Journal of Magnetism and Magnetic Mate

ticaly

. Sc

rger W

rsity

ife Sc

ne 25

In the past, silica sols were used to manufacturelms, membranes, coatings, bulky materials [68]

ARTICLE IN PRESS

Corresponding author. Tel.:+49 241 87 36 27;

or spherically shaped particles [9,10]. The latterproducts are mainly based on the well-known

0304-8853/$ - see front matter r 2005 Published by Elsevier B.V.

doi:10.1016/j.jmmm.2005.01.088

fax:+49 241 87 45 99.

E-mail address: dms@magnamedics.de (D. Muller-Schulte).1. Introduction

Silica solgel technology provides a versatileplatform in material sciences for the manufactur-ing of innovative products such as biomimetricmaterials, biosubstance encapsulated carriers, lu-minescent devices, photochromic coatings, opticalsensors, photonic crystals, separation media andoptical codings systems [14]. Based on the well-known solgel process using either acid- or base-

catalyzed hydrolysis of alkoxysilanes [3,5], hydro-gels or xerogels can be easily obtained by a simplebase addition. By manipulating different para-meters such as composition of the alkoxysilanes,hydrolysis conditions and condensation proce-dures which were concisely reviewed in the past[1,3], products with excellent optical, chemical,and biocompatible characteristics can be obtainedand exploited for the synthesis of a variety ofinnovative products.Abstract

A method for the synthesis of both spherically shaped micro/nano silica particles and silica hybrid particles using a

novel inverse solgel suspension technique was developed. The technique enables the synthesis of beads within seconds

and provides a simple basis for quantum dot and biosubstances encapsulation. The carriers can be used as DNA

adsorbents, individually addressable optical codes for bioassays and biomolecule library screening as well as photonic

crystals.

r 2005 Published by Elsevier B.V.

Keywords: Silica; Synthesis; DNA separation; Encapsulation; Quantum dots; Photonic crystals; Optical codes; Inverse solgel

suspension technique; Silica hybridsUltra-fast synthesis of magnefor versatile bioan

Detlef Muller-Schultea,, TaMagnaMedics GmbH, Martelenbe

bClinic for Radiology, UnivecHogeschool Zuyd Centre of Expertise in L

Available onlirials 293 (2005) 135143

and luminescent silica beadstical applications

hmitz-Rodeb, Paul Bormc

eg 8, D-52066 Aachen, Germany

Hospital, Aachen, Germany

iences, NL-6400 Heerlen, The Netherlands

March 2005

www.elsevier.com/locate/jmmm

necessitate time-consuming and cumbersome mul-

by an ultrasonic treatment. The sol phase issubsequently added to a toluene liquid phasecontaining 3.5 vol% Prisorine 3700 and 0.8 vol%Span 85. Volume ratio organic phase to silica sol7.5:1. After 5 s of stirring using either a conven-tional stirrer or an Ultra-Turrax mixer LR 1000(IKA Werke, Germany), 15 vol% of a 1%ammonia solution is added. After 5 to 10 moreseconds of mixing, this leads to stable solid silicabeads, followed then by several washing steps withmethanol and water. The quantum dots with sizesranging from 3 to 10 nm and the magnetic colloidswere synthesized according to the methods de-scribed elsewhere [2123]. The iron oxide contentcan be adjusted up to maximal 15wt% related to

ARTICLE IN PRESS

ism and Magnetic Materials 293 (2005) 135143tistep preparations.Apart from the Stober process, there are very

few suspension techniques described to producespherical microsized silica beads as encapsulationmatrix [1719]. These techniques are very time-consuming requiring up to 24 h preparation.The present study describes a novel inverse

suspension method which not only enables anultra-fast silica bead synthesis but also offers abroad platform for the encapsulation of magneticcolloids/ferrouids, biosubstances, luminescentagents such as quantum dots and uorescencedyes. The usage of these novel carriers as DNAseparation media, photonic crystals and multi-plexed optical code systems for bioanalysis isdescribed.

