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Evaluation of theophylline imprinted polypyrrole film
Synthetic Metals 209 (2015) 206–211
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Evaluation of theophylline imprinted polypyrrole film Ieva Baleviciutea , Vilma Ratautaitea , Almira Ramanavicienec , Zigmas Baleviciusb , Jeroen Broedersd , Dieter Crouxd , Matthew McDonaldd, Farnoosh Vahidpourd, Ronald Thoelend,e , Ward De Ceuninckd,f , Ken Haenend , Milos Nesladeke, Alfonsas Rezag, Arunas Ramanaviciusa,* a
Department of Physical Chemistry, Vilnius University, Faculty of Chemistry, Naugarduko str. 24, Vilnius, Lithuania Laboratory of Bio-NanoTechnology, State Research Institute—Center for Physical Science and Technology, Gostauto str. 11, Vilnius, Lithuania c NanoTechnas—Centre of Nanotechnology and Materials Science, Vilnius University, Faculty of Chemistry, Naugarduko str. 24, Vilnius, Lithuania d Institute for Materials Research (IMO), Hasselt University, Wetenschapspark 1, B-3590 Diepenbeek, Belgium e Research Centre—IMOMEC, Research Institute—IMEC vzw, Wetenschapspark 1, B-3590 Diepenbeek, Belgium f Xios University College, Agoralaan—gebouw D, 3590 Diepenbeek, Belgium g Department of Optoelectronics, State Research Institute—Center for Physical Science and Technology, Gostauto str. 11, Vilnius, Lithuania b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 May 2015 Received in revised form 5 June 2015 Accepted 13 July 2015 Available online xxx
In this study some affinity and dielectric properties of molecularly imprinted (MIP) conducting polymer— polypyrrole (Ppy) based thin films were evaluated. Films of polypyrrole molecularly imprinted with theophylline (MIP–Ppy) and non-imprinted polypyrrole (NIP–Ppy) were formed on boron doped silicon (Si) substrates in order to evaluate the efficiency of Ppy to bind theophylline. The substrates were modified with boron-doped oxygen terminated nanocrystalline diamond (B:NCD:O) The dielectric properties of B:NCD:O/Ppy-based multi-layered structures were analyzed using spectroscopic ellipsometry and spectrophotometric techniques. Electrochemical impedance spectroscopy was applied for the investigation of kinetics of theophylline interaction with MIP–Ppy and NIP–Ppy. The sensitivity of molecularly imprinted and non-imprinted polymer films was analyzed by injection of different theophylline concentrations. Assuming that Ppy film electrical capacitance change is a result of Ppy dielectric constant change induced by absorbed theophylline molecules, the electrical capacitance change (DC) kinetics at different concentrations of theophylline was analyzed using first pseudo order kinetic equation. The dissociation equilibrium constant KD of MIP–Ppy/theophylline complex at room temperature was calculated as 1.7 108 M, and Gibbs free energy change (DG) of MIP–Ppy/theophylline complex formation was calculated as 43.5 kJ/mol. It was concluded that molecularly imprinted polypyrrole thin film could be used for the detection of theophylline. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Molecular imprints Molecularly imprinted polymers Polypyrrole Theophylline Ellipsometry
Abbreviations: B:NCD:O, boron-doped oxygen terminated nanocrystalline diamond; c, theophylline concentration; Cs, electrical capacitance at steady-state conditions; EIS, electrochemical impedance spectroscopy; F, fill factor; f, frequency; GCE, glassy carbon electrode; ITO, indium tin oxide; k, extinction coefficient; ka, constant of association rate; kd, constant of dissociation rate; KA, association equilibrium constant (KA = ka/kd); KD, dissociation equilibrium constant (KD = 1/KA,KD = kd/ka); KD0, standard dissociation constant for 1 M of theophylline; MIP, molecularly imprinted polymer; MIP–Ppy, molecularly imprinted with theophylline; MIP–Ppy(10%C2H5OH), molecularly imprinted with theophylline formed from polymerization mixture solution containing 10% of ethanol; MIP–Ppy(1%C2H5OH), molecularly imprinted with theophylline formed from polymerization mixture solution containing 1% of ethanol; MSE, mean square error; n, refractive index; N, the density of theophylline molecules bonded by polypyrrole; Neq, concentration of theophylline molecules dispersed in to the PBS; NIP–Ppy, non-imprinted polypyrrole; NIP–Ppy(10%C2H5OH), non-imprinted polypyrrole formed from polymerization mixture solution containing 1% of ethanol; NIP–Ppy(1%C2H5OH), non-imprinted polypyrrole formed from polymerization mixture solution containing 1% of ethanol; Ns, surface density of molecular imprints inside the MIP–Ppy layer; PBS, phosphate buffered saline solution; PGE, pencil-graphite electrode; Ppy, polypyrrole; Rd, Diffuse reflection; Si/B:NCD:O/Ppy, multi-layered structure consisting of silicon:boron doped nanocrystalline diamond:Ppy with or without theophylline imprints; Si/B:NCD: O, multi-layered structure consisting from silicon:boron doped nanocrystalline diamond; t, time; b, stretched exponential changing from 3/7 to 3/5; D, ellipsometric parameter; DC, difference between steady-state capacitance (Cs) and initial capacitance (C0); DC, electrical capacitance change; DG , standard state Gibbs free energy change; t , characteristic time of PBS absorption by MIP; C, ellipsometric parameter; e1, effective dielectric constant of Ppy film affected by PBS but not by the theophylline; e2, dielectric constant of Ppy film impregnated by the theophylline up to concentration; eef, the effective dielectric constant of the medium; ZIm, imaginary part of the impedance. * Corresponding author. E-mail address: [email protected] (A. Ramanavicius). http://dx.doi.org/10.1016/j.synthmet.2015.07.021 0379-6779/ ã 2015 Elsevier B.V. All rights reserved.
