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Synthesis, characterisation and application of silica-magnetite nanocomposites
ARTICLE IN PRESS
Journal of Magnetism and Magnetic Materials 284 (2004) 145–160 www.elsevier.com/locate/jmmm
Synthesis, characterisation and application of silica-magnetite nanocomposites Ian J. Brucea,b,, James Taylorb, Michael Toddb, Martin J. Daviesb, Enrico Borionib, Claudio Sangregorioa, Tapas Senb a
Istituto di Scienze Chimiche, Piazza Rinascimento 6, Universita degli Studi di Urbino, 61029 Urbino, Italy b School of Science, University of Greenwich, Chatham Maritime, Chatham ME6 4BT, Kent, UK Received 19 March 2004; received in revised form 17 May 2004 Available online 2 August 2004
Abstract Silica-magnetite composites were prepared for eventual applications in biomolecular separations (nucleic acids). Their production on large scale has been optimised and they have been extensively characterised in a physical and chemical context. They perform at least as well, if not better than a commercially available equivalent at adsorbing and eluting DNA. Several methods for the preparation of magnetite were compared in order to select one, which produced particles, possessing high magnetic susceptibility, low rate of sedimentation and good chemical stability. Of the main methods studied: (i) oxidative hydrolysis of iron(II) sulphate in alkaline media, (ii) alkaline hydrolysis of iron(II) and iron(III) chloride solutions, and (iii) precipitation from iron(II) and iron(III) chloride solutions by hydrolysis of urea, method (i) produced the ‘best’ magnetite particles. Silica-magnetite composites were prepared using the ‘best’ magnetite, and, for comparison, two methods for depositing silica were used to coat the silica onto magnetite nanoparticles, from silicic acid at pH 10 and by acid hydrolysis of tetraethoxysilane (TEOS) at 90 1C. The best method for yielding silica-magnetite composites that worked well in DNA adsorption and elution proved to be that involving silicic acid and this material could be made in 20 g batch sizes. Silica-magnetite composites from the two methods proved to have distinct and different physical and chemical properties. All magnetite and silica-magnetite samples were fully characterised for their relative chemical composition using Fourier-transform infrared, XRF and thermo-gravimetric analysis. Their physical characteristics were determined using scanning electron microscopy and N2 adsorption and Mossbauer spectroscopy was used to confirm the identity of the iron oxides produced. Selected samples were comparatively tested for their ability to adsorb, and subsequently elute, 2-deoxyguanosine-5monophosphate (GMP) and its non-phosphorylated analogue 2-deoxyguanosine (G) and a range of sequence defined oligonucleotides (NAs) and sheared salmon sperm DNA. It was found that magnetite readily adsorbed GMP via the Corresponding author. Istituto di Scienze Chimiche, Piazza Rinascimento 6, Universita degli Studi di Urbino, 61029 Urbino, Italy.
Tel.: +39-0722-4329; fax: +39-0722-4122. E-mail address: [email protected] (I.J. Bruce). 0304-8853/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2004.06.032
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GMP phosphate anion in water, whereas silica did not, due to electrostatic repulsion between the negatively charged surface of silica and the GMP. Both magnetite and silica magnetite were further tested in adsorption studies of G and GMP in different chaotropic media, 4 M sodium chloride or 4 M ammonium sulphate. The high salt conditions aided binding of GMP silica magnetite but inhibited adsorption to magnetite presumably due to competition for binding sites on the magnetite’s surface by the chaotrope anions. Interestingly, the results from NAs binding studies indicated that sequence appeared to play an important role in adsorption of the different species to silica-magnetite composites. This may indicate a contribution by hydrophobic interactions to the binding mechanism. Multiple depositions of silica onto magnetite performed by deposition from silicic acid at pH 10 did not appear to greatly increase the composite percentage represented by silica whilst composite produced by the acid hydrolysis of TEOS at 90 1C did. However, it appeared that the silica deposited by the first method represented a complete coating of the magnetite core whilst the second method yielded a porous or incomplete coating. In comparison with commercially available silica-magnetite composite in DNA adsorption and elution, the material was observed to perform approximately 10% more efficiently. These findings indicate that it is possible to produce a consistent and cheap silica-magnetite nanoparticle on relatively large scale (greater than 20 g batch size) which is at least as good as, if not better than, a commercially available alternative. r 2004 Elsevier B.V. All rights reserved. PACS: 81.20; 61.46 Keywords: Nanoparticles; Silica; Magnetite
1. Introduction In 1973 Robinson et al. [1] used magnetic separation for the first time in a biotechnology context. In their work, silica-coated magnetic iron oxide and cellulose-coated magnetic iron oxide were used to immobilise two enzymes; a-chymotrypsin and b-galactosidase for applications in bioreactors. Since then, magnetic separation has become an increasingly popular tool for the separation of biological molecules and cells. Materials used this way can be defined as magnetisable particles (i.e. ones that, when under the influence of an external magnetic field, will themselves become magnetic) and can enable the isolation or extraction of a target molecule or substance. The non-magnetic target binds to the surface of the magnetisable solid-phase support (MSPS), either through a specific affinity interaction, or another mechanism, for example, ion exchange or hydrophobic interaction, so that it can then be isolated or extracted by application of an external magnetic field. This type of separation is based on chromatographic principles, except that the solid phase, instead of being packed inside
a column, exists as a suspension. MSPSs are far less susceptible to the negative effects of high viscosity, densities of particulates of the mixtures to be separated, and are more convenient for use in automated processes. Philipse et al. [2] carried out seminal work on the synthesis of silica-magnetite composites in 1994 and MSPSs are commonly between 0.05 and 10 mm in diameter (although MSPSs of larger diameters have been prepared [3]), and comprise of either one or two essential constituents. These can be arranged in three different ways: 1. A magnetisable component that forms the core of the particle that is then encapsulated by a matrix material. 2. A magnetisable component that is evenly dispersed throughout the bulk of a matrix material. 3. A single magnetisable component that also acts as a matrix material. Although many such supports are currently available in a commercial context (Table 1) the aim of this work was to construct silica-magnetite composites on large scale that would be relatively
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Table 1 Commercially available nano- and microparticles [4–8] Name and manufacturer
Diameter (mm)
Matrix material
Surface activation
Biomag (PerSeptive Biosystems, USA)
1
Silica
1
Charcoal
–COOH –NH2 None
Biosphere (Biosource International)
1
Not known
–NH2
Polystyrene
–OS(O2)R Tosyl-activated
Dynabeads M-280 M-450 M-500 (Dynal, Norway)
2.8 4.5 5
Estapor (Prolabo, France).
1
Polystyrene
–COOH –NH2
Iobeads (Immunotech, France)
1
Not known
Not known
M 100 M 104 M 108 (Scigen, UK)
1–10
Cellulose
–OH
MACS MicroBeads (Miltenyi Biotec, Germany)
0.05
Not known
Not known
MagaBeads MagaCell MagAcrolein MagaCharc (Cortex Biochem, USA)
3.2 3 3 3
Polystyrene Cellulose Acrolein Charcoal
–COOH –NH2 Epoxy
Magarose (Whatman International, UK)
20–150
Agarose
None
Magne-Sphere MagneSil (Promega, USA)
o1 5–8.5
None Silica
Not known None
Magnetic beads (ProZyme, USA)
0.8
Latex
Not known
Magnetic microparticles (Polysciences, USA)
1–2
Polystyrene
–COOH –NH2
Magnetic particles (Boehringer, Germany)
1
Polystyrene
Not known
MPG (CPG, USA)
5
Porous glass
None –NH2 Glyceryl Hydrazide
Sera-Mag (Seradyn, USA)
1
Polystyrene
–COOH
SPHERO magnetic particles (Spherotech, USA)
1–4.5
Polystyrene
None –COOH –NH2
XM200 microsphere (Advanced Biotechnologies, UK)
1 1–3.5
Silica Polystyrene
None –COOH
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cheap and yet still be effective in their desired application.
2. Materials 2.1. Reagents Sodium silicate (powder and aqueous solution) and HPLC solvents were purchased from Fisher (Leicestershire, UK). Amberlite ion exchange resin, nucleotides, and nucleosides were purchased from Sigma-Aldrich (Dorset, UK). All other reagents were purchased from Fluka (Dorset, UK). Unless stated otherwise, all water used as deionised using a Purite HP700 laboratory deioniser (Purite Ltd., UK). All samples were stored in polyethylene bottles. 2.2. Oxidative hydrolysis of iron sulphate in alkaline media [9] 2.2.1. Small scale (method 1Ma) Solutions of iron(II) sulphate heptahydrate (17.71 g, 0.06 mol) in 200 mL water, potassium nitrate (10.11 g, 0.1 mol) in 100 mL water, and potassium hydroxide (13.81 g, 0.25 mol) in 50 mL water were prepared. These were added to a 1 L round-bottomed flask in the order given above whilst stirring. The reaction mixture was heated to 90 1C, under nitrogen, and maintained at this temperature for a further 2 h. The nitrogen flow was then turned off, the reaction vessel removed from the heating mantle and the mixture allowed to cool at room temperature for 1 h. After cooling, the black precipitate was washed twice with water (2 L), then once with 1 M nitric acid (2 L), and twice again with water (2 L). The wash procedure was performed using magnetic sedimentation of the solid with the aid of a slab magnet. After removal of the supernatant by aspiration using a water pump, the precipitate was washed by vigorous re-suspension in water. Washing was repeated, if necessary, until a visually clear supernatant was obtained. The final volume was adjusted to 1 L with water. This method regularly yielded 10 g of product.
2.2.2. Large scale (method 1Mb) Iron(II) sulphate heptahydrate (354.2 g, 1.27 mol) was dissolved in 3 L of degassed water in a 5 L reaction vessel equipped with an overhead stirrer, nitrogen inlet and a thermometer. The solution was then heated to 90 1C whilst stirring under nitrogen. Meanwhile, potassium nitrate (80.9 g, 1.25 mol) was dissolved into 1 L of degassed water in a conical flask and potassium hydroxide (188.6 g, 3.36 mol) was added to this solution. The solution was stirred until the reagents had dissolved, then heated to 65 1C in a microwave oven and degassed for a further 5 min before adding to the iron sulphate solution in the 5 L vessel. The resulting mixture was heated, whilst stirring and purging with nitrogen, until a temperature of 92–93 1C had been reached. The stirring was continued for 1 h, keeping the temperature constant at 92–93 1C whilst purging with a slow stream of nitrogen. After 1 h, the mixture was allowed to cool to handling temperature and the precipitate, magnetite, washed with 5 L of water or until the pH of the wash supernatant was neutral. The suspension volume was adjusted to 4 L with water. This method regularly yielded 20 g of product.
