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iomimetic fabrication of hydroxyapatite-coated zirconia–magnesia compositend its application in the separation of proteins
ing Li, Yu-Qi Feng ∗
ey Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China
r t i c l e i n f o
rticle history:eceived 11 June 2009eceived in revised form 9 August 2009ccepted 10 August 2009vailable online 18 August 2009
eywords:
a b s t r a c t
In this work, biomimetic technique was proposed for the first time to prepare chromatographic pack-ings (2HAp-ZM) for protein separation. By mimicking the mineralization procedures in vivo, this type ofmatrix with hydroxyapatite (HAp) coating and zirconia–magnesia (ZM) composite core was fabricated.Systematic characterizations, including scanning electron microscopy (SEM), X-ray photoelectron spec-troscopy (XPS), X-ray diffraction (XRD) and specific surface area analysis, were carried out to investigatethe properties of the material. Results showed that the surface of 2HAp-ZM was composed of bead-like,amorphous or nanocrystalline HAp. The specific surface area, total pore volume, and average pore diam-
eter of the resultant material were 25 m2/g, 0.09 cm3/g, and 14 nm, respectively. Furthermore, 2HAp-ZMexhibited good mechanical stability through repeated testing and its application as stationary phasesfor protein separation was then studied. Five model proteins (bovine serum albumin, trypsin, lysozyme,ribonuclease A, and cytochrome c) were successfully separated on 2HAp-ZM. Finally, the mass recovery
ic loak col
of protein and the dynam93% and 80 �g/mL of blan
. Introduction
Protein separation has been attracting increasing interests withhe development of biology and physiology in recent years. High-erformance liquid chromatography (HPLC) is by far the mostidely utilized technique in this field; its performance is mainly
ased on the packing materials [1]. In this case, some commonriteria have been proposed to dictate the selection of chromato-raphic media for protein separation in a previous review [2], whichre summarized as: (a) chemically and physically stable to with-tand regeneration and cleaning procedures; (b) good mechanicaltrength to allow high flows; (c) not containing groups that bindroteins non-specifically; (d) easy to be derived to allow the
ntroduction of functional groups for interactive chromatographypplications; (e) good control of particle size and pore size; (f) highelectivity and loading capacity; (g) low cost. However, it is oftenot possible to reconcile all the criteria, and therefore compromisesave to be searched and further development of the stationaryhases is required for their application to protein separation.
During the past decades, continuous efforts have been devotedo improve the stationary phases for protein chromatography byhe employment of new packing materials. Zirconia (ZrO2) wasntroduced as a competitive alternative to the traditional silica with
enhanced chemical, thermal or mechanical stability [3]. This advan-tage made it can be cleaned and sterilized using hot acids andbases to remove irreversibly adsorbed proteins [4–6]. Furthermore,this oxide showed ligand-exchange behavior due to the presenceof Lewis acid sites at its surface, which could interact with Lewisbases, such as fluoride, phosphate ions or carboxylic acids, and alsowith the carboxyl groups of proteins. Hence, protein separation wasable to be achieved using a mobile phase containing strong Lewisbase at well defined concentrations [7,8] or on a strong Lewis base-modified ZrO2 [9–14]. Unfortunately, its selectivity and loadingcapacity were relatively limited, owing to the single modal mech-anism of separation and lower surface area, respectively [9,10].