I. Baleviciute et al. / Synthetic Metals 209 (2015) 206–211
1. Introduction Molecularly imprinted polymers (MIPs) are materials that have been extensively studied because of their selective molecular binding characteristics, which is very suitable for sensor design [1,2]. In comparison with biological materials MIPs exhibit affinity properties. MIPs have high mechanical and chemical stability, are easy to prepare and are inexpensive. The MIPs behave as receptors that specifically interact with corresponding ligands [3] and for this reason they can be used in the design of chemical sensors, which are acting similarly to affinity biosensors, which usually are based on immobilized antibodies, receptors or single-stranded DNA [4]. In order to prepare the sensing element the MIP is usually immobilized on the surface of a physical transducer. Several procedures are used in order to deposit MIP films on a signal transducer surface [5], including (1) electropolymerization [4], (2) drop-coating, (3) formation of membranes with entrapped conducting materials such as carbon nanotubes, graphite, or carbon black, (4) in situ chemical polymerization. Each of the mentioned polymerization methods has its advantages, but among many Ppy formation methods, electropolymerization for the preparation of sensors is preferable method resulting in the formation of MIP-based films. Polypyrrole (Ppy) is a conducting polymer which can easily be formed by oxidative polymerization [6] and it can then be imprinted by small and high molecular weight molecules. Ppy based MIPs were formed by electropolymerization on glassy carbon electrodes (GCE) [7], pencil-graphite electrodes (PGE) [8], platinum [9], gold [10] and indium tin oxide (ITO) [11]. It was found that the Ppy film has relatively low stability if it is deposited on GCE [7] and in the case of gold electrode the working electric potential has to be carefully controlled to avoid swelling of the Ppy film and its repulsion from electrode surface [12]. ITO electrodes needs silanization procedure in order to fix polymeric film on the electrode surface firmly [13]. Si-based wafers are very attractive as substrates and even as electrodes, however due to limited adhesion the direct deposition of Ppy onto the surface of Si wafers demonstrated not very good reproducibility between different sensors [14]. Si wafers covered by a thin diamond film could be used as an alternative to bare silicon wafer electrode. Both pure Si and nanocrystalline diamond are not often used for electrochemical applications due to their poor conductivity [15] and doping is applied in order to gain semiconductivity and some other advanced properties. Surface termination of nanocrystalline diamond with oxygen is an additional procedure which is suitable for the regulation of surface adhesive properties [16,17]. It was noted that unlike some other conjugated polymers, the Ppy could form a covalent bond with diamond [18,19], which makes this system more stable and better reproducible. However, according to our best knowledge, analytical properties of the system based on molecularly imprinted polypyrrole deposited on a diamond film modified surface have only been evaluated in a few studies. The novelty of this study also include the polymerization of pyrrole from polymerization mixture containing ethanol. The ethanol presence in the polymerization mixture for preparation of MIP–Ppy is particularly not well studied in literature. The aim of this research was to evaluate the affinity and dielectric properties of MIP–Ppy film deposited on the B:NCD:Omodified Si wafer and to evaluate some properties of such structure in impedimetric theophylline sensors. 2. Experimental 2.1. Chemicals Basic chemicals including salts (NaCl, KH2PO4 and Na2HPO4, theophylline etc.), which were used in preparation of the buffer
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were purchased from Sigma–Aldrich (St. Louis, MO, USA). All these chemicals were of ‘analytical’ or better grade and were used as received from producers unless stated otherwise. Pyrrole of 97% purity was purchased from Sigma–Aldrich (St. Louis, MO, USA) and it was purified further by passing it through a 5 cm length column filled by Al2O3. All solutions were prepared using water purified in a Sartorius Stedim biotech (Goettingen, Germany). All procedures and measurements were carried out at room temperature. 2.2. Formation of B:NCD:O film Boron doped silicon wafer of 0.5 mm thickness was pre-treated by aqueous colloid of an ultra-dispersed (5–10 nm) nanodiamond layer [20,21]. Chemical vapor deposition method was performed in plasma-enhanced ASTEX 6500 microwave plasma reactor. The growth procedure was stopped when the B:NCD reached a thickness of 200 nm. After that it was cooled down by hydrogen flow. During slow cooling from 700 C down to 100 C the hydrogenation of B:NCD by hydrogen plasma was performed. The cleaned B:NCD was oxidized by UV induced ozone treatment for 30 min with digital UV ozone system from Novascan Technologies Inc. (Ames, US) to obtain oxygen terminated boron-doped nanocrystalline diamond (B:NCD:O). The samples and/or electrodes used in further investigations were made as multi-layered structures consisting of Si wafers modified by nanocrystalline diamond film (B:NCD:O) and then additionally coated with molecularly imprinted or non-imprinted polypyrrole films. 2.3. Formation of MIP–Ppy and NIP–Ppy films Preparation of theophylline imprinted polypyrrole (MIP–Ppy) and non-imprinted polypyrrole (NIP–Ppy) on B:NCD:O film was performed by chemical polymerization method. NIP–Ppy was obtained from the solution containing 0.2 M of pyrrole, 390 mM of H2O2, 40 mM of HCl and dissolved in water. MIP–Ppy was obtained from the solution containing 0.2 M of pyrrole, 20 mM of theophylline, 390 mM of H2O2 and 40 mM of HCl dissolved in water. MIP–Ppy and NIP–Ppy films formation was performed at 20 C and the polymerization lasted for 72 h. Si wafers modified by B:NCD:O were immersed to the prepared polymerization mixtures and left there for above-mentioned period of chemical polymerization. By this method MIP–Ppy and NIP–Ppy were formed on the surfaces of B:NCD:O. Then Si wafers modified by B:NCD:O and MIP–Ppy or NIP–Ppy were gently washed by purified water in order to remove the excess of unbounded MIP–Ppy or NIP–Ppy. Additionally ethanol influence to the properties of MIP–Ppy and NIP–Ppy was evaluated by the addition of 1% or 10% of ethanol into the initial polymerization mixture, because in one previous study it was demonstrated that ethanol exhibits strong interaction with nanocrystalline diamond used for deposition of MIP–Ppy and NIP– Ppy [22]. On the other hand, ethanol was selected for the modification of the polymerization mixture due to significant differences of theophylline and pyrrole solubility in pure water and in alcohol. It was analyzed how the addition of this surface active compound is affecting the final properties of the obtained MIP– Ppy. After the modification of electrodes by MIP–Ppy or NIP–Ppy, the electrodes were thoroughly washed by ultra-pure water. Electrochemical impedance spectroscopy (EIS) measurements were performed by a home-made multichannel electrochemical impedance analyzer, as described in previous work [23]. Impedance spectroscopy was performed simultaneously on the MIP–Ppy and NIP–Ppy in a frequency range of 10Hz–100 kHz. Surface area of working electrodes was 28 mm2. Electrochemical experiments were performed in 50 mM phosphate buffered saline solution (PBS), pH 7.0, with 100 mM of NaCl. Investigation of theophylline
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interaction with Ppy was performed in the following way: an electrode modified by Ppy film was incubated in theophylline free PBS and electrochemical impedance was registered. The concentrated theophylline solution in PBS was used to gradually increase the final theophylline concentration in electrochemical cell from 0.1 nM to 1 mM by increasing the concentration in each step by factor of 10. Capacitance from EIS measurements was calculated according to following Eq. (1): C¼
1 2p f Z Im
(1)
here f is frequency and ZIm is the imaginary part of the impedance. Kinetics of capacitance change was monitored until steady-state conditions were achieved. 2.4. Ellipsometry set-up The dielectric properties of multi-layered structures of: (i) silicon, (ii) boron doped nanocrystalline diamond and (iii) Ppy with and without theophylline imprints (Si/B:NCD/PPy) formed from Si wafer coated by boron doped nanocrystalline diamond and polypyrrole layers described above were evaluated by a spectral ellipsometer M2000X from J.A. Woolam Co. (Lincoln, USA) operating in the range from 200 nm to 1000 nm using a focusing probe. Calculation of the optical constants was performed using COMPLETE EASE software from J.A. Woolam Co. (Lincoln, USA). Diffusion reflectance was measured using spectrophotometer UV3600 UV–vis-NIR from Shimadzu (Portland, USA).
ethanol has 4 peaks while for the system based on Ppy formed from initial polymerization mixture solution with 10% of ethanol contained only 3 peaks. Assuming that the mass of the Ppy film was the same for both systems these differences could be explained by different effective optical thickness of the Ppy film. The investigation of Ppy film surface performed by atomic force microscopy demonstrated that the surface roughness of this Ppy film was in the range of 30 nm and not dependent on ethanol concentration. This is of the same order as effective thickness of Ppy film (about 30 nm) as estimated from deposition conditions. It demonstrates the ‘island shaped’ morphology of formed Ppy film. It has to be noted that for such morphology it is difficult to apply a ‘multi-layer approach’, which in some cases is useful for ellipsometry data analysis, due to strong diffusion scattering of the light. In this case more information could be obtained from diffuse reflection measurements. Fig. 2 demonstrates the reflected light intensity vs. wavelength obtained from ellipsometric measurements for two systems where Ppy was formed from initial solutions containing different concentrations of ethanol. The shift of peaks located at 238 nm and 288 nm for Si/B:NCD:O/Ppy10% C2H5OH and 277 nm and 344 nm for Si/B:NCD:O/Ppy1%C2H5OH could be associated with different structures of PPy films. The same shift was obtained in diffuse reflection measurements (Fig. 3). The decrease of reflection at long waves shows that Ppy film in Si/B:NCD:O/Ppy10%C2H5OH formed from initial polymerization mixture solution, which contained 10% of ethanol, is more condensed than the film in Si/B:NCD:O/Ppy1%C2H5OH, which was formed from 1% ethanol. A difference in diffuse reflection the most probably is associated with the increase of density of Ppy film.
