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Porous protein–silica composite formation: manipulation of silicate porosityand protein conformation

Agathe C. Fournier and Kathryn M. McGrath*

Received 20th February 2011, Accepted 15th March 2011

DOI: 10.1039/c1sm05299c

The formation of structured protein–silica composite assemblies is described. The assemblies were

prepared using a convenient one step sol–gel technique developed in our group, from which porous

silica particles, films or monoliths can be synthesized. Two biomolecules were used, bovine serum

albumin (BSA) and lysozyme, to modify the porous structure of the as-synthesized silica materials. The

composite materials were characterized using scanning electron microscopy, N2 sorption isotherms,

FT-IR spectroscopic data, differential scanning calorimetry and thermal gravimetric analysis. The

existence and nature of interactions between the proteins and the silica in the as-synthesized composite

materials were investigated using FT-IR, particularly with respect to the conformation of the proteins.

The conformation of both proteins is modified upon occlusion into the silicate network with

a reduction in the extent of a-helices resulting in a more disordered state. In the case of BSA the

presence of b-sheets is detected. Both BSA and lysozyme establish H-bonding and electrostatic

interactions with the silica. Protein concentrations in the range of 2 to 8 mg mL�1 of condensing

solution were investigated. Upon variation of the concentration of proteins, nitrogen adsorption data

and SEM investigation confirm that, the bimodal porous structure and the uniform pore size

distribution is maintained in the as-synthesized composite materials for all protein concentrations used.

Both the as-synthesized and fully calcined (water and organic components removed) materials show

bimodal porosity of �2 mm and �52 nm (BSA additive) and �5 mm and �57 nm (lysozyme additive).

The sizes of both the nanometre and micrometre sized pores are slightly reduced in the presence of

protein as compared to materials synthesized with no protein due to the interactions between the

biomolecules and the silica.

1. Introduction

Porous silica materials, due to their stability, biocompatibility

and ease of release from the human body are widely used

compounds in vivo electronics,1 imaging2 and in the medical field

for dental restoration,3 replacement bone and as inorganic

carriers for enzyme immobilisation or biologically active

molecular transport.4–6 Investigations focused on the interactions

of biologically relevant solution additives such as oligomers,

amino acids, polymers and macromolecules with silica are

therefore of particular interest. For the past 50 years the

behaviour of silica precursors in the presence of proteins has been

widely surveyed.7 Studies have focused on determining the

catalytic role or the ability to act as templating agents, of bio-

extracts such as silicatein8 or silaffins.9 Numerous experimental

MacDiarmid Institute for Advanced Materials and Nanotechnology,School of Chemical and Physical Sciences, Victoria University ofWellington, Wellington, New Zealand. E-mail: kathryn.mcgrath@vuw.ac.nz

4918 | Soft Matter, 2011, 7, 4918–4927

data point to silica formation as having a dramatic affect on the

behaviour of biomolecules (see, for example, ref. 10–15).

Different sources of silica have been employed in this research.

For instance, Holt et al.16 studied the interaction between

methaemoglobin, insulin, albumin, gelatin or collagen and silicic

acid Si(OH)4 in aqueous solution.16–18 Silicate salts were utilized

as the starting materials for an investigation of the interaction

between silica particles with bovine serum albumin (BSA)19 or

polylysine.13 Commercial colloidal silica dispersions were

used20,21 in silica bio-interaction studies. Besides the variety of

silica precursors employed for preparing silica in the literature,

the process conditions and solvents employed offer an even wider

range of experimental conditions. To consider just one of these,

silica precursor solutions have been buffered to, for example,

pH 713 when Tris-HCl22 or acetate23,24 were used as the reaction

solvents.

Reports describing, upon addition of silica, structural rear-

rangement of biomolecules in the final organic-silica materials

have demonstrated the importance of the global charge of the

bio-additive.21,25 Coradin observed the interaction of lysozyme

and BSA with solutions of sodium silicate at different pH.23 He

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noticed that upon increasing pH, the total charge borne by the

proteins was altered which modified the intensity and/or the

nature of the interactions occurring between the silica and the

biomolecules. In addition, he showed that BSA formed entangled

structures with silica-based materials in vitro.

In a previous publication, we described a method of synthesis

for the formation of porous patterned silicate materials using

responsive soft templates where the porosity was controlled

over two length scales.26 Porosity on the nanometre length scale

is controlled by oil swollen micelles formed via phase separa-

tion while pores micrometres in size are defined by the size of

the emulsion oil droplets. With this method we are able to

control the porosity of the final materials by varying the

concentration of the dispersed phase. Monodisperse emulsions

have been used previously to synthesize porous titania and

zirconia creating pores of a single size,27 or to make individual

silica spheres.28,29

Here we extend our previous study by investigating the

influence of proteins added during the condensation of the

silica/emulsion mixture in defining the porosity of the siliceous

domain during formation of the protein/silica/emulsion

composites. Furthermore we determine the conformation of the

protein molecules occluded into the siliceous domain. Changes

in the conformational state of the proteins arise due to the

introduction of specific interactions between the proteins and

condensing silica. Two widely studied globular proteins; bovine

serum albumin (BSA) and hen egg white lysozyme are used as

the protein additives. Serum albumin is one of the most studied

proteins, particularly the bovine variant, since it is readily

obtained and it displays high structural homology with its

human counterpart (HSA).30 In the work presented here, BSA,

with an isoelectric point of 4.7–4.9,31 bears both positive and

negative charges. In opposition to BSA, we chose lysozyme

since it is widely presents in the human body and plays a basic

counterpart (isoelectric point 9.5–1132) to acidic BSA.

