PAPER www.rsc.org/softmatter | Soft Matter

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Enzyme-induced graft polymerization for preparation of hydrogels: synergeticeffect of laccase-immobilized-cryogels for pollutants adsorption†

Marina Nieto,a Stefania Nardecchia,a Carmen Peinado,b Fernando Catalina,b Concepci�on Abrusci,c

Mar�ıa C. Guti�errez,a M. Luisa Ferrer*a and Francisco del Monte*a

Received 9th March 2010, Accepted 8th April 2010

DOI: 10.1039/c0sm00079e

The use of polyethylene oxide-polypropylene oxide-polyethylene oxide block-copolymers as a mediator

in the laccase-induced graft polymerization of diacrylic derivate of polyethylene glycols resulted in the

formation of PEG-g-F68 hydrogels. The proper oxygen content in the reaction medium to obtain

reasonable polymerization conversions (i.e., on one hand, laccase needs oxygen as substrate whereas, on

the other, oxygen is a strong inhibitor of radical polymerizations) was achieved by the use of an enzymatic

scavenging system consisting of glucose oxidase and glucose. Eventually, laccase was immobilized within

the resulting PEG-g-F68 hydrogel with full preservation of enzyme activity. Laccases have been used for

bioremediation purposes because of their ability to degrade phenolic compounds. Thus, laccase-

immobilized PEG-g-F68 hydrogels were submitted to the ISISA (ice segregation induced self-assembly)

process for preparation of laccase-immobilized PEG-g-F68 cryogels which exhibited a macroporous

structure where immobilized laccase preserved almost total activity (ca. 90%) for a period exceeding three

months after preparation. Synergy between macroporous structure (deriving from the ISISA process),

amphiphilic domains (deriving from graft copolymer) and activity of the immobilized enzyme provided

outstanding adsorption capabilities to the cryogels (up to 235 mg g�1).

Introduction

Hydrogels are three-dimensional, hydrophilic, polymeric

networks capable of imbibing large amounts of water.1 Among

other applications,2 these networks have been used as adsorbents

to remove organic or heavy metal pollutants from wastewater.3

However, simple hydrophilic materials are not efficient adsor-

bents of organic pollutants and it is necessary for them to also

contain hydrophobic pockets which can accommodate and

retain organic substances. Polymer hydrogels containing

hydrophobic units together with the hydrophilic ones are called

amphiphilic polymer hydrogels or amphiphilic polymer networks

and have been the subject of several reviews.4 These networks

swell to a smaller extent than the corresponding hydrophilic

networks but, in addition, they can also swell in organic solvents.

Thus, hydrogels based on block copolymers containing hydro-

philic and hydrophobic domains are excellent candidates for

adsorption purposes since they can interact effectively with water

(through the hydrophilic domains) and they have as well the

capability (through the hydrophobic domains) to extract and

retain any organic substance eventually dissolved in water. The

specific features of hydrophilic and hydrophobic domains

aInstituto de Ciencia de Materiales de Madrid–ICMM, Consejo Superiorde Investigaciones Cient�ıficas–CSIC, Cantoblanco, 28049 Madrid, SpainbInstituto de Ciencia y Tecnolog�ıa de Pol�ımeros– ICTP, Consejo Superiorde Investigaciones Cient�ıficas–CSIC, C/Juan de la Cierva, 3, 28006Madrid, SpaincDepartamento de Biolog�ıa Molecular, Facultad de Ciencias, UniversidadAut�onoma de Madrid-UAM, Cantoblanco, 28049 Madrid, Spain. E-mail:mferrer@icmm.csic.es; delmonte@icmm.csic.es; Fax: +34 91 3720623;Tel: +34 91 334 9033

† Electronic supplementary information (ESI) available: Data resultingfrom fitting experimental adsorption isotherms, SEM and FTIRspectra. See DOI: 10.1039/c0sm00079e

This journal is ª The Royal Society of Chemistry 2010

(e.g. length, the segments arrangement, etc.) will mostly govern

the adsorption performance against a particular solute.5

If one desires to further enhance the adsorption performance of

a certain block-copolymer-based-hydrogel, synergetic moieties

should be included within its structure. Different enzymes, such as

ligninperoxidase, manganese-peroxidase and laccase, have

demonstrated their ability to degrade phenolic compounds6 which

are some of the most recalcitrant pollutants found in wastewaters.

Thus, immobilization of any of these enzymes in the block-

copolymer-based-hydrogels mentioned above could provide

enhanced bioremediation capabilities. However, the challenge

that remains is to immobilize certain unstable enzymes without

compromising their activity and stability.7 The formation of

a hydrogel requires the use of initiators and/or cross-linking

agents (which, ultimately, are incorporated as foreign chemical

functionalities pending from/grafting the polymer chain),8 or

a change of pH, ionic strength, or temperature.9 In every case, the

enzyme can be subjected to suboptimal conditions that may result

in partial or (in the worst scenario) total loss of its catalytic

activity. Enzymatic polymerizations have attracted much atten-

tion lately since they offer mild reaction conditions besides the

occurrence of enzyme immobilization within the formed

hydrogel.10 Suitable enzymes capable of initiating radical reac-

tions comprise peroxidases and laccases.11 The use of laccases

would be interesting for our purposes given that, as mentioned

above, the resulting laccase-immobilized-hydrogels could offer

enhanced bioremediation capabilities. Laccases are especially

efficient for acrylamide polymerizations, but need mediators

(e.g. b-diketones) to polymerize other acrylates.11,12 The oxygen

balance in solution is crucial since, on one hand it is the natural

substrate of laccases while, on the other it is a strong inhibitor of

radical polymerizations.12

Soft Matter, 2010, 6, 3533–3540 | 3533

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In this work, we explored the capability of polyethylene oxide-

