ARTICLE IN PRESS

Journal of Magnetism and Magnetic Materials 277 (2004) 16–23

*Corresp

10-6278248

E-mail1Xin Xie

0304-8853/

doi:10.1016

Preparation and application of surface-coatedsuperparamagnetic nanobeads in the isolation of

genomic DNA

Xin Xiea,1, Xu Zhangb,1, Huan Zhangc, Depu Chend,*, Weiyang Feia

aState Key Laboratory of Extraction Separation Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084,

PR ChinabFaculty of Science, Sichuan Agricultural University, Sichuan 625014, PR China

cDepartment of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USAdDepartment of Chemistry, Tsinghua University, Beijing 100084, PR China

Received 19 May 2003; received in revised form 7 September 2003

Abstract

A novel method was developed to prepare functionalized magnetic beads, in which superparamagnetic Fe3O4 core

was synthesized with an injection-precipitation method and then was coated with functional groups using one-step

suspension polymerization. In the coating and functionalizing process, a unique coupling reagent, bis-(2-hydroxyethyl

methacrylate) phosphate, was introduced so that the monomers polymerized only on the surface of the nanocrystals

without forming separate nuclei. The thickness of the coating layer and the size and density of the coated nanobeads

were controlled by changing the quantity of the coated monomers. The nanobeads were characterized by transmission

electron microscopy, light scattering spectrometry, Fourier transformation infrared spectroscopy, X-ray fluorescence

spectroscopy and the magnetic hysteresis loop determination method. The carboxyl-modified magnetic nanobeads were

employed to simplify the isolation of genomic DNA from human whole blood.

r 2003 Elsevier B.V. All rights reserved.

PACS: 75.50.Tt; 81.20.Ym; 82.35.+t

Keywords: Superparamagnetic nanobeads; Coupling reagent; Carboxyl; Coating; DNA isolation

1. Introduction

Superparamagnetic nanobeads modified withfunctional groups can selectively adsorb biomater-ials of interest. The isolation of genomic DNA

onding author. Tel.: +86-10-62781691; fax: +86-

5.

address: chendp@chem.tsinghua.edu.cn (D. Chen).

and Xu Zhang contributed equally to this work.

$ - see front matter r 2003 Elsevier B.V. All rights reserve

/j.jmmm.2003.09.054

from human whole blood can be greatly simplifiedby manipulating magnetic bead-biomaterial con-jugates with the appropriate magnetic fields [1–3].Furthermore, with their superparamagnetic prop-erty, nanobeads are widely used as magneticcarriers for manipulating biomaterials in a mag-netic–electric biochip.The magnetic beads used for bio-separation

are usually coated with polymer layers [4–14].After coating and modification, the density and

d.

ARTICLE IN PRESS

X. Xie et al. / Journal of Magnetism and Magnetic Materials 277 (2004) 16–23 17

hydrophilic/hydrophobic properties of the beadsare changed to facilitate their binding with thepredetermined biological molecules [15–18]. Manykinds of natural (e.g., proteins and polysacchar-ides), synthetic (e.g., polyelectrolytes), and non-ionic polymers (e.g., polyvinyl alcohol) have beenused for particle coating. Only two coatingstrategies are typically employed. One method isto coat the magnetite cores directly without using acoupling agent [11,12,14]. However, in this caseself-assembly of polymer particles makes it diffi-cult to control the size distribution of the coatedparticles. The other means is to use couplingagents for polymerization coating. Silanizingreagent has been widely used as coupling agent,but it is not very suitable for Fe3O4 crystals due toits weak interaction with Fe3O4 crystals [19].Finding an effective coupling agent applicablefor coating Fe3O4 crystals is very important.In this study, a unique coupling reagent, bis-(2-

hydroxyethyl methacrylate] phosphate, was foundto make the coating process simpler and moreeffective. With the help of the coupling reagent,the nanocrystals were coated with polymer andmodified with carboxyl groups using the one-stepsuspension polymerization process. The functio-nalized magnetic nanobeads were used to developa rapid protocol for extracting and purifyinggenomic DNA from human whole blood.

