Immobilization of Bovine Serum Albumin onto Porous Poly

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Immobilization of Bovine Serum Albumin onto Porous Poly(vinylpyrrolidone)-Modified Silicas Alexander S. Timin,*,† Alexey V. Solomonov,† Irek I. Musabirov,‡ Semen N. Sergeev,‡ Sergey P. Ivanov,§ Evgeniy V. Rumyantsev,† and Alexander Goncharenko† †

Inorganic Chemistry Department, Ivanovo State University of Chemistry and Technology (ISUCT), 7 Sheremetevsky Prospect, 153000 Ivanovo, Russian Federation ‡ Institute for Metals Superplasticity Problems of the Russian Academy of Sciences, St. Khalturina Street 39, 450001 Ufa, Russian Federation § Institute of Organic Chemistry, Ufa Science Centre of the Russian Academy of Sciences, 71 Prospect Oktyabrya, 450054 Ufa, Russian Federation ABSTRACT: Grafting with poly(N-vinyl-2-pyrrolidone) (PVP) was used to functionalize silica. The functionalized silicas were characterized by scanning electron microscopy, nitrogen adsorption, energy-dispersive X-ray analysis, Fourier transform spectroscopy, and thermogravimetric analysis. The Barrett−Joyner−Halenda analysis confirmed that PVP-modified silica samples have a mesoporous structure. Bovine serum albumin (BSA) was immobilized onto the surface of PVP-modified silica systems through physical adsorption. Also it was shown that the adsorption capacity of the tested samples depends on the quantity of PVP inside the silica matrix, which confirmed the formation of a specific complex between BSA and PVP. In addition, the effects of pH, time, and concentration on BSA adsorption were investigated. The maximum BSA adsorption capacity was 71.54 ± 2.0 mg/g. The desorption and reusability of all prepared samples were studied as well. PVP-modified silicas are capable of holding more BSA compared to nonmodified silica. These characteristics indicate that PVP-modified silicas have great potential for efficient protein encapsulation.

1. INTRODUCTION Biomolecule (proteins, drugs, etc.) immobilization onto the surface of organic and inorganic materials (polymers, silica matrixes, etc.) is one of the main and efficient methods which are used in biotechnology. Immobilized molecules have several advantages compared to free molecules. Specifically, they have a wide practical application not only in the nanocomposite field, as drug carriers, in drug screening, in membrane bioreactors, and in biosensor synthesis,1−3 but also in wide branches of modern medical diagnostics, genomics, proteomics,4,5 and affinity chromatography. Due to the significant properties of immobilized molecules, such as a high kinetic and thermodynamic stability and resistance to various environmental factors, they are suitable and valuable for new applications in industry and medicine. A combination of molecules with different properties in a single carrier allows expansion of the action of functional ligands. In the past few years, protein biochips synthesized using immobilization technology have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made in this area.6 Immobilized ligands for effective signaling to maleic anhydride copolymer thin-film coatings enable investigation of cell responses from interface-immobilized ligands.7 Biomarker detection using immunoassays has been widely used as a disease diagnostic tool.8 Immobilization of proteins for single-molecule fluorescence resonance energy transfer is used in measurements of conformation and dynamics methods.9 © XXXX American Chemical Society

A wide variety of immobilization methods are employed to attach proteins for effective surface immobilization, but only three general methods of protein immobilization are the most effective and appropriate (Figure 1). The first method (Figure 1a) is protein immobilization via physical adsorption10 onto glass, alginate beads, or a matrix when the protein is attached to the outside of an inert material. Protein can be immobilized onto the surface via multiple binding sites, which may result in a conformational change and reduction of protein activity.11,12 Using such a technique, the HIV Env antigen (gp41) was adsorbed onto the polymermodified surface to assay antibodies in HIV-infected sera with catalytic silver deposition using gold nanoparticle conjugated secondary antibodies. Xiang et al. designed an “H”-channel glass-covered poly(dimethylsiloxane) (PDMS) chip.13 Immobilization of bovine serum albumin-protected gold nanoclusters was carried out according to this method.14 When physical adsorption is used, serious changes in the structure of the proteins are prevented, which is very important because any serious changes in the structure of the protein may lead to the loss of biofunctional properties and protein denaturation even though these types of interaction might not be strong enough to prevent protein leaching. Another method of protein immobilization (Figure 1b,c) is enzyme entrapment, where the protein is trapped in insoluble Received: May 12, 2014 Revised: July 27, 2014 Accepted: August 9, 2014

A

dx.doi.org/10.1021/ie501915f | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Industrial & Engineering Chemistry Research