3. Results and discussion 4. Dynamics of theophylline interaction with MIP–Ppy
200
model
400
600 (nm)
800
1000
n k
2.6
0.8
B
2.4
0.6
2.2
0.4
2.0
0.2
1.8
200
400
600
800
1000
k
A
75 60 45 30 15 0 -15 -30 -45
For the investigation of theophylline interaction the following Ppy films were used: (i) one sample was prepared without molecular imprints (NIP–Ppy) and (ii) the other samples were made with molecular imprints of theophylline (MIP–Ppy). In additional control-experiments the ethanol was added to the polymerization mixture solution and NIP–Ppy and MIP–Ppy film (NIP–Ppy(1%C2H5OH), MIP–Ppy(1%C2H5OH), NIP–Ppy(10%C2H5OH) and MIP–Ppy(10%C2H5OH)) properties produced from ethanol containing solutions were evaluated. The films prepared from the polymerization mixture solution without ethanol are indicated as NIP–Ppy and MIP–Ppy, from the polymerization mixture with 1% of ethanol as NIP–Ppy(1%C2H5OH) and MIP–Ppy(1%C2H5OH), and from the polymerization mixture with 10% of ethanol as NIP–Ppy(10% C2H5OH) and MIP–Ppy(10%C2H5OH), correspondingly. The change of Ppy electrical capacitance was evaluated as indicator of theophylline interaction dynamics with MIP–Ppy. Electrical capacitance data was obtained from electrochemical impedance spectroscopy. Fig. 4 shows the changes of electrical capacitance in time when
n
45 40 35 30 25 20 15 10 5
(deg.)
(deg.)
In order to characterize the dielectric properties of substrate used for Ppy film deposition, ellipsometric parameters D and C vs. wavelength were measured for two films based system Si/B:NCD: O. Results obtained for this system are presented in Fig. 1A. The solid curve represents the results of calculation assuming the Si as a substrate and B:NCD:O as a film of 166.11 nm thickness, which is characterized by 4 Tauc–Lorentz oscillators. Mean square error (MSE) of this calculation was in the range of 28.7 nm. Optical constants i.e., refractive index (n) and extinction coefficient (k) obtained from this calculation are showed in Fig. 1B and is in agreement with results reported by other authors [19]. The spectroscopic ellipsometry investigations for the system of Si/B:NCD:O/Ppy, which was formed from ethanol-free solution was impossible due to large light diffusion scattering of this film. For this reason only Ppy films formed from initial polymerization mixture solution containing 1% and 10% of ethanol were investigated by spectroscopic ellipsometry. It was observed that dependencies of D and C vs. wavelength for the system based on Ppy formed from initial polymerization solution containing 1% of
0.0
(nm)
Fig. 1. (A) Dependence of ellipsometric parameters C and D on wavelength for Si/B:NCD:O substrate, dotted lines correspond to experimentally measured C and D, solid line correspond to modeling results obtained using 4 Tauc–Laurentz oscillators. In (B) the dashed line corresponds to refractive index dependence on wavelength (n) and the solid line corresponds to extinction coefficient (k) dependence on wavelength obtained after modeling procedure.
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209 b
C ¼ C s þ DC eðt=t Þ
0.10
8
here Cs is electrical capacitance at steady-state conditions, DC is difference between steady-state capacitance (Cs) and initial capacitance (C0) before mounting Ppy film into PBS, t is characteristic time of PBS absorption by MIP–Ppy and b is stretched exponential changing from 3/7 to 3/5. A well fit using Eq. (2) was obtained when Cs = 5.9 mF, DC = 1.96 mF, t = 1236 s, b = 0.55 (Fig. 4 curve 3). The value of parameter b is close to 0.6, which is typical for short range interaction. In case of MIP–Ppy treatment by PBS observed interaction is most probably based on the formation of hydrogen bounds, which are usually formed during the polymer swelling processes [25]. The analysis of electrochemical capacity change due to theophylline interaction was performed at steady-state conditions, which were achieved after treatment of the MIP–Ppy film with PBS. Moreover, it was assumed that theophylline interaction with Ppy takes place by the whole volume of MIP–Ppy. Due to high concentration of theophylline molecules (Neq) dispersed into the PBS, the final concentration of theophylline bounded by Ppy film at the equilibrium conditions have to be equal to Neq. This concentration can be found from the steady-state solution of first pseudo order kinetic Eq. (3) assuming that the concentration of theophylline (c) in solution is much higher than the density of molecular imprints (Ns) inside the MIP–Ppy film:
6
dN ¼ ka c ðNs NÞ kd N dt
4
here N is the density of theophylline molecules bonded by Ppy at a certain time instance and ka and kd are association and dissociation rate constants respectively. Nominating the ratio N/Ns as F (fill
0.08
Intensity
0.07 0.06 0.05 0.04 0.03 200
250
300
350
400
(nm) Fig. 2. Reflectance intensity of multi-layered structure Si/B:NCD:O/Ppy dependence on light wavelength obtained from ellipsometric measurements. Si/B:NCD:O/ Ppy10%C2H5OH were Ppy films formed from polymerization bulk solution containing 10% of ethanol (doted-dashed line), Si/B:NCD:O/Ppy1%C2H5OH were Ppy film formed from polymerization bulk solution containing 1% of ethanol (solid line).
10 c)Si/B:NCD:O/Ppy b)Si/B:NCD:O/Ppy a)Si/B:NCD:O
2 200
400
600
800
10nM
8.0 C0
1000
(3)
3 10 nM
2 10 nM
(nm) 7.0
5.5
As it can be seen from Fig. 4, the prepared MIP–Ppy films (treated and untreated by ethanol) intensively absorb buffer solution up to a certain saturation level. The analysis of capacitance change kinetics in time (t) can be well fitted by stretched exponential law [24]:
1 nM 10 nM 2 10 nM
CS
3 10 nM
2
A
0.5
1 0
1
2
3
4
5
6
time (h)
6.5 6.4 0,1 nM
6.3 1 nM
6.2 6.1
10 nM 2
10 nM
6.0
B 5.9 0
5. Evaluation of theophylline interaction with MIP–Ppy
1.5
1.0
0,1 nM
6.5 6.0
C, F
films were exposed towards the PBS solution containing different concentrations of theophylline. The films were exposed to different concentrations of theophylline gradually increasing by factor of 10 from 0.1 nM up to 1 mM. The arrows in Fig. 4 indicate the time instance at which these concentrations were applied. As can be seen from this figure the MIP–Ppy and MIP–Ppy(10%C2H5OH) films (Fig. 4A, curves 1 and 3) are strongly affected when first exposed to PBS buffer solution free of theophylline. There are no changes of electrochemical capacitance in the case of NIP–Ppy film. Kinetics of capacitance change of MIP–Ppy film formed from ethanol-free solution due to swelling and ion-migration reached steady-state after approximately 150 min. The injection of theophylline molecules induced additional electrochemical capacitance change. From Fig. 4, it is apparent that the MIP–Ppy is more sensitive to the addition of theophylline than the MIP–Ppy(10%C2H5OH) and the NIP–Ppy does not show any sensitivity at all.
C ( F)
7.5 Fig. 3. Diffuse reflection Rd vs. wavelength obtained for different systems: (a) Si/B: NCD:O substrate, (b) Ppy film formed from 1% ethanol containing initial polymerization mixture solution (Si/B:NCD:O/Ppy1%C2H5OH), (c) Ppy film formed from 10% ethanol containing initial polymerization mixture solution (Si/B:NCD:O/ Ppy10%C2H5OH).
1 2 3
3
C ( F)
0.09
Rd
(2)
1
2
3
4
time, h Fig. 4. MIP–Ppy films electrochemical capacity changes vs. time during interaction of theophylline, for: (A) 1—MIP–Ppy film, 2—stretched exponential model for PBS absorption, 3—MIP–Ppy(10%C2H5OH) film formed from 10% ethanol containing solution; arrows indicate time moments when different concentration of theophylline were injected. (B) NIP–Ppy electrochemical capacity changes vs. time during interaction of theophylline. Arrows indicate time moments when films are exposed to different concentrations of theophylline.
