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Tuning Ferritin’s band gap through mixed metal oxide nanoparticle formation

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2017 Nanotechnology 28 195604

(http://iopscience.iop.org/0957-4484/28/19/195604)

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Tuning Ferritin’s band gap through mixedmetal oxide nanoparticle formation

Cameron R Olsen1, Jacob S Embley1, Kameron R Hansen1,Andrew M Henrichsen1, J Ryan Peterson1, John S Colton1 andRichard K Watt2

1Department of Physics and Astronomy, Brigham Young University, Provo UT 84602, United States ofAmerica2Department of Chemistry and Biochemistry, Brigham Young University, Provo UT 84602, United Statesof America

E-mail: rwatt@chem.byu.edu

Received 18 November 2016, revised 15 March 2017Accepted for publication 22 March 2017Published 19 April 2017

AbstractThis study uses the formation of a mixed metal oxide inside ferritin to tune the band gap energyof the ferritin mineral. The mixed metal oxide is composed of both Co and Mn, and is formed byreacting aqueous Co2+ with MnO4

- in the presence of apoferritin. Altering the ratio between thetwo reactants allowed for controlled tuning of the band gap energies. All minerals formed wereindirect band gap materials, with indirect band gap energies ranging from 0.52 to 1.30 eV. Thedirect transitions were also measured, with energy values ranging from 2.71 to 3.11 eV. Tuningthe band gap energies of these samples changes the wavelengths absorbed by each mineral,increasing ferritin’s potential in solar-energy harvesting. Additionally, the success of usingMnO4

- in ferritin mineral formation opens the possibility for new mixed metal oxide cores insideferritin.

Keywords: nanoparticles, synthesis, ferritin, band gaps, metal oxides

(Some figures may appear in colour only in the online journal)

1. Introduction

Ferritin is a hollow spherical protein found in many organ-isms. Its natural role is to control the iron content of cells andblood serum by accepting free iron and oxidizing it to form aferrihydrite mineral (Fe(O)OH) in its hollow interior [1].Recent studies have explored the potential of the ferritinmineral core to act as a charge separation catalyst for solarenergy harvesting, demonstrating its characteristic as asemiconductor with measurable band gap energies [2]. Thebenefits of using ferritin come from its ability to form dif-ferent minerals inside its interior with differing absorptionproperties [2–5]. Additionally, the protein shell can withstanda pH range of 3–10 and temperatures up to 70 °C [6, 7]. Thisshell also adds the benefit of providing solubility and stabilityfor normally insoluble minerals [8].

The native ferrihydrite mineral is easily removed fromthe ferritin protein, and the resulting shell is termed apo-ferritin. As stated previously, apoferritin has the capacity to

form new mineral cores and has long been used as a scaffoldfor controlled nanoparticle synthesis. Many different nano-particles have been formed inside of ferritin, including cobalt[7], manganese [9], and titanium oxides [10]. The solar har-vesting ability of each of these minerals has been exploredthrough measuring their optical absorption properties [11].

Many ferritin nanoparticles are formed by introducingapoferritin to a low-oxidation state transition metal. Apo-ferritin catalyzes the oxidation of the transition metal, withoxygen typically acting as the electron acceptor. The oxidizedmetal migrates towards a nucleation site on the inside offerritin, from which ferritin forms its metal oxide core [1]. Insome cases, for example cobalt ferritin synthesis, oxygen isthermodynamically unable to oxidize the metal, and hydrogenperoxide is used instead [7, 12].

The nanoparticles synthesized within ferritin act assemiconductors and have measurable band gap energies [2–5]. These band gap energies are dependent upon the type andsize of mineral formed inside of ferritin, affecting the

Nanotechnology

Nanotechnology 28 (2017) 195604 (5pp) https://doi.org/10.1088/1361-6528/aa68b0

0957-4484/17/195604+05$33.00 © 2017 IOP Publishing Ltd Printed in the UK1

wavelength of light that can be absorbed [5, 13]. Byexchanging the mineral inside of ferritin, one is able to con-trol the band gap and select the wavelength of light used forsolar energy harvesting.

