Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline

BIOJEC-06634; No of Pages 9 Bioelectrochemistry xxx (2012) xxx–xxx

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Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline Tan-Phat Huynh a, Chandra Bikram K.C. b, Wojciech Lisowski a, Francis D'Souza b,⁎, Wlodzimierz Kutner a, c,⁎⁎ a b c

Institute of Physical Chemistry, Kasprzaka 44/52, 01‐224 Warsaw, Poland Department of Chemistry, University of North Texas, 1155, Union Circle, #305070, Denton TX 76203‐5017, USA Faculty of Mathematics and Natural Sciences, School of Sciences, Cardinal Stefan Wyszynski University in Warsaw, Woycickiego 1/3, 01‐938 Warsaw, Poland

a r t i c l e

i n f o

Article history: Received 4 May 2012 Received in revised form 10 July 2012 Accepted 11 July 2012 Available online xxxx Keywords: Adrenaline molecularly imprinted polymer Bis(2,2′-bithienyl)methane derivatives Differential pulse voltammetry Piezoelectric microgravimetry Capacitive impedometry

a b s t r a c t New chemical sensors for adrenaline Adr were fabricated. For that, a thin film of a molecularly imprinted polymer (MIP) was templated with protonated adrenaline HAdr. Differential pulse voltammetry (DPV), capacitive impedometry (CI), or piezoelectric microgravimetry (PM) was integrated within each chemosensor for analytical signal transduction. First, molecular structure of prepolymerization complex of HAdr with bis(2,2′-bithienyl)methane substituted with the benzo-(18-crown-6) 2 and 4-carboxyphenyl 3 was thermodynamically optimized at the B3LYP/6-31g(d) level. Then, a HAdr-templated MIP film was deposited on the Pt disk electrode and on the Au film electrode of a 10-MHz quartz crystal resonator (QCR) by potentiodynamic electropolymerization from solution of HAdr, 2, 3, and the cross-linking tris([2,2′-bithiophen]-5-yl)methane 4 monomer at the HAdr:2:3:4 = 1:1:1:2 mole ratio. For determination of Adr, the DPV peak current for the K4[Fe(CN)6] redox probe under batch conditions and the resonant frequency and capacity of the electrical double layer under FIA conditions were measured. The lower limits of detection (LOD) were 2 nM, 0.5 μM, and 1.5 μM for the DPV, PM, and CI chemosensor, respectively, indicating suitability of the DPV chemosensor for Adr determination in biological systems (~50 nM). The chemosensors were appreciably selective to Adr in the presence of common interferents. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Adrenaline Adr, (R)-4-(1-hydroxy-2-(methylamino)ethyl)benzene-1, 2-diol, is a hormone and neurotransmitter involved in etiology and symptomatology of several neurological and psychiatric disorders [1]. In human plasma, concentration of adrenaline is quite low (~50 nM) [2]. Therefore, many advanced analytical techniques have been developed for its determination including colorimetry [3], liquid chromatography– mass spectrometry [4], amperometry as well as potentiometry with ion-sensitive field effect transistors (ISFETs) [5]. Moreover, other electroanalytical techniques, and particularly those involving voltammetric measurements [6–12], were employed. Electroanalytical techniques are widely used for this determination because of their superior detectability (at a nM concentration range), reliability (in terms of the potential, current, resistance, or capacitance signal measurements), speed, and simplicity in Adr determination. However, selectivity improvement of the recognition units of the sensors with respect to Adr is much required.

⁎ Corresponding author. ⁎⁎ Correspondence to: W. Kutner, Institute of Physical Chemistry, Kasprzaka 44/52, 01‐224 Warsaw, Poland. Tel.: +48 22 3433217; fax: +48 22 3433333. E-mail addresses: [email protected] (F. D'Souza), [email protected] (W. Kutner).