Furthermore both have been investigated with respect to the

nature of their interaction with condensing silicate mate-

rials.23,33 The condensing solutions, as-synthesized silica mate-

rials and fully calcined silica materials were investigated in

order to gain information on the modification of both the

inorganic and biomolecular phases within this mixed system.

Through this work we confirm that electrostatic interactions

and hydrogen-bonds are formed between both biomolecules

and silica.

The contribution of this work is that it provides information

about the molecular and physicochemical mechanisms at the

origin of the formation of protein–silica porous composite

structures as found in biominerals. Furthermore, we have

established that the proteins become more disordered upon

occlusion into the condensing amorphous silica framework. To

our knowledge, this is the first investigation which utilises our

method of synthesis of porous silica materials in the presence of

proteins.

Fig. 1 Final mixture pH of BSA–silica (A) and lysozyme–silica (B) at

various protein concentrations measured 24 h post mixing.

2. Results and discussion

In order to investigate the silica–biomolecule precursor interac-

tions, physico-chemical analyses were performed on the samples.

This journal is ª The Royal Society of Chemistry 2011

2.1. Formation of siliceous porous-protein composites

2.1.1. pH measurements. Based on the assumption that sili-

cates and proteins mainly interact through electrostatic interac-

tions and hydrogen-bonds, the behaviour of the different

mixtures can be anticipated. Previous studies of protein

adsorption on different polymer surfaces have shown that

protein adsorption is strongly affected by surface charge and

hydrophobicity.34 Equally electrostatics have been found to

dominate in silica/protein interactions.23,33

In Fig. 1 is shown the variation of the final pH of the silica/

emulsion/protein mixtures 24 h after addition of the protein

solution to the silica/emulsion mixtures, once equilibrium had

been reached, as a function of protein concentration. During

silica condensation, the oxide bears negative charges and also

possesses free hydroxyl groups.35 In the presence of BSA and

lysozyme, silica establishes electrostatic and chemical interac-

tions. The positively charged biomolecules and the negatively

charged silica species attract each other and hydrogen bond

formation occurs between silanol groups and proton donors

(such as amines present in the proteins). In the presence of BSA

the final mixture pH steadily increases upon increasing BSA

concentration. At a concentration of 6 and 8 mg mL�1 BSA, the

samples have a pH above the isoelectric point of BSA (pI 4.70).

Although the protein bears a majority of negative charges in

these samples, it will also bear positive charges capable of elec-

trostatically interacting with silica. In contrast when lysozyme is

present the pH reaches a plateau well below the protein isoelec-

tric point (pI 9.5–11) indicating reduced interaction between the

protein and the condensing silica.

2.1.2. Residual concentrations of free proteins. To quantify

the extent of protein occlusion, free BSA and lysozyme concen-

trations in the supernatant were measured as a function of

protein concentration after removal of the insoluble matter by

centrifugation (Fig. 2).

As follows from Fig. 2A and 2B, the free protein concentration

in the supernatant of the BSA–silica and lysozyme–silica samples

increases upon increasing the total protein concentration for all

three compositions of the nonionic emulsions. For BSA-con-

taining samples, the amount of BSA in the supernatant stabilises

to approximately 1 mg mL�1, only a slight increase is noted upon

increasing BSA concentration from 2 mg mL�1 through to 8 mg

mL�1 of protein added initially in the samples and on increasing

dispersed phase, the uptake is decreased. Hence the uptake of

BSA rises upon increasing the initial concentration of the

protein.

Soft Matter, 2011, 7, 4918–4927 | 4919

Fig. 2 Free protein concentration in the supernatant solutions of the

BSA– (A) and lysozyme–silica (B) samples after centrifugation.

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For the systems containing lysozyme, the extent of occlusion

of the lysozyme-silica systems is significantly lower than that for

the BSA–silica composites. While the extent of lysozyme uptake

is an increasing function of initial lysozyme concentration as for

the BSA system, at an initial lysozyme concentration of 8 mg

mL�1, around 5 mg mL�1 remains in the supernatant whereas

only 1 mg mL�1 of BSA was found in the supernatant. Again as

for the pH data this indicates a reduced interaction between the

condensing silica and the added lysozyme.

2.1.3. Morphology of protein–silica composites. The solid

products obtained by centrifugation and freeze-drying the sili-

ceous systems synthesized from emulsion samples 1, 2 and 3 and

of various biomolecule–silica ratio samples were subjected to

SEM analysis to observe the morphology of the as-synthesized

materials (Fig. 3). The composite materials are characterized by

a bimodal porosity. The size of these pores is predominantly

controlled and defined by the characteristics of the emulsion oil

droplets (micrometre) and oil swollen micelles (nanometre). In

the presence of protein the characteristic dimensions corre-

sponding to both the macro and nano pores are slightly

decreased, indicating that the proteins predominantly reside at

the oil–silica interface and control silica wall thickness.

