C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7

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Magnetically separable bimodal mesoporous carbons with alarge capacity for the immobilization of biomolecules

Marta Sevillaa, Patricia Valle-Vigona, Pedro Tartajb, Antonio B. Fuertesa,*

aInstituto Nacional del Carbon (CSIC), Apartado 73, 33080-Oviedo, SpainbInstituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco, 28049-Madrid, Spain

A R T I C L E I N F O

Article history:

Received 12 February 2009

Accepted 11 May 2009

Available online 18 May 2009

0008-6223/$ - see front matter � 2009 Elsevidoi:10.1016/j.carbon.2009.05.004

* Corresponding author: Fax: +34 985 297662.E-mail address: abefu@incar.csic.es (A.B.

A B S T R A C T

A method for the fabrication of carbon-based mesoporous magnetic composites with a

large capacity for the adsorption/immobilization of biomolecules is presented. The com-

posites consist of iron oxide spinel nanoparticles inserted into the pores of templated uni-

modal or bimodal mesoporous carbons. The deposition of the magnetic iron oxide

nanoparticles was carried out following two synthetic routes: (1) the direct incorporation

of nanoparticles into the pores of the templated carbons and (2) the insertion of nanopar-

ticles into the mesopores of the carbon–silica composite followed by the selective removal

of silica framework. The carbon–iron oxide magnetic composites prepared according to

route 2 were found to have better textural properties (larger BET surface areas and pore vol-

umes) and significantly higher capacity for the adsorption of hemoglobin and immobiliza-

tion of lysozyme. The amounts of hemoglobin or lysozyme adsorbed/immobilized by these

materials were 176 mg hemoglobin g�1 support and 131 mg lysozyme g�1 support using

route 1 and 430 mg hemoglobin g�1 support and 322 mg lysozyme g�1 support by route 2.

Furthermore, we have demonstrated that, when no inorganic nanoparticles are deposited,

the bimodal mesoporous carbon shows exceptionally a large immobilization capacity for

hemoglobin (830 mg g�1 support) and lysozyme (510 mg g�1).

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The synthesis of organic (i.e. polymers) or inorganic (i.e. car-

bon, silica, inorganic oxides, etc.) materials for the manipula-

tion (separation, transport, immobilization, release, etc.) of

biomolecules has recently attracted widespread attention

due to the importance of this application in areas related with

biotechnology [1–4]. Many research works are directed to-

wards the encapsulation of biomolecules inside the porosity

of different types of porous materials, mainly mesoporous

carbon or silica matrices [5–10]. In particular, mesoporous car-

bon materials have properties that make them highly suitable

for this type of application: a large specific surface area, a

high pore volume and a porosity made up of uniform mesop-

er Ltd. All rights reservedFuertes).

ores with sizes that can be tuned over a wide range (from

2 nm to >10 nm). Numerous examples of the immobilization

of enzymes and adsorption of biomolecules or drugs on mes-

oporous carbon materials can be found in the literature

[5,9,11–14].

For many biotechnological applications (enzyme immobi-

lization, drug delivery, separation of biomolecules, etc.) the

hybrid systems, which are made up of biomolecules adsorbed

into the pores of the carrier, need to be easily separable/trans-

portable from/through the liquid medium, where they are

normally dispersed. An effective method to manipulate such

hybrid systems is to employ magnetic porous carriers made

up of magnetic nanoparticles dispersed in the porous matrix

of the adsorbent (silica, carbon or polymer) [15–18]. In this

.

