Inverse Opals of Molecularly Imprinted Hydrogels for the Detection of Bisphenol A
C C H 2C
OH H 3C C H 3
O
O H
O H
C H 3 O
OH 3CCCC H 2
H C C H 2C
OH H 3C C H 3
O
O H
O H
C H 3 O
OH 3CCCC H 2
H C
C CH2
OH HO
CH3 O
OH3CCCCH2
C C CH2
OH HO
CH3 O
OH3CCCCH2
C C CH2
OH HO
CH3 O
OH3CCCCH2
Silica spheres Silica extraction
Monomers
UV Removal of BPA
opal Inverse opal hydrogel
after removal of BPA Inverse opal hydrogel
with adsorbed BPA opal hydrogel
C C H 2C
OH H 3C C H 3
O
O H
O H
C H 3 O
OH 3CCCC H 2
H C C H 2C
OH H 3C C H 3
O
O H
O H
C H 3 O
OH 3CCCC H 2
H C
C CH2
OH HO
CH3 O
OH3CCCCH2
C C CH2
OH HO
CH3 O
OH3CCCCH2
C C CH2
OH HO
CH3 O
OH3CCCCH2
Silica spheres Silica extraction
Monomers
UV Removal of BPA
opal Inverse opal hydrogel
after removal of BPA Inverse opal hydrogel
with adsorbed BPA opal hydrogel
70
71
Inverse Opals of Molecularly Imprinted Hydrogels for the Detection of Bisphenol A
Nébéwia Griffete, Hugo Frederich, Agnès Maitre, Serge Ravaine, Mohamed. M. Chehimi and Claire Mangeney
Langmuir, soumis
Objectifs
Etude des caractéristiques structurales des hydrogels (MIPs et NIPs).
Elaboration des opales inverses de polymères à empreintes moléculaires à partir de cristaux collọdaux utilisés comme gabarit et étude de leurs propriétés optiques.
Influence de l’épaisseur des hydrogels sur leurs propriétés de gonflement.
Méthodes
Synthèse de disques d'hydrogels "massifs" (MIPs et NIPs) de MAA-co-EGDMA et étude de leurs propriétés de gonflement en réponse à des variations de pH ou à la présence de BPA.
Elaboration des opales inverses de MIPs et de NIPs par infiltration des hydrogels dans un cristal collọdal utilisé comme gabarit.
Etudes des propriétés optiques des matériaux photoniques macroporeux par réflexion spéculaire à différents angles.
Influence du pH et de la concentration de BPA sur les propriétés optiques des opales inverses et étude de la détection sélective du BPA.
Résultats
Dans un premier temps, les propriétés de gonflement à l'équilibre des hydrogels MIPs et NIPs ont été analysées pour en déduire des paramètres structuraux tels que la taille des nanopores et le taux de réticulation des gels. Cette étude nous a permis de mettre en évidence une augmentation de la taille des nanopores dans les MIPs après extraction du BPA, en accord avec la présence des nanocavités laissées au sein du réseau de polymère par les empreintes de
72 la molécule cible. Les propriétés de gonflement ont également été étudiées afin de mettre en évidence le caractère stimulable des hydrogels et leur capacité à se déformer (par gonflement/dégonflement) en réponse à des stimuli extérieurs tels que le pH ou la présence de BPA.
Puis, les opales inverses des MIPs et des NIPs ont été élaborées pour suivre l'évolution de leurs propriétés optiques en réponse aux mêmes stimuli extérieurs (pH, BPA). Nous avons pu mettre en évidence, par cette étude, l'influence importante de l'épaisseur des films d'hydrogels sur l'amplitude de leur réponse et montrer que des films relativement épais conduisent à une meilleure sensibilité ainsi qu'à des limites de détection plus basses.