2. Materials and methods

2.1. Chemicals and reagents

All chemicals and solvents used were ofanalytical grade and used without further purifica-tion. 2-hydroxyethyl methacrylate, trimethyrol-propanetriacrylate, and methacrylic acid wereobtained from Acros (Acros organics, Geel,Belgium). The high pure water used was preparedby Milli-Q system (Millipore, Bedford, MA). Allsolutions were freshly prepared.

2.2. Experimental instruments

Images of the magnetic nanocrystals wereobtained by a transmission electron microscope

(TEM) H-800 (Hitachi Ltd., Tokyo, Japan)operating at 150 kV. The size distribution ofthe magnetic nanoparticles was obtained bydynamic light scattering (DLS) analysis using alight scattering spectrometer ALV/DLS/SLS-5022F (ALV-Laser Vertriebsgesellschaft m.b.H.,Langen, Germany) at an angle of y ¼ 90�:The initial sample solutions were dilutedusing distilled water, filtered through a 0.22 mmpore size millipore filter and placed in a thermo-stated scattering cell at 25�C. Elementalanalysis of the beads was carried out ona sequential X-ray fluorescence spectrometerXRF-1700 (Shimadzu Corp., Kyoto, Japan)operating at 4 kV and 80mA. IR spectrawere recorded by a FT-IR spectrometer SpectrumGX (Perkin-Elmer, Norwalk, CT). Magnetizationhysteresis loops were obtained on a VibrationSample Magnetometer LDJ 9600 (LDJ ElectronicsInc., Troy, MI). UV absorption spectra wereobtained on an UV-2100S spectrometer fromShimadzu. Agarose gel electrophoresis was per-formed on a Runone Electrophoresis System(Embi Tec., San Diego, CA).

2.3. Preparation of nanocrystal magnetite (Fe3O4)

According to the previous reports [20–24],FeCl3 � 6H2O (4.73 g) and FeCl2 � 4H2O (1.99 g)were dissolved in 55ml of degassed deionizedwater under a stream of nitrogen. The solutionwas adjusted to pH 1.8 to avoid precipitation.The Fe2+/Fe3+ ratio of the mixture was 1/1.75.A 100ml solution of 4M NaOH was addedto a three-necked 250ml flask equipped witha stirrer and a thermostat. The temperaturewas maintained at 63�C. The mixture of Fe2+/Fe3+ was rapidly injected into the flask undervigorous stirring. A large quantity of gray–blackcolloidal precipitate formed immediately.The reaction continued for 30min. The tem-perature was then increased to 82�C, and thereaction continued for another 90min. After thereaction, the nanocrystals were separated througha suitable magnetic field and washed with deio-nized water until the pH of the supernatant wasneutral.

ARTICLE IN PRESS

X. Xie et al. / Journal of Magnetism and Magnetic Materials 277 (2004) 16–2318

2.4. Surface coating and carboxyl functionalization

of superparamagnetic Fe3O4 nanocrystals

Superparamagnetic nanocrystals (3.27 g) wereadded to a three-necked flask containing 500ml oftoluene and 0.81 g sodium lauryl benzene sulfo-nate. The three-necked flask was equipped with astirrer, condenser, and thermostat. The nanocrys-tals were dispersed into toluene with ultrasoundfor 0.5 h and then with vigorous agitation(300 rpm). To the flask was added a mixture of0.15 g initiator benzoylperoxide, 1.2ml monomer2-hydroxyethyl methacrylate, 0.8ml cross-linkingtrimethyrolpropanetriacrylate, 0.4ml couplingagent bis-(2-hydroxyethyl methacrylate) phos-phate and 0.6ml functionalization agentmethacrylic acid. The mixture was stirred vigor-ously for 30min under a nitrogen stream. Thestirring velocity was then lowered to 50 rpm andthe reaction temperature kept at 78�C for 12 hunder a nitrogen stream. The coated superpar-amagnetic nanobeads were then separated with amagnetic field and washed successively withtoluene, acetone, ethanol and deionized water toremove the residual surfactant and unreactedreagents. Finally the coated superparamagneticnanobeads were dispersed into Tris-EDTA buffer(pH 6.5) and stored at 4�C.

