Nature of Interactions of Amino Acids with Bare Magnetite

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On the Nature of Interactions of Amino Acids with Bare Magnetite Nanoparticles Sebastian Patrick Schwaminger, Paula Fraga García, Georg Konrad Merck, Fabian Alexander Bodensteiner, Stefan Heissler, Sebastian Günther, and Sonja Berensmeier J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b07195 • Publication Date (Web): 14 Sep 2015 Downloaded from http://pubs.acs.org on September 19, 2015

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The Journal of Physical Chemistry

On the Nature of Interactions of Amino Acids with Bare Magnetite Nanoparticles Sebastian P. Schwaminger,† Paula Fraga García,† Georg K. Merck,† Fabian A. Bodensteiner,† Stefan Heissler,‡ Sebastian Günther,§ Sonja Berensmeier*,† †

Bioseparation Engineering Group, Technische Universität München, Boltzmannstraße 15,

Garching D-85748, Germany ‡

Karlsruhe Institute of Technology, Institute of Functional Interfaces, Herrmann-von-Helmholtz-

Platz 1, Eggenstein-Leopoldshafen D-76344, Germany §

Chemie Department, Technische Universität München, Lichtenbergstr. 4, Garching D-85748,

Germany

1

ACS Paragon Plus Environment


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ABSTRACT Owing to their chemical and magnetic properties, magnetite nanoparticles are an interesting adsorbing material for biomolecules. The understanding of the interactions of simple biomolecules with inorganic nanoparticles is an important approach for research on the nano-biointerface and can constitute the fundamentals to manifold applications in biotechnology, medicine and catalysis. The aim of the work presented here is to compare the interaction of seven different amino acids (L-alanine, L-cysteine, L-glutamic acid, glycine, L-histidine, Llysine and L-serine) with magnetite nanoparticles in a colloidal system at pH 6. We investigate the influence of the side chain on the adsorption at a magnetite-water interface with incubation experiments. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and simultaneous thermal analysis (STA) reveal deeper insights into the interactions of amino acids with magnetite nanoparticles. The amino acids which contain polar side chains adsorbed on the magnetite nanoparticles to a high degree. Cysteine demonstrated the highest adsorption capacity and the formation of cystine can be observed. ATR-FTIR spectroscopy results indicate a strong influence of the carboxyl group and side chains on the binding mechanism of amino acids at the iron oxide surface. Our investigation offers novel knowledge into adsorption behavior at the bio-nano-interface.

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Introduction Magnetic nanoparticles (MNP) have influenced a great variety of research fields for the last decades. The magnetic properties and size-related behavior of such particles enable interesting applications: catalytic processes,1,2 magnetic storage media3 as well as biotechnological processing4,5 and medicine.6 In medicine, modified particles can be employed in vivo as contrast agents for magnetic resonance imaging7,8 or targeted drug delivery9 and hyperthermia.10 Furthermore, promising applications exist in biological sciences such as support for enzymes,11 cell labeling and cell separation,12 as well as RNA, DNA and protein purification.13,14 The field of coating and functionalization of MNPs for different applications has already been widely reviewed in literature.15,16 However, many aspects of the adsorption process itself still remain unknown. The electrostatic properties of the particles are important for adsorption processes,17 in addition to surface defects, stoichiometry deviations on the surface of MNPs,18 high surface free energy from the high surface-to-volume ratio of nanoparticles19 and the hydroxylation of the surface atoms.20 Different models, such as the charge distribution model,21 force field22 and density functional approaches,23 are known in literature to describe nanoparticle-ligand interaction. However, to our knowledge no specific model exists for magnetite nanoparticles. Owing to their wide biochemical variety, natural amino acids represent a useful reference to improve the understanding of interactions between biomolecules and MNPs.24 They are the building blocks for peptides and proteins and thus for life itself.25 Additionally, amino acids are important for industrial processes, such as solid-phase peptide synthesis,26 the production of pharmaceutical and agrochemical compounds, as well as for applications in biosensors27. Hence, 3



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interaction studies of amino acids with technical surfaces are essential for a deeper understanding of protein adsorption28 and further improvement of separation and purification processes.29,30 Moreover, the adsorption of small organic molecules and condensation of peptide bonds17,31 on minerals and clays might have played an important role for the origin of life32,33 and the biochemical homochirality.34 Hence, a few reviews focus on the adsorption behavior of amino acids on different inorganic surfaces.17,35 Electrostatic interaction plays an important role for the adsorption behavior of zwitterionic molecules such as amino acids and amphoteric hydroxyl groups on metal oxides and is highly dependent on the pH.17 On the other hand, the formation of hydrogen and covalent bonds between amino acids and the surface is also possible.17,33 The formation of covalent anhydrides between the carboxyl groups of amino acids and the surface groups of silicon or aluminum oxide was observed by IR spectroscopy.33 Hydrogen bonding between amino acids and inorganic surfaces, without global electrostatic influences, were also already observed on silica.36,37 Furthermore, the presence of amino acids influences the synthesis of iron oxides and affects the particle size distribution and the colloidal stability in solution.38–40 This matches the complexation observed for carboxyl groups on metal oxides which can be detected by IR spectroscopy.41 The amino acid side chain strongly affects the binding affinity.27,42 Generally, the binding of polar amino acids is influenced by electrostatic effects such as pH change and ionic strength of the liquid phase. However, Gao et al. investigated the binding behavior of the nonpolar amino acids leucine and phenylalanine which demonstrate a higher affinity to hydrophobic surfaces and less dependence on electrostatic effects.27 Even positively charged amino acids like arginine are described to bind through the carboxyl group, although electrochemical investigations of the binding behavior indicate stronger interactions to iron oxide 4