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1.0
(4)
0.8
C/ Cmax
factor) the following solution of this equation is obtained: t 1 F ¼ 1 exp ; t ¼ ka c þ kd t
Fig. 5 shows that the kinetics of theophylline interaction when the PBS influence to the capacity change is negligible could be described by formula (4). At equilibrium condition (when dN/ dt = 0) the Neq value can be obtained using following formula: Neq ¼ Ns
c c þ KD
eef ¼ ð1 FÞe1 þ F e2
(6)
here F is the fill factor showing the ratio between volume of the media having dielectric constant e1 and volume of the material with dielectric constant e2. In our case the F vs. time (t) can be calculated by formula (4). Due to the simple relation between dielectric constant (e) and electrochemical capacity (C) of the film C = e S/d (S is area of the electrodes and d is a distance between them) the e1 and e2 corresponds to the capacitance of the Ppy film at the beginning of interaction (CS) and at the end of the theophylline interaction due to establishment of steady-state conditions (Ceq) respectively when c >> Ns (Fig. 4). Thus then the fill factor F is equal to DC/DCmax = (Ce C1)/(C1 C2), here Ce is effective capacity, which is a function of time. The capacitance
1.0
fill factor
0.8 0.6 0.4 0.2 0.0 -100
100 nM theophyline kinetics
0
0.4
Ceq
C1
C1 C2 c c Kd
0.2 0.0
(5)
here KD = kd/ka; where kd, ka are dissociation and association rate constants respectively. In previous studies affinity constants (the total number of binding sites, Ki) were also calculated [26]. But in contrast to experiments described here an UV–vis spectroscopy was applied in order to analyze affinity of these previously described systems. We assume that experimentally obtained change of Ppy film electrochemical capacitance in the case of theophylline molecules binding has to be associated with changes of Ppy dielectric constant. At the steady-state conditions between Ppy and the solution with theophylline molecules, the Ppy film can be evaluated as ‘effective medium’ consisting of two materials having different dielectric constants e1 and e2. The e1 is effective dielectric constant of Ppy film, which was affected by PBS but not by the theophylline. e2 is the dielectric constant of this film impregnated by the theophylline up to concentration, which is present in Ppy film at steady-state conditions established between PBS containing defined concentration of the theophylline and Ppy film. The effective dielectric constant (eef) of the medium, which is a mixture of materials with dielectric constants e1 and e2, can be found using Drude’s approach:
0.6
0
200
400
600
800
1000
n (nM) Fig. 6. Relative ratio of changes in electrical capacitance obtained at steady-state conditions vs. different concentrations of theophylline.
changes kinetics after the injection of 100 nM theophylline and modeling of results using formula (5) is presented in Fig. 5. The best fitting result was obtained when fitting parameter t was 40 s. The electrochemical capacitance obtained at equilibrium conditions (Ceq) for different concentrations can be calculated using formulas (5) and (6): C eq ¼ C 1
ðC 1 C 2 Þ c c þ Kd
(7)
Fig. 6 shows fitting result (solid line) of experimental data (dots) using formula (6), where DC = Ceq C1 and DCmax = C1 C2. C1 and C2 are the capacitances of Ppy film at the initial phase of theophylline interaction and at the establishment of steady-state conditions, respectively, c— theophylline concentration, KD— affinity dissociation constant (KD = kd/ka), calculated for the interaction of theophylline: kd
MIP PpyðsolidphaseÞ þ theophyllineðdissolvedÞ ? MIP Ppy=theophyllineðsolidphaseÞ
ka
(8)
This modeling provides the possibility to obtain affinity dissociation constant (KD) value, which in this case is equal to 1.7 108 M. Using Eq. (9) it is possible to estimate Gibbs free energy change of theophylline/MIP–Ppy complex formation:
DG ¼
RT lnK 0D
(9)
where the KD0 = KD/c, in this case the c is the concentration of theophylline; Therefore, the KD0 is the value, which is calculated for standard reference concentration of 1 M [27]. It was found that DG = 43.5 kJ/mol for theophylline/MIP–Ppy complex formation, which shows that theophylline/MIP–Ppy complex formation is a thermodynamically favorable reaction. The obtained value of Gibbs free energy change is in the same order as that value calculated by other authors [28] and confirmed in our previous works [27]. These values are close to that which are typical for 2–3 hydrogen bounds formation and electrostatic interactions [29,30]. 6. Conclusions
100 200 300 400 500 600 700
time (s) Fig. 5. MIP–Ppy produced form polymerization bulk solution not containing ethanol, theophylline interaction kinetics using 100 nM concentration. Dots correspond to experimental points, solid line—for model obtained using formula (5).
Theophylline imprinted and non-imprinted polypyrrole thin films were deposited on boron doped nanocrystalline substrate formed on Si wafer. Evaluation of dielectric properties of NIP–Ppy demonstrated that it has low sensitivity to the theophylline solution in PBS. MIP–Ppy showed high affinity towards
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theophylline and therefore it could be used for the sensing of theophylline using impedance spectroscopy method. For low theophylline concentrations in the range from 0.1 to 103 nM more sensitive was the MIP–Ppy film formed from ethanol-free solution. Presence of ethanol in polymerization bulk solution induces changes in structure of MIP–Ppy. Acknowledgments The study was funded from the European Community’s social foundation under Grant Agreement No. VP1-3.1-ŠMM-08-K-01004/KS-120000-1756. We are grateful to Dr. Stoffel Janssens for formation of borondoped nanocrystalline diamond on boron-doped silicon. References [1] K. Haupt, Imprinted polymers-Tailor-made mimics of antibodies and receptors, Chem. Commun. 2 (2003) 171–178. [2] K. Mosbach, Molecular imprinting, Trends Biochem. Sci. 19 (1) (1994) 9–14. [3] A. Cutivet, C. Schembri, J. Kovensky, K. Haupt, Molecularly imprinted microgels as enzyme inhibitors, J. Am. Chem. Soc. 131 (41) (2009) 14699–14702. [4] V. Ratautaite, S.N. Topkaya, L. Mikoliunaite, M. Ozsoz, Y. Oztekin, A. Ramanaviciene, A. Ramanavicius, Molecularly imprinted polypyrrole for DNA determination, Electroanalysis 25 (5) (2013) 1169–1177. [5] P.S. Sharma, A. Pietrzyk-Le, F. D’Souza, W. Kutner, Electrochemically synthesized polymers in molecular imprinting for chemical sensing, Anal. Bioanal. Chem. 402 (10) (2012) 3177–3204. [6] K. Leonavicius, A. Ramanaviciene, A. Ramanavicius, Polymerization model for hydrogen peroxide initiated synthesis of polypyrrole nanoparticles, Langmuir 27 (17) (2011) 10970–10976. [7] L.D. Spurlock, A. Jaramillo, A. Praserthdam, J. Lewis, A. Brajter-Toth, Selectivity and sensitivity of ultrathin purine-templated overoxidized polypyrrole film electrodes, Anal. Chim. Acta 336 (1–3) (1996) 37–46. [8] L. Ozcan, Y. Sahin, Determination of paracetamol based on electropolymerized-molecularly imprinted polypyrrole modified pencil graphite electrode, Sens. Actuators B-Chem. 127 (2) (2007) 362–369. [9] D.R. Albano, F. Sevilla, Piezoelectric quartz crystal sensor for surfactant based on molecularly imprinted polypyrrole, Sens. Actuators B-Chem. 121 (1) (2007) 129–134. [10] B.S. Ebarvia, S. Cabanilla, I.F. Sevilla, Biomimetic properties and surface studies of a piezoelectric caffeine sensor based on electrosynthesized polypyrrole, Talanta 66 (1) (2005) 145–152. [11] W.M. Yeh, K.C. Ho, Amperometric morphine sensing using a molecularly imprinted polymer-modified electrode, Anal. Chim. Acta 542 (1) (2005) 76–82. [12] V. Ratautaite, A. Ramanaviciene, Y. Oztekin, J. Voronovic, Z. Balevicius, L. Mikoliunaite, A. Ramanavicius, Electrochemical stability and repulsion of polypyrrole film, Colloid Surf. A 418 (0) (2013) 16–21. [13] M.C. Blanco-Lopez, M.J. Lobo-Castanon, A.J. Miranda-Ordieres, P. TunonBlanco, Electrochemical sensors based on molecularly imprinted polymers, TRAC—Trends Anal. Chem. 23 (1) (2004) 36–48.
