Ferritine

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ELECTROSPRAYING OF FERRITIN SOLUTIONS FORTHE PRODUCTION OF MONODISPERSE IRONOXIDE NANOPARTICLES

To cite this Article: , 'ELECTROSPRAYING OF FERRITIN SOLUTIONS FOR THEPRODUCTION OF MONODISPERSE IRON OXIDE NANOPARTICLES', ChemicalEngineering Communications, 194:7, 901 - 912To link to this article: DOI: 10.1080/00986440701215531URL: http://dx.doi.org/10.1080/00986440701215531

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Electrospraying of Ferritin Solutions for theProduction of Monodisperse Iron Oxide

Nanoparticles

DAVID GONZALEZ,1 ALBERT G. NASIBULIN,1

HUA JIANG,2 PAULA QUEIPO,1 AND ESKO I.KAUPPINEN1

1Nanomaterials Group, Laboratory of Physics and Center for NewMaterials, Helsinki University of Technology, Espoo, Finland2VTT Nanobiomaterials, Espoo, Finland

Iron oxide nanoparticles with uniform size and narrow size distribution were synthe-sized by electrospraying of ferritin and subsequent heat treatment at 800�, 850�, and900�C. Solutions of ferritin in both water and water=iso-propanol mixture (50:50)were electrosprayed in diverse gaseous environments. Narrow mobility size distribu-tions with a mean mobility diameter of 4.5 nm and a geometric standard deviation<1.2 were obtained at 800�C. The process of aerosol formation involved the thermaloxidation of the ferritin organic shell. The utilization of a water=iso-propanol(50:50) ferritin solution led to the establishment of a more stable electrospray,and, consequently, an increase in the total particle concentration was observed.Furthermore, carbon-coated magnetite (Fe3O4) particles were generated whenCO was used as carrier gas.

Keywords Electrospray; Ferritin; Iron oxide nanoparticles

Introduction

In recent years, nanoparticles have been the subject of intensive research in severaldisciplines since they have unique physical properties (electrical, optical, magnetic,chemical and mechanical) compared to the bulk materials (Pui and Chen, 1997;Kruis et al., 1998; Gupta and Gupta, 2005). Iron oxide nanoparticles have beenstudied as ideal elements for utilization in catalysis, magnetic fluids, biomedicalapplications, etc. (Gupta and Gupta, 2005). For instance, the synthesis of magnetite(Fe3O4) nanoparticles can be of great importance due to their interaction with exter-nal magnetic fields, which can facilitate magnetic resonance imaging for medicaldiagnosis (Bonnemain, 1998). Furthermore, iron oxide nanoparticles present a highchemical accessibility that allows straightforward surface modification. Becausemany properties of these nanoparticles are size dependent, the synthesis ofuniform particles at the nanometric scale is crucial. Wet chemical routes have beenexamined as simple and efficient techniques for the synthesis of monodisperse nano-particles (Smith et al., 2005; Tartaj et al., 2005). Nevertheless, they usually are

Address correspondence to David Gonzalez, Nanomaterials Group, Laboratory ofPhysics and Center for New Materials, Helsinki University of Technology, P.O. Box 1000,FIN-02044 VTT, Espoo, Finland. E-mail: [email protected]

Chem. Eng. Comm., 194:901–912, 2007Copyright # Taylor & Francis Group, LLCISSN: 0098-6445 print/1563-5201 onlineDOI: 10.1080/00986440701215531

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time-consuming methods involving several steps, which restricts their use inapplications demanding continual particle production. For that reason, it is appar-ent that there is a need to exploit other methods for the generation of ultrafineparticles. Aerosol processes are an alternative for the continuous synthesis of parti-cles with desired dimensions and narrow size distribution (e.g., Kodas andHampden-Smith, 1999; Okuyama and Lenggoro, 2003).