Inverse Opals of Molecularly Imprinted Hydrogels for the Detection of Bisphenol A
Nộbộwia Griffete1, Hugo Frederich2, Agnốs Maợtre2, Serge Ravaine3, Mohamed M. Chehimi1 and Claire Mangeney1*
1 ITODYS, Université Paris Diderot (UMR 7086), 15 rue Jean de Bạf, 75013 Paris, France.
E-mail: mangeney@univ-paris-diderot.fr Phone: 33-01-57276878. Fax: 33-01-5727726
2 INSP, UPMC (UMR 7588), 140 rue de Lourmel, 75015 Paris, France.
3 CRPP, Université Bordeaux 1 (UPR 8641), 115 Avenue Schweitzer, 33600 Pessac, France.
[*] Claire Mangeney Corresponding-Author mangeney@univ-paris-diderot.fr
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)
ABSTRACT.
Inverse opal films of molecularly imprinted polymers (MIP) were elaborated using the colloidal crystal template method. The colloidal crystals of silica particles were built by the Langmuir-Blodgett technique allowing a perfect control of the film thickness. Polymerization in the interspaces of the colloidal crystal and removal of the used template provides 3D- ordered macroporous methacrylic acid-based hydrogel films in which nanocavities derived
from the bisphenol A (BPA) are distributed within the thin walls of the inverse opal hydrogel.
The equilibrium swelling properties of the non imprinted (NIPs) and molecularly imprinted polymers (MIPs) were studied as a function of pH and bisphenol A concentration while the molecular structures of the bulk hydrogels were analyzed using a cross-linked network structure theory. This study showed an increase in nanopore (mesh) size in the MIPs after BPA extraction as compared to NIPs, in agreement with the presence of nanocavities left by the molecular imprints of the template molecule. The resulting inverse opals were found to display large responses to external stimuli (pH or BPA) with Bragg diffraction peak shifts depending upon the hydrogel film thickness. The film thickness was therefore shown to be a critical parameter for improving the sensing capacities of inverse opal hydrogel films deposited on a substrate.
INTRODUCTION
The increased global concern about man-made chemicals such as endocrine disruptors (EDCs) stimulates the development of selective and sensitive analytical methods to detect trace amount in environmental, biological and foodstuff samples [1]. Most of the methods to date are based on high performance liquid chromatography (HPLC) [2], gas chromatography (GC) [3] and capillary electrophoresis (CE) [4]. The use of MIPs as selective sorbent materials allows for a customized sample treatment step prior to the final determination. This is of special interest when the sample is complex and the presence of interferences could prevent final quantification via typical chromatographic techniques coupled to common detectors [5]. Nevertheless, these current approaches involve not only expensive instruments, but also a large number of separate analytical procedures, resulting in a complex, time consuming and laborious screening procedure. Thereby, it is highly desirable and useful to develop novel approaches for easy and rapid drug detection without costly instruments and prolix processes, but exhibiting high sensitivity and specificity.
Recently, the group of G. Li proposed an original approach combining molecular imprinting and colloidal crystal to prepare imprinted photonic polymers with 3D, highly-ordered, macroporous structures and specific binding nanocavities for a rapid and self-reporting assay with high sensitivity and specificity [6-8]. Owing to their periodic porous structure, such materials (inverse opals) exhibit fascinating optical properties (Bragg diffraction) and bright structural colors. Particularly, if these highly ordered macroporous materials are made from responsive polymer hydrogels, they are able to swell or shrink in aqueous solution upon molecular recognition or environmental conditions to change the periodic spacing, leading to a change in optical properties.
Combining this approach with the Langmuir-Blodgett (L-B) technique, our group recently elaborated inverse opal hydrogel (IOH) films deposited on a transparent PMMA plate, with thicknesses of 2,8 àm and containing a planar defect layer [9]. The defect layer was shown to enhance the sensing capacities of the optical sensor to detect bisphenol A (BPA), an endocrine disruptor (EDC) [10]. However, many factors can affect the swelling behavior of thin hydrogel films deposited on a substrate and thereby their sensing properties. Among these factors, the adhesion of the film is a critical parameter which hampers the swelling along the plane of the substrate and favors vertical swelling in the vicinity of the surface, as already described by Braun et al. [11]. One could expect that this constraint on swelling should become weaker for thick hydrogel films, far from the substrate. Therefore, the film thickness should be another important factor influencing the swelling properties of the hydrogels. Nevertheless, this parameter has never been investigated so far, probably due to the difficulty to prepare inverse opal hydrogel films with controlled thicknesses. In the present paper, we address this issue by taking advantage of the L-B technique to perfectly control the number of silica particle layers constituting the colloidal crystal template [12-14] and therefore the thickness of the resulting IOH (see Fig. 1). The hydrogel was made of a molecularly imprinted poly(methacrylic acid) reticulated using ethyleneglycol dimethacrylate (EGDMA) as a comonomer and BPA as the template molecule. We first examined the swelling properties of the bulk hydrogels of non imprinted and molecularly imprinted polymers especially in response to pH or various BPA concentration changes. Using a cross-linked network structure theory [15-18], we could obtain insights on the molecular structures of the bulk
hydrogels and on their nanopore (mesh) sizes. Then, the responses of the corresponding inverse opal hydrogels to external stimuli (pH and BPA) were investigated as a function of film thickness, evidencing the importance of this parameter for improving their sensing capacities.