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hard synthetic conditions, which lead to protein denaturation. Therefore, we can use the first method with a minor modification: in our case the main strategy of protein immobilization is focused on application of nanoporous or mesoporous structures. It has been reported that mesoporous or nanoporous structures with well-ordered pores are more promising in the adsorption process compared to polymer monoliths or microporous membranes.21−23The interactions with a large binding partner (e.g., antibody or enzyme) could be prevented by a small pore size or slow diffusion capacity through the porous channel. There are some examples of synthesis of mesoporous materials using different techniques. One of them is a template method for synthesis of porous materials with a suitable pore size.24,25 Such materials have found a wide application in bioimmobilization of proteins. For example, in recent work, the immobilization, stability, and enzymatic activity of albumin and trypsin adsorbed onto nanostructured mesoporous SBA-15 with compatible pore sizes were studied as well.26 It was shown that both proteins can be effectively immobilized in the interior of SBA-15 mesochannels having suitable pore sizes through adsorptive binding. The sol− gel technique is also a very popular and effective method for generating porous materials with the required pore sizes. Some of the benefits of using sol−gels are the excellent enzymatic activity owing to the high encapsulation concentration, mild immobilization conditions,6,4 and optical transparency for imaging. Silica gels obtained by polycondensation of alkoxysilane monomers are basically used in the sol−gel method. Sakai-Kato et al.27 reported a poly(methyl methacrylate) (PMMA) microfluidic enzyme reactor based on silica sol−gel encapsulation of trypsin. The sol−gel material was prepared using tetramethoxysilane (TMOS). TMOS was hydrolyzed to form Si(OH)4−n(OMe)n. After addition of trypsin, a trypsinentrapped sol−gel was formed inside the PMMA chip. The porous structure of silica gels is, however, fragile and may lead to pore collapse, and sometimes offers poor adhesion to the substrate. Thus, the silica surface obtained via the sol−gel method can be treated by different biopolymers that increase the surface properties and porous structure of the silica gel for effective protein encapsulation.28 In our study we used poly(N-vinyl-2-pyrrolidone) (PVP) as a biocompatible polymer which may serve as an effective agent for increasing the surface properties of the final products and adsorption capacity to BSA. A lot of works concerning the application of PVP have been published. Graf et al. reported that PVP is considered as a dispersant to improve the dispersion stability of colloid particles to produce a silica coating in ethanol media.29 PVP can be immobilized onto a broad range of materials, such as metal oxide (TiO2, iron oxide, aluminia),30 polystyrene,31 silica,32 graphite,33 etc. A comparative study of the complexing capacity of BSA and PVP has been performed, but the possible application of PVP as a possible agent in BSA binding within the mesoporous structure of the silica gel has still not been reported. Therefore, PVP can be considered as an agent in sol−gel synthesis for improvement of the textual and surface properties of the final silicas. Furthermore, in a recently published paper,34 the preparation of 5-FU-loaded BSA/PVP nanofibers was described, and it was shown that BSA can be used in targeted delivery of anticancer drugs to tumor tissue as BSA accumulates in solid tumors and hence is an attractive agent for tumor-targeting drug delivery systems. PVP is used for BSA stabilization.

Figure 1. Common surface immobilization methods for heterogeneous assays. Schematic of the immobilization mechanisms: (a) physical adsorption, (b, c) bioaffinity interaction, and (d, e) covalent bonding via a linker.

beads or microspheres, such as calcium alginate beads. An antigen−antibody specific interaction is an example of this immobilization method. Bioaffinity interactions are relatively stronger in comparison with the above-mentioned method, more specific and directed toward protein immobilization.14,15 Yang et al. used aptamer-functionalized magnetic beads in a multilayer PDMS microfluidic device to complete a C-reactive protein (CRP) immunoassay.16 As described in another recently published paper, silica-coated magnetite nanoparticles with immobilized serum albumin were synthesized.17 The last method for efficient protein encapsulation is a crosslinkage (Figure 1d,e) of the protein to the surface via a linker by means of covalent bonding during a chemical reaction. Covalent bonds are frequently used in the immobilization mechanism for microfluidic assays. The immobilization surface is activated via reactive reagents. The activated surface reacts with amino acid residues on the protein exterior and forms an irreversible linkage. One tends to rely on covalent immobilization when high, stable protein coverage is required. Bifunctional spacer molecules are a common approach to form an irreversible bond between proteins and the immobilization surface. In such an approach, one end of a spacer molecule is covalently linked to an activated surface and then a protein is covalently linked to the other end of the spacer. Alternatively, another spacer or protein capture agent (e.g., streptavidin) is cross-linked on the other end. Unreacted active functional groups are blocked or deactivated. A covalent bond can be formed on the active sites of the proteins, resulting in reduced activity. This method allows creation of immunoassay protein chips.18 Covalent immobilization was used to attach albumin onto porous polyethylene membranes using strongly attached polydopamine as a spacer.19 A protein (bovine serum albumin, BSA)-coated polymer was also used as a matrix for enzyme immobilization. This protein-coated polymer provides a novel matrix for covalent immobilization of enzymes.20 Also crosslinkage allows the synthesis of various materials for immunosorbent assays.21 The last two methods of protein immobilization are not always suitable for immobilization of all proteins because of the B



