groups which had been converted to aldehyde groups via treatment with glutaraldehyde and which had been used for the specific capture of complementary single stranded DNA in solution.

Section snippets

Materials

All reagents used were available commercially and were of the highest purity grade. The methanolic coupling solution for the colorimetric assay contained 0.8% (v/v) glacial acetic acid in dry methanol. The hydrolysis solution contained 75 ml H2O, 75 ml MeOH and 0.2 ml glacial acetic acid. (1×)SSC and (13×)SSC buffers were prepared by diluting a stock solution of (20×)SSC buffer (175.3 g NaCl, 88.2 g sodium citrate, 1 l H2O, pH 7.4) with distilled, deionised water, adjusting to pH 7.4 and were

Magnetite and silica-coated magnetite nanoparticles

SEM analysis of the nanoparticles indicated that both the pure magnetite and silica–magnetite nanocomposites were approximately spherical in nature (Fig. 1a and b). However, when viewed under the transmission electron microscope, with higher magnification and better resolution, the particles exhibited a rhombic structure (Fig. 1c and d) with sizes ranging from 30 to 100 nm. Two regions with different electron densities can be distinguished for the rhombic silica-coated magnetite nanoparticles:

Conclusions

Amine-functionalised magnetite and silica–magnetite nanoparticles have been synthesised using APTS as surface modification agent. By variation of the experimental conditions (reaction time and temperature) variants with different surface amine group densities have been obtained and characterised. The performance of these materials in the immobilisation of oligonucleotides and their use in hybrid capture of complementary sequences has been tested where it was observed that efficiency was

Acknowledgements

The authors thank the European Union and FP5 (CHEMAG Project, G5RD-CT-2001-00534) for financial support, Dr. Guidi for the SEM and TEM images and E. Borioni for his help in the experimental work.

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