Fig. 3 SEM images of BSA–silica (A–B) and lysozyme–silica (C–D)

composite materials obtained from sample 1 with a protein concentration

of 8 mg mL�1.

4920 | Soft Matter, 2011, 7, 4918–4927

The BSA- and the lysozyme-containing as-synthesized

composite materials revealed respectively an average micropo-

rosity of 2.0 � 0.5 mm (Fig. 3A—the range of the pore diameter

distribution is from 0.4 to 3 mm) and of 4.8 � 0.7 mm (Fig. 3C—

the range of the pore diameter distribution is from 1 to 9 mm). On

the nanoscale, the average porosities are respectively 42.0 � 15.0

nm (Fig. 3B—with pore diameter interval of 10 to 120 nm) and

57.2 � 10.8 nm (Fig. 3D—with pore diameter interval of 15 to

125 nm) for the BSA- and the lysozyme-based composites.

On average, the size of the pores of the protein-based systems

is smaller than in the absence of protein. This decrease in average

size is attributed to interactions between the biomolecules and

the silica. Furthermore, we observed that the average pore size of

the BSA-containing composites is smaller than the one of the

lysozyme-containing samples, correlating well with the greater

protein uptake in the BSA system. That is the pore dimensions of

the lysozyme-containing silica samples more closely resemble

those of the silica materials made without protein added.

2.2. Characterisation of the systems

2.2.1. Physical properties. Serum albumin undergoes revers-

ible conformational isomerisation with changes in pH. At pH 4

BSA is organised into a conformation called the F form.36 The

viscosity of the BSA-silica condensing mixture, with BSA present

as this conformer, as evident in Fig. 4A for a BSA concentration

of 2 mg mL�1, is much higher than that for the pure siliceous

systems. At a concentration of 4 mg mL�1 and in the range of the

dispersed phases used, the viscosity of the BSA–silica mixture

decreases (Fig. 4A) in parallel with an increase in the pH above

4.3 as represented in Fig. 1A. This is associated with a confor-

mational change of form F to form N of the biomolecule.36

Above pH 4.3, BSA undergoes an abrupt compaction37 to form

the isomer N. For BSA concentrations of 6 and 8 mg mL�1, the

viscosity of the BSA–silica mixture undergoes a minor decrease.

In the case of the lysozyme-based silica mixtures, the viscosity

remains steady upon increasing the concentration of lysozyme,

again this is in agreement with the pH data correlating to the

reduced uptake of the protein into the condensing silica.

From the increase in the viscosity of the samples after addition

of proteins as compared to the pure siliceous systems, it would

seem likely that either the proteins aggregate and/or become

directly associated with the condensing silica. Furthermore the

formation of protein aggregates coupled with the protein com-

plexing with the condensing silica will mostly likely also increase

the amount of surfactant and oil occlusion into the condensing

Fig. 4 Viscosity of silica–BSA (A) and silica–lysozyme (B) samples

prepared at various protein concentrations.

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materials. Collectively this will have the affect of augmenting the

organic component of the final composite materials. This should

be seen in the decomposition of these composites as compared

with those synthesized with no added protein (see section 2.2.2).

These data, in conjugation with the micrographs of the bioma-

terials (Fig. 3), indicate that, protein aggregation is involved in

silica morphology control. Moreover, depending on the nature of

the protein employed, the porosity of the resulting protein-silica

composite differs as has been suggested by Vrieling.38

2.2.2. Thermal analysis. Thermal analyses (DSC and TGA)

were performed on the as-synthesized composite materials to

determine whether the presence of BSA and lysozyme proteins

has any affect on the material properties.

The thermal degradation patterns from DSC scans (Fig. 5)

evidence the presence of BSA or lysozyme in the porous silica.

The silica/emulsion systems show a series of decomposition steps.

Physisorbed free water is removed at �105 �C. Water loss from

Fig. 5 Comparision of thermal degradation patterns as measured by

DSC scans for the as-synthesized composite materials. (A) BSA- and (B)

lysozyme-silica porous composites, synthesized using sample 3 with

different added protein concentrations. Physisorbed free water is

removed at�105 �C followed by loss of water from the pore structure. As

temperature is increased methanol is lost from �200 �C followed by

Triton X-100 (broad peak with an onset of approximately 300 �C).

This journal is ª The Royal Society of Chemistry 2011

the pores then occurs, though this is a very small component of

these systems.

During the sol–gel process of silica condensation liberation of

methanol molecules occurs due to the exchange of hydrogen and

alkoxy groups on the silicon atom. The endothermic peak due to

release of methanol is seen clearly in all materials containing

protein (onset from �200–210 �C), indicating that the presence

of the protein in the siliceous materials aids methanol retention/

occlusion during condensation. This is not readily evident when

no protein is added.

