Colloids and Surfaces B: Biointerfaces 122 (2014) 375–383

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Protein attachment to nanoporous anodic alumina for biotechnological applications: Influence of pore size, protein size and functionalization path Malgorzata Baranowska, Agata J. Slota, Pinkie J. Eravuchira, Gerard Macias, Elisabet Xifré-Pérez, Josep Pallares, Josep Ferré-Borrull, Lluís F. Marsal ∗ Departament d’Enginyeria Electrònica, Elèctrica i Automàtica, Universitat, Rovira i Virgili, Avda. Països Catalans 26, Tarragona 43007, Spain

a r t i c l e

i n f o

Article history: Received 18 March 2014 Received in revised form 4 June 2014 Accepted 15 July 2014 Available online 23 July 2014 Keywords: Nanoporous anodic alumina GTA Silane-PEG-NHS Collagen BSA Reflection interference Fourier transform spectroscopy (RIFTS)

a b s t r a c t Nanoporous anodic alumina (NAA) is a material with great interest in nanotechnology and with promising applications to biotechnology. Obtaining specific and regularly functionalized NAA surfaces is essential to obtain meaningful results and applications. Silane-PEG-NHS (triethoxysilane-polyethylene-glycol-Nhydroxysuccinimide) is a covalent linker commonly used for single-molecule studies. We investigate the functionalization of NAA with silane-PEG-NHS and compared with two common, but not single-molecule, grafting agents, APTMS (3-aminopropylotrimethoxysilane) as an electrostatic linker, and APTMS-GTA (3aminopropylotrimethoxysilane-glutaraldehyde) as covalent. Another outcome of this study is to show how two proteins (collagen and bovine serum albumin, BSA) with different properties differentially arrange for different functionalizations and NAA pore sizes. FTIR is used to demonstrate the surface modification steps and fluorescence confocal microscopy reveals that silane-PEG-NHS results in a more homogeneous protein distribution in comparison to the other linkers. Reflection interference Fourier transform spectroscopy confirms the confocal fluorescence microscopy results and permits to estimate the amounts of linker and linked proteins within the pores. These results permit to obtain uniformly chemical modified NAA supports with a great value in biosensing, drug delivery and cell biology. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Porous materials as nanoporous anodic alumina (NAA) have advantageous features such as low cost fabrication, large surface area, controllable pore structure and well-known optical properties. These unique properties manifest NAA as a useful material for studying biological substances [1–6]. Unlike planar geometries, porous materials offer the possibility to study protein adsorption in three-dimensional arrangements [7]. High sensitivity thin-film detection and NAA provide excellent platform to investigate assembly process [7]. The advantages of functionalized porous materials can be used in many fields such as biosensing, drug delivery systems and cell culture [8,9]. NAA has proved to be excellent as biosensing platform, it can play a role as a selective filter for different molecules by sizeexclusion or surface chemistry selectivity [10]. The advantage of

∗ Corresponding author. Tel.: +34 977 559 625; fax: +34 977 559 605. E-mail address: [email protected] (L.F. Marsal). 0927-7765/© 2014 Elsevier B.V. All rights reserved.

large specific surface area of NAA combined with Reflection interference Fourier transform spectroscopy (RIFTS) can be exploited to develop ultrasensitive biosensor platforms for a broad range of analytes and biological events [11,12]. One of the advantages of NAA over other systems for drug delivery is that their structure is stable and does not degrade nor erode under physiological conditions, simplifying controlled drug release [13,14] over longer time periods. An effective way to control drug release from a porous material is to improve drug–surface interaction by surface chemical functionalization [15]. Another application of functionalized porous material is cell culture. It is known that the cells grow better on functionalized surface and attach better [16]. Functionalizations improve biocompability and hydrophilicity. Surface modification of inorganic material by organic groups is of great interest in various fields: biomedical applications and surface coating [17,18]. Surface modification is a key factor for controlling the activity of the biological molecules. Without functionalization attachment of biomolecules would be not repeatable. Furthermore, by introducing special chemical groups it is possible to orientate the molecules such as antibodies to prevent