2.5. Extraction of genomic DNA from the human

whole blood

The human whole blood was anticoagulatedwith acid citrate dextrose (ACD) (23mM citricacid; 80mM dextrose; 45mM sodium citrate) witha volume ratio of 5:1. The procedure for theisolation of leucocytes and extraction of genomicDNA is described as follows. Anticoagulatedblood (300 ml) was added to a 1.5ml Eppendorftube containing 50 ml of superparamagnetic nano-beads suspension (15 mg/mL, pH 6.5, Tris-EDTAbuffer). The mixture was agitated gently byvortexing for 30 s and incubated at room tempera-ture for 3min. The beads were then immobilizedon a PromegaTM magnetic stand and the super-natant was discarded. Afterwards, 200 ml cell lysissolution (3M NaI; 5M Urea; 40 g/l Triton X-100;10mM EDTA, 25mM Tris-HCl, pH 6.5) was

mixed with the separated nanobeads complexes byvortexing and the suspension then incubated atroom temperature for 2min to lyse the cells.Isopropyl alcohol (200 ml) was added to thesuspension, and the mixture was vortexed gently.After further incubation for 2min, the supernatantwas discarded. The magnetic nanobeads-DNAconjugates were then immobilized on a magneticstand and washed twice with 100 ml solution of11.9M ethanol. After room temperature evapora-tion of ethanol, 50 ml of Tris-EDTA (10mMEDTA, 25mM Tris-HCl, pH 8.0) solution wasadded to the conjugates and incubated at roomtemperature for 5–10min to elute the DNA. Thesuperparamagnetic nanobeads were then immobi-lized on a PromegaTM magnetic stand. The eluantwas collected and analyzed directly by agarose gelelectrophoresis and UV spectroscopy.

3. Results and discussion

3.1. Preparation of Fe3O4 superparamagnetic

nanocrystals

There are generally two main steps involved inthe preparation of nanocrystals—the nucleationand nucleus growth of nanocrystals. It is wellknown that [24,25] the nucleus size is controlled bythe solubility of the precipitate and supersatura-tion of the reaction system in the nucleationprocedure, and the size distribution of nanocrys-tals is determined by the nucleation condition andthe growth rate of nuclei. The Fe3O4 magneticnanocrystals were prepared by injecting Fe3+/Fe2+ into concentrated NaOH solution. To obtainfine nanocrystals, a high concentration of NaOH(4M) was used to increase the supersaturation ofthe system. In fact, the ionic product of Fe2+orFe3+and OH� was much larger than the solubilityproduct of Fe(OH)2 or Fe(OH)3. The supersatura-tion condition was thus guaranteed. Further,almost the same environment for Fe3+/Fe2+ inthe whole nucleation process was required toensure that the reaction continued smoothly andthe nucleus size was controlled. The amount ofNaOH solution added was much more thanneeded, and the mixture of Fe3+/Fe2+ was

ARTICLE IN PRESS

X. Xie et al. / Journal of Magnetism and Magnetic Materials 277 (2004) 16–23 19

injected into superfluous NaOH, instead of theopposite adding sequence. The reason for selectingthe abrupt injection method for introducing Fe3+/Fe2+ was to provide sufficient contact betweenFe3+/Fe2+ and OH�. The nucleation condition ofall particles was thus kept the same during thewhole process. Because Fe2+ is prone to beingoxidized in solution [21], the atomic ratio of Fe2+/Fe3+ was selected to be 1:1.75 rather than thestoichiometric ratio of 1:2 to get the highestmagnetic intensity. The Fe3O4 nanocrystals werefinally obtained by heating the solution at 82�C todehydrate them from hydroxide. Their TEMmicrograph is shown in Fig. 1(a).