2520 C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7

way hybrid systems can be easily manipulated by means of

an external magnetic field. The most practical way to prepare

these magnetic carriers is to incorporate non-toxic iron oxide

spinel (magnetite or maghemite) nanoparticles inside the

pores of the matrix. In spite of silica materials with a meso-

porous network can be easily produced, their surface proper-

ties are not appropriate for the in situ synthesis of magnetic

nanoparticles, which need to be incorporated by means of

complex methods. In these sense, porous carbons constitute

a more suitable support because they allow an easy in situ

synthesis of magnetic nanoparticles with sizes of around

10 nm (superparamagnetic properties). Numerous examples

of magnetic composites made up of iron oxide spinel nano-

particles dispersed in mesoporous carbon matrixes have been

reported [17,18,19–23]. However, these nanoparticles are nor-

mally deposited within the pore channels of the matrix,

which severely reduces the textural properties of the supports

(formation of closed pores) and also restricts accessibility to

the pores. These two drawbacks limit the ability of the meso-

porous carbon–iron oxide magnetic composites to immobilize

or adsorb large molecules. In the present work we propose a

new and facile synthetic strategy to overcome these obsta-

cles. The resulting magnetic mesoporous carbon composites

combine good magnetic properties with an excellent capacity

to immobilize and adsorb large biomolecules. Our synthetic

methodology employs as support a bimodal carbon prepared

by using a mesoporous silica (SBA-15) as sacrificial hard tem-

plate. In a first step, the silica porosity is partially filled with

the carbon precursor. The resulting material is subsequently

carbonized and the magnetic nanoparticles are inserted into

the unfilled pores of the silica. When the silica framework is

selectively removed, the material possesses two pore sys-

tems, one of which contains the deposited magnetic nanopar-

ticles, while the other, created by the dissolution of silica

walls, is completely empty and free of inorganic nanoparti-

cles. This synthetic strategy is schematically illustrated in

Fig. 1 – Illustration of the synthesis scheme used to obtain carbo

composites. (1) Impregnation and carbonization (complete (1a)

precursor); (2) removal of silica framework; (3) insertion of FexO

Fig. 1 and, as we will show below, represents a significant ad-

vance on the method previously reported by our group for

preparing magnetic mesoporous carbons [17–19]. We have

examined the effectiveness of this type of composite by

means of a series of experiments involving the adsorption

of large biomolecules such as hemoglobin and lysozyme. In

order to demonstrate its high performance, we compared its

capacity of adsorption/immobilization with that of magnetic

mesoporous carbons containing directly inserted ferrite

nanoparticles.

2. Experimental section

2.1. Preparation of the mesoporous silica

A mesostructured SBA-15 silica was used as template to pre-

pare the carbon samples and carbon–FexOy composites. The

silica was synthesised according to a procedure reported in

the literature [24]. Briefly, 1.3 g of surfactant Pluronic P123

was dissolved in a mixture of 10 g water and 40 g 2 M HCl,

after which 2.8 g TEOS was added dropwise. The mixture

was stirred for 5 min and then maintained at 35 �C for 20 h.

It was next transferred to an autoclave and heated at 150 �Cfor 24 h. Finally, the sample was recovered by filtration,

washed with abundant distilled water, dried and calcined at

550 �C for 4 h.

2.2. Synthesis of the templated carbons (unimodal andbimodal)

The synthesis of the carbons was performed according to a

procedure reported elsewhere [25]. Briefly, the mesostruc-

tured silica was impregnated with paratoluene sulfonic acid

(>98%, Aldrich) (0.5 M in ethanol) for 1 h, filtered, washed with

a small volume of ethanol and dried at 80 �C. Afterwards, fur-

furyl alcohol (>98%, Merck) was infiltrated into the porosity of

n–silica, templated carbons and carbon–iron-oxide magnetic

and partial (1b) filling of the silica porosity by the carbon

y ferrite nanoparticles.

C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7 2521

silica. Two different degrees of impregnation were used: (a)

the silica pores were filled with a volume of furfuryl alcohol

equal to the pore volume of the silica and (b) approximately

70% of the pore volume of silica was filled with furfuryl alco-

hol. The impregnated samples were cured in air for 8 h at

80 �C in order to polymerise the furfuryl alcohol and to con-

vert it into polyfurfuryl alcohol, which was then carbonised

under vacuum (<0.01 mbar) at 800 �C (3 �C/min). The sample

was held for 1 h at this temperature. Depending on the degree

to which the silica porosity was filled with furfuryl alcohol,

two types of silica–carbon composites were obtained: (a)

CU–S (complete filling of silica porosity) and b) CB–S (filling

of the silica porosity up to 70%). To obtain pure carbon sam-

ples, the resulting carbon–silica composite was immersed in

48% HF at room temperature for 15 h in order to remove the

silica framework. The carbon samples obtained in the form

of an insoluble fraction were washed with distilled water

and then dried in air at 120 �C. These samples are denoted

as CU (from the CU–S composite) and CB (from the CB–S com-

posite) and correspond to carbon materials with one or two

pore systems respectively.

