This content has been downloaded from IOPscience. Please scroll down to see the full text.

Download details:

This content was downloaded by: johnscolton

IP Address: 128.187.112.27

This content was downloaded on 19/04/2017 at 01:23

Please note that terms and conditions apply.

Permanganate-based synthesis of manganese oxide nanoparticles in ferritin

View the table of contents for this issue, or go to the journal homepage for more

2017 Nanotechnology 28 195601

(http://iopscience.iop.org/0957-4484/28/19/195601)

Home Search Collections Journals About Contact us My IOPscience

Permanganate-based synthesis ofmanganese oxide nanoparticles in ferritin

Cameron R Olsen1, Trevor J Smith2, Jacob S Embley1, Jake H Maxfield3,Kameron R Hansen1, J Ryan Peterson1, Andrew M Henrichsen1,Stephen D Erickson1, David C Buck2, John S Colton1 and Richard K Watt2

1Department of Physics and Astronomy, Brigham Young University, Provo, Utah 84602, United States ofAmerica2Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, UnitedStates of America3Department of Exercise Science, Brigham Young University, Provo, Utah 84602, United States ofAmerica

E-mail: rwatt@chem.byu.edu

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

AbstractThis paper investigates the comproportionation reaction of MnII with MnO4

- as a route formanganese oxide nanoparticle synthesis in the protein ferritin. We report that MnO4

- serves asthe electron acceptor and reacts with MnII in the presence of apoferritin to form manganese oxidecores inside the protein shell. Manganese loading into ferritin was studied under acidic, neutral,and basic conditions and the ratios of MnII and permanganate were varied at each pH. Themanganese-containing ferritin samples were characterized by transmission electron microscopy,UV/Vis absorption, and by measuring the band gap energies for each sample. Manganese coreswere deposited inside ferritin under both the acidic and basic conditions. All resultingmanganese ferritin samples were found to be indirect band gap materials with band gap energiesranging from 1.01 to 1.34 eV. An increased UV/Vis absorption around 370 nm was observed forsamples formed under acidic conditions, suggestive of MnO2 formation inside ferritin.

Keywords: nanoparticles, synthesis, ferritin, band gaps, manganese oxide

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

1. Introduction

The study and synthesis of nanoparticles is an area of sig-nificant interest across many different fields [1–6]. Recentinvestigations have investigated using protein nanostructuresto aid in the formation of these nanoparticles [7–9]. Theprotein ferritin has been shown to be an effective scaffold fornanoparticle formation [7, 8, 10, 11], which nanoparticleshave been studied for use in biomedical imaging [1, 4],electronics [10, 12], and redox enzymatic activity [13].Manganese nanoparticles in particular, including ones formedin ferritin, have been investigated for their magnetic[4, 14, 15] and semiconducting properties [3]. Currentmethods for forming manganese nanoparticles includehydrothermal [16, 17], molten salt [18], and microemulsionmethods [19]. The use of ferritin allows one to form stable

nanoparticles with a narrow range of size distribution at lowerreaction temperatures, and allows for insoluble materials to behandled in solution [11]. Manganese nanoparticles have pre-viously been formed in ferritin; however, they have beenrestricted to a single manganese moiety, namely Mn(O)OH[20–22]. Using the comproportionation reaction betweenMnII and MnO ,4

- we aim to form new manganese nano-particles within ferritin that have different semiconducting,magnetic, and reduction–oxidation properties, while stillretaining the advantages that come through using a protein-based shell.

Ferritin is a spherical iron storage protein found in mostorganisms from bacteria through vertebrates. Ferritin has amolecular mass of 450 kDa and is composed of 24 subunits,which assemble into a sphere measuring 12 nm in diameterwith an 8 nm hollow interior. Its primary role in cells is to

Nanotechnology

Nanotechnology 28 (2017) 195601 (10pp) https://doi.org/10.1088/1361-6528/aa68ae

0957-4484/17/195601+10$33.00 © 2017 IOP Publishing Ltd Printed in the UK1

store iron as a ferrihydrite mineral. Ferritin iron acquisitionoccurs using a catalytic site called the ferroxidase center thatfacilitates iron oxidation by oxygen [23, 24]. The ferroxidasecenter of ferritin has been exploited for materials applicationsby using chemical techniques to replace the native iron oxidecore (or more technically, iron oxyhydroxide, Fe(O)OH) withnon-native metal oxides and oxyhydroxides as nanoparticleswithin ferritin [22, 25–27]. The spherical shell of ferritinallows for the stable production of these nanoparticles with auniform size distribution and helps maintain the solubility ofthe resulting nanoparticles. The protein shell can withstandtemperatures up to 70 °C and variations in pH rangingfrom pH 4–10 [26, 28]. Additionally, ferritin can be arrangedinto ordered 2D architectures [29–31] and shows potentialin bio-battery [32, 33] and light harvesting applications[3, 12, 34, 35].

Ferritin metal oxide cores are traditionally synthesizedin vitro by first removing the native iron oxide core—theresulting protein shell being termed apoferritin—and thenreacting a transition metal in a low oxidation state withoxygen in the presence of apoferritin. For example, ferrousiron binds to the ferroxidase center and is oxidized beforemigrating into the interior and forming the native Fe(O)OHmineral. Hydrogen peroxide can be used to facilitate oxida-tion in cases where diatomic oxygen is unable to oxidize themetal [26, 34]. Using oxygen or hydrogen peroxide as theoxidant has allowed for the synthesis of many differentnanoparticles in ferritin [22, 25–27]. The protein shell is alsoknown to allow diffusion of oxo-anions [27, 36–41] anddivalent metals [42–46] into the ferritin cavity. Metal oxo-anion minerals can thus be deposited inside ferritin by addingoxo-anions to the reaction buffer during iron loading [27, 36].Using these methods, Mn nanoparticles have previously beenformed inside of ferritin [3, 21, 22].