Hydroxyapatite [HAp: Ca10(PO4)6(OH)2] was another promisingmedium for protein separation. It had unique bioaffinity and bio-compatibility, which could adsorb and desorb biomolecules such asproteins and nucleic acids without denaturation [15–17]. Its under-lying mechanism of separation was multimodal [1,18–23]: cationexchange took place at PO4
3− (P) sites; anion exchange as wellas ligand exchange took place at Ca2+ (C) sites. Nevertheless, theoriginal crystalline HAp was too fragile to withstand high packingpressure and flow rates [2]. One way to solve this problem wasthe preparation of ceramic HAp, which was sintered into a spher-
ical particle with large pores [24]. The other was the fabricationof HAp-based composites. Majumder and Ramesh [25] suspendedphosphorylated silica in 2 M CaCl2 solution and then in 1 M phos-phate buffer to obtain a HAp substitute, which could be employedfor open column LC as well as for HPLC purpose. Though it was
uccessfully used in the purification of topoisomerase I and II fromheat germ, no detailed information on its morphological features
r stability were reported. Later on, Honda et al. [26,27] reportedwo kinds of HAp-coated composite particles, which were madef orthodox fragile HAp (or cattle bone powder) and polyethyleneeads by the dry impact blending method. Irreversible adsorptionf proteins onto the columns was observed during the chromato-raphic running.
Considering the inherent properties of the packing materialsisted above, we aimed at preparing a type of matrix that consistedf a HAp surface and a ZrO2-based core for protein chromatogra-hy. Through integrating unique bioaffinity and biocompatibility,nd excellent thermal and mechanical stability as a whole, this sta-ionary phase was expected to provide better separation efficiencynd larger application scale. Thus, it is of great importance to selectn appropriate method to achieve this objective.
Biological systems are a rich source of ideas for individuals con-erned with the synthesis of new materials and processes [28], fromhich the biomimetic technique was derived and developed. The
iomimetic technique was a low-cost and facile process to coatmplants (usually used in orthopedic surgery) with calcium phos-hate [29–34]. It was performed by soaking substrates under mildH and temperature conditions into simulated body fluid (SBF)olutions that have a similar inorganic content as human bloodlasma [35]. Once the apatite nuclei formed on the surface of theubstrate, they could spontaneously grow by consuming the cal-ium and phosphate ions from the fluid [36]. It was worthwhile tooint out that the functional groups on the surface of the substrateere important to induce the nucleation of apatite [37]. Wen et al.
38] reported that HAp could deposit and grow on oxide layers ofitanium in a modified SBF solution (supersaturated calcificationolution, SCS), indicating TiO2 were favorable to induce apatite for-ation in the body environment. Fortunately, ZrO2 possessed the
imilar surface chemical properties as TiO2 [39], which inspireds that biomimetic technique would be a feasible pathway to pre-are HAp-coated zirconia–magnesia (a ZrO2-based composite) astationary phase for protein chromatography.
In the present work, HAp-coated zirconia–magnesia (ZM)icrospheres were prepared via biomimetic technique by incu-
ating the substrates in SCS and applied to the separation ofroteins with HPLC. This was, to the best of our knowledge, therst demonstration of using biomimetic technique to fabricatehromatographic packings for biological analysis. Five proteins,.e. bovine serum albumin, trypsin, lysozyme, ribonuclease A, andytochrome c, were employed as model analytes to evaluate theeparation performance of the resultant materials. Enhanced sepa-ation efficiency and dynamic loading capacity, as well as improvedechanical stability, were observed when compared with unmod-
fied ZM and HAp, respectively.
. Experimental
.1. Chemicals and materials
ZM microspheres (8–10 �m) were synthesized by sol–gelethod in our laboratory [40]. Tiselius type HAp, NaCl, KCl, anhy-
rous Na2HPO4 (NaP), CaCl2, HCl, trihydroxymethylaminomethaneTris), anhydrous K2HPO4 (KP) and H3PO4 were purchased fromhanghai General Chemical Reagent Factory (Shanghai, China).
ysozyme was purchased from Shanghai Bio Life Science & Tech-ology (Shanghai, China). Trypsin was purchased from AmrescoSolon, OH, USA). Bovine serum albumin (BSA), ribonuclease ARNase A), cytochrome c (Cyt c), ovalbumin and �1-acid glyco-rotein (AGP) were purchased from Sigma (St. Louis, MO, USA).ouble-distilled water was used for all experiments.