2. Methods and materials

All chemicals used were of analytical gradesupplied by Fluka, Germany, and without furtherpurication. The preparation procedure is outlinedelsewhere [20]. A typical silica sol is formed by anaqueous suspension consisting of 71 vol% alkox-ysilane (e.g. tetraethoxysilane, tetramethoxysilane)and 29 vol% 0.05M HCl. Hydrolysis leading to anhomogeneous sol is assisted by a conventionalultrasonic treatment for 10 to 15min. After solformation, a water-based magnetic colloid/ ferro-uid and/or the substances to be encapsulated areadded (concentration ranges: 520 vol%) and thenStober process [11] resulting exclusively in nano-particles. However, particles based on this methodshow unfavourable optical properties; they areopaque, and the encapsulation of biosubstances orthe usage in DNA separation, which is one of themost prominent applications of silica carriers, hasnot been described so far. Encapsulation ofmagnetic colloids to render the particles magneticresults in particles with an uneven colloid coating[12]. If these particles are applied as uorescenceor dye-labelled tags e.g. for biomolecule detection,chemical coupling procedures using functionalizeduorescence dyes [13,14] or core-shell coatingtechniques [15,16] are required. All techniques

D. Muller-Schulte et al. / Journal of Magnet136mixed for a few seconds with the sol phase assistedthe SiO2 content in the nal polymer matrix.The ow chart for the preparation of the silica

spheres is shown in Fig. 1.Apart from synthesizing the basic silica bead,

the technology described above also enables asimple possibility to synthesize functionalizedcarriers to which bioligands such as DNAfragments, peptides or proteins can be covalentlycoupled. For this purpose 5 to 20mol% functio-nalized silanes carrying either amino-, epoxy and/or mercapto groups such as 3-aminopropyl-triethoxysilane, 3-glycidyloxypropyltrimethoxysi-lane, 3-mercaptopropyltrimethoxysilane areadded to the original alkoxysilane mixture. Thesubsequent suspension procedure which leads tothe nal beads follows the same procedure as for

Fig. 1. Schematic ow diagram of the synthesis steps for silica-sphere preparation.

formation after sol preparation does not necessi-

ARTICLE IN PRESS

ism atate more than 120 s including ve washing steps.The viscosity of the silica sol preferably in therange of 530mPa s and the continuous phase(0.72mPa s) which contains carefully selected15wt% suspension stabilizers are the crucialparameters for obtaining ideally shaped particles.As the viscosity of the organic and the sol phase islow, the size range of the silica beads can be easilycontrolled by the stirring speed. Stirring speedsbetween 500 and 1500 rpm in general lead tomicrobeads covering a size range from 20 to200 mm, whereas stirring speeds between 2000 and10,000 rpm lead to sizes in the range of 1020 mm.Particle sizes in the range of 510 mm, 25 mm,13 mm and nanosized particles (500 to 1000 nm)can be prepared using a high-speed suspension toolthe basic silica carriers. Standard coupling proto-cols for the attachment of bioligands using e.g.carbodiimide, glutaraldehyde, BrCN can be ap-plied [24].Ion exchange particles carrying diethylami-

noethyl groups for DNA binding were preparedusing a standard procedure [20]: 100mg vacuumdried silica beads were activated for 3 h underreux in 4ml distilled toluene (dried over sodiummetal) to which 0.5ml 3-glycidyloxypropyltri-methoxysilane was added. The ethanol- andwater-washed carrier was subsequently incubatedwith 3ml diethylamine overnight at room tem-perature, followed by several washing steps usingethanol and water.

3. Results and discussion

A novel inverse suspension technique thatenables an ultra-fast synthesis was developed toobtain spherically shaped silica micro and nano-particles platform for biosubstances and pigment/colloid encapsulation. The ow diagram outlinedin Fig. 1 schematically shows the diverse prepara-tion steps required to obtain the silica particles.The whole procedure starting with the hydrolysisof the alkoxysilanes to the nal magnetic silicabeads including diverse encapsulation steps, takesno more than 10 to 15min. The actual silica bead

