Synthesis of Novel Porous Magnetic Silica Microspheres as Adsorbents forIsolation of Genomic DNA

Zhichao Zhang, Liming Zhang, Lei Chen, Ligong Chen, and Qian-Hong Wan*College of Pharmaceuticals and Biotechnology, Tianjin University, Tianjin 300072, China

An improved procedure is described for preparation of novel mesoporous microspheres consistingof magnetic nanoparticles homogeneously dispersed in a silica matrix. The method is based ona three-step process, involving (i) formation of hematite/silica composite microspheres by urea-formaldehyde polymerization, (ii) calcination of the composite particles to remove the organicconstituents, and (iii) in situ transformation of the iron oxide in the composites by hydrogenreductive reaction. The as-synthesized magnetite/silica composite microspheres were nearlymonodisperse, mesoporous, and magnetizable, with as typical values an average diameter of3.5 µm, a surface area of 250 m2/g, a pore size of 6.03 nm, and a saturation magnetization of9.82 emu/g. These magnetic particles were tested as adsorbents for isolation of genomic DNAfrom Saccharomyces cereVisiae cells and maize kernels. The results are quite encouraging asthe magnetic particle based protocols lead to the extraction of genomic DNA with satisfactoryintegrity, yield, and purity. Being hydrophilic in nature, the porous magnetic silica microspheresare considered a good alternative to polystyrene-based magnetic particles for use in biomedicalapplications where nonspecific adsorption of biomolecules is to be minimized.

Introduction

Magnetic particles or microspheres consisting of magneticnanoparticles embedded or encapsulated in an inorganic oxidematrix have attracted much attention in recent years owing totheir potential applications in the fields of chemical industry,biotechnology, and medicine (1-6). Among the inorganic oxidesstudied, silica is most commonly used as a dispersion medium,providing magnetic particles with several benefits for use inbiomedical applications such as biocompatibility in biologicalsystems, high stability against aggregation, versatility in surfacemodification, and hydrophilic character. Other desirable proper-ties of silica-based magnetic particles include strong magneticsusceptibility, minimal residual magnetism, large surface area,and narrow particle size distribution. Several approaches (3,5-8) have been proposed for the preparation of such magneticsilica particles, including in situ formation of magnetic nano-particles within the pores of the silica matrix (often referred toas backfilling) and microemulsion polymerization of silicaprecursors with magnetic nanoparticles. While the backfillingapproach risks clogging of the matrix pores and reducing thesurface area available, the microemulsion approach often leadsto magnetic particles with low magnetic susceptibility. It istherefore still of great interest to develop methods for preparationof magnetic silica particles that fulfill the criteria mentionedabove for biomedical applications such as separation of bio-molecules/organisms.

Recently, we reported a polymerization-induced particleformation method for preparation of maghemite (γ-Fe2O3)/silicacomposite microspheres that possess some favorable propertiesfor use in bioseparations (9). In this method, a urea-formalde-hyde (UF) polymerization process is used to coacervate silicacolloids together with iron oxide nanoparticles, resulting inmonodispersed inorganic/organic hybrid microspheres. The silica

and iron oxide nanoparticles are homogeneously dispersed inthe composite particles thus formed. The UF polymer matrixthat served as a shape template is then burned off to afford ironoxide/silica composite microspheres with open pore structures.Calcination of the resulting particles at elevated temperatureleads to partial phase transformation of iron oxide and in situformation of magnetic maghemite (10). A distinct advantageof this approach is that the magnetic particles produced are ofnearly monodisperse.size and spherical shape, thus eliminatingthe need for costly and tedious particle size classification duringa postsynthesis phase (11-13). However, characterization ofthe composite particles by X-ray diffraction shows that only asmall portion of nonmagnetic hematite (R-Fe2O3) was trans-formed into maghemite during the heat treatment, giving riseto poor response to an external magnetic field.

