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Nano-hard template synthesis of pure mesoporous NiO and its application for streptavidin

protein immobilization

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2014 Nanotechnology 25 165701

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Nanotechnology

Nanotechnology 25 (2014) 165701 (7pp) doi:10.1088/0957-4484/25/16/165701

Nano-hard template synthesis of puremesoporous NiO and its application forstreptavidin protein immobilizationMohammad A Wahab and Farzana Darain

Australian Institute of Bioengineering and Nanotechnology (AIBN) of the University of Queensland, 75,Corner of College and Cooper Roads, St Lucia, Brisbane, QLD 4072, Queensland, Australia

E-mail: m.wahab@uq.edu.au

Received 31 August 2013, revised 20 December 2013Accepted for publication 21 February 2014Published 26 March 2014

AbstractA simple and efficient immobilization of streptavidin protein (with hexa-histidine tag) onto thesurface of mesoporous NiO is described. Before immobilization of streptavidin protein (withhexa-histidine tag) onto the surface of mesoporous NiO, we first synthesized well-organizedmesoporous NiO by a nanocasting method using mesoporous silica SBA-15 as the hardtemplate. Then, the well-organized mesoporous NiO particles were characterized by smallangle x-ray diffraction (XRD), wide angle XRD, nitrogen adsorption/desorption, andtransmission electron microscopy (TEM). TEM and small angle XRD suggested the formationof mesoporous NiO materials, whereas the wide angle XRD pattern of mesoporous NiOindicated that the nickel precursor had been transformed into crystalline NiO. The N2 sorptionexperiments demonstrated that the mesoporous NiO particles had a high surface area of281 m2 g−1, a pore volume of 0.51 cm3 g−1 and a pore size of 4.8 nm. Next, theimmobilization of streptavidin protein (with hexa-histidine tag) onto the surface ofmesoporous NiO was studied. Detailed analysis using gel electrophoresis confirmed that thisapproach can efficiently bind his-tagged streptavidin onto the surface of mesoporous NiOmaterial since the mesoporous NiO provides sufficient surface sites for the binding ofstreptavidin via non-covalent ligand binding with the histidine tag.

Keywords: mesoporous materials, nanocasting hard templating approach, NiO, streptavidinprotein

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

1. Introduction

Along with the rapid development of biomedical and othernanobiotechnology strategies, among the metal nanoparticles,in particular, Ni nanoparticles (NiPs) have received a greatdeal of research interest because of their unique physical andchemical properties for a wide range of applications [1–7].According to a recent literature survey, several studies onsmall amounts of incorporation have been reported andfound that NiPs/NiPs-containing mesoporous structures havefound applications that include biomolecule immobilization orseparation, biosensors, magnetic resonance imaging (MRI),

targeted drug delivery, biocatalysis, toxic gas detection,and advanced anodic electrodes for energy storage [7–14].Meanwhile, Mirkin and co-workers have first coated atomicforce microscopy (AFM) tips with a thin layer of Ni onSi(100) wafers by thermal evaporation, then bioactive proteinnanoarrays on nickel oxide surfaces were formed by dip-pennanolithography without applying a potential [8]. Sometimelater, Lee et al [9] reported the synthesis of partially NiO-coated porous particles for selective binding and magneticseparation of histidine-tagged proteins.

Toward this direction, not the pure mesoporous NiOmaterials but rather mesoporous silica SBA-15 spheres

0957-4484/14/165701+07$33.00 1 c© 2014 IOP Publishing Ltd Printed in the UK

Nanotechnology 25 (2014) 165701 M A Wahab and F Darain

partially functionalized with NiPs have been used for themagnetic separation of protein molecules from a proteinmixture solution [10]. In a separate study, multifunctionalmesoporous core/shell structures with a mixture of metalparticles (Fe3O4@NiSiO3) have also been designed toselectively separate and efficiently enrich biomolecules [11].Besides the use of mesoporous silicate particles/nanostructuresfor the immobilization of biomolecules [12], Gaffney et al [13]have recently grafted the cyclam moiety on the surface ofmesoporous SBA-15 silica and finally NiPs were again graftedonto cyclam-grafted SBA-15 silica, which has subsequentlybeen used for tailoring the adsorption of His6-tagged proteinbut not the pure mesoporous NiO materials. However, thesenanostructures, although promising, have the drawback ofrequiring a complex multi-step fabrication strategy andyet still have a poor binding affinity. Furthermore, noneof these reports demonstrated effective binding/affinitybetween pure mesoporous NiO and protein molecules, eventhough NiO porous material presents a high surface area,large pore volume and uniform pore size, magnetic andopto-electronic properties, and excellent surface affinitytowards biomolecules. To date, to the best of our knowledge,pure ordered mesoporous NiO has not been used for theimmobilization of proteins or any other biomolecules, eventhough the NiO surface shows good affinity to poly-histidines.On the basis of this discovery, we envisioned that the synthesisof ordered pure mesoporous NiO material which shows highaffinity towards histidine-tagged biomolecules may be furthermodified for biomedical applications.