M. Baranowska et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 375–383

a decrease in their biological activity. Organosilanes (R SiO R) are well known molecules that can provide bond between inorganic and organic materials. In order to link the organic part, they deliver a suitable functional group on the functionalized surface, changing its properties by making them chemically active to attach molecules electrostatically or covalently [19,20]. APTMS (3-aminopropylotrimethoxysilane) reacts with the free hydroxyl groups ( OH) of oxidized metals by exchange of the silane methoxy (CH3 O Si ) groups with the ( Si O ) silanol oxygen, with loss of methanol (CH3 OH). The free positively charged amino group ( NH3 + ) on another side of the molecule is very active with negatively charge carboxyl group ( COO− ) of the amino acids in proteins. These two groups make electrostatic interaction between protein and APTMS [21]. To obtain covalent bond between protein and linker, APTMS can be activated with glutaraldehyde (GTA) [22]. As a comparison to the glutaraldehyde linker, we use silane-PEG-NHS, which has the ability to attach only one molecule of protein. This propriety is important in single molecule studies as well as to obtain homogeneous layers of proteins. N-hydroxysuccinimide (NHS) is a reactive group which reacts with primary amine groups to form stable bonds [23]. Furthermore, up to our knowledge this linker has not been previously reported as functionalization in NAA. Bovine serum albumin and collagen are two common proteins used in testing the functionalization of other inorganic materials [24–28]. BSA is a small size protein (few nm in length) and is used for many applications in immunohistochemistry, cell culture and assays. In contrast, collagen is one of the long, fibrous structural proteins (around 300 nm long); commonly used to coat surfaces to stimulate cell adhesion [24]. This protein is biocompatible and hydrophilic, making it a very sustainable, cost-effective material for modification [25]. Collagen has been used in medicine and dentistry for a long time as a wound healing tissue [26,27]. The researchers have shown that cells grow better on surfaces coated with collagen [28]. In this study we analyze different modifications of the NAA surface chemistry and the influence of such functionalization on the protein attachment into the inner pore surfaces. We use two proteins with different size and charge properties and immobilize them onto NAA with various pore sizes. Characterization of the samples carried out by fluorescence confocal microscopy, reflection interference Fourier transform spectrophotometry and FTIR. Studies made by confocal microscopy will show the correlation of protein distribution and functionalization of the NAA while spectrophotometry studies permit to study organization of protein within pores. The aim of this study is to provide information for successful surface functionalization for further applications such as biosensing, cell growth and drug delivery.

2.2. Nanoporous anodic alumina production Nanoporous anodic alumina (NAA) samples were fabricated using a two-step anodization procedure [12]. Aluminum foils were electropolished in a 1:4 mixture of perchloric acid (HClO4 ) and ethanol (EtOH) for 4 min with voltage 20 V. After the samples were thoroughly rinsed with water and ethanol follow by drying to avoid any residues of acid. Afterwards the first step anodization was carried out in 0.3 M oxalic acid (H2 C2 O4 aqueous) at 40 V and 5 ◦ C for 20 h and in 1% phosphoric acid (H3 PO4 ) in ethanol: water mixture (1:4) at 194 V and 0 ◦ C for 24 h. Subsequently, the alumina layer with disordered pores was dissolved through wet chemical etching in a mixture of phosphoric acid (H3 PO4 ) 0.4 M and chromic acid (H2 CrO7 ) 0.2 M for 3 h at 70 ◦ C. Then, the second step of anodization was performed under the same conditions as the first step. The time of anodization was controlled by the charge to obtain layers with 5 ␮m thickness for NAA anodized with oxalic acid and 600 nm thickness for NAA anodized with phosphoric acid. Finally, the pore diameter was widened by wet chemical etching with 5% H3 PO4 and resulting in pores with 70 nm of diameter for NAA anodized with oxalic acid and 200 nm of diameter for NAA anodized with phosphoric acid. 2.3. APTMS functionalization In order to develop a surface able to attach a protein, the NAA was treated with boiling hydroxyperoxide (H2 O2 ) to obtain hydroxyl ( OH) groups on the surface. The substrates were washed with water and followed by ethanol and dried with nitrogen. The surfaces need to be absolutely dry as any trace of water may make silane aggregation and avoid attachment to the surface. Samples were kept in nitrogen atmosphere through all reaction. 20 ml of dry toluene was added to the samples followed by of 0,2 ml 3-aminopropyltrimethoxysilane (APTMS) to obtain a 1% (v/v) concentration of the silane in the mixture. Then, the solution was stirred for 1 h followed by sonication for 1 h to remove any polymerized APTMS. After this process, samples washed with toluene, ethanol and water, then dried with nitrogen and incubated overnight in the oven at 110 ◦ C to crosslink the silane. 2.4. APTMS-GTA functionalization After the first step functionalization with APTMS, in the second step of chemical functionalization, APTMS-modified NAA was immersed in 10% (v/v) glutaraldehyde (GTA) for 1 h at room temperature and after this time washed with ethanol and water and dried. The result is aldehyde ending ( CHO) modified surface that allows to attach biomolecules covalently. APTMS and GTA are molecules with small size (0.7–0.8 nm) and can infiltrate easily into the pores of NAA.