The electrospray technique (Zeleny, 1914) is considered as the only spray tech-nology capable of atomizing a liquid into ultrafine droplets. This method is based onthe natural formation of sharp liquid cones when a meniscus supported at one end ofa capillary tube is charged to several kilovolts with respect to an electrode. A thinand steady jet is then ejected from the apex of the cone and breaks up further down-stream to disperse into a fine spray of highly charged droplets. This regime of sprayoperation is usually referred to as cone-jet mode (Cloupeau and Prunet-Foch, 1989;Cloupeau, 1994). A detailed review of the theory and applications of the electrospraycan be found in J. Aerosol Sci. 25 (Special Issue) (1994). Electrospray has been exam-ined for the production of nanoparticles with narrow size distributions (Lenggoroet al., 2000; Nakaso et al., 2003). Lenggoro et al. (2002) have also reported on sizingof colloidal nanoparticles by means of electrospray combined with aerosol techni-ques. Nasibulin et al. (2002) used electrospray and a high-resolution differentialmobility analyzer to select individual polyethylene glycol molecules with a mass dif-ference of 44 amu. More recently, Suh et al. (2005) have shown the production ofhighly charged monodisperse aerosol nanoparticles from electrosprayed gold nano-particle suspensions. Further investigations for the production of monodispersenanoparticles in the range below 10 nm are still desirable.

Ferritins are a class of iron storage proteins found throughout the animal, plant,and microbial kingdoms (Yang et al., 1998). Mammalian ferritins are composed of24 polypeptide chains that form a nearly spherical hollow shell and encapsulate acrystalline core of hydrous ferric oxide (Fe2O3.9H2O). The protein shell of ferritinhas an outer diameter of 12–13 nm and is capable of storing up to �4500 iron atomsinside a 5–8 nm central cavity (Massover, 1993). Apart from the biological applica-tions of ferritin as a natural source of iron (Harrison and Arosio, 1996), someauthors have already taken advantage of the sharp size distribution and propertiesof this protein for their utilization in technological processes. For instance, carbonnanotubes were synthesized using the discrete particle cores of ferritin as catalyst(Li et al., 2001; Bonard et al., 2002; Kim et al., 2002).

Here, we study the applicability of the electrospray technique for the productionof aerosol monodisperse iron oxide nanoparticles using ferritin as the precursor. Forthis purpose, ferritin solutions were electrosprayed in diverse gaseous environments.After droplet formation and solvent evaporation, particles were carried into a high-temperature reactor where the protein organic shell decomposed, thus producing aerosoliron oxide nanoparticles. The production of monodisperse nanoparticles was assayed bymeans of differential mobility analyzer (DMA) measurements and transmission electronmicroscopy (TEM) analysis. Additionally, the synthesis of carbon-coated iron oxidenanoparticles was also investigated by using CO as carrier gas.

Experimental Section

The experimental setup used in this study is shown in Figure 1. It comprises anelectrospray (ES) source, a tubular flow reactor, and particle size and concentration

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measurement equipment. The liquid was electrosprayed inside a stainless-steel elec-trospray chamber provided with glass windows in order to observe the shape of theliquid meniscus through a 30� microscope. The solution was placed in a 1 cm3

polypropylene vial and pushed through a silica needle (40 mm i.d., 358 mm o.d.;Polymicro Technologies) using compressed nitrogen. The tip of the needle wastapered conically down to nearly zero thickness for better control of the sprayshape. A positive high voltage of 3–4 kV was applied to the capillary by using aDC voltage power supply together with a proportional HV DC converter (EMCOHigh Voltage Corp.) for the establishment of the necessary electric field. Separationbetween the capillary and the counter electrode was 3 cm. The formed spray parti-cles were carried in a gaseous surrounding, and the solvent was evaporated whileexiting the ES chamber. Different gaseous environments of CO2 (2.5 L=min), amixture of CO2=O2 (2.5 L=min=0.09 L=min), and CO (0.5 L=min) were used.

Electrosprayed molecules were passed through a reactor consisting of a cer-amic tube with a 22 mm inner diameter, heated with a dual zone furnace with atotal heated length of 40 cm. All the experiments were done at atmospheric pres-sure, and the reactor temperature was varied between 800� and 900�C. Residencetimes in the heated zone were estimated to be between 1 and 5 s. The number sizedistribution was measured by a differential mobility analyzer system consisting of aclassifier (modified Hauke, analyzer length 11 cm, sheath air and excess flow23 L=min) and a condensation particle counter (CPC, TSI 3027). An electrostaticprecipitator was used to collect particles on a carbon-coated copper grid, andthe morphology and the primary particle size of generated particles were investi-gated by means of a field emission transmission electron microscope (PhilipsCM200 FEG).