D. Muller-Schulte et al. / Journal of Magnet(e.g. Ultra-Turrax mixer, IKA-Werke, Germany)adjusting the stirring speed to 11,000, 13,000,16,000 and 22,000 rpm, respectively.When compared to established procedures such

as the Stober process [11] or other previouslydescribed W/O suspension methods [12,1418], thenewly developed inverse suspension techniqueoffers the following advantages and features: theparticles are spherical and totally transparent, thesizes are adaptable in the range from 0.5 to1000 mm, synthesis can be performed withinminutes and encapsulation of both magnetic andluminescent substances is easily performed dispen-sing with any multistep coating or core-shelltechnique.A selection of the substances used for encapsu-

lation into the silica bead matrices are listed inTable 1. All products listed can be alternativelyencapsulated in combination with up 10wt% ironcontent. As can be seen from Table 1, the quantityof the magnetic colloid can be varied in a broadconcentration range. This variation in the mag-netic colloid content enables a precise control ofthe magnetic attraction properties so that anoptimal adaptation to the appropriate beadapplication is easily possible. This particularlyapplies to an automated DNA separation wherehigh separation speeds are required which can onlybe obtained using beads with a high magneticportion resulting in strong magnetic attractionforces. The reverse is the case when using themagnetic particles in immuno assays where longsuspension periods are required without mechan-ical mixing. For such purposes lower magneticcolloid contents are needed to adapt the specicdensity of the beads close to the density of theaqueous suspension phase. In Fig. 2. microscopicimages of the basic magnetic micro and nanopar-ticles and their respective magnetic behaviourusing a conventional hand magnet are shown.As nucleic acid separation plays an ever-grow-

ing role in biotechnology and bioanalysis, high-performance separation media and especiallymagnetic ones which can full the increasingdemand for automatization in DNA separationare asked for. It is not surprizing that thedevelopment of magnetic adsorbents exhibitinghigh DNA binding capacities combined with

nd Magnetic Materials 293 (2005) 135143 137distinct magnetic properties is of vital interest.

ARTICLE IN PRESS

ssful

ma)

maxim

e bea

ism aIn a rst test series, anion exchanger magneticsilica particles were used to study the basic DNAadsorption properties. For this purpose, diethyla-minoethyl groups were introduced into the silicaby reacting the 3-glycidyloxypropyltrimethoxysi-lane activated matrix with diethylamine (seeSection 2). The adsorption performance of the

Table 1

List of selected substances and bioproducts, respectively, succe

adjusted between 0.5600mm

Silica sample Encapsulated producta

A (Fig. 2) Magnetic colloid [22]

B (Fig. 4,5) CdSe/Zn quantum dots

C (Fig. 4c) Rhodamin B

D Yeast cells

E (Fig. 4d) Methylene blue

F Herring sperm DNA

G Green uorescent protein

H Fluorescein

I Gold colloid (size 1723 nm, Sig

K X-CyclodextrinL Silica nanoparticles [12]

M (Fig. 6) Polystyrene latex (200 nm)

N Polyvinyl alcohol (Mw: 48 kDa)

aAll listed products can be encapsulated simultaneously withbThe amount of substance which can be encapsulated into thcNot determined.

D. Muller-Schulte et al. / Journal of Magnet138modied silica carrier was examined and com-pared with a commercial anion silica resin (Qia-gen, Germany) (for details see legend of Fig. 3.).As can be concluded from test results usingstandard ethidium bromide staining, the modiedsilica carriers show a distinctly better adsorptionperformance of the magnetic silica beads than thecommercial carrier under identical test conditions.Among the encapsulated substances listed in

Table 1, luminescent particles such as quantumdots and uorescent dyes are certainly of parti-cular interest from the bioanalytical or diagnosticpoint of view. In recent years semiconductornanocrystallites, so-called quantum dots, mainlycomposed of elements belonging to II and VIgroup such as CdS, ZnS, CdSe, have gained muchinterest because of their exceptional luminescentproperties and chemical stability [25]. Research inthis eld has focused primarily on the synthesis,quantum connement effect and their size-tune-able optical features [26]. Interesting approacheshave recently been made to use quantum dots forbiosensing and biolabelling [27]. Apart from usingthe nanosized basic quantum dots for cell- andbiomolecule labelling, efforts have been made toencapsulate the nanocrystals into a polymercarriers such as polystyrene and mesoporous silicaparticles [28,29]. Both matrices, however, exhibit

ly encapsulated into the silica matrix. Bead size range can be

Max. concentrationb (related to SiO2 content)

45wt% (35 vol%)