Scheme 1: Schematic illustration of the procedure used for the preparation of a molecularly imprinted inverse opal hydrogel (IOH) film.
EXPERIMENTAL SECTION Materials:
Methacrylic Acid (MAA), ethylene glycol dimethacrylate (EGDMA) and BPA were purchased from Aldrich. 2,2’-azobis(isobutyronitrile), (AIBN) was obtained from Fluka.
Tetraethoxysilane (TEOS, Fluka), ammonia (29% in water, J. T. Baker), styrene (Aldrich), azodiisobutyramidine dihydrochloride (Aldrich), and aminopropyltriethoxysilane (Aldrich) were used without purification. Ethanol and chloroform were purchased from Prolabo.
Cyclohexane (J. T. Baker, France, 99%) and dibutyltin dilaurate (DBTDL, Sigma-Aldrich, 98%) were used as received. Tolylene-2,4-diisocyanate (TDI,à Sigma-Aldrich) was purified by distillation under vacuum. Ethylene glycol (EG, Sigma-Aldrich, 99%) and monohydroxyterminated polydimethylsiloxane (PDMS-OH), 4670 g ã mol-1, (Sigma-Aldrich,
C C C H2
OH H3C C H3
O
O H
O H
C H3 O
OH3CCCC H2
H C C C H2
OH H3C C H3
O
O H
O H
C H3 O
OH3CCCC H2
H C
C CH2
OH O H
CH3 O
OH3CCCCH2
C C CH2
OH O H
CH3 O
OH3CCCCH2
C C CH2
OH O H
CH3 O
OH3CCCCH2
Silica spheres Monomers Silica extraction
UV Removal of BPA
opal Inverse opal hydrogel
after removal of BPA Inverse opal hydrogel
with adsorbed BPA opal hydrogel
C C C H2
OH H3C C H3
O
O H
O H
C H3 O
OH3CCCC H2
H C C C H2
OH H3C C H3
O
O H
O H
C H3 O
OH3CCCC H2
H C
C CH2
OH O H
CH3 O
OH3CCCCH2
C C CH2
OH O H
CH3 O
OH3CCCCH2
C C CH2
OH O H
CH3 O
OH3CCCCH2
Silica spheres Monomers Silica extraction
UV Removal of BPA
opal Inverse opal hydrogel
after removal of BPA Inverse opal hydrogel
with adsorbed BPA opal hydrogel
99%) were used as received. PMMA slides with 40*30*3 mm3 for photonic film supports were cleaned with anhydrous ethanol.
Preparation of non imprinted and molecularly imprinted hydrogels
The typical preparation of hydrogel of MAA-co-EGDMA was as follows: 10 g of MAA, 0,05 g of EGDMA, 10 ml of ethanol were mixed thoroughly. To this solution was added 0.1 g AIBN. After mixing completely, the obtained clear solution was deoxygenated and kept for at least 30 minutes. The hydrogel precursor solution was irradiated in a photochemical reactor operating with 120 volts. After polymerisation, the hydrogels were cut into cylindrical spheres with a diameter of 20 mm and a thickness of 10 mm. Hydrogels were kept in distilled water for at least 24 hours, replacing the water daily to remove any impurities, such as monomer, initiator, etc. For the MIP preparation, 0,1 g of BPA was added. BPA was removed from the polymer thanks to a 2.0 M acetic acid solution during 1h.