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Figure 2. EDX spectra of PVP-modified silicas.

contained different quantities of PVP in the range from 0.08 to 0.24 g to prepare four solutions with different quantities of PVP (Table 1). Then the first solution was added to the second solution under stirring. This obtained final mixture was stirred for 2 h. A 0.05 mL volume of ammonia solution (pH 8) was added every 20 min as a base catalyst until the formation of ultradisperse silica particles. After that, the final product was transferred into a Petri dish for solvent evaporation at room temperature. The obtained samples were washed with ethanol and then with deionized water. Finally, they were separated by centrifugation and dried at 95 °C under vacuum for 3 days. 2.3. Immobilization of BSA onto Mesoporous PVPModified Silica. BSA was immobilized into PVP-modified silica by physical adsorption. A series of experiments to evaluate the amount of adsorbed BSA at different pH values was performed. The isoelectric point of BSA is equal to 4.7. At a pH below its isoelectric point, the protein surface has a positive charge;35 above its isoelectric point, the protein surface has a negative charge. Therefore, the values of pH must be optimized for better explanation of interactions between BSA and PVP. Experimentally, 65 mg of PVP-modified silica was suspended in 5 mL of BSA solution (4 mg/mL) by varying the pH (2.3, 3.2, 4.9, 7.4, and 8.1) of potassium acetate and phosphate buffers. To optimize the time it takes to reach equilibrium adsorption,

In this paper, we have described the preparation and characterization of PVP-functionalized silicas and the application of these materials in BSA immobilization. The Brunauer− Emmett−Teller (BET)/Barrett−Joyner−Halenda (BJH) analysis, the morphology of the final products, the thermal stability of the incorporated PVP, and the adsorption properties have been examined.

2. EXPERIMENTAL SECTION 2.1. Reagents and Materials. TEOS (tetraethoxysilane, Si(OC2H5)4; 98%) was obtained from Ecos-1 (Russian Federation). PVP (Mw = 10 000 g/mol) and BSA (Mw = 66 430 g/mol) were supplied by Sigma-Aldrich (United States). Absolute ethanol and ammonia solution (25 wt % ammonia) were obtained from Ecos-1. The chemicals were of analytical grade and were used without further purification. Deionized water was used for all preparations. 2.2. Synthesis of PVP-Modified Silicas. A typical sol−gel synthesis of nonfunctionalized silica particles is described in ref 35 and is based on a mixture of two solutions at room temperature. The first solution was a mixture of 8 g of TEOS and 20 g of ethanol, and the second solution was a mixture of 0.1 mL of 25 wt % ammonia and 2.8 g of deionized water. In the case of PVP-functionalized silicas, the second solution also C



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Table 1. Amount of Reagents and Precursors Used in the Sol−Gel Synthesis and EDX Analysis Results of PVP-Modified Silicas elemental analysis (concn, wt %) sample SiO2 Si-PVP-1 Si-PVP-2 Si-PVP-3 Si-PVP-4

sol−gel formulation (mass ratio, g) TEOS:PVP:C2H5OH:H2O TEOS:PVP:C2H5OH:H2O TEOS:PVP:C2H5OH:H2O TEOS:PVP:C2H5OH:H2O TEOS:PVP:C2H5OH:H2O

= = = = =

C

8:0:21:2.8 8:0.08:21:2.8 8:0.1:21:2.8 8:0.16:21:2.8 8:0.24:21:2.8

3.28 4.32 6.64 9.48

V (Co − C) m

O

Si

0.64 0.84 1.29 1.84

53.25 51.78 51.29 50.19 48.84

46.75 44.30 43.56 41.88 39.84

gelation time 1−2 1−2 1−2 1−2 1−2

days days days days days

Fourier transform infrared (FTIR) spectra of the samples were obtained with a Nicolet 4700 FTIR spectrometer (Nicolet, United States) using the KBr technique. Thermoanalyses of the obtained samples were performed on a TG 209 F1 (Netzsch, Germany) using platinum crucibles in an argon atmosphere with a heating rate of 10 °C/min. The accuracy of the sample mass measurement was 1 × 10−6 g, and the accuracy of the temperature measurement was 0.1 °C. The particle size analysis was performed on a laser diffraction analyzer (Analysette 22 Micro TecPlus, Fritsch, Germany).