20wt%

5wt%

b

8wt%

18wt%

c

10wt%

20 vol%

25wt%

35 vol%

35 vol%

20wt%

al 20wt% of the magnetic colloid.

ds at 495%.

nd Magnetic Materials 293 (2005) 135143unfavourable basic optical properties due to theautouorescence (polystyrene) and opaque proper-ties (silica beads), thereby substantially restrictingthe luminescent intensities of the carries [29]. Thesilica bead technology introduced here provides anexciting basis to circumvent both the opticalimpairments as well as the cumbersome prepara-tion procedures of established methods. By directencapsulation of the quantum dots or alternativelyuorescent dyes into the silica matrix, an almostunlimited amount of luminescent substances canbe applied thus circumventing all particle- andpore size-related restrictions during the dopingprocess of the polymers with the quantum dots. InFig. 4. some real colour uorescent images ofCdSe/ZnS quantum dots, rhodamine B andmethylene blue-doped silica particles are shown.As demonstrated, by varying the type of the

embedded uorescent substance or dye, the wholespectral range can be covered and by varying thequantity of the encapsulated substances the colour

ARTICLE IN PRESS

ism aD. Muller-Schulte et al. / Journal of Magnetbrightness adjusts accordingly. In addition to themonochromatic carriers, the silica technology alsooffers a simple means to synthesize multicolouredbeads by simultaneously encapsulating eitherquantum dots having different sizes or mixturesof different uorescent dyes. This combinatorialencapsulation approach using different lumines-cent agents with different concentration amountsresults in silica beads which can be favourablyused as addressable optical systems for theindividual encoding of biomolecules such asnucleic acids or proteins. Han et al. [28] have

Fig. 2. Light microscopy images of typical magnetic silica micro (a,b)

(b,d). Aligning occurs within 2 s after magnet application; magnet app

the applied magnetic eld. All images were taken with a confocal lase

using a transmission mode. Scale bar: mm.nd Magnetic Materials 293 (2005) 135143 139described a theoretical model for such multiplexedoptical coding using quantum dots-doped poly-styrene particles. On the basis of n colourintensities and m different colours nm1, indivi-dual codes can be generated whose large numbersare, however, limited in practise by spectraloverlapping.Apart from the widely tuneable parameters used

to control the optical properties of the luminescentsilica particles, the present technology offers anintriguing approach to simultaneously encapsulatea magnetic colloid and a uorescent substance.

and nanoparticles (c,d) in response to an external magnetic eld

lied: neodymiumboroniron. Arrows indicate the direction of

r scanning microscope Zeiss Axioplan 2, Carl Zeiss, Germany,

ARTICLE IN PRESS

ism aThis provides an additional parameter to extendthe application of such particles for multiplexedoptical coding of biomolecules [29].

Fig. 3. DNA adsorption capacities of cationic silica particles.

(a): Magnetic silica beads (mean diameter 26 mm) (b): DEAE-silica, Qiagen, (c): Ethidium bromide blind test with 200mlstarting DNA concentration. 30-ml settled carrier volume wereincubated for 10min with 200ml 0.01 phosphate buffer, pH 5.5,containing 300 ng phage l DNA (48502 base pairs, MBIFermentas, Germany). Elution was conducted with 200ml10mM TrisHCl/1M NaCl/ 1mM EDTA buffer, pH 8.0;

elution time 2min.Visualization using ethidium bromide

follows standard protocols.

D. Muller-Schulte et al. / Journal of Magnet140In Fig. 5 CdSe/ZnS quantum dots embeddedmagnetic particles are exemplarily depicted. Ap-plication of a neodymiumboroniron magnetleads to a pronounced magnetic response withina fraction of a second showing the typical aligningprocess (Fig. 5b). The pictures unambiguouslyshow that when compared with the non-magneticspheres (Fig. 4a) the luminescent properties arepractically not impaired by the encapsulatedmagnetic colloid. Similar applies to rhodamine B,uorescein and green uorescence protein (GFP)encapsulated beads (not shown) which exhibitsimilar magnetic and luminescent properties.Exploiting the combinatorial principles of lumi-

