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Applied Surface Science 298 (2014) 130–136

Contents lists available at ScienceDirect

Applied Surface Science

journa l h om epa ge: www.elsev ier .com/ locate /apsusc

agneto-fluorescent hybrid of dye and SPION with ordered andadially distributed porous structures

adhulekha Gogoi, Pritam Deb ∗

ezpur University (Central University), Napaam, 784028 Tezpur, Assam, India

r t i c l e i n f o

rticle history:eceived 15 November 2013eceived in revised form 22 January 2014ccepted 23 January 2014vailable online 31 January 2014

eywords:icroporous

a b s t r a c t

We have reported the development of a silica based magneto-fluorescent hybrid of a newly synthe-sized dye and superparamagnetic iron oxide nanoparticles with ordered and radially distributed porousstructure. The dye is synthesized by a novel yet simple synthetic approach based on Michael additionbetween dimer of glutaraldehyde and oleylamine molecule. The surfactant used for phase transformationof the dye from organic to aqueous phase, also acts as a structure directing agent for the porous structureevolution of the hybrid with radial distribution. The evolution of the radially distributed pores in thehybrids can be attributed to the formation of rod-like micelles containing nanoparticles, for concentra-

ybridagneto-fluorescent

tion of micelles greater than critical micelle concentration. A novel water extraction method is applied toremove the surfactants resulting in the characteristic porous structure of the hybrid. Adsorption isothermanalysis confirms the porous nature of the hybrids with pore diameter ∼2.4 nm. A distinct modificationin optical and magnetic property is observed due to interaction of the dye and SPION within the silicamatrix. The integration of multiple structural components in the so developed hybrid nanosystem resultsinto a potential agent for multifunctional biomedical application.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Hybrid nanosystems are multifunctional in nature as theyombine different functionalities in a single stable construct. Forxample, mesoporous silica based hybrid of magnetic and fluores-ent nanoparticles find diverse applications in magnetic resonancemaging [1], hyperthermia [2], magnetic separation [3], drug deliv-ry [4] and as fluorescent labels in biology for selective recognition,ensitive imaging and reporting [5]. The design of such hybrids isn intricate process that requires different steps [6]. Since organicyes usually have high quantum yield and enhanced biocompat-

bility than inorganic fluorescent probes, therefore integration ofagnetic nanoparticles with dye to develop the hybrid, may open

ew vistas in biotechnology for achieving high signal amplificationn ultrasensitive bioanalysis [7]. The high fluorescence intensityf such hybrids is because of presence of large number of dyeolecules within the matrix. Moreover, the extraordinary photo-

tability, hydrophilicity and biocompatibility rendered by the thickilica coating to the hybrid, made it as an excellent candidate foriomedical applications. But synthesizing the organic-dye-doped

∗ Corresponding author. Tel.: +91 3712 267007/8/9x5560; fax: +91 3712 267006.E-mail address: pdeb@tezu.ernet.in (P. Deb).

169-4332/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.apsusc.2014.01.140

silica particles is difficult due to the highly hydrophobic nature oforganic dyes as compared to the hydrophilic surface of silica. Now,once this primary challenge is overcome, the next will be to achievethe stable suspension of the system containing hydrophobic dye, inwater.

In this paper, a new synthetic approach has been adoptedto develop an organic fluorescent dye. This is followed bythe stabilization of the dye using the surfactant cetyl-trimethylammonium bromide (CTAB) and forming an oil-in-water emul-sion to develop a hybrid of dye and superparamagnetic ironoxide nanoparticles (SPION) in silica matrix. Further, we haveemployed a novel water extraction method to remove the watersoluble surfactant templates, resulting in porous structure and sol-vents after formation of the hybrid. Herein, we show that thehybrid formed by templated route, possess unique microstruc-ture consisting of radially distributed porous channels normalto the periphery. The porous microstructure has been revealedby high resolution transmission electron microscope (HR-TEM)and N2-sorption analysis. This hybrid is stable in water dis-persion under physiological condition without any possibility of

leakage of the hydrophobic dye, during application. Also, theinteraction of the dye and SPION with the silica matrix hasbeen investigated through detailed magnetic and optical propertystudy.

