method of fluorescence detection. Fig. 1: Synthesis principle of multifunctional magnetic nanoparticles for life science applications (schematic view). Here we ...
European Cells and Materials Vol. 3. Suppl. 2, 2002 (pages 81-83)
ISSN 1473-2262
MULTIFUNCTIONAL SUPERPARAMAGNETIC NANOPARTICLES FOR LIFE SCIENCE APPLICATIONS S. Rudershausen, C. Grüttner, M. Frank, J. Teller, & F. Westphal micromod Partikeltechnologie GmbH, Friedrich-Barnewitz-Str. 4, 18119 Rostock, Germany METHODS: Magnetic nanoparticles can be divided in two sections given by the kind of application: nanoparticles separable by permanent magnets (a) and nanoparticles separable by a high gradient magnetic field (b). Nanoparticles with the magnetic properties (a) or (b) can be synthesized via two different synthesis strategies: either by precipitation of iron oxide in the presence of polymers or by coating of iron oxide with polymers according to the core-shell principle (Fig.2) [1].
INTRODUCTION: Superparamagnetic nanoparticles have a high potential as carriers for oligonucleotides and biomolecules (e.g. proteins, antibodies, enzymes, and nucleic acids) in different life science applications (e.g. immunoassays, magnetic resonance imaging, magnetic cell separation, magnetic oligonucleotide and nucleic acid separation). Beside the magnetic separation and targeting of nanoparticles, different methods for their detection become more and more important. Up to now the detection of magnetic nanoparticles was mainly realized by their magnetic relaxation properties in resonance techniques. A multifunctional fluorescent superparamagnetic nanoparticle separable with a conventional permanent magnet and tagged with a biomolecule (Fig. 1) allows for magnetic separation and magnetic targeting in life science applications in combination with the sensitive method of fluorescence detection.
Fig. 2: Strategies for the synthesis of monodisperse superparamagnetic nanoparticles and resulting size ranges.
Furthermore, the technique of high pressure homogenization can be used to move the borderline between (a) and (b) to smaller nanoparticle diameters. Fluorescence labelling of superparamagnetic nanoparticles can be done by covalent coupling of fluorescent dyes to the matrix polymer covering the magnetic core of the particle. Regarding the applications of our nanoparticles, we used the biocompatible and biodegradable biopolymer dextran as a matrix polymer benefitting additionally by its huge number of functionalities (hydroxyl groups) for the covalent coupling of fluorescence markers and biochemical compounds. For instance three commonly used fluorescence markers like aminofluorescein (employed as the reactive dichlorotriazinyl derivative DTAF), rhodamine B or DAPI (4’,6-diamidino-2-phenylindol) can be attached covalently to the surface of the superparamagnetic dextran nanoparticle nanomag® -D (Fig.3). In the case of DAPI a bifunctional carboxylic acid spacer is used to
Fig. 1: Synthesis principle of multifunctional magnetic nanoparticles for life science applications (schematic view)
Here we report on the synthesis, magnetic properties, and the chemical and biochemical functionalization of superparamagnetic nanoparticles. In a modular way the magnetic properties of the particles are combined with the introduction of fluorescence and the immobilization of proteins, antibodies, enzymes, nucleic acids and oligonucleotides on the particle surface.
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couple the fluorescent dye via one of its amino functionalities to the dextran.
chosen depending on the desired application and the special kind of biomolecule.
In a typical experiment, a suspension of nanomag® -D in borate buffer (pH = 9) is shaken with DTAF overnight. Unreacted dye is then removed by repeated magnetic washing (principle of separation and resuspension) of the particles with water.
RESULTS & DISCUSSION: Superparamagnetic nanoparticles having a matrix of the biodegradable and biocompatible polymer dextran (nanomag® -D) were synthesized in three different sizes: 50 and 100 nm (separable by high gradient magnetic fields) and 250 nm (separable by conventional permanent magnets) according to Grüttner et al [1]. To get the advantages of smaller superparamagnetic nanoparticles like prolonged blood circulation and higher surface area, and to keep the advantage of separability by a permanent magnet, we applied high pressure homogenization on nanomag® -D with a diameter of 250 nm. The resulting nanoparticles had a diameter of about 130 nm - and were still separable by a conventional permanent magnet - which speeds up different coupling procedures of biomolecules on the particle surface with many washing steps and allows for magnetic targeting, for example.
Fig. 3: Covalent coupling of aminofluorescein, rhodamine B and DAPI to the biodegradable particle matrix polymer dextran To use nanomag® -D as a carrier biomolecules like proteins, antibodies, enzymes, nucleic acids and oligonucleotides can be immobilized additionally to the fluorescence marker on the surface of the superparamagnetic dextran nanoparticle because of the high number of hydroxyl groups of the dextran matrix polymer. In general three activation procedures were applied for the coupling of biomolecules to the dextran matrix of the nanoparticle: periodate activation (a), cyanogen bromide activation (b) and NHS (Nhydroxysuccinimide) / carbodiimide activation (c) (compare Fig.4). The appropriate method can be
Fig. 4: Covalent coupling of biomolecules to the biodegradable particle matrix polymer dextran Fluorescence detection is a very common method and is highly sensitive. Lots of devices for the detection of fluorescently-labelled compounds,
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REFERENCES: 1C. Grüttner, J. Teller, W. Schütt, F. Westphal, C. Schümichen, and B.R. Paulke (1997) Preparation and Characterization of Magnetic Nanospheres for In Vivo Application in Scientific and Clinical Applications of Magnetic Carriers (eds U.O. Häfeli, W. Schütt, J. Teller, and M. Zborowski) Plenum Press, New York, pp 5368.
already established on the market, can also be used for the fluorescence detection of superparamagnetic nanoparticles nanomag® -D labelled with a fluorescent dye. Fluorescence filters are available, which are compatible for the emission wavelengths of aminofluorescein, rhodamine B and DAPI. So we coupled these dyes covalently to the dextran matrix polymer of nanomag® -D benefitting from the large number of hydroxyl groups on the particle surface deriving from the dextran. Aminofluorescein was employed as its reactive trichlorotriazinyl derivative DTAF, which easily reacts with the hydroxyl groups under alkaline conditions. Rhodamine B was coupled via its carboxylic acid group by using the method of carbodiimide activation. The attachment of DAPI took place after the introduction of a bifunctional carboxylic acid spacer on the particle surface. Spacer and DAPI were both coupled by using carbodiimide activation. Because of the covalent bonding of the fluorescent dyes to the matrix polymer dextran, no leakage of the dye from the nanoparticle surface into the surrounding medium is observed. Since dextran has so many hydroxyl groups a biochemical functionalisation of already fluorescently-labelled nanomag® -D can additionally be done giving a nano-carrier. For instance antibodies or proteins like streptavidin or protein A were covalently bound to nanomag® -D using coupling methods a) to c). The method of choice depends on the kind of the biomolecule and also on the application of the carrier. We applied a periodate activation (a in Fig.4), a cyanogen bromide activation (b) and a NHS / carbodiimide activation with good results. In first experiments multifunctional fluorescent superparamagnetic nanoparticles nanomag® -D were loaded with biomolecules, moved into cells, and were visualized there because of their fluorescence emission and had a special effect on cell growth and behavior, for example. CONCLUSIONS: Superparamagnetic nanoparticles of different adjusted sizes from 50250 nm were fluorescently-labelled with commonly used dyes and tagged with biomolecules like antibodies or streptavidin. This allows for complex applications in life sciences combining magnetic separation and targeting with fluorescence detection and a specific biological effect, for example.
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