3.2. Surface coating and functionalization of

superparamagnetic Fe3O4 nanocrystals

The surface coating of the magnetic Fe3O4

nanocrystals was carried out by suspension poly-merization in toluene solution. The shell layerthickness of coated nanobeads was controlled bychanging the quantity of the coating monomers. Inan ideal coating process, monomers should com-pletely polymerize on the surface of the magneticnanocrystals and never form polymer particles.

Fig. 1. TEM images of (a) Fe3O4 magnetic nanobeads; (b) coated Fe3O

of the naked beads, in which data were statistically calculated from

distribution of coated magnetic beads, in which data were statistically c

for round I from Fig. 1(b); (f) magnified picture for round II from F

However, because the surface polarity of themagnetic Fe3O4 core is very strong and that ofthe polymer shell is rather weak, it is thereforedifficult for just the polymerization reaction tooccur on the surface of the magnetic nanocrystalswithout the formation of polymer particles. Tosolve this problem, a coupling agent, bis-(2-hydroxyethyl methacrylate) phosphate, was foundto be very effective to facilitate the polymerizationreaction. It was added to the monomer solution.One terminal of the coupling agent was aphosphoryl group with a polarity capable ofbinding strongly to ferric oxide, and the otherterminal was a double bond that facilitatedcopolymerization with the monomers. With thehelp of this new coupling agent, the monomerseventually polymerized in situ to form an evenlayer of polymer network on the surface of thenanocrystals to obtain the original nanobeads.Upon the completion of the coating, the resultingnanobeads were separated by a magnetic field. Noseparate polymer particles were observed, indicat-ing that the polymerization reaction happenedonly on the surface of the Fe3O4 nanocrystals. Inthe absence of the coupling agent, the monomersdid not polymerize on the surface of the Fe3O4

4 magnetic nanobeads; (c) the histogram of the size distribution

the particle size in Fig. 1(a); (d) and the histogram of the size

alculated from the particle size in Fig. 1(b); (e) magnified picture

ig. 1(b).

ARTICLE IN PRESS

X. Xie et al. / Journal of Magnetism and Magnetic Materials 277 (2004) 16–2320

nanocrystals, but form separate polymer particles.If an appropriate reagent with both double bondand functional carboxyl group can be introducedinto the system simultaneously with the coatingmonomers, the coating process and functionaliza-tion process could be accomplished in one step.Furthermore, nearly every single bead was coatedthrough this method. The coating layer is visible inthe magnified TEM images (Fig. 1(b), (e) and (f)).

3.3. Size determination by TEM and DLS analysis

TEM and DLS were applied to determine themorphological parameter of the beads. The size ofthe uncoated nanocrystals was 9.771.8 nm andthat of the coated nanobeads was 18.573.2 nm, asmeasured from TEM micrographs. DLS analysisyielded an apparent uncoated particle size of 28–69 nm and coated particle size of 62–128 nm (asshown in Fig. 2). Despite the aggregation, both thesize distributions of the uncoated particles and thecoated particles was relatively narrow with anaverage of 44 and 90 nm, respectively. Comparedwith the results of the TEM micrographs (asshown in Fig. 1), a noticeable discrepancy appears,with the particle size from DLS analysis obviouslylarger than those of the TEM graphs. Accordingto Perchec et al. and Mendenhall et al. [14,26], theparticle size measured by the DLS methodis determined primarily by the magnetite radius,the hydrated polymer at the magnetite surface,and partly by the particle complex, which consistsof the magnetic particle, the associated water,