0 (2009) 889–894
2.2. Preparation of SCS
The SCS was prepared according to the method describedby Kokubo et al. [41] with a little modification. NaCl, KCl,anhydrous NaP and CaCl2 were dissolved one by one indistilled water, the concentrations of each ion were listedas follows: [Na+] = 136.8 mM, [K+] = 3.71 mM, [Cl−] = 144.5 mM,[Ca2+] = 3.10 mM, and [HPO4
2−] = 1.86 mM. The SCS was bufferedat pH 7.4 with HCl and Tris at 36.5 ◦C. After preparation, it waspreserved at 5–10 ◦C in a refrigerator, and was used within 30 days.
2.3. Preparation of the HAp-coated ZM microspheres
The preparation of HAp-coated ZM was composed of foursuccessive steps: activation, pre-calcification, incubation, and cal-cination.
ZM microspheres were firstly dispersed in 1 M NaOH at roomtemperature overnight, then washed to neutral and dried at160 ◦C for 8 h. Pre-calcification treatment was then performed byimmersing the activated ZM in 0.5 M NaP solution at 100 ◦C for12 h, followed by suspending them in freshly prepared saturatedCa(OH)2 solution for 2 h. The particles were thoroughly rinsed withdistilled water and dried at 120 ◦C. Afterwards, the pretreated ZMwere soaked in SCS in a 36.5 ◦C water bath to produce HAp coatingson their surfaces. The SCS was replaced every 2 days. In this study,all the HAp-coated ZM were denoted as HAp-ZM, ZM obtained afterone incubation in SCS were denoted as 1HAp-ZM, after two incu-bations in SCS were denoted as 2HAp-ZM, and so forth. Finally,the calcination procedure was carried out by heating at a rate of0.5 ◦C/min to 600 ◦C and holding for 2 h to age the HAp coating.
2.4. Instrumentation
Surface morphology of the particles was displayed by aQUANTA-200 scanning electron microscopy (SEM) system (FEI,Eindhoven, the Netherlands). Ca/P ratios of the HAp coatings weredetermined by an XSAM800 X-ray photoelectron spectroscopy(XPS) system (Kratos, Manchester, UK), with Mg K� radiation as theexciting source. Crystal structure of HAp-ZM was determined withan XRD-6000 X-ray diffraction (XRD) system (Shimadzu, Kyoto,Japan) using Cu K� radiation and a rotating anode operating at40 kV and 30 mA. Specific surface area, pore volume, and pore sizewere measured by nitrogen sorption at 77 K using a SA3100 Plussurface area analyzer (Beckman, Fullerton, CA, USA).
2.5. Chromatographic conditions
Unmodified ZM and 2HAp-ZM were both slurry-packed into150 mm × 4.6 mm I.D. stainless steel columns with 0.3 M sodiumphosphate buffer (pH 6.8) at 6000 psi.
All chromatographic measurements were carried out at roomtemperature on an Agilent 1100 HPLC system (Agilent Technolo-gies, Palo Alto, CA, USA) including a Rheodyne Model 7125 injectorwith a 20 �L loop, a quaternary pump, a micro-vacuum degasserand a diode array detector (DAD). Separation of proteins was per-formed at a flow rate of 1 mL/min. The gradient elution was firstlywater for 3 min, and then transferred to 300 mM, pH 6.8 NaP bufferin 1 min, delayed for 12 min, at last transferred to 500 mM, pH 6.8KP buffer in 1 min, and delayed for 18 min. The eluent was moni-tored at 220 nm. When investigating the dynamic loading capacityof the 2HAp-ZM stationary phase, 1 mL injection loop was used.
2.6. Protein recovery studies
The protein mass recovery was measured according to a previ-ously reported method [9,10], by comparing the amount of protein
T. Li, Y.-Q. Feng / Talanta 80 (2009) 889–894 891
Fig. 1. Typical SEM photographs of unmodified ZM and HAp-ZM: (a), (b) unmodified ZM; (c), (d) pre-calcified ZM; (e), (f) 2HAp-ZM; (g), (h) 10HAp-ZM.