Many nanoparticles formed in ferritin act as semi-conducting materials [47, 48], and band gap energies forsome previously synthesized nanoparticles have been repor-ted, including indirect and direct transitions for Co-, Mn-, andTi-oxyhydroxides [3] and in nanoparticles where the nativeFe(O)OH has been co-deposited with PO ,4

3- MO ,42-

WO ,42- and MnO4

- [27]. In the latter paper, Smith et al alsoobserved that ferritin iron loading in the presence of MnO4

-

produces a core containing both iron and manganese [27].This observation indicates a possible new route for manga-nese loading into ferritin, where MnO4

- may be able toreplace oxygen as the oxidant and subsequently be incorpo-rated into the ferritin core. Additionally, the use of MnO4

-

might allow the formation of manganese cores with higheroxidation states in the resulting mineral when compared to thetypical +3 oxidation state formed in the reaction with oxygenat the ferroxidase center [20, 22].

In this paper we investigate the reaction of MnII withMnO4

- as a new route to synthesize manganese oxide coreswithin ferritin. We report the successful synthesis of manga-nese oxide nanoparticles inside ferritin while using MnO4

- asthe oxidant. We also report that these ferritin samples possessdifferent manganese cores than the previously reported

ferritin manganese minerals, as confirmed by differences intheir absorbance profiles and band gap energies.

2. Experimental

2.1. Potential synthesis pathways

The first reported manganese loading into ferritin was per-formed by reacting MnII with apoferritin in a basic solutioncontaining oxygen [22]. Under these conditions, a Mn(O)OHmineral was formed [20]. This synthesis required the pH ofthe reaction solution to be greater than 9 before the oxidationof MnII became thermodynamically favorable and manganeseloading occurred [49]. By replacing oxygen with MnO ,4

- weproposed the possibility of forming manganese ferritin atdifferent pH values and of forming manganese minerals withhigher oxidations states within ferritin. Because of thedependence of manganese reactions on pH, we tested theability of MnO4

- to influence manganese loading at acidic,neutral, and basic conditions.

We proposed that the reaction of MnII and MnO4- with

apoferritin could follow two different pathways. The firstpathway was based on the traditional synthesis method inwhich two MnII atoms bind to the ferroxidase center [22]. Thetwo MnII atoms would be oxidized by MnO ,4

- which wouldaccept one electron from each MnII atom, resulting in twoMnIII ions. These MnIII would follow the typical path into theprotein shell and form Mn(O)OH minerals inside ferritin [20].This reaction is portrayed by equation (1a). The inclusion ofMnV on the product side is written to account for MnO4

-

accepting two electrons, though the actual formation of aMnV product is unlikely in aqueous solutions [49, 50]. Thereis the possibility that the permanganate species participates intwo rounds of the reaction, where after two cycles at theferroxidase center (FC), MnO4

- would have accepted a totalof four electrons and forms a MnIII ion that enters ferritin, asshown in equation (1b). The given equations show only themanganese species as the reactants and products. Theequations are purposefully left unbalanced because reactionswere studied under acidic, neutral and basic conditions.

a

Apoferritin 2Mn MnO

2Mn O OH ferritin Mn , 1

II4

V

+ + +

-

( ) ‐ ( )

bApoferritin 4Mn MnO

5Mn O OH ferritin 2 FC cycles . 1

II4+ +

-

( ) ‐ ( ) ( )

The second pathway involves MnII and MnO4- diffusing

into ferritin before reacting. Once inside ferritin, MnII andMnO4

- can undergo comproportionation to form MnO2, asshown in equation (2):

Apoferritin 3Mn 2MnO 5MnO ferritin. 2II4 2+ + - ‐ ( )

Even though MnO2 seems a likely product when the stoi-chiometry is as given by equation (2), the comproportionationof MnII and MnO4

- is known to produce multiple, differentend-products [51]. Therefore, the result is likely to be morecomplicated than shown in equation (2), and the resulting Mn

2

Nanotechnology 28 (2017) 195601 C R Olsen et al

product that actually forms might exist with an oxidation statebetween +2 and +4.

We used the ratios between MnII and MnO4- outlined in

equations (1a), (1b), and (2) as a basis for our synthesismethods and fixed the total amount of Mn added into thesolution. By varying the ratio between the reactants, wehoped to maximize the loading conditions into ferritin usingMnO4

- as the oxidant, as well as shed light on whichmechanism the reactions would follow.

We acknowledge that the chemistry involved surround-ing the various oxidation states of manganese is complex andthat the result may likely be a combination of the separatepathways occurring simultaneously rather than the formationof a single mineral. While we included these two pathways aspossible mechanisms for how loading occurs under thesesituations, the primary purpose of this work was to demon-strate a new method for manganese incorporation into ferritinand if possible to describe the nature of the reaction pathway.The two proposed pathways are summarized in figure 1. Inaddition to these two pathways, we recognize the possibilitythat permanganate will oxidize and denature some of theprotein, complicating our ability to confirm which mechanismis followed in these reactions [52].

2.2. Synthesis methods

In order to determine the effect of MnO4- on Mn loading into

ferritin, we first prepared apoferritin through dialyzing horsespleen ferritin against thio-glycolic acid. This removes theferrihydrite core and readies the ferritin for metal loading[27, 36]. Reaction solutions were prepared by adding 3.0 mg(6.67 nmol) of apoferritin to 1.0 ml of one of the followingthree buffered solutions: 1 M pH 5.4 2-(N-morpholino)

ethanesulfonic acid buffer, 1 M pH 7.4 imidazole buffer, or1 M pH 9.4 N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid buffer. MnCl2 and KMnO4

were then added to the buffered apoferritin solutions asdescribed in the next paragraph to synthesize the manganeseferritin cores. As stated above, the reactions were tested inacidic, neutral, and basic conditions (pH 5.4, 7.4 or 9.4)because manganese redox reactions are pH-dependent.