Protonated adrenaline (HAdr) is specifically recognized in nature at pH values below that of pKa = 8.55 of Adr [13] with five different amino acids, i.e., Ser204, Ser207, Phe290, Asn293, and Asp113, of the β2-adrenergic receptor via five-point binding. These involve the hydrogen bonds formed at the hydroxy and protonated amine groups as well as π–π interactions at the benzene ring of HAdr. If nature is to be mimicked for artificial sensitive and selective determination of Adr, this binding implies fabrication of a chemosensor featuring as many as possible recognition sites. Herein, we mimicked natural adrenaline recognition by engineering an artificial receptor. Fabrication of new and efficient synthetic materials for chemical recognition has largely developed since 1940 when the theory of biological specificity based on the antibody and antigen interactions was first described [14]. In 1949, first specific adsorbents with inherent memory were prepared. These absorbents were capable of selective recognition of an azo dye analyte in the presence of other azo compounds by using a silica gel matrix [15]. After decades, this idea of artificial selective recognition was revolutionized with the synthesis of molecularly imprinted polymers (MIPs) [16,17]. In these syntheses, functional and cross-linking monomers were co-polymerized in the presence of a target analyte, initially serving as the template. For several recent years, electrochemical polymerization has extensively been explored for that purpose [18–22]. Subsequent removal of the

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Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003

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template rendered a recognition material capable of the analyte selective binding. Among different chemosensor signal transductions, piezoelectric microgravimetry (PM) and several electroanalytical techniques play a very important role [23]. MIP based chemosensors for neurotransmitters, including catecholamines, histamine, tryptamine, etc., have widely been described [24–27]. For example, the lower limit of detection (LOD) of 10 nM dopamine was reached by using a MIP film integrated with a PM transducer [27]. However, only a few studies have applied MIPs for determination of Adr. In one study, a film of MIP based on the methacrylic acid (MAA) functional monomer was used to imprint Adr [28,29]. The MIP film of the MAA-HAdr complex was deposited by spin coating of the MAA-HAdr:poly(vinyl chloride) (1:1, w:w) suspension in tetrahydrofuran on the electrode of a 9-MHz quartz crystal resonator (QCR). Although LOD of the resulting acoustic chemosensor was quite low, being 20 nM, a major disadvantage of this chemosensor included low mechanical stability of the MIP film because of its poor adherence to the electrode surface. Besides, there were some attempts of theoretical optimizations of structures of

complexes of HAdr with different recognition compounds, such as 12-crown-4 [30], 15-crown-5 [31], as well as formate and its derivatives [32]. However, these studies focused on interaction of a single recognition site of the complex analyte using a single functional monomer rather than multiple functional monomers for different binding sites of the analyte to achieve a multi-point binding, which would result in higher selectivity. In continuation of our research on determination of biogenic amines using MIP chemosensors [25–27], the present work aims at fabrication of a selective MIP chemosensor for Adr. Toward that, two electroactive functional monomers based on bis(2,2′-bithienyl) methane, i.e., bis(2,2′-bithienyl)-benzo-[18-crown-6]methane 2 and bis(2,2′-bithienyl)-(4′-carboxyphenyl)methane 3, were engaged to construct a multi-point pre-polymerization complex of HAdr (Scheme 1a). Then, a MIP film was deposited by potentiodynamic electropolymerization. The advantage of this polymerization procedure includes fast and easy control over the film thickness with the number of potential cycles as well as over the effective electrode area with the careful selection of a porogenic solvent and supporting

Scheme 1. (a) Structural formula and (b) the B3LYP/6-31g(d) optimized structure of the complex of HAdr bound to 2 and 3. Compound 4 was used as a cross-linking monomer.

Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003

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electrolyte. Finally, three different transduction schemes were applied to the chemosensors for determination of HAdr and their analytical parameters were compared. That is, differential pulse voltammetry (DPV) using a K4[Fe(CN)6] redox probe was applied for indirect Faradaic determination of HAdr relaying on the change of the DPV peak current of the probe under batch analysis conditions. Moreover, PM and capacitive impedometry (CI) were utilized for this determination under flow-injection analysis (FIA) conditions with respect to the change of resonant frequency and double layer capacity, respectively. Adr is electroactive in the potential range of thiophene electropolymerization [33]. In case of an electroactive template, at least two procedures are available to avoid imprinting of products of this electro-oxidation instead of the genuine template itself. These involve application of either an electroinactive close analog of the template [34] or a barrier underlayer film [25]. In the present work, we have overcome this difficulty in another way, which involved careful control of film thickness during electropolymerization of the pre-polymerization complex. 2. Experimental 2.1. Reagents and chemicals Acetonitrile, L-adrenaline, ascorbic acid, L-3,4-dihydroxyphenylalanine (L-DOPA), catechol, and all chemicals (except of 60% perchloric acid) for synthesis were purchased from Sigma-Aldrich. Trifluoroacetic acid (TFA) and tetra-n-butylammonium perchlorate [(TBA)ClO4] were supplied by Fluka. Ethanol, toluene, potassium hexacyanoferrate(II) [K4Fe(CN)6], sodium hydroxide (NaOH), potassium fluoride (KF), and potassium nitrate (KNO3) were from CHEMPUR. 60% Perchloric acid was purchased from Fisher. The functional monomer 2 was prepared according to the described procedure [25]. Procedures of syntheses of 3 and 4 are described in Supporting Information. 2.2. Instrumentation An AUTOLAB computerized electrochemistry system of Eco Chemie (Utrecht, The Netherlands), equipped with expansion cards of the PGSTAT 12 potentiostat and the FRA2 frequency response analyzer and controlled by the GPES 4.9 software of the same manufacturer, was used for the potentiodynamic, cyclic voltammetry (CV), DPV, and electrochemical impedance spectroscopy (EIS) or CI measurements. The X-ray photoelectron spectroscopy (XPS) spectra were recorded on a PHI 5000 VersaProbe (ULVAC-PHI) scanning ESCA Microprobe using monochromatic Al-Kα radiation (hν = 1486.6 eV). The Casa XPS software was used to evaluate the XPS data. Background was subtracted using the Shirley method and peaks were fitted with (Gaussian– Lorentzian)-shaped profiles. The binding energy (BE) scale was referenced to the C 1s peak with BE = 284.6 eV. The UVISEL spectroscopic ellipsometer of HORIBA Jobin Yvon (Longjumeau, France) was used for the MIP film surface characterization based on the polarization of light in the spectral range of 245 to 2100 nm. The contact angle between the incident light beam and the sample surface was 70°. The Forouhi Bloomer model of optical dispersion (see Supporting Information) using the DP2 software of HORIBA was applied for fitting optical parameters in order to determine thickness of the MIP films. A model EQCM 5610 and EQCM 5710 [35] quartz crystal microbalance, controlled by the EQCM 5710-S2 software, all of the Institute of Physical Chemistry (Warsaw, Poland), was used to perform the PM experiments under the FIA and batch analysis conditions, respectively. The resonant frequency change was measured with 1-Hz resolution using a 14-mm diameter, AT-cut, plano-plano, QCR of 10-MHz resonant frequency with 5-mm diameter and 100-nm thick Au film electrodes evaporated over a

3

Ti underlayer film on both its sides. However, only one QCR side was wetted by a working solution and the Au film electrode of this side was used both as the working electrode and the substrate for the recognition MIP film. The resonators were cleaned for 5 min with ethanol before electropolymerization. A large-volume radial-flow thin-layer electrochemical cell [36] was used to perform the CI experiments under FIA conditions controlled by the AUTOLAB system. The 1-mm diameter Pt disk working electrode was axially mounted opposite to the inlet capillary tip at the distance of 100 μm. A Pt wire and an Ag/AgCl electrode were used as the auxiliary and reference electrodes, respectively. During measurements, this cell was completely filled (~ 35 mL) with the 0.1 M KF carrier solution.

2.3. Fabrication of the molecularly imprinted polymer (MIP) film Preparation of the HAdr-templated MIP (MIP-HAdr) films involved electropolymerization performed under potentiodynamic conditions over the potential range of 0.50 to 0.95 V vs. Ag/AgCl at the potential scan rate of 20 mV/s. The growth of the films, on the 1-mm diameter Pt disk electrode and on the Au electrode of 10-MHz QCR, was controlled by the number of potential cycles. After electropolymerization, the MIP-HAdr films were rinsed with the abundant acetonitrile solvent in order to remove excess of the supporting electrolyte. Then, the HAdr template was extracted with 0.01 M NaOH for 2 h at 60 °C. Completeness of the extraction was confirmed by the XPS measurements. A non-imprinted polymer (NIP) control film was deposited from the template-free solution using the same electropolymerization procedure.