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surfaces for glycine and glutamic acid than for arginine and lysine.43 Another possible binding mechanism is the dissociative adsorption which is described for formic acid on magnetite.44 A special case in the binding behavior is cysteine, which can also form a bond through the thiol group to iron oxide surfaces as observed in spectroscopic and thermogravimetric studies.38 Another possible binding mechanism for cysteine on iron oxides is the formation of cystine, which is able to bind through two carboxyl groups to the iron oxide surface as suggested by Viera et al.45 Nevertheless, other inhibiting factors concerning the adsorption behavior such as nanoparticle agglomeration play an important role in such systems and have also to be considered.46,47 In this article, we compare the adsorption behavior of seven different amino acids on MNPs at pH 6. We focus on the interactions of proteinogenic amino acids with the iron oxide surface. Therefore we use model amino acids for different side chain properties which are illustrated in table 1. Table 1: Side chain properties of amino acids deployed for adsorption experiments with magnetite nanoparticles. Side chain Properties Alanine

CH3

nonpolar

Cysteine

CH2SH

thiole group

Glutamic acid C2H4COO- polar, negatively charged Glycine

H

no side chain

Histidine

C4H5N2

imidazole group

Lysine

C4H8NH3

polar, positively charged

Serine

CH2OH

polar, uncharged

Adsorption is investigated by IR spectroscopy and X-ray photoelectron spectroscopy (XPS) as well as thermogravimetry. By discussing the common and particular results from these methods 5



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for each amino acid, a deeper understanding of the binding behavior and also the validation of the side chain effects is achieved. The goal of our investigation is to extrapolate specific biomolecule-surface interactions to more complex systems such as the adsorption of proteins to nanoparticles. If such interactions can be derived, it is possible to predict the formation of protein coronas and design proteins with specific binding characteristics.28,29 Furthermore, these findings can be used for medical applications10 or the synthesis of nanoparticles where amino acids are often used as stabilizers.38

Materials and methods Synthesis L-Alanine, L-cysteine, L-glutamic acid, glycine, L-histidine, L-lysine and L-serine were selected for the interaction study. L-Cysteine was obtained from Sigma-Aldrich Co. (Germany) while the other amino acids were purchased from SERVA Electrophoresis GmbH (Germany). The synthesis and characterization of magnetite nanoparticles applied has been described elsewhere.48 To 50 mL of iron oxides suspensions (10 g L-1), which were adjusted and equilibrated to a pH of 6, amino acid powders were added according to a concentration 25 mmol L-1. The pH of the mixtures was adjusted to 6 with hydrochloric acid and sodium hydroxide and the concentration of salt ions does not exceed 0.005 mol L-1. The samples were incubated for 24 hours at room temperature and under continuous shaking. The supernatant was decanted from the residue by magnetic separation. The residue was lyophilized by an ALPHA 1-2LDplus (Martin Christ Gefriertrocknungsanlagen GmbH Germany) at a pressure of around 0.02 mbar and -60°C for 3 days, before further characterizations were accomplished. Each experiment was conducted in triplicate. 6


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Characterization Infrared spectroscopy The dried mixtures of MNPs and amino acids were characterized by ATR-IR spectroscopy. A Bruker Optics Tensor 27 spectrometer (Bruker Optics, Ettlingen, Germany), equipped with a Bruker Optics Platinum ATR accessory (diamond crystal, 1 mm2 area with single reflection) and a room temperature deuterated triglycine sulfate (RT-DTGS) detector was applied for all experiments. Each sample was recorded twice against air background (from 4000 cm−1 to 400 cm−1 with a spectral resolution of 4 cm−1), and averaged 64 times. The powder samples were mixed and so the measured position changed on the ATR crystal in between two measurements. For all spectra an atmospheric compensation, an ATR correction (experimental ATR correction factor for magnetite 1.91) and a concave rubber band baseline correction were applied with the software OPUS 7.2. Furthermore, a maximum/minimum normalization was accomplished without consideration of the CO2 region. The spectra of dissolved 0.01 mol L-1 amino acids in water were measured at pH 4, 6 and 8 for comparison purposes and are shown in the Supporting Information (SI) (S1-S8). X-ray photoelectron spectroscopy Chemical analysis was accomplished by X-ray Photoelectron Spectroscopy with a LeyboldHeraeus LHS 10 XPS system in ultra-high vacuum (UHV) hosting a nonmonochromatized AlKα-source (1486.7 eV). The powder samples were fixed on a vacuum compatible copper foil adhesive tape. The spectra were recorded at a constant pass energy mode set to 100 eV and a full width at half maximum (FWHM) of ~1.1 eV. The C 1s (284.5 eV) peak corresponding to adventitious carbon was used as energy reference to compensate energy shifts due to charging. 7