Here we describe an improved approach to mesoporous silica-based magnetic microspheres that exhibit strong magneticsusceptibility, minimal residual magnetism, and narrow particlesize distribution. The preparation method consists of threesteps: (i) formation of hybrid microspheres composed of silicaand hematite nanoparticles via a UF condensation-polymeriza-tion, (ii) calcination of the hybrid microspheres to remove theorganic constituents, and (iii) in situ transformation of hematiteto magnetite (Fe3O4) in the composite particles by chemicalreduction (14, 15). The morphological, structural, and magneticproperties of the resulting magnetic silica microspheres werecharacterized by a range of techniques including X-ray diffrac-tion, scanning electron microscopy, BET analysis, and vibratingsample magnetometry. The utility of such magnetic micro-spheres in bioseparations is demonstrated by isolation ofgenomic DNA fromSaccharomyces cereVisiaecells and maizekernels.

Materials and Methods

Materials. All chemicals used for synthesis of magnetic silicaparticles were of reagent grade unless otherwise specified. Silica

* To whom correspondence should be addressed. Ph:+86-22-27403650.Fax: +86-22-27403650. Email: qhwan@tju.edu.cn.

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10.1021/bp050400w CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical EngineersPublished on Web 03/03/2006

sol (30% SiO2, 10-15 nm) was obtained from GuolianChemical Co. (Jiangyin, China). Wild-typeS. cereVisiae cellsused were a gift from Mr. Yonggang Zhang of TianjinUniversity. Fresh corn used to provide maize kernels wasobtained from a local fruit market. Agarose of molecular biologygrade was from Lianxing Biotechnology Co. (Tianjin, China).Molecular weight marker 1 kb DNA step ladder was fromDingguo Biotechnology Co. Ltd. (Beijing, China). Snailase wasfrom Hope Biotechnology Co. Ltd. (Tianjin, China). Otherreagents used in DNA isolation and analysis were of analyticalgrade and were obtained from various vendors.

Synthesis.The flowchart for the synthesis of mesoporousmagnetic silica microspheres is shown in Figure 1. In a typicalpreparation, hematite suspension was obtained by adding 5.1 gof NaHCO3 to 7.2 g of FeCl3‚6H2O dissolved in 50 mL ofdistilled water and stirring the mixture vigorously for 10 min,yielding a reddish solution with pH∼2. The silica suspensionwas prepared separately by adding dropwise concentrated HNO3

to 100 mL of silica sol until a pH 2 was reached. The hematiteand silica suspensions thus obtained were then mixed undervigorous stirring. To the suspension was added 3.0 g of ureaand 4.0 mL of formaldehyde (37%) under stirring, and themixture was allowed to stand at ambient temperature overnight.The resulting composite microspheres were collected by cen-trifugation at 2000 rpm for 10 min and washed successivelywith water, water-methanol (1:1), methanol, and acetone. Theywere dried at 60°C under vacuum overnight to yield 10.3 g offree-flowing, yellowish microspheres. To remove the polymertemplate and enhance the mechanical strength of the porousparticles, the hybrid particles were subjected to staged heatingtreatments at 300°C for 2 h, then at 600°C for 2 h, and finallyat 800°C for 2 h. After removal of the chippings generated in

the above process by centrifugation at 800 rpm, 5.8 g of free-flowing, brownish red microspheres were obtained in a yieldof 18.3 wt % based on the initial hematite and silica colloidsused.

The iron oxide/silica composite particles obtained that wereonly weakly magnetic responsive due to partial formation ofmaghemite during the heating process were subjected toreduction treatment in order to enhance their magnetizationproperties. To this end, a quartz tube of 750 mm in length and25 mm in inner diameter mounted in a tube furnace with atemperature control facility was built in-house and used for thereductive reaction. Two grams of the hematite/silica micro-spheres was placed in the quartz tube, and the tube was purgedby nitrogen gas for 30 min at 40 mL/min to remove the oxygenentrapped in the system. This was followed by hydrogen gasalso at 40 mL/min as soon as the nitrogen gas flow was stopped.Then the furnace temperature was increased from ambient to282°C over 1 h. The reductive reaction was allowed to proceedfor 3 h, giving rise to 1.6 g of free flowing, black microspheresthat can be readily attracted by a permanent magnet.