polypropylene oxide-polyethylene oxide block-copolymers

(e.g. PEO80-PPO27-PEO80, F68) to be used as mediators in

laccase-induced polymerizations. Generation of long-lived mac-

roradicals in oxygen-free media has been widely explored for

PEO and PPO polymers,13 but never via enzymatic. The mono-

mer of choice was a diacrylic derivate of polyethylene glycol

(e.g. PEG550-DA) so that PEG-g-F68 hydrogels were obtained

by grafting of PEG550-DA onto F68 followed by cross-linking

of the acrylic moieties remaining after grafting. Glucose oxidase

(GOX) and glucose (GLU) were also added to obtain the

oxygen-free media required for radical polymerization. Hydro-

gels were thereafter subjected to a unidirectional freezing process

that, by ice-induced-self-assembly-segregation (ISISA) of the

matter homogeneously distributed within the gel and subsequent

high-vacuum sublimation of ice,14 promoted the formation of

macroporous structures while preserving the activity of immo-

bilized biological entities. Freeze-dried hydrogels were called

cryogels from the Greek kryos (krcos, meaning frost of ice).15

The morphology of PEG-g-F68 cryogels was studied by scanning

electron microscopy (SEM). Polymerization conversions were

calculated using the cryogel’s weight after soaking in distilled

water (to wash un-reacted acrylates) divided by their original

weight before washing. FTIR spectroscopy was used to analyze

the composition of cryogels before and after washing. Swelling

experiments were carried out in both water and toluene to

confirm the amphiphilic nature (consisting of hydrophilic and

hydrophobic domains) of washed cryogels. The adsorption of

Brilliant Blue dye Remazol (BBdR) as model of organic

pollutant was used to test the adsorption capability of the cry-

ogels. Adsorption isotherms were carried out with different

samples to assess the synergetic contribution of the activity of the

immobilized laccase besides the macroporous structure and the

amphiphilic nature of the cryogel to the overall adsorption

performance of these materials.

Experimental part

In a typical batch, F68 (30 mg), PEG550-DA (60 mg), laccase

(0.88 mg, 20 units) and glucose oxidase (GOX, 0.25 mg, 90 units)

were dissolved in a phosphate buffered solution (1.8 mL) also

containing glucose (GLU, 5 mM) for a polymer content of 7 wt%

Table 1 Experimental data used for preparation of graft copolymers at 37 �CF68(2 : 1-7)-lac1N2 which was deaerated under N2 flow. Sample PEG-g-F68(2coming from the use of GOX/Glu. Experimental data used for preparation oare also included

Sample FA68 PEG550DA LACAS

F68(7)-lac1 90 mg – 20UPEG(7)-lac1 – 90 mg 20UPEG-g-F68(2 : 1-7)-lac1 30 mg 60 mg 20UPEG-g-F68(2 : 1-7)-lac1N2 30 mg 60 mg 20UPEG-g-F68(2 : 1-7)-lac1c 30 mg 60 mg 20UPEG-g-F68(2 : 1-7)-lac2 30 mg 60 mg 80UPEG-g-F68(2 : 1-7)-lac3 30 mg 60 mg 200UPEG-g-F68(1 : 1-7)-lac1 45 mg 45 mg 20UPEG-g-F68(3 : 1-7)-lac1 23 mg 69 mg 20UPEG-g-F68(1 : 1-10)-lac1 60 mg 60 mg 20UPEG-g-F68(2 : 1-10)-lac1 40 mg 80 mg 20U

3534 | Soft Matter, 2010, 6, 3533–3540

(see Table 1). The solution was gently stirred for homogeniza-

tion, placed in an insulin syringe and stored 17 h at 37 �C for

gelation. The resulting hydrogel was called PEG-g-F68(2 : 1-7)h-

lac1, where h stands for hydrogel, 2 : 1 stands for PEGDA/F68

weight ratio, 7 for the weight percentage of polymers (F68 +

PEGDA) in solution and lac1 for laccase concentration (20 units)

used for macroradical generation. An identical procedure was

followed for preparation of samples described in Table 1 except

for the use of different PEGDA to F68 weight ratios (1 : 0, 0 : 1,

1 : 1 or 3 : 1), total polymer content (10 wt%) or laccase

concentration (80 units for samples called lac2 and 200 units for

samples called lac3). Moreover, PEG-g-F68(2 : 1-7)-lac1N2

hydrogels were prepared as described above, except for the use of

N2 flow to deaerate the sample instead of using the GOX/GLU

system. Meanwhile, PEG-g-F68(2 : 1-7)-lac1c hydrogels were

prepared using the GOX/GLU system coupled with catalase for

elimination of H2O2 resulting as a by-product of GOX/GLU

system (see Scheme 1). The freshly gelled hydrogels were unidi-

rectionally frozen by dipping the insulin syringes at a constant

rate of 5.9 mm min�1 into a liquid nitrogen bath (77 K). Samples

were freeze-dried using a ThermoSavant Micromodulyo freeze-

drier. The resulting cryogels were monoliths keeping both the

shape and the size of the insulin syringes. Particular cryogels were

called as described above for hydrogels except for the use of c

instead of h superscript (e.g. PEG-g-F68(2 : 1-7)c-lac1 for

a freeze-dried hydrogel prepared with 2 : 1 PEGDA to F68

weight ratio, 7 wt% total polymer content and 20 units of

laccase).

Laccase activity was determined following the oxidation of

ABTS at 420 nm (3 ¼ 3.6 � 104 cm�1 M�1 for the oxidation

product). The reaction mixture contained 100 mL of the enzyme

solution and 900 mL of ABTS (1 mM) in acetate/citrate buffer

(0.05 M, pH 5.0) at 30 �C. One unit of enzyme activity is defined

as the amount of enzyme that oxidizes 1 mmol ABTS in 1 min.

Polymer conversions were determined gravimetrically. Cry-

ogels were immersed in distilled water for 72 h at 25 �C to extract

unreacted monomers and subsequently freeze-dried. Washed

cryogels are called cryogels except for the use of wc instead of c

superscript (e.g. PEG-g-F68(2 : 1-7)wc-lac1 for a washed PEG-g-

F68(2 : 1-7)c-lac1 cryogel). Conversions were calculated (in %)

using the dry gel weight divided by the initial weight of monomer

in the sample. Swelling experiments were carried out by

. GOX/Glu was used as an oxygen scavenger except for sample PEG-g-: 1-7)-lac1c included catalase to address any eventual role played by H2O2

f bare PEG and F68 cryogels (PEG(7)-lac1 and F68(7)-lac1, respectively)

SE GOX GLU CATALASE Conversion

90U 5 mM – No gel90U 5 mM – 56%90U 5 mM – 58%– – – 48%90U 5 mM 250U 57%90U 5 mM – 59%90U 5 mM – 61%90U 5 mM – 50%90U 5 mM – 60%90U 5 mM – 61%90U 5 mM – 63%

This journal is ª The Royal Society of Chemistry 2010

Scheme 1 Use of F68 as mediator in laccase induced polymerization of

PEGDA for the formation of graft copolymers. The ideal oxygen balance

in solution (oxygen is the laccase substrate but it is also a strong inhibitor

of radical polymerizations) was provided by the enzymatic GOX/GLU

system.