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M. Gogoi, P. Deb / Applied Su

. Experimental procedure

.1. Synthesis

Ferric nitrate, cetyl-trimethyl ammonium bromide (CTAB),etraethylorthosilicate (TEOS) and ammonia solution are procuredrom Merck. Dimercaptosuccinic acid (DMSA) and oleylamine arerocured from Aldrich. Stearic acid from Loba Chemie and glu-araldehyde from Himedia are used during preparation. The organicolvents used are of analytical reagent grade.

The dye is prepared by mixing 0.5 ml of ethanol, 10 �l of glu-araldehyde and 17 �l of oleylamine in a beaker. The mixture isllowed to stand for 12 h. After that, the blood red colored dye isormed which is then dispersed in 0.5 ml of chloroform. Now inrder to prepare CTAB template based silica hybrid system, first ofll, the hybrid components need to be CTAB stabilized. For that, aTAB solution in water is made with concentration of 1 mg/ml. BothTAB (3 ml) and dye solutions are mixed and stirred vigorously for

h. An emulsion is formed which is kept in oven for 15 min at 60 ◦Co evaporate chloroform. Hence an aqueous solution of the CTABtabilized fluorescent dye is obtained.

Stearic acid capped SPIONs are prepared by a procedureeported by our group previously [8]. Then CTAB stabilization isone with 1.6 mg of SPION in the same way as with the dye. For theormation of the silica hybrid of dye and SPION, sol–gel method ispplied [9]. The method comprises of mixing together the SPIONnd dye solutions followed by addition of 20 ml water. Then 2 mlthylacetate, 0.48 ml NH4OH and 0.14 ml TEOS are added to itequentially with stirring. Stirring is continued further for 30 s andhen the reaction mixture is allowed to stand for 12 h. The pink-sh precipitate is collected through centrifugation and repeated

ashing with water and ethanol.To remove the CTAB templates and other organic solvents from

he hybrid, calcination cannot be done in this case as it leads toecomposition of the organic dye. Therefore, we have applied aovel water extraction procedure consisting of adding 34 mg of theybrid in 15 ml of water mixed by sonication for 30 min and thenefluxing at 90 ◦C for 12 h along with stirring. Finally, centrifugations done to separate out the hybrid from water solution. By this wayhe water soluble CTAB and ethyl acetate molecules are removed.

.2. Characterization

The size, shape and morphology of the developed hybrids arexamined by SEM (JSM-6390 LV, Jeol), HR-TEM (Jeol JEM-2200FS

Scheme 1. Glutaraldehyde dimer (Structure I) formation at basic pH and it

cience 298 (2014) 130–136 131

operated at 200 kV) and atomic force microscope (AFM) (Nanon-ics Multiview 2000). The BET surface area and pore morphology ofthe hybrid are analyzed with the help of NOVA 1000e surface areaand pore size analyzer. The magnetic properties of the hybrids areanalyzed by PPMS Dynacool (Quantum Design USA) and the fluo-rescence micrographs are taken in an inverted microscope LEICADMI6000B. The compositional and surface characteristics are stud-ied by energy dispersive X-ray microanalysis (EDX 7582, OxfordInstrument, UK), GC–MS spectroscopy (PerkinElmer) and Fouriertransform infra-red spectroscopy (FTIR) (NICOLET-IMPACT) respec-tively. The streaming potential measurement of the samples wascarried out using the charge titration system stabilizer (ParticleMetrix GmbH, Germany).The optical properties of the dye andhybrid are analyzed by UV–vis absorption (Shimadzu, UV-2450)and photoluminescence (PerkinElmer, LS55) spectroscopic tech-niques.