65 mg of the tested sample was mixed with 5 mL of BSA solution (4 mg/mL) and the resulting mixture was stirred at room temperature followed by centrifugation at 6000 rpm. To evaluate the optimal amount of BSA that can be immobilized inside the PVP-modified silica matrix, 65 mg of PVP-modified silica was mixed with 5 mL of BSA solution at different concentrations: 0.5, 1, 2, 2.5, 3.5, and 4 mg/mL. The BSA concentrations were determined by a UV−vis spectrophotometer (SF-104, Aquilon, Russian Federation) at 280 nm (BSA ε280 = 41 000). The pH values were measured by a pH meter (U-500, Aquilon, Russian Federation). The experiments were performed in replicates of three. For each set of data presented, standard statistical analysis was used to determine the mean values and standard deviations (SDs). Error bars were used for every adsorption experiment to represent the SDs. The amount of adsorbed BSA (mg/g) was calculated by the following equation: q=

N

3. RESULTS AND DISCUSSION 3.1. Characterization of the Obtained Materials. 3.1.1. SEM−EDX Analysis. EDX spectra of the PVP-modified silicas were collected and compared to control the content of PVP in the silica matrix (Figure 2). The presence of N and C can be clearly seen from the EDX spectra of PVP-modified silicas. The EDX measurements show the elemental analysis (wt %) of our samples (Table 1). Elemental analysis of pure silica showed that the concentration of silicon was 53.2 wt % and that of oxygen was 46.8 wt % corresponding to completely condensed silica.36 The percentage of nitrogen increased as the amount of PVP used in sol−gel synthesis also increased. The EDX analysis confirmed that PVP was successfully incorporated into silica via sol−gel synthesis. To investigate the influence of PVP incorporated into the silica matrix on the morphology and particle size of modified silicas, all samples with different quantities of PVP were examined by scanning electron microscopy. It has been reported35 that the morphology of nonfunctionalized silica (the same synthesis) is found to be spherical in shape. Figure 3 represents the typical SEM images of PVP-modified silicas. From the obtained images, it can be seen that all samples exhibit a hierarchical microstructure and also contain big clusters of agglomerated particles. These big clusters of agglomerated particles consist of uniform spherical nanoparticles (Figure 3). Also the SEM images of Si-PVP-1 show the presence of spherical particles. However, other samples with larger quantities of PVP are characterized by nonuniform particles and particle agglomerates. We can conclude that incorporation of PVP into the silica matrix leads to the formation of different interconnected globules. This is mostly because of a high linking ability of PVP. During the formation of the silica core after hydrolysis and polycondensation of TEOS, PVP molecules interact with the silica particles via electrostatic and intermolecular hydrogen bonds between the OH groups of silica and CO groups of PVP.37 As a result, the increasing concentration of PVP used in the sol−gel process leads to the formation of big particle clusters consisting of uniform silica nanoparticles (Figure 3), and therefore, growth of the particles is observed as evidenced from particle size analysis (see section 3.1.2). Such an influence of the polymer on the morphology and particle size is in agreement with the

(1)

where Co and C are the initial and residual BSA concentrations in solution (mg/mL), V is the volume of the BSA solution (mL), and m is the mass of the adsorbent (g). 2.4. Desorption and Repeated Use. The desorption of BSA was studied in a buffer solution (1 mol/L NaCl). The tested sample (65 mg) was placed in this desorption medium (5 mL of buffer solution), and the resulting mixture was stirred at 6000 rpm at room temperature. The BSA concentration was analyzed by a UV spectrophotometer. The adsorption− desorption cycle was repeated several times using the same silica particles with a washing procedure to investigate the stability of BSA in the silica matrix. As for the adsorption process, all experiments were repeated three times for determination of average data and standard deviations. 2.5. Characterization Techniques. Scanning electron microscopy (SEM) was performed using a high-resolution scanning electron microscope (Tescan Mira-3LMH) operating at an accelerating voltage of 20 kV. All samples were dissolved in ethanol before examination. The obtained emulsion was coated onto the polished surface of an aluminum table. The obtained samples were kept for a day under vacuum (10−2 Pa). Elemental analysis was performed using an energy-dispersive Xray (EDX) probe attached to the same scanning electron microscope described above. The specific surface area, total pore volume, and average pore diameter were measured by nitrogen adsorption−desorption isotherms at 77 K on a Micromeritics ASAP 2020 instrument (Micromeritics Instrument Corp., Norcross, GA). The pore size was calculated on the adsorption branch of the isotherms using the BJH method, and the specific surface area was calculated using the BET method. The errors in determining the BET surface area and pore volume are estimated to be within 5%. D



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Figure 3. SEM micrographs of PVP-modified silicas. E



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Figure 4. Particle size distributions of PVP-modified silicas and pure silica (insets).