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immersion of PEG-g-F68wc-lac1 cryogels in an excess amount of

either deionized water or toluene at 20 �C. The swollen samples

were weighed at various time intervals. Experiments were con-

ducted in triplicate.

The adsorption isotherms were measured in a Variant Cary

4000 spectrophotometer by taking fixed amounts of adsorbant

(20 mg), which were submerged in 5 mL of a dye solution at

different concentrations (ranging from 50 to 900 mg L�1 for any

of PEG-g-F68-lac0 and PEG-g-F68-lac1 hydrogels/cryogels,

from 50 to 1500 mg L�1 for any of PEG-g-F68-lac2 cryogels and

from 50 to 3500 mg L�1 for any of PEG-g-F68-lac3 cryogels) and

left to adsorb for 72 h at 20 �C. Afterwards, the scaffolds were

removed from the solution and the remaining contaminant

concentration was measured by absorption spectrophotometry.

Experiments were conducted in triplicate. Averaged data was

used for the representation of Langmuir and Freundlich

isotherms. The standard deviation was less than 5%.

Cryogel morphology was studied by SEM using a Zeiss DSM-

950 microscope. Samples were mounted onto the SEM sample

holder and gold sputter coated (for 2 min, to obtain a ca. 5 nm

gold film) prior to SEM observation. The accelerating voltage

was 15–20 kV and the work distance was 14–9 mm. FTIR spectra

were carried out in a FTIR Bruker IFS60v spectrometer.

Fig. 1 SEM micrographs of cross-sectioned PEG-g-F68(2 : 1-7)c-lac1

(a–c) and PEG-g-F68(2 : 1-7)c-lac3 (d). Scale bars are 400 mm in (a),

100 mm in (b) and 40 mm in (c,d).

Results and discussion

The ISISA process is a simple and versatile methodology based

on the unidirectional freezing of a hydrogel by its immersion (at a

rate typically ranging from 0.7 mm min�1 to 9.1 mm min�1) into

a liquid nitrogen bath (at �196 �C).16 Upon freezing, the ice

formation (hexagonal form) causes the hydrogel network

(originally occupying the entire hydrogel volume) to become

segregated and concentrated between adjacent ice crystals.

Freeze-drying resulted in the formation of structures consisting

of micrometre-sized pores corresponding to the empty areas

where ice crystals originally resided. Our interest in the appli-

cation of the ISISA process for the formation of cryogels was

double. First, it was our belief that the achievement of a highly

open microchanneled structure could favour mass transport and

accessibility of small molecules to hydrophilic and hydrophobic

domains distributed throughout the inner structure and thus,

This journal is ª The Royal Society of Chemistry 2010

a potential enhancement to the adsorption performance of these

materials. The second interesting feature of the ISISA process is

that freeze-drying/lyophilization processes are widely used in

biology for preservation and storage of proteins. This feature,

besides the overall biocompatibility of the whole ISISA process

(i.e. it begins from an aqueous suspension and runs in the absence

of further chemical reactions, avoiding potential complications

associated with by-products or purification procedures), can

eventually provide near full preservation of the original activity

of enzymes17 (and even of biological like membrane structures18)

after immobilization within the macroporous structure of the

cryogels. In the particular case of laccase immobilized within

PEG-g-F68 cryogels, almost 90% of activity was preserved over

a period longer than three months (see data below).

With regard to the first issue, the application of the ISISA

process to PEG-g-F68 hydrogels indeed resulted in the formation

of monolithic cryogels exhibiting macropores homogeneously

distributed throughout the whole cross-sectioned structure

(Fig. 1). The structural pattern consisted of well-aligned mac-

ropores in the freezing direction as a consequence of the unidi-

rectional freezing that characterizes the ISISA process

(Fig. S1, ESI†). The macroporous structures of PEG-g-F68

cryogels prepared from different PEGDA:F68 ratios (e.g. 1 : 1,

2 : 1 and 3 : 1), total polymer content (e.g. 7 and 10 wt%) and

laccase concentration were also analyzed (see Fig. 1, 2 and 3) and

no significant differences were observed among the different

samples. SEM micrographs of F68(7)-lac1 and PEG(7)-lac1

cryogels are also depicted in Fig. 4 and Fig. S2, ESI†, for

comparison. Interestingly, F68(7)-lac1 cryogels exhibited much

wider macropores than PEG(7)-lac1 cryogels and wider than any

of the graft copolymer based cryogels mentioned before.

Previous works on cryogels have related the decrease of pore size

to the cross-linking degree of the hydrogels submitted to the

ISISA process.19 Obviously, a lack of cross-linking occurred in

Soft Matter, 2010, 6, 3533–3540 | 3535

Fig. 3 SEM micrographs of cross-sectioned PEG-g-F68(1 : 1-10)c-lac1

(a) and PEG-g-F68(2 : 1-10)c-lac1 (b). Bars are 50 mm.

Fig. 2 SEM micrographs of cross-sectioned PEG-g-F68(1 : 1-7)c-lac1 (a)

and PEG-g-F68(3 : 1-7)c-lac1 (b). Scale bars are 200 mm in (a) and (b), and

20 mm in inset of (a).

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F68(7)-lac1 because of the absence of acrylate moieties. This

event was confirmed by the determination of the polymer

conversion, that is, the fraction of monomer incorporated into

the hydrogel network (see experimental for details). For this

purpose, cryogels were soaked in distilled water for 72 h (to wash

out unreacted monomers) and, subsequently, freeze-dried and

weighed, and their weight correlated to the initial weight of

monomer in the sample. Thus, F68(7)-lac1 cryogels were

completely dissolved when immersed in distilled water resem-

bling a lack of polymer network formation (null conversion).

