Novel Humic Acid-Bonded Magnetite Nanoparticles for Protein Immobilization Mevlut Bayrakci, Orhan Gezici, Salih Zeki Bas, Mustafa Ozmen, Esra Maltas PII: DOI: Reference:

S0928-4931(14)00349-X doi: 10.1016/j.msec.2014.05.066 MSC 4690

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

22 January 2014 15 April 2014 30 May 2014

Please cite this article as: Mevlut Bayrakci, Orhan Gezici, Salih Zeki Bas, Mustafa Ozmen, Esra Maltas, Novel Humic Acid-Bonded Magnetite Nanoparticles for Protein Immobilization, Materials Science & Engineering C (2014), doi: 10.1016/j.msec.2014.05.066

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1 Novel Humic Acid-Bonded Magnetite Nanoparticles for Protein Immobilization

Nigde University, Ulukisla Vocational School, 51100 Ulukisla Nigde/Turkey

c

Nigde University, Department of Chemistry, 51100 Campus Nigde/Turkey

RI

b

Selcuk University, Department of Chemistry, 42031 Campus Konya/Turkey

SC

a

PT

Mevlut Bayrakcia,*, Orhan Gezicib,†, Salih Zeki Basc, Mustafa Ozmenc, Esra Maltasc

NU

Abstract

The present paper is the first report that introduces (i) a useful methodology for chemical

MA

immobilization of humic acid (HA) to aminopropyltriethoxysilane-functionalized magnetite iron oxide nanoparticles (APS-MNPs) and (ii) human serum albumin (HSA) binding to the obtained

D

material (HA-APS-MNPs). The newly prepared magnetite nanoparticle was characterized by

TE

using Fourier Transform Infrared Spectroscopy (FTIR), Transmission Electron Microscope

AC CE P

(TEM), Scanning Electron Microscopy (SEM), Thermogravimetric Analysis (TGA), and elemental analysis. Results indicated that surface modification of the bare magnetite nanoparticles (MNPs) with aminopropyltriethoxysilane (APS) and HA were successfully performed. The protein binding studies evaluated in batch mode exhibited that HA-APS-MNPs could be efficiently used as a substrate for the binding of HSA from aqueous solutions. Usually, recovery values higher than 90% were found to be feasible by HA-APS-MNPs, while that value was around 2% and 70% in the cases of MNPs and APS-MNPs, respectively. Hence, the capacity of MNPs was found to be significantly improved by immobilization of HA. Furthermore, thermal degradation of HA-APS-MNPs and HSA bonded HA-APS-MNPs was evaluated in terms of the Horowitz-Metzger equation in order to determine kinetic parameters for thermal decomposition. *

Corresponding author. Tel.: +90 388 2252106 fax: +90 388 2250180; e-mail: [email protected] Present address for O. Gezici: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich 8093, Zurich, Switzerland. †

ACCEPTED MANUSCRIPT 2 Activation energies calculated for HA-APS-MNPs (20.74 kJmol-1) and HSA bonded HA-APSMNPs (33.42 kJmol-1) implied chemical immobilization of HA to APS-MNPs, and tight

PT

interactions between HA and HA-APS-MNPs.

RI

Keywords: Humic substances; Human serum albumin (HSA); Magnetite nanoparticle; Protein

SC

binding; Horowitz-Metzger method.

NU

1. Introduction

MA

Interactions between solid substrates and proteins (and other biomolecules) are important in various applications such as protein purification and separation, peptide synthesis, and

D

diagnostic assays [1,2]. Despite the fact that interaction of proteins with material surfaces is a

TE

complicated phenomenon, protein binding to a solid surface leads development and design of different types of tools and techniques such as biochips, biosensors, bioreactors and diagnostic

AC CE P

techniques [3]. In a number of disciplines such as biology, biotechnology, biochemical engineering and biomedicine, interaction between proteins and surfaces play an important role [4]. For instance in biomedicine, protein binding on polymer particles includes the following: Artificial tissues and organs, drug delivery systems, biosensors, solid-phase immunoassays, immunomagnetic cell separations, and immobilized enzymes [5-8]. In recent years, separation and purification of target protein and elucidation of protein function are some of the major tasks facing researchers. Thus, development of tools to enhance protein studies is critical. Many tools have been developed to immobilize, purify or separate individual proteins from biological matrices. With this respect, application of magnetite nanoparticles, MNPs, (microspheres, nanospheres and ferrofluids) in the mentioned processes purification, separation and immobilization of protein and enzymes is straightforward [9-11].

ACCEPTED MANUSCRIPT 3 Iron oxide magnetite particles are a group of the paramagnetic nanoparticles which are usually modified with various functional groups such as epoxy, amine and aldehyde in order to

PT

improve their efficiency [12-14]. Immobilization of biomolecules to a material possessing

RI

magnetic feature leads quick, easy and gentle separation of biological molecules and macromolecules by using an external magnetic field gradient [15-17]. Although some

SC

nanomaterials have excellent physical and chemical bulk properties, they do not possess suitable

NU

surface properties for specific applications. Consequently, it may be necessary to modify the surface of such materials properly [18-19]. Modification of the surface of magnetite nanoparticles

MA

with functional groups enhances recognition properties, and affinity of MNP towards target biomolecule. The most common way is to attach suitable organic groups to the surface.