2.3. Precipitation of magnetite by the alkaline hydrolysis of iron(II) and iron(III) chloride solutions [2] (method 2M) Solutions of 1 M iron(III) chloride hexahydrate (FeCl3 6H2O) and 2 M iron(II) chloride tetrahydrate (FeCl2 4H2O) were prepared by dissolving FeCl3 6H2O (10.8120 g, 0.04 mol) in 40 mL H2O and FeCl2 4H2O (3.9762 g, 0.02 mol) in 10 mL 2 M HCl, respectively. The two solutions were then mixed together prior to their addition to 500 mL of 0.7 M aqueous ammonia solution with continuous ‘mechanical’ stirring. After stirring for 30 min, the precipitate was washed twice with water (1 L), by magnetic sedimentation. In all, 60 mL of 1 M tetramethylammonium hydroxide (TMA), diluted from a 25% TMA stock (2.75 M), was added and the final volume made up to 320 mL with water.
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2.4. Precipitation of magnetite from iron(II) and iron(III) chloride solutions by hydrolysis of urea [10] (method 3M)
solution (pH 10, 2 L) and then with water (2 L) until the pH of the supernatant was neutral. The product was resuspended in 1 L of water.
Iron(III) chloride hexahydrate (13.51 g, 0.05 mol) and urea (180.18 g, 3 mol) were dissolved in water (200 mL) in a 1 L round-bottomed flask. The solution was degassed for 30 min with oxygen-free nitrogen. Iron(II) chloride tetrahydrate (4.07 g, 0.02 mol) was added and degassing continued for a further 15 min. The vessel was sealed to prevent atmospheric oxygen entering the solution, and the temperature raised to 98 1C. The mixture was then stirred for 90 min using an overhead stirrer. The resulting precipitate was washed with boiling water (6 200 mL), or until the pH of the wash supernatant was reduced to 7. The final total volume for the suspension was adjusted to 300 mL using water.
2.5.2. Large scale, with aqueous sodium silicate (waterglass method 2SM) In all, 83.0 g of aqueous sodium silicate (27% SiO2) was dissolved in water to a total volume of 2 L. A column containing 110 g of ‘‘Amberlite IR120’’ ion exchange resin was regenerated with 1 L each of the following: hot water (around 70 1C), 3 M HCl, and finally cold water. Sodium silicate solution was passed down the column, allowing the first 100 mL to pass uncollected. In all, 1800 mL of the eluate (now in the form of silicic acid) was taken and its pH immediately raised to 12 using aqueous TMA (25%) to prevent homogeneous silica nucleation. In all, 900 mL of magnetite suspension (approximately 23 g L1, method 1Ma or b) was mixed with 500 mL water and titrated to pH 12 with TMA whilst stirring. With continued stirring, the sodium silicate eluate (at pH 12) was added to the magnetite suspension. This mixture was then slowly titrated to pH 10.0 (from 12) using 0.5 M HCl over approximately 1 h. Titration to pH 10.7 was performed over the first 30 min, and then from pH 10 to 10.7 over the last 30 min. The mixture at pH 10.0 was stirred for a further 2 h before transferring to a 5 L flask. The silica-magnetite particles were then washed once with 2 L of TMA solution at pH 10.0, and then five times with 2 L of deionised H2O by magnetic sedimentation. The pH was then tested to ensure the supernatant was at neutral pH (below pH 8). After washing, the silica magnetite was resuspended in 1 L of water. This method regularly yielded 20 gm of product.
2.5. Deposition of silica from silicic acid solution at pH 10 [2,11] 2.5.1. Small scale, with sodium silicate powder (method 1SM) Sodium silicate powder (8.3 g, 67% SiO2) was dissolved in water (200 mL). This solution (170 mL) was passed through a column (44 mm diameter) containing 25 g of ‘‘Amberlite IR-120’’ ion exchange resin which had been previously regenerated with 200 mL of each of the following: hot water, 3 M HCl, and finally cold water. The first 20 mL was allowed to pass uncollected to flush the column. After collecting the remaining eluate (now in the form of silicic acid or ‘active’ silica), its pH was raised to 9.5 using some of the stock sodium silicate solution. The resulting silicic acid solution (133 mL) was added to a volume of stirred magnetite suspension containing 1.5 g of magnetite (from either method 1Ma or b) in a 1 L beaker [prior to addition of the silicic acid all magnetite samples other than that prepared by method 1.3 were mixed with 1 M TMA (19 mL)]. The pH of the resulting suspension was around 12.5. The pH was carefully lowered to 10.0 by titrating dropwise with 0.5 M HCl over a period of approximately 1 h. The suspension was then stirred for 2 h before washing with TMA
2.5.3. Consecutive depositions of silica using methods 1SM and 2SM Up to four consecutive depositions as described above were carried out. Between each deposition, the silica magnetite was washed with water until the wash supernatant was at neutral pH. Prior to each successive deposition the suspension volume was adjusted with water to that of the original magnetite suspension. An aliquot of suspension (50 mL) was retained for analysis following each deposition.
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2.6. Deposition of silica by acid hydrolysis of tetraethoxysilane at 90 1C in glycerol [11] (method 3SM) In all, 100 mL of a suspension of magnetite prepared by method 1Ma or b (containing 2.3 g magnetite) was placed into a 500 mL roundbottomed flask and the supernatant aspirated. A 10% solution of aqueous tetraethoxysilane (TEOS) (230 mL) was added to the flask. Glycerol (200 mL) was added, whilst stirring with an overhead stirrer, and the pH of the suspension lowered to pH 4.5 using glacial acetic acid. The mixture was then heated to 90–95 1C and stirred at this temperature for 2 h under nitrogen. After cooling to handling temperature, the suspension was washed once with water (500 mL), twice with methanol (500 mL), and finally five times with water (500 mL). The final suspension volume was adjusted to 230 mL with water. 2.6.1. Consecutive depositions of silica using method 3SM Up to five depositions of silica have been carried out. Previously coated silica magnetite from method 3SM was used as starting material in further coating by the reaction above. An aliquot of suspension (115 mL) was retained for analysis following each deposition. Reaction volumes were scaled down by the appropriate amounts in each successive deposition, as shown in Table 2. 2.7. Rate of dissolution of silica-magnetite composites in acid Silica is soluble at pHs above 10 whilst magnetite is soluble in acid. If silica magnetite is
placed in strong acid the rate of dissolution can be taken as an indirect measure of the degree to which the magnetite is coated with silica and can also provide an indication about the nature of the silica coating. This was measured in the following way: 0.5 g samples of magnetite or silica magnetite were suspended in 100 mL of 8.6 M HCl (3:1 concentrated HCl to water by volume) and the mixture stirred gently. After the desired time interval (0, 30 s, 1, 2, 4, and 8 min), 0.1 mL of supernatant was removed and added to a 10 mL volumetric flask containing 9 mL water. The flask was then made up to the mark with additional deionised water. To construct a standard curve for magnetite assay solutions were prepared as follows: 0.5 g magnetite was dissolved in 90 mL HCl (8 M) and made up to 100 mL with HCl (8 M). The standard solution (1 mL) was then removed and diluted with water to 100 mL in a volumetric flask to give a 50 g L1 standard. Subsequent dilutions were performed with 0.8, 0.6, 0.4, 0.2, and 0 mL of stock solution to give standards of 40, 30, 20, 10, and 0 g L1, respectively. The standards and samples were analysed for their absorbance using a UV spectrophotometer set at a wavelength of 333 nm. 2.8. G, GMP, and oligonucleotides (NA) adsorption and elution studies A solution of the NA at a concentration of 0.05 g L1 was prepared in the desired binding medium. Triplicate aliquots of nanoparticle suspension, containing the required equivalent ‘dry’ weight of support (determined by heating a known volume of homogeneous particle suspension to
Table 2 Volumes (in mL) of reagents and sample used to deposit 1–5 layers of silica Sample
Volume suspension used
Volume TEOS
Volume glycerol
Final volume
1 2 3 4 5
280 (magnetite) 805 (silica magnetite) 690 575 460
920 805 690 575 460
800 700 600 500 400
920 805 690 575 460
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100 1C for 2 days or until no further weight loss was observed), were pre-washed twice with binding medium and placed in three 0.5 mL microcentrifuge tubes and the supernatant removed after magnetic immobilisation of the support. NA solution (0.2 mL) was then added to the supports in binding medium and the suspension gently agitated to permit binding. The supernatant was subsequently removed and stored on ice in a separate 0.5 mL microcentrifuge tube ready for analysis by HPLC. The nanoparticles were washed in 70% v/v aqueous ethanol (which was retained for HPLC assay) and 0.2 mL of the appropriate eluant was added to the supports and the suspension agitated for 2 min to elute the adsorbed species. The supernatant (eluate) was then removed and kept on ice for analysis by HPLC. HPLC analyses of 2-deoxyguanosine (G) and 2deoxyguanosine-5-monophosphate (GMP) were carried out at 260 nm using a Kontron 325 system connected to a Kontron 332 detector and a Kontron integrator 3.90 (Kontron Instrments Spa, Milano, Italy). A Genesis C18 (reversed phase) HPLC column (150 mm 4.6 mm, 4 mm particle size) was used. All samples were loaded into a Rheodyne injection valve fitted with a 50 mL sample loop. All solvents (water, methanol, acetonitrile [AcN]) were HPLC grade, and any chemicals used in the mobile phase were also of HPLC grade quality. HPLC analysis of nucleosides and nucleotides involved the use of two mobile phases ((A) KH2PO4 (0.02 M) and (B) methanol [MeOH]) at a flow rate of 2.0 mL min1. Samples were injected into a mobile phase of 100% KH2PO4 (0.02 M) and eluted using a gradient consisting of between 0% and 60% MeOH (100–40% KH2PO4) over 3 min (gradient change of 20% MeOH min1). Prior to analysis of each sample, the column was rinsed with 100% solvent B for 5 min and then with 100% solvent A for 5 min, or until a steady stable baseline was observed. HPLC analysis of oligonucleotides was at the same wavelength as above and involved the use of two mobile phases ((A) ammonium acetate buffer (0.1 M) and (B) AcN) at a flow rate of 2.0 mL min1. Samples were injected in a mobile
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phase of 3% AcN. NAs were then eluted isocratically with a mobile phase of 25% AcN: 75% ammonium acetate (0.1 M) for 2.5 min. Prior to analysis of each sample, and between samples, the column was washed with 100% solvent B for 5 min and then with 97% solvent A (3% solvent B) for 5 min, or until a steady stable baseline was observed. 2.9. Salmon sperm DNA binding assays In all, 50 mg of sheared, salmon sperm DNA (produced by repeated passage through a hypodermic syringe needle) was resuspended in 400 ml of TEN buffer (100 mM Tris–HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl) and 400 ml of polyethylene glycol (PEG, Mr 8000) in 4 M NaCl and added to 2 mg of silica magnetite (previously washed in sterile water) in a sterile Eppendorf tube. The mixture was incubated with gentle agitation for 5 min at 25 1C after which the support material was immobilised using a Promega magnetic stand (Promega Corp., Madison, USA). The supernatant was retained for assay at OD260 nm. The support material was then washed in 1 mL of ice cold 70% v/v aqueous ethanol by gentle agitation for 5 min at room temperature. The support was immobilised magnetically and the wash solution removed and retained for UV assay. DNA was eluted by washing the support sequentially, twice, in sterile deionised water (200 ml each) for 5 min each at room temperature. Each 200 mL was assayed independently for its OD at 260 nm to estimate its DNA content. A standard curve was constructed for comparison purposes using a series of salmon sperm DNA solutions of known concentration. 2.10. Fourier-transform infrared All samples were characterised by Fouriertransform infrared (FT-IR) spectroscopy (SHIMADZU 8300, Shimadzu Corp., Japan) and prior to compacting as pellets all samples and KBr were dried at 95 1C for 48 h. Each sample (5 mg) was thoroughly mixed and crushed with 500 mg of KBr using a mortar and pestle. The mixture (80 mg) was placed in a pellet former and 10 tons of
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pressure was applied for 2 min to form the KBr pellet. The FT-IR spectrum was collected after 50 scans of the region between 400 and 4000 cm1.