Meanwhile, conversions ranging 55 to 65% were obtained for

cross-linked cryogels. Thus, the grafting mechanism that is

conducive to the formation of hydrogels seems to be governed by

the efficient formation of F68 macroradicals (enzymatically

induced) which, thereafter, promote PEGDA polymerization

Fig. 4 SEM micrographs of cross-sectioned F68(7)c-lac1 (a, scale bars

are 500 mm and 20 mm in the inset) and PEG(7)c-lac1 (b, scale bars are

200 mm and 20 mm in the inset).

3536 | Soft Matter, 2010, 6, 3533–3540

and cross-linking (Scheme 1). The above described mechanism

was also valid for the formation of PEG(7)-lac1 hydrogels from

bare PEGDA via enzymatic generation of PEO based macro-

radicals. Actually, the generation of long-lived macroradicals in

oxygen-free media has been demonstrated for both PEO and

PPO based polymers via non-enzymatic processes.13

The conversions reported in Table 1 were slightly below those

previously reported for laccase induced polymerizations (up to

90% for MMA or acrylamide polymerizations carried out at

50–60 �C),11,12 although it is worth noting that, in our case,

temperature was lower (e.g. 37 �C). It is worth noting that the use

of low temperatures may decrease the conversion but it was

crucial for the activity preservation of the immobilized

enzyme.12,20 Furthermore, the macromolecular nature of both

the radicals and the monomers could also play a role in the

achievement of lower conversions than those obtained using

b-diketones as the mediator and methylmethacrylate or acryl-

amide as monomers. In our case, the use of GOX was also crucial

to provide an ideal balance of oxygen concentration in the

solution; enough as substrate for optimum (or near-optimum)

laccase activity but below that which would inhibit radical

polymerization. Similar conversions were obtained in absence of

GOX/GLU only when the solution was thoroughly deaerated

under nitrogen flow (Table 1). Otherwise, hydrogels were not

obtained. It is also worth noting that GLU degradation by GOX

generates low concentrations of H2O2 as a byproduct (Scheme 1).

The role played by H2O2 in the mechanism depicted in Scheme 1

is still unclear though preliminary studies seem to suggest that its

presence has no influence on PEGDA grafting onto F68. In fact,

the use of an enzymatic oxygen-scavenging system21 based on

coupled GOX and catalase in a buffer containing millimolar

GLU provided quite similar conversions to those where there

was no catalase (see Table 1).

At this stage, we considered that the FTIR spectra of cryogels

before and after washing (e.g. PEG-g-F68c and PEG-g-F68wc,

respectively), and also of the water that was used for washing

them could provide interesting information about cryogel

composition. FTIR spectroscopy confirmed that PEG-g-F68wc

cryogels (i.e. after extraction of unreacted acrylates) were

composed of both PPO segments from F68 (bands at ca.

2950 cm�1 ascribed to CH3 symmetric and antisymmetric

stretching modes)22 and PEO segments from both F68 and

PEGDA (Fig. 5); i.e. pure PEO has strong bands at 844 and

950 cm�1, and a shoulder at 962 cm�1. FTIR spectrum of PEG-

g-F68wc cryogels also confirmed the grafting of PEGDA onto

F68 by the shift of the peak of carbonyl stretching resonance

from 1724 (conjugated with acrylic groups) to 1734 cm�1 (non-

conjugated) upon cross-liking.23 The decrease in the relative

intensity of the peak assigned to C ¼ C vibration (at 1636 cm�1)

versus that of the peak assigned to C ] O stretching (within the

1724–1734 cm�1 range) in PEG-g-F68wc cryogels revealed effi-

cient PEGDA grafting and polymerization onto F68. On the

basis of the data obtained from FTIR spectra, one can conclude

that PEG-g-F68wc cryogels were composed of both F68 and

PEG (thus, of amphiphilic nature), while non-reacted species

mostly corresponded to PEGDA monomers. FTIR spectra of

PEG-g-F68(1 : 1-7)-lac1 and PEG-g-F68(3 : 1-7)-lac1 cryogels

(either before or after washing) exhibited a similar trend

(Fig. S3, ESI†).

This journal is ª The Royal Society of Chemistry 2010

Fig. 5 FTIR spectra of PEG-g-F68(2 : 1-7)-lac1 cryogels before (PEG-

g-F68(2 : 1-7)c-lac1, middle line) and after (PEG-g-F68(2 : 1-7)wc-lac1,

top line) extraction of un-reacted monomers (by keeping in distilled water

at 25 �C for 72 h). The FTIR spectrum of the extracted fraction is also

represented (H2O[PEG-g-F68(2 : 1-7)-lac3], bottom line).

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The amphiphilic nature of cryogels obtained after extraction

of non-reacted species was also corroborated by their swelling

capability in both water and toluene. As mentioned in the

introduction, amphiphilic networks containing both hydro-

phobic and hydrophilic domains swell to a smaller extent than

the corresponding hydrophilic networks, but they can swell in

organic solvents as well.4 The swelling plots of PEG-g-

F68(1 : 1-7)wc-lac1, PEG-g-F68(2 : 1-7)wc-lac1 and PEG-g-

F68(3 : 1-7)wc-lac1 cryogels in water and toluene are depicted in

Fig. 6. These six plots exhibited a trend in which there is an initial

burst release followed by a stationary swelling phase. In water,

initial bursts are characteristic of amphiphilic gels where first

water molecules entering the hydrogel matrix would preferen-

tially hydrate hydrophilic rather than hydrophobic domains.

Subsequent hydration and swelling of hydrophobic domains

occurs at the expense of hydrophilic ones. The lower hydration

capacity of hydrophobic domains (as compared to hydrophilic

ones) results in the ultimate reduction of swelling ratio observed

at the stationary phase. In toluene, this swelling mechanism also

applies; i.e., toluene molecules swell first hydrophobic domains

whereas hydrophilic domains swell later on at the expense of the

hydrophobic ones. This behavior is almost negligible on

Fig. 6 Time evolution of relative swelling degree (expressed as grams of

solvent per gram of dry PEG-g-F68) obtained after immersion of PEG-g-

F68(1 : 1-7)wc-lac1 (squares), PEG-g-F68(2 : 1-7)wc-lac1 (circles) and

PEG-g-F68(3 : 1-7)wc-lac1 (diamonds) cryogels in an excess amount of

either deionized water (open symbols) or toluene (solid symbols) at 20 �C.