D

Humic acid (HA), which occurs naturally by decomposition of mainly plant residues, is a

TE

plenty of natural macromoleculer organic matter on earth [20]. HA has a high interaction

AC CE P

capability with different types of species owing to its multifunctional macromolecular structure containing carboxyl, phenolic hydroxyl, carbonyl, methoxyl, alcoholic hydroxyl, ether and amino groups covalently bonded to a hydrophobic framework [21-23]. Although HA has a multifunctional character and thus constitutes a suitable environment for various species, some drawbacks, such as its high solubility and difficulties encountered to separate it from its suspensions, etc., restricts utilization of solid HA as a substrate [24]. For this reason, immobilization of HA to a suitable solid support with good mechanical properties is deemed important in order to take the benefit of its multifunctional character [25]. In the past three decades, different types of inorganic and organic backbones, such as anion-exchange resins [26], hematite particles [27], alginate gels [28], synthetic polymers [29] and silica particles [30-34] were used as solid support for immobilization of HA. At this context, it is a good tentative idea to combine the multifunctional character of HA and magnetic properties of iron oxide particles

ACCEPTED MANUSCRIPT 4 through a careful immobilization of HA [35]. Despite the fact that there is a growing interest on HA-based magnetite nanoparticles, the concept of preparation of HA-based magnetite

PT

nanoparticles are usually based on co-precipitation and/or physical interactions between

RI

magnetite nanoparticle and HA. This leads materials with poor mechanical strength and thus makes it difficult to utilize the obtained materials over a wide range of pH. Although the

SC

environmental applications of magnetite nanoparticles based HA have been extensively studied

NU

[36-38], yet, little or no literature is available on HA-protein interaction. So, HA-bonded magnetite nanoparticles are expected to be a suitable linker system for protein immobilization.

MA

Therefore, the aim of this study is to explore the applicability of a new methodology for immobilization of HA to aminopropyl trietoxysilane modified superparamagnetite ironoxide

D

nanoparticle via covalent bond formation and to explore protein binding efficiency of the

AC CE P

2. Experimental

TE

obtained material toward Human Serum Albumin (HSA).

2.1. Materials and Methods

Ferric chloride hexahydrate (FeCl3·6H2O, >99%), ferrous chloride tetrahydrate (FeCl2·4H2O, >99%), tris(hydroxymethyl)-aminomethane (99.8–100.1%), ammonium hydroxide (25%, w/w), sodium hydroxide (≥97%), hydrochloric acid (37%) and ethanol (>99.2%) were obtained from Merck (Darmstadt, Germany). (3-Aminopropyl)-triethoxysilane (APS, 99%), Albumin from human serum (97–99%) and phosphate–buffered saline solution (PBS: pH 7.4, 0.1M) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium form of Aldrich HA was used as the source of HA after a simple purification process [25,32] that involves dissolution under alkaline conditions, filtration of undissolved particles and re-precipitation of HA by addition of acid. In this way, the material was also turned into its protonated form. HA was turned into

ACCEPTED MANUSCRIPT 5 humyl chloride (HUM-Cl) by reaction of oxalyl chloride (Merck). Dimethylformamide (DMF, Merck), triethylamine (TEA, Merck), acetone (Merck) and dichloromethane (Merck) were the

PT

solvents and/or reagents used during the immobilization of HA to APS-MNPs and subsequent

RI

purification steps.

All aqueous solutions were prepared with deionized water that had been passed through a

SC

Millipore Milli-Q Plus water purification system. All chemicals were of analytical grade and used

NU

as received.

MA

2.2. Instruments

Elemental analyses were performed by a Leco CHNS–932 analyzer, and changes in elemental

D

composition were used to evaluate the efficiency of surface modifications. IR spectra were

TE

recorded on a Perkin–Elmer spectrum 100 FTIR spectrometer with an ATR compartment. The spectra were compared with each other to understand mechanism of surface modifications and

AC CE P

protein binding to HA-APS-MNPs. Thermogravimetric analysis (TGA) was carried out with a Setaram SETSYS thermal analyzer at temperature range of 25-950ºC at a heating rate of 10ºC min-1 under argon atmosphere with a gas flow rate of 20 mLmin-1. The size and shape of the NPs were determined by transmission electron microscope (TEM, FEI Company-TecnaiTM G2 Spirit/Biotwin). SEM images were obtained using a Zeiss LS-10 field emission SEM instrument equipped with an Inca Energy 350 X-Max (Oxford Instruments) spectrometer. Samples were sputter-coated with Au (60%) and Pd (40%) alloy using a Q150R (Quorum Technologies) instrument. Images were obtained at 3×10−4 Pa working pressure and 15 keV accelerating voltage using InLens detection mode (2 mm working distance). Fluorescence spectra of HSA were recorded on a Perkin-Elmer LS-55 fluorescence spectrometer, and the instrument was used to

ACCEPTED MANUSCRIPT 6 determine the concentration of HSA in solutions. Finally, a combination Orion 410 A+ pH meter

2.3. Magnetite Nanoparticles and Surface Modifications

PT

was used for the pH measurements.

RI

Bare MNP were prepared according to the method described [9]. Briefly, 3.1736 g of

SC

FeCl2·4H2O (0.016 mol) and 7.5684 g of FeCl3·6H2O (0.028 mol) were dissolved in 320 mL of deionized water, such that Fe2+/Fe3+ = 1/1.75. The mixed solution was stirred under N2 at 80°C

NU

for 1 h. Then, 40 mL of NH3·H2O was injected into the mixture rapidly, stirred under N2 for

MA

another 1 h and then cooled to room temperature. The precipitated particles were washed five times with hot water until the supernatant reached pH 7 and separated by magnetic decantation.

D

Finally, magnetic nanoparticles were dried under vacuum at 70°C.