A solution of ferritin from horse spleen in 150 mM of NaCl was purchased fromFluka Chemie GmbH. Protein concentration in solution was 69 mg=mL. Statisticalmeasurements of the particle size distribution of the cores of the ferritin as receivedwere estimated by measuring the diameter from TEM micrographs. For this pur-pose, a drop of the ferritin solution was placed onto a carbon-coated copper grid,which had been previously wetted in methanol for better dispersion of the moleculeson the grid film.

Figure 1. Schematic of the experimental setup.

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For electrospray investigations, two different solvents were used. Ferritin wasdissolved in both deionized water and a mixture of deionized water and isopropanol(50:50). The final concentration of the solutions was adjusted to be 6.5�10�5 mol=L.

Results and Discussion

Characterization of the Ferritin as Received

Number size distribution of the ferritin as received is illustrated in Figure 2. Itshowed a narrow distribution with a geometric number mean diameter of 6.9 nmand a standard deviation of 1.08. TEM imaging of the protein and statisticalmeasurements clearly confirmed the high level of monodispersity of the cores.

Electrospraying of Ferritin Solutions at Ambient Temperature

As presented in Figure 3(a), electrospraying of ferritin solutions at ambient tempera-ture resulted in a bimodal mobility size distribution regardless the solvent used. Thisbehavior can be understood on the basis of the known mechanism of droplet forma-tion and evaporation in electrosprays. The electrospray source generates highlycharged droplets. Solvent evaporation leads to drop shrinkage and an increase inthe electrical field normal to the surface of the droplets. At some given radius belowthe Rayleigh limit (Kebarle and Peschke, 2000), the electrostatic repulsion of thesurface charges overcome the surface tension force of the liquid, and, in consequence,droplets disintegrate due to the occurrence of a series of coulombic explosions. Theresult of this process is a generally bidisperse spray of droplets, some relatively large

Figure 2. Number size distribution and TEM image of the ferritin as received.


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and some relatively small (Kebarle and Peschke, 2000; Ku et al., 2004). In our inves-tigations, this idea is additionally supported by the absence of neutralizing sourceimmediately after droplet formation, which facilitates the formation of the highlycharged droplets followed by droplet disintegration. Therefore, the peak observed inFigure 3(a) at diameters above 10 nm corresponds to particles formed after the com-plete solvent evaporation of the large drops containing several molecules of ferritin.However, solvent evaporation of the small droplet leads to the formation of particlessmaller in size. Hence, when the droplet diameter is reduced to dimensions similar tothe ferritin molecule size, isolated ferritin molecules can be obtained after evaporation,and, consequently, a signal at diameters below 10 nm is observed. Additionally, the for-mation of multiply charged ferritin droplets after electrospraying may also contributeto the presence of this second mode in the size distribution.

Figure 3. (a) Number size distribution of particles generated after electrospraying the waterand water=isopropanol ferritin solutions in CO2 at ambient temperature; TEM images ofthe electrosprayed ferritin solution in water=isopropanol showing (b) discrete inorganic coresof ferritin and (c) aggregates consisting of several ferritin molecules.


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TEM observation corroborated the results deduced from the electrical mobilityspectra. Thus, for instance, TEM images of the ferritin solution in water=isopropanol showed both discrete inorganic cores of ferritin (Figure 3(b)), withappearance similar to those observed from the protein as received (Figure 2), andaggregates consisting of several ferritin molecules (Figure 3(c)).

Electrospraying of Ferritin Solutions and Posterior Treatmentat High Temperature

Experiments of Ferritin Solution in Water Using CO2 as Carrier GasMobility size distributions of electrospraying the ferritin solution in water followedby heat treatment at 800�, 850�, and 900�C are shown in Figure 4(a). When the tem-perature of the reactor was 800�C, a remarkably narrow mobility peak at about4.5 nm with a geometric standard deviation of 1.19 was observed. A progressiveincrease of the temperature up to 900�C led to an increase of the mean size of theparticles, the broadness of the size distribution and the total particle concentration.A low-intensity mobility signal at diameters above 10 nm was also found, whichseems to decrease in intensity as the temperature of the reactor increases from800� to 900�C.