Preparation of hydrogel inverse opals
The method employed for the synthesis of silica particles and corresponding colloidal crystal was similar to the one described in a previous work [12-14]. Prior to fabrication of a photonic imprinted film, homogeneous monomer mixture of MAA (0.4g), EGDMA (0.02g), AIBN (0.04g) and BPA (0.04g) in ethanol (0.4g) was degassed under nitrogen for 3 min and dropped on a silica colloidal crystal.
Glass slides with a colloidal-crystal film were coated with a PMMA slide (40*30*3mm3) and held together to retain the above-mentioned precursor mixture. Once the colloidal crystal of the formed sandwich structure became transparent, a successful infiltration process was completed. After the removal of excess precursors, photopolymerization was performed under
UV light at 365 nm for 2 h. The sandwich structure was immersed in 4% hydrofluoric acid solution for 3 h to separate double slides and completely etch the silica colloids.
The polymer film on the PMMA substrate was soaked in a 2 M acetic acid solution for 2h and rinsed with deionised water in order to remove the BPA. The photonic non-imprinted polymer was prepared using the same procedure but in the absence of BPA.
Recognitive Studies.
Batch adsorption experiments were carried out by allowing a weighed amount of polymer to reach equilibrium with BPA solution of known concentration. In typical experiments, MIPs and NIPs (3-5 mg) were placed in 20 mL of 0.1 mol/L bisphenol A solution in distilled water.
After 30 hours incubation, the residual concentration of BPA in the aqueous solution was determined by UV-vis spectroscopy.
Instrumentation
SEM micrographs were obtained with a Cambridge 120 apparatus fitted with a zirconated tungsten filament. The acceleration voltage was set at 20 kV. All specimens were coated with gold prior to analysis in order to minimize static charging effects. A Cary Win 500 spectrophotometer in specular geometry was used for studying the optical properties of the inverse opal hydrogels. A bare PMMA slide, without any colloidal assemblies, was used as a reference.
Determination of equilibrium swelling ratio
Swelling experiments were performed on non imprinted and imprinted hydrogels in phosphate buffer at three different pH (2, 4 and 6) at 25°C.
The % swelling ratio (% SR) and the % equilibrium swelling (% ES) were calculated as:
Where mt and md are weights of swollen hydrogels for a given time and dried hydrogels, respectively. Here, ms is the weight of the swollen hydrogel at equilibrium.
Initial weight of the hydrogel, after drying, was taken over a single-pan digital microbalance (sensitive to ±0.01 mg). The polymer volume fraction in the gel immediately after preparation (relaxed state), 2r, and the polymer volume fraction of the swollen gels (swollen state), 2s, were determined using eqs 3 and 4:
where mr is the weight of the hydrogel after preparation, 1 and 2 are densities of the solvent and polymer network, respectively.
THEORETICAL BASIS
The pH-responsive swelling behavior of the hydrogels was analyzed within the framework of the Flory-Rehner theory of swelling [15]. According to the Flory-Rehner theory, the osmotic
%SR = ì 100mt_ md md
%SR = ì 100mt_ md
md (1)
(2)
2r = 1 + mr md
_ 1 2
1
_1
2r = 1 + mr md
_ 1 2
1
_1
2s = 1 + ms md
_ 1 2
1
_1
2s = 1 + ms md
_ 1 2
1
_1
(3)
(4)
%ES = ì 100ms_md md
%ES = ì 100ms_md md
pressure π of a hydrogel during swelling is given as the sum of the pressures due to polymer–
solvent mixing (mix), due to deformation of network chains to a more elongated state (el), and due to the nonuniform distribution of mobile counterions between the hydrogel and the external solution (ion):
The mixing term is satisfactorily represented by Flory-Huggins type expression of the form [19]:
where R is the gas constant and T is the temperature. The most important feature of crosslinked polymers that differs from the uncrosslinked polymer solutions of the same chemical nature may be a memory effect of the initial condition. Once a gel is formed, its structure is more or less fixed depending on the initial condition, resulting in an emergence of the frozen structure. Thus, one should also take into account the parameters at the initial condition for the study of gels [20]. To describe the elastic contribution πel to the swelling pressure, we use here the phantom network model [21]:
where is the number branches originating from a crosslinking site.