placed in the TGA furnace and heated at a rate of 10 1C min1 to 1000 1C in static air. 2.15. Magnetic susceptibility
2.11. Scanning electron microscopy Nanoparticle size and morphology were characterised by scanning electron microscopy (SEM). The scanning electron micrographs were recorded on a Cambridge Instruments Stereoscan 90 (Cambridge Instruments Ltd., Cambridge, UK). The samples were prepared by sprinkling the powder materials onto double-sided sticky tape which was mounted on a microscope stub. All samples were coated with a thin gold film under vacuum prior to microscopy. 2.12. XRF The silica concentration in various silica-coated magnetite samples were determined using an Oxford/Link XRD200 (Oxford Instruments plc, Oxon, UK). A standard curve was constructed for comparative purposes with sample measurements using quantities of silica gel with known silica concentration. 2.13. Surface area measurement The surface area of all samples (dried at 100 1C overnight) was determined by the BET method (nitrogen gas as an adsorbent) using a Micrometrics Instrument Corporation Gemini 2375 V4.01 (Norcross, USA). Each dried sample (0.1–0.2 g) was weighed accurately up to four decimal places and placed in a sample tube (reference an empty tube). Analysis was performed using an automatic five-point adsorption programme, measuring the volume of nitrogen adsorbed by the sample at the following pressures: 76, 114, 152, 190, and 228 mmHg. 2.14. Thermo-gravimetric analysis Thermo-gravimetric analysis (TGA) of samples was performed using a Dupont 2000 instrument (Wilmington, USA). Dried sample (10–20 mg) was
Magnetite susceptibility measurements were carried out using a superconducting quantum interference device (SQUID) magnetometer.
3. Results (magnetite) 3.1. Visual characterisation Iron oxides can vary quite significantly in colour and this can be used as an aid in their identification. It may also be used as a rule of thumb guide to purity in some cases. Table 3 indicates the different colours for each known iron oxide [12,13] and the oxides produced in this project. Magnetite produced by oxidative hydrolysis of iron(II) sulphate in alkaline media was jet black and therefore expected to be pure magnetite. The other methods generated products of various colours, from medium brown to very dark brown indicating a mixture of iron oxides. FT-IR and Mossbauer spectroscopy, plus TGA were used to analyse these products. All of the methods used for producing magnetite yielded products that reacted to a magnet to a certain degree, but methods 1Ma Table 3 Colour characterisation of different iron oxides Iron oxide
Colour
Magnetite Maghemite Hematite Feroxyhyte Ferrihydrite Lepidocrocite Goethite Akaganeite
Jet black Brownish-red Red Reddish-brown Reddish-brown Orange Yellowish-brown Yellowish-brown
Product
Colour
Method Method Method Method
1Ma 1Mb 2M 3M
Jet black Jet black Brown to very dark brown Brown
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and b produced material which reacted the quickest (results not shown). Method 3M produced what appeared to be a brown coloured solid that left sticky brown residues when contacted with glass or plastic ware surfaces which could not be removed, even after extensive washing. Methods 2M and 3M were not subsequently used for the production of magnetite for use in silica-magnetite composite manufacture. 3.2. Fourier-transform infrared FT-IR spectroscopy was used for analysis of products from the three methods. Table 4 shows the reported values for absorbance of various iron oxides [12,13] and those obtained for products of the various methods. Methods 1Ma and b were observed to produce very pure magnetite with little or no other iron oxide impurities. Magnetite prepared by method 2M appeared to be a mixture of magnetite and maghemite. The FT-IR spectrum for magnetite produced by method 3M was too complex in the magnetite, maghemite, and hematite absorption region (500–700 cm1) to allow definition of the product.
Fig. 1. SEM micrographs of magnetite synthesised by method IMb.
Confirmation was obtained by Mossbauer spectroscopy of the products of methods 1Ma and b which indicated that they possessed a hyper magnetic field of 518 kG and were therefore magnetite. An SEM image of the magnetite is provided in Fig. 1. 3.3. Thermo-gravimetric analysis
Table 4 IR bands of various iron oxides Iron oxide
IR bands (cm1)
Magnetite Maghemite Hematite Feroxyhyte Lepidocrocite Goethite Akaganeite
590 (400) 630, 590, 570, 450 (400) 540, 470 (345) 1110, 920, 790, 670 1026, 1161, 753 890, 797 840, 640
Product
Identity and bands observed
Method 1Ma Method 1Mb Method 2M
Magnetite (583) Magnetite (583) Magnetite (580)+maghemite (631, 570, 453) Magnetite, hematite, and maghemite (570, 476)
Method 3M
Note: Values in brackets indicate IR absorptions which could not be measured accurately.
TGA analysis of magnetite produced by methods 1Ma and b indicated decreases in weight of the sample as the temperature rose to 120 1C, and from 257 to 321 1C. These are likely to be due to loss of adsorbed water and dehydration of internal OH groups, respectively. The increase in weight observed from 120 to 257 1C is most likely due to the oxidation of magnetite to maghemite. These observations support the FT-IR and Mossbauer data that methods 1Ma and b produced effectively pure magnetite. 3.4. Particle size analysis Table 5 indicates the results from SEM size analysis of magnetite/iron oxide produced by the various methods employed. The only difference in magnetite produced from methods 1Ma and b is the particle size distribution and shape. SEM
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Table 5 Particle sizes of magnetite from SEM Method and particle size distribution
Particle size (nm)
Method 1Mb Minimum Maximum Mean
140 170 150
Method 2M Minimum Maximum Mean
100 200 140
Method 3M Minimum Maximum Mean
80 120 100
analysis has indicated that method 1Ma produced magnetite particles with a narrower apparent size range distribution than method 1Mb but with a larger mean particle size.
of 5.18 g cm3. Whereas materials produced by methods 2M and 3M contain a mixture of what is principally magnetite and maghemite (density 4.87 g cm3), so would possess a density somewhere between the values for magnetite and maghemite. For the sake of simplicity, it was assumed that methods 2M and 3M were a 50:50 mixture of magnetite and maghemite and therefore possessed a density of 5.03 g cm3. The results for the surface area and estimated particle diameter with comparison to the literature values are summarised in Table 6. Using this approach it appears that magnetite prepared by method 1Mb possessed the largest mean particle diameter at 79.3 nm whilst magnetite prepared by method 1Ma is slightly smaller at 54.4 nm. This is approximately twice the value obtained by SEM.
4. Results (silica-magnetite composites)
3.5. Surface area analysis and correlation with particle size
4.1. FT-IR and XRF analyses of silicic acid and TEOS deposed composites
For a non-porous spherical particle, the relative surface area (as defined by surface area to mass ratio) is inversely proportional to its diameter. By knowing the density of the material of which the particles are composed, their sizes can be calculated, through the manipulation of the following formulae:
The presence of silica in all silica-magnetite composites was confirmed by FT-IR and XRF and results are shown in Tables 7 and 8 and Figs. 2 and 3. For FT-IR, the quantitative results describe the relative peak heights at the indicative spectral points for magnetite and silica. It was observed that silica:magnetite molar ratio increases with successive rounds of silica deposition. In fact, the relative amount of silica deposited using the TEOS method is greater per deposition than that achieved using the silicic acid approach. One reason for the slow rate of accumulation of silica on subsequent rounds of deposition to the surface of the silica-magnetite composite produced via acid titration of silicic acid is likely to be due to the fact that for each successive deposition the material’s pH is raised to 14 prior to titration with HCl. This could mean that at least a part of the existing surface silica would be resolubilised and lost. This would effectively mean that on each deposition very little (as observed) extra silica might be deposited.
density ðsÞ ¼
mass ðmÞ ; volume ðvÞ
relative surface area ðaÞ ¼
area ðAÞ ; mass
area of sphere ¼ 4pr2 ; volume of a sphere ¼ 43 pr3 : These equations can be arranged to give sa1 r¼ : 3 Magnetites produced by methods 1Ma and 1b are effectively pure and so should have a density
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Table 6 Surface areas of iron oxide products and calculate particle diameters Sample
Method Method Method Method
1Ma 1Mb 2M 3M
Relative surface area (m2 g1)
Estimated particle size (nm)
Observed
Reported value
Calculated
Reported value
21.30 14.60 108.81 25.46
— o4 [13] — —
54.4 79.3 11.0 46.9
100 [9] 50–200 10 [2] 800 [10]
Table 7 FT-IR absorbance ratios of silica/magnetite for silicic acid and TEOS deposition methods (with principal measurement points) Sample
Principal adsorption bands (cm1)
% relative response
Silicic acid (2SM) 1 layer –2 layers –3 layers –4 layers
Magnet. Magnet. Magnet. Magnet.
(584)+silica (579)+silica (586)+silica (583)+silica
(895, (861, (884, (872,
1023) 988) 1035) 1023)
15.61 17.36 20.31 27.76
TEOS (3SM) 1 layer –2 layers –3 layers –4 layers –5 layers
Magnet. Magnet. Magnet. Magnet. Magnet.
(578)+silica (582)+silica (582)+silica (582)+silica (582)+silica
(798, (798, (795, (795, (798,
900, 900, 897, 897, 900,
23.34 26.00 35.36 49.23 64.70
1050) 1088) 1113) 1103) 1100)
Table 8 XRF results for silica-magnetite composites Sample
No. of counts
% element
At%
Silicic acid (2SM) 1 layer 29 2 layer 35 3 layers 43 4 layers 47
0.027 0.029 0.030 0.033
0.053 0.058 0.060 0.066
TEOS (3SM) 1 layer 45 2 layers 48 3 layers 69 4 layers 118 5 layers 139
0.027 0.034 0.043 0.077 0.094
0.053 0.067 0.085 0.154 0.187 Fig. 2. FT-IR of relative silica composition of silica-magnetite composites.
