Magnetic Hydroxyapatite

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Synthesis of Cu2+ chelated cellulose/magnetic hydroxyapatite particles (Cell/MHAP) hybrid beads and its potential for high specific adsorption of histidine-rich proteins Kaifeng Du, Xiaohong Liu, Shikai Li, Liangzhi Qiao, and Hao Ai ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01699 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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ACS Sustainable Chemistry & Engineering

Synthesis of Cu2+ Chelated Cellulose/magnetic Hydroxyapatite Particles (Cell/MHAP) Hybrid Beads and its Potential for High Specific Adsorption of Histidine-rich Proteins

Kaifeng Du*, Xiaohong Liu, Shikai Li, Liangzhi Qiao, Hao Ai

Department of Pharmaceutical & Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, P. R. China

Corresponding author: Dr. Kaifeng Du Department of Pharmaceutical & Biological Engineering, School of Chemical Engineering, Sichuan University, No.24 South Section 1, Yihuan Road, Chengdu 610065, China Tel.: +86-28-85405221; Fax: +86-28-85405221 Email: [email protected]

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ABSTRACT: A novel method is described for facile fabrication of immobilized Cu2+ chelated cellulose/magnetic hydroxyapatite hybrid beads (denoted as Cu2+ Cell/MHAP) by an emulsification technique. In this process, the particles of MHAP, as main component of adsorbent, were prepared by simply mixing iron oxide with N-(phosphonomethyl)-iminodiacetic acid (PM-IDA) and followed by hydroxyapatite (HAP) deposition in aqueous solution. The obtained MHAP particles were suspended in cellulose solution and emulsified into Cell/MHAP hybrid beads. Finally, chelating Cu2+ on Cell/MHAP through oxygen ions of PO43- groups gave the immobilized metal affinity adsorbent. The combination of immobilized Cu2+ ligands, MHAP particles, and magnetic response together with large size of spherical cellulose support endowed the adsorbent with excellent adsorption selectivity, high adsorption capacity and easy recovery of adsorbent from solvent, respectively. By physical characterization, Cu2+ Cell/MHAP possessed large specific surface area of 94.2 m2 g-1 and uniform spherical shape in size of about 137.4±19.5 µm. The adsorption evaluation indicated that Cell/MHAP has high static adsorption capacity (4533.1 mg g-1) of bovine hemoglobin (BHb). Obviously, high adsorption capacity is ascribed to small size and well dispersion of Cu2+ chelated HAP particles in spherical cellulose supports, which provided sufficient specific surface area and suitable volume for protein adsorption. The practical separation potential of Cell/MHAP was evaluated by using diluted bovine blood as probe and high adsorption selectivity was confirmed by SDS-PAGE. Together with superparamagnetism, large size, and excellent adsorption performance, the adsorbent of Cell/MHAP has great potential in the field of histidine-rich protein 2


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purification. Keywords: hydroxyapatite particles; cellulose beads; selectivity; immobilized Cu2+ adsorbent; histidine-rich proteins.

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INTRODUCTION Histidine (his)-rich proteins are characteristic biomolecules that play crucial roles in various physiological processes and the enrichment and purification of such proteins are currently a hot topic.1 As a result, varied methods for purifying histidine (his)-rich proteins from biological samples have attracted great attentions in therapeutic and diagnostic field.2,3 Traditionally, immobilized metal affinity chromatography (IMAC) is one of the most widely ways for separation of his-rich proteins. The popularity lies that the histidine residuals on proteins can interact with the immobilized metal ions to create strong and reversible bonding between aimed proteins and adsorbent.4,5 However, a major limitation of traditional immobilized metal affinity adsorbent is its complicated and time-consuming synthesis process and low surface metal ion density, which restricts its practical application in protein separation. In this context, it is necessary to develop simpler and more efficient method for constructing novel immobilized metal affinity adsorbent for separation of his-rich proteins. Synthetic hydroxyapatite (denote as HAP) is the main mineral constituent of bone, which possesses low cost, tunable porous structure, chemical stable and biocompatible properties.6-8 Moreover, HAP consists of positively charged pairs of calcium ions and clusters of six negatively charged phosphate sites. These rich surface active sites conduce HAP to a kind of adsorbent for heavy metal ions and protein adsorption.9-12 While the HAP-type adsorbent shows poor selectivity for protein purification due to its complex surface.13,14 As for hydroxyapatite, there 4