TE

In order to improve the efficiency of surface modification, it is a very common way to attach

AC CE P

proper linkers to the surface before immobilization of a target macromolecule [9]. Surface silanization with aminopropyltriethoxysilane (APS) is one of the useful methods used for this purpose. Aminopropyl groups on the surface of the obtained product (APS-MNPs) are good linkers for the attachment of HA via amide bond formation mechanism. The same mechanism was used for immobilization of HA to aminopropyl silica, and the obtained materials were found to be stable [25,32]. By turning carboxylic acid groups of HA into their respective acid chlorides (Humyl chloride, HUM-Cl) [39], the efficiency of HA immobilization onto an aminopropylfunctionalized surface can be improved. Therefore, surface modification of MNPs with HA was performed on the basis of reaction between APS-MNPs and HUM-Cl. APS-MNPs were prepared according to the methods described in the literature [40,41]. Briefly, 4.2252 g MNPs were sonicated in 150 mL ethanol/water (volume ratio, 1:1) solution for

ACCEPTED MANUSCRIPT 7 30 min to get uniform dispersion. Then 16.1600 g of APS was added to solution under N2 atmosphere at 40°C for 2 h. After that the solution was cooled to room temperature. The prepared

PT

APS-MNPs were collected with a magnet, and washed with ethanol, followed by deionized water

RI

for three times. Finally, APS-MNPs was dried under vacuum at 70°C.

SC

HUM-Cl was prepared by adding 25 mL of anhydrous oxalyl chloride to 1.50 g of dried HA in 50 mL of anhydrous DMF with stirring. The reaction mixture was refluxed for 72 h, and the

NU

resulting mixture was evaporated to dryness under reduced pressure. Finally, obtained crude

MA

product was used immediately for the next step without further purification. 1.5 g of APS-MNPs was interacted with the prepared HUM-Cl in presence of 1.00 mL of TEA in 50 mL of anhydrous

D

DMF for 72 h at 60°C. After magnetic separation, obtained magnetite nanoparticles were washed

TE

in sequence three times with warm DMF, dichloromethane, acetone and distilled water. Acid chloride groups of immobilized HUM-Cl turn rapidly into their respective carboxylic acid (and/or

AC CE P

carboxylate) groups through the final washing step, yielding HA-APS-MNPs (Scheme 1). The product was dried under vacuum at 100 C for 3 h and kept in a desiccator before use. According to the results of thermal analyses, amount of HA bonded onto APS-MNPs was found to be approximately 151 mg HA/g of magnetite nanoparticle. IR νmax (ATR)/cm-1: 3110; 2933; 1714; 1505; 1204; 1137; 549. Please insert here Scheme 1. 2.4. Protein Binding Different amounts of HA-APS-MNPs ranging from 5 to 30 mgmL-1 were mixed with 0.1 mgmL-1 of HSA solution in 20 mM×Tris (pH 7.4). Each mixture was tumbled for 2 h at 4°C [9,42]. Protein bounded HA-APS-MNPs was separated from supernatant by means of a magnet.

ACCEPTED MANUSCRIPT 8 Unbounded protein from HA-APS-MNPs was removed by centrifugation at 5000 rpm for 5 minutes at 4°C. Supernatant was kept at 4°C for analysis of protein concentration. HSA bounded

PT

support was washed three times with 20 mM×Tris buffer. It was also washed with ethanol for

RI

chemical characterization.

The amounts of residual HSA in the supernatant after the immobilization of HSA on HA-

SC

APS-MNP were determined by using fluorescence spectroscopy. For this purpose, intrinsic

NU

fluorescence of protein was recorded at 280 and 342 nm of excitation and emission wavelengths, respectively [42,43]. Unbounded protein concentration was calculated from linear calibration

MA

curve (r=0.998) obtained via analysis of external standard solutions of HSA having different

D

concentrations using the same instrument.

TE

3. Results and Discussions

AC CE P

3.1. Surface Modifications

The performance of the surface modifications was evaluated in terms of elemental analysis, FTIR, TGA, TEM and SEM. Elemental percentage of a material usually changes after a surface modification, and this property can be used to evaluate surface modification of MNP with the linker APS and the target macromolecule HA. Percentages of C, H, and N in APS-MNPs and HA-APS-MNPs are seen in Table 1. As an effect of HA immobilization to APS-MNP, percentage of C-atom seems to be almost doubled (i.e. 7.20 and 14.31% for APS-MNP and HAAPS-MNP, respectively). Amount of aminopropyl groups per 1.0 g of APS-MNPs was calculated on the basis of elemental abundance of N atom in APS-MNPs and found as 0.95 mmol APS/g APS-MNPs. Immobilization yield for HA was calculated from thermograms of APS-MNPs and HA-APS-MNPs, and found as 151 mg HA/g HA-APS-MNPs (on the dry material basis).

ACCEPTED MANUSCRIPT 9 Please insert here Table 1 and Fig. 1 ATR-FTIR spectroscopy was used to elaborate the structure of MNPs, APS-MNPs and

PT

HA-APS-MNPs. In FTIR spectra (Fig. 1), the bands related with vibrations of Fe-O bonds are

RI

seen at low wave numbers (≤700 cm-1). Hence, band around 550 cm-1 was ascribed to the

SC

stretching vibration mode of Fe-O bonds in magnetite nanoparticle(MNP). The introduction of aminopropyl groups on the surface of MNPs was confirmed by two broad bands (at around 3300

NU

and 1605 cm-1) which were assigned to N–H stretching vibration and NH2 bending mode of free

1

MA

NH2 group, respectively. In the FTIR spectra of HA-APS-MNPs, the bands at 1505 and 1700 cmwere attributed to the stretching vibrations of C=O bond in carboxylate and carboxylic acid

D

groups of HA, respectively [20,23]. The band around 1031 cm-1 was ascribed to the deformation

TE

vibration of C-H bond of benzene ring. Also, the presence of the anchored propyl group of the solid support and -CH3 and -CH2 groups in structure of HA were confirmed by C–H stretching