4.2. Rate of dissolution of silica-magnetite composites in acid The resistance to acid dissolution of the two silica magnetites is different (Fig. 4).
For the silicic acid deposed material it is clear that there is no real difference in acid resistance between 1 and 4 coatings of silica. All the silicic
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Fig. 3. XRF estimation of silica-magnetite composites silica content. Fig. 4. Acid dissolution characteristics for magnetite and silicamagnetite composites.
acid-coated forms were more or less equally resistant to dissolution. The likely conclusion is that even the first round of deposition must give good silica coverage, which is presumably less porous or more complete than that generated using TEOS. In the case of TEOS-coated magnetite the first silica layer appears to afford no protection to the magnetite from dissolution by acid, as the dissolution curves for both magnetite and this material are virtually identical. The second coating offers some resistance but it is only after the third and fourth coatings that similar levels of resistance to those observed with silicic acid deposed magnetite are reached. The implication of these results is that even though the TEOS method is efficient in depositing more silica per round of coating than the silicic acid method, the nature of the silica deposited or the type of coverage of the iron oxide core must be different. It could be that the TEOS silica coating is porous or incomplete. In either case, the differences between the two composites are likely to have implications on their abilities to be used in bioseparative processes because their surfaces will represent different chemistries. The former hypothesis is largely supported by data from surface area analysis of the materials using N2 (data not shown) where the TEOS deposed material has a significantly larger comparative surface area to that of the silicic acid deposed material.
4.3. Adsorption and elution of G, GMP, and oligonucleotides to magnetite and silica-magnetite composites When the ability to adsorb and elute G and GMP to and from the surface of magnetite and silicic acid and TEOS magnetite composites is compared these differences are highlighted (see Fig. 5). 4.3.1. Magnetite In water magnetite, in contrast to silica, adsorbs most GMP, whilst little or no G is adsorbed (data not shown). This strongly suggests an electrostatic mechanism mediated by the phosphate group as the attractive force. From the results indicated in Fig. 6 (effect of pH on adsorption) the adsorption of GMP to magnetite can readily be explained on the basis of the degree of protonation of surface Fe–OH groups and phosphate group possessed by the GMP. Effectively two competing effects occur with respect to these groups and pH. As the pH is lowered so the phosphate group of GMP is more likely to be protonated leading to a diminished negativity, whilst at the same time the surface Fe–O groups also become increasingly protonated. The reverse is true with increasing pH. Adsorption of GMP by magnetite from salt solutions results in a reduction of the amount of
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These results are summarised in the diagram in Fig. 5 for GMP and were obtained using the protocols described in Section 2. Silica used in the comparison was produced by the same method as for silicic acid deposition of silica to magnetite with the difference that silica gel was used as the core material.
Fig. 5. GMP adsorption characteristics for magnetite, silica, and silica-magnetite composites in various media.
Fig. 6. Adsorption of GMP by magnetite at various pHs.
GMP bound which is presumably due to competition between the salt negative ion species and the charged phosphate group present in GMP. (The corresponding nucleoside shows little binding to magnetite under any condition.) In contrast, silica shows greatest binding of GMP under chaotropic conditions. The silicic acid-deposited magnetite mimics silica in its GMP adsorption characteristics whilst magnetite deposited with silica using the TEOS method mimics magnetite with respect to its GMP binding when coated with a single layer. Interestingly as more silica is added to the magnetite using the TEOS method (as consecutive rounds of deposition are performed) its characteristics were observed to become more like that of silica and the silicic acid-deposited material.
4.3.2. Silica-magnetite (method 2SM) adsorption characteristics for oligonucleotides Table 9 illustrates the results of adsorption studies involving NAs and silica-magnetite composite. Mono-base characterised NAs were observed to bind to silica magnetite in water in a way which appeared to be dependent on the base type, with purine base constituted NAs, (dA)10 and (dG)10, binding more than the corresponding pyrimidine base constituted NAs, (dC)10 and (dT)10. This may be a reflection of the fact that purine bases tend to be more prone to hydrophobic interactions than pyrimidine bases, which may indicate that, even in water, hydrophobic interactions play an important role in binding to silica. Alternatively, this increased degree of binding could have been due to the higher corresponding molecular weight of the purine base NAs, which would have resulted in a lower molarity for the solution of these NAs. However, this is contradicted by the results from the di-base constituted NAs, where (dTA)5 bound almost as much as (dA)10, and more than (dT)10. This also suggests that the purine bases may make a greater Table 9 Percentage adsorption of GMP and oligonucleotides to silica magnetite Oligonucleotide
(dG)10 (dC)10 (dA)10 (dT)10 (dTA)5 (dGC)5 GMP
% binding H2O
4 M sodium chloride
4M ammonium sulphate
17.1 11.1 19.2 13.2 17.8 6.4 7.5
80.4 92.2 49.2 21.5 30.3 32.9 21.6
75.0 80.3 90.4 66.2 92.3 50.0 12.9
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contribution to binding than pyrimidine bases. (dGC)5 was observed to bind much less than all others. Both these NAs have similar molecular weights. NA binding to silica magnetite was also performed in 4 M sodium chloride and 4 M ammonium sulphate and in both cases binding was significantly better than in water and appeared to demonstrate a chaotrope and sequence dependent effect. NAs bind better to silica under chaotropic conditions as a consequence of electrostatic shielding of the negatively charged groups of the silica surface and phosphate groups and by promotion of hydrophobic interactions [14]. Binding is greater in 4 M ammonium sulphate, for (dA)10, (dT)10, (dTA)5, and (dGC)5, possibly as a consequence of it being a stronger chaotrope. It is unclear as to why there is such a large difference between the effect of sodium chloride and ammonium sulphate on the relative adsorption of adenine and thymine containing NAs to those containing guanine and cytosine. Perhaps heterocyclic base polarity (hydrophobicity) affects NA adsorption in different chaotropic salts. G and C are more polar than A and T. 4.3.3. Silica-magnetite (method 2SM) elution characteristics for oligonucleotides Table 10 indicates the recovery efficiencies for NAs absorbed to silicic acid-deposited magnetite in water under various salt conditions.
All NAs could be desorbed in water after adsorption under chaotropic conditions with high efficiency. Once again chaotrope and sequence differences were observed. 4.4. Adsorption and elution characteristics for salmon sperm DNA using method 2SM silica-magnetite composites Fig. 7 illustrates the results from experiments involving the use of sheared, denatured salmon sperm DNA binding and elution from silica magnetite (silicic acid deposition method) and a comparison with a commercially available equivalent material. See Section 2 for a full description of the protocol. Experiments were conducted in triplicate in two different batches of silica magnetite and an average value calculated. These data indicate that the performance of the material produced by us does not improve radically with each successive deposition of silica. It also indicates that the material is very efficient at allowing the recovery of all, or almost all, of the DNA adsorbed and that the material performed better in the assay than the commercial equivalent (approximately 10% better). Although the quantity of DNA adsorbed and eluted from the composite does not significantly increase with increasing silica coating it was observed that the quality of the eluted DNA does. In restriction digestions of eluted DNA, it has
Table 10 Percentage recovery of GMP and oligonucleotides bound to four silica magnetite Oligonucleotide
(dG)10 (dC)10 (dA)10 (dT)10 (dTA)5 (dGC)5 GMP
% recovery after binding in salt and elution in water In 4 M sodium chloride
In 4 M ammonium sulphate
35.8 55.6 50.2 64.9 64.5 63.6 39.3
33.5 17.5 69.1 67.3 77.3 85.9 58.5
Fig. 7. Sheared salmon sperm adsorption and elution characteristics of magnetite and its silica-coated forms (1–4 coated) and a commercial equivalent (Merck GmbH).
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M(emu/g)
80
T=2.5K
0
0 5 4 3 2 1
-80
-5
0
5
H(Tesla)
Fig. 8. Magnetic hysteresis of magnetite and 1–5 coated silicamagnetite nanocomposites.
been found that DNA from the four coated material performs better (data not shown). The reason for this is unknown. 4.5. Magnetic measurement The temperature dependence of the static magnetic susceptibility of pure magnetite and silicamagnetite nanocomposites (synthesised by the silicic acid method) was measured and the hysteresis loop (Fig. 8) was collected at 2.5 K. Coercivities and reduced remanences (MH=0/Msat) were also measured and the saturation magnetisation (Ms) was centred at 88 emu g1 at 2.5 K and around 82 emu g1 at 300 K. These values are close to those of bulk magnetite (90 emu g1 at 300 K and 85 emu g1 at 4.2 K) confirming the presence of large size magnetic particles with respect to the single domain limit. Neither material reach the saturation of magnetisation at the largest measuring field at 6.5 T while the values of M r =M s ratio decreases from 0.4 at 2.5 K to 0.3 at 300 K. Such behaviour is characteristic of nanometre size particles. These results suggest that silica coating did not change the magnetic property of the particles.
5. Conclusion From the present study, we have obtained a good understanding of the structure–function–
159
chemistry involved with silica-magnetite composites and the best method for producing such a material with utility in nucleic acid adsorption and elution. If a silica-magnetite composite with solely silica surface characteristics with respect to nucleic acid binding and elution is required then the silicic acid deposition method (2SM) is the best route to achieve it, but the TEOS (3SM) actually deposits more silica—and more rapidly. It may be that further scale-up, and speed, with which the composite material can be produced will involve the manufacture of a hybrid material possessing an initial silicic acid-deposited layer followed by a layer mediated by TEOS. This is currently being investigated. However, the composite described here represents a cheap and efficient alternative to existing materials for the bioseparation and purification of nucleic acids with what appears to be better performance than at least one potential competitor material.
Acknowledgements The authors would like to thank the European Union and specifically the 5th Framework Programme for funding this work (CHEMAG contract no. G5RD-CT-2001-00534) and Dr. George Fern for performing the Mossbauer analyses. References [1] P.J. Robinson, P. Dunnill, M.D. Lilly, Biotechnol. Bioeng. 15 (1973) 603. [2] A.P. Philipse, M.P.B. Vanbruggen, C. Pathmamanoharan, Langmuir 10 (1994) 92. [3] J. Ugelstad, P. Stenstad, L. Kilaas, W.S. Prestvik, R. Herje, A. Berge, E. Hornes, Blood Purification 11 (1993) 349. [4] I. Safarik, M. Safarikova, J. Chromatogr. B 722 (1999) 33. [5] B. Sinclair, Scientist 12 (1998) 16 et seq. [6] P.R. Levison, S.E. Badger, J. Dennis, P. Hathi, M.J. Davies, I.J. Bruce, D. Schimkat, J. Chromatogr. A 816 (1998) 107. [7] D. White, B. Butler, D. Creswell, C. Smith, Promega Notes (1999) 12–14. [8] R.A. Whitehead, S. Chagnon, E.V. Groman, L. Josephson, Magnetic particles for use in separations, United
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States Patent 4554088, Advanced Magnetics Inc., US, 1985. [9] T. Sugimoto, E. Matijevic, J. Colloid Interface Sci. 74 (1979) 227. [10] K. Matsuda, M. Sumida, K. Fujita, S. Mitsuzawa, Bull. Chem. Soc. Jpn. 60 (1987) 4441. [11] M.D. Butterworth, S.A. Bell, S.P. Armes, A.W. Simpson, J. Colloid Interface Sci. 183 (1996) 91.