Peaks of the endothermic heat released due to thermal

oxidation of Triton X-100 appear between 270 �C and 360 �C in

the pure silica system.26 These peaks are generally shifted to

higher temperatures, with onsets as high as 300 �C and are

broadened, indicating release from a variety of different local

environments in the presence of protein. These data indicate

a direct association between the surfactant and the protein, as is

the case with methanol, aiding occlusion of organic material into

the silica framework. Degradation of the organic surfactant is

initiated in approximately the same range of temperatures for

both proteins.

The intensity of the Triton X-100 peak increases in parallel

with the increase of the concentration of the proteins, as would

be expected if the enhanced surfactant uptake is moderated by

the protein. Consider for example the lysozyme data. Here it is

evident that even a modest increase in the uptake of lysozyme

results in a substantial increase in the extent of Trion X-100

uptake.

The differences observed between the two protein DSC curves

are due to the unique sequence of the physicochemical reaction

that occurs over specific temperature ranges and heating rates

and are a function of the molecular structure of the biomolecules.

Thermogravimetric analysis (TGA) of the as-synthesized

composites was performed in triplicate over the temperature

range of 0–900 �C at a rate of 5 �C min�1 in an air atmosphere

(ESI Fig. 1). All materials, both as-synthesized and post-calci-

nation protein-silica macrocomposites are white, the latter indi-

cating that all protein, surfactant and oil components were

successfully removed and that the materials are completely open

porous networks, i.e., they are bicontinuous.

The TGA curves of the pure proteins contain peaks at 578 and

641 �C, respectively for pure BSA and pure lysozyme curves

which were not observed for the protein–silica samples. This

modification of the TGA pattern of the protein can be explained

by the occlusion of the protein within the silica domain of the

materials and the subsequent destabilisation of BSA and lyso-

zyme upon binding to the silica leading to conformational

modifications (see below). Total degradation of the pure proteins

occurs respectively at 628 �C and at 652 �C for BSA and

lysozyme.

The TGA curves for the samples containing protein show

a significant enhancement in the weight loss related to partial

degradation of the surfactant template (initiated at �275 �C) ascompared to the siliceous material containing no protein. This is

consistent with the increased occlusion of Triton X-100 into the

final composite materials when protein is present. As such, the

TGA data for the protein–silica samples varied in a systematic

fashion with increasing protein concentration and thereby

increased occlusion of organics (Fig. 6). The higher the initial

Soft Matter, 2011, 7, 4918–4927 | 4921

Fig. 6 Comparison of weight loss from BSA– and lysozyme–silica

mesoporous composites, synthesized using sample 3 and different protein

concentrations.

Fig. 7 Peak fitting of the truncated amide I region of the BSA-silica FT-

IR spectrum performed for the 2 mg mL�1 BSA with sample 1. Solid line

represents original spectrum. Solid line with dots represents theoretical

spectrum which is the sum of all fitted peaks. Fitted peaks are shown by

dotted lines.

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concentration of protein in the samples the greater the observed

weight loss irrespective of whether BSA or lysozyme was

considered. Both proteins showed increasing incorporation of

protein upon increasing the initial protein concentration (Fig. 2)

resulting in increased occlusion of methanol and Triton X-100

into the final composite materials.

Similarly the importance of the weight loss occurring during

the second transition peak increases upon increase of the initial

concentration of the proteins which also relates this peak to the

degradation of the organic template but now in association with

loss of the proteins. This transition is shifted to higher temper-

atures when the initial concentration of the proteins is increased.

For example, when the concentration of BSA was increased from

2 to 8 mg mL�1, the maximum was shifted from 527 to 534 �C.And in the case of lysozyme, the maximum was moved from 559

to 598 �C. The presence of lysozyme enhanced the thermal

stability of the biomaterials, most likely due to the increased

occlusion of Triton X-100 compared to when the protein is BSA.

In addition, a higher concentration of protein provided

a stronger resistance of the biocomposites to thermal treatment.

2.2.3. Surface area measurements. The structure evolution of

the silica porous films in the presence of proteins has been studied

using nitrogen gas adsorption.

The data indicate that the bio-siliceous composites, yield type

IV isotherms typical of micro/mesoporous materials.39 After

addition of proteins, the size of the pores as well as the surface

areas of the new bio-siliceous systems diminished. This is typical

of an uptake of organic material, as previously detected by TGA

measurements. Nitrogen adsorption data confirm that the

porous structure of the synthesized compounds, as described by

the pore sizes and the surfaces areas, is maintained with Si–BSA

mole ratio of 2.74 � 103 to 1.10 � 104 and Si–lysozyme ratio of

6.10 � 102 to 2.43 � 103.

In the range of concentrations of dispersed phase studied, the

average pore size of the samples prepared from BSA (12.1 � 0.5

nm) is insignificantly smaller than that obtained for the samples

synthesized from lysozyme (14.9 � 2.00 nm). The interactions of

BSA and lysozyme with silica produced the partial or complete

filling of some pores on the outer surface of the silica particles by

the proteins which provoked a reduction of both the surface area

and the diameter of the pores of the final materials.