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are abundant phosphate sites on its framework, which provide lots of chelating sites for metal ions immobilization.15,16 Via the metal immobilization, not only surface heterogeneity of original HAP is partly eliminated but also the immobilized metal ions can serve as high-selectivity affinity ligands for his-rich proteins adsorption. However, the current HAP adsorbent is often constructed in form of small particles.16-18 Although these particles exhibit large specific surface area for high adsorption capacity, small size makes the adsorbent unsuitable for treating large-volume effluent and limits its spread in industrial use. It is well known that large-size beads have the ability of processing industrial effluent since large size allows easy recovery of adsorbent from complex biological effluent by a simple filtration operation in batch adsorption.19 Plus, if being packed into chromatographic column, the large beads still endow the chromatographic bed with relatively high porosity, which would contribute low backpressure for the reduced operation cost.20 Inspired by this, we proposed a novel approach toward the fabrication of magnetic hydroxyapatite (MHAP) based spherical adsorbent. The proposed adsorbent combines the structure characteristics of both small MHAP particles and large-size beads for high-performance adsorption, in which the entrapped small MHAP particles contributed to high protein adsorption capacity and large beads size contributed to easy recovery of adsorbent from effluent. Specially, the magnetic response is more efficient for fast recovery of adsorbent and low operation coat when feed liquid is very little. Finally, chelating Cu2+ ions on 5



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MHAP gave an affinity spherical adsorbent, which would exhibit high adsorption selectivity for aimed proteins. After the preparation of immobilized Cu2+ affinity adsorbent, its physical properties are characterized in detail by varied methods of optical

microscopy

(OM),

scanning

electron

microscopy

(SEM),

N2

sorption/desorption analysis, and vibrating sample magnetometer (VSM). Furthermore, histidine-rich proteins and diluted blood were applied to evaluate the adsorption performance of the proposed affinity adsorbent and demonstrated its practical potential for protein purification.

MATERIALS AND METHODS Chemicals. N-(phosphonomethyl) iminodiacetic acid (PM-IDA) was obtained from TCI Delelopment (Shanghai, China). Microcrystalline cellulose was provided from Shanhe Medicinal Accessary Material (Anhui, China). 1-butyl-3-methylimidazolium chloride (ionic liquid, IL) was purchased from TCI (Shanghai, China). Sorbitantrioleate (Span 85) and transformer oil were obtained from Runhua (Guangdong, China). Bovine hemoglobin (BHb) and bovine serum albumin (BSA) were obtained from Sigma (MO, USA). Other regents, such as FeCl3, CuSO4, FeCl2, Ca(OH)2, Na2HPO4, and epichlorohydrin et al., were of analytical grade from local source. Preparation of magnetic hydroxyapatite (MHAP) particles. The MHAP particles were fabricated by coating hydroxyapatite (HAP) on surface of magnetic iron oxide nanoparticles. The whole synthesis process is described as 6


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follows. First, magnetic iron oxide nanoparticles were synthesized by alkali-assisted coprecipitation (Seeing Supporting Information). Then, 1 g Fe3O4 nanoparticles and 1g PM-IDA were mixed in 500 mL NaOH solution (pH 11). After stirring for 20 min, the resultant dark particles were collected and immersed quickly into 1000 mL 0.01 M Ca(OH)2 (ethanol/water: 1:1,v/v) solution. Then, 500 mL 0.012 M Na3PO4 was added into the suspension solution and kept stirring for 8 h at 50oC, which initiated the HAP precipitation on iron oxide. Finally, the magnetic hydroxyapatite particles (MHAP) were collected with a magnet and dried in a vacuum oven at 30℃ for 10 h. Preparation of immobilized Cu2+ cellulose/MHAP hybrid beads (Cu2+ Cell/MHAP). Cu2+ Cell/MHAP was prepared by water/oil emulsification and followed by chelating Cu2+ ions on MHAP.21 Typically, 10 g microcrystalline cellulose was immersed in 133 g ionic liquid (1-butyl-3-methylimidazolium chloride) and kept in 90 oC oil bath for complete dissolution of cellulose powder. Then, 8 g MHAP was suspended in a 100 mL cellulose solution and the suspension was emulsified with an oil phase (400 mL of transformer oil and 18 mL of Span 85) by stirring at 1000 rpm. Lowering the temperature precipitated the Cell/MHAP hybrid beads from ionic liquid through the regenerated hydrogen bonds between the cellulose fibers. The Cell/MHAP hybrid beads were thoroughly washed with deionized water and screened out with a standard sieve. Finally, the collected Cell/MHAP hybrid beads were dispersed in 2 M 50 mL CuSO4 solution to chelate Cu2+ ions, which were denoted as Cu2+ Cell/MHAP. 7