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Detail spectra of the C 1s, O 1s and Fe 2p regions were acquired by repeatedly scanning the same region 30 times in order to reduce statistical noise. In the case of the N 1s region, 50 repetitions were used. All spectra were recorded in an UHV at a pressure below 5x10-8 mbar. The core level spectra were fitted by a mix of Gaussian and Lorentzian functions (Gaussian line width (0.7 eV) and Lorentzian line width (0.3 eV). Shirley backgrounds were subtracted from all N 1s spectra. The spectra in the Fe 2p, O 1s, and C 1s regions are shown in the SI (S9-S11) as well as the reference N 1s spectra of amino acid powders (S12-S19). Simultaneous thermal analysis The binding behavior of amino acids on magnetite nanoparticles was analyzed gravimetrically by a simultaneous thermal analysis system (STA 449C Jupiter, Netzsch Gerätebau GmbH, Germany). The weight loss and the heat transfer of the solid samples were recorded at a heating rate of 10 K min-1 (303 K-1173 K) under nitrogen atmosphere. The gas phase was analyzed by a mass spectrometry system (QMS 403 Aëolos, Netzsch Gerätebau GmbH, Germany). The following mass signals (amu) were recorded to identify decomposition fragments: 16 (O and NH2), 17 (OH and NH3), 18 (H2O), 30 (CH2-NH2), and 44 amu (CO2). Additionally, specific mass fragments for each amino acid were recorded and are shown in the SI (S20-S27). Elemental analysis Elemental analysis of solids was carried out by CHNS combustion analysis using a Euro EA elemental analyzer (HEKAtech GmbH, Germany) and the results are shown in the SI (table S1). Calculation of residue loading The weight loss determined by the STA measurement was up-scaled to the entire batch of each experiment. Here, the percentage of residue at 880°C is divided by the percentage of residue of the magnetite reference and represents the magnetite share in the entire batch. Furthermore, the 8


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oxidation of amino acids by surface oxide groups of the magnetite to CO2, SO2/SO3 and NO/NO2 is considered in our calculations. The residue corresponding to the elemental analysis was determined by the mass content of nitrogen, carbon and sulfur. For the XPS loading, the relative ratio of N 1s:Fe 2p is used for comparison to the other methods. The average value obtained from triplicate experiments was used for the residue analysis by XPS, STA and elemental analysis. The calculations of residue loading are shown in the SI.

Results and Discussion Magnetite nanoparticles The magnetite (Fe3O4) particles used for these experiments were already characterized by Roth et al. with regard to their size and their chemical, crystallographic and physical properties.48 The particles show a uniform size distribution around 14 nm and a spherical shape, evidenced by TEM measurements. Their specific surface area accounts for 101.5 m2 g-1 (S28). The crystal phase is cubic as evidenced by XRD results and Mössbauer spectroscopy states the presence of Fe2+ ions.48 The vibrational levels of Fe-O did not change during the experiments as demonstrated by IR and Raman spectroscopy. The experiments were conducted pH 6, in order to have a controlled positively charged surface and to compare the adsorption of zwitterionic amino acids with positively and negatively charged ones. The point of zero charge was determined by potentiometric acid-base titrations and is shown in Figure 1 and in the SI (S29). Spectra of amino acids (0.1 M) at different pH-values have been analyzed for comparison purposes and are shown in the SI (S1-S7).To visualize the acid-base characteristics of the amino acids in water and explain the content of the IR spectra in the SI, Figure 1 summarizes these states at different pH values. 9



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PZC

Magnetite

7.8

positive pK1 = 2.3

Alanine

negative pK2 = 9.7

H2Ala+ pK1 = 2.0

HAla AlapK2 = 8.2 pK3 = 10.1

Cysteine

H2Cys+ HCys pK1 = 2.2 pK2 = 4.3

Glutamic Acid

H3Glu+ H2Glu pK1 = 2.3

Glycine

H2Gly+ pK1 = 1.8 H3His2+ H2His+ pK1 = 2.2

Lysine

H3Lys2+ pK1 = 2.2

Serine

H2Ser+ 1

2

CyspK3 = 9.7 HGlu-

Glu2GlypK3 = 9.2

HHis HispK2 = 9.0 pK3 = 10.5 H2Lys+

HLys pK2 = 9.2

4

5

LysSer-

HSer

3

Cys2-

pK2 = 9.6 HGly pK2 = 6.0

Histidine

0

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6

7

8

9 10 11 12 13 14

pH

Figure 1: A comparison of the charged states of the magnetite nanoparticle surface (S29) and the acid-base characteristics of deployed amino acids over the pH.49 Positively charged species are illustrated black, while zwitterionic amino acids are displayed hatched and negatively charged species white, respectively. IR characterization Figure 2a shows the IR spectra of magnetite and mixtures with Gly, Lys, Ser and Glu in the region of carboxyl stretching and amino deformation vibrations. The peak in the blank sample spectrum can be attributed to a δs(OH) bending mode of adsorbed water.37 The bands of functional groups of amino acids investigated are contrasted to IR spectra of amino acids dissolved in water at pH 6 in Table 2 and 3. The spectra of the MNP amino acid mixtures, which were lyophilized, diverge significantly from dissolved amino acids (S1-S8). The broadening of the carboxylic stretch vibrations in particular indicates adsorption on the surface.42 This behavior can be attributed to the complexation of the iron oxide surface by the carboxylate group.50 Also, the wavenumber splitting of asymmetric and symmetric stretching of carboxyl group, ∆(νas(COO-) νs(COO-)) is greater compared to ATR-IR spectra of uncoordinated amino acids, indicating a bridged or ionic 10