AC CE P

vibrations that appeared around 2890-2900 cm−1. Compared the spectra of APS-MNPs and HAAPS-MNPs, both appearance of new broad bands and disappearance of free NH2 bands indicated that the surface modification reaction successfully occurred as proposed. It should be noted that complex macromolecular structure of HA restricts gathering further information about immobilization of HA to APS-MNPs from FTIR spectra. Both TG and SEM and TEM analyses revealed immobilization of humic macromolecules onto APS-MNPs. 3.2. Protein Binding to HA-APS-MNPs It is well-known that HA has a skeleton of alkyl and aromatic units that attach with carboxylic acid, phenolic hydroxyl, and quinone functional groups. Owing to its multifunctional nature, HA is an efficient substrate for toxic metal cations and neutral organic molecules [44,45]. Owing to its hydrophobic network and abundant hydrophilic groups including ionizable groups,

ACCEPTED MANUSCRIPT 10 HA easily interacts with metal ions, oxides and some poisonous organic matters through dipoledipole interactions, ion-dipole attraction, charge-transfer interactions, hydrophobic interactions,

PT

chelating, ion exchange reactions and hydrogen bonding [46]. Proteins are expected to be

RI

attracted by HA through one or a combination of different types of interactions. Therefore, we have performed some preliminary evaluations to investigate binding efficiency of HA-APS-

SC

MNPs for HSA by using solid-phase extraction. Unbounded HSA was determined by using

NU

fluorescence spectroscopic method after the removal of HSA from particles. Amount of HSA binding to magnetic nanoparticles was measured at 280 nm of excitation and emission

MA

wavelengths (Fig. 2). The results showed that HSA could be extracted from aqueous solution at

Please insert here Fig. 2

TE

D

pH 7.4 with a good efficiency. The results are summarized in Table 2 and Fig. 3.

AC CE P

Please insert here Table 2 and Fig. 3

Results showed that protein binding slightly increased with increasing of support concentrations for HSA. From the obtained results, maximum percentage of HSA binding was observed in the case of HA-APS-MNPs (99.74%) at pH 7.4 (1×Tris-HCl) at a concentration of 30 mgmL-1. Because of the functional groups present in their structure, proteins can interact with surface of magnetite nanoparticles through covalent or non-covalent interactions. In the case of HA-APS-MNPs, HSA is expected to interact through weak interaction forces such as π-π interactions, dipole-dipole bonding, and/or electrostatic attraction may be another important contributions to the interaction between HA-APS-MNPs and HSA [47]. Hence, macromolecular structure of HA constitutes a suitable medium for proteins having aliphatic amino acid

ACCEPTED MANUSCRIPT 11 components favors attachment of proteins to HA [48-51]. Another possible explanation of the observed behavior is the presence of a very large number of reactive carboxylic groups of HA

PT

which suitable to covalently bind the protein through an amide bond. This behavior can be

RI

explained taking into account hydrogen bonding interaction between adjacent carboxylic groups which lead to an increase in the polarization of the C=O bond, making the carbon atom more

SC

positive and so favoring the nucleophilic attack by the NH2 termination of the protein [52]. This

NU

possible amide bond formation between protein and carboxylic groups of HA on the surface of magnetite nanoparticles was supported by FTIR (ATR) analysis. When the spectra of the HSA-

MA

HA-APS-MNPs are compared with that of the HA-APS-MNPs, the new broadened and shifted

AC CE P

TE

Please insert here Fig. 4 and 5

D

bands around 1650 cm–1 and 1500-1550 cm–1 can be attributed to the possible amide bonds [52].

Furthermore, protein immobilization onto HA-APS-MNPs was characterized by SEM, TEM and TGA (Fig. 4, Fig. 5 and Fig. 6). In the SEM result of protein immobilized nanoparticle, the characteristic protein crystals, which were adhered to nanoparticle surface, were clearly detectable in the mixture, thus, confirming the presence of human serum albumin protein (Fig. 4). In order to receive more direct information on the particle size and morphology, transmission electron microscopy (TEM) micrographs of pure Fe3O4 nanoparticles and HSA-HA-APS-MNPs were investigated (Fig. 5). Observing the TEM micrographs, nanoparticles formed dense aggregates due to the lack of any repulsive force between the magnetic nanoparticles. This force is mainly due to the nano-size of the Fe3O4, which is about 10 ± 2 nm. After protein immobilization, the dispersion of particles was improved greatly, which can easily be explained by the electrostatic repulsion force and steric hindrance between protein species on the surface of

ACCEPTED MANUSCRIPT 12 Fe3O4 nanoparticles. Additionally, the grafted protein units which have many hydrophilic units that are able to cause aggregation resulted with increasing particle size of MNPs after

PT

immobilization of HSA. Since organic solvents may denaturate proteins, it is desirable to use

RI

only water as a solvent in most of the bioapplications including protein interactions with solid substrates. However, sometimes, solid substrates exhibiting high hydrophobicity may not suitably

SC

disperse in aqueous solutions, resulting in poor efficiency towards analytes to be interacted with.

NU

So, dispersibility of such type of substrates in water is deemed important. With this respect, HAAPS-MNP particles were found to be well-dispersed in pure water by forming no aggregates in

MA

aqueous solutions. Finally, MNP, APS-MNP and HA-APS-MNP could easily be separated from its suspensions to the wall of the containers within by using an external magnet. HSA binding is a

D

kind of model system to show protein binding on the surface of magnetite nanoparticles

TE

containing HA via HSA composed of basic twenty amino acids. These amino acids provide

AC CE P

similar chemical properties to all proteins which consist of various sequences and numbers of amino acids, resulting in different types of proteins such as enzyme, receptor, glycoprotein and lipoprotein with specific functions in cell. The thermal stability of HA-APS-MNPs and HSA-HAAPS-MNPs was evaluated by thermo gravimetric method and the TG curves were shown in Fig. 6. The initial weight loss (about 1.3%) from both materials up to 130ºC is due to the removal of physically adsorbed water on the material and surface hydroxyl groups [53]. The weight loss of HA-APS-MNPs appears about 20% in a broad temperature range between 180-900ºC which is attributed to thermal decomposition of the 3-aminopropyl groups and HA [54,55]. The weight loss of HSA-HA-APS-MNPs is about 22% in the same temperature range. A difference of about 2% in the weight loss between HA-APS-MNPs and HSA-HA-APS-MNPs is attributed to the thermal decomposition of HSA molecules immobilized on HA-APS-MNPs. According to this result, it was concluded that HSA was successfully immobilized on HA-APS-MNPs.