4922 | Soft Matter, 2011, 7, 4918–4927

The surface areas increased from 1.0 to 2.2 m2 g�1 for the BSA-

based systems and from 1.3 to 11.9 m2 g�1 for the lysozyme-based

systems when the concentration of the dispersed phase was

altered from 0.5 to 2 wt. %. The presence of the biomolecules did

not inhibit the positive effect of the concentration of the

dispersed phase on the value of the surfaces areas.

In order to explore the proteins properties within the inorganic

framework, infrared spectroscopy measurements were per-

formed on the solid-state samples.

2.2.4. Study of protein conformation in the presence of silica.

A typical peak-fitted FT-IR spectrum of one of the BSA–silica

composites prepared with 2 mg mL�1 BSA is shown in Fig. 7. For

the calculation of the percentage of secondary structures content,

the amide I peak group (1700–1600 cm�1) was quantified. For the

interpretation of the results, the amide II region (1600–1500

cm�1) was also considered.

In the case of the BSA-based composites, we observed that the

peak area of the amide I and II peaks increase in concert with the

increase of the initial biomolecule concentration. This is true for

all emulsion concentrations tested in this work. It confirms that

the more protein added, the bigger the uptake of BSA by silica.

The interacting sites of the oxide of silicon for the biomolecule

show no saturation in the range of protein concentrations used.

This is in agreement with the Bradford protein essay results

(Fig. 2A). For the lysozyme-containing samples, we did not

notice such a trend. As anticipated with the measurements of the

concentration of the unbound protein in the supernatants

(Fig. 2B), silica shows a limited uptake of lysozyme. Addition of

further protein to the initial sample appears as further unbound

protein present in the supernatant.

After the amide I band contour was decomposed into its

underlying components, the secondary structure of the native

proteins in the absence of silica was determined to be primarily a-

helices, at 67.0 and 55.4%, respectively for BSA and lysozyme.

These data are supported by the literature.40–42 Both native

proteins are significantly arranged as well in short-segment

chains connecting a-helical segments of the biomolecules at 16.34

This journal is ª The Royal Society of Chemistry 2011

† We note that the initial concentration of the TMOS solution is twicethat used in the original paper. This is required to ensure that the finalsilica concentration is the same as that described in the originalmanuscript, since here dilution results from the addition of the proteinsolution. Similarly, for direct comparison, in order to achieve the samefinal amount of dispersed phase, the original surfactant concentrationswere doubled.

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and 11.67% respectively for BSA and lysozyme. Peak-fitting of

the amide I region of native lysozyme reflects that the protein

also contains disordered structures at 10.91%.

The comparison of the spectra of native BSA and lysozyme

with the spectra of the proteins (at different concentrations)

interacting with the silica matrix directly shows that the

secondary structure of the biomolecules is modified. Clearly, the

a-helix content is lower for proteins bound to silica for all three

dispersed phase concentrations used when compared to free

proteins in similar solutions. The a-helix content drops off,

depending on the concentration of the dispersed phase used,

to �45 to 53% for BSA and to �44 to 52% for lysozyme.

However, this structure remains dominant for both proteins.

This is in agreement with previous studies which stipulated that

when BSA interacts with charged molecules41,42 or desorbed from

SiO2 surface,43,44 the a-helix content decreases to approximately

50%.

For the BSA-containing systems, we also observed that the

interactions with silica associated with an increase of pH, causes

part of the polypeptide chain of the BSA macromolecule to be

transformed into the b-form with intermolecular association

between (partially) unfolded protein molecules as represented in

Fig. 8 A to C. A significant proportion of BSA secondary

structures formed b-sheets and random coils; at the highest

concentration of BSA, this part corresponds to 28.99, 43.11 and

21.76% for respectively the samples 1 to 3.

In the range of amide II vibrations, for all concentrations of

dispersed phase tested, the spectra acquired at different protein

concentrations reveal a maximum at 1545 cm�1 and a shoulder at

1520 cm�1 distinguishing the a-helical content of the unbound

protein.45 Another recurrent maximum is observed in all the

spectra at 1570 cm�1. It is typical of the presence of deprotonated

glutamic acid side chains. This corroborates the results of the pH

data. Under our conditions, the carboxyl functions of the side

chains contained in the sequence of BSA are deprotonated and

thus propitious for developing chemical interactions with the

protonated silanol groups bear by the silica network. The FT-IR

spectrum of sample 2 also shows a maximum at 1530 cm�1 which

illustrates the formation of hydrogen-bonds between the oxygen

acceptor of the silanol groups present at the surface of silicon

dioxide and the hydrogen of the amino groups of BSA.

Despite its good stability documented in the past,46 lysozyme

suffered a slight variation of its a-helical content following

association with the silica although to a less extent than BSA.

The fundamental amide I band at 1658 cm�1 related to a-helical

secondary structure of the protein has a low-frequency shoulder

at 1647 cm�1 corresponding to the random coils of lysozyme.

This type of organisation becomes the second most important

one with all the dispersed phase concentrations utilized (Fig. 8 D

to F). It reaches �22.31 � 2.65% of the secondary structure

content of lysozyme.