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coordination between the carboxyl anions and the surface cations.26,42,51 The monodentate species is also connected to an increase of wavenumber splitting in literature,42 yet the wavenumber splitting typically exhibits a much greater gap (∆) than the bands of ionic complexes.52 Moreover, the peak corresponding to the δs(NH3+) band is weakened or disappears after the adsorption for all amino acids but cysteine. This effect has been observed by other groups and related to the interaction of the amino group to the surface.24 On the other hand, Begonja et al. observed no weakening of the δs(NH3+) band for the adsorption of cysteine on a titanium oxide surface and related the charged amino group to an electrostatic adsorption where the amino group plays a significant role.53 Another possibility is an attenuation of the δs(NH3+) band due to a dissociative adsorption process44,54 where the magnetite surface is hydroxylated by the amino group. This dissociative adsorption via formation of an anionic amino acid state has already been stated for the adsorption of lysine on montmorillionite55 and silica56. Glycine demonstrates the greatest shift compared to a glycine solution in water at pH 6. The overlapping bands of δas(NH3+) and νas(COO-), which cannot be separated in the spectra acquired, shift from 1600 to 1591 cm-1. Such a signal indicates strong electrostatic interaction as the vibration is influenced by the ionic strength and also shifts are reported for both bands at higher pH values.26 While the symmetric deformation band δs(NH3+) shifts just from 1513 to 1510 cm-1, the symmetric νs(COO-) band shifts from 1412 to 1378 cm-1. This indicates an interaction of magnetite and glycine through the carboxyl group leading to the additional assumption that this amino acid binds in ionic or bidentate coordination to the surface.52 As glycine is the basic element for other amino acids, we expect a similar adsorption behavior for lysine, glutamic acid, and serine.

11



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With lysine, both asymmetric deformation stretching modes δas(NH3+) (main and side chain) shift significantly from 1600 cm-1 to a broad band at 1584 cm-1 overlapping with the carboxyl mode νas(COO-) which shifted from 1600 to 1584 cm-1. This behavior can be inferred from surface complexation occurring through amino groups, eventually.24,26,57 Although the position of the symmetric deformation δs(NH3+) does not change with the adsorption, the symmetric carboxyl stretch shifts from 1412 to 1393 cm-1 . Thus, the positively charged amino acid lysine rather binds through the carboxyl group to the surface than through the amino group. With serine ∆(νas(COO-) νs(COO-)) is increased by a shift of the symmetric stretch vibration of 23 cm-1. This presents an excellent indication for the formation of ionic or bidentate bridging complexes of serine on the surface.51,52 Interestingly, the band of the symmetric amino stretch vibration cannot be observed in the adsorbed state, and the alcohol group of the side chain δ(OH) shows band broadening and a blueshift to 1036 cm-1 indicating hydrogen bonding.52 Glutamic acid demonstrates, beside the largest bands, also the strongest wavenumber splitting, which is in good agreement with the bands described by Roddick-Lanzillotta et al. for glutamic acid adsorbed on TiO2 at a pH of 6.42 This effect is associated with an adsorption mechanism which can occur through the amino acid side chain, the alpha carboxyl group or a combination of both.58 As those bands are usually overlapping, a broadening of bands indicates differences in the force constants - meaning that only one carboxyl group is adsorbed or they are adsorbed in different states or orientations. The band at 1540 cm-1 can also be attributed to an asymmetric carboxyl group beside the asymmetric stretch at 1597 cm-1.42 Hence, glutamic acid indicates an involvement of the side chain in its adsorption towards magnetite. While there is a possibility for both carboxyl groups to be involved in the adsorption of one single amino acid at the same time, it seems more likely that glutamic acid is able to bind through its side chain carboxyl group or 12


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alpha carboxyl group. The shifts indicate a bridging mechanism of the carboxyl group to the iron ions.42,51,52 Figure 2b compares amino acids with nonpolar side chains. Here, alanine and histidine show a similar adsorption behavior as described for glycine while the spectrum of cysteine indicates a different adsorption behavior. Alanine demonstrates the smallest band broadening as well as the lowest intensities, but still an increase in ∆(νas(COO-) νs(COO-)) can be observed. While the asymmetric carboxyl and amino bands overlap in one peak in the spectrum for alanine at pH 6, the symmetric δs(NH3+) deformation cannot be detected. The symmetric νs(COO-) stretch shifts from 1413 to 1388 cm-1 and states an ionic or bridged bidentate binding of alanine to magnetite.51,52 We observed that cysteine adsorbed in a completely different mechanism than all other amino acids. Viera et al. discuss the formation of cystine by reducing the Fe3+ ions on the surface leading to S-S bond formation of two cysteines.45 2 Fe + 2 cys → 2 Fe + cis + 2 H

(1)

Another group reports the formation of Fe-S bonds for a MNP synthesis with cysteine as surfactant.38 For TiO2, electrostatic interactions with cysteine have been reported.53 In our IR spectrum only the bands corresponding to cystine can be recognized. Furthermore, the bands are slightly shifted compared to solid state cystine while no cysteine could be detected.59 The shifting behavior of the carboxyl stretch and amino deformation vibrations in particular were already observed by Viera et al..45 While the asymmetric carboxyl stretch νas(COO-) shifted from 1578 to 1587 cm-1 and the symmetric carboxyl stretch νs(COO-) shifted from 1403 to 1409 cm-1, a shift for the symmetric δs(NH3+) deformation (1482 to 1489 cm-1) could be observed as well. Other vibrations such as δs(C-H) deformation were not influenced upon adsorption. This 13



indicates that the formed cystine is adsorbed on the surface by carboxyl groups due to electrostatic interactions. The formation of cystine is further evidenced by Raman spectroscopy and the strong S-S vibration found at 500 cm-1, which is shown in figures S30 and S31. Thus, an electrostatic adsorption of cystine on the magnetite surface seems reasonable. The histidine spectrum shows a great band broadening for the bands corresponding to the carboxyl stretch vibrations. Additionally, the wavenumber splitting between asymmetric and symmetric carboxyl stretch ∆(νas(COO-) νs(COO-)) increases as νas(COO-) shifts from 1607 to 1597 cm-1 and νs(COO-) shifts from 1408 to 1382 cm-1. Furthermore, an overlapping and broadening of imidazole ring vibrations can be observed at the band 1083 cm-1.Thus, we assume a contribution of the imidazole ring to the binding behavior of histidine to magnetite nanoparticles. a)

b) Mag-Glu Mag-Ser Mag-Gly Mag-Lys Blank

νas(COO-)