[12] R.M. Cornell, The Iron Oxides: Structure, Properties, Reactions, Occurrences and Uses, Wiley, New York, USA, ISBN 3527302743, 2003. [13] U. Schwertzman, R.M. Cornell, Iron Oxides in the Laboratory—Preparation and Characterisation, VCH Verlagsgesellschaft, Weiden, Germany, 1991. [14] K.A. Melzak, C.S. Sherwood, R.F.B. Turner, C.A. Haynes, J. Colloid Interface Sci. 181 (1996) 635.
Journal of Magnetism and Magnetic Materials 284 (2004) 145–160 www.elsevier.com/locate/jmmm
Synthesis, characterisation and application of silica-magnetite nanocomposites Ian J. Brucea,b,, James Taylorb, Michael Toddb, Martin J. Daviesb, Enrico Borionib, Claudio Sangregorioa, Tapas Senb a
Istituto di Scienze Chimiche, Piazza Rinascimento 6, Universita degli Studi di Urbino, 61029 Urbino, Italy b School of Science, University of Greenwich, Chatham Maritime, Chatham ME6 4BT, Kent, UK Received 19 March 2004; received in revised form 17 May 2004 Available online 2 August 2004
Abstract Silica-magnetite composites were prepared for eventual applications in biomolecular separations (nucleic acids). Their production on large scale has been optimised and they have been extensively characterised in a physical and chemical context. They perform at least as well, if not better than a commercially available equivalent at adsorbing and eluting DNA. Several methods for the preparation of magnetite were compared in order to select one, which produced particles, possessing high magnetic susceptibility, low rate of sedimentation and good chemical stability. Of the main methods studied: (i) oxidative hydrolysis of iron(II) sulphate in alkaline media, (ii) alkaline hydrolysis of iron(II) and iron(III) chloride solutions, and (iii) precipitation from iron(II) and iron(III) chloride solutions by hydrolysis of urea, method (i) produced the ‘best’ magnetite particles. Silica-magnetite composites were prepared using the ‘best’ magnetite, and, for comparison, two methods for depositing silica were used to coat the silica onto magnetite nanoparticles, from silicic acid at pH 10 and by acid hydrolysis of tetraethoxysilane (TEOS) at 90 1C. The best method for yielding silica-magnetite composites that worked well in DNA adsorption and elution proved to be that involving silicic acid and this material could be made in 20 g batch sizes. Silica-magnetite composites from the two methods proved to have distinct and different physical and chemical properties. All magnetite and silica-magnetite samples were fully characterised for their relative chemical composition using Fourier-transform infrared, XRF and thermo-gravimetric analysis. Their physical characteristics were determined using scanning electron microscopy and N2 adsorption and Mossbauer spectroscopy was used to confirm the identity of the iron oxides produced. Selected samples were comparatively tested for their ability to adsorb, and subsequently elute, 2-deoxyguanosine-5monophosphate (GMP) and its non-phosphorylated analogue 2-deoxyguanosine (G) and a range of sequence defined oligonucleotides (NAs) and sheared salmon sperm DNA. It was found that magnetite readily adsorbed GMP via the Corresponding author. Istituto di Scienze Chimiche, Piazza Rinascimento 6, Universita degli Studi di Urbino, 61029 Urbino, Italy.
Tel.: +39-0722-4329; fax: +39-0722-4122. E-mail address: [email protected] (I.J. Bruce). 0304-8853/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2004.06.032
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GMP phosphate anion in water, whereas silica did not, due to electrostatic repulsion between the negatively charged surface of silica and the GMP. Both magnetite and silica magnetite were further tested in adsorption studies of G and GMP in different chaotropic media, 4 M sodium chloride or 4 M ammonium sulphate. The high salt conditions aided binding of GMP silica magnetite but inhibited adsorption to magnetite presumably due to competition for binding sites on the magnetite’s surface by the chaotrope anions. Interestingly, the results from NAs binding studies indicated that sequence appeared to play an important role in adsorption of the different species to silica-magnetite composites. This may indicate a contribution by hydrophobic interactions to the binding mechanism. Multiple depositions of silica onto magnetite performed by deposition from silicic acid at pH 10 did not appear to greatly increase the composite percentage represented by silica whilst composite produced by the acid hydrolysis of TEOS at 90 1C did. However, it appeared that the silica deposited by the first method represented a complete coating of the magnetite core whilst the second method yielded a porous or incomplete coating. In comparison with commercially available silica-magnetite composite in DNA adsorption and elution, the material was observed to perform approximately 10% more efficiently. These findings indicate that it is possible to produce a consistent and cheap silica-magnetite nanoparticle on relatively large scale (greater than 20 g batch size) which is at least as good as, if not better than, a commercially available alternative. r 2004 Elsevier B.V. All rights reserved. PACS: 81.20; 61.46 Keywords: Nanoparticles; Silica; Magnetite
1. Introduction In 1973 Robinson et al. [1] used magnetic separation for the first time in a biotechnology context. In their work, silica-coated magnetic iron oxide and cellulose-coated magnetic iron oxide were used to immobilise two enzymes; a-chymotrypsin and b-galactosidase for applications in bioreactors. Since then, magnetic separation has become an increasingly popular tool for the separation of biological molecules and cells. Materials used this way can be defined as magnetisable particles (i.e. ones that, when under the influence of an external magnetic field, will themselves become magnetic) and can enable the isolation or extraction of a target molecule or substance. The non-magnetic target binds to the surface of the magnetisable solid-phase support (MSPS), either through a specific affinity interaction, or another mechanism, for example, ion exchange or hydrophobic interaction, so that it can then be isolated or extracted by application of an external magnetic field. This type of separation is based on chromatographic principles, except that the solid phase, instead of being packed inside
a column, exists as a suspension. MSPSs are far less susceptible to the negative effects of high viscosity, densities of particulates of the mixtures to be separated, and are more convenient for use in automated processes. Philipse et al. [2] carried out seminal work on the synthesis of silica-magnetite composites in 1994 and MSPSs are commonly between 0.05 and 10 mm in diameter (although MSPSs of larger diameters have been prepared [3]), and comprise of either one or two essential constituents. These can be arranged in three different ways: 1. A magnetisable component that forms the core of the particle that is then encapsulated by a matrix material. 2. A magnetisable component that is evenly dispersed throughout the bulk of a matrix material. 3. A single magnetisable component that also acts as a matrix material. Although many such supports are currently available in a commercial context (Table 1) the aim of this work was to construct silica-magnetite composites on large scale that would be relatively
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Table 1 Commercially available nano- and microparticles [4–8] Name and manufacturer
Diameter (mm)
Matrix material
Surface activation
Biomag (PerSeptive Biosystems, USA)
1
Silica
1
Charcoal
–COOH –NH2 None
Biosphere (Biosource International)
1
Not known
–NH2
Polystyrene
–OS(O2)R Tosyl-activated
Dynabeads M-280 M-450 M-500 (Dynal, Norway)
2.8 4.5 5
Estapor (Prolabo, France).
1
Polystyrene
–COOH –NH2
Iobeads (Immunotech, France)
1
Not known
Not known
M 100 M 104 M 108 (Scigen, UK)
1–10
Cellulose
–OH
MACS MicroBeads (Miltenyi Biotec, Germany)
0.05
Not known
Not known
MagaBeads MagaCell MagAcrolein MagaCharc (Cortex Biochem, USA)
3.2 3 3 3
Polystyrene Cellulose Acrolein Charcoal
–COOH –NH2 Epoxy
Magarose (Whatman International, UK)
20–150
Agarose
None
Magne-Sphere MagneSil (Promega, USA)
o1 5–8.5
None Silica
Not known None
Magnetic beads (ProZyme, USA)
0.8
Latex
Not known
Magnetic microparticles (Polysciences, USA)
1–2
Polystyrene
–COOH –NH2
Magnetic particles (Boehringer, Germany)
1
Polystyrene
Not known
MPG (CPG, USA)
5
Porous glass
None –NH2 Glyceryl Hydrazide
Sera-Mag (Seradyn, USA)
1
Polystyrene
–COOH
SPHERO magnetic particles (Spherotech, USA)
1–4.5
Polystyrene
None –COOH –NH2
XM200 microsphere (Advanced Biotechnologies, UK)
1 1–3.5
Silica Polystyrene
None –COOH
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cheap and yet still be effective in their desired application.
2. Materials 2.1. Reagents Sodium silicate (powder and aqueous solution) and HPLC solvents were purchased from Fisher (Leicestershire, UK). Amberlite ion exchange resin, nucleotides, and nucleosides were purchased from Sigma-Aldrich (Dorset, UK). All other reagents were purchased from Fluka (Dorset, UK). Unless stated otherwise, all water used as deionised using a Purite HP700 laboratory deioniser (Purite Ltd., UK). All samples were stored in polyethylene bottles. 2.2. Oxidative hydrolysis of iron sulphate in alkaline media [9] 2.2.1. Small scale (method 1Ma) Solutions of iron(II) sulphate heptahydrate (17.71 g, 0.06 mol) in 200 mL water, potassium nitrate (10.11 g, 0.1 mol) in 100 mL water, and potassium hydroxide (13.81 g, 0.25 mol) in 50 mL water were prepared. These were added to a 1 L round-bottomed flask in the order given above whilst stirring. The reaction mixture was heated to 90 1C, under nitrogen, and maintained at this temperature for a further 2 h. The nitrogen flow was then turned off, the reaction vessel removed from the heating mantle and the mixture allowed to cool at room temperature for 1 h. After cooling, the black precipitate was washed twice with water (2 L), then once with 1 M nitric acid (2 L), and twice again with water (2 L). The wash procedure was performed using magnetic sedimentation of the solid with the aid of a slab magnet. After removal of the supernatant by aspiration using a water pump, the precipitate was washed by vigorous re-suspension in water. Washing was repeated, if necessary, until a visually clear supernatant was obtained. The final volume was adjusted to 1 L with water. This method regularly yielded 10 g of product.