nescence and magnetism, the newly developed silicaparticles as optical signature provide versatileencoding possibilities which may open up broadperspectives for multiplexed assays, biochemistrysensing, protein library and/or pathogen (e.g. tumorcell) screening and drug delivery systems.A further outstanding feature of the silica

carriers in comparison to conventional syntheticpolymer beads is the ultimate heat stability. Heattreatments at 500 1C and above are feasible with-out destroying the bead structure. This specialproperties can now be exploited to manufacturepore size-selective silica beads. By encapsulatingbiomolecules/-substances such as nucleic acids,proteins, cells or viruses possessing a denedspacial structure and subsequent calcination ofthese embedded particles at temperatures4500 1C, silica replica with dened mesoporestructures are formed. Figs. 6a and b exemplarilyshow a polystyrene latex encapsulated silica carrierwhose pores were created by calcination of thesample at 800 1C (Fig. 6b). Using this combinedencapsulation and calcination technique, a varietyof differently structured porous silica matrices canbe designed which may serve as afnity separation,tetoxication medium or decontamination agentfor heavy metals.Apart from the bioanalytical application there is

a great interest in manufacturing novel photoniccrystals for light modulation, optical switches ormirrors. Xu et al. [30] recently described a novelapproach to synthesise magnetically controllablephotonic crystals using ferromagnetic polystyrenenanoparticles incorporated into a acrylamide gelmatrix. The silica bead technology described herewith its simple encapsulation of nanoparticles,magnetic colloid and/or quantum dots provides afavourable platform to produce such photoniccrystals. The quantum dots encapsulated magneticsilica beads depicted in Fig. 5 can serve as such abasic photonic crystal. Using a rotating neody-miumboroniron magnet exerting a magneticeld of E0.8 T, a maximal induced oscillation ofapprox. 120Hz can be achieved. This represents analmost 60% higher frequence than recentlyachieved with a polystyrene-doped crystallinecolloidal array [30]. When applying photoniccrystal sizes o10 mm, the respective response ratescan be even increased up to approx. 300Hz (notshown). Hence the silica technology with itsvariability regarding size, selection and concentra-tion of embeddable agents as well as opticalproperties, offers an excellent basis for the devel-opment of tuneable photonic crystals which couldbe used as magneto-optical device whose diffrac-tion and transmission can be externally controlled

nd Magnetic Materials 293 (2005) 135143by a magnetic eld.

ARTICLE IN PRESS

ism aD. Muller-Schulte et al. / Journal of Magnet4. Conclusion

Nano and microsized silica particles can bemanufactured on the basis of a novel inversesuspension technology. By controlling the viscosityof the sol phase in combination with low-viscosityorganic suspension phases, a broad variety ofmagnetic and non-magnetic beads can be synthe-sized within a short time frame requiring just a fewminutes. Due to the high suspension stability ofthe silica sol when mixed with a broad range ofdiverse biological and uorescent substances and

Fig. 4. Fluorescence microscope image of differently doped silica be

610 nm), (b): CdSe/ZnS quantum dot (size 3 nm, lmax 522 nm), (c):images were taken with a confocal laser scanning microscope Zeiss A

mode (d).nd Magnetic Materials 293 (2005) 135143 141pigments e.g. proteins, quantum dots, cells anduorescent dyes, stable silica beads in which thesesubstances can be encapsulated are able to beformed with the aid of an extremely simpleprocess.The special features of the technology and

particles are: (i) ultra-fast synthesis of sphericallyshaped particles, (ii) adjustable bead size ranges,(iii) total transparency (iv) an almost unlimitedencapsulation potential for bio and uorescentsubstances. Exploiting the combinatorial encapsu-lation potential the silica carries can offer

ads; (a): CdSe/ZnS quantum dot (size 7 nm, lex: 470 nm, lmaxrhodamine B (lmax 595 nm; (d): methylene blue. Fluorescencexioplan 2 operating in a luminescent (a,b,c) and transmission

ARTICLE IN PRESS

ism and Magnetic Materials 293 (2005) 135143D. Muller-Schulte et al. / Journal of Magnet142intriguing perspectives for the design of multi-plexed optical coding for biomolecule or pathogen,tuneable photonic devices, nano and micromotorsand/or drug releasing systems.