2.3. Synthesis of the carbon–iron oxide composites

Two different procedures were employed to synthesise the

magnetic mesoporous carbon–iron oxide nanocomposites.

2.3.1. Direct insertion of magnetic iron oxide nanoparticlesinto the mesoporous carbonsTo incorporate the magnetic iron oxide spinel nanoparticles

into the pores of the templated mesoporous carbons (i.e. CU

and CB samples), the samples were impregnated with a

mixture of 2 g Fe(NO3)3.9H2O (Aldrich) in 1 g ethanol, which

was added dropwise until incipient wetness and then the

sample was dried at 60 �C under vacuum. This process was re-

peated several times until the amount of iron nitrate infil-

trated was equivalent to around 25 wt.% of the iron oxide in

the carbon–Fe3O4 composite. The impregnated iron nitrate

was converted into iron oxide ferrite nanoparticles by means

of a procedure described elsewhere [26]. In brief, the impreg-

nated sample is exposed to propionic acid vapours (at 80 �Cfor 15 h) followed by thermal treatment under N2 for 2 h at

a temperature of 300 �C. The resulting carbon–iron oxide com-

posites were denoted as CU–FexOy and CB–FexOy.

2.3.2. Carbon–iron oxide materials prepared by incorporatingmagnetic nanoparticles into the silica–carbon compositesTo synthesise this type of material we used the CB–S com-

posite prepared as described in Section 2.1. As the first step,

iron oxide magnetic nanoparticles were inserted into the

open pores of the CB–S sample according to the procedure de-

scribed in the previous section (the amount of iron nitrate

infiltrated was equivalent to 25 wt.% of the iron oxide in the

composite). Subsequently, the CB–S/iron oxide sample was

immersed in a NaOH 2 M solution for 15 h in order to remove

the silica framework. The resulting carbon–iron oxide com-

posite was denoted as CBO–FexOy. Fig. 1 schematically illus-

trates the methodology used to prepare both types of

templated carbons (unimodal and bimodal) and the magnetic

carbon–iron oxide composites.

2.4. Adsorption of hemoglobin and immobilization oflysozyme

A certain amount of the support (�4 mg for the experi-

ments with bovine hemoglobin and �10 mg for the experi-

ments with lysozyme) was dispersed (using an orbital stirrer)

at room temperature in 10 mL of bovine hemoglobin (Aldrich)

solution (initial concentration: 0.4 mgÆmL�1, pH 6 buffer solu-

tion) or 10 mL of lysozyme (Fluka) solution (initial concentra-

tion: 0.56 mg mL�1, pH 7 buffer solution) in a closed vessel in

order to avoid evaporation. The amount of protein or enzyme

immobilised in the support was evaluated by measuring the

concentration of hemoglobin or lysozyme in the solution with

a UV–vis spectrophotometer (Shimadzu UV-2401PC) using UV

absorption at 410 and 280 nm, respectively.

2.5. Characterization

X-ray diffraction (XRD) patterns were obtained on a Sie-

mens D5000 instrument operating at 40 kV and 20 mA, using

CuKa radiation. The microstructure of the samples was

examined by scanning (SEM, Zeiss DSM 942) and transmis-

sion (TEM, JEOL-2000 FXII) electron microscopy. Nitrogen

adsorption and desorption isotherms were performed at

�196 �C in a Micromeritics ASAP 2020 volumetric adsorption

system. The BET surface area was deduced from the analysis

of the isotherm in the relative pressure range of 0.04–0.20. The

total pore volume was calculated from the amount of nitro-

gen adsorbed at a relative pressure of 0.99. The pore size dis-

tribution (PSD) was obtained by means of the Kruk-Jaroniec-

Sayari method [27]. The magnetization curves (up to a field

of 5 T) of the samples were recorded on a vibrating sample

magnetometer (MLVSM9 MagLab 9 T, Oxford Instrument).