2. Materials and methods

2.5. Silane-PEG-NHS functionalization

2.1. Materials

Hydroxylated NAA were immersed in freshly prepared 1% (w/v) of silane-PEG-NHS in Dimethyl sulfoxide (DMSO) solution. Samples were kept for 1 h at room temperature. Silane-PEG-NHS left for long time polymerizes and looses activity. After one hour the surface was washed with DMSO and water and dried with nitrogen.

Aluminum foils (99,999%) were purchased from Goodfellow Cambridge Ltd. (UK). Glutaraldehyde (anhydrous 10% in EtOH) was purchased from Electron Microscopy Science (US). Silane-PEG-NHS was obtained from Nanocs (US). Oxalic acid, phosphoric acid, perchloric acid, chromic acid, hydroxyperoxyde, 3-aminopropylotrimethoxysilane, collagen type I from bovine achilles tendon, bovine serum albumin (BSA), rhodamine B isothiocyanate, phosphate buffer saline (PBS), toluene, dimethylsulfoxide and ethanol were purchased from Sigma–Aldrich (Spain). Mili-Q-water (18 M) was used for rinsing and preparation of solutions.

2.6. Protein immobilization In order to study the effect of the pore size onto the attachment of biomolecules in the inner pore walls and on the upper NAA surface, two model proteins were used: collagen and bovine serum albumin. Collagen type I was used as it is the most common type in the human body and it is mostly used in cell adhesion experiments.

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Immobilization of proteins was made by incubation of functionalized substrate with appropriate protein solution (5 mg/ml in PBS pH 7.4) overnight in high humidity chamber at 4 ◦ C. BSA and collagen were labeled with rhodamine isothiocyanate B. Rhodamine isothiocyanate B was dissolved in DMSO (concentration 1 mg/ml). 100 ␮l of fluorophore solution was added drop by drop to 1 ml of protein solution (5 mg/ml in PBS). Mixture was stirred for 1 h at room temperature and deployed on the surface. 2.7. Confocal microscopy Confocal fluorescence microscopy images were obtained using a NIKON Eclipse TE 2000 from Nikon Instruments (US). The laser excitation at 543 nm and emission window at 590–650 nm was chosen for Rhodamine isothiocyanate (excitation  = 543 nm; emission  = 550–650 nm). Confocal images were captured with inverted microscope equipped with a 100× oil immersion objective. Quantification of pictures was made using Image J. ( 2.8. Spectrophotometry The reflectance spectra of the NAA were measured in a Perkin Elmer UV–visible–IR spectrophotometer model lambda 950 at room temperature. Provided the porous alumina layer thickness is small enough, Fabry–Pérot interferences give oscillations on the reflectance spectrum [29]. The effective optical thickness (EOT = 2nL) [4] of the layer can be estimated from the positions of the reflectance maxima in the spectrum, using formula 2nL cos() = mm , where n is the effective refractive index. L is the film physical thickness, m is the wavelength of the m-th order maximum and  is the propagation angle inside the film. The value of EOT can be estimated by analyzing the fast Fourier transform (FFT) of the reflectance spectrum and locating the peak associated with the Fabry–Pérot oscillations. Surface modification due to functionalization with different molecules or protein attachment can be detected through changes on the effective optical thickness (EOT). 2.9. Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy (FTIR) was used in order to characterize the presence of specific chemical groups bonded to the chemically modified surfaces after each step. FTIR spectra were obtained in the wavenumber range from 400 to 4000 cm−1 . All spectra were collected with 16 scans and 4 cm−1 resolution. The FTIR measurements were carried out in FTIR spectrometer Bruker Vertex 70 equipped with Tungsten and Globar sources for NIR–MIR range. 3. Results and discussion 3.1. Confocal microscopy Figs. 1 and 2 present confocal images of collagen and BSA on functionalized NAA, respectively. In each of these figures, the first row (images a and b) corresponds to APTMS functionalization, the second row (images c and d) to APTMS-GTA while the third row (images e and f) to silane-PEG-NHS. Furthermore, in each of the figures, the left column (images a, c and e) corresponds to NAA with 70 nm pore diameter, while the right column (images b, d and f) corresponds to NAA with 200 nm pore diameter. Concerning the results for the APTMS functionalization, it has to be taken into account that both proteins interact electrostatically with APTMS, with the strength of binding depending on the molecule charge [21,30]. Both proteins were dissolved in PBS at pH