A new method for mineral formation in ferritin hasrecently been discovered by replacing the oxidant (oxygen orperoxide) with permanganate [14]. Permanganate allows forthe formation of new nanoparticles inside ferritin with alteredband gap energies. This discovery led us to investigate usingpermanganate to form a mixed metal oxide nanoparticle inferritin, which would contain both manganese and a secondmetal of choice. This kind of formation of mixed metal oxidesinside ferritin is a new field of study. As such, there are only afew cases where these minerals have been recorded in ferritin.In these instances, the cores formed contain both Fe and Mnand were formed by introducing both metals in a low oxi-dation state to the apoferritin molecule [5, 15].

In this study, we use permanganate to oxidize Co2+ inthe presence of apoferritin to form a mixed metal core insideof ferritin. By using different ratios, we are able to tune theband gap of the resultant mineral and increase the potentialfor ferritin to act as a solar light-harvesting material. We showthat permanganate is sufficient to oxidize cobalt, leading todiffering mixed metal cores with a range of measurable bandgap energies.

2. Experimental methods

We prepared apoferritin for nanoparticle synthesis by firstremoving the native ferritin core. This was done through thestandard methods of dialyzing the native ferritin againstthioglycolic acid [1, 16].

The synthesis of the ferritin cores was performed byadding 3 mg apoferritin to 1 ml of a buffer solution containing1M pH 8.5 N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid. This pH value was chosenaccording to the values tested by Douglas et al in order togive an accurate comparison to previously synthesizedcobalt–ferritin [7]. The apoferritin-buffer solution was placedon a magnetic stir plate and stirred at a medium speed.Synthesis of the mixed cobalt/manganese cores was achievedby adding Co(NO3)2 and KMnO4 to our buffered solution ofapoferritin. Both salts were prepared at 53.3 mM, and enoughvolume of each salt was added to insert 160 metal atoms perround of injections. This was repeated 9 additional times (i.e.10 times total, targeting 1600 metal atoms added per ferritin)while waiting 10 min between each round of injections. Dif-ferent Co:Mn ratios were targeted, ranging from 1600:0 to0:1600 Co:Mn per ferritin. For each ratio, the total metalcount was held constant at 1600 metal atoms/ferritin, and thenumbers of both metal types were varied in increments of±200. The values for each sample are shown in table 1. As anexample, sample 5 targeted 800 cobalt and 800 manganeseper ferritin. To accomplish this, we added 10 μl of both theCo2+ and permanganate solutions, each 53.3 mM to thebuffered solution for each injection round. Due to the fact thatoxygen is unable to oxidize cobalt from its +2 state to its +3

state, the effect of oxygen was negligible and all sampleswere synthesized aerobically.

After synthesis, all samples were centrifuged at 3100×gfor 10 min and the supernatant was decanted. Each samplewas then passed over a Sephadex G-100 filtration column(12.5 cm×1 cm) to separate the ferritin from unboundmanganese and cobalt metals. The eluent was collected in1 ml fractions, and the absorbance was measured for eachfraction. The absorption at 280 nm was used to determine thepresence of either the protein or the metal salt, both of whichabsorb strongly at that wavelength. Each fraction was alsomeasured for metal content by running each sample on aninductively coupled plasma mass spectrometer (ICP-MS).The absorption and metal concentration data were used toconstruct elution profiles for each sample. Fractions con-taining ferritin were combined for each sample and the pro-tein concentration was determined by the standard Bradfordprotein assay [17]. The metal content of the combined frac-tions for each sample was also determined by use of the ICP-MS. The protein and metal concentrations were used to cal-culate the average number of metal atoms loaded per ferritin.Each sample was then loaded onto a 3 mm carbon meshcopper grid for imaging on a Tecnai TF-20 transmissionelectron microscope (TEM).

Once core formation was established through thesemethods, the band gap energies were determined by opticalabsorption spectroscopy, as outlined by Colton et al [2]. Twolight sources were used to measure transmission: a tungstenhalogen lamp for measuring visible and IR transmission, anda xenon arc lamp for measuring transmission in the UV range.Light from these lamps passed through a monochromator anda mechanical chopper (allowing for lock-in detection) beforepassing through the sample, which was placed in a UV–viscuvette. The transmitted intensity at each wavelength wasmeasured with a photodiode detector. This was repeated witha control, which consisted of the buffer solution placed in asimilar cuvette. The transmissions from the controls and thesamples were used to calculate the absorbances and produceTauc plots, from which the indirect and direct band gapenergies were determined.