The saturation magnetization (Ms) and coercivity field values

(Hc) were calculated from the magnetization curves. Ms values

were obtained from 1/H extrapolation at high fields (Langevin

approach) since the magnetization curves did not appear to

be blocked (Hc = 0). Fourier transform infrared spectra for

proteins loaded on magnetic composite were recorded on a

Nicolet 8700 spectrometer fitted with a diffuse reflection

attachment.

3. Results and discussion

3.1. Physical properties of the templated mesoporouscarbons

The structural properties of the SBA-15 silica, the templated

carbon materials and the carbon–FexOy composites were

investigated by SEM, TEM, X-ray diffraction analysis and gas

adsorption measurements. Both the templated carbon and

the corresponding composites retain the morphology of

SBA-15 silica particles, which have a platelet-like shape with

a size of approximately 1 lm as illustrated by the SEM images

(see Fig. 2a and b). Furthermore, the SBA-15 silica obtained by

hydrothermal treatment at 150 �C exhibits a well-ordered

porosity, as can be deduced from the XRD pattern at the low

angle range (see Fig. 2c) and is made up of uniform mesopores

with a size centred at 11 nm (see Fig. 2d). The silica–carbon

composite (CU–S) obtained by complete infiltration of the

2 Theta (º)

Inte

nsity

(a. u

.)

0

200

400

600

800

0.5 1.5 2.5 3.5 4.5 0.0 0.2 0.4 0.6 0.8 1.0

Relative pressure (p/po)

Ads

orbe

d vo

lum

e, (c

m3

STP/

g)

0

4

8

12

2 4 6 8 10 12 14 16 18 20Pore size (D), nm

dV/d

log(

D),

cm3 /g

11 nm dc (100)

(110)

(200)

a b

5 µm 2 µm

Fig. 2 – Structural properties of SBA-15 silica obtained by hydrothermal treatment at 150 �C. (a, b) SEM microphotographs, (c)

XRD patterns at the low angle range and (d) nitrogen sorption isotherm and pore size distribution (Inset). This material has a

BET surface area of 490 m2/g and a pore volume of 1.18 cm3/g.

2522 C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7

silica porosity hardly contains any mesopores and the tex-

tural properties measured can be ascribed almost exclusively

to the micropores of the carbon framework (see Fig. 3). How-

ever, in the case of the silica–carbon composite (CB–S) ob-

0

50

100

150

200

250

300

350

0.0 0.2 0.4 0.6 0.8 1.0Relative pressure (p/po)

Ads

orbe

d vo

lum

e, (c

m3

STP

/g)

CU-S (Complete filling of the porosity)

CB-S (Filling of the porosity up to 70%)

0.0

0.5

1.0

1.5

1 10 100Pore size (D), nm

dV/d

log(

D),

cm3 /g

10.7 nm

Fig. 3 – Nitrogen sorption isotherms and pore size

distributions (inset) of silica–carbon composites prepared by

complete (CU–S) and partial (CB–S, 70%) filling of the pore

volume of SBA-15 silica with the carbon precursor (furfuryl

alcohol).