2.2.2. Large scale (method 1Mb) Iron(II) sulphate heptahydrate (354.2 g, 1.27 mol) was dissolved in 3 L of degassed water in a 5 L reaction vessel equipped with an overhead stirrer, nitrogen inlet and a thermometer. The solution was then heated to 90 1C whilst stirring under nitrogen. Meanwhile, potassium nitrate (80.9 g, 1.25 mol) was dissolved into 1 L of degassed water in a conical flask and potassium hydroxide (188.6 g, 3.36 mol) was added to this solution. The solution was stirred until the reagents had dissolved, then heated to 65 1C in a microwave oven and degassed for a further 5 min before adding to the iron sulphate solution in the 5 L vessel. The resulting mixture was heated, whilst stirring and purging with nitrogen, until a temperature of 92–93 1C had been reached. The stirring was continued for 1 h, keeping the temperature constant at 92–93 1C whilst purging with a slow stream of nitrogen. After 1 h, the mixture was allowed to cool to handling temperature and the precipitate, magnetite, washed with 5 L of water or until the pH of the wash supernatant was neutral. The suspension volume was adjusted to 4 L with water. This method regularly yielded 20 g of product.
2.3. Precipitation of magnetite by the alkaline hydrolysis of iron(II) and iron(III) chloride solutions [2] (method 2M) Solutions of 1 M iron(III) chloride hexahydrate (FeCl3 6H2O) and 2 M iron(II) chloride tetrahydrate (FeCl2 4H2O) were prepared by dissolving FeCl3 6H2O (10.8120 g, 0.04 mol) in 40 mL H2O and FeCl2 4H2O (3.9762 g, 0.02 mol) in 10 mL 2 M HCl, respectively. The two solutions were then mixed together prior to their addition to 500 mL of 0.7 M aqueous ammonia solution with continuous ‘mechanical’ stirring. After stirring for 30 min, the precipitate was washed twice with water (1 L), by magnetic sedimentation. In all, 60 mL of 1 M tetramethylammonium hydroxide (TMA), diluted from a 25% TMA stock (2.75 M), was added and the final volume made up to 320 mL with water.
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2.4. Precipitation of magnetite from iron(II) and iron(III) chloride solutions by hydrolysis of urea [10] (method 3M)
solution (pH 10, 2 L) and then with water (2 L) until the pH of the supernatant was neutral. The product was resuspended in 1 L of water.
Iron(III) chloride hexahydrate (13.51 g, 0.05 mol) and urea (180.18 g, 3 mol) were dissolved in water (200 mL) in a 1 L round-bottomed flask. The solution was degassed for 30 min with oxygen-free nitrogen. Iron(II) chloride tetrahydrate (4.07 g, 0.02 mol) was added and degassing continued for a further 15 min. The vessel was sealed to prevent atmospheric oxygen entering the solution, and the temperature raised to 98 1C. The mixture was then stirred for 90 min using an overhead stirrer. The resulting precipitate was washed with boiling water (6 200 mL), or until the pH of the wash supernatant was reduced to 7. The final total volume for the suspension was adjusted to 300 mL using water.
2.5.2. Large scale, with aqueous sodium silicate (waterglass method 2SM) In all, 83.0 g of aqueous sodium silicate (27% SiO2) was dissolved in water to a total volume of 2 L. A column containing 110 g of ‘‘Amberlite IR120’’ ion exchange resin was regenerated with 1 L each of the following: hot water (around 70 1C), 3 M HCl, and finally cold water. Sodium silicate solution was passed down the column, allowing the first 100 mL to pass uncollected. In all, 1800 mL of the eluate (now in the form of silicic acid) was taken and its pH immediately raised to 12 using aqueous TMA (25%) to prevent homogeneous silica nucleation. In all, 900 mL of magnetite suspension (approximately 23 g L1, method 1Ma or b) was mixed with 500 mL water and titrated to pH 12 with TMA whilst stirring. With continued stirring, the sodium silicate eluate (at pH 12) was added to the magnetite suspension. This mixture was then slowly titrated to pH 10.0 (from 12) using 0.5 M HCl over approximately 1 h. Titration to pH 10.7 was performed over the first 30 min, and then from pH 10 to 10.7 over the last 30 min. The mixture at pH 10.0 was stirred for a further 2 h before transferring to a 5 L flask. The silica-magnetite particles were then washed once with 2 L of TMA solution at pH 10.0, and then five times with 2 L of deionised H2O by magnetic sedimentation. The pH was then tested to ensure the supernatant was at neutral pH (below pH 8). After washing, the silica magnetite was resuspended in 1 L of water. This method regularly yielded 20 gm of product.
2.5. Deposition of silica from silicic acid solution at pH 10 [2,11] 2.5.1. Small scale, with sodium silicate powder (method 1SM) Sodium silicate powder (8.3 g, 67% SiO2) was dissolved in water (200 mL). This solution (170 mL) was passed through a column (44 mm diameter) containing 25 g of ‘‘Amberlite IR-120’’ ion exchange resin which had been previously regenerated with 200 mL of each of the following: hot water, 3 M HCl, and finally cold water. The first 20 mL was allowed to pass uncollected to flush the column. After collecting the remaining eluate (now in the form of silicic acid or ‘active’ silica), its pH was raised to 9.5 using some of the stock sodium silicate solution. The resulting silicic acid solution (133 mL) was added to a volume of stirred magnetite suspension containing 1.5 g of magnetite (from either method 1Ma or b) in a 1 L beaker [prior to addition of the silicic acid all magnetite samples other than that prepared by method 1.3 were mixed with 1 M TMA (19 mL)]. The pH of the resulting suspension was around 12.5. The pH was carefully lowered to 10.0 by titrating dropwise with 0.5 M HCl over a period of approximately 1 h. The suspension was then stirred for 2 h before washing with TMA
2.5.3. Consecutive depositions of silica using methods 1SM and 2SM Up to four consecutive depositions as described above were carried out. Between each deposition, the silica magnetite was washed with water until the wash supernatant was at neutral pH. Prior to each successive deposition the suspension volume was adjusted with water to that of the original magnetite suspension. An aliquot of suspension (50 mL) was retained for analysis following each deposition.
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2.6. Deposition of silica by acid hydrolysis of tetraethoxysilane at 90 1C in glycerol [11] (method 3SM) In all, 100 mL of a suspension of magnetite prepared by method 1Ma or b (containing 2.3 g magnetite) was placed into a 500 mL roundbottomed flask and the supernatant aspirated. A 10% solution of aqueous tetraethoxysilane (TEOS) (230 mL) was added to the flask. Glycerol (200 mL) was added, whilst stirring with an overhead stirrer, and the pH of the suspension lowered to pH 4.5 using glacial acetic acid. The mixture was then heated to 90–95 1C and stirred at this temperature for 2 h under nitrogen. After cooling to handling temperature, the suspension was washed once with water (500 mL), twice with methanol (500 mL), and finally five times with water (500 mL). The final suspension volume was adjusted to 230 mL with water. 2.6.1. Consecutive depositions of silica using method 3SM Up to five depositions of silica have been carried out. Previously coated silica magnetite from method 3SM was used as starting material in further coating by the reaction above. An aliquot of suspension (115 mL) was retained for analysis following each deposition. Reaction volumes were scaled down by the appropriate amounts in each successive deposition, as shown in Table 2. 2.7. Rate of dissolution of silica-magnetite composites in acid Silica is soluble at pHs above 10 whilst magnetite is soluble in acid. If silica magnetite is
placed in strong acid the rate of dissolution can be taken as an indirect measure of the degree to which the magnetite is coated with silica and can also provide an indication about the nature of the silica coating. This was measured in the following way: 0.5 g samples of magnetite or silica magnetite were suspended in 100 mL of 8.6 M HCl (3:1 concentrated HCl to water by volume) and the mixture stirred gently. After the desired time interval (0, 30 s, 1, 2, 4, and 8 min), 0.1 mL of supernatant was removed and added to a 10 mL volumetric flask containing 9 mL water. The flask was then made up to the mark with additional deionised water. To construct a standard curve for magnetite assay solutions were prepared as follows: 0.5 g magnetite was dissolved in 90 mL HCl (8 M) and made up to 100 mL with HCl (8 M). The standard solution (1 mL) was then removed and diluted with water to 100 mL in a volumetric flask to give a 50 g L1 standard. Subsequent dilutions were performed with 0.8, 0.6, 0.4, 0.2, and 0 mL of stock solution to give standards of 40, 30, 20, 10, and 0 g L1, respectively. The standards and samples were analysed for their absorbance using a UV spectrophotometer set at a wavelength of 333 nm. 2.8. G, GMP, and oligonucleotides (NA) adsorption and elution studies A solution of the NA at a concentration of 0.05 g L1 was prepared in the desired binding medium. Triplicate aliquots of nanoparticle suspension, containing the required equivalent ‘dry’ weight of support (determined by heating a known volume of homogeneous particle suspension to
Table 2 Volumes (in mL) of reagents and sample used to deposit 1–5 layers of silica Sample
Volume suspension used
Volume TEOS
Volume glycerol
Final volume
1 2 3 4 5
280 (magnetite) 805 (silica magnetite) 690 575 460
920 805 690 575 460
800 700 600 500 400
920 805 690 575 460
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100 1C for 2 days or until no further weight loss was observed), were pre-washed twice with binding medium and placed in three 0.5 mL microcentrifuge tubes and the supernatant removed after magnetic immobilisation of the support. NA solution (0.2 mL) was then added to the supports in binding medium and the suspension gently agitated to permit binding. The supernatant was subsequently removed and stored on ice in a separate 0.5 mL microcentrifuge tube ready for analysis by HPLC. The nanoparticles were washed in 70% v/v aqueous ethanol (which was retained for HPLC assay) and 0.2 mL of the appropriate eluant was added to the supports and the suspension agitated for 2 min to elute the adsorbed species. The supernatant (eluate) was then removed and kept on ice for analysis by HPLC. HPLC analyses of 2-deoxyguanosine (G) and 2deoxyguanosine-5-monophosphate (GMP) were carried out at 260 nm using a Kontron 325 system connected to a Kontron 332 detector and a Kontron integrator 3.90 (Kontron Instrments Spa, Milano, Italy). A Genesis C18 (reversed phase) HPLC column (150 mm 4.6 mm, 4 mm particle size) was used. All samples were loaded into a Rheodyne injection valve fitted with a 50 mL sample loop. All solvents (water, methanol, acetonitrile [AcN]) were HPLC grade, and any chemicals used in the mobile phase were also of HPLC grade quality. HPLC analysis of nucleosides and nucleotides involved the use of two mobile phases ((A) KH2PO4 (0.02 M) and (B) methanol [MeOH]) at a flow rate of 2.0 mL min1. Samples were injected into a mobile phase of 100% KH2PO4 (0.02 M) and eluted using a gradient consisting of between 0% and 60% MeOH (100–40% KH2PO4) over 3 min (gradient change of 20% MeOH min1). Prior to analysis of each sample, the column was rinsed with 100% solvent B for 5 min and then with 100% solvent A for 5 min, or until a steady stable baseline was observed. HPLC analysis of oligonucleotides was at the same wavelength as above and involved the use of two mobile phases ((A) ammonium acetate buffer (0.1 M) and (B) AcN) at a flow rate of 2.0 mL min1. Samples were injected in a mobile
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phase of 3% AcN. NAs were then eluted isocratically with a mobile phase of 25% AcN: 75% ammonium acetate (0.1 M) for 2.5 min. Prior to analysis of each sample, and between samples, the column was washed with 100% solvent B for 5 min and then with 97% solvent A (3% solvent B) for 5 min, or until a steady stable baseline was observed. 2.9. Salmon sperm DNA binding assays In all, 50 mg of sheared, salmon sperm DNA (produced by repeated passage through a hypodermic syringe needle) was resuspended in 400 ml of TEN buffer (100 mM Tris–HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl) and 400 ml of polyethylene glycol (PEG, Mr 8000) in 4 M NaCl and added to 2 mg of silica magnetite (previously washed in sterile water) in a sterile Eppendorf tube. The mixture was incubated with gentle agitation for 5 min at 25 1C after which the support material was immobilised using a Promega magnetic stand (Promega Corp., Madison, USA). The supernatant was retained for assay at OD260 nm. The support material was then washed in 1 mL of ice cold 70% v/v aqueous ethanol by gentle agitation for 5 min at room temperature. The support was immobilised magnetically and the wash solution removed and retained for UV assay. DNA was eluted by washing the support sequentially, twice, in sterile deionised water (200 ml each) for 5 min each at room temperature. Each 200 mL was assayed independently for its OD at 260 nm to estimate its DNA content. A standard curve was constructed for comparison purposes using a series of salmon sperm DNA solutions of known concentration. 2.10. Fourier-transform infrared All samples were characterised by Fouriertransform infrared (FT-IR) spectroscopy (SHIMADZU 8300, Shimadzu Corp., Japan) and prior to compacting as pellets all samples and KBr were dried at 95 1C for 48 h. Each sample (5 mg) was thoroughly mixed and crushed with 500 mg of KBr using a mortar and pestle. The mixture (80 mg) was placed in a pellet former and 10 tons of
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pressure was applied for 2 min to form the KBr pellet. The FT-IR spectrum was collected after 50 scans of the region between 400 and 4000 cm1.