Regarding the amide II region, we can distinguish two

considerable peak vibrations at 1545 and 1530 cm�1. These bands

are characteristic respectively of significant a-helix content in the

protein and of the presence of hydrogen-bonds, thus confirming

the conclusions drawn from the data in the amide I area.

During the adsorption in the thin slit-like pores of silica, BSA

biomacromolecules whose diameter in an aqueous solution is d¼6.2 nm47 are forced to drastically change their secondary

This journal is ª The Royal Society of Chemistry 2011

structure also due to a substantial rise in the fraction of planar b-

fragments of the polypeptide chain. This is materialised, at raised

concentrations (typically from 6 mg mL�1), with pronounced

shoulders in the amide I peak at 1621 and 1646 cm�1 character-

istic of the formation of intermolecular antiparallel b-sheet,

accompanied by a less pronounced shoulder at 1688 cm�1

attributed to the formation of hydrogen bonded COOH. A

similar trend is observed with the lysozyme-based systems. It

indicates that during interactions, denaturation of the proteins

takes place; i.e. part of the a-helix is lost because of unfolding of

the proteins. Due to more pronounced binding forces between

BSA and silica than those between lysozyme and silica, the BSA

structure changes more than that of lysozyme.

3. Experimental

3.1. Materials

BSA (98–99% pure) and lysozyme ($90%) were purchased from

Sigma. Ethanol (>99.8%) was purchased from Pure Science

(Porirua, New Zealand) and diluted to 70% with double distilled

water. n-Hexane (95%) was obtained from Lab-Scan (Auckland,

New Zealand). n-Octadecane ($99%) was purchased from Fluka

Analytical (Auckland, New Zealand). Tetramethoxysilane

(TMOS) (>98%) was supplied by Merck-Schuchardt (Hohen-

brunn, Germany). 1 M hydrochloric acid (HCl) pH 2 buffer was

prepared from a commercial solution obtained from the Scien-

tific and Chemical Suppliers Ltd (Bilston, England). Triton X-

100 (C8H17-(C6H4)-(OCH2CH2)n-OH), the nonionic surfactant

(where n on average is equal to 10), was supplied by the Aldrich

Chemical Company (Auckland, New Zealand). Water was

purified by a Millipore Milli-Q system. All reagents were used

without further purification.

3.2. Methods

3.2.1. Sample preparation. Siliceous inorganic–organic

composite materials were synthesized using Triton X-100/octa-

decane/water emulsions as the scaffold material and tetrame-

thoxysilane (TMOS) as the silica precursor according to the

procedure described previously.26 Briefly, a first solution con-

sisting of a triton X-100/octadecane/water emulsion was

prepared with an octadecane to Triton X-100 wt. % ratio of 5 : 1.

A second solution, the silica precursor, was prepared by adding

the appropriate amount of TMOS in 1 M HCl to obtain a 3.0 M

TMOS solution, which was left to hydrolyze for 20 min.†

The amount of dispersed phase in the emulsion solution, here

octadecane, directly controls the porosity and surface area of the

final composite materials (increasing the volume fraction of the

dispersed phase increases the total porosity of the final silicate

material), as such this is a free parameter which may be varied

over a considerable range. Emulsions having 2.5 to 70 wt. % oil

may be prepared.48 The concentration of TMOS however is fixed.

Soft Matter, 2011, 7, 4918–4927 | 4923

Fig. 8 Contribution of different amide vibration bands to the area of the amide I band, determined by means of peak fitting for samples prepared from

BSA–silica systems (A to C) or lysozyme–silica systems (D to F) with sample 1 (A andD), sample 2 (B and E) or sample 3 (C and F). Side chain vibrations

1613 cm�1; b-sheets 1620 cm�1; b-strands 1630 cm�1; random coils 1646 cm�1; a-helices 1654 cm�1 and turns/H-bonded COOH 1680–1690 cm�1.

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For comparison purposes we have limited ourselves to just three

initial Triton X-100 concentrations; 1, 2 and 4 wt. % (full

composition information for the emulsion solutions is given in

Table 1).

The two solutions, the emulsion and the pre-hydrolyzed

TMOS, were mixed together under continuous stirring, in

a volume ratio of 1 : 2, emulsion to silica precursor at 70 �C. Themixture was left under constant stirring at 70 �C for�2 h, during

Table 1 Composition of the emulsion solution

Triton X-100(wt. %)

Oil(wt. %)

Water(wt. %) Water (mL)

Sample 1 1.0 5.0 94 14.10Sample 2 2.0 10.0 88 13.20Sample 3 4.0 20.0 76.0 11.40

4924 | Soft Matter, 2011, 7, 4918–4927

which time condensation was initiated. The mixture was then

cooled to room temperature. To 5 mL of this room temperature

mixture, 10 mL of an organic hydrophobic solvent, n-hexane,

was added. The mixture was rested for a total of 60 min allowing

condensation to continue. The mixture is a low viscosity fluid

during this entire processing. The pH of the resulting mixture was

3.00 � 0.15 independent of the dispersed phase concentration.