ACCEPTED MANUSCRIPT 13 Please insert here Fig. 6 Please insert here Table 3

PT

The thermodynamic and kinetic parameters for the thermal decomposition of HA-APS-

RI

MNPs and HSA-HA-APS-MNPs were determined from analysis of TG curves by using the Horowitz-Metzger equation [55]. The method is useful to derive significant information about

SC

protein binding to solid substrates by using thermograms. The rate of a thermal decomposition

NU

process can be described as the product of two separate functions of temperature and conversion [56,57].

MA

d / dt  k (T ) f ( )

(1)

where α is the fraction decomposed at time t, k(T) is the temperature dependent function and f(α)

TE

D

is the conversion function dependent on the mechanism of decomposition. The rate constant k is normally expressed by the Arrhenius equation:

AC CE P

k  A exp(E * / RT )

(2)

where E* is the activation energy (kJ mol-1), A is the preexponential factor and R is the gas constant (J mol-1 K-1). Substituting Eq. (2) into Eq. (1), we get:

d / dT  A /  exp(E * / RT ) f ( )

(3)

where φ is the heating rate dT/dt. On integration and approximation, this equation can be obtained in the following form:

ln g( )  E * / RT  ln( AR / E * )

(4)

where g(α) is a function of α dependent on the mechanism of reaction. For the evaluation of decomposition kinetics, the Horowitz-Metzger equation is an illustrative of the approximation methods. According to the Horowitz-Metzger method for a first-order (n = 1) kinetic process,

lg(lg( w / w )  E * / 2.303RTs2  lg 2.303

(5)

ACCEPTED MANUSCRIPT 14 θ = T – Ts, wγ = wα – w, wα : mass loss at the completion of the decomposition, w : mass loss up to time t, T : the temperature in Kelvin at any instant, Ts : the DTG peak temperature. The plot

PT

lg[lg(wα/wγ)] versus θ (inset of Fig. 6) should give a straight line whose slope is E*/2.303RTs2. The preexponential factor (A) is calculated from Eq.(6):

SC

RI

E* / RTs2  A /( exp(E* / RTs )

(6)

The other thermodynamic parameters can be calculated as follows:

NU

S   R ln( Ah / kBTs )

MA

H   E  RTs

G  H   Ts S 

(7) (8) (9)

D

where ΔS* is the entropy of activation (Jmol-1K-1), kB is the Boltzmann constant, h is the Planck

TE

constant, ΔH* is the enthalpy of activation (kJmol-1) and ΔG* is the Gibbs free energy of

AC CE P

activation (kJmol-1). The thermodynamic and kinetic parameters evaluated by the HorowitzMetzger method were listed in Table 3. From the results obtained, the activation energies for HAAPS-MNPs and HSA-HA-APS-MNPs were calculated to be 20.74 kJmol-1 and 33.42 kJmol-1, respectively. These E* values indicate that HA-APS-MNPs are a more stable material than HSAHA-APS-MNPs. As can be seen from Table 3, ΔG* is positive for both materials while ΔH* is positive and ΔS* is negative. The positive sign of the activation enthalpy change (ΔH*) indicates that the decomposition stages are endothermic processes. The positive sign of Gibbs free energy (ΔG*) of activation reveals that the decomposition stages of the materials are non-spontaneous processes. The negative value of the entropy of activation (ΔS*) indicates that both materials has more ordered structures.

ACCEPTED MANUSCRIPT 15 4. Conclusion In conclusion, a new type of magnetite nanoparticles was obtained by chemical

PT

immobilization of HA to iron oxide magnetite nanoparticles. Protein binding to HA-APS-MNP

RI

was studied by using HSA as model protein, and HA-APS-MNP was found to be an efficient medium for protein extraction from aqueous medium with recovery values usually higher than

SC

90%. The results were compared with bare magnetite nanoparticle and it was observed that HSA

NU

binding was higher in the case of HA-coated material, indicating the role of HA immobilization on adsorption capacity So, the concept seems to be promising in separation and purification of

MA

proteins from aqueous solutions. For example, HSA is one of the most abundant proteins in human fluids and its high abundance usually complicates analyses of less-abundant proteins. So,

D

removal of HSA is deemed important, and utilization of protein depletion materials are very

TE

common for this purpose. High recoveries obtained in the present study makes HA-APS-MNP

AC CE P

promising, because it may offer efficient and inexpensive manners for protein depletion. However, further studies are needed to show its efficiency and applicability in protein depletion studies in detail.

Acknowledgement Authors wish to thank Nigde University and Selcuk University for the facilities provided. One of the authors (O. Gezici) wishes to thank TUBITAK for scholarship provided through 2219International Post-Doctoral Research Fellowship Programme, and ETH Zurich for library facilities.

ACCEPTED MANUSCRIPT 16

References [1]

H. Shi, W.B. Tsai, M.D. Garrison, S. Ferrari, B.D. Ratner, Template-imprinted

J. Yakovleva, R. Davidsson, M. Bengtsson, T. Laurell, J. Emneus, Microfluidic enzyme

RI

[2]

PT

nanostructured surfaces for protein recognition, Nature 398 (1999) 593-597.

SC

immunosensors with immobilised protein A and G using chemiluminescence detection, Biosens. Bioelec. 19 (2003) 21-34.