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Synthetic Metals journal homepage: www.elsevier.com/locate/synmet
Evaluation of theophylline imprinted polypyrrole film Ieva Baleviciutea , Vilma Ratautaitea , Almira Ramanavicienec , Zigmas Baleviciusb , Jeroen Broedersd , Dieter Crouxd , Matthew McDonaldd, Farnoosh Vahidpourd, Ronald Thoelend,e , Ward De Ceuninckd,f , Ken Haenend , Milos Nesladeke, Alfonsas Rezag, Arunas Ramanaviciusa,* a
Department of Physical Chemistry, Vilnius University, Faculty of Chemistry, Naugarduko str. 24, Vilnius, Lithuania Laboratory of Bio-NanoTechnology, State Research Institute—Center for Physical Science and Technology, Gostauto str. 11, Vilnius, Lithuania c NanoTechnas—Centre of Nanotechnology and Materials Science, Vilnius University, Faculty of Chemistry, Naugarduko str. 24, Vilnius, Lithuania d Institute for Materials Research (IMO), Hasselt University, Wetenschapspark 1, B-3590 Diepenbeek, Belgium e Research Centre—IMOMEC, Research Institute—IMEC vzw, Wetenschapspark 1, B-3590 Diepenbeek, Belgium f Xios University College, Agoralaan—gebouw D, 3590 Diepenbeek, Belgium g Department of Optoelectronics, State Research Institute—Center for Physical Science and Technology, Gostauto str. 11, Vilnius, Lithuania b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 May 2015 Received in revised form 5 June 2015 Accepted 13 July 2015 Available online xxx
In this study some affinity and dielectric properties of molecularly imprinted (MIP) conducting polymer— polypyrrole (Ppy) based thin films were evaluated. Films of polypyrrole molecularly imprinted with theophylline (MIP–Ppy) and non-imprinted polypyrrole (NIP–Ppy) were formed on boron doped silicon (Si) substrates in order to evaluate the efficiency of Ppy to bind theophylline. The substrates were modified with boron-doped oxygen terminated nanocrystalline diamond (B:NCD:O) The dielectric properties of B:NCD:O/Ppy-based multi-layered structures were analyzed using spectroscopic ellipsometry and spectrophotometric techniques. Electrochemical impedance spectroscopy was applied for the investigation of kinetics of theophylline interaction with MIP–Ppy and NIP–Ppy. The sensitivity of molecularly imprinted and non-imprinted polymer films was analyzed by injection of different theophylline concentrations. Assuming that Ppy film electrical capacitance change is a result of Ppy dielectric constant change induced by absorbed theophylline molecules, the electrical capacitance change (DC) kinetics at different concentrations of theophylline was analyzed using first pseudo order kinetic equation. The dissociation equilibrium constant KD of MIP–Ppy/theophylline complex at room temperature was calculated as 1.7 108 M, and Gibbs free energy change (DG) of MIP–Ppy/theophylline complex formation was calculated as 43.5 kJ/mol. It was concluded that molecularly imprinted polypyrrole thin film could be used for the detection of theophylline. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Molecular imprints Molecularly imprinted polymers Polypyrrole Theophylline Ellipsometry
Abbreviations: B:NCD:O, boron-doped oxygen terminated nanocrystalline diamond; c, theophylline concentration; Cs, electrical capacitance at steady-state conditions; EIS, electrochemical impedance spectroscopy; F, fill factor; f, frequency; GCE, glassy carbon electrode; ITO, indium tin oxide; k, extinction coefficient; ka, constant of association rate; kd, constant of dissociation rate; KA, association equilibrium constant (KA = ka/kd); KD, dissociation equilibrium constant (KD = 1/KA,KD = kd/ka); KD0, standard dissociation constant for 1 M of theophylline; MIP, molecularly imprinted polymer; MIP–Ppy, molecularly imprinted with theophylline; MIP–Ppy(10%C2H5OH), molecularly imprinted with theophylline formed from polymerization mixture solution containing 10% of ethanol; MIP–Ppy(1%C2H5OH), molecularly imprinted with theophylline formed from polymerization mixture solution containing 1% of ethanol; MSE, mean square error; n, refractive index; N, the density of theophylline molecules bonded by polypyrrole; Neq, concentration of theophylline molecules dispersed in to the PBS; NIP–Ppy, non-imprinted polypyrrole; NIP–Ppy(10%C2H5OH), non-imprinted polypyrrole formed from polymerization mixture solution containing 1% of ethanol; NIP–Ppy(1%C2H5OH), non-imprinted polypyrrole formed from polymerization mixture solution containing 1% of ethanol; Ns, surface density of molecular imprints inside the MIP–Ppy layer; PBS, phosphate buffered saline solution; PGE, pencil-graphite electrode; Ppy, polypyrrole; Rd, Diffuse reflection; Si/B:NCD:O/Ppy, multi-layered structure consisting of silicon:boron doped nanocrystalline diamond:Ppy with or without theophylline imprints; Si/B:NCD: O, multi-layered structure consisting from silicon:boron doped nanocrystalline diamond; t, time; b, stretched exponential changing from 3/7 to 3/5; D, ellipsometric parameter; DC, difference between steady-state capacitance (Cs) and initial capacitance (C0); DC, electrical capacitance change; DG , standard state Gibbs free energy change; t , characteristic time of PBS absorption by MIP; C, ellipsometric parameter; e1, effective dielectric constant of Ppy film affected by PBS but not by the theophylline; e2, dielectric constant of Ppy film impregnated by the theophylline up to concentration; eef, the effective dielectric constant of the medium; ZIm, imaginary part of the impedance. * Corresponding author. E-mail address: [email protected] (A. Ramanavicius). http://dx.doi.org/10.1016/j.synthmet.2015.07.021 0379-6779/ ã 2015 Elsevier B.V. All rights reserved.