Aqueous stock solutions of BSA and lysozyme (16 mg mL�1)

were prepared by dissolving the relevant protein powder in

distilled deionised water directly before use. The as-prepared

solutions had a pH of 7.04 and 3.94, BSA and lysozyme,

respectively.

A series of silica/emulsion/protein solutions were prepared

using the as-described silica/emulsion solutions. To the room

temperature rested silica/emulsion mixtures different volumes of

fresh biomolecule stock solution were added. Upon addition of

This journal is ª The Royal Society of Chemistry 2011

Table 2 Range of silica–protein mole ratios used/103

Silica:BSA Silica:lysozyme

11.0 2.432.74 0.610

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the aqueous protein solution the mixtures were vigorously stirred

for 60 s using a vortex mixer and left to age at room temperature

(20� 0.2 �C) for 24 h. During this time condensation of the silica

continued, now in the presence of protein. Since condensation

induces changes to the physical characteristics of the mixture,

such as increased viscosity, the kinetics of the condensation

process can be followed and the time at which equilibrium is

established determined. The ageing time of 24 h used here was

chosen on the basis of such preliminary kinetic experiments.‡

The parameters followed were mixture pH, viscosity and particle

size which had all stabilised after 24 h of monitoring, indicating

that equilibrium had been reached. At this time silica conden-

sation was complete and the association or uptake of the protein

with or by the silica had been optimised by the system. The final

silica concentration in the silica–protein solutions was 0.33 M,

and the protein concentration was varied from 0 to 8 mg mL�1 in

steps of 2 mg mL�1. The ranges of final silica to protein mole

ratios are given in Table 2.

Isolation of the as-synthesized inorganic–organic composite

materials was achieved using centrifugation. A minimum of three

centrifugations were required to isolate the water insoluble

composite materials which were finally rinsed with double

distilled water.

3.2.2. Analysis of condensing mixtures: silica/emulsion and

silica/emulsion/protein samples. The pH of the stock protein

solutions, the silica/emulsion mixture and the silica/emulsion/

protein samples during the full 24 h ageing period was measured

using a pH-meter Seven Easy S20 (Mettler Toledo, Greifensee,

Switzerland) and a temperature sensor. The pH probe was cali-

brated using three IUPAC buffers (pH 4.005, 7.001, and 10.008).

The absolute error of the pH measurements was � 0.1.

Viscosity measurements of the silica/emulsion/protein samples

during the 24 h ageing period were performed on samples diluted

10 times in distilled water for ease of measurement at 25 � 2 �Cusing an AND SV-10 vibro-viscometer (KSV Instruments, Ltd.,

NSW, Australia) with a gold-coated transducer and temperature

sensor. The readings of the viscosity were acquired in 10 mL

plastic cells with fixed size and position.

Dynamic light scattering of the samples was performed using

a Zetasizer Nano ZS from Malvern Instruments (Lucas Heights,

Australia). The measurements were performed at 25 �C. Samples

were diluted (<0.02 wt. %) using distilled water and housed in a 1

cm poly(methylmethacrylate) cuvette to optimise the extent of

scattering. The monitoring of the scattering intensity fluctuations

was performed at an angle of 90�.

‡ It was not the aim of this investigation to undertake a full kineticanalysis, but rather to determine the result of the protein silicainteraction on modifying (1) the silica condensation and therefore thenanometre and micrometre pore system and (2) the proteinconformation.

This journal is ª The Royal Society of Chemistry 2011

3.2.3. Measurement of the concentration of free protein. The

free BSA or lysozyme concentrations in the supernatant of the

silica/emulsion/protein samples were measured by a Bradford

total protein concentration assay.49 Absorbance measurements

were performed on the supernatant, after the silica/emulsion/

protein mixture had been centrifuged at 3000 rpm for 10 min to

remove any siliceous solids, using a NanoDrop 1000 spectro-

photometer V3.3.0 (ThermoFisher Scientific, Auckland, New

Zealand). An aliquot of 0.1 mL of the sample supernatant was

diluted in 9.9 mL of distilled deionised water. A volume of 1.0

mL of the resulting solution was mixed with 5.0 mL of the

Bradford reagent containing 0.1 g L�1 Coomassie Brilliant Blue

G-250, 5.0% ethanol, and 8.5% phosphoric acid (filtered before

use) according to the method described in ref. 49. The absor-

bance of the solutions was integrated for 15 s at a wavelength of

595 nm 5 min after mixing and the protein concentration was

calculated from the calibration curve obtained with BSA or

lysozyme solutions of known concentration.

3.2.4. Analysis of isolated as-synthesized and calcined sili-

ceous materials. The isolated solid as-synthesized silica

composite materials and calcined silica materials were mounted

on SEM stubs and coated with platinum and carbon using

a standard procedure. The morphology of the samples was

characterised utilising a JEOL 6500 F field-emission gun scan-

ning electron microscope operating at an accelerating voltage

of 15 kV.