3. Result and discussion

The first step, in characterizing the hybrid is to identify the newdye molecule and to understand its formation mechanism. In thisregard it is worthwhile to mention that glutaraldehyde is knownto be an efficient cross linker [10]. The structure of glutaraldehydeis dependent on the pH of the aqueous solution [11]. For exam-ple, it remains as free aldehyde, in hydrated form or as its cyclichemiacetal form in acidic or neutral pH conditions. But in alka-line aqueous solution, glutaraldehyde exists as polymer or as thehydrated form of the polymer. Also there is evidence of forma-tion of dimer of glutaraldehyde by aldol condensation between twomolecules with the elimination of one water molecule in alkalineaqueous medium [12]. From GC–MS data of the dye molecule, themajor mass fraction is obtained to be m/z = 170 (Supplementary Fig.1), which corresponds to the dimer of Scheme 1 glutaraldehyde(Structure I) formed by aldol condensation. This is possible in theless polar solvent, ethanol, in the presence of amine bases. Hence,the plausible mechanism for crosslinking of glutaraldehyde witholeylamine will be through Michael addition reaction. Such adducts(Structure II) are stable and no reduction is necessary to stabilize[11]. The reactions of dimer formation of glutaraldehyde and thenits addition with oleylamine are shown below. The Structure II isnamed as oleylamine complex or O-complex.

The microporous silica hybrid of the newly synthesized dyemolecule and SPION with ordered, radially distributed pore chan-nels are fabricated by using CTAB as the structure directing agent. Asshown in Scheme 2(a), the phase transformation of O-complex and

s Michael addition with oleylamine to yield O-complex (Structure II).

132 M. Gogoi, P. Deb / Applied Surface Science 298 (2014) 130–136

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cheme 2. Schematic representation of (a) the phase transformation of the O-complf porous structure.

PION from oil to water phase is carried out with the help of cationicurfactant CTAB. Initially, an oil-in-water emulsion is formed byTAB micelle formation [diagram II in Scheme 2(a)] and then oilhase i.e. chloroform is evaporated out to obtain CTAB stabilized O-omplex and SPION [diagram III in Scheme 2(a)]. During the silicaybrid formation, rod-like surfactant micelles containing nanopar-icles in the core are formed in the reaction mixture due to vigorousgitation for CCTAB > CMC [diagram I in Scheme 2(b)] [13]. Then, theegatively charged hydrolysis products of TEOS start attaching tohe positively charged head groups of CTAB micelles and form a

ayer over it [as shown in the enlarged diagram II in Scheme 2(b)].hese rod-like micelles coated with SiO2 layer are organized intolusters under the influence of van der Waals forces [13] to formilica particles of size ∼100 nm [diagram III in Scheme 2(b)]. Then

SPIONs from oil to water phase and (b) the hybrid formation with radial distribution

with CTAB removal by water extraction method, the micelles arewashed out to yield the silica particles with radial distribution ofpore channels. In the meanwhile, the O-complexes and SPIONsremained attached to the walls of the pores and in some cases thelong chain molecule O-complex can penetrate to the silica layer[diagram IV in Scheme 2(b)].

The so developed hybrids are observed to be spherical in shapefrom electron microscopy results. A distribution of particle sizeis observed in the range of 100–200 nm from the SEM image inFig. 1(a) and AFM image of the hybrid (in Supplementary Fig. 3).

In (b), the representative HR-TEM image of single silica particleis shown. The enlarged image of the portion of (b) indicated bygreen box is shown in Fig. 1(c), from which the radial distributionof the pore channels normal to the particle surface as proposed in

M. Gogoi, P. Deb / Applied Surface Science 298 (2014) 130–136 133

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ig. 1. (a) SEM image, (b) HR-TEM image of single hybrid particle with radial dinterpretation of the references to colour in this figure legend, the reader is referred

chematic 2(b), is clearly evident. In order to confirm the CTAB tem-late removal by water extraction procedure, EDX characterizationf the hybrid is performed. The EDX spectrum (in Supplementaryig. 4) shows no presence of Br, which can be corroborated to theuccessful removal of CTAB from the hybrid.