Characterization. Optical microscopy (OM) observation was conducted on Nikon ECLPSE600 microscope. The microscopic morphology of Cu2+ Cell/MHAP was investigated by scanning electron microscopy (SEM) (Philips, Netherlands). Pore size distribution and pore volume were analyzed by using the adsorption branch of N2 isotherm by BET and BJH method (Quantanchrome, USA), and the information of chemical bonds of samples were collected by Fourier-transform infrared spectroscopy (FTIR, Bruker). X-ray diffraction (XRD) patterns of the samples were analyzed on X-ray diffraction (XRD, Rigaku) with a scan range between 20º and 70º. The magnetic properties of iron oxide and MHAP were measured using a vibrating sample magnetometer (VSM) at room temperature. Protein adsorption equilibrium. Static adsorption of BSA and BHb were conducted with two types of Cell/MHAP hybrid beads with and without Cu2+ chelation by the following process. After equilibration with phosphate solution (pH 6.5), 50 mg adsorbent for both BSA and BHb was mixed with 8 mL protein solutions at different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.5, and 2.0 mg mL-1) and incubated at 200 rpm for 180 min. After membrane filtration, the residual liquid was collected to determine the protein concentrations with UV/Vis spectrophotometer, in which BSA concentrations were detected at 280 nm and BHb concentrations at 410 nm. In addition, buffer solutions with varied pH values (6.0, 6.5, 7.0, 7.5, 8.0) were used to evaluate the effect of pH on equilibrium adsorption capacity of 8


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proteins on adsorbent. The experimental adsorption capacity for protein was determined based on Equation 1. The experimental adsorption data were fitted by Langmuir model (Equation 2) and Freundlich model (Equation 3) as follows. qୣ =

(௖బ ିୡ౛)౒

qୣ =

(1)

୑ ୯ ౣ ୡ౛

(2)

୩ౚ ାୡ౛ ଵ/୬

q ୣ = K ୊ cୣ

(3)

where qe (mg g-1) and qm (mg g-1) are experimental adsorption capacity and fitted equilibrium adsorption capacity for proteins, respectively; co and ce(mg mL-1) are the initial and equilibrium proteins concentrations, respectively; V (L) is the volume of adsorbed solution, and M (g) is mass of adsorbent; kd (mg L-1) is the dissociation constant of Langmuir model; KF is the characteristic constant related to the adsorption capacity; n is a constant related to the adsorption intensity. Protein adsorption kinetics. Adsorption kinetics experiments for BSA and BHb were conducted on the adsorbent in a 10-mL centrifuge tube, as described by Du et al.22 Prior to the adsorption operation, varied protein solutions were prepared at different concentrations (BHb: 0.1, 1.0, 6.0 mg mL-1; BSA:0.4, 0.8, and 1.5 mg mL-1). Then, after equilibrating with equilibrium buffer (pH6.5), a known amount of adsorbent (50 mg adsorbent for BSA, 5mg adsorbent for BHb) was added to 8 mL protein solution for dynamic adsorption. At the defined time intervals, 1 mL sample was periodically collected to determine protein concentration, which returned to the centrifuge tube again. By this procedure, the 9



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adsorption capacities (qt) on adsorbent at each defined time were calculated via mass balance. These adsorption capacities with real time served as adsorption kinetics data. Fitting these adsorption data into pseudo first-order model and second order model gave the corresponding kinetic parameters (k1 and k2).

where

qt = qe (1 − e − k1t )

(4)

qt = k2 qe 2 t/ (1 + k2 q 2 t)

(5)

qe and qt are adsorption capacity of proteins at equilibrium (mg mL-1)

and at a real time ( t ), respectively; k1 and k2 are pseudo-first- and second-order adsorption kinetic parameters, respectively. Evaluation of adsorption selectivity and real separation. The adsorption selectivity of Cu2+ Cell/MHAP towards BHb was investigated by using 100-fold dilution bovine blood, which was dissolved in a phosphate buffer (50 mM, pH 7.4) with 0.15 M NaCl. In the typical procedure, 5 mg Cu2+ Cell/MHAP was poured into 8 ml diluted bovine blood, and the mixture was shaken at room temperature for 180 min for achieving adsorption equilibrium. Then the adsorbent was washed with 50 mM pH 8.0 phosphate buffer and followed by the elution by using 50 mM pH 8.0 phosphate buffer containing 0.5 M imidazole and 0.5 M NaCl. The elution BHb solution was collected and further analyzed with 12% SDS poly(acrylamide) gel electrophoresis (SDS-PAGE).