Swelling of PEG(7)wc-lac1 in both water (open triangles) and toluene

(solid triangles) was also included for comparison. The swollen samples

were weighed at various time intervals. Experiments were conducted in

triplicate.

This journal is ª The Royal Society of Chemistry 2010

non-amphiphilic hydrogels which gradually swell up to reach

equilibrium (as depicted in Fig. 6 for PEG(7)wc-lac1).

Interestingly, the equilibrium degree of swelling decreased with

the increase of the cross-linking density of hydrogels,24 that is,

with the amount of PEGDA grafted onto F68 (e.g. from PEG-g-

F68(1 : 1-7)wc-lac1 to PEG-g-F68(2 : 1-7)wc-lac1 up to PEG-g-

F68(3 : 1-7)wc-lac1 cryogels). The equilibrium degree of swelling

of PEG(7)wc-lac1 cryogels (without F68 and, hence, with the

highest ratio of acrylate units) further corroborated this trend.

Actually, the high cross-linking density of PEG(7)wc-lac1 cryogels

was also reflected in the close macroporous structure observed in

SEM micrographs depicted in Fig. 4b.

After cryogel characterization, we focused on potential

applications where one could exploit the synergy between func-

tionality (provided by both the amphiphilic nature of the

hydrogel and the immobilization of laccase) and structure

(provided by the macroporous structure resulting from

the application of the ISISA process). The biocompatibility of

the ISISA process also allows laccase immobilization within the

resulting cryogels with nearly total preservation of original

enzyme activity so that resulting cryogels are suitable for biore-

mediation purposes. Based on these features, we explored the use

of cryogels for adsorption of recalcitrant pollutants found in

wastewaters. In particular, we carried out equilibrium experi-

ments in aqueous solutions of Brilliant Blue dye Remazol

(BBdR, as model of organic pollutant). The adsorption

isotherms were fitted to Langmuir and Freundlich adsorption

models (Table 2 and Table S1, ESI†).25 In the Langmuir equa-

tion, the constant b is related to the energy of adsorption, Ceq is

the equilibrium concentration of the dye in solution, q is the

amount of adsorbed dye on the adsorbent surface and the

constant Qmax represents the maximum binding at the complete

saturation of adsorbent binding sites. In the Freundlich equa-

tion, KF and n are the Freundlich adsorption isotherm constants

characteristic of the system indicating the extent of adsorption

and degree of non-linearity between solution concentration and

adsorption, respectively. Data obtained from fitting (including

correlation coefficients, R2) are listed in Table 2 and Table S1,

ESI†. In every case, the Langmuir model provided better corre-

lation data than the Freundlich model. The dimensionless

constant separation factor or equilibrium parameter (RL, in

Table 2) was < 1, which is indicative of favorable adsorption.25

To estimate the benefits that may eventually derive from the

macroporous structure, we compared the adsorption capability

of PEG-g-F68(2 : 1-7)-lac1 in its wet (e.g. hydrogel) and freeze-

dried (e.g. cryogel) states, that is, before and after the application

of the ISISA process (samples represented in Table 2 as PEG-g-

F68(2 : 1-7)h-lac1 and PEG-g-F68(2 : 1-7)c-lac1, respectively).

The equilibrium experiments were performed by immersion of

cryogels in aqueous solutions of BBdR for 24 h to ensure full dye

adsorption. The maxima monolayer adsorption capacities (Qmax)

found for PEG-g-F68(2 : 1-7)h-lac1 and PEG-g-F68(2 : 1-7)c-lac1

were 40.6 and 74.5 mg g�1 respectively which confirms the

enhanced performance provided by the macroporous structure

of the cryogel. A similar trend was observed for hydrogels and

cryogels which had been prepared with increased amounts of

laccase and hence, with an increased concentration of immobi-

lized laccase (see PEG-g-F68(2 : 1-7)h-lac3 and PEG-g-F68(2 : 1-

7)c-lac3 in Table 2).

Soft Matter, 2010, 6, 3533–3540 | 3537

Table 2 Averaged data (experiments were conducted in triplicate) resulting from experimental plots fitting to Langmuir equation. The standarddeviation was less than 5%a

Sample Variable b/L mg�1 Qmax/mg g�1 R2 RL

PEG-g-F68(2 : 1-7)h-lac1 Hydrogels versus cryogels 0.008 40.6 0.985 0.116PEG-g-F68(2 : 1-7)c-lac1 0.014 74.5 0.997 0.070PEG-g-F68(2 : 1-7)h-lac3 0.006 328.6 0.992 0.052PEG-g-F68(2 : 1-7)c-lac3 0.007 450.2 0.994 0.039

PEG(7)c-lac1 PEG content 0.009 50.1 0.994 0.105PEG-g-F68(3 : 1-7)c-lac1 0.005 99.6 0.998 0.174PEG-g-F68(2 : 1-7)c-lac1 0.014 74.5 0.997 0.070PEG-g-F68(1 : 1-7)c-lac1 0.006 55.8 0.997 0.150

PEG-g-F68(3 : 1-7)c-lac1 Original cryogels versus washedcryogel

0.005 99.6 0.998 0.174PEG-g-F68(3 : 1-7)wc-lac1 0.020 51.8 0.990 0.050PEG-g-F68(1 : 1-7)c-lac1 0.006 55.8 0.997 0.150PEG-g-F68(1 : 1-7)wc-lac1 0.007 32.2 0.995 0.131PEG-g-F68(2 : 1-7)c-lac1 0.014 74.5 0.997 0.070PEG-g-F68(2 : 1-7)wc-lac1 0.012 40.0 0.989 0.082H2O[PEG-g-F68(2 : 1-7)-lac1]1 0.013 32.9 0.996 0.075PEG-g-F68(2 : 1-7)c-lac3 0.007 450.2 0.994 0.039PEG-g-F68(2 : 1-7)wc-lac3 0.006 235.6 0.996 0.052H2O[PEG-g-F68(2 : 1-7)-lac3]1 0.007 202.1 0.991 0.045