νs(COO-)

Absorbance [a.u.]

νs(COO-)

-

νas(COO )

Absorbance [a.u.]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Mag-Cys Mag-His Mag-Ala Blank

1800 1700 1600 1500 1400 1300 1200 1100 1000

1800 1700 1600 1500 1400 1300 1200 1100 1000

Wave Number [cm-1]

Wave Number [cm-1]

Figure 2: ATR-IR spectra of freeze-dried mixtures of different amino acids adsorbed on MNPs after 24 hours of incubation at pH 6 and bare MNPs (blank) Table 2: Samples measured by ATR-IR spectroscopy after adsorption on MNPs and reference bands of the functional groups of the amino acids glutamic acid, glycine, lysine and serine. Reference spectra of dissolved amino acids were recorded in water at pH 6 (S1-S4) Glu

Gly

Lys

Ser 14


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Bands [cm-1]

Ref (S1)

adsorbed Ref (S2)

adsorbed Ref (S3)

adsorbed Ref (S4)

adsorbed

+

1598

1597

1600

1591

1600

1584

1602

1603

+

δs(NH3 )

1558

1513

1510

1521

1523

1517

νa(COO-)

1598

1597

1600

1591

1600

1584

1602

1603

1405

1399

1412

1378

1412

1396

1407

1385

δa(NH3+) 1521 δs(NH3+) 1521

δa(NH3+) δ(OH) 1510 1054 δs(NH3+) 1510

δa(NH3 )

-

νs(COO )

Side chain ν(C=O) vibrations 1719

νa(COO-) 1540 νs(COO-) 1322

δ(OH) 1036

Table 3: Samples measured by ATR-IR spectroscopy after adsorption on MNPs and reference bands of the functional groups of the amino acids alanine, cysteine and histidine as well the disulfide cystine. The cystine sample was measured in solid state and is shown in SI (S6), while the other references derive from amino acids dissolved in water at pH 6 (S5-S7). Ala Ref (S5)

+

1596

+

δs(NH3 )

1519

νa(COO-)

1596 1413

-1

Bands [cm ] δa(NH3 )

-

νs(COO )

adsorbed

Cys Ref (S6)

Cis Ref (S6)

adsorbed

His Ref (S7)

adsorbed

1592

1603

1622

1624

1607

1595

1517

1482

1489

1521

1497

1592

1603

1578

1587

1607

1595

1388

1399

1403

1409

1408

1382

1089

1083

Ring vibrations

XPS characterization The XP spectra in the N1s region are shown in Figure 3. Since this spectroscopy method presents an atomic probe, the N1s spectral intensity reflects the amount of nitrogen in the specimen and can thus be employed to identify the amount of the adsorbed amino acid. In order to remove inaccuracies by reproducing absolute intensities, we use the intensity ratio of the N 1s:Fe 2p core levels, which should be directly proportional to the amount of adsorbed molecules as long as 15



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molecular loadings are small and no significant difference of the Fe2p signal occurs. Indeed, the Fe2p region shows no significant differences among the several adsorption experiments (S9). The O 1s spectra (S10) demonstrate a broad peak shape, indicating Fe-O and surface hydroxide chemical shifts which has been reported for magnetite surface exposed to water.60,61 The chemical shifts in the XP spectra shown in Figure 3 indicate an uncharged amine group with the N 1s binding energy ranging between 399.2 and 399.4 eV.62 The direct bonding of the amine group to the iron ion, which would correspond to a N 1s binding energy in this region,63 seems unlikely, since higher amounts of nitrogen are evidenced in the XP spectra for glutamic acid than lysine. Another possibility is the formation of hydrogen bonds of the amine group to surface hydroxyl groups and adsorbed water leading to a chemical shift of 1 eV to 400.3 eV.64,65 On the other hand, charged amino groups would be observable, as for adsorbed amino acids on TiO2, if the amine group is involved in the binding mechanism.62,66 Since no charged amino group, which should be found at 401 -404 eV, is visible in the XP spectra of most adsorbed amino acids except for cysteine, we assume that the amino acids do not form an ionic bond to the magnetite surface through the amino group. Furthermore, some authors discuss the possibility of a multilayer formation, which could not be evidenced by XPS in this investigation.67 Hence, we expect that if amino groups interact with the surface of magnetite, the most likely possibility is that this will occur through the formation of hydrogen bonds.68 The cysteine sample shows the broadest peak in the N 1s region and is the only species where a charged amino group could be detected. Besides the reduction of surface iron ions, the adsorption mechanism of cysteine/cystine seems to be different from the other amino acids. The interaction of amino groups with the magnetite surface and a multilayer adsorption cannot be excluded for this amino acid. 16