RESULTS AND DISCUSSION Physical properties of Cu2+ Cell/MHAP hybrid beads. The synthesis route 10


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of immobilized Cu2+ cellulose/magnetic hydroxyapatite hybrid beads (Cu2+ Cell/MHAP) is illustrated in Figure 1. In the typical process, the iron oxide nanoparticles were first prepared by alkali-assisted coprecipitation of ferric and ferrous salts and followed by coating with N-(phosphonomethyl) iminodiacetic acid (PM-IDA) to produce the carboxyl groups modified Fe3O4 nanoparticles. When getting in touch with Ca2+ and PO43- ions in reaction system, the immobilized

carboxyl

terminals

directed

the

uniform

deposition

of

hydroxyapatite on magnetic iron oxide particles (MHAP).23,24 The obtained MHAP particles were then suspended into cellulose solution and emulsified into cellulose/MHAP hybrid beads with large size. The appealing feature of proposed synthesis strategy is that the prepared hybrid beads integrate both high adsorption capacity of small MHAP particles and easy recovery of adsorbent from effluent due to their large size and magnetic response. It is well known that the surface of hydroxyapatite is rich in oxygen ions due to the existence of phosphate groups, which provide lots of active sites for chelating Cu2+ ions. Given this, to improve the adsorption selectivity, the Cu2+ ions were chelated on MHAP toward the generation of Cu2+ Cell/MHAP hybrid spherical adsorbent. As shown in Figure 1, it is expected that the affinity adsorbent of Cu2+ Cell/MHAP has great potential for high-performance separation of his-rich proteins from complex solution in laboratory and industrial use.

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Figure 1 Schematic illustration of fabrication route of Cu2+ Cell/MHAP hybrid beads and the adsorption mechanism for histidine-rich proteins

The magnetization of Cu2+ Cell/MHAP originates from the entrapped MHAP in adsorbent, which facilitates easy recovery of adsorbent from effluent. To explore the magnetic response property, the particles of bare iron oxide and MHAP were evaluated via a vibrating sample magnetometer. It found that magnetic saturation values of bare iron oxide and MHAP were 64.4 and 29.2 emu g-1, respectively, from the characterization results (Figure 2a). The big difference in magnetism is ascribed to the structure difference between iron oxide and MHAP. The bare iron oxide exhibited irregular spherical morphology and relatively small particles size of about 10-15 nm by the TEM observation (Seeing Figure S1 in Supporting Information). When hydroxyapatite was coated on iron oxide, the particles MHAP reached to about 4 µm in diameter (Figure 2b). Obviously, the decrease of magnetic saturation values was ascribed to the high 12


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content of hydroxyapatite (HAP) on iron oxide. Though the magnetization value of MHAP decreased to 29.2 emu g-1, the magnetic response to a magnetic field was strong still and enough for the complete separation from solution with 20 seconds, which is of great importance in the separation process. The external morphology of Cu2+ Cell/MHAP was observed by both optical microscopy (OM) and scanning electron microscope (SEM) before and after drying in air, respectively. It found from OM image (Figure 2c) that all these beads in swollen state exhibited perfect spherical morphology and relatively smooth surface with an average diameter of about 137.4±19.5 µm. Moreover, the Cu2+ Cell/MHAP seemed to rather dark by the OM observation. It can be explained by that lots of MHAP particles were entrapped heavily in cellulose network and blocked the permeation of light through the adsorbent. To explore the structure characteristic more clearly, Cu2+ Cell/MHAP was dried in air and further observed by SEM (Figure 2d). One can see that the dried Cu2+ Cell/MHAP displayed still excellent spherical morphology and relatively rough surface, as a result of contraction difference between cellulose gel and MHAP particles on the drying process. By analyzing quantitatively the size change, it revealed that the dried Cu2+ Cell/MHAP reduced from 137.4±19.5 µm to 67±11.2 µm. The contraction in bead size is due to that lots of entrapped water was drained-off from cellulose support after drying. These results indicated that there was large porosity within the adsorbent, which would accommodate the aimed proteins for adsorption and fast mass transfer. 13