For the first time, we herein report a procedure that isa simple and efficient immobilization of streptavidin protein(with hexa-histidine tag) onto the surface of mesoporousNiO with crystalline walls. In this study, mesoporous NiOshows excellent affinity to bind streptavidin (with a poly-histidine tail) onto the surface of pure mesoporous NiO. TheXRD, TEM and Brunauer–Emmett–Teller (BET) analysesshow the formation of a pure ordered mesoporous NiOstructure. The well-dried pure mesoporous NiO was used forthe immobilization of his-tagged streptavidin protein. Lithiumdodecyl sulfate–polyacrylamide gel electrophoresis (LDS–PAGE) was used to investigate the efficient immobilizationof his-tagged streptavidin onto the surface of mesoporous NiOmaterial.

2. Experimental details

Materials: Pluronic surfactant P123 (EO20PPO70EO20),tetraethyl orthosilicate (TEOS), nickel(II) nitrate hexahydrateNi(NO3)2·6H2O and ethanol were used as received for thesynthesis work. Streptavidin protein (hexa-histidine-tagged,17 kDa) was purchased from Abcam R©.

2.1. The synthesis of mesoporous SBA-15 silicate hardtemplate

2 g of Pluronic surfactant P123 (EO20PPO70EO20) was stirredin 60 ml of 2 M HCl at 38 ◦C for 2 h. Then 4.2 g of TEOS wasadded slowly to the surfactant solution with vigorous stirring.

The mixture was stirred for only 6–8 min, then the solutionwas left to stand for another 24 h at 38 ◦C. The mixturewas subsequently heated at 100 ◦C for another 24 h in anautoclave. The as-synthesized SBA-15 template was collectedby filtration, dried and then calcined at 550 ◦C for 6 h in air.

2.2. Synthesis of mesoporous NiO particles with crystallinepore walls by a nanocasting method

The mesoporous NiO particles were replicated from meso-porous SBA-15 silicate using a nanocasting method, as shownin figure 1(a). In a typical procedure, 0.87 g of Ni(NO3)2·6H2Oprecursor (Ni source) was dissolved in 15 ml ethanol toform a transparent solution. Then, 0.45 g of dry mesoporousSBA-15 silicate/silica samples was added slowly to the Nisource containing transparent solution. After stirring for12 h, the solvent was evaporated at room temperature for12 h and then at 50 ◦C for another 12 h. The obtainedNi-impregnated solid SBA-15 materials were ground andsintered at 400 ◦C for 2 h. The impregnation procedure wasrepeated once more to obtain the nickel precursor/mesoporousSBA-15 silicate composites. After removing the solventusing the aforementioned procedure, the impregnated nickelprecursor/SBA-15 silicate materials were then calcined at600 ◦C for 6 h, then the SBA-15 silica templates were removedby stirring with hot 2 M sodium hydroxide solution (80–90 ◦C)twice for 12 h to obtain the mesoporous NiO. The final wettedmesoporous NiO was collected after washing with ethanol anddistilled water and dried in an oven at 50 ◦C.