results of our previous studies,35,38 where we have shown that incorporation of a polymer which is capable of interacting with the Si−OH and/or Si−O− groups causes the formation of big clusters of interconnected particles during the sol−gel process. 3.1.2. Particle Size Analysis and Surface Properties. Laser diffraction analysis was performed to analyze the influence of PVP on the particle size distribution. As shown in Figure 4, pure silica has a relatively narrow size distribution with an average size of 6.2 ± 0.1 μm, whereas the PVP-modified silicas are characterized by a bimodal particle size distribution. The average size with SD was determined and is presented in Table 2. As is evidenced, with an increase of the PVP content in silica, the average particle size shifted toward a higher value, which

can be associated with formation of big interconnected globules between silica particles and PVP during the sol−gel process as discussed above. Nitrogen adsorption−desorption isotherms at 77 K of all prepared samples are reported in Figure 5. As can be seen from Figure 5, all samples exhibit type IV adsorption isotherms (Brunauer definition). The typical hysteresis loops are observed for all PVP-modified silicas and are attributed to the presence of mesopores. The shapes of the hysteresis loops for Si-PVP-1 and Si-PVP-2 are very similar and correspond to the H1 type, indicating a cylindrical pore shape. The hysteresis loops of SiPVP-3 and Si-PVP-4 are assigned to the H3 type. Type H3 hysteresis is usually found on solids with a very wide distribution of pore size as evidenced from pore size distribution curves for Si-PVP-3 and Si-PVP-4. The isotherm of pure silica exhibits a very gradual increase of adsorbed nitrogen and is characterized by a small volume (Table 2). In this case no hysteresis loop is observed, showing an important effect of PVP on the silica porosity. The specific surface area of all samples along with their total pore volumes and average pore sizes calculated according to the BJH methods are listed in Table 2. The obtained results show that the surface properties of modified silicas are clearly different from those of pure silica. Pure silica is characterized by a low specific surface area (∼40 m2/g). An increase in the surface area, pore size, and pore volume is observed after incorporation of PVP into the silica matrix. The specific surface area and average pore size of Si-PVP-1 are 264 m2/g and 9.4 nm, while the specific surface area and average pore size of Si-

Table 2. Structure−Adsorption Characteristics and Average Particle Sizes of the Obtained Samples sample

BET surface areaa (m2/g)

total pore volb (cm3/g)

av pore sizec (nm)

SiO2 Si-PVP-1 Si-PVP-2 Si-PVP-3 Si-PVP-4

40 264 240 170 140

0.015 0.56 0.55 0.58 0.68

2.5 9.4 9.6 15.6 21.1

av particle sized (μm) 6.2 6.3 6.9 8.7 11.5

± ± ± ± ±

0.1 0.1 0.1 0.1 0.1

a

BET specific surface area measured in the 0.05−0.25 range of relative pressures. bThe total pore volume was calculated at a relative pressure of about 0.99. cThe average pore size was calculated from the BJH method. dThe average particle size was determined by laser diffraction analysis. F



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Figure 5. Nitrogen adsorption−desorption isotherms at 77 K of the obtained samples with the corresponding pore size distributions (insets).

PVP-2 are 240 m2/g and 9.6 nm, respectively. Also these samples are characterized by a narrow pore size distribution. However, the large content of PVP in the silica matrix leads to a decrease in the specific surface area (the specific surface area of Si-PVP-3 is 170 m2/g, and that of Si-PVP-4 is 140 m2/g) and simultaneously an increase in the pore size. PVP is a long-chain polymer which may combine and form large micelles at high concentration.39 As a result, the prepared samples with a high concentration of PVP have a larger pore size because since the PVP is added during sol−gel synthesis, it is possible that a sol− gel structure is formed around the PVP micelles, causing the formation of different holes (or larger spaces) in the silica and composite as well. The mean pore volume increases from 0.558 to 0.676 cm3/g as the quantity of PVP increases. The mean pore diameter of Si-PVP-4 is 21.14 nm according to BJH analysis.