7.4. As the isoelectric point (IEP) of collagen is 7.8 [28] and of BSA is 4.7 [31], in this pH collagen will have slightly positive charge while BSA will be negatively charged. On the other hand, APTMS makes the NAA surface positively charged. From the images is possible to observe that the amount of attached BSA is greater than of collagen. This can be due to the convenient charge of the surface and BSA. Furthermore, BSA is spread all over the surfaces with some uncovered areas, while collagen aggregates are observed due to repulsive forces between protein and substrate. The influence of the pore size on the protein immobilization with APTMS can be deduced by comparing Figs. 1(a) and 2(a) (70 nm pore diameter NAA) with Figs. 1(b) and 2(b) (200 nm pore diameter NAA), respectively. As the BSA is a much smaller protein than collagen, it is possible to observe different protein immobilization results: images for collagen on NAA with smaller pores show big aggregates on the NAA surface (indicated by an intensity 63% higher in some areas with respect the minimum intensity in the image), while the image for BSA shows a more uniform distribution with black spots. The collagen aggregates indicate it cannot infiltrate in pores because of both a size effect and a charge effect. Instead, the black spots observed for the BSA image may indicate that the protein forms a thin film onto the inner walls of the pores. On the other hand, the images for collagen on NAA with bigger pores do not show aggregates and the underlying pore arrangement can be observed. This demonstrates the protein infiltrates almost fully inside the pores. Fig. 1(c)–(f) illustrates covalent binding of collagen and Fig. 2(c)–(f) covalent attachment of BSA. Figs. 1(c), (d) and 2(c), (d) represent APTMS-GTA functionalization and collagen and BSA attachment, respectively. Figs. 1(c) and 2(c) demonstrate attachment of proteins on 70 nm pore diameter NAA and Figs. 1(d) and 2(d) show attachment of proteins on 200 nm pore diameter NAA. In Fig. 1(c) it is possible to notice improvement in attachment of collagen to APTMS-GTA in NAA with smaller pores as compared to electrostatic adsorption through APTMS. Surface is almost fully covered with protein although there still remain some aggregates. On the other hand, Fig. 1(d) shows that collagen is spread all over the NAA with bigger pores surface and infiltrates inside pores (the image shows the underlying pattern of NAA). Both images show darker regions that can be related to a non-uniform collagen attachment (with a decrease in intensity of 48% with respect to the brightest part for 70 nm pores NAA and 37% for 200 nm pore NAA). This is probably caused by the use of GTA as covalent linker, since as it was suggested by Kiernan [32] the free aldehyde groups introduced by glutaraldehyde fixation cause problems like non-specific binding of proteinaceous reagents. Glutaraldehyde allows to make covalent imine ( C N ) bonding between the biomolecule and grafting agent. This molecule is a common covalent linker for protein but it has the disadvantage of showing non-specific bonding, and consequently produces non-homogenous covered surfaces what may disturb further studies. Comparing collagen organization and BSA is possible to observe some changes. BSA covers uniformly all surface of 70 nm pores NAA (Fig. 2(c)) while deposition of BSA on 200 nm pores NAA (Fig. 2(d)) shows changes in intensity around 50% between darker and brighter areas which can be due to differences in the number of layers of attached proteins. Figs. 1(e), (f) and 2(e), (f) represent results of covalent linking proteins to silane-PEG-NHS NAA. Fig. 1(e) and (f) establishes organization of collagen on silane-PEG-NHS NAA. On 70 nm pore NAA (Fig. 1(e)) the protein is spread homogenously on all surfaces, in good agreement with Michel [23] who studied attachment of this silane on glass and with DNA and concluded that silane-PEGNHS attach only one biomolecule. The intensity of fluorescence is unvarying (with a maximum variation of 14%) which confirms regular protein attachment. On Fig. 1(f) (200 nm pores NAA) the proteins