Characterization. The different phases of the iron oxidepresent in the particles were identified by X-ray diffraction(X’Pert PRO, PANalytical Instrument Ltd., The Netherlands)using Co KR (λ ) 0.1789 nm) radiation. The morphology andsize of the magnetic microspheres were examined by scanningelectron microscopy (X-650, Hitachi, Japan). The surface areaand pore size of the particles were measured by Sorptometer(NOVA 2000, Quantachrome, FL). The iron content of theparticles was determined by polarized Zeeman atomic absorptionspectrophotometer (AAS 180-80, Hitachi, Japan). The magneticproperties of the particles were recorded on a vibrating samplemagnetometer (LDJ 9600-1, LDJ Electronics, MI).

Extraction of Genomic DNA from Yeast Cells. Theprotocol used to extract genomic DNA from wild-typeS.cereVisiaewas based on a modification to previously reportedprocedures (16, 17). Briefly, 4 mL of S. cereVisiae culturesolution was centrifuged in a 7-mL tube to yield a 0.12 g ofwet pellet. Into the tube was added 1 mL of 0.1% ME-PB buffer(0.1% â-mercaptoethanol, 25 mM Na2HPO4, 175 mM NaH2-PO4, 0.8 M sorbitol, pH 5.8). Following 10 s of vortexing, themixture was transferred into a 1.5-mL microcentrifuge tube andincubated at 30°C for 30 min. The mixture was centrifuged at3500 rpm for 5 min, and the supernatant was discarded. Intothe tube was added 1 mL of 1% snailase-PBS buffer (pH 7.5),and the suspension was gently agitated at 30°C for 3 h. Theresulting suspension was centrifuged at 4000 rpm for 20 min,and the supernatant was removed again. Into the tube were added1 mL of lysis buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mMEDTA, 1% Triton-X-100, pH 8.0) and 0.2 mL of 10% sodiumdodecyl sulfate (SDS), and the mixture was incubated at 55°Cfor 10 min. Then 1 mL of binding buffer (20% PEG 8000, 2 MNaCl) was added, followed by 0.2 mL of magnetic microspheressuspension (0.05 g/mL). The suspension was agitated gently atroom temperature for 10 min, and the magnetic particles wereimmobilized using a MagneSphere Technology magnetic sepa-ration stand (Promega, WI). The supernatant was removed, andmagnetic particles were washed with 80% 2-propanol, followedby 70% ethanol. After the removal of the supernatant, theadsorbed DNA was eluted from the magnetic particles byaddition of 0.2 mL of TE buffer (10 mM Tris-HCl, 1 mMEDTA, pH 7.8) and incubation with gentle agitation at 25°Cfor 10 min. The magnetic microspheres were immobilized again,and the eluate was collected and analyzed by UV spectroscopy(UV 2450, Shimadzu, Japan). A 10-µL aliquot of the eluted

Figure 1. Schematic showing the synthesis of porous magnetic silicamicrospheres.

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DNA was separated by agarose gel electrophoresis on a 1%agraose gel in Tris-acetate buffer. Genomic DNA isolated fromS. cereVisiaeculture solution by a traditional phenol-chloroformextraction method was used as positive control (18).

Extraction of Genomic DNA from Maize Kernels. Adifferent protocol was adapted for isolation of genomic DNAfrom maize kernels (19). After treatment of liquid nitrogen, 1g of maize kernels was thoroughly ground with a pestle in amortar. A portion of ground maize kernels (30 mg) was placedin a 1.5-mL microcentrifuge tube and suspended in 0.9 mL lysisbuffer (100 mM Tris-HCl, 50 mM EDTA, 500 mM NaCl, 1.5%SDS, pH 8.0). The suspension was vortexed and incubated at65 °C for 30 min. To the suspension was added 100µL of 3 Msodium acetate, and the sample was vortexed once again. Thetube was placed on ice for 10 min, and the resulting suspensionwas centrifuged at 10 000 rpm for 10 min. The supernatant wastransferred to a fresh 1.5-mL microcentrifuge tube and subjectedto magnetic separation as for yeast cells. The purified corn DNAsamples were analyzed using UV spectrometer and gel elec-trophoresis and were compared with those isolated by thephenol-chloroform extraction method.