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1. Introduction Molecularly imprinted polymers (MIPs) are materials that have been extensively studied because of their selective molecular binding characteristics, which is very suitable for sensor design [1,2]. In comparison with biological materials MIPs exhibit affinity properties. MIPs have high mechanical and chemical stability, are easy to prepare and are inexpensive. The MIPs behave as receptors that specifically interact with corresponding ligands [3] and for this reason they can be used in the design of chemical sensors, which are acting similarly to affinity biosensors, which usually are based on immobilized antibodies, receptors or single-stranded DNA [4]. In order to prepare the sensing element the MIP is usually immobilized on the surface of a physical transducer. Several procedures are used in order to deposit MIP films on a signal transducer surface [5], including (1) electropolymerization [4], (2) drop-coating, (3) formation of membranes with entrapped conducting materials such as carbon nanotubes, graphite, or carbon black, (4) in situ chemical polymerization. Each of the mentioned polymerization methods has its advantages, but among many Ppy formation methods, electropolymerization for the preparation of sensors is preferable method resulting in the formation of MIP-based films. Polypyrrole (Ppy) is a conducting polymer which can easily be formed by oxidative polymerization [6] and it can then be imprinted by small and high molecular weight molecules. Ppy based MIPs were formed by electropolymerization on glassy carbon electrodes (GCE) [7], pencil-graphite electrodes (PGE) [8], platinum [9], gold [10] and indium tin oxide (ITO) [11]. It was found that the Ppy film has relatively low stability if it is deposited on GCE [7] and in the case of gold electrode the working electric potential has to be carefully controlled to avoid swelling of the Ppy film and its repulsion from electrode surface [12]. ITO electrodes needs silanization procedure in order to fix polymeric film on the electrode surface firmly [13]. Si-based wafers are very attractive as substrates and even as electrodes, however due to limited adhesion the direct deposition of Ppy onto the surface of Si wafers demonstrated not very good reproducibility between different sensors [14]. Si wafers covered by a thin diamond film could be used as an alternative to bare silicon wafer electrode. Both pure Si and nanocrystalline diamond are not often used for electrochemical applications due to their poor conductivity [15] and doping is applied in order to gain semiconductivity and some other advanced properties. Surface termination of nanocrystalline diamond with oxygen is an additional procedure which is suitable for the regulation of surface adhesive properties [16,17]. It was noted that unlike some other conjugated polymers, the Ppy could form a covalent bond with diamond [18,19], which makes this system more stable and better reproducible. However, according to our best knowledge, analytical properties of the system based on molecularly imprinted polypyrrole deposited on a diamond film modified surface have only been evaluated in a few studies. The novelty of this study also include the polymerization of pyrrole from polymerization mixture containing ethanol. The ethanol presence in the polymerization mixture for preparation of MIP–Ppy is particularly not well studied in literature. The aim of this research was to evaluate the affinity and dielectric properties of MIP–Ppy film deposited on the B:NCD:Omodified Si wafer and to evaluate some properties of such structure in impedimetric theophylline sensors. 2. Experimental 2.1. Chemicals Basic chemicals including salts (NaCl, KH2PO4 and Na2HPO4, theophylline etc.), which were used in preparation of the buffer
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were purchased from Sigma–Aldrich (St. Louis, MO, USA). All these chemicals were of ‘analytical’ or better grade and were used as received from producers unless stated otherwise. Pyrrole of 97% purity was purchased from Sigma–Aldrich (St. Louis, MO, USA) and it was purified further by passing it through a 5 cm length column filled by Al2O3. All solutions were prepared using water purified in a Sartorius Stedim biotech (Goettingen, Germany). All procedures and measurements were carried out at room temperature. 2.2. Formation of B:NCD:O film Boron doped silicon wafer of 0.5 mm thickness was pre-treated by aqueous colloid of an ultra-dispersed (5–10 nm) nanodiamond layer [20,21]. Chemical vapor deposition method was performed in plasma-enhanced ASTEX 6500 microwave plasma reactor. The growth procedure was stopped when the B:NCD reached a thickness of 200 nm. After that it was cooled down by hydrogen flow. During slow cooling from 700 C down to 100 C the hydrogenation of B:NCD by hydrogen plasma was performed. The cleaned B:NCD was oxidized by UV induced ozone treatment for 30 min with digital UV ozone system from Novascan Technologies Inc. (Ames, US) to obtain oxygen terminated boron-doped nanocrystalline diamond (B:NCD:O). The samples and/or electrodes used in further investigations were made as multi-layered structures consisting of Si wafers modified by nanocrystalline diamond film (B:NCD:O) and then additionally coated with molecularly imprinted or non-imprinted polypyrrole films. 2.3. Formation of MIP–Ppy and NIP–Ppy films Preparation of theophylline imprinted polypyrrole (MIP–Ppy) and non-imprinted polypyrrole (NIP–Ppy) on B:NCD:O film was performed by chemical polymerization method. NIP–Ppy was obtained from the solution containing 0.2 M of pyrrole, 390 mM of H2O2, 40 mM of HCl and dissolved in water. MIP–Ppy was obtained from the solution containing 0.2 M of pyrrole, 20 mM of theophylline, 390 mM of H2O2 and 40 mM of HCl dissolved in water. MIP–Ppy and NIP–Ppy films formation was performed at 20 C and the polymerization lasted for 72 h. Si wafers modified by B:NCD:O were immersed to the prepared polymerization mixtures and left there for above-mentioned period of chemical polymerization. By this method MIP–Ppy and NIP–Ppy were formed on the surfaces of B:NCD:O. Then Si wafers modified by B:NCD:O and MIP–Ppy or NIP–Ppy were gently washed by purified water in order to remove the excess of unbounded MIP–Ppy or NIP–Ppy. Additionally ethanol influence to the properties of MIP–Ppy and NIP–Ppy was evaluated by the addition of 1% or 10% of ethanol into the initial polymerization mixture, because in one previous study it was demonstrated that ethanol exhibits strong interaction with nanocrystalline diamond used for deposition of MIP–Ppy and NIP– Ppy [22]. On the other hand, ethanol was selected for the modification of the polymerization mixture due to significant differences of theophylline and pyrrole solubility in pure water and in alcohol. It was analyzed how the addition of this surface active compound is affecting the final properties of the obtained MIP– Ppy. After the modification of electrodes by MIP–Ppy or NIP–Ppy, the electrodes were thoroughly washed by ultra-pure water. Electrochemical impedance spectroscopy (EIS) measurements were performed by a home-made multichannel electrochemical impedance analyzer, as described in previous work [23]. Impedance spectroscopy was performed simultaneously on the MIP–Ppy and NIP–Ppy in a frequency range of 10Hz–100 kHz. Surface area of working electrodes was 28 mm2. Electrochemical experiments were performed in 50 mM phosphate buffered saline solution (PBS), pH 7.0, with 100 mM of NaCl. Investigation of theophylline
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interaction with Ppy was performed in the following way: an electrode modified by Ppy film was incubated in theophylline free PBS and electrochemical impedance was registered. The concentrated theophylline solution in PBS was used to gradually increase the final theophylline concentration in electrochemical cell from 0.1 nM to 1 mM by increasing the concentration in each step by factor of 10. Capacitance from EIS measurements was calculated according to following Eq. (1): C¼
1 2p f Z Im
(1)
here f is frequency and ZIm is the imaginary part of the impedance. Kinetics of capacitance change was monitored until steady-state conditions were achieved. 2.4. Ellipsometry set-up The dielectric properties of multi-layered structures of: (i) silicon, (ii) boron doped nanocrystalline diamond and (iii) Ppy with and without theophylline imprints (Si/B:NCD/PPy) formed from Si wafer coated by boron doped nanocrystalline diamond and polypyrrole layers described above were evaluated by a spectral ellipsometer M2000X from J.A. Woolam Co. (Lincoln, USA) operating in the range from 200 nm to 1000 nm using a focusing probe. Calculation of the optical constants was performed using COMPLETE EASE software from J.A. Woolam Co. (Lincoln, USA). Diffusion reflectance was measured using spectrophotometer UV3600 UV–vis-NIR from Shimadzu (Portland, USA).
ethanol has 4 peaks while for the system based on Ppy formed from initial polymerization mixture solution with 10% of ethanol contained only 3 peaks. Assuming that the mass of the Ppy film was the same for both systems these differences could be explained by different effective optical thickness of the Ppy film. The investigation of Ppy film surface performed by atomic force microscopy demonstrated that the surface roughness of this Ppy film was in the range of 30 nm and not dependent on ethanol concentration. This is of the same order as effective thickness of Ppy film (about 30 nm) as estimated from deposition conditions. It demonstrates the ‘island shaped’ morphology of formed Ppy film. It has to be noted that for such morphology it is difficult to apply a ‘multi-layer approach’, which in some cases is useful for ellipsometry data analysis, due to strong diffusion scattering of the light. In this case more information could be obtained from diffuse reflection measurements. Fig. 2 demonstrates the reflected light intensity vs. wavelength obtained from ellipsometric measurements for two systems where Ppy was formed from initial solutions containing different concentrations of ethanol. The shift of peaks located at 238 nm and 288 nm for Si/B:NCD:O/Ppy10% C2H5OH and 277 nm and 344 nm for Si/B:NCD:O/Ppy1%C2H5OH could be associated with different structures of PPy films. The same shift was obtained in diffuse reflection measurements (Fig. 3). The decrease of reflection at long waves shows that Ppy film in Si/B:NCD:O/Ppy10%C2H5OH formed from initial polymerization mixture solution, which contained 10% of ethanol, is more condensed than the film in Si/B:NCD:O/Ppy1%C2H5OH, which was formed from 1% ethanol. A difference in diffuse reflection the most probably is associated with the increase of density of Ppy film.