We tested the reaction shown in equation (1b) by adding12 μl of 66.7 mM MnCl2 (0.8 μmol) to each buffered solutioncontaining 3 mg (6.67 nmol) of apoferritin, followed by 3 μlof 66.7 mM KMnO4 (0.2 μmol), giving us a 4:1 ratio betweenMnII and MnO .4

- These additions were repeated 9 times (i.e.10 times total) targeting 1200 MnII and 300 MnO4

- per fer-ritin, or 1500 Mn atoms in total. We waited 10 min betweenadditions to allow time for manganese to be incorporated intoferritin. Each synthesis was performed both aerobically andanaerobically to test the role of MnO4

- as the oxidant. Similarprocedures were followed using the 2:1 and the 3:2 ratiosbetween MnII and MnO4

- (equations (1a) and (2), respec-tively), also targeting 1500Mn atoms per ferritin. In addition,control reactions without apoferritin were run for each ratioand in each buffer to observe the reaction of MnCl2 andMnO4

- in solution.In addition to the comproportionation reactions, a sepa-

rate control reaction was performed to determine whetherMnO4

- loads into ferritin in the absence of MnCl2. Reactionscontaining only MnO4

- were performed by inserting tenadditions of 15 μl of 66.7 mM KMnO4 (1.0 μmol) into eachof the three buffered solutions described above, with eachsolution containing 3 mg (6.67 nmol) of apoferritin. Welikewise waited 10 min between additions, targeting a total of

Figure 1. Manganese loading into ferritin using MnO .4- (a) The first proposed pathway, utilizing the ferroxidase center. MnII binds at the

ferroxidase center (FC) and is oxidized to MnIII by MnO .4- After oxidation the MnIII ions migrate to the ferritin interior and become

mineralized as Mn(O)OH. This proposed pathway follows the reactions given in equations (1a) and/or (1b). (b) The comproportionationpathways. MnII and MnO4

- first enter ferritin and then react to form a new manganese oxide mineral. This diagram follows equation (2).

3

Nanotechnology 28 (2017) 195601 C R Olsen et al

1500 Mn atoms/ferritin. These permanganate only reactionswere run both aerobically and anaerobically. Finally, a Mn(O)OH sample was synthesized according to methods similar tothe ones outlined by Erickson et al [3], and is termed thetraditional sample. Control reactions were also run at eachpH without apoferritin.

After the additions were complete, the samples werestirred for an additional two to three hours and a noticeablydark brown precipitate was observed for each sample. Eachsample was then centrifuged at 3100×g for 10 min and thesupernatant containing ferritin decanted. The resulting solu-tion was a medium-brown color. The ferritin samples wereloaded onto a 12.5 cm×1 cm Sephadex G-100 size exclu-sion column, which was buffered with 25 mM pH 8.5 TRISbuffer, no salt, to prevent core degradation. The sample wascollected in 1 ml fractions and the optical absorption mea-sured for each fraction. The fractions containing ferritin elutedfirst and were collected and characterized for manganesecontent by methods described below.

2.3. Characterization

Protein and metal analyses were completed using standardmethods [27, 35]; protein analysis was performed using theBradford protein assay [53], and metal analysis was per-formed using inductively coupled plasma mass spectrometry(ICP-MS). The two molar concentrations were used to findthe average number of manganese per ferritin.

The manganese oxide ferritin was prepared for viewingon a transmission electron microscope (TEM) by depositiononto a carbon type-b 300 mesh copper grid. A uranyl acetatenegative stain was applied to the samples to help visualize theprotein shell surrounding the metal-oxide interior. Bright fieldimages and diffraction patterns were captured using a Tecnai-F20 TEM. Images were also collected on the same TEMwhile in scanning transmission electron microscopy (STEM)mode. The images were used to determine the number offerritin molecules containing a ferritin core, and this fillingfraction was used to adjust the atoms calculated per ferritin torepresent a more accurate count. The diameters of the ferritinnanoparticle were measured and used to create a histogram ofthe nanoparticle size distribution.

X-ray powder diffraction studies were performed using aBruker-Nonius FR591 single crystal diffractometer with arotating Cu anode. Data was collected in transmission modex-ray diffraction (XRD) with a polyimide capillary that con-tained the manganese ferritin solutions.

Band gaps were measured using absorption spectroscopyas described by Colton et al [12], and absorbance profileswere taken with an HP 8453 UV/Vis Spectrophotometer.

3. Results and discussion

3.1. Evidence for core formation inside of ferritin

As mentioned above, all samples were centrifuged and passedover a filtration column after synthesis to separate ferritin

from the unbound metals. In each case, manganese wasobserved to migrate with ferritin as the samples were elutedover the gel filtration column. The elution profile for theMnO4

- only sample synthesized at pH 5.4 is displayed infigure 2 and is representative of all the elution profiles. Theabsorbance of each eluted fraction was recorded at 255 and420 nm. The absorption at 255 nm indicates the presence ofeither the protein or manganese, while absorption at 420 nmindicates the presence of manganese only. ICP-MS resultsshowed high manganese content in the fractions containingferritin (fractions 4, 5, and 6), while low amounts of man-ganese eluted later in fractions 9 through 15. The elution ofmanganese with ferritin followed by a separate elution peakof manganese demonstrates that manganese was depositedinside ferritin and remained associated with the protein shellwhile unbound manganese was separated from ferritin by thegel filtration column.

The comproportionation of MnII and MnO4- with apo-

ferritin at pH 5.4 and 9.4 successfully resulted in core for-mation for all ratios (see table 1). No significant coreformation was observed in the neutral buffer (pH 7.4), andsignificant precipitation was present during the reactions atthis pH. Control reactions without apoferritin present pro-duced a dark-brown manganese precipitate and resulted in aclear solution after centrifugation; a similar precipitate wasobserved in small amounts for all samples containing ferritin,suggesting some manganese precipitation occurred outside offerritin. The pH 9.4 samples all retained a medium-browncolor even after centrifugation and filtration over the gel-exclusion column. The pH 5.4 samples also retained theircolor but were noticeably darker. The presence of color in thefractions containing ferritin after gel filtration indicatesmanganese core formation inside of ferritin.