M. Baranowska et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 375–383

Fig. 1. Confocal microscopy images of labeled collagen on functionalized NAA with 70 nm diameter pores (left column) and with 200 nm diameter pores (right column). (a) NAA (70 nm) functionalized with APTMS; (b) NAA (200 nm) functionalized with APTMS; (c) NAA (70 nm) functionalized with APTMS-GTA; (d) NAA (200 nm) functionalized with APTMS-GTA; (e) NAA (70 nm) functionalized with silane-PEG-NHS; (f) NAA (200 nm) functionalized with silane-PEG-NHS. The scale bar in all images corresponds to 10 ␮m.

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Fig. 2. Confocal microscopy images of labeled BSA on functionalized NAA with 70 nm diameter pores (left column) and with 200 nm diameter pores (right column). (a) NAA (70 nm) functionalized with APTMS; (b) NAA (200 nm) functionalized with APTMS; (c) NAA (70 nm) functionalized with APTMS-GTA; (d) NAA (200 nm) functionalized with APTMS-GTA; (e) NAA (70 nm) functionalized with silane-PEG-NHS; (f) NAA (200 nm) functionalized with silane-PEG-NHS; The scale bar in all images corresponds to 10 ␮m.


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Fig. 3. FTIR transmission spectra of NAA with pore size 70 nm after functionalization with APTMS-GTA (blue solid line) and silane-PEG-NHS (red dashed line): (a) low wavenumber range 1750–1000 cm−1 ; (b) high wavenumber range 3300–2800 cm−1 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

spread regularly and enter into pores and follow NAA pattern. BSA deposition on 70 nm pores NAA (Fig. 2(e)) also follow the underlying NAA pattern while on bigger pores size NAA (Fig. 2(f)) protein spreads regularly (maximally 11% changes in intensity between darker and brighter zone). The confocal images prove that silane-PEG-NHS results in a more uniform spread of both tested proteins on the surface. The linker is most suitable for collagen but as well for BSA. This is especially relevant for the case of BSA, as this protein has tendency to agglomerate easily. The specific bonding properties of this linker avoid disorderly attaching of proteins and empty places on the surface. 3.2. Fourier transform infrared spectroscopy The FTIR spectra of functionalized surfaces are displayed in Figs. 3 and 4. Fig. 3 shows transmittance spectra while Fig. 4 shows reflectance spectra. Figs. 3(a) and 4(a) represent low wavenumbers spectra while Figs. 3(b) and 4(b) correspond to high wavenumbers. Solid blue line curve shows results of APTMS-GTA functionalization while red dash line curve represents silane-PEG-NHS. According to literature [33] is possible to distinguish some vibrations peaks belonging to particular molecular groups. The measurement were done with NAA as produced as a background in that result the peaks belong only to functionalized surface not to NAA. The measurement was made in both transmittance and reflection modes as some vibrations can be seen in the first mode while others in the second. In transmittance spectra at low wavenumbers (Fig. 3(a)) of APTMS-GTA (solid blue curve) is possible to recognize bending