Fig. 5. Fluorescence microscopy image of CdSe/ZnS quantum

dot (size 6 nm) and magnetic colloid encapsulated silica beads

without (a) and with (b) external magnetic eld (neodymium

boroniron magnet). Magnetic colloid content 6wt%.

References

[1] J. Livage, T. Coradin, C. Roux, J. Phys.: Condens. Matter

13 (2001) R673.

[2] M.I. Samoilovich, A.F. Belyanin, E.P. Grebennikov, et al.,

Nanotechnology 13 (2002) 763.

[3] I. Gill, A. Ballesteros, TIBTECH 18 (2000) 282.

[4] C. Stemmer, M. Beau-Faller, E. Pencreach, et al., Clin.

Chem. 49 (2003) 1953.

Fig. 6. Microscopic image ( 400) of silica particles doped withpolystyrene latex (particle size 200 nm) (a) and after calcination

for 30min at 700 1C (b).

[5] D.L. Meixner, P.N. Dyer, J. SolGel Sci. 14 (1999) 223.

[6] J.R. Premkumar, O. Lev, R. Rosen, et al., Adv. Mater. 13

(2001) 1773.

[7] T. Jesionowski, J. Mat. Sci. 37 (2002) 5275.

[8] H. Schmidt, G. Jonschker, S. Goedicke, et al., J. SolGel

Sci. Technol. 19 (2000) 39.

[9] H. Nishimori, M. Tatsumisago, T. Minami, J. SolGel Sci.

Technol. 9 (1997) 25.

[10] S.M. Yang, N. Coombs, G.A. Ozin, Adv. Mater. 12 (2000)

1940.

[11] W. Stober, A. Fink, E. Bohn, J. Colloid Interface Sci. 26

(1968) 62.

[12] Q. Liu, Z. Xu, J.A. Finch, et al., Chem. Mater. 10 (1998)

3936.

[13] A. van Blaaderen, A. Vrij, Langmuir 8 (1992) 2921.

[14] M. Bele, O. Siiman, E. Matijevic, J. Colloid Interface Sci.

254 (2002) 274.

[15] C. Graf, W. Schartl, K. Fischer, et al., Langmuir 15 (1999)

6170.

[16] K.P. Velikov, A van Blaaderen, Langmuir 17 (2001) 4779.

[17] J.-H. Park, C. Oh, S. Shin, et al., J. Colloid Interface Sci.

266 (2003) 107.

[18] M. Aizawa, S. Kitajima, M. Ohsawa, et al., J. SolGel Sci.

Technol. 19 (2000) 329.

[19] T.A. Kavassalis, F. M. Winnik, US Patent 5,209,998.

[20] D. Muller-Schulte, WO 03/083481 A1.

[21] D. Muller-Schulte, F. Fussl, M. DeCuyper, in: U. Hafeli,

W. Schutt, J. Teller, M. Zborowski (Eds.), Scientic and

Clinical Applications of Magnetic Carriers, Plenum Press,

New York, 1997.

[22] M. Shinkai, H. Honda, T. Kobayashi, Biocatalysis 5

(1991) 61.

[23] B.O. Dabbousi, J. Rodriguez-Viejo, F.V. Mikulec, et al.,

J. Phys. Chem. B 101 (1997) 9463.

[24] G.T. Hermanson, in: Bioconjugate Techniques, Academic

Press, New York, 1996.

[25] A.P. Alivisatos, Science 271 (1996) 933.

[26] C.J. Murphy, Anal. Chem. 74 (2002) 520A.

[27] E.R. Goldman, A.R. Clapp, G.P. Anderson, et al., Anal.

Chem. 76 (2004) 684.

[28] M. Han, X. Gao, J.Z. Su, et al., Nature Biotechnol. 19

(2001) 631.

[29] X. Gao, S. Nie, J. Phys. Chem. 107 (2003) 11575.

[30] X. Xu, S.A. Majetich, S.A. Asher, J. Am. Chem. Soc. 124

(2002) 13864.

ARTICLE IN PRESS

D. Muller-Schulte et al. / Journal of Magnetism and Magnetic Materials 293 (2005) 135143 143

Ultra-fast synthesis of magnetic and luminescent silica beads for versatile bioanalytical applicationsIntroductionMethods and materialsResults and discussionConclusionReferences