Figure 2. A typical elution profile of manganese–ferritin passed overa Sephadex G-100 column, indicating the separation of amanganese–ferritin from unbound manganese. The y-axis for theabsorbance measured at 255 nm (black squares) and at 420 nm (redcircles) is on the left. The manganese concentration in each fractionwas measured by ICP-MS (blue triangles) and is displayed on thesecondary y-axis on a log scale on the right. The profile shown hereis for the permanganate only sample run at pH 5.4, sample 5.

4

Nanotechnology 28 (2017) 195601 C R Olsen et al

Manganese cores were successfully synthesized using thecomproportionation reactions under both aerobic and anae-robic conditions. The number of metals loaded per ferritin arereported in table 1, as are the measured band gaps energies foreach sample.

Table 1 also identifies the amount of protein lost duringthe reaction. As MnO4

- is a strong oxidizing species and isknown to oxidize proteins, the fact that ferritin was notimmediately oxidized and denatured in the presence ofMnO4

- demonstrates the remarkable stability of ferritin. Onemajor difference noted between the aerobic and anaerobicsamples was the amount of protein lost during the acidicreactions (see table 1, samples 1-8). More protein was lost inthe aerobic samples and for those with higher MnO4

- content.Additionally, the formation of manganese cores despite theabsence of oxygen demonstrates the ability of MnO4

- to actas the oxidant for Mn2+.

Adding MnO4- into the buffered apoferritin solution (i.e.

no MnII added) also resulted in core formation for both theacidic and basic conditions. A brown precipitate was formedin all samples but was removed by centrifugation. The pH 5.4buffered sample resulted in a dark-brown color once passedover the gel-exclusion column, while the pH 9.4 sampleresulted in a light brown–yellow color, indicative of loweramounts of manganese loading into the protein interior. ThepH 7.4 sample resulted in core formation, but it was unstableand both the core and protein precipitated after several hours.

In the absence of ferritin, a brown precipitate likewise formedfrom the reactants and the solution was clear after removal ofthe precipitate by centrifugation.

Images taken with the TEM show the presence ofnanoparticles inside ferritin. Representative bright field ima-ges for samples 3 and 12 from table 1 are shown infigures 3(a) and (c). TEM analysis of other manganese ferritinsamples showed similar core formation in each ferritin sam-ple. The metal cores are observed in the interior of ferritin asdark spots. The uranyl acetate stain was used to providecontrast for the protein shell, and can be seen as the light ringin the bright field images and the dark ring in the STEMimages (figures 3(b) and (d)). Analysis of the TEM imagesrevealed a ferritin filling fraction of 90%–95% and nano-particles with a mean diameter of 6.7±1.7 nm. A histogramis plotted in figure 3(d) inset.

3.2. Band gap measurements of the manganese ferritinminerals

Band gap measurements were performed on all of the samplesvia optical absorption spectroscopy to assist in determiningwhether a new manganese core had formed. The manganeseminerals formed by the above methods were found to beindirect band gap semiconductors, which was also observedin the Mn(O)OH ferritin minerals studied by Erickson et al[3]. The band gaps for each of these samples are listed in

Table 1. Results of both the permanganate only samples (samples 1, 5, 10, and 14) and the comproportionation samples (samples 2–4, 6–8,11–13, and 14–17). Each section of data represents either the acidic or basic buffer solution run either aerobically or anaerobically. Thelisting of ‘traditional method’ under the ratio of MnII:MnO4

- column refers to the method originally discovered for manganese–ferritinsynthesis using MnII and oxygen [22]. The band gap energies for samples 9 and 10 are left blank due to the core size being too small for bandgap detection.

SampleRatio ofMnII:MnO4

- pHAnaerobicor aerobic

MnII

added(Mn/ferritin)

MnO4-

added(Mn/ferritin)

Metalsloaded(Mn/ferritin)

Proteinlost (%)

Presenceof 370 nmabsorption

Indirectbandgap (eV)

Direct bandgap (eV)

1 0 5.4 Anaerobic 0 1500 796±70 53 Yes 1.34±0.07 2.69±0.022 3:2 5.4 Anaerobic 900 600 699±110 32 Yes 1.26±0.08 2.66±0.013 2:1 5.4 Anaerobic 1000 500 496±70 27 Yes 1.21±0.09 2.64±0.014 4:1 5.4 Anaerobic 1200 300 294±28 26 Yes 1.17±0.09 2.63±0.015 0 5.4 Aerobic 0 1500 644±17 77 Yes 1.30±0.08 2.70±0.056 3:2 5.4 Aerobic 900 600 551±21 57 Yes 1.21±0.09 2.68±0.027 2:1 5.4 Aerobic 1000 500 612±69 39 Yes 1.23±0.08 2.66±0.028 4:1 5.4 Aerobic 1200 300 494±61 30 Yes 1.19±0.10 2.64±0.029 Traditional

method5.4 Aerobic 1500 0 57±1 17 — — —

10 0 9.4 Anaerobic 0 1500 84±7 32 — — —

11 3:2 9.4 Anaerobic 900 600 268±22 18 No 1.10±0.05 2.56±0.0212 2:1 9.4 Anaerobic 1000 500 451±37 27 No 1.05±0.05 2.76±0.0313 4:1 9.4 Anaerobic 1200 300 913±124 27 No 1.09±0.05 2.99±0.0314 0 9.4 Aerobic 0 1500 142±11 29 Yes 1.30±0.09 2.72±0.0115 3:2 9.4 Aerobic 900 600 169±12 26 No 1.21±0.05 2.56±0.0216 2:1 9.4 Aerobic 1000 500 164±8 24 No 1.16±0.07 2.65±0.0217 4:1 9.4 Aerobic 1200 300 314±24 28 No 1.19±0.05 2.52±0.0218 Traditional

method9.4 Aerobic 1500 0 490±28 9 No 1.01±0.05 2.51±0.01

5

Nanotechnology 28 (2017) 195601 C R Olsen et al

table 1. Our manganese–ferritin samples were measured tohave indirect band gap energies ranging from 1.01 to 1.34 eVand direct band gap energies ranging from 2.51 to 2.99 eV.The number of metals in samples 9 and 10 are insufficient toform a true mineral core in ferritin, and hence their band gapmeasurements provided no data showing the presence of anyband gap.