Fig. 4. FTIR reflection spectra of NAA with pore size 70 nm after functionalization with APTMS-GTA (blue solid line) and silane-PEG-NHS (red dashed line): (a) low wavenumber range 1800–900 cm−1 ; (b) high wavenumber range 3300–2800 cm−1 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

vibration from Si O at 1202 cm−1 . Instead, this peak is not visible in silane-PEG-NHS (dashed red curve) as there appears a wide peak with maximum at 1138 cm−1 corresponding to bending vibrations from C N . Peaks for the C H group at 1300–1383 cm−1 are visible for both functionalization procedures as well as bending vibration of C Si is possible to notice at 1449 cm−1 . In APTMS-GTA spectrum vibrations at 1673 cm−1 represent imine bond ( C N ) and presence of aldehyde group ( CHO) is confirmed at 1727 cm−1 . In silane-PEG-NHS is there are additional bending vibrations from N H amide at 1525 cm−1 and carbonyl group at 1617 cm−1 . In higher wavenumbers (Fig. 3b) is recognizable presence of stretching vibrations from ( C H ) at 2934 cm−1 , C H at 2872 cm−1 and Si C at 2981 cm−1 . In silane-PEG-NHS these vibrations are not very visible due to the spectrum oscillations caused by Fabry–Pérot interferences in the NAA layer. In silanePEG-NHS is possible to observe additional stretching vibrations from N H with maximum at 3223 cm−1 . In reflectance spectra (Fig. 4) some vibrations are more visible. In Fig. 4(a) at 995 cm−1 is possible to notice bending vibrations from Si O in both spectra. In APTMS-GTA (solid blue line) there is visible peak at 1082 cm−1 vibration from Si O Al . There present vibrations from C H at 1350–1478 cm−1 in both spectra. In APTMS-GTA spectrum there are vibrations from aldehyde group CHO at 1741 cm−1 . In silane-PEG-NHS (dashed red) are additional vibrations from, Si C at 1475 cm−1 , N H at 1583 cm−1 and carbonyl group ( C O) at 1704 cm−1 . In spectrum at higher wavenumbers (Fig. 4(b)) is present very significantly vibrations of CH , C H and C Si in APTMS-GTA. In silane-PEG-NHS cause are large molecule this vibrations are not visible.

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Fig. 5. Detail of the reflectance spectrum and of the corresponding FFT for two representative samples after each step in the process: blue lines correspond to as-produced NAA, red lines to the same sample after functionalization with APTMS-GTA and green lines to the same sample after the attachment of two different proteins: (a) collagen, and (b) BSA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

3.3. Spectrophotometry The different surface functionalization procedures and the protein attachment were monitored by recording at visible-NIR wavelength reflectance spectra and obtaining the EOT of the NAA. Results of the obtained spectra for a particular sample and process are shown in Fig. 5. The blue curve in Fig. 5(a) corresponds to the reflectance spectrum for an as-produced NAA sample while the red curve corresponds to the same sample after functionalization with APTMS-GTA. Finally, the green spectrum corresponds to the same sample after the collagen immobilization procedure. The upper image on Fig. 5(a) shows a gradual shift of the reflectance maxima toward longer wavelengths [10], indicating that the silane and the protein attached to the pore surfaces replace air within the pore volume and thus increase effective optical thickness. The down image shows the fast Fourier transform maxima corresponding to each of the samples. The EOT of the layer is taken from the position of such maxima. Fig. 5(b) shows the same series of samples functionalized with APTMS-GTA and BSA as attached protein. Table 1 summarizes all the results obtained from the analysis of the reflectance spectra. The table reports the percent change in effective optical thickness after each process step (silanization and subsequent protein attachment): EOTfunct. (%) =

EOTfunct. − EOTas-produced

EOTprotein (%) =

EOTas-produced EOTprotein − EOTfunct. EOTfunct.