3. Results and discussion 4. Dynamics of theophylline interaction with MIP–Ppy
200
model
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For the investigation of theophylline interaction the following Ppy films were used: (i) one sample was prepared without molecular imprints (NIP–Ppy) and (ii) the other samples were made with molecular imprints of theophylline (MIP–Ppy). In additional control-experiments the ethanol was added to the polymerization mixture solution and NIP–Ppy and MIP–Ppy film (NIP–Ppy(1%C2H5OH), MIP–Ppy(1%C2H5OH), NIP–Ppy(10%C2H5OH) and MIP–Ppy(10%C2H5OH)) properties produced from ethanol containing solutions were evaluated. The films prepared from the polymerization mixture solution without ethanol are indicated as NIP–Ppy and MIP–Ppy, from the polymerization mixture with 1% of ethanol as NIP–Ppy(1%C2H5OH) and MIP–Ppy(1%C2H5OH), and from the polymerization mixture with 10% of ethanol as NIP–Ppy(10% C2H5OH) and MIP–Ppy(10%C2H5OH), correspondingly. The change of Ppy electrical capacitance was evaluated as indicator of theophylline interaction dynamics with MIP–Ppy. Electrical capacitance data was obtained from electrochemical impedance spectroscopy. Fig. 4 shows the changes of electrical capacitance in time when
n
45 40 35 30 25 20 15 10 5
(deg.)
(deg.)
In order to characterize the dielectric properties of substrate used for Ppy film deposition, ellipsometric parameters D and C vs. wavelength were measured for two films based system Si/B:NCD: O. Results obtained for this system are presented in Fig. 1A. The solid curve represents the results of calculation assuming the Si as a substrate and B:NCD:O as a film of 166.11 nm thickness, which is characterized by 4 Tauc–Lorentz oscillators. Mean square error (MSE) of this calculation was in the range of 28.7 nm. Optical constants i.e., refractive index (n) and extinction coefficient (k) obtained from this calculation are showed in Fig. 1B and is in agreement with results reported by other authors [19]. The spectroscopic ellipsometry investigations for the system of Si/B:NCD:O/Ppy, which was formed from ethanol-free solution was impossible due to large light diffusion scattering of this film. For this reason only Ppy films formed from initial polymerization mixture solution containing 1% and 10% of ethanol were investigated by spectroscopic ellipsometry. It was observed that dependencies of D and C vs. wavelength for the system based on Ppy formed from initial polymerization solution containing 1% of
0.0
(nm)
Fig. 1. (A) Dependence of ellipsometric parameters C and D on wavelength for Si/B:NCD:O substrate, dotted lines correspond to experimentally measured C and D, solid line correspond to modeling results obtained using 4 Tauc–Laurentz oscillators. In (B) the dashed line corresponds to refractive index dependence on wavelength (n) and the solid line corresponds to extinction coefficient (k) dependence on wavelength obtained after modeling procedure.
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209 b
C ¼ C s þ DC eðt=t Þ
0.10
8
here Cs is electrical capacitance at steady-state conditions, DC is difference between steady-state capacitance (Cs) and initial capacitance (C0) before mounting Ppy film into PBS, t is characteristic time of PBS absorption by MIP–Ppy and b is stretched exponential changing from 3/7 to 3/5. A well fit using Eq. (2) was obtained when Cs = 5.9 mF, DC = 1.96 mF, t = 1236 s, b = 0.55 (Fig. 4 curve 3). The value of parameter b is close to 0.6, which is typical for short range interaction. In case of MIP–Ppy treatment by PBS observed interaction is most probably based on the formation of hydrogen bounds, which are usually formed during the polymer swelling processes [25]. The analysis of electrochemical capacity change due to theophylline interaction was performed at steady-state conditions, which were achieved after treatment of the MIP–Ppy film with PBS. Moreover, it was assumed that theophylline interaction with Ppy takes place by the whole volume of MIP–Ppy. Due to high concentration of theophylline molecules (Neq) dispersed into the PBS, the final concentration of theophylline bounded by Ppy film at the equilibrium conditions have to be equal to Neq. This concentration can be found from the steady-state solution of first pseudo order kinetic Eq. (3) assuming that the concentration of theophylline (c) in solution is much higher than the density of molecular imprints (Ns) inside the MIP–Ppy film:
6
dN ¼ ka c ðNs NÞ kd N dt
4
here N is the density of theophylline molecules bonded by Ppy at a certain time instance and ka and kd are association and dissociation rate constants respectively. Nominating the ratio N/Ns as F (fill
0.08
Intensity
0.07 0.06 0.05 0.04 0.03 200
250
300
350
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(nm) Fig. 2. Reflectance intensity of multi-layered structure Si/B:NCD:O/Ppy dependence on light wavelength obtained from ellipsometric measurements. Si/B:NCD:O/ Ppy10%C2H5OH were Ppy films formed from polymerization bulk solution containing 10% of ethanol (doted-dashed line), Si/B:NCD:O/Ppy1%C2H5OH were Ppy film formed from polymerization bulk solution containing 1% of ethanol (solid line).
10 c)Si/B:NCD:O/Ppy b)Si/B:NCD:O/Ppy a)Si/B:NCD:O
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As it can be seen from Fig. 4, the prepared MIP–Ppy films (treated and untreated by ethanol) intensively absorb buffer solution up to a certain saturation level. The analysis of capacitance change kinetics in time (t) can be well fitted by stretched exponential law [24]:
1 nM 10 nM 2 10 nM
CS
3 10 nM
2
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1 0
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6.5 6.4 0,1 nM
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B 5.9 0
5. Evaluation of theophylline interaction with MIP–Ppy
1.5
1.0
0,1 nM
6.5 6.0
C, F
films were exposed towards the PBS solution containing different concentrations of theophylline. The films were exposed to different concentrations of theophylline gradually increasing by factor of 10 from 0.1 nM up to 1 mM. The arrows in Fig. 4 indicate the time instance at which these concentrations were applied. As can be seen from this figure the MIP–Ppy and MIP–Ppy(10%C2H5OH) films (Fig. 4A, curves 1 and 3) are strongly affected when first exposed to PBS buffer solution free of theophylline. There are no changes of electrochemical capacitance in the case of NIP–Ppy film. Kinetics of capacitance change of MIP–Ppy film formed from ethanol-free solution due to swelling and ion-migration reached steady-state after approximately 150 min. The injection of theophylline molecules induced additional electrochemical capacitance change. From Fig. 4, it is apparent that the MIP–Ppy is more sensitive to the addition of theophylline than the MIP–Ppy(10%C2H5OH) and the NIP–Ppy does not show any sensitivity at all.
C ( F)
7.5 Fig. 3. Diffuse reflection Rd vs. wavelength obtained for different systems: (a) Si/B: NCD:O substrate, (b) Ppy film formed from 1% ethanol containing initial polymerization mixture solution (Si/B:NCD:O/Ppy1%C2H5OH), (c) Ppy film formed from 10% ethanol containing initial polymerization mixture solution (Si/B:NCD:O/ Ppy10%C2H5OH).
1 2 3
3
C ( F)
0.09
Rd
(2)
1
2
3
4
time, h Fig. 4. MIP–Ppy films electrochemical capacity changes vs. time during interaction of theophylline, for: (A) 1—MIP–Ppy film, 2—stretched exponential model for PBS absorption, 3—MIP–Ppy(10%C2H5OH) film formed from 10% ethanol containing solution; arrows indicate time moments when different concentration of theophylline were injected. (B) NIP–Ppy electrochemical capacity changes vs. time during interaction of theophylline. Arrows indicate time moments when films are exposed to different concentrations of theophylline.