tained by infiltrating �70% of the silica pore volume, a large

fraction of the porosity is made up of uniform mesopores cen-

tred at �10.7 nm (see Fig. 3). Fig. 4a shows a TEM image of the

CB–S composite with bright regions that can be adscribed to

the silica pores only partially filled with carbon. These results

indicate that, in the case of the CB–S sample, while some of

the pores of the template silica sample remain empty, others

are completely filled with carbon. Obviously, removal of the

silica framework in the CU–S composite will lead to a tem-

plated carbon with only one pore system (CU sample). In

the case of the CB–S composite, however, this will result in

a carbon material with two pore systems (CB sample). This

can be confirmed by comparing the nitrogen sorption iso-

therms and pore size distributions obtained for the CU (see

Fig. 5a) and CB samples (see Fig. 5b). The pore system centred

at around 5 nm (primary mesopores), common to both types

of carbons (CU and CB), is created by the dissolution of the sil-

ica walls. The CB carbon contains an additional pore system,

centred at around 20 nm (secondary mesopores), which was

formed by the coalescence of the unfilled silica pores with

the voids resulting from the dissolution of the adjacent silica

walls. The textural properties (BET surface area, pore volume

and pore sizes) of both types of templated carbons are pre-

sented in Table 1. The nanostructure of the CU and CB car-

bons is clearly illustrated in the TEM images taken

perpendicular to the direction of the hexagonal pore arrange-

ment (Fig. 4b and c). What remarkable is that, whereas the

nanostructure of the CU sample is made up of uniform

nanorods characteristic of the CMK-3 carbon (Fig. 4b), in the

Fig. 4 – TEM images of the carbon–silica composite (a: CB–S), the templated carbons with one (b: CU sample) and two (c: CB

sample) pore systems, and the carbon–iron oxide composites (d: CU–FexOy; e: CBO–FexOy; f: CB–FexOy). In Figure 3c, the P-

arrow indicates a primary mesopore and the S-arrow a secondary mesopore.

C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7 2523

case of the CB carbon some of the nanorods appear to be bro-

ken, resulting in large voids (Fig. 4c). Clearly, these voids cor-

respond to the secondary mesopores of the CB sample. Fig. 4c

shows the primary and secondary mesopores which are

marked by means of arrows.

3.2. Structural characteristics of mesoporous magneticcomposites

As mentioned in the experimental section and illustrated

in Fig. 1, the magnetic mesoporous carbon–iron oxide com-

posites were fabricated by one of two synthetic strategies:

3.2.1. Strategy 1The magnetic iron oxide nanoparticles are directly incor-

porated into the pores of the templated carbons (CU or CB).

In this way, the nanoparticles are deposited in both the pri-

mary and secondary mesopores. This strategy was applied

to synthesise the CU–FexOy and CB–FexOy samples (see Fig. 1).

3.2.2. Strategy 2The magnetic nanoparticles are first inserted in the mes-

opores of the CB–S composite and the silica framework is

selectively removed. In this case, the iron oxide nanoparticles

are incorporated exclusively into the secondary mesopores.

0

200

400

600

800

1000

0.0 0.2 0.4 0.6 0.8 1.0

Relative pressure (p/po)

Ads

orbe

d vo

lum

e, (c

m3

STP

/g)

CUCU-FexOy

0

2

4

6

8

0 2 4 6 8 10Pore size (D), nm

dV/d

log(

D), c

m3/g

5.1 nm

0

200

400

600

800

1000

1200

0.0 0.2 0.4 0.6 0.8 1.0Relative pressure (p/po)

Ads

orbe

d vo

lum

e, (c

m3

STP/

g)

CBCBO-FexOyCB-FexOy

0

2

4

6

1 10 100Pore size (D), nm

dV/d

log(

D), c

m3 /g

5.3 nm

20 nm

a

b

Fig. 5 – Nitrogen sorption isotherm and pore size

distributions (Inset) for: (a) unimodal and (b) bimodal

mesoporous carbons and the magnetic composites.

Table 1 – Physical properties of the mesoporous carbons and thhemoglobin and lysozyme adsorbed.

Codesample

SBET

(m2 g�1)Vp (cm3 g�1)a Pore size

(nm)bdXRD (nm) MS (em

CB 1310 1.79 (0.70/0.93) 5.3, 20 –

CBO–FexOy 950 1.14 (0.52/0.53) 4.8, 20 8.5 6.7

CB–FexOy 800 0.97 (0.47/0.40) 4.8, 22 13.4 13.8

CU 1350 1.35 5.1 –

CU–FexOy 850 0.85 5.0 13.3 13.9

a The pore volumes corresponding to the two pore systems (small size/l

b Maximum/a of the pore size distribution.

2524 C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7

This strategy was employed to obtain the CBO–FexOy sample

(see Fig. 1).