× 100

× 100,

where the subscript ‘funct.’ refers to the EOT after one of the three different functionalizations while the subscript ‘protein’ refers to the EOT after the attachment of one of the two proteins. Percent

change in optical thickness permits to compare between samples with different as-produced EOT. The results after the functionalization show different behaviors of the EOT variation depending on the pore size of NAA. For the APTMS functionalization, there is a remarkable difference between the two electrolytes, while for the APTMS-GTA and the silane-PEGNHS such difference is smaller. On the other hand, it can also be noticed that the biggest EOT variation corresponds to the APTMSGTA process. Such behaviors can be explained by assuming that the optical effect of the functionalization can be modeled by the formation of a thin layer of molecules on the pore inner surfaces. The explanation involves considering the average pore diameter (70 nm for oxalic-prepared NAA and 200 nm for phosphoric-prepared NAA) and the thickness and effective refractive index of the functionalization layer. The two EOT values corresponding to the APTMS process agree with the formation of a thin single-molecule layer on the NAA surface but dense enough to show a high degree of interaction with light (and thus a high refractive index). Such a thin layer and high refractive index, combined with the differences in pore diameter between the two kinds of NAA result in the observed EOT. Instead, the two EOT for APTMS-GTA indicate that this functionalization results in layer with a similar refractive index as the obtained for APTMS but thicker. Such bigger thickness explains the small observed difference between the results for oxalic and phosphoric NAA. Finally, to explain the two values of EOT obtained for the silane-PEG-NHS it is necessary to assume that the layer has a lower effective refractive index but a thickness of the same order as that obtained for APTMS-GTA. Table 1 also shows the results of percent EOT after protein infiltration and attachment. Depending of the protein different behaviors can be observed, mainly because of the protein size.


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Table 1 Percent increment in effective optical thickness after functionalization and after protein attachment. Functionalization path




EOT (%) after functionalization

Attached protein

EOT (%) after protein attachment





Collagen BSA Collagen BSA

0.2 1.8 2.5 0.5





Collagen BSA Collagen BSA

0.5 1.8 1.4 0.2





Collagen BSA Collagen BSA

0.2 1.1 1.6 1.3

NAA Pore size (nm)

The small values of EOT for collagen in 70 nm pore NAA indicate this protein infiltrates very slightly inside the pores, whatever the functionalization procedure. Instead, for 200 nm pore NAA the values show collagen infiltration. The bigger EOT for APTMSfunctionalized NAA and the smaller and similar values for the other two functionalizations indicate a complete filling of the pore with the protein. For BSA infiltration it can be observed that the EOT depends on both the size of the pore and the thickness of the functionalization layer. Thus, smaller pores result in a bigger EOT since probably all the remaining pore volume is full with the BSA. Furthermore, BSA tends to aggregate in the APTMS functionalized NAA because of the electrostatic forces. On the other hand, APTMS-GTA functionalized NAA show a similar EOT to APTMS functionalized NAA, in this case probably non-specific binding of proteinaceous reagents by glutaraldehyde [31]. Instead, for bigger pores, the EOT is smaller as only a layer of BSA is covering the functionalization layer. All these observations are in good agreement with the confocal microscopy results. Attachments of collagen to silane-PEG-NHS demonstrate similar arrangement as with APTMS-GTA. This protein not infiltrates in NAA with smaller pores but fills NAA with 200 nm diameter pores. BSA infiltrates both NAA giving similar results in EOT. 4. Conclusions We have presented three methods of functionalization of nanoporous anodic alumina and we have studied how two different kinds of proteins attach and arrange on the functionalized NAA. Furthermore, the effect of the size of the NAA pore on the protein arrangement has been also studied, by fabricating NAA with 70 and 200 nm pore diameter. We compared two common functionalization agents for NAA (APTMS and GTA-activated APTMS) and the single-molecule-linker silane-PEG-NHS. The functionalization process and the protein attachment and distribution have been studied by fluorescence confocal microscopy, FTIR spectroscopy and reflectance Fourier-transform interference UV–vis spectroscopy. From the analysis of the confocal microscopy images, it can be concluded that after functionalization with APTMS, BSA deposits homogeneously while collagen has a tendency to form aggregates. Concerning the APTMS-GTA, an improvement in collagen arrangement was observed because of the covalent linking mechanism, although some aggregates still remain because each GTA linker can attach several proteins. Silane-PEG-NHS resulted in the most homogeneous distribution of both proteins because of the covalent linking and of the one-to-one protein-linker attachment. Reflectance UV–vis spectra show that there is a correlation between spectrophotometry measurements and confocal images. Comparison of the spectra for the as-produced samples and the same samples after functionalization indicates that APTMS