The ionic contribution πion to the swelling pressure is caused by the concentration difference of counterions between the hydrogel and the outer solution. The ideal Donnan theory gives πion as the pressure difference of mobile ions inside and outside the hydrogel:
ion = RT i2c2 4I
ion = RT i2c22
4I
2
(5)
(6)
(7)
(8)
mix= RT
_
V1 [ln (1 _ 2s) + 2s+2s2]
mix= RT
_
V1 [ln (1 [ln (1 __ 2s2s) +) + 2s2s++2s2s22]]
2/3 1/3
Mc
el= _ RT
(1 _2/)22/3 1/32r 2s Mc
el= _ RT
(1 _2/)22r 2s
= mix+ el + ion
In the above equation, i is the degree of ionization, I is the ionic strength of the swelling medium, and c2 is the concentration of the ionizable polymer (mol.cm-3). c2 can be written in terms of polymer structural parameters for copolymeric hydrogels as:
where fi is the mol fraction of the ionic unit in the gel system and is the average molar volume of polymer repeat units. Combining eqs 8 and 9 we obtain:
For the hydrogels with monoprotic acid moieties, there is only one equilibrium:
where [HA] is the concentration of undissociated polymer chains, [A-] is the concentration of dissociated polymer chains, and [H+] is the concentration of hydrogen ions. i is defined as follows:
With eq 12, eq 13 may be rewritten as follows:
Ka= [H+] [A-] Ka= [H[HA]+] [A-] [HA]
i = = [A-] [AH] + [A-]
[A-] [AH]
1 + [A-] [AH]
i = = [A-] [AH] + [A-]
[A-] [AH]
1 + [A-] [AH]
i = = = Ka/[H+] 1 + Ka/[H+]
Ka [H+] + Ka
Ka 10_ pH+ Ka i = = = Ka/[H+]
1 + Ka/[H+]
Ka [H+] + Ka
Ka 10_ pH+ Ka
(9)
(10)
(11)
(12)
(13) c2 = fi 2s
Vr c2 = fi 2s
Vr
ion = RT I22s fi2 4IVr
2
ion = RT I22s f2i2
4IVr
2 2
Vr Vr
If we substitute in eq 13, the following equation can be obtained for πion:
The complete equilibrium expression, which accounts for the mixing, elastic-retractive, and ionic contributions to the osmotic pressure of monoprotic polymeric networks, is given below:
RESULTS AND DISCUSSION
Swelling Studies.
Swelling in deionized water
Characterization of the cross-linked structures of the MIPs and NIPs samples was achieved by equilibrium swelling studies in distilled water. The swelling experiments for MIPs were performed after extraction of BPA. The swelling at equilibrium was observed to be significantly higher for the BPA-free MIP gels with SR = 378 % compared to the 256 % measured for the NIPs (see Figure 1).
(14)
(15)
ion= _ Ka 10_pH + Ka
RTV12s fi2 4IVr2
2 2
ion= _ Ka 10_pH + Ka
RTV12s fi2 4IVr2
2 2
(1_ 2/)V12r 2s RT Ka _2s ln(1 _ 2s) _2s_1 =
10_pH+ Ka
V1fi2 4IVr2
2 2
2/3 -5/3
Mc
_2 (1_ 2/)V12r 2s
RT Ka _2s ln(1 _ 2s) _2s_1 =
10_pH+ Ka
V1fi2 4IVr2
2 2
2/3 -5/3
Mc
_2
2 cm
2 cm
0 1000 2000 3000 4000 5000 6000
0 200 400 600 800
% SR
Time (min)
0 1000 2000 3000 4000 5000 6000
0 200 400 600 800
% SR
Time (min)
2 cm
2 cm
0 1000 2000 3000 4000 5000 6000
0 200 400 600 800
% SR
Time (min)
0 1000 2000 3000 4000 5000 6000
0 200 400 600 800
% SR
Time (min)
Figure 1: % Swelling ratio of a) non imprinted hydrogel and b) imprinted hydrogel in distilled water (FULL line) and in buffer solutions at pH 2.0 (dashed line) and 6.0 (dotted line), at fixed I = 0.10 M. Insets shows photographs of the BPA-free imprinted hydrogels after exposure to pH 2 and pH 6 phosphate buffers.