As was previously mentioned, the droplets generated by electrospray undergosolvent evaporation while being transported through the reactor by the carriergas. Once the ferritin molecules are introduced in the reactor, either as individualsor forming aggregates, the organic shell surrounding the core of the protein maydecompose due to the oxidizing atmosphere created at high temperature, allowingthe release of the monodisperse iron oxide cores in the gas phase. Since the particlecores can carry electrical charges, the coagulation and growth of particles are mostlysuppressed due to mutual electrostatic repulsions. Consequently, the final particlespreserve their original high degree of monodispersity.

On the basis of this model, size distribution obtained after experiments at 800�Ccan be simply attributed to the presence of discrete ferritin cores released when theprotein shell is removed. Moreover, as was predicted, a narrow size distribution witha geometric standard deviation of 1.19 was obtained, indicating the production ofquite monodisperse particles. A TEM image of these particles generated at 800�Cis shown in Figure 4(a). Additionally, crystallographic studies, using electron diffrac-tion and high-resolution TEM images, were carried out on a synthesized particle(Figure 4(b)). This particle was identified as c-Fe2O3.

Furthermore, the appearance of a low-intensity peak at higher mobility dia-meters indicates that the organic shell decomposition process could be unfinishedat 800�C, which means that a reasonable quantity of particle cores could still remainencapsulated inside their corresponding organic shells after treatment at this tem-perature. With regard to the tendency of the size distributions obtained at tempera-tures above 800�C, a conclusive interpretation of either the shifting in the particlediameter or the increase in the particle concentration cannot as yet be proposed.

As can be seen, there is an evident difference between the mean diameterestimated by DMA of the particles obtained at 800�C, 4.5 nm, and that estimatedby TEM of the protein as received, 6.9 nm (Figure 2). This can be explained interms of the discrepancy between size distributions measured by these two differentmethods (Nasibulin et al., 2001). Rudyak et al. (2002) have already studied thisphenomenon with particles smaller than 10 nm.


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Experiments of Ferritin Solution in Water Using CO2=O2 as Carrier GasIn order to enhance the decomposition of the ferritin organic shell at high temperature,experiments were also carried out using a mixture of CO2 (2.5 L=min) and O2

(0.09 L=min) as carrier gases. Mobility size distributions of particles generated after elec-trospraying and subsequent heat treatment are shown in Figure 5. At 800�C, a narrowdistribution was observed with a mean mobility diameter of 4.5 nm and a geometricstandard deviation of 1.18. A similar size distribution was previously obtained whenthe gas was just CO2 (Figure 4(a)) and, as in that case, is attributed to the presence ofsingle isolated particle cores of ferritin, as was observed by TEM (Figure 5).

Figure 4. (a) Number size distributions of particles generated after electrospraying the water-ferritin solution in CO2 and subsequent heat treatment at 800�, 850�, and 900�C; TEM imageshowing single isolated ferritin cores obtained at 800�C. (b) High-resolution TEM image of anisolated ferritin core, including lattice vectors, synthesized at 800�C.


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Furthermore, as expected, the utilization of a better oxidant atmosphere facili-tated a higher effective decomposition of the protein shell, increasing the numberof individual particle cores in the gas phase. Thus, the total particle concentrationsignificantly increased from 103 #=cm3 to 104–105 #=cm3 in the whole range of tem-peratures studied. A gradual heating of the reactor led to larger particles as well asbroader size distributions than those obtained when no oxygen was added to thereactor. TEM pictures of particles generated at 900�C plainly revealed the formationof partially sintered agglomerates of several ferritin cores (Figure 5).

Experiments of Ferritin Solution in Water=Isopropanol Using CO2 as Carrier GasFigure 6 illustrates the mobility size distributions of particles generated using water=isopropanol as solvent. In general, these showed an analogous tendency to thatobserved from the experiments carried out with the water solution (Figure 4(a)).Thus, a narrow mobility peak corresponding to the synthesis of monodisperse par-ticles with a mean mobility diameter of 4.9 (rg ¼ 1.19) was obtained at 800�C,whereas larger particles were generated as the temperature increased. However,the use of water=isopropanol as solvent led to a noticeably higher total particleconcentration at all temperatures. This difference in the concentration can beexplained by the establishment of a more stable cone-jet mode at the tip of theneedle, presumably due to the lower surface tension of the ferritin solution.