covers the inner NAA pore surface with a thin single molecular layer while GTA and silane-PEG-NHS generated thicker molecular layers. Results show that after protein incubation collagen cannot infiltrate in the smallest pores while it completely fills the biggest. On the other hand, BSA can be infiltrated for both pore sizes. This work analyses and establishes the most suitable functionalization path for NAA is the silane-PEG-NHS because it promotes a homogeneous attachment of proteins for all protein sizes and pore sizes. These results are useful for several applications such as bio and chemical sensing, cell growth and drug delivery systems. Acknowledgments This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) under grant number TEC201234397 and by Catalan Government under project AGAUR 2014 SGR 1344. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at 2014.07.027. References [1] L.A. DeLouise, P.M. Kou, B.L. Miller, Anal. Chem. 77 (2005) 3222. [2] E.J. Szili, A. Jane, S.P. Low, M. Sweetman, P. Macardle, S. Kumar, R.S.C. Smart, N.H. Voelcker, Sensor. Actuat. B 160 (2011) 341. [3] A. Santos, V.S. Balderrama, M. Alba, P. Formentín, J. Ferré-Borrull, J. Pallarès, L.F. Marsal, Adv. Mater. 24 (2012) 1050. [4] A. Santos, G. Macias, J. Ferré-Borrull, J. Pallarès, L.F. Marsal, ACS Appl. Mater. Interfaces 4 (2012) 3584. [5] R. Urteaga, L.N. Acquaroli, R.R. Koropecki, A. Santos, M. Alba, J. Pallares, L.F. Marsal, C.L.A. Berli, Langmuir 29 (2013) 2784. [6] G. Macias, L.P. Hernández-Eguía, J. Ferré-Borrull, J. Pallares, L.F. Marsal, ACS Appl. Mater. Interfaces 5 (2013) 8093. [7] T.D. Lazzara, I. Mey, C. Steinem, A. Janshoff, Anal. Chem. 83 (2011) 5624. [8] M. Hartmann, Chem. Mater. 17 (2005) 4577. [9] T. Orita, M. Tomita, K. Kato, Colloids Surf. B 84 (2011) 187. [10] R.C. Bailey, M. Parpia, J.T. Hipp, Mater. Today 8 (2005) 46. [11] A. Santos, V.S. Balderrama, M. Alba, P. Formentín, J. Ferré-Borrull, J. Pallarès, L.F. Marsal, Nanoscale Res. Lett. 7 (2012) 370. [12] A. Santos, T. Kumeria, D.T. Losic, Anal. Chem. 44 (2013) 25. [13] M. Vallet-Regi, A. Ramila, R.P. Del Real, J. Perez-Pariente, Chem. Mater. 13 (2001) 308. [14] J. Chakraborty, S.D. Sarkar, S. Chatterjee, M.K. Sinha, D. Basu, Colloids Surf. B 66 (2008) 295. [15] S. Kapoor, R. Hegde, A.J. Bhattacharyya, J. Control. Release 140 (2009) 34. [16] S.P. Low, K.A. William, L.T. Canham, N.H. Voelcker, Biomaterials 27 (2006) 4538. [17] D.A. Javaid, D.M. Krapchetov, J. Ford, J. Membr. Sci. 246 (2005) 181. [18] J. Salonen, V.P. Lehto, Chem. Eng. J. 137 (2008) 162. [19] H.S. Mansur, R.L. Oréfice, W.L. Vasconcelos, Z.P. Lobato, L.J.C. Machado, J. Mater. Sci.: Mater. Med. 16 (2005) 333. [20] X.L. Xie, C.Y. Tang, X.P. Zhou, R.K.Y. Li, Z.Z. Yu, X.Q. Zhang, Y.W. Mai, Chem. Mater. 16 (2004) 31.

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Protein attachment to nanoporous anodic alumina for biotechnological applications: influence of pore size, protein size and functionalization path.

Nanoporous anodic alumina (NAA) is a material with great interest in nanotechnology and with promising applications to biotechnology. Obtaining specif...
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