Results and Discussion

In the present work, magnetic silica microspheres with openpore structure and narrow particle size distribution were preparedthrough integration of polymer templated synthesis with in situmagnetic transformation. To verify phase changes brought aboutby the reductive reaction, X-ray diffraction (XRD) analysis wasperformed on the particle samples obtained before and afterhydrogen reduction treatment. Figure 2 shows the XRD patternsfrom which phases of iron oxide in the composite microspherescan be readily identified. While the broad peaks at 2θ ) 23-27° are ascribed to amorphous silica, the rest of the peaks ofthe diffractograms obtained before and after the reduction areidentified as corresponding to hematite (R-Fe2O3) and magnetite(Fe3O4), respectively. The sharp peaks are characteristic of largenanoparticles from which a mean crystallite diameter can becalculated using the Scherrer equation (20). The mean crystallitediameters are 31 nm forR-Fe2O3 as estimated from (104)reflection and 48 nm for Fe3O4 from the (311) reflection,pointing to an increase in the crystallite size when the iron oxideundergoes phase transition by the reductive reaction.

The morphology of the as-synthesized magnetite/silica com-posite particles was examined by scanning electron microscopy(SEM). As shown in Figure 3, the particles are largely spherical

and essentially free from clustering. The average diameter ofthe particles was estimated from SEM micrographs to be∼3.5µm.

The specific surface area and pore structure parameters ofthe particle sample were determined by the nitrogen adsorption-desorption method. As is evident from Figure 4, the magneticparticles display characteristic type IV adsorption isotherm witha distinct hysteresis in the desorption isotherm at relativepressure (P/Po) in the range 0.4-0.9 (21). This behavior isindicative of mesoporous structure of the tested sample. Themagnetic particles exhibit a BET surface area of 250 m2/g withan average pore diameter of 6.03 nm and a total pore volumeof 0.374 cm3/g as determined by the Barret-Joyner-Halendamethod (BJH) from the desorption branch.

Elemental analysis of the particle sample was performed usingan atomic absorption spectrophotometer, which typically givesabout 9 wt % Fe or, equivalently, 12 wt % Fe3O4.

The magnetic properties of the magnetite/silica microsphereswere measured on a vibrating sample magnetometer. Figure 5shows a hysteresis loop registered at room temperature. Themeasured saturation magnetization (Ms), the coercivity (Hc),and the reduced remanence (Mr/Ms) are 9.822 emu/g, 150.9Oe, and 0.219, respectively. The Ms value, when normalizedto the iron oxide content, is found to be 81.85 emu/g, which ishigher than that of Fe3O4 particles with diameter of 30 nm(73∼78 emu/g) and less than that of the bulk Fe3O4 (90.0 emu/g). These results confirm the presence of larger size magneticnanoparticles with respect to the single domain limit (D < 30nm) as revealed by the XRD measurements (15).

Magnetic particles have been widely employed in thepreparation of nucleic acid templates as they promise to simplify,expedite, and automate the nucleic acid extraction process forhigh throughput analysis (22, 23). DNA and RNA are anionsat neutral pH and highly hydrated in aqueous solutions. Underchaotropic conditions or in the presence of high sodium chlorideand poly(ethylene glycol) (PEG) concentrations, however, thenucleic acids in a crude cell lysate can be selectively precipitatedonto the surfaces of silica or magnetite while the proteins andother cellular constituentes remain in the supernatant (19, 24,25). Due to the use of chaotropic reagents, the sample solutioncan become very viscous, making it troublesome to separatethe magnetic particles from the suspension. As shown above,the magnetic particles prepared by our approach exhibit

Figure 2. XRD patterns of iron oxide/silicamicrospheres taken beforeand after hydrogen reductive treatment on a X’Pert PRO X-raydiffractometer using Co KR (λ ) 0.1789 nm) radiation source.

Figure 3. Scanning electron micrograph of porous magnetic silicami-crospheres taken by a Hitachi X-650 scanning electron microscopeoperating at an acceleration voltage of 20 kV.

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relatively high saturation magnetization. Using a strong rareearth magnet as employed in the Promega magnetic separationstand, which has typical maximum energy product above 30MGOe, they are immobilized from a suspension typically inless than 10 s, leaving a clear supernatant that can be readilyremoved by aspiration or decantation. As such, the magnetic

silica particles were considered well suited for extraction ofgenomic DNA from biological matrices such as maize kernelsandS. cereVisiae cells.