892 T. Li, Y.-Q. Feng / Talanta 80 (2009) 889–894
cwp
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r
3
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3
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acde
Fig. 2. XPS spectrums of HAp-ZM.
ollected in a control experiment in which the column was replacedith a zero dead volume pipe to that obtained with the column inlace. The mass recovery was calculated as follows:
ass recovery% = area (column)area (pipe)
× 100%
The proteins were eluted isocratically, and the analysis wasepeated three times for each protein.
. Results and discussion
By definition, biomimetic technique for HAp deposition ishe mimic formation of inorganic compounds in vivo (biomin-ralization). Its mechanism is complicated and the process isime-consuming. Hence, pre-calcification was proposed to acceler-te the precipitation rate of HAp [38]. In this procedure, phosphatenions and calcium cation were adsorbed onto ZM surface one afterhe other; and a nonstoichiometric calcium phosphate was formed.
hen the pre-calcified ZM were immersed in SCS, some compo-ents are released from the material into the fluid and, thereby,
ncrease the ionic activity product of the apatite, which can acceler-te apatite formation. Additionally, SCS rather than SBF was utilizedn this study because the former one provided higher concentrationf phosphate and calcium ions, which could consequently speed uphe deposition rate of apatite [42].
.1. Characterization of the HAp-ZM
Direct observation of the surface morphology of unmodifiedM and HAp-ZM is provided by the SEM micrographs illustratedn Fig. 1. Fig. 1b shows a smooth surface of unmodified ZM, andig. 1d, f and h shows the modified surfaces of ZM after pre-alcification, 2 and 10 repetitions of incubation in SCS, respectively.t can be seen that rod-like nanoparticles are formed on the sub-trate after pre-calcification (Fig. 1d), which is speculated to beomposed of nonstoichiometric calcium phosphate. After repeat-ng the immersion in SCS, the rod-like nanoparticles grow morend bigger (bead-like, Fig. 1f) gradually, and finally a dense andniform HAp coating emerged via attraction and attachment of theanoparticles (Fig. 1h).
The composition of HAp coating on the surfaces of ZM was char-cterized by XPS. Results are shown in Fig. 2. It can be found thatonsiderable amount of Ca and P elements has existed on ZM after 4ays immersing in SCS. Increasing the immersion time, tiny differ-nces in peak area of Ca, P and Zr are observed between 2HAp-ZM
Fig. 3. XRD patterns of (A) HAp and (B) 10HAp-ZM. Peaks (1–3) around 32–34◦ wereascribed to the diffraction of 2 1 1, 3 0 0 and 2 0 2 planes of HAp crystal. The strongpeaks in (B) were engendered by the ZM matrix.
and 10HAp-ZM. This means that a HAp coating was basically formedat the stage of 2HAp-ZM; long immersion time was avoided becauseof the introduction of pre-calcification treatment, which stimulatesthe apatite nucleation and accelerates the growth rate of the HAplayer. Furthermore, the Ca/P ratio of the resultant coating is cal-culated to be 1.59, close to the Ca/P ratio of HAp 1.66. Therefore,we considered that this coating was mainly composed of HAp and2HAp-ZM was selected for the following experiments.
The X-ray diffraction patterns of 2HAp-ZM were displayed inFig. 3 in comparison with that of Tiselius type HAp. Only small typi-cal peaks (around 32◦ and 34◦), which are ascribed to the diffractionof 2 1 1, 3 0 0 and 2 0 2 planes of HAp crystal, are observed, sug-gesting the poor crystallinity of the HAp on 2HAp-ZM. Combinedwith the SEM photos, we can suppose that the coating is composedof an amorphous or nanocrystalline apatite. This phenomenon isconsistent with the previously reported results [36,43].
The specific surface area, total pore volume, and average porediameter of 2HAp-ZM are 25 m2/g, 0.09 cm3/g, and 14 nm, respec-tively, calculated using the standard BET method.
3.2. Chromatography of proteins
The feasibility of the HAp-coated ZM used as HPLC station-ary phase for protein chromatography was evaluated using BSA,trypsin, lysozyme, RNase A and Cyt c as model analytes. The physicaland chemical properties of these proteins are listed in Table 1.