H. Chen, Y.S. Kim, J. Lee, S.J. Yoon, D.S. Lim, H.-J. Choi, K. Koh, Enhancement of BSA

NU

[3]

[4]

MA

binding on au surfaces by calix[4] bisazacrown monolayer, Sensors 7 (2007) 2263-2272. K. Nakanishi, T. Sakiyama, K. Imamura, On the adsorption of proteins on solid surfaces, a

J.H. Kim, J.Y. Yoon, Protein adsorption on polymer particles, Encyclopedia of Surface and

TE

[5]

D

common but very complicated phenomenon, J. Biosci. Bioeng. 91 (2001) 233-244.

Colloid Science, Marcel Dekker, New York 2002, pp 4272-4381. J. Leaver, Protein Adsorption onto latex particles. In surfaces of nanoparticles and porous

AC CE P

[6]

materials; Schwarz, J.A. Contescu, C.I. Eds.; Marcel Dekker: New York, 1999, pp 743-761. [7]

L.B. Bangs, New developments in particle-based immunoassays: Introduction, Pure Appl. Chem. 68 (1996) 1873-1879.

[8]

J.L. Brash, T.A. Horbett, Proteins at interfaces: An overview. In proteins at interfaces II: Fundamentals and applications; T.A. Horbett, J.L. Brash, Eds.; ACS Symposium Series 602, American Chemical Society: Washington DC, 1995, pp 1-23.

[9]

K. Can, M. Ozmen, M. Ersoz, Immobilization of albumin on aminosilane modified superparamagnetic magnetite nanoparticles and its characterization, Coll. Surf. B:Biointer. 71 (2009) 154-159.

ACCEPTED MANUSCRIPT 17 [10] Y. Zhang, N. Kohler, M. Zhang, Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake, Biomater. 23 (2002) 1553-1561.

PT

[11] J. Choi, Y. Jun, S. I. Yeon, H.C. Kim, J. S. Shin, J. Cheon, Biocompatible heterostructured

RI

nanoparticles for multimodal biological detection, J. Am. Chem. Soc. 128 (2006) 1598215983.

SC

[12] K. Yan, P. Li, H. Zhu, Y. Zhou, J. Ding, J. Shen, Z. Li, Z. Xu, P. K. Chu, Recent advances

NU

in multifunctional magnetic nanoparticles and applications to biomedical diagnosis and treatment, RSC Adv. 3 (2013) 10598-10618.

MA

[13] H. Qu, H. Ma, A. Riviere, W. Zhou, C. J. O'Connor, One-pot synthesis in polyamines for preparation of water-soluble magnetite nanoparticles with amine surface reactivity, J.

D

Mater. Chem. 22 (2012) 3311-3313.

TE

[14] J.S. Jiang, Z.F. Gan, Y. Yang, B. Du, M. Qian, P. Zhang, A novel magnetic fluid based on

AC CE P

starch-coated magnetite nanoparticles functionalized with homing peptide, J. Nanopart. Res. 11 (2009) 1321-1330.

[15] A.K. Gupta, M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications, Biomater. 26 (2005) 3995-4021. [16] J. Krizzova, A. Spanova, B. Rittich, D. Horak, Magnetic hydrophilic methacrylate-based polymer microspheres for genomic DNA isolation, J. Chromatogr. A 1064 (2005) 247-253. [17] T. Neuberger, B. Schopf, H. Hofmann, M. Hofmann, B. Rechenberg, Superparamagnetic nanoparticles for biomedical applications: Possibilities and limitations of a new drug delivery system, J. Magn. Magn. Mater. 293 (2005) 483-496. [18] E. Ruckenstein, Z.F. Li, Surface modification and functionalization through the selfassembled monolayer and graft polymerization, J. Coll. Inter. Sci. 113 (2005) 43-63.

ACCEPTED MANUSCRIPT 18 [19] M.A. Neouze, U Schubert, Surface modification and functionalization of metal and metal oxide nanoparticles by organic ligands, Monatsh. Chem. 139 (2008) 183-195.

PT

[20] X. Zhang, P. Zhang, Z. Wu, L. Zhang, G. Zeng, C. Zhou, Adsorption of methylene blue

RI

onto humic acid-coated Fe3O4 nanoparticles, Coll. Surf. A:Physicochem. Engi. Asp. 435 (2013) 85-90.

SC

[21] K. Yang, L. Zhu, B. Lou, B. Chen, Correlations of nonlinear sorption of organic solutes

NU

with soil/sediment physicochemical properties, Chemosphere 61 (2005) 116-128. [22] E. Tipping, Cation Binding by Humic Substances, Cambridge University Press, West

MA

Nyack, NY, USA, 2002.

[23] W.L. Huang, M.A. Schlautman, W. J. Weber, A distributed reactivity model for sorption by

D

soils and sediments .5. The influence of near-surface characteristics in mineral domains,

TE

Environ. Sci. Technol. 30 (1996) 2993-3000.

AC CE P

[24] O. Gezici, H. Kara, M. Ersoz, Y. Abali, The sorption behavior of a nickel-insolubilized humic acid system in a column arrangement, J. Coll. Inter. Sci. 292 (2005) 381-391. [25] O. Gezici, H. Kara, Towards multimodal HPLC separations on humic acid-bonded aminopropyl silica: RPLC and HILIC behavior, Talanta 85 (2011) 1472-1482. [26] D. Heitkamp, K. Wagner, New aspects of uranium recovery from sea-water, Ind. Eng. Chem. Process Des. Dev. 21 (1982) 781-784. [27] C.H. Ho, N.H. Miller, Effect of humic-acid on uranium uptake by hematite particles, J. Coll. Inter. Sci. 106 (1985) 281-288. [28] H. Seki, A. Suzuki, Adsorption of lead ions on immobilized humic-acid, J. Coll. Inter. Sci. 134 (1990) 59-65. [29] A. Okada, A. Usuki, The chemistry of polymer-clay hybrids, Mater. Sci. Eng. C:Mater. Bio. App. 3 (1995) 109-115.