The analysis of structure−adsorption parameters (surface area, pore volume, and pore size) confirmed deep surface and porous changes of silica after modification. Thus, we can conclude that the PVP molecules have an influence on the surface area as was reported previously36 and also that PVP can serve as a template to form a large pore size in the silica matrix during the sol−gel synthesis. 3.1.3. Thermoanalysis of the Prepared Samples. All prepared PVP-modified silicas were characterized by thermogravimetric analysis. Figure 6 shows the thermograms of the weight loss as a function of temperature for PVP-modified silicas with a heating rate of 10 °C/min from room temperature (20 °C) to 600 °C in an argon atmosphere. As can be seen in Figure 6, the initial weight loss for all samples starts at round 64 °C associated with the loss of physically adsorbed water and ethanol molecules from the silica surface40 and ranges from G



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Figure 6. TGA/DTG thermograms of PVP-modified silicas and pure nonmodified silica (insets) obtained in an argon atmosphere up to 600 °C. The heating rate was 10 °C/min. The uncertainties in TGA measurements were 0.3% for temperature and 0.5% for weight.

amount of grafted PVP ranges from 0.26 to 1.55 mg/m2, which is typical for such a technique.41 3.2. Effect of pH on the Adsorption of BSA. The pH is one of the major factors that has an impact on the adsorption of proteins. The isotherms of BSA adsorbed on nonmodified and PVP-modified silicas at different pH values ranging from 2.3 to 8.1 are shown in Figure 7. The results show that the adsorption

3.95% to 8.96%. The second weight loss between 150 and 300 °C is observed only for pure nonmodified silica, a considerable weight loss of about 16.28%, and is associated with removal of organic residues and further polymerization of the silica networks.34 The main weight loss for PVP-modified silicas starts around 300 °C, continues up to 489 °C, and corresponds to the thermal degradation of PVP. From the analysis of differential thermogravimetry (DTG) curves, it is evident that the decomposition peaks for PVPmodified silicas corresponding to the thermal destruction of PVP in the silica matrix shifted to higher temperature with increasing amount of incorporated PVP, which indicates enhanced thermal stability. Therefore, we can conclude that the thermal stability of PVP-modified silicas increased as the quantity of incorporated PVP increased due to formation of different noncovalent and hydrogen interactions between the silica matrix and polymer providing a more stable structure. The contents of silica (by weight) and PVP in the composite were calculated using thermogravimetric analysis (TGA) curves, and the grafting amount of PVP on the silica surfaces was also determined. All results are shown in Table 3. The estimated content of PVP in the composite is higher than expected. This is particularly true for polymer-grafted silica obtained via the sol−gel method because of incomplete hydrolysis and polycondensation of TEOS, and therefore, the mass of the final products is lower than could be obtained. The

Figure 7. Effect of pH on the adsorption of BSA onto pure and PVPmodified silicas. The data presented are the average value ± SD of n = 3 experiments.

Table 3. TGA Parameters of Pure Silica and PVP-Modified Silicas sample SiO2 Si-PVP-1 Si-PVP-2 Si-PVP-3 Si-PVP-4

agent mass ratio (g) TEOS:PVP TEOS:PVP TEOS:PVP TEOS:PVP TEOS:PVP

= = = = =

8:0.00 8:0.08 8:0.10 8:0.16 8:0.24

PVP concna (wt %)

silica concn (wt %)

amount of grafted PVPb (mg/m2)

5.33 7.14 11.42 17.14

83.72 85.71 84.32 82.03 78.91

0.26 0.35 0.82 1.55

behavior of BSA was strongly affected by the value of the pH. Each isotherm shows a sharp middle rise. The amount of adsorbed BSA increases from 3.3 to 5 and then decreases in the pH range from 7.4 to 8.1. As can be seen from Figure 7, the maximum adsorption capacity is reached at pH 4.9, which corresponds to the isoelectric point of BSA.40 Moreover, the amount of BSA is increased with a high content of PVP incorporated into the silica matrix. The unbound electron pairs on either the amine or carbonyl groups in the PVP fragment located on the five-membered ring can act as weak Lewis bases.42 As a result, PVP can form a water-soluble complex with BSA via electrostatic forces and high complexity of BSA. Moreover, the adsorption of proteins on the silica surface is a complicated process not only driving electrostatic forces and the complexity of the used protein but also involving van der Waals, hydrophobic, and hydrophilic forces and the porosity of the used materials.

a

Determined as the difference in weight loss between the initial and final stages of thermal decomposition of PVP in silica. bmPVP (mg/m2) = ((ΔWinitial − ΔWfinal)/(ΔW600S)) × 1000, where ΔWinitial and ΔWfinal are the weight loss ratios of the initial and final stages of thermal degradation of PVP, respectively, ΔW600 is the residual weight ratio of PVP-grafted silica at 600 °C, and S is the specific surface area of PVP-modified silica. H



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3.3. Effect of Time on the Adsorption of BSA. The effect of time on BSA adsorption at pH 5 by pure and PVPmodified silicas was studied and is illustrated in Figure 8. As can

Figure 9. Effect of the equilibrium BSA concentration on the adsorption of BSA on nonmodified and PVP-modified silicas. Vtotal = 5 mL, mads = 68 mg, time = 120 min, and T = 26 °C. The data presented are the average value ± SD of n = 3 experiments.