The extraction protocols used consist of cell lysis to releasegenomic DNA and binding of the DNA to magnetic particles,followed by elution of the purified DNA. While cell lysis wasachieved by grinding with a pestle in a mortar for maize kernels,the preparation of spheroplasts by enzymatic treatment of cellsand a subsequent lysis of cellular membrane by osmotic shockwas adapted for yeast cells. The integrity, yield, and purity ofthe extracted DNA were checked with agarose gel electrophore-sis and spectrophotometry. As shown in Figure 6, the extractedgenomic DNAs are of large molecular sizes with molecularweights greater than 8 kb. Smeared bands observed in theelectropherogram of the corn DNA are most likely due to thebreakdown of genomic DNA during the grinding process. Thequantity and quality of the extracted DNA were estimated onmeasurements of the absorbance at 260 nm (A260) and theabsorbance ratio at 260 nm/280 nm (A260/A280), respectively.About 6µg of genomic DNA was extracted from 1 mL of yeastcells cultured overnight and about 7µg of DNA from 30 mg ofmaize kernels. The yields of the DNA isolated using the newmagnetic particles are comparable to those obtained withcommercial kits. TheA260/A280 ratios of the DNA samplesobtained from yeast cells and maize kernels were 1.86 and 1.79,respectively, suggesting that the extracted nucleic acid templatesare of relatively high purity.

The mechanisms of selective binding and elution of DNAonto and from the silica particles have been extensively studied.Vasilevskaya et al. (26) have shown that nucleic acids can existeither in hydrated coil form or in compact globule form. In thepresence of PEG and salts, DNA undergoes a phase transitionfrom coil to globule and binds to the silica particles throughhydrogen bonding (27, 28). In this way, DNA is separated fromproteins, cellular debris, and other cellular constituents thatremain in the supernatant. As a key component of the bindingbuffer, PEG not only induces the phase transition of DNA butreduces the adsorption of protein as well by dynamically coatingthe silica particles (29). With a PEG-free and low salt buffer asthe elution buffer, all of the bound DNA is released from thesilica particles in its initial hydrated coil form, free from proteincontamination and suitable for further manipulation.

Figure 4. Nitrogen adsorption/desorption isotherms of porous magneticsilica microspheres obtained on a Quantochrome NOVA 2000 sorp-tometer operating at adsorption temperature of-195.6 °C (liquidnitrogen).

Figure 5. Room-temperature magnetization curves for porous magneticsilica microspheres obtained on a LDJ 9600 vibrating sample magne-tometer operating at maximum magnetic field of 15 kOe.

Figure 6. 1% Agarose gel electrophoresis of genomic DNA isolated from (a)Saccharomyces cereVisiaecells and (b) maize kernels. Lane 1, 1 kbDNA ladder; lane 2, DNA isolated by phenol-chloroform extraction; lane 3, DNA isolated using porous magnetic silica microspheres.

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Conclusions

The work presented above demonstrates that magnetite/silicacomposite microspheres can be prepared by a three-step processinvolving bead formation, calcinations, and reduction. Theparticles produced by this method are essentially monodisperse,mesoporous, and magnetically retrievable. These propertiesmake them an ideal carrier for use in biomedical applicationssuch as isolation of genomic DNA from biological matrices.The magnetite/silica composite particles offer many advantagesover polystyrene-based magnetic particles. First, the silica-basedmagnetic particles carry a large number of hydrophilic andionizable hydroxyl groups on their surface. The hydrophiliccharacter of these functional groups allows the minimizationof the nonspecific adsorption caused by hydrophobic interac-tions. The presence of negatively charged hydroxyl groups mightintroduce significant unwanted interactions with positivelycharged biomolecules such as proteins, but this can be preventedby means of surface modifications such as dynamic coating orchemical bonding. Second, the surface hydroxyl groups on bothmagnetite and silica can be readily reacted with silylationreagents, providing them with a variety of surface functionalgroups and thus selectivity benefits. Furthermore, the silica-based magnetic particles have an open-pore structure that allowsaccess to novel applications in which interplay of size exclusionand magnetic interactions may be exploited (8).

Acknowledgment

This work was supported by the National Natural ScienceFoundation of China (no. 20375027).

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Accepted for publication February 8, 2006.

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