The incorporation of nanoparticles within the pores of a

solid causes a reduction of the textural properties (surface

area and pore volume) due to: (a) the incorporation of a sub-

stance that is non-porous and (b) the blockage of a number

of pores, leading to the formation of closed porosity. In addi-

tion, when these nanoparticles are deposited, pore constric-

tions are created, as a result of which the diffusion rate of

the large molecules is reduced. Table 1 shows the textural

properties of the magnetic composites prepared according

to the two strategies. It should be noted that the CBO–FexOy

sample which was prepared according to Strategy 2 exhibits,

in relation to the CU–FexOy and CB–FexOy composites pre-

pared by following Strategy 1, higher BET surface area and

pore volume values. Fig. 5 shows the nitrogen sorption iso-

therms and pore size distributions of the magnetic compos-

ites. A comparison of the sorption isotherms of the bimodal

materials (Fig. 5b) clearly reveals that the CBO–FexOy sample

exhibits a larger N2 adsorption uptake than CB–FexOy, thereby

confirming the data presented in Table 1. These results cor-

roborate our hypothesis and show that the synthetic method

based on Strategy 2 prevents the incorporation of inorganic

nanoparticles into the primary mesopore system, which re-

mains unobstructed, thus facilitating the diffusion, adsorp-

tion or immobilization of large molecules.

Proof of the formation of ferrite nanoparticles was ob-

tained by X-ray diffraction. The XRD patterns in Fig. 6 reveal

that the deposited nanoparticles consist of iron oxides with

an inverse cubic spinel structure, comprising both magnetite

(Fe3O4) and maghemite (c-Fe2O3). The magnetic properties of

these iron oxides are very similar. Maghemite only differs

from magnetite in that all the Fe cations are in the trivalent

state. Because the exact nature of the crystalline phase (mag-

netite or maghemite) of the iron oxide ferrite nanoparticles is

difficult to discern from the XRD patterns, we used the gen-

eral formula FexOy. The size of the crystallites deduced by

applying the Scherrer equation to the (3 1 1) peak of the XRD

patterns is between around 8.5 and 13 nm (see Table 1).

Fig. 4 contains the TEM images of the carbon–iron oxide com-

posites. It can be seen that the inserted nanoparticles are uni-

formly distributed throughout the carbonaceous porosity. In

general, the crystallite sizes obtained by means of XRD anal-

e magnetic nanocomposites, together with the amounts of

u g�1) Hc

(Oe)Immobilization of biomolecules (mg g�1 support)

Lysozyme Hemoglobin

510 830

0 322 430

0 131 176

116 372

0 77 327

arge size) are shown in parentheses.

20 30 40 50 60 702 Theta (º)

Inte

nsity

(a. u

.)

(220)

(311)

(400)

(422)(511)

(440)

CBO-FexOy

CB-FexOy

CU-FexOy

Fig. 6 – XRD patterns of the mesoporous carbon–iron oxide

composites.

C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7 2525

ysis are slightly smaller than the diameters of the ferrite

nanoparticles deduced from TEM inspection (see Fig. 4d, e

and f). This suggests that the ferrite nanoparticles have been

formed by the aggregation of several crystallites.

The magnetization reversal process was recorded at room

temperature (RT) in order to determine the magnetic moment

of the mesoporous carbons and to check for superparamag-

netic behaviour in the samples (Fig. 7). None of the samples

have magnetic memory (zero coercivity field) as evidenced

by the inset (b) in Fig. 7. The saturation magnetization values

(MS) of the mesoporous carbons correlate well with the size of

iron oxide spinel nanocrystals (the composites have a similar

iron oxide content). Consequently, the MS values were compa-

rable for those composites containing spinels with similar

crystallite sizes. In addition, the MS values increase with the

increase in crystallite size (Table 1, Fig. 7). Due to the presence

Fig. 7 – Magnetization curves (room temperature) of the

magnetic mesoporous carbons. CU–FexOy and CB–FexOy

carbons display similar magnetization curves. Inset (a)

shows an example of magnetic separation for CBO–FexOy

particles loaded with hemoglobin. Inset (b) is a low-field

zoom showing the absence of magnetic memory in all of the

composites (zero coercivity field, Hc = 0).

of nanosized iron oxide spinels, theMS values normalized to the

iron oxide content were in all cases (from 25 to 55 emu g�1)

lower than those of magnetite (75–85 emu g�1).