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With histidine, in addition to hydrogen bound amines, two different nitrogen species, N-H2 and C-N-C, can be evidenced by XPS. Here, compared to the spectrum of histidine adsorbed on metallic surfaces,69 only the C-N-C chemical shift for histidine adsorbed on oxide surfaces is observed.70 This observation leads to the assumption that the imidazole side chain of histidine might play an important role in its adsorption process as stated for the adsorption of histidine on TiO2.24 Thus, we assume that all amino acids investigated adsorb through carboxyl groups on the magnetite surface and in addition to that, the imidazole group of histidine is also involved in the binding mechanism. For the analysis of the peak areas, the two amine groups of lysine and the imidazole ring of histidine have to be considered. While the adsorbed cysteine demonstrates the largest ratio of N 1s peak integral to Fe 2p peak integral in XPS corresponding to the highest load, the other ratios of N 1s to Fe 2p peak areas appear in the following order: Glu>Ser~Gly~His>Lys>Ala. Since in these amino acids the nitrogen content per molecule is identical, or in the case of lysine and histidine normalized, the intensities reflect the adsorption capacity of the MNPs. This order seems to reflect the polarity of the amino acids leading to the assumption that electrostatic interaction between amino acid and surface is stronger than a van-der-Waals interaction. Hence, local interactions must play a dominant role, as proposed previously for other zwitterion adsorption systems.26,71 Figure 4 presents a comparison of the amount of bound amino acids on magnetite, calculated by STA and elemental analysis which is compared to the relative ratio of N 1s:Fe 2p peak integrals, measured by XPS. The discrepancy in some values can be explained by the different coking behavior of amino acids which could not fully be considered for the calculations as well as possible inhomogeneities of the XPS samples.

17



a)

C-NH2

b)

C-N-C C-NH2

C-NH3+ C-NH2 H Bonds C-N-C

H Bonds Cumulative Fit

H Bonds C-NH2

H Bonds C-NH3+

Mag-Ser

Mag-Lys

Mag-Gly

Cumulative Fit Mag-His

Intenyity [a.u.]

Intensity [a.u.]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Mag-Cys

Mag-Glu

Mag-Ala Magnetite

Magnetite 404

402

400

398

396

Binding Energy [eV]

404

402

400

398

396

Binding Energy [eV]

Figure 3: XP Spectra in the N 1s region of bound amino acids on magnetite and reference

STA characterization Additionally to the XPS measurements, the loading of amino acids on magnetite was determined by STA-MS, as shown in Figure 4. The results demonstrate a similar trend compared to the peak integrals obtained by XPS when the mass of each amino acid is considered. Figure 5 presents the normalized values for the XPS, STA and elemental analysis loadings of adsorbed amino acids on the surface. During the STA analysis, all materials released adsorbed water from 100 till 300°C, which is evidenced by the recorded data exhibited for mass 18 (S20-S27). The mass loss of pure magnetite stops at 400°C with only one significant decomposition step and almost no evidence of organic material. The alanine-magnetite serine-magnetite and glycinemagnetite samples decompose in three steps: at 200°C and 350°C. Typical and specific decomposition products are formed and especially the detection of CO2 can be observed in each step. The decomposition behavior of magnetite coated with tyrosine, histidine or tryptophane has already been reported to be similar.39,40

18


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Furthermore, lysine decomposes in 5 steps. In addition to the 2 steps observed for each amino acid, further lysine fragments decompose at 500, 600 and 700°C. These results can be interpreted by the gradual decomposition of the long carbon skeleton of lysine. The alanine-magnetite mixture demonstrates its last decomposition step at around 460°C without formation of CO2 but an indication of nitrogen containing products evidenced by an increase in the mass signal at 30 amu. For the cysteine-magnetite mixture similar steps can be observed. The first decomposition step, after the desorption of water, starts at 260°C and a second step at 340°C, where fragments of cystine are detected, can be observed. This result also confirms the theory of Viera et al. who stated a formation of cystine by reduction of cysteine through the surface iron ions.45 Besides the enormous discrepancy in the amount of adsorbed cysteine, as derived by STA with respect to the other samples, this particular system demonstrated two further significantly higher decomposition temperatures at 700 and 800°C (indicated by the formation of CO2). This observation could be attributed to a strong complex formation between the carboxyl groups and the surface. Another possibility is the formation of a Fe-S bond, which also breaks around 800°C.72 However, the latter explanation is unlikely, as only the formation of an S-S bond was observed by Raman spectroscopy (S30 and S31). Additionally, the same decomposition temperatures as described above with smaller amounts of weight loss were observed for glutamic acid magnetite samples. Histidine shows different decomposition behavior as the other amino acids. Here, additionally to the decomposition steps at 200 and 350°C, extremely sharp mass losses occur at 300 and 500°C. This behavior might be explained by the decomposition of the imidazole ring and is an indicator for the involvement of the imidazole ring upon adsorption of histidine on magnetite.73 19



Blank Mag-Ala Mag-Gly Mag-Ser Mag-Glu Mag-Lys Mag-His Mag-Cys

Weightloss

1.00

0.95

0.90

0.85

0.80 100 200 300 400 500 600 700 800

Temperature [°C]

Figure 4: Thermogravimetric analysis of all amino acid magnetite mixtures and a reference

1.4

0.030

1.2

Thermal analysis Elemental analysis 0.025 XPS

1.0

0.020

0.8 0.015 0.6 0.010

0.4

0.005

0.2 0.0

0.000

Cys

Glu

Ser

His

Gly

Lys

Ala

Ratio of area increments N1s:Fe2p [a.u.]

sample.