While the band gaps listed represent the primaryabsorption edge of the materials, in all samples there is someabsorption even below the reported band gaps. We attributethis primarily to two factors. First, there may be a defect stateslightly lower in energy than the band gap. This has pre-viously been reported in native ferritin (with an Fe(O)OHnanoparticle) [12], and we see similar features in the below-gap absorption in these samples. Secondly, the size variationin nanoparticles leads to a distribution of band gaps (larger

nanoparticle cores tend to have smaller band gaps [3]), sosome long wavelength absorption undoubtedly arises fromthe tail of the size distribution, at energies below where theabsorption begins for the size distribution peak.

Previous work by our group [3] showed an indirect bandgap of 1.574 eV for a sample similar in size and synthesismethod to sample 18. The value we report here for sample 18is lower, primarily due to an increased ability to measure longwavelengths. In the previous work it had appeared that thetransmission plateaued around 700–800 nm, which weattributed to scattering, leading us to normalize the trans-mission to the average value in that range. However in ourmore recent measurements we have found that transmissioncontinues to increase at even longer wavelengths, so we haveremoved that normalization step. This results in smallerdeduced indirect band gap values.

Figure 3. TEM analysis of stained manganese-loaded ferritin synthesized using a 2:1 ratio of MnII and permanganate. (A) Bright field imageof sample 3, run under anaerobic and acidic conditions. (B) STEM image of sample 3. (C) Bright field image of sample 12, run underanaerobic and basic conditions. (D) STEM image of sample 12. Inset: histogram showing size distribution of the ferritin nanoparticles.

6

Nanotechnology 28 (2017) 195601 C R Olsen et al

3.3. Permanganate control reactions

As stated above, manganese was observed to load into ferritinin the control reactions with MnO ,4

- where no MnII wasadded. We observed that the acidity of the solution greatlyaffected the manganese incorporation into ferritin, wheresignificantly more metal ions were loaded in the acidic con-dition (see table 1, comparing samples 1 and 5 with 10 and14). In the acidic solution, the presence of oxygen increasedthe protein loss from 53% to 77%, while simultaneouslydecreasing the total number of metals loaded from 716 Mn/ferritin to 580 Mn/ferritin. This dependence on the pH of thesolution, as well as the presence of oxygen, is consistent withthe reaction mechanism of MnO4

- in acid [54, 55], as shownin the following equation:

4MnO 4H 4MnO 3O 2H O. 34 2 2 2+ + +- + ( )

Equation (3) explains why anaerobic conditions favor thereaction, as O2 is a product and would slow the reaction bybeing present on the reactant side of the equation. Thedependence on pH as well as the effect of oxygen on thereaction likewise suggest the formation of MnO2 inside fer-ritin. Under these circumstances, MnO4

- likely migrates intothe ferritin interior before interacting with ferritin and pro-ducing manganese oxide, though some precipitation wouldlikely still occur outside of ferritin as well.

The absorption profiles of these permanganate controlsamples, displayed in figure 4, show increased absorptionnear 370 nm. This absorption peak is observed in other MnO2

compounds, and gives evidence that some amount of MnIV

forms inside ferritin under these reaction conditions [55, 56].Note how this increased absorption is absent in the traditionalMn(O)OH–ferritin, demonstrating the formation of a newmanganese mineral in ferritin. The overall absorption of the

samples also increases due to the increase in manganese ionsper ferritin.

The band gap energies for the MnO4- only samples all

fall around 1.30 eV (see samples 1, 5, and 14; sample 10would have been included in this category, but the band gapwas unable to be measured due to core size). This agreesperfectly with the measurements for α-MnO2 as reported byCockayne and Li [57], and further strengthens the notion forthe formation of MnO2.

3.4. Comproportionation reactions

As mentioned previously, the reaction of MnO4- and MnII in

the presence of apoferritin resulted in the formation of amanganese mineral inside of ferritin. Manganese loadingoccurred in all anaerobic samples, despite the absence ofoxygen. For example, in the anaerobic sample 13, moremanganese loaded per ferritin than possible due to MnO4

-

alone (822 Mn atoms were loaded with a maximum of 300being from MnO4

-), meaning that some amount of MnII wasoxidized by MnO4

- and incorporated into the core. Thisdemonstrates the ability of MnO4

- to act as the oxidant in theabsence of oxygen.

In each trial, up to half of the total metals were depositedinto ferritin, as shown in table 1. The loss of metal is due tothe precipitation that occurred outside of ferritin, which pre-cipitate was removed from the solution during centrifugation.As mentioned previously, this precipitate resulted from MnII

and MnO4- reacting before MnII diffusion into and interaction

with ferritin.XRD and electron diffraction were both used in an

attempt to characterize the mineral and to determine whichproposed mechanism the reactions follow. Results showed thepresence of an amorphous manganese core in each case,making it impossible to determine the mineral type throughthese methods. This result is not entirely unexpected as fer-ritin often forms amorphous cores in its interior [20, 58].