0 20 40 60 80 100 120 1400.0

0.2

0.4

0.6

0.8

1.0naked beadscoated beads

Inte

nsi

ty

Hydrodynamic diameter (nm)

Fig. 2. DLS analysis of magnetic nanobeads.

solvated ions, and counterions on which somepolymer is attached. The results of the DLSanalysis show that both the naked beads and thecoated ones aggregate to some extent. However, itis proposed by Levison et al. [6] that theaggregation of the magnetic nanobeads will notaffect their efficiency in purifying DNA from thehuman whole blood. The efficiency of the beads toextract DNA is mainly determined by surfacecharacteristics and magnetic properties.The results of TEM and DLS analysis also

confirm the high efficiency of the coupling agent,with which free polymer particles are rarelyobserved after the coating process [11,12]. More-over, compared with the wide size distribution(50–500 nm) of the nanobeads prepared throughthe direct coating method without the use of thecoupling agent [12], the size distribution of thecoated beads in this study is narrower, and theuniformity is remarkably improved.

3.4. Analysis of magnetic property

The specific magnetism ss; the remanence sr;and the coercivity Hc are generally used tocharacterize the magnetic properties of magneticmaterials. The remanence sr is the remainingmagnetization of nanobeads when the appliedmagnetic field is switched off from saturation. Thecoercivity Hc is the reversed field required toreduce the magnetization of nanoparticles to zerofrom saturation.Magnetization hysteresis loops of both super-

paramagnetic Fe3O4 nanocrystals and coatedsuperparamagnetic Fe3O4 nanobeads are shownin Fig. 3(a) and (b) respectively. The specificmagnetism ss is 64.4 emu/g, remanence sr 3.9 emu/g, and the coercivity Hc 30.5Oe for the uncoatedFe3O4 magnetic nanocrystals, while ss is 48.9 emu/g, sr 0.5 emu/g, and Hc 4.5Oe for the coated Fe3O4

magnetic nanobeads. The ss of the preparedmagnetic nanocrystals is high, and the sr and Hc

are low. It can be seen from Fig. 3 that ss; sr andHc decrease after the particles are coated. Thedecrease of the magnetization is due to the coatingpolymer on the surface of the magnetite. In ourexperiment the coated magnetic nanobeads wereimmobilized on the wall of a test tube for 10 s by

ARTICLE IN PRESS

-6000 -4000 -2000 2000 4000 6000

-80

-60

-40

-20

0

20

40

60

80

ba

b

H (O e)

σ(em

u/g)

a

Fig. 3. Magnetization hysteresis loops of (a) Fe3O4 magnetic

nanocrystals and (b) coated Fe3O4 magnetic nanoparticles.

Fig. 4. FT-IR spectra of (a) Fe3O4 magnetic nanocrystals and

(b) coated Fe3O4 magnetic nanoparticles.

Fig. 5. Element content of magnetic nanobeads (3.2 g) coated

with different quantities of organic monomers: (1) 2ml; (2) 3ml;

(3) 6ml; (4) 10ml.

X. Xie et al. / Journal of Magnetism and Magnetic Materials 277 (2004) 16–23 21

applying a magnetic field through a PromegaTM

magnetic stand. In other words, after coating, thess value of the nanobeads is still strong enough toaccomplish the bio-separation.

3.5. FT-IR spectra of the superparamagnetic

nanobeads

The IR spectra of uncoated nanocrystals andnanobeads coated with polyacrylate and func-tionalized with carboxyl groups are shown inFig. 4(a) and (b) respectively. From Fig. 4 it can beseen that a series of absorption bands that do notshow up in Fig. 4(a) occur in Fig. 4(b). The peaksof 1728 and 1155 cm�1 are characteristic absorp-tion bands of carbonyl groups from acrylic esters.The band at 3390 cm�1 might be attributed to thehydroxyl group from 2-hydroxyethyl methacrylateor acylic acid.