Residue [mmolg-1]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 5: Comparison between XPS (relative ratio of N 1s:Fe 2p), STA and elemental analysis data of the amino acid residues on magnetite nanoparticles.

Binding mechanisms The analysis of the final state of adsorbed amino acids on nanoparticles by ATR-IR spectroscopy, XPS and STA offers us insights towards the binding mechanisms. The existence of surface hydroxyl groups is evidenced by ATR-IR and XP spectroscopy. Positively charged 20


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amino groups can only be observed for cysteine where the formation of the disulfide cystine is evidenced by Raman spectroscopy (S30 and S31). Cysteine reacts with the surface iron ions of magnetite reducing ferric ions to ferrous ions and forming cystine.45 The most probable binding mechanism for all amino acids is the formation of an ionic interaction between the negatively charged carboxyl group and the positively charged surface at pH 6 (S29). This observation is emphasized by the wavenumber splitting of the COO- bands in the ATR-IR spectra which is in the region for ionic and bridged bidentate bonds.52 The broad peak shapes indicate an overlap of those binding states. Especially the broadest νs(COO-) band of glutamic acid which is slightly blueshifted compared to the other adsorbed amino acids indicates this overlapping of different binding states. As the wavenumber splitting is smaller for glutamic acid we reason a stronger electrostatic binding component than for alanine and glycine. The partly formation of strong coordination bonds is furthermore suggested by STA where the release of CO2 components is shown at temperatures above 700°C (S20-S27). The absence of peaks corresponding to positively charged amino groups in XP and ATR-IR spectra can indicate a dissociative adsorption where the amino group is deprotonated by the magnetite surface. Such an adsorption of an anionic amino acid species is in good agreement with literature.55,56 Conjoint with an ionic binding and bridging mechanism between the carboxyl groups and the surface, the formation of hydrogen bonds between the amino groups and surface hydroxyl groups or other amino acids or physisorbed water plays a role in the adsorption process as indicated by our XPS results. The most probable binding states at pH 6 according to our findings are summarized in Scheme 1. These results match the observations for the adsorption on different metal oxides by other groups.26,36,42,56,57 These findings do not exclude lateral interactions which we presume for cysteine since the surface coverage is 5.53 molecules nm-2 (table S2). While the area required for 21



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carboxyl groups to adsorb is around 0.16 nm2,74 a lateral interaction between molecules and a multilayer adsorption is probable. Furthermore, the existence of a charged amino species in XPS and ATR-IR analysis confirms the hypothesis of multilayer adsorption.67 The absence of the amino groups (NH3+) in the XPS spectra and the lower surface coverages for the other amino acids (table S2) leads us to the conclusion that the interaction is with the surface only. Bridging coordination

R

Ionic coordination

Coordination and H-bonding

NH2

O

O

Fe2+/Fe3+

Fe2+/Fe3+

Magnetite

Serine

Glutamic acid NH3

NH2 O H OH Fe2+/Fe3+

O

O

Fe2+/Fe3+

Fe2+/Fe3+

Magnetite

O

O

O

Magnetite

Histidine

Cystine

H3N

NH3 S

Magnetite

O

Fe2+/Fe 3+ Fe2+/Fe3+ Fe 2+/Fe3+ Fe2+/Fe 3+

S

O

O

O

O

Fe2+/Fe3+

Fe2+/Fe3+

Fe2+/Fe3+

Fe2+/Fe3+

Magnetite

Scheme 1: Structure of different binding states of amino acids on a magnetite surface and possible interactions of side chains with the surface in water. The binding states of amino acids side chains are drawn in bridging coordination to iron ions but can also occur in ionic coordination or through surface hydroxyl groups.

Conclusion The binding behavior of amino acids on magnetite nanoparticles in water follows complex mechanisms which are influenced by the side chains. Our data suggests different binding 22


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mechanisms for cysteine, glutamic acid, histidine and serine. The binding mechanism of glutamic acid can either include alpha or side chain carboxyl group as well as the binding of both carboxyl groups at the same time. Furthermore, the IR spectra of histidine and serine indicate an involvement of the imidazole and the hydroxyl group in the bonding to magnetite nanoparticles respectively. For histidine this observation can be further emphasized by XPS data. These side chain effects can be utilized for the prediction of the binding behavior of larger molecules like proteins to magnetic nanoparticles and therefore enable new possibilities in biotechnology and medicine.28 The polarity of the amino acids is also decisive for the adsorption, as already observed for amino acids on some metal oxide surfaces from water by other authors.17,27 Therefore, an order of decreasing adsorption capacity can be inferred from our results: Cys>Glu>Ser~His~Gly>Lys>Ala. For cysteine, the high amount of 5.53 molecules nm-2 is adsorbed on the particles, which is an indicator for multilayer adsorption as is emphasized by XPS data. The other amino acids demonstrate an adsorption capacity between 1.84 and 0.66 molecules nm-2 which is in good agreement with the few existing literature values for the adsorption of small molecules on inorganic nanoparticles.21,24 The carboxyl group is the most important factor for the binding of amino acids though unipolar interactions occur on the surface of MNPs as well. Cysteine represents a special case with a particular binding form, which leads to significantly higher capacity than for the rest of the studied amino acids. Moreover, the formation of an S-S bond is evidenced instead of bonding through the thiole group to magnetite. The overall analysis of our data shows that besides carboxyl and amino groups, the reactivity of the magnetite nanoparticle surface and the redox chemistry of Fe3+ and Fe2+ ions play an important role upon adsorption of biomolecules. The surface charge of magnetite nanoparticles exerts a decisive influence on ionic interactions and the formation of hydrogen bonds. In order to 23



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correlate the adsorption behavior to the surface charge further investigations are required. We demonstrate the versatility of adsorption mechanisms of biomolecules to MNPs which is dependent on the sidechains and the molecular skeleton as similarities for nonpolar as well as positively and negatively charged amino acids exist at the bio-nano-interface.