We also measured the absorption profiles for each of thecomproportionation samples to help differentiate our newmanganese–ferritin from the traditionally synthesized man-ganese–ferritin (sample 18). The spectra of samples 2 and 13are plotted alongside the traditional manganese–ferritin sam-ple in figure 5, and all samples were prepared at 0.1 mg ml−1

ferritin. All acidic samples had absorption profiles similar tothe sample 2 as shown in figure 5, and the basic samples weresimilar to the one selected for the same figure (sample 13).The presence of oxygen had no apparent effect on theabsorption profiles other than to change the strength of theabsorption by changing the number of metals loaded. Thesesamples were chosen because they had the highest metalloading in the basic and acidic groups and helped highlightthe differences between these two groups.

In the absorption profiles for these samples, part of theabsorption at 280 nm comes from the protein itself, while theabsorption tailing into the visible wavelengths arises solelyfrom the metal-oxide cores. An increase in absorption near370 nm appears in the profiles of the acidic comproportio-nation samples, a trait which was absent in both the basic

Figure 4. The absorption spectra of manganese-loaded ferritinsynthesized using MnO4

- only. Each sample was prepared at0.10 mg ml−1 ferritin, and the traditional manganese–ferritin andapoferritin are included for comparison. The legend follows thesamples from top to bottom. Note how samples 1, 5, and 14 have anincreased absorption shoulder near 370 nm, indicative of a newmineral formation.

7

Nanotechnology 28 (2017) 195601 C R Olsen et al

samples and the traditional sample but present in the MnO4-

control reactions. This increased absorption confirms thepresence of a new manganese mineral within ferritin. Asmentioned in the MnO4

- control section, this increase inabsorption near 370 nm has been observed with MnO2 for-mation and is suggestive of some amount of MnIV formation.Such a formation would propose the use of the secondpathway, outlined in equation (2).

The basic samples, on the other hand, appear quitesimilar to the traditional sample, and suggest that the mineralformed through a similar reaction mechanism. If true, thisresult would suggest that the basic conditions make use of theferroxidase center, as outlined in equations (1a) and (1b). Inthe aerobic trials, MnO4

- competes with oxygen and is lessable to assist in loading at the ferroxidase center. Hence it isleft to react with MnII in solution, and fewer metals areavailable to load into ferritin. This effect is seen by comparingthe total metal loading in the anaerobic and aerobic samplessynthesized under basic conditions (compare samples 11–13with samples 15–17). However, in the absence of oxygen,MnO4

- is allowed to oxidize MnII at the ferroxidase centerrather than precipitating in solution, nearly doubling the totalnumber of metals loaded. Despite these possibilities, how-ever, it is difficult to pin down which reaction occurs in eachcondition due to protein loss and the amorphous nature of themineral.

As noted above, samples synthesized using only MnO4-

had band gaps near 1.3 eV and have the highest indirect bandgap energy in each set of conditions. As the amount ofMnO4

- decreases and the amount of MnII increases, theindirect bang gap energies decreases in almost every scenario.The traditional sample had the lowest indirect band gapenergy at 1.01 eV. The change in band gap energy within

each set of conditions is attributed to a change in the mineralformed under each circumstance. It is interesting to note thatthe band gap energies for samples 11–13 did not change asmuch as their aerobic counterparts (samples 14–17). This islikely due to the fact that, in the absence of oxygen, the role ofoxidant was played by MnO4

- only, leaving little MnO4-

available to undergo other pathways and form differentminerals. Hence, the indirect band gap values for samples11–13 do not differ significantly from the traditional methodin sample 18, which used no MnO4

- in its synthesis. Samples14–17, on the other hand, were exposed to oxygen, leaving asufficient amount of MnO4

- in solution to form different Mnminerals, resulting in higher band gap energies. Lastly, thepresence of oxygen had little effect on the band gap energyfor the acidic samples, where each sample is comparable to itscounterpart (i.e. samples 3 and 7). This suggests it does notmuch affect the type of mineral formed in the reaction,affecting only the size of the final mineral formed.

4. Conclusion

Ferritin is a unique protein that allows for the synthesis of awide variety of nanoparticles. The comproportionation reac-tion between MnII and MnO4

- in the presence of apoferritinresulted in the formation of manganese compounds within theprotein shell. In the absence of oxygen, MnO4

- acts as theoxidant and is able to oxidize MnII to form the manganeseoxide core. Manganese loading into ferritin was establishedby ICP-MS and TEM imaging. This reaction leads to theformation of a new manganese oxide core inside ferritin.Differences between the traditional samples and the newsamples include changes in the indirect band gap energies byup to 0.3 eV and significant changes in the absorption profile.These differences also suggest that the comproportionation ofMnII and MnO4

- in an acidic solution form a core likelycontaining some amount of MnO2 inside ferritin (a +4 oxi-dation state), as opposed to the Mn(O)OH core that forms inthe traditional sample (a +3 oxidation state). In addition todetermining optimal conditions for the formation of thesedifferent cores inside ferritin, we also discovered that throughmaking changes in pH, the presence of oxygen, or the ratiobetween the reactants, we are able to alter both the mineraland its properties.

The development of biohybrid materials continues to bea field where science can utilize the tools of nature toachieve its goals. Our aim was to use ferritin to add to thatrepertoire of materials by finding a new method for syn-thesizing manganese oxide nanoparticles. Due to the ferritinencapsulation, we were able to create manganese oxidenanoparticles in solution and without using high tempera-tures, as was done in the hydrothermal and molten saltmethods [16–18]. The use of ferritin also allows for thesenanoparticles to potentially coat surfaces in 2D arrays andalso allows for normally insoluble materials to be handled insolution [10, 31]. In addition, the change in mineral typewithin ferritin resulted in new band gap energies, expandingthe possible wavelengths in which ferritin can act as a

Figure 5. Absorption spectra of manganese-loaded ferritin samples,comparing two samples synthesized through the comproportionationreactions at both the acidic and basic conditions. The traditionalmanganese–ferritin and apoferritin are included for comparison. Thelegend follows the samples from top to bottom. Note how sample 2has a markedly increased absorption near 370 nm, indicative of anew mineral formation.