PEG-g-F68(2 : 1-7)wc-lac1 Aging 0.012 40.0 0.989 0.082PEG-g-F68(2 : 1-7)awc-lac1 0.011 38.1 0.997 0.085

PEG-g-F68(2 : 1-7)wc-lac0 Concentration of immobilizedenzyme

0.013 23.2 0.997 0.093PEG-g-F68(2 : 1-7)wc-lac1 0.012 40.0 0.989 0.082PEG-g-F68(2 : 1-7)wc-lac2 0.009 126.3 0.998 0.075PEG-g-F68(2 : 1-7)wc-lac3 0.006 235.6 0.996 0.052

a Superscript notation: h stands for hydrogel, c stands for cryogels, wc stands for washed cryogel and awc stands for aged washed cryogel. 1H2O[PEG-g-F68]refers to water recovered after washing a particular cryogel (eventually described into brackets).

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We also considered whether the use of amphiphilic polymers

containing hydrophobic and hydrophilic domains (coming from

PPO and PEO units, respectively) can enhance the absorption

performance. For this purpose, we studied the adsorption

capacity of graft copolymer cryogels resulting from PEGDA

grafting onto F68 (having different PEGDA/F68 ratios) and

bare PEGDA cryogels (e.g. PEG-g-F68(3 : 1-7)c-lac1, PEG-g-

F68(2 : 1-7)c-lac1, PEG-g-F68(1 : 1-7)c-lac1 and PEG(7)c-lac1,

respectively). The presence of PPO units within the cryogel

invariably improved the adsorption performance of the cryogels.

Interestingly, such increase of the absorption capacity was not

related directly to the increase of PPO units (at least, within the

studied range) in the hydrogel (the adsorption capacity decreased

from 99.6 to 74.5 up to 55.8 mg g�1 for PEG-g-F68(3 : 1-7)c-lac1,

PEG-g-F68(2 : 1-7)c-lac1 and PEG-g-F68(1 : 1-7)c-lac1, respec-

tively). This trend should mostly be ascribed to a somehow

favored arrangement of hydrophilic and hydrophobic domains

(at least with respect to the particular absorption experiments

carried out in this work) when the PEO/PPO units ratio increases

(again, within the studied range). Further work based on NMR

studies is currently in progress to assess this possibility.

The use of the ISISA process also offers the possibility to store

samples over extended periods of time. To avail of this, we

washed the cryogels for 72 h and freeze-dried them again (as we

did for conversion calculation) to extract any unreacted mono-

mers detrimental to enzyme stability. The Qmax measured for the

washed cryogel (PEG-g-F68(2 : 1-7)wc-lac1 in Table 2) and the

water used for washing (H2O[PEG-g-F68(2 : 1-7)-lac1] in

3538 | Soft Matter, 2010, 6, 3533–3540

Table 2) were respectively 40.0 and 32.9 mg g�1, which was in

agreement with the Qmax found for unwashed cryogel

(e.g. 74.5 mg g�1) and the polymer conversion depicted in Table 1

for this cryogel (58%). A close correlation between conversions

and absorption capacities of washed and unwashed cryogels was

also found for samples prepared with different PEG/F68 ratios

(e.g. PEG-g-F68(1 : 1-7)-lac1 and PEG-g-F68(3 : 1-7)-lac1) and

laccase concentration (e.g. PEG-g-F68(2 : 1-7)-lac3). This data

revealed that upon washing, enzyme units dissolved in unreacted

monomers were also removed from cryogel. Attempts to

decrease enzyme release will be the subject of our future work in

order to improve the efficiency of an eventual bioreactor based

on cryogels.6b,6c With regard to the stability issue, the Qmax

measured for washed cryogels after storage of over 5 months in

regular laboratory conditions (PEG-g-F68(2 : 1-7)awc-lac1 in

Table 2) was 38.1 mg g�1, so that less than 10% of enzyme activity

was lost over this period.

Finally, we studied the performance enhancement eventually

provided by laccase immobilization. It is worth noting that lac-

case concentration remained unmodified in every one of the

studies described above so that different trends were only related

to polymer features (hydrogels versus cryogels, presence versus

absence of PPO units, aging, etc.). In this case, we determined the

adsorption capabilities of washed cryogels having three

different enzyme concentrations (PEG-g-F68(2 : 1-7)wc-lac1,

PEG-g-F68(2 : 1-7)wc-lac2 and PEG-g-F68(2 : 1-7)wc-lac3,

Table 2). For comparison, the adsorption efficiency of bare PEG-

g-F68 cryogels (e.g. without active laccase) was also studied

This journal is ª The Royal Society of Chemistry 2010

Fig. 7 Experimental (symbols), Langmuir (solid line) and Freundlich

(dash line) isotherms for adsorption of Brilliant Blue dye Remazol on

PEG-g-F68(2 : 1-7)wc-lac0 (bottom half solid circles), PEG-g-

F68(2 : 1-7)wc-lac1 (solid circles), PEG-g-F68(2 : 1-7)wc-lac2 (open circles)

and PEG-g-F68(2 : 1-7)wc-lac3 (bottom half open circles). Lac0 stands for

0 units, lac1 stands for 20 units lac2 stands for 80 units and lac3 for 200

units. Langmuir and Freundlinch equations, and data resulting from

Langmuir equation fitting are also included.

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(e.g. PEG-g-F68(2 : 1-7)wc-lac0). Since the block copolymer

based cryogels used in this study cannot be obtained in absence

of laccase, PEG-g-F68(2 : 1-7)wc-lac0 was obtained by submis-

sion of one of the PEG-g-F68 cryogels having immobilized lac-

case (e.g. PEG-g-F68(2 : 1-7)wc-lac1) to a thermal treatment

consisting of 60 �C for 24 h for laccase inactivation.12,20 Fig. 7

shows the adsorption isotherms fitted Langmuir and Freundlich

adsorption models as well as the data resulting from fitting (also

depicted in Table 2 and Table S1, ESI†). The Qmax found for

PEG-g-F68(2 : 1-7)wc-lac0 was 23.2 mg g�1 and increased linearly

with the concentration of immobilized laccase (from 40.0 mg g�1

for PEG-g-F68(2 : 1-7)wc-lac1, to 126.3 mg g�1 for PEG-g-

F68(2 : 1-7)wc-lac2, up to 235.6 mg g�1 for PEG-g-F68(2 : 1-7)wc-

lac3, see Fig. 7). Such enhancement of the adsorption capacity

confirms the favorable synergetic effect between the amphiphilic

polymer and the immobilized enzyme for adsorption purposes.