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Figure 2 Magnetic hysteresis loops (a) of iron oxide and MHAP, SEM (b) of MHAP, OM (a) and SEM (d) of Cu2+ Cell/MHAP

Brunauer-Emmett-Teller (BET) method was conducted for evaluation of the surface area of MHAP particles. Figure 3 displayed the N2 adsorption-desorption isotherms and the corresponding pore-size distribution curve for MHAP particles. With BET analysis, the BET surface area and pore volume of MHAP were determined to be 94.2 m2 g-1 and 0.377 cm3 g-1, respectively. It indicated that the prepared MHAP possessed relatively higher specific surface area compared with other similar adsorbent.

According

to

IUPAC

classification,

the

N2

adsorption/desorption curve of MHAP particles exhibits a typical IV-type isotherm with a H1 type hysteresis loop at a relative pressure p/p0=0.80-0.98, 14


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indicating a typical mesoporous material. Derived from N2 desorption curve, the pore size distribution of MHAP was determined as a single peak and the pore center was at about 14.8 nm (inset of Figure 3). In addition, the nitrogen uptake increased sharply and did not clearly exhibit any adsorption plateau at relative pressures close to unity, which proved the pore size distribution of MHAP extended from mesopores to macropores.

Figure 3 N2 adsorption/desorption isotherm and its pore size distribution (inset) of MHAP particles.

The XRD patterns of Fe3O4 and MHAP were shown in Figure 4. It revealed that

pure

Fe3O4

exhibited

the

characteristic

diffraction

peaks

of

face-centered-cubic structure according to the index of JCPDS card No. 19-0629. After coating with HAP, the sample of MHAP showed new diffraction peaks at 15



25.8, 28.1, 31.7, 32.2, 34, 38.1, 39.7, 46.6, 49.4, 51.2, which were indexed as (002), (102), (211), (112), (202), (220), (310), (222), (213), and (410) of hydroxyapatite, as indicative in JCPDS No.19-0432. In addition, both of the samples showed the characteristic diffraction peaks of Fe3O4, indicating that the coated hydroxyapatite did not change the phase structure of Fe3O4, confirming the reliability of this coating technique. As a result, a layer of HAP was successfully coated on surface of Fe3O4 via the chelating interaction.

Figure 4 XRD patterns of Fe3O4 (a) and MHAP particles (b).

To further verify the presence of HAP coating on Fe3O4 nanoparticles, the detailed investigation of both Fe3O4 and MHAP particles was performed using FTIR spectra (Figure 5). It revealed that Fe3O4 possessed several characteristic absorbance peaks, which were assigned to various chemical bonds. Among them, 16


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a typical strong and broad peak was observed at 3140 cm−1, which corresponded to the O−H bending vibration that belonged to hydrogen bonds. The weak peaks at 1619 cm-1 and 576 cm−1 were assigned to the stretching vibration of OH groups and the stretching vibration of Fe−O bonding, respectively. As for the sample of MHAP, the presence of the absorption peaks at 1113 cm-1, 563 and 1035 cm-1 were all ascribed to the molecular vibration of the PO43- groups. It proved that the HAP was deposited successfully onto the surface of Fe3O4. Based on the combined XRD and FTIR results, the composite adsorbent with magnetic hydroxyapatite have been successfully synthesized via such simple and facile approach.

Figure 5 FTIR spectra of Fe3O4 (a) and MHAP (b).