It is reported that there are two types of interaction site onHAp. An acidic protein, such as BSA, mainly interacts with the Csites, while a basic protein, such as lysozyme, mainly interacts withthe P sites [27]. Hence, both acidic and basic proteins were ableto be resolved within a single run of HPLC on the basis of multi-modal mechanism of separation [49]. In this study, five proteinswere successfully separated on the 2HAp-ZM stationary phases in
gradient elution mode using pH 6.8 phosphate buffer as mobilephase, as shown in Fig. 4. Compared to the unmodified ZM, onwhich lysozyme and RNase A were eluted in one peak withoutsufficient resolution, it can be concluded that the HAp coating is
T. Li, Y.-Q. Feng / Talanta 80 (2009) 889–894 893
Table 1Properties of proteins investigated in this study.
Protein Source MW pIa Rsb (nm)
BSA Bovine serum 66 000 4.7 3.39c
Trypsin Bovine pancreas 23 300 10.1 2.40d
Lysozyme Chicken egg white 14 700 11.0 1.95e
RNase A Bovine pancreas 13 700 9.6 1.93c
Cyt c Horse heart 12 384 9.8 1.70c
Ovalbumin Chicken white 44 287 4.7 3.02c
AGP Bovine serum 41 000 2.7 3.64f
a Isoelectric point.b The Stokes radii (Rs) values of proteins whose intrinsic viscosity ([�]) is known
from literature were calculated using the relationship proposed by Tanford [44]:[�] = (2.5 Na/MW)(4/3�R3
s ), where Na is Avogadro’s number and MW is the proteinmolecular weight.
eiapt�fhp(metn
iwcbwebpcc
Fmp3
3.4. Protein recovery and loading capacity
c Ref. [45].d Ref. [46].e Ref. [47].f Ref. [48].
ffective to improve the selectivity of 2HAp-ZM, mainly result-ng from its unique bioaffinity toward biomolecules. Compared to
commercial HAp packing [27], although the retention of basicroteins on 2HAp-ZM is comparable, the retention of acidic pro-eins varies. BSA as well as other acidic proteins (ovalbumin and1-acid glycoprotein) has been tested on this column under dif-
erent chromatographic conditions. Unfortunately, they were allardly retained even eluted with pure water as mobile phase. Com-aring the Stocks radii of acidic and basic proteins in our studyTable 1), the relatively small pore size of the stationary phases
ay be one possible explanation. Another explanation may be thelectrostatic repulsion between the negatively charged Zr–OH onhe uncoated matrices and the acidic proteins, which were alsoegatively charged at pH 6.8.
The dependence of protein retention on pH values was alsonvestigated on 2HAp-ZM. Three pH values (pH 6.0, 6.8 and 7.8)
ere employed; lower or higher pH was avoided in view of itshemical instability at acidic conditions and lack of selectivity atasic conditions. Results are shown in Fig. 5. Retention increasesith decreasing pH for all proteins except BSA, implying that cation
xchange provides significant contributions to the retention of
asic proteins. Higher pH of the mobile phase may deprotonate theroteins. Accordingly, basic proteins, which were less positivelyharged at higher pH, are less retained on 2HAp-ZM via weakeration exchange interaction.
ig. 4. Separation of five model proteins on unmodified ZM and 2HAp-ZM. Chro-atographic conditions: NaP–KP buffer at pH 6.8, 1 mL/min, the gradient of mobile
hase was outlined in Section 2.5; detection at 220 nm. Peaks: 1 = BSA; 2 = trypsin;= RNase A; 4 = lysozyme; 5 = Cyt c.
Fig. 5. Separation of five model proteins at three pH values on 2HAp-ZM column.Other conditions were the same as those in Fig. 4. Two forms of Cyt c (oxidized andreduced) were separated on at pH 6.0.