8

Nanotechnology 28 (2017) 195601 C R Olsen et al

photosubstrate in light-harvesting techniques. Manganesenanomaterials are currently being explored for theirmagnetic properties [4, 15] and as water splitting and oxy-gen reducing catalysts [59–61]. The minerals formed in thisproject will likely have differing magnetic and reduction–oxidation properties from their ferritin pre-cursors, thoughthese will have to be determined by future experiments.Lastly, the success of this synthesis method suggests thepossibility of using other comproportionation reactions (e.g.between Cr(IV) and a lower valence chromium ion) for theformation of new nanoparticles within ferritin.

Acknowledgments

Partial funding for this project was provided by the UtahOffice of Energy Development through the Governor’sEnergy Leadership Scholars Program, Brigham Young Uni-versity’s Department of Physics and Astronomy, BrighamYoung University’s Department of Chemistry and Biochem-istry, and by Strategic Environmental Research and Devel-opment Program (SERDP)—Weapons Platform (WP) projectWP-2142. Assistance in operating the TEM was provided byPaul Minson, from BYU’s TEM Facilities. Assistance withoperating the x-ray powder diffraction instrument was pro-vided by Dr Stacey Smith, from BYU’s Chemistry and Bio-chemistry Department. Assistance in operating the ICP-MSwas provided by Anna Nielson, a graduate student in theDepartment of Chemistry and Biochemistry at BYU.

References

[1] Fan K et al 2012 Magnetoferritin nanoparticles for targetingand visualizing tumour tissues Nat. Nanotechnol. 7 459–64

[2] Li C et al 2015 Phase and composition controllable synthesisof cobalt manganese spinel nanoparticles towards efficientoxygen electrocatalysis Nat. Commun. 6 7345

[3] Erickson S D et al 2015 Non-native Co-, Mn-, and Ti-oxyhydroxide nanocrystals in ferritin for high efficiencysolar energy conversion Nanotechnology 26 015703

[4] Crich S G et al 2015 Targeting ferritin receptors for theselective delivery of imaging and therapeutic agents to breastcancer cells Nanoscale 7 6527–33

[5] Chinen A B et al 2015 Nanoparticle probes for the detection ofcancer biomarkers, cells, and tissues by fluorescence Chem.Rev. 115 10530–74

[6] Chernousova S and Epple M 2013 Silver as antibacterial agent:ion, nanoparticle, and metal Angew. Chem., Int. Ed. 521636–53

[7] Maity B, Fujita K and Ueno T 2015 Use of the confined spacesof apo-ferritin and virus capsids as nanoreactors for catalyticreactions Curr. Opin. Chem. Biol. 25 88–97

[8] Abe S, Maity B and Ueno T 2016 Design of a confinedenvironment using protein cages and crystals for thedevelopment of biohybrid materials Chem. Commun. 526496–512

[9] Ueno T and Watanabe Y 2013 Coordination Chemistry inProtein Cages: Principles, Design, and Applications (NewYork: Wiley)

[10] Jutz G N et al 2015 Ferritin: a versatile building block forbionanotechnology Chem. Rev. 115 1653–701

[11] Zeth K, Hoiczyk E and Okuda M 2016 Ferroxidase-mediatediron oxide biomineralization: novel pathways tomultifunctional nanoparticles Trends Biochem. Sci. 41190–203

[12] Colton J S et al 2014 Sensitive detection of surface- and size-dependent direct and indirect band gap transitions in ferritinNanotechnology 25 135703

[13] Liu X et al 2012 Apoferritin–CeO2 nano-truffle that hasexcellent artificial redox enzyme activity Chem. Commun.48 3155–7

[14] Leal M P et al 2015 Long-circulating PEGylated manganeseferrite nanoparticles for MRI-based molecular imagingNanoscale 7 2050–9

[15] Felton C et al 2014 Magnetic nanoparticles as contrast agentsin biomedical imaging: recent advances in iron-andmanganese-based magnetic nanoparticles Drug Metab. Rev.46 142–54

[16] Park S-K et al 2014 In situ hydrothermal synthesis of Mn3O4

nanoparticles on nitrogen-doped graphene as high-performance anode materials for lithium ion batteriesElectrochim. Acta 120 452–9

[17] Kim M et al 2014 Hydrothermal synthesis of metalnanoparticles using glycerol as a reducing agent J. Supercrit.Fluids 90 53–9

[18] Sui N et al 2009 Large-scale preparation and catalyticproperties of one-dimensional α/β-MnO2 nanostructuresJ. Phys. Chem. C 113 8560–5

[19] Wang N et al 2007 λ-MnO2 nanodisks and their magneticproperties Nanotechnology 18 475605

[20] Mackle P et al 1993 Characterization of the manganese core ofreconstituted ferritin by x-ray absorption spectroscopyJ. Am. Chem. Soc. 115 8471–2

[21] Meldrum F C et al 1995 Reconstitution of manganese oxidecores in horse spleen and recombinant ferritins J. Inorg.Biochem. 58 59–68

[22] Meldrum F C et al 1991 Synthesis of inorganic nanophasematerials in supramolecular protein cages Nature 349 684–7

[23] Chasteen N D and Harrison P M 1999 Mineralization inferritin: an efficient means of iron storage J. Struct. Biol. 126182–94

[24] Chasteen N D, Antanaitis B C and Aisen P 1985 Irondeposition in apoferritin—evidence for the formation of amixed-valence binuclear iron complex J. Biol. Chem. 2602926–9 http://www.jbc.org/content/260/5/2926.abstract

[25] Klem M T et al 2008 Photochemical mineralization ofeuropium, titanium, and iron oxyhydroxide nanoparticles inthe ferritin protein cage Inorg. Chem. 47 2237–9

[26] Douglas T and Stark V T 2000 Nanophase cobaltoxyhydroxide mineral synthesized within the protein cage offerritin Inorg. Chem. 39 1828–30