3. Results and discussion

The reaction of Co2+ and permanganate in the presence ofapoferritin successfully resulted in the formation of metaloxide cores for nearly all tested ratios. The average number ofmetal atoms loaded into ferritin is shown in table 1 as anindication of nanoparticle size. Core formation was indicatedby an olive green–brown color in the apoferritin solution thatpersisted even after filtration over the Sephadex G-100 col-umn. A precipitate formed during synthesis and was removedthrough centrifugation. The amount of precipitate increased aspermanganate increased, and no precipitate was evident in thesample with no permanganate (sample 1). This precipitate islikely an insoluble compound formed during the reactionmixed with protein lost due to oxidation by permanganate.

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A representative elution profile for sample 6 from table 1is shown in figure 1 and demonstrates the separation of fer-ritin-bound cobalt and manganese from free metal salts dis-solved in the solution. Manganese and cobalt were observedto migrate with the protein ferritin and eluted in fractions 4–6,as shown by the peak on the left. Unbound manganese andcobalt eluted later in samples 13–20 (the right peak). Thisfigure confirms the separation of ferritin with its metal corefrom unreacted or free manganese and cobalt.

Representative images taken on the TEM are shown infigure 2 for samples 6 and 4. The dark circles represent theprotein shell, while the surrounding white halos come fromthe uranyl acetate stain. The metal oxide core is shown by thebright core inside the protein shell. These images, combinedwith the elemental analysis information obtained by the ICP-MS, confirm the formation of metal oxide cores inside offerritin. Optical absorption measurements were performed onall samples, and samples 3–9 were found to be semi-conducting materials with an indirect band gap. Samples 1and 2 showed no detectable band gap energies due to low

amounts of mineral formation. The results of the band gapenergy measurements for all other samples are included intable 1 and the energies are plotted versus the sample numberin figure 3.

Table 1 shows that the ratio of Co2+ and MnO4- greatly

affects the formation of the mixed metal oxide cores withinferritin. The number of atoms loaded per ferritin variedwidely from less than 30 atoms (see sample 1) to 1102 atomsper ferritin, almost 70% of our target loading (see sample 6).However, for all samples, the final amount of metal loadedinto ferritin ended up less than the targeted amount of 1600atoms per ferritin. This reduced metal loading is attributed inpart to the reaction between Co2+ and MnO4

- outside offerritin. Additionally, the elution profiles in figure 1 show thatunreacted cobalt atoms remain in solution after the reactionhas ended, as indicated by the concentration that eluted infractions 14–20. Samples 1 and 2 show very high amounts ofcobalt in solution, consistent with their low levels of metalloading. Lastly, table 1 shows negligible amounts of Mn insample 1 and Co in sample 9. Neither metal was included inthe respective synthesis for each reaction, and the reportedvalues are likely a result of noise inherent in the metal con-centration measurements performed on the ICP-MS.

More protein was lost during the reactions when highamounts of permanganate and low amounts of cobalt werepresent (i.e., samples 7–9) due to an insufficient supply of theelectron donor. When the ratio of permanganate to cobaltexceeded 1.67 (as in sample 6), the amount of protein lossincreased dramatically and the efficiency of manganeseloading decreased (comparing sample 6 to samples 7–9). Wealso note that sample 8 loaded nearly twice as much man-ganese as sample 9, even though more permanganate wasadded to sample 9 than sample 8. The 200 cobalt atoms perferritin added to sample 8 allows for a much greater increasein manganese loading and demonstrates permanganate’sdependence on cobalt as an electron source.

Samples with little to no permanganate present showedminimal loading into ferritin despite high levels of cobalt (seesamples 1 and 2, where less than 100 atoms loaded per fer-ritin). In the absence of MnO ,4

- the apoferritin is unable tooxidize cobalt to its 3+ state, preventing incorporation into

Table 1. Table of the cobalt–manganese samples synthesized at pH 8.5. Samples 1 and 2 did not form large enough cores for band gapdetection due to the relative or complete lack of oxidizing agent.