placed in the TGA furnace and heated at a rate of 10 1C min1 to 1000 1C in static air. 2.15. Magnetic susceptibility
2.11. Scanning electron microscopy Nanoparticle size and morphology were characterised by scanning electron microscopy (SEM). The scanning electron micrographs were recorded on a Cambridge Instruments Stereoscan 90 (Cambridge Instruments Ltd., Cambridge, UK). The samples were prepared by sprinkling the powder materials onto double-sided sticky tape which was mounted on a microscope stub. All samples were coated with a thin gold film under vacuum prior to microscopy. 2.12. XRF The silica concentration in various silica-coated magnetite samples were determined using an Oxford/Link XRD200 (Oxford Instruments plc, Oxon, UK). A standard curve was constructed for comparative purposes with sample measurements using quantities of silica gel with known silica concentration. 2.13. Surface area measurement The surface area of all samples (dried at 100 1C overnight) was determined by the BET method (nitrogen gas as an adsorbent) using a Micrometrics Instrument Corporation Gemini 2375 V4.01 (Norcross, USA). Each dried sample (0.1–0.2 g) was weighed accurately up to four decimal places and placed in a sample tube (reference an empty tube). Analysis was performed using an automatic five-point adsorption programme, measuring the volume of nitrogen adsorbed by the sample at the following pressures: 76, 114, 152, 190, and 228 mmHg. 2.14. Thermo-gravimetric analysis Thermo-gravimetric analysis (TGA) of samples was performed using a Dupont 2000 instrument (Wilmington, USA). Dried sample (10–20 mg) was
Magnetite susceptibility measurements were carried out using a superconducting quantum interference device (SQUID) magnetometer.
3. Results (magnetite) 3.1. Visual characterisation Iron oxides can vary quite significantly in colour and this can be used as an aid in their identification. It may also be used as a rule of thumb guide to purity in some cases. Table 3 indicates the different colours for each known iron oxide [12,13] and the oxides produced in this project. Magnetite produced by oxidative hydrolysis of iron(II) sulphate in alkaline media was jet black and therefore expected to be pure magnetite. The other methods generated products of various colours, from medium brown to very dark brown indicating a mixture of iron oxides. FT-IR and Mossbauer spectroscopy, plus TGA were used to analyse these products. All of the methods used for producing magnetite yielded products that reacted to a magnet to a certain degree, but methods 1Ma Table 3 Colour characterisation of different iron oxides Iron oxide
Colour
Magnetite Maghemite Hematite Feroxyhyte Ferrihydrite Lepidocrocite Goethite Akaganeite
Jet black Brownish-red Red Reddish-brown Reddish-brown Orange Yellowish-brown Yellowish-brown
Product
Colour
Method Method Method Method
1Ma 1Mb 2M 3M
Jet black Jet black Brown to very dark brown Brown
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and b produced material which reacted the quickest (results not shown). Method 3M produced what appeared to be a brown coloured solid that left sticky brown residues when contacted with glass or plastic ware surfaces which could not be removed, even after extensive washing. Methods 2M and 3M were not subsequently used for the production of magnetite for use in silica-magnetite composite manufacture. 3.2. Fourier-transform infrared FT-IR spectroscopy was used for analysis of products from the three methods. Table 4 shows the reported values for absorbance of various iron oxides [12,13] and those obtained for products of the various methods. Methods 1Ma and b were observed to produce very pure magnetite with little or no other iron oxide impurities. Magnetite prepared by method 2M appeared to be a mixture of magnetite and maghemite. The FT-IR spectrum for magnetite produced by method 3M was too complex in the magnetite, maghemite, and hematite absorption region (500–700 cm1) to allow definition of the product.
Fig. 1. SEM micrographs of magnetite synthesised by method IMb.
Confirmation was obtained by Mossbauer spectroscopy of the products of methods 1Ma and b which indicated that they possessed a hyper magnetic field of 518 kG and were therefore magnetite. An SEM image of the magnetite is provided in Fig. 1. 3.3. Thermo-gravimetric analysis
Table 4 IR bands of various iron oxides Iron oxide
IR bands (cm1)
Magnetite Maghemite Hematite Feroxyhyte Lepidocrocite Goethite Akaganeite
590 (400) 630, 590, 570, 450 (400) 540, 470 (345) 1110, 920, 790, 670 1026, 1161, 753 890, 797 840, 640
Product
Identity and bands observed
Method 1Ma Method 1Mb Method 2M
Magnetite (583) Magnetite (583) Magnetite (580)+maghemite (631, 570, 453) Magnetite, hematite, and maghemite (570, 476)
Method 3M
Note: Values in brackets indicate IR absorptions which could not be measured accurately.
TGA analysis of magnetite produced by methods 1Ma and b indicated decreases in weight of the sample as the temperature rose to 120 1C, and from 257 to 321 1C. These are likely to be due to loss of adsorbed water and dehydration of internal OH groups, respectively. The increase in weight observed from 120 to 257 1C is most likely due to the oxidation of magnetite to maghemite. These observations support the FT-IR and Mossbauer data that methods 1Ma and b produced effectively pure magnetite. 3.4. Particle size analysis Table 5 indicates the results from SEM size analysis of magnetite/iron oxide produced by the various methods employed. The only difference in magnetite produced from methods 1Ma and b is the particle size distribution and shape. SEM
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Table 5 Particle sizes of magnetite from SEM Method and particle size distribution
Particle size (nm)
Method 1Mb Minimum Maximum Mean
140 170 150
Method 2M Minimum Maximum Mean
100 200 140
Method 3M Minimum Maximum Mean
80 120 100
analysis has indicated that method 1Ma produced magnetite particles with a narrower apparent size range distribution than method 1Mb but with a larger mean particle size.
of 5.18 g cm3. Whereas materials produced by methods 2M and 3M contain a mixture of what is principally magnetite and maghemite (density 4.87 g cm3), so would possess a density somewhere between the values for magnetite and maghemite. For the sake of simplicity, it was assumed that methods 2M and 3M were a 50:50 mixture of magnetite and maghemite and therefore possessed a density of 5.03 g cm3. The results for the surface area and estimated particle diameter with comparison to the literature values are summarised in Table 6. Using this approach it appears that magnetite prepared by method 1Mb possessed the largest mean particle diameter at 79.3 nm whilst magnetite prepared by method 1Ma is slightly smaller at 54.4 nm. This is approximately twice the value obtained by SEM.
4. Results (silica-magnetite composites)
3.5. Surface area analysis and correlation with particle size
4.1. FT-IR and XRF analyses of silicic acid and TEOS deposed composites
For a non-porous spherical particle, the relative surface area (as defined by surface area to mass ratio) is inversely proportional to its diameter. By knowing the density of the material of which the particles are composed, their sizes can be calculated, through the manipulation of the following formulae:
The presence of silica in all silica-magnetite composites was confirmed by FT-IR and XRF and results are shown in Tables 7 and 8 and Figs. 2 and 3. For FT-IR, the quantitative results describe the relative peak heights at the indicative spectral points for magnetite and silica. It was observed that silica:magnetite molar ratio increases with successive rounds of silica deposition. In fact, the relative amount of silica deposited using the TEOS method is greater per deposition than that achieved using the silicic acid approach. One reason for the slow rate of accumulation of silica on subsequent rounds of deposition to the surface of the silica-magnetite composite produced via acid titration of silicic acid is likely to be due to the fact that for each successive deposition the material’s pH is raised to 14 prior to titration with HCl. This could mean that at least a part of the existing surface silica would be resolubilised and lost. This would effectively mean that on each deposition very little (as observed) extra silica might be deposited.
density ðsÞ ¼
mass ðmÞ ; volume ðvÞ
relative surface area ðaÞ ¼
area ðAÞ ; mass
area of sphere ¼ 4pr2 ; volume of a sphere ¼ 43 pr3 : These equations can be arranged to give sa1 r¼ : 3 Magnetites produced by methods 1Ma and 1b are effectively pure and so should have a density
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Table 6 Surface areas of iron oxide products and calculate particle diameters Sample
Method Method Method Method
1Ma 1Mb 2M 3M
Relative surface area (m2 g1)
Estimated particle size (nm)
Observed
Reported value
Calculated
Reported value
21.30 14.60 108.81 25.46
— o4 [13] — —
54.4 79.3 11.0 46.9
100 [9] 50–200 10 [2] 800 [10]
Table 7 FT-IR absorbance ratios of silica/magnetite for silicic acid and TEOS deposition methods (with principal measurement points) Sample
Principal adsorption bands (cm1)
% relative response
Silicic acid (2SM) 1 layer –2 layers –3 layers –4 layers
Magnet. Magnet. Magnet. Magnet.