Figure 8. Effect of time on the adsorption of BSA at pH 5 by nonmodified and PVP-modified adsorbents. The data presented are the average value ± SD of n = 3 experiments.

results of PVP-modified silica, sample Si-PVP-4 is characterized by the highest percentage of nitrogen (Table 1), and therefore, the largest amount of PVP was grafted onto the silica surface. Thus, Si-PVP-4 and Si-PVP-3 have better adsorption capacities even though their surface areas are lower than for Si-PVP-1 and Si-PVP-2. In the silica matrix, the mesopores and channels are full of PVP with the potential to interact with BSA, so we can observe that the adsorption capacity depends on the amount of PVP incorporated into the silica matrix. The large mesopores in PVP-modified silicas allow the BSA molecules to diffuse into the material and interact with incorporated PVP via electrostatic and H-bond interactions that provide additional binding with BSA. Also hydrophobic/hphydrophilic forces positively influence BSA immobilization.40,43 The nonmodified silica is characterized by a low adsorption capacity compared to PVPmodified silicas due to the low surface area and pore size. Only Si−OH/Si−O− groups on the inner walls of the silica channels facilitate protein immobilization. To show the stability and determine the maximum content of grafted PVP, silica samples with different contents of PVP (5−26 wt %) were extensively washed in buffer solution (pH 7.4) for 1 h. Then they were centrifuged and removed from the solution. After that, the solution was analyzed using UV spectroscopy because PVP can be detected in water solution in the ultraviolet range, with a maximum at 213.5 nm.44 It turns out that such a sol−gel method of synthesis allows silicas with a content of PVP not exceeding 17 wt % to be obtained. In other cases when the content of PVP is more than 17%, the release of polymer is observed. From the literature focused on protein (BSA) immobilization, we can compare the adsorption capacity of PVP-modified silicas with those of some other affinity adsorbents. For example, Chase45 reported that CB-attached Sepharose CL-6B has an adsorption capacity of 14 mg/g for BSA. It has been46 reported that Cibacron Blue F3GA nanospheres can be successfully used in BSA separation and has an adsorption capacity of 109 mg/g. In a recent study,47 an agarose−nickel (Ag−Ni) composite matrix was prepared for efficient BSA immobilization. The authors announced that Ag−Ni exhibited a high adsorption rate and also a higher binding capacity of BSA (31.4 mg/g). We summarize that the adsorption capacity of PVP-modified silicas is also good in comparison with those of

be seen from the figure, the adsorption curves of BSA adsorbed within nonmodified and modified silicas as a function of the contact time show different times to reach equilibrium conditions. The adsorption capacity of the modified adsorbents is higher than for pure silica. It is obvious that the adsorption kinetics of pure amorphous silica was faster than for the modified silicas. The time required to reach equilibrium conditions was approximately 60 min in the case of pure amorphous silica, whereas the adsorption time of Si-PVP-1 and Si-PVP-2 was ∼90 min and that of Si-PVP-3 and Si-PVP-4 was ∼120 min. These results indicate that the kinetic adsorption rate of BSA depends on the amount of PVP incorporated into the silica matrix and the diffusion process inside the mesopores. We can conclude that more time is required to reach equilibrium conditions in the case of PVP-modified silicas because of the high complexation and geometric affinity between PVP and BSA, which is determined by the influence of specific van der Waals interactions, hydrogen bonding, and electrostatic forces. A wide range of adsorption rates have been reported in the literature. From an analysis of the literature,1−20 it can be concluded that the time required for BSA adsorption ranges from 1 to 2 h. In this study, it can be concluded that the time to reach equilibrium conditions seems to be quite suitable. 3.4. Effect of the Equilibrium Concentration of BSA. Experiments involving the adsorption of BSA onto the silica surface at room temperature were conducted for a range of initial concentrations (Co) of BSA from 0.5 to 4 mg/mL. As can be observed from Figure 9, the adsorption values increased as the concentration of BSA increased. The maximum amount of BSA adsorbed on nonmodified silica was 26 ± 2.1 mg/g, whereas using PVP as an agent for surface modification, 71.54 ± 2.0 mg/g was achieved. It is also an interesting fact that silica systems with small specific surface areas (Si-PVP-3 and Si-PVP4) are characterized by better BSA adsorption ability than in the case of Si-PVP-1 and Si-PVP-2, which have higher surface areas. These results strongly support that incorporation of PVP into the silica matrix plays an important role in protein immobilization. However, the location of PVP within the silica matrix is an important issue. Only surface-located PVP has the ability to bind BSA. According to the EDX surface analysis I



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Figure 10. FTIR spectral patterns of the nonmodified and PVP-modified silicas (a) and BSA/PVP-modified silicas (b). The FTIR spectra were recorded using a Nicolet 4700 FTIR spectrometer (Nicolet, United States). The FTIR patterns have been shifted for clarity.