3.3. Immobilization of biomolecules

In order to evaluate the ability of the mesoporous carbons

and magnetic composites to manipulate biomolecules, we

investigated their capacities to adsorb a large protein (bovine

hemoglobin) and to immobilize an enzyme (lysozyme).

Hemoglobin is a globular heme protein that has a high molec-

ular weight (MW = 64,500), a large size (Dimensions:

5.3 · 5.4 · 6.5 nm3) and an isoelectric point of pI 6.8. It was

chosen because of its importance for the fabrication of NO,

NO�2 and H2O2 sensors [28,29] and also as an electrocatalyst

[30]. Lysozyme (MW: 14,300, Dimensions: 3.0 · 3.0 · 4.5 nm3

and pI 11) has been widely employed as a reference substance

in order to test the enzyme immobilization capacity of meso-

porous substrates (mainly carbon and silica matrises) [7–

9,13,14,31]. These biomolecules were recently used by our

group to demonstrate the ability of silica- and carbon-based

porous magnetic materials to adsorb proteins and to immobi-

lize enzymes [19,26].

Fig. 8 shows the change with time in the amount of hemo-

globin adsorbed (Fig. 8a) and lysozyme immobilized (Fig. 8b)

by the templated carbons and the magnetic composites. Table

1 contains the equilibrium adsorption amounts of hemoglo-

bin and lysozyme. Exceptional high biomolecule loadings

were achieved by the CB bimodal carbon (830 mg hemoglobin

g�1 support and 510 mg lysozyme g�1 support). These values

are significantly larger than those of the CU unimodal carbon

(372 mg hemoglobin g�1 support and 116 mg lysozyme g�1

support). To the best of our knowledge, the amount of lyso-

zyme immobilized by the CB bimodal carbon constitutes the

largest adsorption uptake achieved by this model enzyme

over porous carbon supports. In fact, the amount of lysozyme

immobilized by the CB carbon is even higher than that re-

cently reported by Vinu et al. [14] for a unimodal mesoporous

carbon (CKT-3(A) sample, SBET = 1600 m2 g�1, Vp = 2.1 cm3 g�1

and a pore size of 5.2 nm), which is claimed to have a large

immobilization capacity (�360 mg lysozyme g�1). Taking into

account that the adsorption of large biomolecules within por-

ous matrixes is commonly limited by steric effects, the pres-

ence in the CB sample of large secondary mesopores (approx.

20 nm) greatly enhances the diffusion and loading of bulky

biomolecules. This may explain the greater adsorption up-

takes for the CB sample with respect to the CU or CKT-3(A)

carbons, which have a unimodal porosity made up of mesop-

ores of around 5 nm, a size similar to that of lysozyme. These

differences in pore structure also explain the trends observed

in the adsorption rates. Indeed, examination of the immobili-

zation curves of the different samples reveals that the mate-

rials with two pore systems exhibit higher adsorption rates

than those of the unimodal samples (see Fig. 8). This is espe-

cially true for the adsorption of hemoglobin (see Fig. 8a),

which has a larger size than lysozyme.

As previously pointed out, the incorporation of iron oxide

nanoparticles induces a reduction of textural properties and

the appearance of pore constrictions. Clearly, this will cause

a decrease in the quantities adsorbed by the carbon–iron

0

100

200

300

400

500

600

700

800

900

0 10 20 30 40 50 60 70 80 90 100Time, h

Hem

oglo

bin

adso

rbed

, mg.

g-1 s

uppo

rt

0

100

200

300

400

500

600

0 10 20 30 40 50 60 70 80 90 100Time, h

Lyso

zym

e im

mob

ilize

d, m

g. m

g-1

sup

port

b

a

CBO-FexOy

CB-FexOy

CB

CU

CU-FexOy

CBO-FexOy

CB

CB-FexOy CU

CU-FexOy

Fig. 8 – Variation with the time of the amounts of (a)

hemoglobin and (b) lysozyme adsorbed by the mesoporous

carbon materials and the magnetic composites.