Cu2+ immobilization on Cell/MHAP. Many literatures have reported that 17



hydroxyapatite has ability of binding copper ions by the chelating interaction between PO43- and Cu2+. It is noted that the chelated Cu2+ can eliminate the surface heterogeneity of HAP and then conduce to the improvement of adsorption selectivity for his-rich protein due to the coordination interaction between the chelated Cu2+ and the imidazole rings on his-rich proteins. In this context, the most optimized amount of Cu2+ on support is especially crucial for fabricating Cu2+ Cell/MHAP with high adsorption efficiency. For this purpose, a series of tests were carried out to study the effect of pH on the adsorption amount of Cu2+ on Cell/MHAP (Figure 6). As shown in here, the adsorbed amount of Cu2+ on Cell/MHAP maintained a relatively stable value of 50.7±0.1 mg mg-1 in a widely pH range (pH 4-8), indicating that solution pH affected slightly on Cu2+ immobilization at such pH range, which was ascribed to the strong chelation between PO43+ and Cu2+. In addition, at solution pH 3, the Cu2+ adsorption capacity reached minmum, which was due to the fierce competition between positively H3O+ and Cu2+ on hydroxyapatite. Together with these results, the stable Cu2+ chelation on Cell/MHAP in a widely pH range endowed the adsorbent with stable structure and made it possibility for his-rich proteins adsorption in a wide pH range.

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Figure 6 Effect of pH on Cu2+ adsorption capacity on Cell/MHAP

Proteins adsorption evaluation. To explore the feasibility of adsorption selectivity for aimed proteins, BSA and BHb were chosen as probes to evaluate Cu2+ Cell/MHAP at varied pH values (6.0-8.0), and the results were shown in Figure 7. With the same batch adsorption conditions, it revealed that Cu2+ Cell/MHAP possessed maximum adsorption capacity of about 728.7 mg g-1 for BHb at pH 6.5. And, the adsorption capacity of BHb reduced gradually with higher and lower solution pH. This kind of adsorption behavior is ascribed to the structure characteristic of BHb, which is possessed of 20 surface-exposed histidine residues. At pH 6.5, more histidine residues are exposed on surface of BHb, which lead to stronger chelation between BHb and immobilized Cu2+ ions and contribute to larger adsorption capacity. And, BSA displays negative charge when solution pH is higher over 4.8. Given this, with pH increasing from pH 6 to pH 8, the BSA adsorption capacities decreased dramatically due to the increased 19



electrostatic repulsion between BSA and adsorbent. It is expected that the adsorption difference of Cu2+ Cell/MHAP along with varied pH values made it possible of adsorption selectivity toward special proteins.

Figure 7 Effect of pH on adsorption capacities of BHb and BSA on Cu2+ Cell/MHAP

To elucidate clearly the selectivity, a series of binary protein mixtures were made up with different mass ratios (mBHb : mBSA=4:1, 2:1, 1:1, 1:2, 1:4) and then were incubated with Cu2+ Cell/MHAP for adsorption equilibrium (Figure S3 in Supporting Information). It was observed that, even at very high ratio of BSA (1.2 mg mL-1), Cu2+ Cell/MHAP exhibited very low adsorption capacity for BSA (39.5 mg g-1), while relatively high adsorption capacity of 365.7 mg g-1 was obtained for BHb. The competitive adsorption verified that the prepared affinity adsorbent of Cu2+ Cell/MHAP possessed excellent adsorption selectivity for BHb with the coexistence of BSA. The adsorption equilibrium isotherm is a fundamental technique that describes the adsorption behavior of proteins on adsorbent. In this context, two 20


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isotherm models (Langmuir and Freundlich) were applied to analyze the adsorption data of BHb and BSA on Cell/MHAP before and after Cu2+ chelation, respectively. The fitted curve and the according isotherm adsorption parameters were shown in Figure 8 and Table 1. As seen in Figure 8, Langmuir model exhibited a better fit than Freundlich, as a result of the adsorption of proteins on adsorbent with energetically uniform surface. Based on Langmuir model, Cu2+ Cell/MHAP showed the maximum adsorption capacity reached about 728.7 mg g-1 for BHb, which was 7.2 times larger than BSA (101.7 mg g-1). Adsorbent dosage is also considered as one of important parameters. In this way, the effect of adsorbent dosage on static adsorption capacities of both BHb and BSA was studied and the results were shown in Figure S4 of Supporting Information. The results showed that, with the fixed protein solutions, less dosage of adsorbent (5 mg) gave higher static adsorption capacity of BHb (about 4533.1 mg g-1), which was higher obviously than the adsorption capacity (728.7 mg g-1) with more dosage of adsorbent (50 mg). With further reducing the adsorbent dosage below 5 mg, the static adsorption capacity of BHb on Cu2+ Cell/MHAP increased slightly, as a result of adsorption saturation. The phenomenon can be explained by the following reason. Before the adsorption saturation, the same amount of BHb was distributed on adsorbent with fewer amounts and therefore led to in the increase of adsorption capacity on unit mass adsorbent.