2.3. Immobilization of his-tagged streptavidin onto the surfaceof the mesoporous NiO scaffold

For immobilization of his-tagged streptavidin onto the meso-porous NiO, 150 µl of 0.5 µg ml−1 of his-tagged streptavidinwas added to 300 µg of mesoporous NiO and kept overnightat 4 ◦C on a shaker operating at 400 rpm. Next, themesoporous NiO was washed three times with phosphatebuffer saline (PBS, pH 7.4, Sigma) under centrifugation at10 000 g for 3 min at room temperature. The mesoporousNiO was separated by centrifugation and the supernatant wasused for protein determination using a NanoDrop R© ND-1000spectrophotometer at 280 nm. The schematic procedure forimmobilizing his-tagged streptavidin onto the surface ofmesoporous NiO is shown in figure 1(b). The binding amountsof his-tagged streptavidin on mesoporous NiO were measuredby shaking 500 µl of each his-tagged protein solution atdifferent initial concentrations for 12 h with 0.5 mg of themesoporous NiO. The binding amount was then determinedby comparing the his-tagged streptavidin concentration in thesupernatant before and after adding the mesoporous NiO, usinga NanoDrop R© ND-1000 spectrophotometer. A leaching testwas carried out with protein-immobilized mesoporous NiOsuspended in PBS at pH 7.4, which was continuously shakenat 400 rpm at 4 ◦C overnight. After centrifuging at 10 000 g for3 min at room temperature, the amount of protein leakage wasinvestigated using a NanoDrop R© ND-1000 spectrophotometerat 280 nm. All experiments were done in triplicate.

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Nanotechnology 25 (2014) 165701 M A Wahab and F Darain

Figure 1. Synthesis of (a) mesoporous NiO via mesoporous SBA-15 silicate and (b) subsequent binding of his-tagged streptavidin protein.On the right side the arrow with the cartoon mark shows the Ni–histidine coordination bonds.

2.4. Characterization techniques

The mesoporous SBA-15 silica template and pure meso-porous NiO samples were characterized by small XRD,N2 absorption–desorption isotherms, and TEM. The synthe-sized mesoporous SBA-15 silica samples were characterizedby powder x-ray diffraction (XRD, Bruker Radiation D8Advanced Diffractometer, Cu Kα radiation). For mesoporousNiO, wide angle XRD patterns were recorded under identicalconditions on a Rigaku Miniflex diffractometer (Japan) usingCu Kα radiation at a scanning rate of 2◦ min−1 in the 2θrange from 10◦ to 80◦. The BET surface areas and texturalstructure were measured using an automated adsorptionanalyzer (Autosorb-1C, Quantachrome, USA). The Barrett-Joyner-Halenda (BJH) method was used to calculate thepore-size distribution curve using the desorption isotherms.Transmission electron microscope (TEM) observation was car-ried out on an FEI Tecnai 20 microscope with an acceleratingvoltage 200 kV. The TEM samples were prepared by dippingultrasonically dispersed samples in ethanol. The solution wasallowed to settle and carbon TEM grids were used to scoopup some of the clear liquid and then allowed to dry at roomtemperature for half an hour. This was done so that the smallestparticles could be mounted on the grid for better TEM analysis.For gel electrophoresis, 3.3µl of lithium dodecyl sulfate (LDS)sample buffer (Invitrogen, Australia) was added to 10 µlof each sample, followed by heating at 70 ◦C for 10 min.10 µl of supernatant was immediately taken after heating andloaded on NuPAGE R© Novex 4–12% Bis-Tris Gels (Invitrogen,Australia). The gel was stained with SYPRO R© Ruby ProteinStain (Invitrogen, Australia) according to the manufacturer’sinstructions for protein analysis. The gel was read using aTyphoonTM 9400 (GE Healthcare, UK) variable mode imager.

3. Results and discussion

In order to prepare pure mesoporous NiO, first a porous SBA-15 template as a highly ordered mesoporous silicate structurethroughout was prepared from the non-ionic surfactant P123under acidic conditions [15–17]. The low angle XRD patternsin figure 2(a) show a very intense diffraction peak and twoadditional peaks, assigned to the 100, 110 and 200 planes,respectively, which are characteristic of a material with ahighly ordered hexagonal structure (p6 mm), consistent withother previously reported highly ordered mesoporous SBA-15silicate [15–17]. The TEM image in figure 2(b) shows well-organized nanochannels, which is evidence of the formation ofhighly ordered mesoporous SBA-15 silicate. The surface-porestructural properties of mesoporous SBA-15 silicate in table 1were investigated by N2 adsorption–desorption measurements.The specific surface area (SBET), total pore volume and poresize of the mesoporous SBA-15 silicate were 719 m2 g−1,1.12 cm3 g−1 and 9.1 nm, respectively, which is consistentwith previous results on highly ordered mesoporous SBA-15silicate [15–17].