ACCEPTED MANUSCRIPT 19 [30] G. Szabo, S.L. Prosser, R.A. Bulman, Determination of the adsorption coefficient (KOC) of some aromatics for soil by RP-HPLC on 2 immobilized humic-acid phases, Chemosphere

PT

21 (1990) 777-788.

RI

[31] G. Szabo, G. Farkas, R.A. Bulman, Evaluation of silica-humate and alumina-humate HPLC

aromatics, Chemosphere 24 (1992) 403-412.

SC

stationary phases for estimation of the adsorption coefficient, KOC, of soil for some

NU

[32] L.K. Koopal, Y. Yang, A.J. Minnaard, P.L.M. Theunissen, W.H. Van Riemsdijk, Chemical immobilisation of humic acid on silica, Coll. Surf. A:Physicochem. Engi. Asp. 141 (1998)

MA

385-395.

Engi. Asp. 203 (2002) 47-54.

D

[33] M. Klavins, L. Eglite, Immobilisation of humic substances, Coll. Surf. A: Physicochem.

TE

[34] A.G.S. Prado, B.S. Miranda, J.A. Dias, Attachment of two distinct humic acids onto silica

AC CE P

gel surface, Coll. Surf. A:Physicochem. Engi. Asp. 242 (2004) 137-143. [35] S.T. Yang, P.F. Zong, X.M. Ren, Q. Wang, X.K. Wang, Rapid and highly efficient preconcentration of Eu(III) by core-shell structured Fe3O4@humic-acid magnetic nanoparticles, ACS Appl. Mater. Inter. 4 (2012) 6890-6899. [36] P. Janos, M. Kormunda, O. Zivotsky, V. Pilarova, Composite Fe3O4/humic acid magnetic sorbent and its sorption ability for chlorophenols and some other aromatics compounds, Sep. Sci. Technol. 48 (2013) 2028-2035. [37] L. Chekli, S. Phuntsho, M. Roy, H.K. Shon, Characterisation of Fe-oxide nanoparticles coated with humic acid and Suwannee River natural organic matter, Sci. Total Environ. 461 (2013) 19-27.

ACCEPTED MANUSCRIPT 20 [38] J.F. Liu, Z.S. Zhao, G.B. Jiang, Coating Fe3O4 magnetic nanoparticles with humic acid for high efficient removal of heavy metals in water, Environ. Sci. Technol. 42 (2008) 6949-

PT

6954.

RI

[39] D. Luo, Q.W. Yu, H.R. Yin, Y.Q. Feng, Humic acid-bonded silica as a novel sorbent for solid-phase extraction of benzo[a]pyrene in edible oils, Anal. Chim. Acta 588 (2007) 261-

SC

267.

NU

[40] E. Maltas, M. Ozmen, H. Cingilli Vural, S. Yildiz, M. Ersoz, Immobilization of albumin on magnetite nanoparticles, Mater. Lett. 65 (2011) 3499-3501.

MA

[41] M. Bayrakci, E. Maltas, S. Yigiter, M. Ozmen, Synthesis and application of novel magnetite nanoparticle based azacrown ether for protein recognition, Macromol. Res. 21

D

(2013) 1029-1035.

TE

[42] L.N. Zhang, F.Y. Wu, A.H. Liu, Study of the interaction between 2,5-di-[2-(hydroxyl-

AC CE P

phenyl)ethylene]-terephthalonitril and bovine serum albumin by fluorescence spectroscopy, Spectrochim. Acta Part A:Mol. Biomol. Spect. 79 (2011) 97-103. [43] Y.J. Hu, Y. Liu, X.S. Shen, X.Y. Fang, S.S. Qu, Studies on the interaction between 1hexylcarbamoyl-5-fluorouracil and bovine serum albumin, J. Mol. Struct. 738 (2005) 143147.

[44] A. Amirbahman, T.M. Olson, Transport of humic matter-coated hematite in packed-beds, Environ. Sci. Technol. 27 (1993) 2807-2813. [45] P.Matus, K. Gardosova, M. Bujdos, P. Matus, Sorption of humic acids onto fungal surfaces and its effect on heavy metal mobility, Water Air Soil Pollut. 225 (2014) 1839-1845. [46] R.A. Alvarez-Puebla, C. Valenzuela-Calahorro, J.J. Garrido, Retention of Co(II), Ni(II) and Cu(II) on a purified brown humic acid. Modeling and characterization of the sorptiın process, Langmuir 20 (2004) 3657-3664.

ACCEPTED MANUSCRIPT 21 [47] H.Y. Wang, J.J. Wang, M.H. Fan, Extraction of ionic liquids from aqueous solutions by humic acid:an environmentally benign, inexpensive and simple procedure, Chem. Comm.

PT

48 (2012) 392-394.

RI

[48] X. Zang, J.D.H. van Heemst, K.J. Dria, P.G. Hatcher, Encapsulation of protein in humic

sediment, Org. Geochem. 31 (2000) 679-695.

SC

acid from a histosol as an explanation for the occurrence of organic nitrogen in soil and

NU

[49] M. Sander, J.E. Tomaszewski, M. Madliger, R.P. Schwarzenbach, Adsorption of insecticidal Cry1Ab protein to humic substances.1.Experimental approach and mechanistic

MA

aspects, Environ. Sci. Technol. 46 (2012) 9923-9931. [50] W.F. Tan, L.K. Koopal, L.P. Weng, W.H. van Riemsdijk, W. Norde, Humic acid protein

D

complexation, Geochim. Cosmochim. Acta 72 (2008) 2090-2099.