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Figure 8 Adsorption isotherms of BHb and BSA on Cell/MHAP with and without Cu2+ chelation

Compared with other similar adsorbents, Cu2+ Cell/MHAP still exhibits advantage in adsorption capacity for BHb purification (Table 2).25-29 High adsorption capacity of BHb originates from two reasons. One is that Cu2+ Cell/MHAP possessed both suitable porous structure and large chelated Cu2+ amount on support (50.7 mg g-1), which provided more active sites for protein adsorption. The other is ascribed to the accessible number of histidines terminals on BHb. In general, each BHb molecule has 20 water-accessible histidines terminals with suitable geography for easy adsorption. But there are only 5 histidines terminals for BSA molecule, which strongly limit BSA adsorption on adsorbent. Hence, Cu2+ Cell/MHAP exhibited a tremendous adsorption capacity for BHb and a rather low capacity for BSA. In addition, compared with Cell/MHAP without Cu2+ chelation, Cu2+ Cell/MHAP displayed greater adsorption capacities and smaller dissociation constants for both proteins (Table 22


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1). Obviously, the immobilized Cu2+ on Cell/MHAP enhanced the interaction between adsorbent and proteins, which then conduced to high protein adsorption capacity.

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Table1. Adsorption isotherm parameters of BHb and BSA on Cell/MHAP with and without Cu2+ chelation BHb isotherm models

BSA

parameters Cu2+ Cell/MHAP Cell/MHAP

Cu2+ Cell/MHAP

Cell/MHAP

qm (mg g-1)

728.7

84.0

101.7

59.2

qexp (mg g-1)

677.9

71.2

93.3

50.5

kd (mg L-1)

0.1368

0.3658

0.1087

0.2288

R2

0.993

0.994

0.997

0.995

KF (mg g-1)

631.3

59.2

92.5

46.9

n (mg g-1)

2.833

2.458

4.261

3.218

Langmuir

Freundlich

24


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R2

0.949

0.982

25


0.923

0.950


Table 2 Adsorption properties of difference adsorbents for BHb purification25-29 adsorbents

adsorption capacity (mg g-1)

Ni2+-NH2-SiO2-Fe3O4

1054.3

Cu2+-EDTA-Fe3O4

1250

BhB imprinted MPs

37.58

Fe3O4@SiO2@IL

2150

CuFe2O4 MNCs

4475

Cu2+ Cell/MHAP (this work)

4533.1

Adsorption kinetics is an important method to evaluate the adsorbent in terms of its adsorption efficiency and help to explore the adsorption mechanism. In this context, two typical kinetics models, pseudo-first-order and pseudo-second-order equations, were applied to analyze the time-dependent adsorption data of BHb and BSA on Cu2+ Cell/MHAP. The fitted kinetic curves and the according parameters were shown in Figure 9 and Table 3. It revealed that the adsorption capacity of both proteins on Cu2+ Cell/MHAP increased rapidly in the first 60 min and almost reached equilibrium after 80 min. This relatively fast adsorption equilibrium time was probably due to highly porous structure of cellulose network, which nearly showed no significant effects on mass transfer of solute through surface of adsorbent. In addition, the values of k1and k2 for both BHb and BSA decreased regularly along with the increase of initial proteins concentrations, and the correlation coefficients (R2) of pseudo-first-order model were higher than that of pseudo-second-order one (Table 3), which 26


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demonstrated that the adsorption process was better described by pseudo-first-order adsorption model. According to the analysis of pseudo-first-order model, we concluded that the proteins adsorption only occurred on pore surface of adsorbent and the adsorption mechanism was ascribed to the sharing or exchanging of electrons between the Cu2+and the histidines groups on proteins.