3.6. Sequential X-ray fluorescence analysis of

coated magnetic nanobeads

The particle size distribution and contents ofthe elements in the coated magnetic nanobeadsare controlled by the quantity of organic mono-mer. The amount of the magnetic nanocrystalswas kept at 3.2 g, and the quantity of thecoating monomer was chosen to be 2, 3, 6and 10ml, respectively, in four parallel coating

experiments. The composition of the monomersolution was kept the same in all experiments,as described above in the section on the prepa-ration of magnetic nanocrystals. The contentsof the elements Fe, O, C and P are analyzedon a sequential X-ray fluorescence spectrometer.The results are shown in Fig. 5. It was foundthat the organic groups are chemically boundto the magnetic nanocrystals. The carbon content

ARTICLE IN PRESS

Fig. 6. Agarose gel electrophoresis of genomic DNA. Lanes1,2:

5 ml DNA by using phenol/chloroform method; 3,4: 5 ml DNAby using our method; Lane M: 1 ml l phage DNA/Hind III

digest marker.

X. Xie et al. / Journal of Magnetism and Magnetic Materials 277 (2004) 16–2322

increased and the ferrum content decreased withincreasing coating monomer for the coated mag-netic nanobeads.All the results obtained above from TEM

imaging, dynamic light scattering, magnetic prop-erties, IR examination and element analysis by X-ray fluorescence spectrometry indicate that thecoating and functionalization of the magneticnanocrystals were successful.

3.7. Isolation of genomic DNA using functionalized

magnetic nanobeads

The functionalized superparamagnetic nano-beads can be applied to different fields in bio-separations through specific molecular binding.For example, they can be used to capture antigenswhen the corresponding antibodies are connectedto the surface of the nanobeads. They can also beused to immobilize biotinylated biomolecules oncethe nanobeads are coated with streptavidin (datanot shown).However, the extraordinary characteristic of the

nanobeads is its unique surface properties thathave effective application in bio-separations suchas DNA preparation through nonspecific molecu-lar binding. Nanobead usage simplifies the wholeprocedure for DNA preparation and makespracticable the construction of miniaturized elec-tric–magnetic biochips.Genomic DNA isolated from 300 ml of the

human whole blood utilizing different amount ofmagnetic nanobeads were examined by agarose gelelectrophoresis and the results are shown in Fig. 6.When the phenol/chloroform method [27] is usedto extract genomic DNA from 300 ml anticoagu-lated human whole blood, the extraction required150min for a yield of approximately 5.46 mg andOD260/OD280 ratio of 1.87. With the use of 50 mlmagnetic nanobeads suspension, it took 20min toyield approximately 2.87 mg DNA and OD260/OD280 ratio of 1.85. Compared with the phenol/chloroform method, the nanobeads method couldextract genomic DNA with comparable purity buta lower yield. The most important advantage forthe nanobeads method is that it is simple and fast.This makes the automated isolation of DNA fromwhole blood with high throughput possible with

only a slight decrease in the yield of DNA. Theabove method has been successfully used inour Lab to purify DNA from complex bio-logical matrices, such as the whole blood, saliva,and bacterial culture. This method has shownexcellent reproducibility through hundreds ofexperiments.

4. Conclusion

In this article magnetic nanocrystals wereprepared with narrow size distribution by injec-tion-precipitation method. These nanocrystalswere coated with a polymer shell and thenfunctionalized with carboxyl groups to obtain theexpected superparamagnetic nanobeads. Themonomers participated only in the surface poly-merization process on the nanocrystals withoutforming separate nuclei after the coupling agentbis-(2-hydroxyethyl methacrylate) phosphate wasintroduced. The coated nanobeads are suitable forfast extraction of genomic DNA without involvingthe conventional centrifugation procedure and theuse of hazardous agents. It is a promising methodfor high-throughput automated extraction ofgenomic DNA from whole blood samples at adecreased yield.