ASSOCIATED CONTENT Supporting Information: Full range IR spectra and IR spectra of dissolved amino acids at different pH values. XP spectra of magnetite and magnetite amino acid spectra at regions Fe2p, O1s and C1s. XP Spectra at the region N1s for pressed amino acid powder pellets and the experimental details for amino acid samples. Mass spectroscopy data of thermogravimetic analysis for magnetite and amino acid magnetite mixtures. Experimental details on the determination of the specific surface area and the point of zero charge. Experimental details of Raman spectroscopy measurements and Raman spectra of magnetite, cysteine, cystine and magnetite cysteine mixture. Elemental analysis data, calculations for loads and surface coverages and ATR-IR spectra of solid state amino acids. This material is available free of charge through the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes 24


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The authors declare no competing financial interest. ACKNOWLEDGMENT The authors would like to express their gratitude to Florian Hein and Korbinian Huber for synthesis of magnetite particles. Furthermore, we are particularly appreciative for the financial support of this work by the Federal Ministry of Education and Research (Grant number 031A173A). References (1) Gawande, M. B.; Branco, P. S.; Varma, R. S. Nano-Magnetite (Fe3O4) as a Support for Recyclable Catalysts in the Development of Sustainable Methodologies. Chem. Soc. Rev. 2013, 42, 3371–3393. (2) Polshettiwar, V.; Luque, R.; Fihri, A.; Zhu, H.; Bouhrara, M.; Basset, J.-M. Magnetically Recoverable Nanocatalysts. Chem. Rev. 2011, 111, 3036–3075. (3) Sun, S. Monodisperse FePt Nanoparticles and Ferromagnetic FePt Nanocrystal Superlattices. Science 2000, 287, 1989–1992. (4) Berensmeier, S. Magnetic Particles for the Separation and Purification of Nucleic Acids. Appl. Microbiol. Biotechnol. 2006, 73, 495–504. (5) Heyd, M.; Franzreb, M.; Berensmeier, S. Continuous Rhamnolipid Production with Integrated Product Removal by Foam Fractionation and Magnetic Separation of Immobilized Pseudomonas Aeruginosa. Biotechnol. Prog. 2011, 27, 706–716. (6) Colombo, M.; Carregal-Romero, S.; Casula, M. F.; Gutiérrez, L.; Morales, M. P.; Böhm, I. B.; Heverhagen, J. T.; Prosperi, D.; Parak, W. J. Biological Applications of Magnetic Nanoparticles. Chem. Soc. Rev. 2012, 41, 4306. (7) Qiao, R.; Yang, C.; Gao, M. Superparamagnetic Iron Oxide Nanoparticles: From Preparations to In Vivo MRI Applications. J. Mater. Chem. 2009, 19, 6274. (8) Lee, N.; Hyeon, T. Designed Synthesis of Uniformly Sized Iron Oxide Nanoparticles for Efficient Magnetic Resonance Imaging Contrast Agents. Chem. Soc. Rev. 2012, 41, 2575–2589. (9) Mikhaylov, G.; Mikac, U.; Magaeva, A. A.; Itin, V. I.; Naiden, E. P.; Psakhye, I.; Babes, L.; Reinheckel, T.; Peters, C.; Zeiser, R.; et al. Ferri-Liposomes as an MRI-Visible Drug-Delivery System for Targeting Tumours and their Microenvironment. Nat. Nanotechnol. 2011, 6, 594–602. (10) Mahmoudi, M.; Serpooshan, V.; Laurent, S. Engineered Nanoparticles for Biomolecular Imaging. Nanoscale 2011, 3, 3007–3026. (11) Wei, H.; Wang, E. Nanomaterials with Enzyme-Like Characteristics (Nanozymes): Next-Generation Artificial Enzymes. Chem. Soc. Rev. 2013, 42, 6060–6093. (12) Pan, Y.; Du, X.; Zhao, F.; Xu, B. Magnetic Nanoparticles for the Manipulation of Proteins and Cells. Chem. Soc. Rev. 2012, 41, 2912–2942. (13) Liu, G.; Gao, J.; Ai, H.; Chen, X. Applications and Potential Toxicity of Magnetic Iron Oxide Nanoparticles. Small 2013, 9, 1533–1545.

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Table of Contents

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Scheme 1: Structure of different binding states of amino acids on a magnetite surface and possible interactions of side chains with the surface in water. The binding states of amino acids side chains are drawn in bridging coordination to iron ions but can also occur in ionic coordination or through surface hydroxyl groups. 97x116mm (600 x 600 DPI)


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Figure 1: A comparison of the charged states of the magnetite nanoparticle surface (S29) and the acid-base characteristics of deployed amino acids over the pH.49 Positively charged species are illustrated black, while zwitterionic amino acids are displayed hatched and negatively charged species white, respectively. 24x20mm (600 x 600 DPI)


Nature of Interactions of Amino Acids with Bare Magnetite

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