TE

[51] W.F. Tan, L.K. Koopal, W. Norde, Interaction between humic acid and lysozyme studied

AC CE P

by dynamic light scattering and isothermal titration calorimetry, Environ. Sci. Technol. 43 (2009) 591-596.

[52] P. Rivolo, S.M. Severino, S. Ricciardi, F. Frascella, F. Geobaldo, Protein immobilization on nanoporous silicon functionalized by RF activated plasma polymerization of acrylic acid, J. Coll. Inter. Sci. 416 (2014) 73-80. [53] M.Z. Kassaee, H. Masrouri, F. Movahedi, Sulfamic acid-functionalized magnetic Fe3O4 nanoparticles as an efficient and reusable catalyst for one-pot synthesis of alpha-amino nitriles in water, Appl. Cataly. A:General 395 (2011) 28-33. [54] A.G.S. Prado, J.D. Torres, P.C. Martins, J. Pertusatti, L.B. Bolzon, E.A. Faria, Studies on copper(II)- and zinc(II)-mixed ligand complexes of humic acid, J. Hazard. Mater. 136 (2006) 585-588.

ACCEPTED MANUSCRIPT 22 [55] H.H. Horowitz, G. Metzger, A new analysis of thermogravimetric traces, Anal. Chem. 35 (1963) 1464-1468.

PT

[56] T.A. Yousef, O.A. El-Gammal, S.E. Ghazy, G.M. Abu El-Reash, Synthesis, spectroscopic

RI

characterization, pH-metric and thermal behavior on Co(II) complexes formed with 4-(2pyridyl)-3-thiosemicarbazide derivatives, J. Mol. Struct. 1004 (2011) 271-283.

SC

[57] M.S. Al-Amoudi, M.S. Salman, A.S. Megahed, M.S. Refat, Synthesis and characterization

NU

of nanocomposites having catalytic activities using microwave techniques, Eur. Chem.

AC CE P

TE

D

MA

Bull. 1 (2012) 293-304.

ACCEPTED MANUSCRIPT 23

AC CE P

TE

D

MA

NU

SC

RI

PT

FIGURE CAPTIONS

Scheme 1. Synthetic illustration of preparation of humic acid coated magnetite nanoparticles (HA-APS-MNPs and HSA-HA-APS-MNPs)

Table 1. Results of elemental analysis for APS-MNPs and HA-APS-MNPs. Compounds

C(%)

H(%)

N(%)

APS-MNPs

7.20

1.11

1.33

HA-APS-MNPs

14.31

2.21

1.24

ACCEPTED MANUSCRIPT 24

Table 2. Amount of HSA bounded to MNPs, APS-MNPs and HA-APS-MNPs at different amounts of nanoparticlesa Binding amount (mg)

PT

Amount of support MNPsb

APS-MNPsb

5

-

0.403

10

-

15

-

20

0.008

25

0.011

MA

NU

SC

RI

(mg)

HA-APS-MNPs 0.729

0.417

1.280

0.431

1.450

0.436

1.730

0.441

1.740

Averages of the data obtained from three independent protein binding experiments.

b

[41]

AC CE P

TE

D

a

Table 3. Kinetic parameters for the thermal decomposition of HA-APS-MNPs and HSA-HAAPS-MNPs evaluated by Horowitz-Metzger method. Compound

HA-APS-MNPs HSA-HA-APS-MNPs

*

Parameter -1

E (kjmol ) 20.74 33.42

*

ΔH (kjmol-1) 16.11 28.77

ΔS* (jmol-1) -213.22 -231.89

ΔG* (kjmol-1) 134.73 158.23

ACCEPTED MANUSCRIPT

TE

D

MA

NU

SC

RI

PT

25

AC CE P

Fig. 1. FTIR spectra of magnetite nanoparticles (a)HSA-HA-APS-MNPs, (b)MNPs, (c)APSMNPs, (d)HA-APS-MNPs.

ACCEPTED MANUSCRIPT 26

1200

PT

Fluorescence Intensity

1000

800

SC

RI

600

400

300

350

400

450

500

MA

0 250

NU

200

Wavelength, nm

D

Fig. 2. Emission spectra of HSA at various concentrations between 10-50×10-5 gmL-1 at 280 and

AC CE P

TE

342 nm of excitation and emission wavelengths.

2.0

Immobilized HSA, mg

1.6

1.2

0.8

0.4

0.0 0

5

10

15

20

25

30

HA-APS-MNPs, mg

Fig. 3. Plots of amount of bounded HSA in the presence of increasing amounts of magnetite nanoparticles.

ACCEPTED MANUSCRIPT

NU

SC

RI

PT

27

MA

Fig. 4. SEM images of magnetite nanoparticles (a) HA-APS-MNPs and (b) HSA-HA-APS-

AC CE P

TE

D

MNPs.

Fig. 5. TEM images of pure Fe3O4 nanoparticles (a) and HSA-HA-APS-MNPs (b).

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

28

Fig. 6. TG curves of HA-APS-MNPs and HSA-HA-APS-MNPs. Inset: Horowitz-Metzger plots of HA-APS-MNPs and HSA-HA-APS-MNPs.

ACCEPTED MANUSCRIPT 29

Graphical abstract Novel Humic Acid-Bonded Magnetite Nanoparticles for Protein Immobilization

AC CE P

TE

D

MA

NU

SC

RI

PT

Mevlut Bayrakcia, , Orhan Gezicib, , Salih Zeki Basc, Mustafa Ozmenc, Esra Maltasc

___________________________________________________________________________

ACCEPTED MANUSCRIPT

RESEARCH HIGHLIGHT

PT

30

SC

RI

► A new magnetite nanoparticle based humic acid was prepared for the first time.

NU

► Protein binding studies of magnetite nanoparticle based humic acid were performed.

AC CE P

TE

D

MA

► Kinetic parameters of protein and/or humic acid bonded nanoparticles were evaluated.