ARTICLE IN PRESS

X. Xie et al. / Journal of Magnetism and Magnetic Materials 277 (2004) 16–23 23

Acknowledgements

This work was supported by the project of theNational Key Basic Research Development Pro-gram (G19990116) and the National ScienceFoundation of China (39989001, 39825108).

References

[1] C.H. Setchell, J. Chem. Technol. Biotechnol. 35B (1985)

175.

[2] M.J. Davies, J.I. Taylor, N. Sachsinger, I.J. Bruce, Anal.

Biochem. 262 (1998) 92.

[3] P.R. Levison, S.E. Badger, P. Hathi, M.J. Davies, I.J.

Bruce, V. Grimm, J. Chromatogr. A 827 (1998) 337.

[4] K. Furusawa, K. Nagashima, C. Anzai, Colloid Polym.

Sci. 272 (1994) 1104.

[5] A. Kondo, H. Fukuda, Colloid Surf. A—Physicochem.

Eng. Asp. 153 (1999) 435.

[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] J.M. Rodriquez-Paris, K.V. Nolta, T.L. Steck, J. Biol.

Chem. 268 (1993) 9110.

[8] L.A. Perrin-Cocon, S. Chesne, I. Pignot-Paintrand, P.N.

Marche, C.L. Villiers, J. Magn. Magn. Mater. 225 (2001)

161.

[9] B. Yoza, M. Matsumoto, T. Matsunaga, J. Biotechnol. 94

(2002) 217.

[10] A. Wooding, M. Kilner, D.B. Lambrick, IEEE Trans.

Magn. 24 (1988) 1650.

[11] N.A.D. Burke, H.D.H. Stover, F.P. Dawson, J.D. Lavers,

P.K. Jain, H. Oka, IEEE Trans. Magn. 37 (2001) 2660.

[12] J. Chatterjee, Y. Haik, C.-J. Chen, J. Magn. Magn. Mater.

246 (2002) 382.

[13] J. Lee, T. Isobe, M. Senna, Colloid Surf. A—Physicochem.

Eng. Asp. 109 (1996) 121.

[14] I. Dumazet-Bonnamour, P.L. Perchec, Colloid Surf. A—

Physicochem. Eng. Asp. 173 (2000) 61.

[15] D. Tanyolac, A.R. Ozdural, React. Funct. Polym. 45

(2000) 235.

[16] M.D. Butterworth, L. Illum, S.S. Davis, Colloid Surf. A—

Physicochem. Eng. Asp. 179 (2001) 93.

[17] T.L. Hawkins, T. O’Connor-Morin, A. Roy, C. Santillan,

Nucleic Acids Res. 22 (1994) 4543.

[18] J.I. Taylor, C.D. Hurst, M.J. Davies, N. Sachsinger, I.J.

Bruce, J. Chromatogr. A 890 (2000) 159.

[19] L.-D. Zhang, J.-M. Mou, Nano-Materials and Nano-

Structures, Science China Press, Beijing, 2001, p. 140.

[20] N. Feltin, M.P. Pileni, Langmuir 13 (1997) 3927.

[21] S.E. Khalafalla, G.W. Reimers, IEEE Trans. Magn. 16

(1980) 178.

[22] T. Sato, T. Iijima, M. Seki, N. Inagaki, J. Magn. Magn.

Mater. 65 (1987) 252.

[23] E. Tronc, P. Belleville, J.P. Jolivet, J. Livage, Langmuir 8

(1992) 313.

[24] K.J. Davies, S. Wells, S.W. Charles, J. Magn. Magn.

Mater. 122 (1993) 24.

[25] T. Sugimoto, Adv. Colloid Interface Sci. 28 (1987) 65.

[26] G.D. Mendenhall, Y. Geng, J. Hwang, J. Colloid Interface

Sci. 184 (1996) 519.

[27] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY, 1989.