(584)+silica (579)+silica (586)+silica (583)+silica
(895, (861, (884, (872,
1023) 988) 1035) 1023)
15.61 17.36 20.31 27.76
TEOS (3SM) 1 layer –2 layers –3 layers –4 layers –5 layers
Magnet. Magnet. Magnet. Magnet. Magnet.
(578)+silica (582)+silica (582)+silica (582)+silica (582)+silica
(798, (798, (795, (795, (798,
900, 900, 897, 897, 900,
23.34 26.00 35.36 49.23 64.70
1050) 1088) 1113) 1103) 1100)
Table 8 XRF results for silica-magnetite composites Sample
No. of counts
% element
At%
Silicic acid (2SM) 1 layer 29 2 layer 35 3 layers 43 4 layers 47
0.027 0.029 0.030 0.033
0.053 0.058 0.060 0.066
TEOS (3SM) 1 layer 45 2 layers 48 3 layers 69 4 layers 118 5 layers 139
0.027 0.034 0.043 0.077 0.094
0.053 0.067 0.085 0.154 0.187 Fig. 2. FT-IR of relative silica composition of silica-magnetite composites.
4.2. Rate of dissolution of silica-magnetite composites in acid The resistance to acid dissolution of the two silica magnetites is different (Fig. 4).
For the silicic acid deposed material it is clear that there is no real difference in acid resistance between 1 and 4 coatings of silica. All the silicic
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Fig. 3. XRF estimation of silica-magnetite composites silica content. Fig. 4. Acid dissolution characteristics for magnetite and silicamagnetite composites.
acid-coated forms were more or less equally resistant to dissolution. The likely conclusion is that even the first round of deposition must give good silica coverage, which is presumably less porous or more complete than that generated using TEOS. In the case of TEOS-coated magnetite the first silica layer appears to afford no protection to the magnetite from dissolution by acid, as the dissolution curves for both magnetite and this material are virtually identical. The second coating offers some resistance but it is only after the third and fourth coatings that similar levels of resistance to those observed with silicic acid deposed magnetite are reached. The implication of these results is that even though the TEOS method is efficient in depositing more silica per round of coating than the silicic acid method, the nature of the silica deposited or the type of coverage of the iron oxide core must be different. It could be that the TEOS silica coating is porous or incomplete. In either case, the differences between the two composites are likely to have implications on their abilities to be used in bioseparative processes because their surfaces will represent different chemistries. The former hypothesis is largely supported by data from surface area analysis of the materials using N2 (data not shown) where the TEOS deposed material has a significantly larger comparative surface area to that of the silicic acid deposed material.
4.3. Adsorption and elution of G, GMP, and oligonucleotides to magnetite and silica-magnetite composites When the ability to adsorb and elute G and GMP to and from the surface of magnetite and silicic acid and TEOS magnetite composites is compared these differences are highlighted (see Fig. 5). 4.3.1. Magnetite In water magnetite, in contrast to silica, adsorbs most GMP, whilst little or no G is adsorbed (data not shown). This strongly suggests an electrostatic mechanism mediated by the phosphate group as the attractive force. From the results indicated in Fig. 6 (effect of pH on adsorption) the adsorption of GMP to magnetite can readily be explained on the basis of the degree of protonation of surface Fe–OH groups and phosphate group possessed by the GMP. Effectively two competing effects occur with respect to these groups and pH. As the pH is lowered so the phosphate group of GMP is more likely to be protonated leading to a diminished negativity, whilst at the same time the surface Fe–O groups also become increasingly protonated. The reverse is true with increasing pH. Adsorption of GMP by magnetite from salt solutions results in a reduction of the amount of
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These results are summarised in the diagram in Fig. 5 for GMP and were obtained using the protocols described in Section 2. Silica used in the comparison was produced by the same method as for silicic acid deposition of silica to magnetite with the difference that silica gel was used as the core material.
Fig. 5. GMP adsorption characteristics for magnetite, silica, and silica-magnetite composites in various media.
Fig. 6. Adsorption of GMP by magnetite at various pHs.
GMP bound which is presumably due to competition between the salt negative ion species and the charged phosphate group present in GMP. (The corresponding nucleoside shows little binding to magnetite under any condition.) In contrast, silica shows greatest binding of GMP under chaotropic conditions. The silicic acid-deposited magnetite mimics silica in its GMP adsorption characteristics whilst magnetite deposited with silica using the TEOS method mimics magnetite with respect to its GMP binding when coated with a single layer. Interestingly as more silica is added to the magnetite using the TEOS method (as consecutive rounds of deposition are performed) its characteristics were observed to become more like that of silica and the silicic acid-deposited material.
4.3.2. Silica-magnetite (method 2SM) adsorption characteristics for oligonucleotides Table 9 illustrates the results of adsorption studies involving NAs and silica-magnetite composite. Mono-base characterised NAs were observed to bind to silica magnetite in water in a way which appeared to be dependent on the base type, with purine base constituted NAs, (dA)10 and (dG)10, binding more than the corresponding pyrimidine base constituted NAs, (dC)10 and (dT)10. This may be a reflection of the fact that purine bases tend to be more prone to hydrophobic interactions than pyrimidine bases, which may indicate that, even in water, hydrophobic interactions play an important role in binding to silica. Alternatively, this increased degree of binding could have been due to the higher corresponding molecular weight of the purine base NAs, which would have resulted in a lower molarity for the solution of these NAs. However, this is contradicted by the results from the di-base constituted NAs, where (dTA)5 bound almost as much as (dA)10, and more than (dT)10. This also suggests that the purine bases may make a greater Table 9 Percentage adsorption of GMP and oligonucleotides to silica magnetite Oligonucleotide
(dG)10 (dC)10 (dA)10 (dT)10 (dTA)5 (dGC)5 GMP
% binding H2O
4 M sodium chloride
4M ammonium sulphate
17.1 11.1 19.2 13.2 17.8 6.4 7.5
80.4 92.2 49.2 21.5 30.3 32.9 21.6
75.0 80.3 90.4 66.2 92.3 50.0 12.9
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contribution to binding than pyrimidine bases. (dGC)5 was observed to bind much less than all others. Both these NAs have similar molecular weights. NA binding to silica magnetite was also performed in 4 M sodium chloride and 4 M ammonium sulphate and in both cases binding was significantly better than in water and appeared to demonstrate a chaotrope and sequence dependent effect. NAs bind better to silica under chaotropic conditions as a consequence of electrostatic shielding of the negatively charged groups of the silica surface and phosphate groups and by promotion of hydrophobic interactions [14]. Binding is greater in 4 M ammonium sulphate, for (dA)10, (dT)10, (dTA)5, and (dGC)5, possibly as a consequence of it being a stronger chaotrope. It is unclear as to why there is such a large difference between the effect of sodium chloride and ammonium sulphate on the relative adsorption of adenine and thymine containing NAs to those containing guanine and cytosine. Perhaps heterocyclic base polarity (hydrophobicity) affects NA adsorption in different chaotropic salts. G and C are more polar than A and T. 4.3.3. Silica-magnetite (method 2SM) elution characteristics for oligonucleotides Table 10 indicates the recovery efficiencies for NAs absorbed to silicic acid-deposited magnetite in water under various salt conditions.
All NAs could be desorbed in water after adsorption under chaotropic conditions with high efficiency. Once again chaotrope and sequence differences were observed. 4.4. Adsorption and elution characteristics for salmon sperm DNA using method 2SM silica-magnetite composites Fig. 7 illustrates the results from experiments involving the use of sheared, denatured salmon sperm DNA binding and elution from silica magnetite (silicic acid deposition method) and a comparison with a commercially available equivalent material. See Section 2 for a full description of the protocol. Experiments were conducted in triplicate in two different batches of silica magnetite and an average value calculated. These data indicate that the performance of the material produced by us does not improve radically with each successive deposition of silica. It also indicates that the material is very efficient at allowing the recovery of all, or almost all, of the DNA adsorbed and that the material performed better in the assay than the commercial equivalent (approximately 10% better). Although the quantity of DNA adsorbed and eluted from the composite does not significantly increase with increasing silica coating it was observed that the quality of the eluted DNA does. In restriction digestions of eluted DNA, it has
Table 10 Percentage recovery of GMP and oligonucleotides bound to four silica magnetite Oligonucleotide
(dG)10 (dC)10 (dA)10 (dT)10 (dTA)5 (dGC)5 GMP
% recovery after binding in salt and elution in water In 4 M sodium chloride
In 4 M ammonium sulphate
35.8 55.6 50.2 64.9 64.5 63.6 39.3
33.5 17.5 69.1 67.3 77.3 85.9 58.5
Fig. 7. Sheared salmon sperm adsorption and elution characteristics of magnetite and its silica-coated forms (1–4 coated) and a commercial equivalent (Merck GmbH).
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M(emu/g)
80
T=2.5K
0
0 5 4 3 2 1
-80
-5
0
5
H(Tesla)
Fig. 8. Magnetic hysteresis of magnetite and 1–5 coated silicamagnetite nanocomposites.
been found that DNA from the four coated material performs better (data not shown). The reason for this is unknown. 4.5. Magnetic measurement The temperature dependence of the static magnetic susceptibility of pure magnetite and silicamagnetite nanocomposites (synthesised by the silicic acid method) was measured and the hysteresis loop (Fig. 8) was collected at 2.5 K. Coercivities and reduced remanences (MH=0/Msat) were also measured and the saturation magnetisation (Ms) was centred at 88 emu g1 at 2.5 K and around 82 emu g1 at 300 K. These values are close to those of bulk magnetite (90 emu g1 at 300 K and 85 emu g1 at 4.2 K) confirming the presence of large size magnetic particles with respect to the single domain limit. Neither material reach the saturation of magnetisation at the largest measuring field at 6.5 T while the values of M r =M s ratio decreases from 0.4 at 2.5 K to 0.3 at 300 K. Such behaviour is characteristic of nanometre size particles. These results suggest that silica coating did not change the magnetic property of the particles.
5. Conclusion From the present study, we have obtained a good understanding of the structure–function–
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chemistry involved with silica-magnetite composites and the best method for producing such a material with utility in nucleic acid adsorption and elution. If a silica-magnetite composite with solely silica surface characteristics with respect to nucleic acid binding and elution is required then the silicic acid deposition method (2SM) is the best route to achieve it, but the TEOS (3SM) actually deposits more silica—and more rapidly. It may be that further scale-up, and speed, with which the composite material can be produced will involve the manufacture of a hybrid material possessing an initial silicic acid-deposited layer followed by a layer mediated by TEOS. This is currently being investigated. However, the composite described here represents a cheap and efficient alternative to existing materials for the bioseparation and purification of nucleic acids with what appears to be better performance than at least one potential competitor material.
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