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1.0
(4)
0.8
C/ Cmax
factor) the following solution of this equation is obtained: t 1 F ¼ 1 exp ; t ¼ ka c þ kd t
Fig. 5 shows that the kinetics of theophylline interaction when the PBS influence to the capacity change is negligible could be described by formula (4). At equilibrium condition (when dN/ dt = 0) the Neq value can be obtained using following formula: Neq ¼ Ns
c c þ KD
eef ¼ ð1 FÞe1 þ F e2
(6)
here F is the fill factor showing the ratio between volume of the media having dielectric constant e1 and volume of the material with dielectric constant e2. In our case the F vs. time (t) can be calculated by formula (4). Due to the simple relation between dielectric constant (e) and electrochemical capacity (C) of the film C = e S/d (S is area of the electrodes and d is a distance between them) the e1 and e2 corresponds to the capacitance of the Ppy film at the beginning of interaction (CS) and at the end of the theophylline interaction due to establishment of steady-state conditions (Ceq) respectively when c >> Ns (Fig. 4). Thus then the fill factor F is equal to DC/DCmax = (Ce C1)/(C1 C2), here Ce is effective capacity, which is a function of time. The capacitance
1.0
fill factor
0.8 0.6 0.4 0.2 0.0 -100
100 nM theophyline kinetics
0
0.4
Ceq
C1
C1 C2 c c Kd
0.2 0.0
(5)
here KD = kd/ka; where kd, ka are dissociation and association rate constants respectively. In previous studies affinity constants (the total number of binding sites, Ki) were also calculated [26]. But in contrast to experiments described here an UV–vis spectroscopy was applied in order to analyze affinity of these previously described systems. We assume that experimentally obtained change of Ppy film electrochemical capacitance in the case of theophylline molecules binding has to be associated with changes of Ppy dielectric constant. At the steady-state conditions between Ppy and the solution with theophylline molecules, the Ppy film can be evaluated as ‘effective medium’ consisting of two materials having different dielectric constants e1 and e2. The e1 is effective dielectric constant of Ppy film, which was affected by PBS but not by the theophylline. e2 is the dielectric constant of this film impregnated by the theophylline up to concentration, which is present in Ppy film at steady-state conditions established between PBS containing defined concentration of the theophylline and Ppy film. The effective dielectric constant (eef) of the medium, which is a mixture of materials with dielectric constants e1 and e2, can be found using Drude’s approach:
0.6
0
200
400
600
800
1000
n (nM) Fig. 6. Relative ratio of changes in electrical capacitance obtained at steady-state conditions vs. different concentrations of theophylline.
changes kinetics after the injection of 100 nM theophylline and modeling of results using formula (5) is presented in Fig. 5. The best fitting result was obtained when fitting parameter t was 40 s. The electrochemical capacitance obtained at equilibrium conditions (Ceq) for different concentrations can be calculated using formulas (5) and (6): C eq ¼ C 1
ðC 1 C 2 Þ c c þ Kd
(7)
Fig. 6 shows fitting result (solid line) of experimental data (dots) using formula (6), where DC = Ceq C1 and DCmax = C1 C2. C1 and C2 are the capacitances of Ppy film at the initial phase of theophylline interaction and at the establishment of steady-state conditions, respectively, c— theophylline concentration, KD— affinity dissociation constant (KD = kd/ka), calculated for the interaction of theophylline: kd
MIP PpyðsolidphaseÞ þ theophyllineðdissolvedÞ ? MIP Ppy=theophyllineðsolidphaseÞ
ka
(8)
This modeling provides the possibility to obtain affinity dissociation constant (KD) value, which in this case is equal to 1.7 108 M. Using Eq. (9) it is possible to estimate Gibbs free energy change of theophylline/MIP–Ppy complex formation:
DG ¼
RT lnK 0D
(9)
where the KD0 = KD/c, in this case the c is the concentration of theophylline; Therefore, the KD0 is the value, which is calculated for standard reference concentration of 1 M [27]. It was found that DG = 43.5 kJ/mol for theophylline/MIP–Ppy complex formation, which shows that theophylline/MIP–Ppy complex formation is a thermodynamically favorable reaction. The obtained value of Gibbs free energy change is in the same order as that value calculated by other authors [28] and confirmed in our previous works [27]. These values are close to that which are typical for 2–3 hydrogen bounds formation and electrostatic interactions [29,30]. 6. Conclusions
100 200 300 400 500 600 700
time (s) Fig. 5. MIP–Ppy produced form polymerization bulk solution not containing ethanol, theophylline interaction kinetics using 100 nM concentration. Dots correspond to experimental points, solid line—for model obtained using formula (5).
Theophylline imprinted and non-imprinted polypyrrole thin films were deposited on boron doped nanocrystalline substrate formed on Si wafer. Evaluation of dielectric properties of NIP–Ppy demonstrated that it has low sensitivity to the theophylline solution in PBS. MIP–Ppy showed high affinity towards
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theophylline and therefore it could be used for the sensing of theophylline using impedance spectroscopy method. For low theophylline concentrations in the range from 0.1 to 103 nM more sensitive was the MIP–Ppy film formed from ethanol-free solution. Presence of ethanol in polymerization bulk solution induces changes in structure of MIP–Ppy. Acknowledgments The study was funded from the European Community’s social foundation under Grant Agreement No. VP1-3.1-ŠMM-08-K-01004/KS-120000-1756. We are grateful to Dr. Stoffel Janssens for formation of borondoped nanocrystalline diamond on boron-doped silicon. References [1] K. Haupt, Imprinted polymers-Tailor-made mimics of antibodies and receptors, Chem. Commun. 2 (2003) 171–178. [2] K. Mosbach, Molecular imprinting, Trends Biochem. Sci. 19 (1) (1994) 9–14. [3] A. Cutivet, C. Schembri, J. Kovensky, K. Haupt, Molecularly imprinted microgels as enzyme inhibitors, J. Am. Chem. Soc. 131 (41) (2009) 14699–14702. [4] V. Ratautaite, S.N. Topkaya, L. Mikoliunaite, M. Ozsoz, Y. Oztekin, A. Ramanaviciene, A. Ramanavicius, Molecularly imprinted polypyrrole for DNA determination, Electroanalysis 25 (5) (2013) 1169–1177. [5] P.S. Sharma, A. Pietrzyk-Le, F. D’Souza, W. Kutner, Electrochemically synthesized polymers in molecular imprinting for chemical sensing, Anal. Bioanal. Chem. 402 (10) (2012) 3177–3204. [6] K. Leonavicius, A. Ramanaviciene, A. Ramanavicius, Polymerization model for hydrogen peroxide initiated synthesis of polypyrrole nanoparticles, Langmuir 27 (17) (2011) 10970–10976. [7] L.D. Spurlock, A. Jaramillo, A. Praserthdam, J. Lewis, A. Brajter-Toth, Selectivity and sensitivity of ultrathin purine-templated overoxidized polypyrrole film electrodes, Anal. Chim. Acta 336 (1–3) (1996) 37–46. [8] L. Ozcan, Y. Sahin, Determination of paracetamol based on electropolymerized-molecularly imprinted polypyrrole modified pencil graphite electrode, Sens. Actuators B-Chem. 127 (2) (2007) 362–369. [9] D.R. Albano, F. Sevilla, Piezoelectric quartz crystal sensor for surfactant based on molecularly imprinted polypyrrole, Sens. Actuators B-Chem. 121 (1) (2007) 129–134. [10] B.S. Ebarvia, S. Cabanilla, I.F. Sevilla, Biomimetic properties and surface studies of a piezoelectric caffeine sensor based on electrosynthesized polypyrrole, Talanta 66 (1) (2005) 145–152. [11] W.M. Yeh, K.C. Ho, Amperometric morphine sensing using a molecularly imprinted polymer-modified electrode, Anal. Chim. Acta 542 (1) (2005) 76–82. [12] V. Ratautaite, A. Ramanaviciene, Y. Oztekin, J. Voronovic, Z. Balevicius, L. Mikoliunaite, A. Ramanavicius, Electrochemical stability and repulsion of polypyrrole film, Colloid Surf. A 418 (0) (2013) 16–21. [13] M.C. Blanco-Lopez, M.J. Lobo-Castanon, A.J. Miranda-Ordieres, P. TunonBlanco, Electrochemical sensors based on molecularly imprinted polymers, TRAC—Trends Anal. Chem. 23 (1) (2004) 36–48.
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