To realize porous morphology of the hybrid, pore size, BETurface area and pore volume are determined by using N2-orption technique at −196 ◦C. The BET surface area is estimatedith relative pressure in the range of 0.05–0.90. The pore sizeistribution is calculated from the adsorption branch by usingarett–Joyner–Halenda (BJH) method. Fig. 2(a) shows the N2-orption isotherm of the hybrid. The isotherm is observed to bef type II, indicating that the silica hybrid is mainly composed oficroporous structures [14]. The BET surface area of the system is

ound to be 105.86 m2/gm. The pore size distribution plot of theilica hybrid is shown in Fig. 2(b). From this plot, the average poreiameter is observed to be ∼2.4 nm and from the BJH data, aver-ge pore surface area and volume are obtained as 68.8 m2/gm and.14 cc/gm respectively. The BET analysis confirms the microporousharacteristic of the developed hybrid.

The optical and surface characteristics of the hybrid are stud-ed and the results are shown in Fig. 3. From the comparison plotf UV–vis spectra of the O-complex, SPION and hybrid in Fig. 3(a),t can be inferred that the characteristic peaks of glutaraldehyden O-complex are present at 249 nm and 302 nm respectively, cor-esponding to n → �* and n → �* transitions for aldehydic group15,16]. But in the hybrid, these peaks have got suppressed due to

he thick silica coating whereas the other peaks are almost unal-ered. Also, the broad absorption hump of SPION covering the wholeisible range is observed in Fig. 3(a). This is due to the allowed tran-ition of 5T2g → 5Eg in divalent iron [17]. From the comparative plot,

tion of pore channels and (c) the enlarged image of the small portion of (b).(Fore web version of this article.)

it is understood that due to the intense absorption by SPION in thevisible range the overall absorption of the hybrid increases than thepristine dye. On the other hand, in the PL spectra of Fig. 3(b), thehybrid emission peak seems to be quenched than the pristine dyewhereas only SPION shows no significant emission. This is becausethe transitions arising from excited triplet states in divalent ironare spin forbidden resulting in weaker bands [17]. In addition tothis, SPION absorbs the visible emission from dye molecules in thehybrid leading to the observed quenching in emission from thehybrid. The emission peak of the dye, centered around 459 nm, isbroad in nature (Fig. 3(b)). This is the characteristic of dye moleculebecause of its complexity, which ultimately leads to line splittingproducing broad absorption and emission curves. The slight redshift in the emission peak in the hybrid is occurring may be due tononradiative transition of electrons from excited dye molecules tonew states on hybrid formation. Fig. 3(c) shows the fluorescenceimage of the hybrid excited with UV light. From this, the bright flu-orescent property of the hybrid is evidenced corroborating to thePL spectrum, which is desirable for making the hybrid a potentialimaging probe. From the FTIR spectra of Fig. 3(d), the silica peaks areobserved for Si O Sistretch at 1087 cm−1, Si OHstretch at 957 cm−1,Si O Sibend at 801 cm−1, longitudinal optical mode of SiO2 at1236 cm−1 and O Si Obend at 457 cm−1 respectively [18,19]. How-ever, the broad peak at 3424 cm−1 is assigned to N Hstretch becausethe O Hstretch at similar range would have been more intense thanthe silica peaks. The 1652 cm−1 peak is assigned to the N Hbendvibration of amine group in the O-complex [20]. The symmetric and

antisymmetric C H and C Hscissoring bands of long alkyl chain of theO-complex are observed at 2854, 2931 and 1470 cm−1 respectively[8]. Presence of Si OH groups over the hybrid surface is furthercorroborated from the streaming potential measurement plot of

134 M. Gogoi, P. Deb / Applied Surface Science 298 (2014) 130–136

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Fig. 2. (a) N2-sorption isotherm and (

ig. 3(e). Due to the ionization of silanol groups, the overall sur-ace charge is observed to be negative in the whole pH range. Ateutral pH, the streaming potential of the hybrid is measured toe −81 mV. This observation also points towards the deep embed-

ent of the constituent components within silica network and to

he stability of the dispersion of the hybrid, containing hydrophobicye molecules and SPION, in water medium.

ig. 3. (a) UV–vis absorption spectra, (b) photoluminescence spectra, (c) fluorescence mnd hybrid and (e) streaming potential plot of the hybrid.

e size distribution plot of the hybrid.