In order to produce a material with highly orderedmesoporous NiO, it is essential to achieve a high degreeof infiltration and wetting of the precursor solution withinthe pore structure of the hard template SBA-15. This wasachieved in the case of pure mesoporous NiO by infiltrating asolution of Ni(NO3)2 in ethanol into the pores of mesoporousSBA-15 silicate. In this way, it was possible to achieve avery high degree of pore filling. The intense small angleXRD peak at 1.0◦ for mesoporous NiO along with thebroad secondary reflection near 2.0◦ in figure 3 confirms thatmesoporous NiO materials have been successfully replicatedon the mesoporous SBA-15 silicate template. The d100 spacingof mesoporous NiO is 8.83 nm. The wide angle XRD

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Nanotechnology 25 (2014) 165701 M A Wahab and F Darain

Figure 2. Small angle XRD pattern (a) and TEM image (b) ofmesoporous SBA-15 silicate.

pattern of mesoporous NiO shown in the inset of figure 3(b)exhibits diffraction peaks at 21.4◦, 43.25◦, 45.46◦, 50.64◦,59.79◦, 66.29◦, 69.12◦and 74.5◦(2θ ), indicating that the nickelprecursor has been transformed into crystalline NiO. Thesediffraction peaks are consistent with JPCDS No 001-1239.The N2 adsorption–desorption results outlined in figure 4(a)and table 1 correspond well with the formation of mesoporousNiO [12–23]. The N2 adsorption–desorption isotherms ofmesoporous NiO in figure 4(a) show two slopes, which arelocated at relative pressures of 0.43 and above 0.9, which arecharacteristic of a bimodal pore-size distribution of the finalproduct. The BJH pore-size distribution of the mesoporousNiO is found to be 4.8 in figure 4(b). The presence of a narrowpore-size distribution is attributed to uniformly ordered poresof mesoporous NiO. The small number of pores in the rangeabout 30–45 nm could be related to the interspacing betweenNiO secondary particles or disordered mesopores of the finalproduct [21, 22, 24]. The pore-size distribution obtained formesoporous NiO from N2 adsorption is compared with that forSBA-15 in table 1. The result indicates that the NiO is an almostregularly formed mesoporous structure with a narrow pore-sizedistribution centered at 4.8 nm, while the pore-size distributionfor pure SBA-15 is centered at 9.1 nm. This nanostructure isformed by the pore filling method as shown in figure 1, where

Figure 3. Small angle XRD pattern of mesoporous NiO in the mainfigure. Inset shows the higher angle XRD patterns.

Table 1. Physicochemical properties obtained from the nitrogenadsorption analysis for the mesoporous materials used in this study.

Sample codeSurface area(m2 g−1)

Pore volume(cm3 g−1)

Pore size(nm)

Mesoporous SBA-15 719 1.12 9.1Mesoporous NiO 281 0.52 4.8

ordered mesoporous SBA-15 silica is used as a template,the final removal of which leaves behind an ordered NiOframework. As clearly shown in figure 1, the nanostructureof the NiO prepared for this study is composed of orderedmesoporous NiO, which was formed inside the cylindricalpores/nanochannels of the SBA-15 silica template via theimpregnation method. Once the template has been completelyremoved by washing with 2 M NaOH, the ordered mesoporousNiO is interconnected into an ordered hexagonal array by Nispacers, which are formed inside the complementary poresbetween adjacent cylinders. The obtained results are consistentwith previously reported porous carbon materials whereSBA-15 silica has been used as a template. Similar shapes andhysteresis loops for other mesoporous transition metal oxidessynthesized by the hard template route have been reported[13, 14, 22]. In addition, we studied the mesoporous NiOmaterials by TEM and the images in figures 5(c) and (d) showa well-ordered pore structure of mesoporous NiO, consistentwith previous replicated mesoporous systems [13, 14, 22, 24].The resulting textural porosity is caused by an incompleteand/or non-uniform filling of the template pores with the NiOprecursor, which may result in some unfilled interconnectingpores.

The functionalization of mesoporous NiO with strep-tavidin is advantageous for further applications because ofits extremely high affinity to biotin, as biotin can be easilyconjugated to different proteins so that a variety of assayscan be developed for the detection of different analytes [8–10,24–27]. The binding of streptavidin (his-tag) to mesoporousNiO was analyzed with denaturing protein gel electrophoresis

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Nanotechnology 25 (2014) 165701 M A Wahab and F Darain

Figure 4. Nitrogen adsorption–desorption isotherms (a) and thePSD (b) of mesoporous NiO.