3.3. Stability
Mechanical stability is a crucial criterion for stationary phaseof HPLC, and also the primary objective for us to prepare theHAp-coated ZM. The backpressure as well as the separation per-formance was thus monitored on 2HAp-ZM throughout the wholeexperiment. It was demonstrated that this particle could withstandthe packing pressure (approximately 6000 psi) and allow the useof high flow rate of mobile phase (1 mL/min). Moreover, duringthe 3-month experiment, the backpressure of 2HAp-ZM columnremained stable; and the separation performance was also pre-served, as shown in Fig. 6.
The goal of this work was to synthesize a viable support forprotein chromatography. To be well suited for this application,
Fig. 6. Chromatograms of proteins on 2HAp-ZM column at different times: (A) oncein the experiment; (B) 3 months later (approximately 100 injections had been oper-ated within this period); (C) 6 months later (stored in refrigerator at about 4 ◦C).Other conditions were the same as those in Fig. 4.
894 T. Li, Y.-Q. Feng / Talanta 8
Table 2Protein recovery study on 2HAp-ZM column.
Protein Mobile phase % Recovery (n = 3)
BSA 5 mM NaP buffer, pH 6.0 99.7 ± 3.1Trypsin 5 mM NaP buffer, pH 6.8 93.3 ± 3.5Lysozyme 150 mM NaP buffer, pH 6.8 102.6 ± 0.9RNase A 300 mM NaP buffer, pH 7.8 97.0 ± 3.7Cyt c 500 mM KP buffer, pH 6.8 100.2 ± 1.1
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ig. 7. Study of loading capacity of the 2HAp-ZM column. Experimental conditions:socratic elution with 150 mM NaP mobile phase at pH 6.8; 1 mL/min; detection at20 nm. Symbols: (�) capacity factor; (�) peak width.
ery high recoveries are essential. Therefore, protein recovery wastudied on 2HAp-ZM with results listed in Table 2. In order tonsure accurate peak integration, mobile phases were deliberatelyelected to obtain reasonable retention time and suitable peakidth [6]. It can be found that the 2HAp-ZM column gave good
ecovery for all tested proteins, with the average recovery higherhan 93%, indicating little irreversible adsorption of proteins.
The dynamic loading capacity of the 2HAp-ZM column wasnvestigated by monitoring the column performance with increas-ng sample quantity. It is reported that the retention time and peak
idth will remain constant when the sample quantities are belowhe loading capacity of the column, while overloading will cause
decrease in retention time and an increase in peak width [9].ysozyme was chosen as a tested sample, and the results are shownn Fig. 7. The peak width is somewhat more sensitive to sampleoading than the capacity factor. The dynamic loading capacity ofhe 2HAp-ZM-600 column was estimated to be about 80 �g/mL oflank column volume, based on the amount of sample which caused10% increase in peak width. This is higher than those reported foruoride- and phosphate-modified zirconia (60 �g/mL) [9,10], but
ower than that of a particular high-performance hydroxyapatiteupport (120 �g/mL) [50].
. Conclusions
In this paper, biomimetic technique was demonstrated to be aow-cost and facile approach to prepare biocompatible and stableolumn packings (2HAp-ZM) for protein chromatography. System-tic characterization showed that bead-like HAp was successfullyeposited on the surface of zirconia–magnesia microspheres afterre-calcification and 4 days incubation in SCS. Using this mate-ial as stationary phase, improved separation efficiency, resulting
rom the unique selectivity of HAp coating, was confirmed by theeparation of lysozyme and RNase A (their physical and chemicalroperties were similar) in comparison with an unmodified ZMolumn. And following experiments also indicated that 2HAp-ZMxhibited good mechanical stability, relatively high recovery, and
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0 (2009) 889–894
acceptable dynamic loading capacity, which made it a promisingHPLC support in the separation of proteins or other biomolecules,but there remained much to be understood and optimized beforeroutine use.
Acknowledgments
This work was partly supported by the National Science Fund forDistinguished Young Scholars (No. 20625516), the Science Fund forCreative Research Groups (No. 20621502), NSFC, and the National973 project of China (2007CB914200).
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