Experiments of Ferritin Solution in Water Using CO as Carrier GasIn this work, it was also considered important to study the feasibility of electrospray-ing ferritin solutions for the production of carbon-coated iron oxide nanoparticles.

Figure 5. Number size distributions of particles generated after electrospraying the water-ferritin solution in CO2=O2 and subsequent heat treatment at 800�, 850�, and 900�C and TEMimages of particles formed at 800� and 900�C.


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These coated particles can be of great interest for many applications since they main-tain the favorable properties of the nanoparticles but are protected from undergoingother undesired reactions (Carpenter et al., 2003). For this purpose, electrosprayedparticles were transported to the reactor using CO as carrier gas with a flowrate of 0.5 L=min. It is well known that CO undergoes a simple disproportionationreaction at high temperatures (Nasibulin et al., 2006; Moisala et al., 2003):

COðgÞ þ COðgÞ () CðsÞ þ CO2ðgÞ; DH ¼ �171 kJ=mol: ð1Þ

As can be observed from the size distributions of Figure 7, mean mobilitydiameters measured by DMA were typically between 28 and 36 nm with wide distri-butions for all the temperatures studied. For instance, geometric standard deviationsof 1.47, 1.39, and 1.60 were obtained at 800�, 850�, and 900�C, respectively. Accord-ing to these results, it is clear that considerably larger particles than those producedin CO2, are generated when CO is used as the carrier gas. It is also worth mentioningthat a collateral effect derived from the use of CO was particle charge losses at 900�Cas deduced from the unobservable size distribution at that temperature.

An explanation for this significant particle growth is found not only in the longerresidence times of the particles in the reactor, which in turn favors particle coagu-lation, but also in the disproportionation of CO at high temperature. This reactionleads to the liberation of carbon, which subsequently deposits onto the surface ofthe particles. A TEM image of a carbon-coated particle generated after electrospray-ing at 800�C is also shown in Figure 7. Furthermore, a detailed crystallographic studyfrom high-resolution TEM images showed that particles produced were in the form ofmagnetite (Fe3þ [Fe2þFe3þ ]O4) (Cornell and Schwertmann, 1996). This implies thatthe iron oxide cores of the ferritin (Feþ3) were partially reduced.

Figure 6. Number size distributions of particles generated after electrospraying the ferritin sol-ution in water=isopropanol (50:50) in CO2, and subsequent treatment at 800�, 850� and 900�C.


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Conclusions

Monodisperse iron oxide nanoparticles have been generated from ferritin solutionsusing the electrospraying technique and high-temperature treatment. Particles wereproduced via thermal decomposition of the ferritin organic shell and subsequentrelease of the protein cores. Mobility size distributions and TEM observationsshowed that single isolated cores were obtained at 800�C regardless the type of sol-vent employed. However, using a mixture of water=isopropanol led to a considerablyhigher particle concentration that was likely due to the occurrence of a more stablecone-jet mode at the tip of the needle. A gradual increase of the temperature up to900�C resulted in an increase in particle mobility diameter and broader mobilitydistributions. This effect was particularly remarkable when oxygen was added tothe reactor, causing the generation of particles with a mean mobility diameter of17.1 nm and a geometric standard deviation of 1.46.

Finally, carbon-coated magnetite particles with mobility diameters in the rangebetween �29 and �36 nm were obtained when the carrier gas was CO. This is aconsequence of the deposition of carbon, produced by the disproportionation reac-tion of CO at high temperature, onto the surface of the particles.

Acknowledgments

Mr. Raoul Jarvinen is gratefully acknowledged for assistance in building theexperimental system. The authors also thank Dr. Unto Tapper and Dr. David

Figure 7. Number size distributions of particles generated after electrospraying the ferritin sol-ution in water in CO, and subsequent treatment at 800�, 850�, and 900�C; high-resolutiontransmission electron micrograph of Fe3O4 (magnetite) particle generated at 800�C.


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P. Brown for interesting discussions and proofreading the manuscript, respectively.This work was supported by the Academy of Finland and the European CommunityResearch Training Network ‘‘Nanocluster’’ (grant No. HPRN-CT-2002-00328).

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