other prepared materials. The adsorption capacity depends on the structure, functional groups, ligand loading, and surface area as well. Therefore, using different ligands, we can reach the maximum adsorption ability for protein or other analogues with similar functional groups. Infrared spectroscopy was employed to gain information on the PVP interaction with the silica surface and the surface sites present on the materials before and after adsorption of BSA. The spectra of pure and PVP-modified silicas are characterized by a broad intensive band at around 3482−3420 cm−1 which is assigned to the Si−OH stretching mode (ν(Si−OH)) of surface silanols hydrogen-bonded to molecular water (SiO− H···H2O) and O−H stretching vibrations of hydrogen-bonded water molecules (H−O−H···H).48−50 In the case of PVPmodified silicas, the peak between 3482 and 3420 cm−1 is slightly shifted and more intense (Figure 10a). The signals at 2944 cm−1 are attributed to the asymmetric C−H stretching modes of −CH2 in PVP. Only Si-PVP-3 and Si-PVP-3 are characterized by a signal in the range of 1480−1350 cm−1 corresponding to the asymmetric and symmetric bending modes of −CH2. A very intense band at 1095−1085 cm−1 is assigned to the Si−O−Si asymmetric stretching vibrations.48 The signals at 1648 cm−1 are observed only for PVP-modified silicas and attributed to the CO stretching vibration band in the PVP fragment.37 The FTIR spectra of pure and PVP-modified silicas with physically adsorbed BSA are presented in Figure 10b and compared with the FTIR spectra of all samples before BSA adsorption. The FTIR spectra of BSA-containing samples show two very intense peaks at 1656 and 1533 cm−1 corresponding to the amide I and amide II bands, respectively.51 This fact confirmed the successful grafting of BSA onto the surface of PVP-modified silicas via physical adsorption. 3.5. Desorption Study. The desorption factor of the immobilized BSA was studied by evaluating the amount of BSA after several desorption cycles of strong mixing of all samples in buffer solution (1 mol/L NaCl). Figure 11 shows the BSA desorption capacity as a function of time for all prepared samples. As can be seen the amount of desorbed BSA is rapidly

Figure 11. BSA desorption capacity as a function of time for pure and PVP-modified silicas. Vtotal = 5 mL, mads = 68 mg, time = 70 min, and T = 26 °C.

increased in a period of time from 0 to 20 min. The equilibrium amount of desorbed BSA for nonmodified silica was about 54%, whereas it was about 25−19% for PVP-modified silicas. It appears that immobilization of BSA onto the silica surface is not strong enough to prevent the release of a large amount of BSA from nonmodified silica. The release of a large quantity of BSA from the silica matrix was reduced using PVP. However, since the immobilization of BSA was performed at pH 5 via physical adsorption, we suggest that desorption of BSA occurs due to an increase of the ionic strength, which leads to a decrease of electrostatic interaction between PVP and BSA. This behavior indicates the successful application of PVP in sol−gel synthesis for effective immobilization of BSA.

4. CONCLUSIONS The synthesis of PVP-modified silicas was carried out via the sol−gel process. EDX and FTIR analysis results showed that PVP was successfully encapsulated in the modified materials. J



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The morphological structure and surface properties of the final products were tunable by the amount of incorporated PVP. The incorporation of a large amount of PVP into silica leads to a decrease in the surface area and a heterogeneous pore size distribution. The effects of the pH, time, and quantity of incorporated PVP on the adsorption capacity for BSA were investigated. We demonstrated that incorporation of PVP greatly increases the amount of BSA that can be immobilized onto the silica surface. The optimum content of PVP for such a method of synthesis was found to be 17 wt %. The PVPmodified silicas showed a good adsorption capacity of BSA in comparison with other analogues reported in the literature. Taking into account the high ability of BSA to form macromolecular complexes, we suggest that such BSA immobilized composites have good potential as efficient sorbents for removing toxic agents, in microfluidic assays, and in immunoferment analysis. We also believe that the obtained results concerning BSA adsorption onto the surface of modified silica with a limited content of PVP can be effectively applied to immobilization of other biomolecules which have the same functional groups and chemical properties as BSA (antigen, antibodies, other proteins, DNA, RNA, etc.).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +7 (910) 669-36-43. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We are grateful for support via a stipend from the President of the Russian Federation for study abroad in 2014−2015. This work was supported by a grant from the President of the Russian Federation (Mκ-287.2014.3) (2014−2015) and a bursary from the President of the Russian Federation (SP6898.2013.4) for young scientists and graduate students engaged in advanced research and development in priority directions of modernization of Russian economics (2013− 2015).

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Immobilization of Bovine Serum Albumin onto Porous Poly

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