Sample

Co2+/FTNadded

MnO4-/FTN

addedFinal

Co/FTNFinal

Mn/FTNFinal

Metal/FTNProteinloss (%)

Indirect bandgap (eV)

Direct bandgap (eV)

1 1600 0 28±2 1±1 29±3 0 K K2 1400 200 50±5 41±4 91±9 4 K K3 1200 400 71±3 111±5 182±8 5 0.52±0.07 3.11±0.034 1000 600 129±10 285±23 414±34 8 0.63±0.06 2.90±0.025 800 800 214±25 568±66 782±90 7 0.68±0.05 2.86±0.026 600 1000 264±48 838±151 1102±199 9 0.76±0.06 2.80±0.027 400 1200 193±25 899±115 1092±140 23 0.86±0.06 2.80±0.018 200 1400 92±9 936±95 1028±95 35 1.08±0.08 2.73±0.019 0 1600 6±1 538±99 544±100 33 1.30±0.07 2.71±0.05

Figure 1.A representative elution profile, specifically for sample 6 intable 1. The absorbance is measured at 320 nm (blue triangles). Theconcentration of manganese and cobalt were measured by ICP-MSand are shown by the black squares and red diamonds, respectively.

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Nanotechnology 28 (2017) 195604 C R Olsen et al

the protein’s interior. During the reaction, manganese is alsoincorporated into the ferritin mineral, as detected by ICP-MSanalysis. Both manganese and cobalt loaded most efficientlyin sample 6, which had the highest total amount of metalsatoms loaded while losing less than 10% of total protein.Efficient loading of cobalt and manganese into ferritin thusrequires both metal atoms to be present, with an optimal rationear 1.67 between manganese and cobalt (sample 6).

The ratio of cobalt and permanganate in the final ferritincore greatly affects the band gap energies of the samples. Theresults followed our hypothesis that changing the ratio

between manganese and cobalt shifts the band gap energy. Asthe relative amount of manganese in each sample increases,the indirect band gap energies increase while the direct bandgap energies decrease. The sample synthesized using entirelypermanganate (sample 9) shows an indirect band gap energyof 1.30 eV; and as the amount of cobalt increases, the indirectband gap energy decreases until the lowest energy is mea-sured at 0.52 eV (sample 3). The direct gaps show a reversetrend, with band gap energies ranging from 2.73 up to3.04 eV. These trends of the band gap energy for each sampleare apparent in figure 3.

Sample 9, which was synthesized using solely MnO ,4-

had the highest indirect band gap energy at 1.30 eV. Thisvalue is similar to the 1.30 eV reported by Olsen et al for asample synthesized in similar conditions using only per-manganate [14]. Sample 3, which was synthesized using aCo:Mn ratio of 3:1, has an indirect band gap value near0.52 eV. Between the two endpoints, the trend in band gaps isfairly monotonic as expected.

4. Conclusion

Adding Co2+ and MnO4- to a solution containing apoferritin

successfully resulted in nanoparticle formation inside of ferritin.The ferritin core contained both cobalt and manganese, andcobalt and manganese loading was established by both ICP-MSand TEM imaging. Maximum loading occurred near a 1.67 ratiobetween MnO4

- and Co2+ and resulted in a core size of 1102metal atoms per ferritin. Beyond this point, protein lossincreased to greater than 20% and loading efficiency decreased.The band gap energies proved to be dependent upon the ratio of

Figure 2. (a) Scanning transmission electron microscope (STEM) image of cobalt–manganese ferritin (sample 6). (b) STEM image ofsample 4.

Figure 3. A graph of the band gap energies for samples 3–9 (fromleft to right) from table 1. Core sizes were too small for detectableband gap energies in samples 1 and 2.

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Nanotechnology 28 (2017) 195604 C R Olsen et al

metals deposited inside ferritin. Indirect band gap energies ofthese samples ranged from 0.52 to 1.30 eV and increased as theratio of manganese to cobalt increased, while the direct band gapenergies decreased from 3.11 to 2.71 eV. The ability to changethe indirect and direct band gaps by 0.78 eV and 0.40 eV,respectively, demonstrates the potential to tune the band gaps ofthese ferritin minerals and increase their utility in solar energyharvesting. Additionally, the formation of a mixed cobalt–manganese oxide core suggests the possibility that this methodfor combining two metals (with a high and low oxidation state,respectively) may be used to create new and unexploredminerals in ferritin.

Acknowledgments

Funding for this project was provided by the Utah Office ofEnergy Development through the Governor’s Energy Leader-ship Scholars Program, by Brigham Young University’sDepartment of Physics and Astronomy, and by Brigham YoungUniversity’s Department of Chemistry and Biochemistry.Assistance in operating the TEM was provided by Paul Minson,from BYU’s TEM Facilities, and assistance in operating theICP-MS was provided by Anna Nielson, a graduate student inthe Department of Chemistry and Biochemistry at BYU.

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