Thermogravimetric analysis (TGA) (Shimadzu TGA-50H,

Japan) was used to follow the pyrolysis of all samples. Approx-

imately 5 mg of as-synthesized material was pyrolysed under a 50

mL min�1 air flow at a heating rate of 5 �C/min. The samples

were first conditioned at 110 �C for 10 min in order to remove

physically adsorbed free water. The temperature of the samples

was kept constant at 110 �C for 5 min. A second heating of the

samples was performed at the same heating rate, from 110 �C to

900 �C. All measurements were verified at least three times for

each sample and samples were run in triplicate. Their validity was

reflected by the very low standard deviation observed over the

range of concentrations used (�3.14%). An air flow rate of 50 mL

min�1 ensures an inert atmosphere above the sample during the

run, while the small amount of sample and the slow heating rate

ensures that the heat transfer limitations can be ignored.

Nitrogen adsorption isotherms were recorded using helium as

the carrier gas at liquid nitrogen temperature, and using

a commercial instrument (Micromeritics Gemini V Series, Nor-

cross, GA, USA). Prior to obtaining the gas adsorption

isotherms, outgassing of the adsorbent was executed in a thin

glass tube. The adsorbent is exposed to the vacuum at 110 �C for

a minimum of 24 h (Micromeritics Flowprep 060) in order to

remove previously adsorbed gases, essentially water vapour,

from the surface. The specific surface areas of the samples were

calculated using the multipoint analysis method included in the

instrument software. The BET surface area was calculated from

the adsorption branches of the isotherms in the relative pressure

range 0.06–0.99,50 and the total pore volume was evaluated at

a relative pressure of approximately 0.60.51

3.2.5. Fourier-transformed transmission infrared spectros-

copy. Room temperature IR measurements of the as-synthesized

Soft Matter, 2011, 7, 4918–4927 | 4925

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samples were performed on a FT-IR spectrophotometer (Perkin-

Elmer, USA, interferometer speed 0.4747 cm�1). Spectra were

taken in KBr pellets. For each spectrum, 64 interferograms were

collected, coadded and Fourier transformed, yielding a resolu-

tion of 2 cm�1. For secondary structure assessment, the smoothed

and base-lined spectra were used between 1720 and 1490 cm�1

(amide I and II bands). Peak positions corresponding to different

structural elements of the protein were obtained from the liter-

ature,52,53 their variation being limited to �2 cm�1 during the fit.

Six peaks were fitted to the amide I feature, corresponding to

vibrations attributed to side chain vibrations (1613 cm�1), b-

sheets (1620 cm�1), b-strands (1630 cm�1), random coils (1646

cm�1), a-helices (1654 cm�1) and turns/H-bonded COOH (1680–

1690 cm�1).

4. Conclusions

In this work, the adaptability of a method of synthesis for the

formation of porous patterned silicate materials using responsive

soft templates previously developed in our group is exposed. So

far, the method has been used to produce pure siliceous systems.

We prove here its robustness by applying it to the synthesis of

porous bio-silica composites where BSA and lysozyme are the

model biomolecules of varying charge employed at different

concentrations.

The bio-silica materials produced are homogeneous and have

a bimodal porosity as observed by SEM and N2 gas adsorption

measurements, in similarity to the pure silica macroporous

monoliths. Microscale porosity is centred at �5 mm and is

controlled by the oil droplets while the shorter hierarchical length

scale of porosity, comprised between�27 and 53 nm, is set by the

oil swollen micelles.

The final porosity and therefore surface area of the bio-sili-

ceous macroporous monoliths are controlled via the emulsion

constituent concentrations and the concentration ratio between

the emulsion and the silica employed.

Protein uptake by the silica is increased upon increasing

the initial concentration of the biomolecule, as showed by the

measurement of the protein remaining in the supernatant of the

samples.

The formation of the hybrid protein-silica materials results

from a cooperative effect between hydrogen-bonding interac-

tions originating from silanol groups and electrostatic interac-

tions between the acidic amino acids side chain functional groups

and silica. Such interactions presumably lead to the partial or

complete filling of some outer surface pores by the proteins since

after addition of proteins, the size of the pores as well as the

surface areas of the new bio-siliceous systems are slightly

diminished compared with materials synthesized with no protein.

Thermogravimetric analysis corroborates the data obtained

from the Bradford protein assay. The protein content in the final

composites increases upon increase of the initial protein

concentration. However this trend is less marked for lysozyme

than it is for BSA. This could be due to the preferential associ-

ation of silica with BSA as compared with lysozyme as suggested

by the higher viscosity of the BSA-based samples than the

viscosity of the lysozyme-based samples. Furthermore, DSC data

demonstrate the increase of the stability of the bio-silica

4926 | Soft Matter, 2011, 7, 4918–4927

materials; the thermal degradation of the pure siliceous systems

was delayed upon occlusion of proteins.

Infrared spectroscopy revealed the silica-dependent confor-

mation of BSA adsorbed to silica monoliths. The structural

rearrangement in BSA and lysozyme molecules upon interaction

with silica was probed by the a-helix content. With both proteins

the a-helix content decreases upon interaction, resulting in the

proteins becoming more disordered upon occlusion into the silica

framework. This effect was less pronounced for lysozyme than

for BSA.

Acknowledgements

The work was supported by the MacDiarmid Institute for

Advanced Materials and Nanotechnology.

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