2526 C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7

oxide composites, as is evidenced by the adsorption amounts

listed in Table 1. This is confirmed by the results obtained for

the samples with a bimodal porosity. The bimodal mesopor-

ous carbon CB adsorbs very large amounts of hemoglobin

(830 mg g�1 support) and lysozyme (510 mg g�1). Compared

to these adsorption uptakes, we observed a notable reduction

for CB–FexOy sample (176 mg hemoglobin g�1 support and

131 mg lysozyme g�1 support). However, in the case of the

composite prepared according to Strategy 2 (CBO–FexOy) the

decrease is not as great (430 mg hemoglobin g�1 support

and 322 mg lysozyme g�1 support). It can be concluded, there-

fore, that Strategy 2 is a better method than Strategy 1 when

synthesising magnetic mesoporous carbon composites for

immobilizing large biomolecules. These findings are coherent

with the results obtained for the textural properties (see Sec-

tion 3.2) and confirm our hypothesis that the synthetic meth-

od based on Strategy 2 prevents the inorganic nanoparticles

from entering the primary mesopore system, thereby facili-

tating the immobilization of large biomolecules. In addition,

a number of secondary mesopores in the CBO–FexOy sample

will remain unoccupied and therefore available for the

adsorption/diffusion of biomolecules. It is the combination

of both these effects that explains the superior performance

of the CBO–FexOy sample in immobilizing biomolecules. It

should be also noted that an important advantage of using

magnetic composites to immobilize biomolecules is that

these substances can be easily recovered from the reaction

media by means of an external magnetic field. This is illus-

trated in Fig. 7 (inset (a)) which shows CBO–FexOy particles

loaded with hemoglobin.

In order to analyze the structural stability of the immo-

bilized biomolecules (hemoglobin and lysozyme), FTIR spec-

tra of the free biomolecules and magnetic composite (CBO–

FexOy) loaded with these substances were recorded (Fig. S1

in Supporting information). The spectrum of the free bio-

molecules exhibits two characteristic peaks at �1650 cm�1

and at �1530 cm�1 denoted as Amide bands I and II respec-

tively. Amide I vibration arise mainly from the C = O

stretching vibration with minor contributions from the

out-of-phase CN stretching vibration, the CCN deformation

and the NH in-plane bend. Amide II vibration is the out-

of-phase combination of the NH in-plane bend and the

CN stretching vibration with smaller contributions from

the CO in-plane bend and the CC and NC stretching vibra-

tions [9,14,32,33]. These bands also appear in the spectrum

corresponding to the biomolecules adsorbed by the mag-

netic carbon composite (CBO–FexOy). This result evidences

the presence of these substances in the magnetic compos-

ites and also that the secondary structure of the proteins

remains intact after immobilization.

4. Conclusions

In summary, a novel methodology for the fabrication of

magnetically separable templated mesoporous carbons with

a large capacity for the adsorption/immobilization of bio-

molecules is presented. The effectiveness of this synthetic

strategy is based on being able to insert magnetic iron oxide

nanoparticles within the mesopores of only one of the pore

systems present in a bimodal mesoporous carbon. Thus,

the larger mesopores (pores of around 20 nm) are partially

occupied by the magnetic nanoparticles, leaving the other

pore system (mesopores of around 5 nm) completely free

of inorganic nanoparticles. Compared to magnetic compos-

ites made up of bimodal mesoporous carbon with uni-

formly distributed iron oxide nanoparticles, the bimodal

mesoporous carbon composites fabricated using our meth-

odology exhibits better textural properties (higher BET sur-

face areas and larger pore volumes) and a significantly

larger capacity for the adsorption of hemoglobin and the

immobilization of lysozyme.

Acknowledgment

The financial support for this research work provided by the

Spanish MCyT (MAT2008-00407, MAT2008-03224) is gratefully

acknowledged.

C A R B O N 4 7 ( 2 0 0 9 ) 2 5 1 9 – 2 5 2 7 2527

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.carbon.2009.05.004.

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