As mentioned above, synergy must result from the ability of

laccase to degrade phenolic compounds.26 Thus, BBdR adsorbed

on the hydrated block-copolymer based cryogel would, after

adsorption, be degraded by immobilized laccase. Dye degrada-

tion would result in partial renewal of adsorption domains at

block-copolymer based hydrogel allowing for further BBdR

adsorption. It is worth noting that byproducts of enzymatic

BBdR degradation remained adsorbed (they were not detected in

solution by UV-Vis spectroscopy, see Fig. S4, ESI†). Considering

that the Langmuir isotherm assumes a monolayer coverage and

uniform activity distribution on the adsorbent surface, the

enhancement of the adsorption capacity must be explained in

terms of availability of adsorption domains for BBdR byprod-

ucts (due their size, charge and/or hydrophilic-hydrophobic

nature), which were not initially available for original BBdR.27

Conclusions

Summarizing, PEG-g-F68 hydrogels have been obtained via (1)

laccase-generation of long-lived macroradicals in PEO-PPO

segments of F68, and (2) macroradical induced grafting of

PEG550DA onto F68. The use of GOX/GLU provided the

proper balance of oxygen in the reaction medium (i.e., on one

This journal is ª The Royal Society of Chemistry 2010

hand, laccase needs oxygen as a substrate while, on the other,

oxygen is a strong inhibitor of radical polymerizations) to obtain

reasonable polymerization conversions. Eventually, laccase was

immobilized within the resulting PEG-g-F68 hydrogel with full

preservation of enzyme activity (90% of activity was still

preserved three months after preparation). Laccase-immobilized

PEG-g-F68 hydrogels were submitted to the ISISA process for

preparation of laccase-immobilized PEG-g-F68 cryogels, which

exhibited outstanding adsorption capabilities (up to 235 mg g�1).

This eminent suitability of cryogels for adsorption of recalcitrant

pollutants found in wastewaters resided in the synergetic effect of

(1) the macroporous structure that allow easy mass transport to

adsorption sites, (2) the amphiphilic nature of the polymer that

provides hydrophilic and hydrophobic domains for pollutants

adsorption, and (3) the immobilized enzyme that degrades

adsorbed pollutant thus allowing further adsorption on renewed

sites.

Acknowledgements

This work was supported by MICINN (MAT2009-10214,

MAT2009-09671 and PET2008-0168-01) and CSIC

(200660F011). M. N. and S. N. thank CSIC for a research

contract and a PhD fellowship, respectively. C. A. thanks

MICINN for a Ramon y Cajal contract. We thank F. Pinto for

assistance with SEM.

References

1 (a) D. J. Lloyd, Colloid Chemistry. ed. J. Alexander, The ChemicalCatalogue Co., New York, 1926, vol. 1, p. 767; (b) P. Flory,Faraday Discuss. Chem. Soc., 1974, 57, 7; (c) L. A. Estroff andA. D. Hamilton, Chem. Rev., 2004, 104, 1201–1217.

2 (a) K. Y. Lee and D. J. Mooney, Chem. Rev., 2001, 101, 1869–1879;(b) A. S. Hoffman, Adv. Drug Delivery Rev., 2002, 54, 3–12.

3 (a) Y. S. Ho and G. McKay, Process Biochem., 1999, 34, 451–465;(b) B. Adhikari, G. Palui and A. Banerjee, Soft Matter, 2009, 5,3452–3460; (c) Y. Chujo, K. Sada and T. Saegusa, Macromolecules,1993, 26, 6315–6319; (d) H. Kasxg€oz, S. €Ozg€um€u�s and M. Orbay,Polymer, 2003, 44, 1785–1793.

4 C. S. Patrickios and T. K. Georgiou, Curr. Opin. Colloid InterfaceSci., 2003, 8, 76–85.

5 V. Bekiari and P. Lianos, Chem. Mater., 2006, 18, 4142–4146.6 (a) G. Palmieri, P. Giardina and G. Sannia, Biotechnol. Prog., 2005,

21, 1436–1441; (b) G. Palmieri, P. Giardina and G. Sannia, EnzymeMicrob. Technol., 2005, 36, 17–24; (c) V. Arantes andA. M. F. Milagres, Enzyme Microb. Technol., 2007, 42, 17–22.

7 Q. Wang, Z. Yang, Y. Gao, W. Ge, L. Wanga and B. Xu, Soft Matter,2008, 4, 550–553.

8 A. Barbetta, M. Massimi, L. C. Devirgiliis and M. Dentini,Biomacromolecules, 2006, 7, 3059–3068.

9 (a) L. Mart�ın, M. Alonso, M. M€oller, J. C. Rodr�ıguez-Cabello andP. Mela, Soft Matter, 2009, 5, 1591–1593; (b) R. E. Sallach, W. Cui,J. Wen, A. Martinez, V. P. Conticello and E. L. Chaikof,Biomaterials, 2009, 30, 409–422.

10 (a) J. S. Dordick, Trends Biotechnol., 1992, 10, 287–29; (b) A. Singhand D. L. Kaplan, Adv. Polym. Sci., 2006, 194, 211–224;(c) S. Kobayashi and A. Makino, Chem. Rev., 2009, 109, 5288–5353.

11 (a) D. Teixeira, T. Lalot, M. Brigodiot and E. Mar�echal,Macromolecules, 1999, 32, 70–72; (b) R. Ikeda, H. Tanaka,H. Uyama and S. Kobayashi, Macromol. Rapid Commun., 1998, 19,423–425; (c) T. Tsujimoto, H. Uyama and S. Kobayashi, Macromol.Biosci., 2001, 1, 228–232; (d) B. Kalra and R. A. Gross, GreenChem., 2002, 4, 174–178.