This indicates that the presence of BPA nanocavities in the polymer network after extraction of the template molecules leads to the formation of a more nanoporous structure. In order to obtain a better insight on the structural properties of the hydrogels, the equilibrium swelling data were used to evaluate their cross-linked structure. Typically, the number average molecular weight between cross-links, was calculated [22]. This parameter is an indication of the cross-linked nature of the hydrogel, as high values of imply loosely cross-linked hydrogel. The results of this study are presented in Table 1 [23]. They indicate that the value of the non-imprinted polymer is significantly lower (780 g.mol-1 corresponding to ~ 9 methacrylic acid units) than that of the BPA-free imprinted polymer (2100 g.mol-1 corresponding to ~ 24 methacrylic acid units), resulting in a lower crosslink density (e = 2/Mc ) [24] for the later polymer.
Hydrogels 2s (g.mol-1)b Na ξ (Å) e 104
NIPs 0.21 780 9 42 16.6
BPA-free MIPs 0.16 2100 24 75 6.3
Table 1: Structural parameters of the MIP and NIP hydrogels. aN corresponds to the number of methacrylic acid units between crosslinks.
These differences in crosslink densities between the MIPs and NIPs are probably due to the inclusion of the template molecule during polymerization, which creates additional free space or vacuoles of which the polymeric network must form around. The higher penetrant uptake of recognitive polymers compared with control polymers can therefore be attributed to more porous networks.
Mc Mc Mc
Mc
Mc Mc
Mc Mc
Mesh Size and Micro- and Nanoporous Structural Analysis. The free spaces inside the hydrogel networks are often regarded as the “pores”. Depending upon the size of these pores, hydrogels can be macroporous, microporous, or nonporous (also often named nanoporous). A structural parameter that can be used to describe the size of the recognitive pores is the correlation length, ξ, which is defined as the linear distance between two adjacent crosslinks and can be calculated using the following equation:
Here, is the elongation ratio of the polymer chains in any direction and (ro2)1/2 is the root- mean-square, unperturbed, end-to-end distance of the polymer chains between two neighboring cross-links.
Scheme 2: Schematic representation of the mesh size in a molecularly imprinted hydrogel.
For isotropically swollen hydrogel, the elongation ratio, , can be related to the swollen polymer volume fraction, 2s, using eq 17.
( ro2)1/2
( ro2)1/2 (16)
(17)
= 2s _ 1/3
Mc
Mc Mc
The unperturbed end-to-end distance of the polymer chain between two adjacent cross-links can be calculated using eq 18, where Cn is the Flory characteristic ratio, l is the length of the bond along the polymer backbone (for methacrylates polymers, 1.54 Å), and N is the number of links per chain that can be calculated by eq 19.
In eq 19, Mr is the molecular weight of the repeating units from which the polymer chain is composed. Finally, when one combines eqs 16-19, the correlation distance between two adjacent cross-links in a swollen hydrogel can be obtained:
The network mesh size, ξ was calculated using eq 20. In this expression, the Flory characteristic ratio Cn, was CnPMAA = 14.6 [25]. The results of these calculations are presented in Table 1 and are quite revealing of the modifications of porosity induced by the imprinting process, with ξ values noticeably higher for BPA-free MIPs as compared to NIPs.
Swelling in buffer solutions at various pHs
The pH-responsive properties of the hydrogels were studied by dynamic swelling experiments in PBS solutions with pH values varying from 2 to 6 (at a fixed ionic strength of 0.1M).
Figure 1 shows that the equilibrium swelling percents of both MIPs and NIPs become higher
( ro2 )1/2 = l (CnN)1/2
N= 2Mc Mr N= 2Mc
Mr
(18)
(19)
(20)
= 2s_1/3 2CnMc l Mr
1/2
= 2s_1/3 2CnMc l Mr
1/2