Figure 9 Adsorption kinetics of BHb and BSA on Cu2+ Cell/MHAP

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ACS Sustainable Chemistry & Engineering 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

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Table 2 Adsorption kinetic constants of BHb and BSA adsorption on Cu2+ Cell/MHAP initial BHb concentration ( mg mL-1)

initial BSA concentration ( mg mL-1)

adsorption kinetics models 0.5

1.0

6.0

0.4

0.6

0.8

qe,exp (mg g-1)

695.9

1403.2

3950.9

56.5

78.6

92.1

Pseudo-first-order

qe, cal (mg g-1)

674.8

1374.6

3801.8

57.3

78.7

91.1

model

k1 (h-1)

0.120

0.069

0.054

0.034

0.044

0.062

R2

0.991

0.996

0.995

0.992

0.996

0.998

qe,exp (mg g-1)

695.9

1403.2

3950.9

56.5

78.6

92.1

Pseudo-second-order

qe, cal (mg g-1)

731.6

1512.5

4412.7

71.1

93.6

101.3

model

k1 (×10-4 h-1)

0.261

0.690

0.146

4.690

5.213

8.851

R2

0.989

0.972

0.984

0.975

0.977

0.975

28


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Separation of BHb from real sample. To explore the adsorption selectivity and practical potential, Cu2+ Cell/MHAP was applied to purify selectively BHb from 100-fold diluted fetal bovine serum. Figure 10 presented a series of optical microscopy images and UV adsorption spectrum, in which the real separation process and adsorption efficiency were tracked along with time. It revealed that the color of diluted fetal bovine serum solution gradually changed from red to pink with increasing the adsorption time, as a result of fast and successful adsorption of BHb on Cu2+ Cell/MHAP. By the UV analysis, when the adsorption was completed, the characteristic peak at 414 nm from BHb becomes weakened while peak at 280 nm reduced slightly, indicating most of BHb proteins were adsorbed on adsorbent. The elution solution from adsorbent was further analyzed by SDS-PAGE gel electrophoresis (Figure S5 in Supporting Information). It confirmed that nearly 95% of total BHb was captured by Cu2+ Cell/MHAP, and the other proteins including BSA were almost completely retained in the supernatant. All these results demonstrated that the affinity adsorbent of Cu2+ Cell/MHAP had the excellent ability of purifying BHb from complex proteins mixture.

29



Figure 10 Photographs of separation (upper) and its UV-Vis spectrum (lower) of BHb from 100-fold diluted fetal bovine serum by Cu2+ Cell/MHAP with increasing time.

CONCLUSIONS In summary, we presented a facile approach to fabricate a novel affinity adsorbent of Cu2+ Cell/MHAP by encapsulating magnetic hydroxyapatite (MHAP) particles into cellulose beads and followed by Cu2+ chelation on MHAP. This metal chelated affinity adsorbent integrated the advantages of magnetic large beads (easy recovery of adsorbent), particles with high specific surface area (large adsorption capacity) and Cu2+ chelated modification (high adsorption selectivity). The selectivity and adsorption capacity of Cu2+ Cell/MHAP were evaluated by using BHb, BSA and diluted bovine blood. The most distinct feature of Cu2+ Cell/MHAP is its high maximum adsorption capacity of about 728.7 mg g-1 for BHb, which is ascribed to the suitable porosity of Cu2+ Cell/MHAP and the coordination between immobilized Cu2+ 30


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and accessible histidine terminals of BHb. In addition, Cu2+ Cell/MHAP was successfully employed to selectively separate BHb from bovine blood sample. All of these splendid properties demonstrated that the prepared Cu2+ Cell/MHAP can be used as novel, highly efficient immobilized metal affinity sorbent for his-rich proteins separation from biological sample.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthetic methods and adsorption evaluation, TEM of iron oxide nanoparticles (Figure S1), Particles sizes of Cu2+ Cell/MHAP (Figure S2); Competitive adsorption of BHb and BSA on Cu2+ Cell/MHAP (Figure S3), Effect of adsorbent dosage on static adsorption capacity of BHb (Figure S4); SDS-PAGE analysis (Figure S5).

AUTHOR INFORMATION Corresponding Author *Kaifeng Du: E-mail: [email protected]. Tel.:0086 028 85405221. Fax: 0086 028 85405221. Notes The authors declare no completing financial interest.

31



ACKNOWLEDGMENTS The work was funded by Natural Science Foundation of China (21676170 and 21476144). And, we would like to thank the Analytical & Testing Center of Sichuan University for analyzing the external morphology of the prepared samples and we would be grateful to Shuping Zheng for her help of SEM images.

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Table of Contents Only

Synopsis The prepared adsorbent is mainly composed of cellulose and hydroxyapatite, which are all natural and sustainable materials.

35



Schematic illustration of fabrication route of Cu2+Cell/MHAP hybrid beads and the adsorption mechanism for histidine-rich proteins 331x164mm (144 x 144 DPI)


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Magnetic Hydroxyapatite

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