In order to investigate the role of SPION in the developed hybrid,its magnetic properties are studied through M–H and M–T charac-terization techniques. From the M–H characterization results, it isobserved that the superparamagnetic character of SPION is retained

after hybrid formation with silica and O-complex, though thereis a decrease in the saturation magnetization after hybrid forma-tion (Fig. 4(a) and (b)). This is due to the diamagnetic contribution

icrograph of hybrid excited with UV-light (Filter A), (d) FTIR spectra of O-complex

M. Gogoi, P. Deb / Applied Surface Science 298 (2014) 130–136 135

. M–T

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Fig. 4. M–H curves of (a) SPION and (b) hybrid at 300 K

rom the thick silica coating as well as from the organic dye, O-omplex. Also, it can be correlated with the surface spin disorder ofPION induced by silica coating [21]. From the M–T characterizationurves of SPION and its hybrid, the superparamagnetic property isurther confirmed with splitting of FC and ZFC curves at a temper-ture above blocking temperature, TB. It is also observed that TB ofPION has decreased from 218 K to 145 K on hybrid formation. Thisecrease is attributed to the reduction of the average effective vol-me, Veff of the �-Fe2O3 nanoparticles due to interfacial interactionetween SiO2 and outer layer of the iron oxide [22] which leads toisordered Fe spins near the surface having diamagnetic contribu-ion, as stated earlier. From the relation of blocking temperature,B = KVeff/25kB, the decrease in TB can also be corroborated [23].

Based on the above observations, the formation of the sta-le aqueous phase magneto-fluorescent hybrid with radiallyistributed porous structure is realized. The CTAB stabilized O-omplex and SPION are coloaded within the silica network. Usef cationic surfactant CTAB leads to the generation of microporousorphology of the hybrid.

. Conclusion

In conclusion, hybrid of a new fluorescent dye and SPION inilica matrix is synthesized. Prior to that the dye developed by aovel approach has been stabilized with CTAB for incorporation

n the hybrid which later acts as a structure directing agent. Afterormation of the hybrid, a novel water extraction method has beenmployed for removal of CTAB templates resulting in a stable hybridith radial distribution of the pore channels. Such stable hybrid sys-

ems rule out any possibility of leakage of the organic dye from thickilica network. The distribution of silica particles is observed to be inhe range ∼ 100–200 nm with average pore diameter, surface areand volume as 2.4 nm, 68.8 m2/gm and 0.14 cc/gm respectively. At

eutral pH, the streaming potential of the hybrid is measured to be81 mV. The optical property of the dye is found to be almost unal-

ered after hybrid formation. Also, the fluorescence image of theybrid corroborates to the bright fluorescent property of the hybrid.

curves of (c) SPION and (d) hybrid measured at 500 Oe.

Further, magnetic characterization results exhibit the superpara-magnetic characteristic of the hybrid. The saturation magnetizationas well as TB has decreased on hybrid formation, which is caused bythe silica coating leading to disordered Fe spin near the surface ofSPION. This magneto-fluorescent hybrid with porous morphologyforms stable water dispersion and is having the potential to becomean efficient multifunctional agent in biomedical application.

Acknowledgements

The authors acknowledge Department of Atomic Energy (DAE),Govt. of India for providing financial support through the researchgrant No. 20 07/20/34/04-BR NS/1865. MG acknowledges Dept. ofScience & Technology (DST), Govt. of India for INSPIRE Fellowship.The authors acknowledge SAIF-NEHU for extending HR-TEM facil-ity and Prof. R. C. Deka, Department of Chemical Science, TU forextending BET characterization facility.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.apsusc.2014.01.140.

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