(LDS–PAGE), as shown in figure 6(a). To denature proteinsall samples are treated with lithium dodecyl sulfate (LDS)sample buffer and heated at 70 ◦C for 10 min. By usingthis technique, any proteins bound to NiO are expectedto dissociate and enter the gel. The migration of proteinsdepends on the mass/charge ratio of the denatured proteins.For this study, we systematically prepared seven samples:molecular weight marker, supernatant after incubation ofmesoporous NiO with his-tagged streptavidin (as control 1),his-tagged streptavidin immobilized mesoporous NiO, his-tagged streptavidin standard, his-tagged streptavidin modifiedmesoporous NiO after imidazole treatment, supernatant afterincubation of streptavidin (without his-tag) with mesoporousNiO (as control 2), and mesoporous NiO after streptavidin(without his-tag) incubation (as control 3), as shown infigure 6(a) (Lanes M–7, from the right). A pronounced bandappeared for the his-tagged streptavidin-bound mesoporousNiO in Lane 2 at exactly the same mobility as the standard

Figure 5. TEM images of different areas of mesoporous NiO (a)and (b).

his-tagged streptavidin (Lane 3). This result indicates thathis-tagged streptavidin was attached to mesoporous NiO andthen released from the nanoparticles after heating with thedenaturing reagent. On the other hand, the supernatant of themixture without mesoporous NiO (as control experiment 1)was clear after intensive washing and no corresponding bandwas observed, as shown in Lane 1. The incubation of imidazolewith his-tagged streptavidin-bound mesoporous NiO causedthe his-tagged streptavidin to be released from the NiO onthe basis of ligand exchange and therefore no band appearedin Lane 4. As another control experiment, mesoporous NiOwas incubated with streptavidin without his-tag (controlexperiment 2). A pronounced band was observed for thesupernatant of this mixture in Lane 5, at the same position asthe standard in Lane 6, which means streptavidin without thehis-tag does not bind, or only weakly binds to mesoporousNiO. It is worth noting that no band appeared for themesoporous NiO incubated with streptavidin without his-tagafter intensive washing (control experiment 3) in Lane 7. That

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Nanotechnology 25 (2014) 165701 M A Wahab and F Darain

Figure 6. (a) LDS–PAGE for the immobilization of his-taggedstreptavidin on mesoporous NiO. Lane M: molecular weight marker(BenchMarkTM), Lane 1: supernatant after incubation of his-taggedstreptavidin with mesoporous NiO, Lane 2: his-tagged streptavidinimmobilized mesoporous NiO, Lane 3: his-tagged streptavidinstandard, Lane 4: his-tagged streptavidin modified mesoporous NiOafter imidazole treatment, Lane 5: supernatant after incubation ofstreptavidin (without his-tag) on mesoporous NiO, Lane 6:streptavidin standard (without his-tag), Lane 7: mesoporous NiOafter streptavidin incubation. (b) Binding of histidine-taggedstreptavidin on mesoporous NiO. The error bars indicate thestandard error of the mean.

means weakly bound (if any) streptavidin (without his-tag) wasremoved and NiO did not contain any protein after washing.Considering the observations from controls 1, 2 and 3, it isevident that his-tagged streptavidin was strongly bound tomesoporous NiO. Figure 6(b) shows the corresponding curvebetween the amount of added his-tagged streptavidin andthe amount of bound his-tagged streptavidin on mesoporousNiO. As expected, the figure shows a sharp initial riseat lower solution concentrations, then slows to reach aplateau, indicating a high affinity between the histidine-taggedstreptavidin on the NiO surface. An important considerationis to investigate whether the immobilized protein is leachingfrom the NiO support. A study of histidine-tagged streptavidinon mesoporous NiO confirmed that there is no or negligibleleaching of protein from the support to the buffered mediumusing a NanoDrop R© ND-1000 spectrophotometer at 280 nmand may find potential applications in various fields.

4. Conclusion

In this work, for the first time, streptavidin was successfullyimmobilized on mesoporous NiO via non-covalent bindingusing a hexa-histidine tag. Importantly, this work was carriedout to investigate protein immobilization via metal–ligandcoordination bonding, as the histidine tag is functional in thisprotein for complexing with Ni rather than adsorption or boundinto the mesoporous interior. This kind of binding can providecontrol over the uniformity of protein binding without directcontact between the active area of protein and the substratesurface. Potentially, biotinylated protein could also be attachedon the mesopore surface via the affinity interaction for furtherapplication. We believe that this approach may open a newavenue for the immobilization of proteins on mesoporousNiO for applications in bioassays, including biosensors andnanobiotechnology areas.

Acknowledgments

This work has been supported by the University ofQueensland Grant Number RM 2010001818 and ARC Centrefor Functional Nanomaterials, AIBN. UQ.

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