12 F. Hollmann, Y. Gumulya, C. T€olle, A. Liese and O. Thum,Macromolecules, 2008, 41, 8520–8524.

13 (a) J. Yamauchi, A. Yamaoka, K. Ikemoto and T. Matsui, J. Appl.Polym. Sci., 1991, 43, 1197–1203; (b) L. Bromberg, J. Phys. Chem. B,

Soft Matter, 2010, 6, 3533–3540 | 3539

Dow

nloa

ded

by C

entr

o de

Quí

mic

a O

rgán

ica

"Lor

a T

amay

o" o

n 25

Nov

embe

r 20

10Pu

blis

hed

on 2

0 Ju

ly 2

010

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/C0S

M00

079E

View Online

1998, 102, 10736–10744; (c) L. Bromberg, J. Phys. Chem. B, 1998, 102,1956–1963; (d) L. Bromberg, Langmuir, 1998, 14, 5806–5812;(e) Y. Zhang and Y. M. Lam, J. Colloid Interface Sci., 2007, 306,398–404; (f) S.-P. Zhao, L.-M. Zhang, D. Ma, C. Yang and L. Yan,J. Phys. Chem. B, 2006, 110, 16503–16507.

14 (a) S. R. Mukai, H. Nishihara and H. Tamon, Chem. Commun., 2004,874; (b) H. Nishihara, S. R. Mukai and H. Tamon, Carbon, 2004, 42,899; (c) Zhang, I. Hussain, M. Brust, M. F. Butler, S. P. Rannard andA. I. Cooper, Nat. Mater., 2005, 4, 787; (d) S. Deville, E. Saiz,R. K. Nalla and A. P. Tomsia, Science, 2006, 311, 515; (e) S. Bandiand D. A. Schiraldi, Macromolecules, 2006, 39, 6537–654;(f) M. C. Gutierrez, M. J. Hortig€uela, R. Jimenez, J. M. Amarilla,M. L. Ferrer and F. del Monte, J. Phys. Chem. C, 2007, 111, 5557;(g) A. Abarrategui, M. C. Gutierrez, M. J. Hortig€uela, V. Ramos,J. L. Lopez-Lacomba, M. L. Ferrer and F. del Monte, Biomaterials,2008, 29, 94; (h) Q. Shi, H. Liang, D. Feng, J. Wang andG. D. Stucky, J. Am. Chem. Soc., 2008, 130, 5034; (i) F. Khan,D. Walsh, A. J. Patil, A. W. Perriman and S. Mann, Soft Matter,2009, 5, 3081–3085.

15 M. B. Dainiak, A. Kumar, I. Yu. Galaev and B. Mattiasson, Proc.Natl. Acad. Sci. U. S. A., 2006, 103, 849–854.

16 (a) M. C. Gutierrez, Z. Y. Garcia-Carvajal, M. Jobbagy, L. Yuste,F. Rojo, M. L. Ferrer and F. del Monte, Adv. Funct. Mater., 2007,17, 3505–3513; (b) M. C. Gutierrez, M. L. Ferrer and F. del Monte,Chem. Mater., 2008, 20, 634–648.

17 M. C. Gutierrez, M. Jobbagy, N. Rapun, M. L. Ferrer and F. delMonte, Adv. Mater., 2006, 18, 1137.

18 (a) M. L. Ferrer, R. Esquembre, I. Ortega, C. R. Mateo and F. delMonte, Chem. Mater., 2006, 18, 554; (b) M. C. Gutierrez,

3540 | Soft Matter, 2010, 6, 3533–3540

Z. Y. Garcia-Carvajal, M. Jobbagy, F. Catalina, C. Abrusci,L. Yuste, F. Rojo, M. L. Ferrer and F. del Monte, Chem. Mater.,2007, 19, 1968; (c) M. C. Gutierrez, Z. Y. Garcia-Carvajal,M. J. Hortig€uela, L. Yuste, F. Rojo, M. L. Ferrer and F. delMonte, J. Mater. Chem., 2007, 17, 2992.

19 X. Wu, Y. Liu, X. Li, P. Wen, Y. Zhang, Y. Long, X. Wang, Y. Guo,F. Xing and J. Gao, Acta Biomater., 2010, 6, 1167–1177.

20 A. Rek�uc, J. Bryjak, K. Szymanska and A. B. Jarzebski, ProcessBiochem., 2009, 44, 191–198.

21 S. C. Blanchard, H. D. Kim, R. L. GonzalezJr., J. D. Puglisi andS. Chu, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 12893–12898.

22 (a) C. Guo, H. Liu, J. Wang and J. Chen, J. Colloid Interface Sci.,1999, 209, 368–373; (b) C.-H. Choi, J.-H. Jung, T.-S. Hwang andC.-S. Leezz, Macromol. Res., 2009, 17, 163–167.

23 (a) Y. Wu, F.-B. Che and J.-H. Chen, J. Appl. Polym. Sci., 2008, 110,1118–1128; (b) D. Biswal and J. Z. Hilt, Polymer, 2006, 47, 7355–7360.

24 (a) D. S. Achilleos, T. K. Georgiou and C. S. Patrickios,Biomacromolecules, 2006, 7, 3396–3405; (b) F. M. Goycoolea,A. Heras, I. Aranaz, G. Galed, M. E. Fern�andez-Valle andW. Arg€uelles-Monal, Macromol. Biosci., 2003, 3, 612–619.

25 See, for instance: K. Vijayaraghavan, S. W. Won, J. Mao andY.-S. Yun, Chem. Eng. J., 2008, 145, 1–6.

26 P.-P. Champagne and J. A. Ramsay, Appl. Microbiol. Biotechnol.,2007, 77, 819–823.

27 (a) L. Pereira, A. V. Coelho, C. A. Viegas, C. Ganachaud, G. Iacazio,T. Tron, M. P. Robalo and L. O. Martinsa, Adv. Synth. Catal., 2009,351, 1857–1865; (b) Y. Sugano, Y. Matsushima, K. Tsuchiya,H. Aoki, M. Hirai and M. Shoda, Biodegradation, 2009, 20,433–44.

This journal is ª The Royal Society of Chemistry 2010