[27] Smith T J et al 2014 Tuning the band gap of ferritinnanoparticles by co-depositing iron with halides or oxo-anions J. Mater. Chem. A 2 20782–8

[28] Kim M et al 2011 pH-dependent structures of ferritin andapoferritin in solution: disassembly and reassemblyBiomacromolecules 12 1629–40

[29] Ikezoe Y et al 2009 Random number generation by a two-dimensional crystal of protein molecules Langmuir 254293–7

[30] Huang C H et al 2011 Optical absorption characteristic ofhighly ordered and dense two-dimensional array of siliconnanodiscs Nanotechnology 22 105301

[31] Bellido E et al 2010 Nanoscale positioning of inorganicnanoparticles using biological ferritin arrays fabricated bydip‐pen nanolithography Scanning 32 35–41

[32] Varadan V K et al 2004 Fabrication of cell structures forbionanobattery Proc. SPIE 5389 443–51

[33] Watt G D et al 2012 A protein-based ferritin bio-nanobatteryJ. Nanotechnol. 2012 1–9

9

Nanotechnology 28 (2017) 195601 C R Olsen et al

[34] Hosein H A et al 2004 Iron and cobalt oxide and metallicnanoparticles prepared from ferritin Langmuir 20 10283–7

[35] Keyes J D et al 2010 Ferritin as a photocatalyst and scaffold forgold nanoparticle synthesis J. Nanopart. Res. 13 2563–75

[36] Polanams J, Ray A D and Watt R K 2005 Nanophase ironphosphate, iron arsenate, iron vanadate, and iron molybdateminerals synthesized within the protein cage of ferritinInorg. Chem. 44 3203–9

[37] Johnson J L et al 1999 Forming the phosphate layer inreconstituted horse spleen ferritin and the role of phosphatein promoting core surface redox reactions Biochemistry 386706–13

[38] Huang H et al 1993 Role of phosphate in iron(2+) binding tohorse spleen holoferritin Biochemistry 32 1681–7

[39] Treffry A and Harrison P M 1978 Incorporation and release ofinorganic-phosphate in horse spleen ferritin Biochem. J. 171313–20

[40] Hilton R J et al 2012 Anion deposition into ferritin J. Inorg.Biochem. 108 8–14

[41] Watt R K, Petrucci O D and Smith T 2013 Ferritin as a modelfor developing 3rd generation nano architecture organic/inorganic hybrid photo catalysts for energy conversionCatalysis Sci. Technol. 3 3103–10

[42] Zhang B et al 2005 Kinetic and thermodynamic characterizationof the cobalt and manganese oxyhydroxide cores formed inhorse spleen ferritin Inorg. Chem. 44 3738–45

[43] Theil E C 2003 Ferritin: at the crossroads of iron and oxygenmetabolism J. Nutrition 133 1549s–53s

[44] Price D J and Joshi J G 1984 Ferritin: protection of enzymaticactivity against the inhibition by divalent metal ions in vitroToxicology 31 151–63

[45] Price D and Joshi J G 1982 Ferritin—a zinc detoxicant and azinc ion donor. Proc. Natl Acad. Sci. USA 79 3116–9

[46] Jacobs D L et al 1989 Redox reactions associated with ironrelease from mammalian ferritin Biochemistry 28 1650–5

[47] Ensign D, Young M and Douglas T 2004 Photocatalyticsynthesis of copper colloids from Cu(II) by the ferrihydritecore of ferritin Inorg. Chem. 43 3441–6

[48] Nikandrov V V et al 1997 Light induced redox reactionsinvolving mammalian ferritin as photocatalyst J. Photochem.Photobiol. B 41 83–9

[49] Pisarczyk K 2000 Manganese compounds Kirk-OthmerEncyclopedia of Chemical Technology (New York: Wiley)

[50] Simandi L I et al 1985 Kinetics and mechanism of thepermanganate ion oxidation of sulfite in alkaline-solutions—the nature of short-lived intermediates J. Am. Chem. Soc.107 4220–4

[51] Villalobos M et al 2003 Characterization of the manganeseoxide produced by pseudomonas putida strain MnB1Geochim. Cosmochim. Acta 67 2649–62

[52] Hopwood D 1969 Fixation of proteins by osmium tetroxide,potassium dichromate and potassium permanganate. Modelexperiments with bovine serum albumin and bovine gamma-globulin Histochemie 18 250–60

[53] Bradford M M 1976 A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding Anal. Biochem. 72 248–54

[54] Webster A H and Halpern J 1957 Kinetics of the reduction ofpermanganate in aqueous solution by molecular hydrogenTrans. Faraday Soc. 53 51

[55] Fujimoto T et al 2001 Sonolytical preparation of various typesof metal nanoparticles in aqueous solution Scr. Mater. 442183–6

[56] Butterfield C N et al 2013 Mn(II,III) oxidation and MnO2

mineralization by an expressed bacterial multicopperoxidase Proc. Natl Acad. Sci. 110 11731–5

[57] Cockayne E and Li L 2012 First-principles DFT + U studies ofthe atomic, electronic, and magnetic structure of α-MnO2

(cryptomelane) Chem. Phys. Lett. 544 53–8[58] Li M, Viravaidya C and Mann S 2007 Polymer-mediated

synthesis of ferritin-encapsulated inorganic nanoparticlesSmall 3 1477–81

[59] Meng Y et al 2014 Structure–property relationship ofbifunctional MnO2 nanostructures: highly efficient, ultra-stable electrochemical water oxidation and oxygen reductionreaction catalysts identified in alkaline media J. Am. Chem.Soc. 136 11452–64

[60] Mette K et al 2012 Nanostructured manganese oxide supportedon carbon nanotubes for electrocatalytic water splittingChemCatChem 4 851–62

[61] Kuo C-H et al 2015 Robust mesoporous manganese oxidecatalysts for water oxidation ACS Catalysis 5 1693–9

10

Nanotechnology 28 (2017) 195601 C R Olsen et al