2.4. Calculations and measurements Structures of the complexes formed between HAdr and different bis(2,2′-bithienyl)methane functional monomers were optimized and association energy changes were evaluated at the B3LYP/ 6-31g(d) level using the Gaussian-09 software [37]. For the batch analysis experiments, the 1-mm diameter Pt disk electrodes coated with the HAdr-extracted MIP-HAdr films were soaked for 10 min in solutions of different concentrations of HAdr or interfering compounds. Then, DPV curves were recorded for solution of 0.1 M K4[Fe(CN)6] in 0.1 M KNO3. The potential range, potential step, pulse amplitude, and pulse duration were 0 to 0.5 V, 5 mV, 25 mV, and 50 ms, respectively. For the FIA experiments involving PM detection, water was used as the carrier liquid. It was pumped at the 20 μL/min flow rate with the model KDS100 syringe pump of KD Scientific (Holliston MA, USA). A model 7725i rotary six-port valve of Rheodyne (Cotati CA, USA) was used to inject the sample solution of the volume of 200 μL. These FIA conditions were selected such that the dispersion coefficient was equal to one. Samples of the analyte and interferents were dissolved in solution of the same composition as that of the carrier liquid, i.e., water for PM determinations. For these determinations, the Au electrode of 10-MHz QCR, coated with the HAdrextracted MIP-HAdr film, was examined using the flow-through EQCM 5610 holder. The FIA conditions of determination of Adr by CI were the same as those of PM, however, the flow-through EQCM 5610 holder was replaced by the large-volume radial-flow thin-layer electrochemical cell. The applied potential was kept constant, Eappl = 0.40 V vs. Ag/AgCl, at the value of the absence of any Faradaic process. Moreover, frequency was kept low and constant, f = 20 Hz. Samples of the analyte and interferents were dissolved in solution of the same composition as that of the carrier solution, i.e., 0.1 M KF.

Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003

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3. Results and discussion 10

3.1. Molecular modeling of interactions between binding sites of protonated adrenaline (HAdr) and recognizing sites of different derivatives of bis(2,2′-bithienyl)methane

3.2. Deposition of the MIP-HAdr film by potentiodynamic electropolymerization In accordance with the results of the quantum chemistry modeling, an MIP was synthesized. For that, the 0.1 mM Adr, 0.1 mM 2, 0.1 mM 3, 0.2 mM 4, 0.02 mM TFA (pH = 4.0), and 0.1 M (TBA)ClO4 acetonitrile– toluene (4:1, v:v) solution for electropolymerization was prepared. Compounds 2 and 3 played the role of functional monomers and 4 that of the cross-linking monomer. The solution was spiked with toluene to dissolve completely 3 and 4. That was because of their non-polar nature incurred by the bithiophene moieties. The MIP-HAdr film was deposited by potentiodynamic electropolymerization on the working electrode from the above solution. This electropolymerization at the Pt working electrode was manifested by the anodic peak at 1.10 V vs. Ag/AgCl and the shoulder at 1.50 V (curves 2 and 3, respectively, in Fig. 1). Moreover, the anodic peak of HAdr oxidation in the presence of the functional monomers was negatively shifted, because of complex formation, by 0.10 V with respect to that of HAdr, which was present at ~0.75 V in a blank solution (curve 1 in Fig. 1). Presumably, the complex of the product of adrenaline oxidation was

Table 1 Calculated values of thermodynamic functions of formation of a complex of the protonated adrenaline, HAdr, and two derivatives of bis(2,2′-bithienyl)methane bearing different recognition sites. Complex formation Hydrogen bonding of the catechol moiety of Adr to carboxy group of 3 Inclusion binding of 1-hydroxy-2-(methylammonium) moiety of HAdr to benzo-[18-crown-6] entity of 2 Total

ΔH/kJ mol−1

ΔG/kJ mol−1

−48.0

−3.7

−175.1

−106.7

−223.1

−110.4

ΔH and ΔG are the enthalpy and free energy change due to formation of the complex of the MIP cavity and the HAdr molecule.

I / μA

6

3 2

4

2

1 0 0.4

0.6

0.8

1.0

1.2

1.4

1.6

E / V (vs. Ag/AgCl) Fig. 1. Cyclic voltammetry curves, recorded at the 1-mm diameter Pt disk working electrode, for the solution of (1) 0.1 mM HAdr, (2) 0.1 mM 2, 0.1 mM 3, and 0.2 mM 4, and (3) 0.1 mM HAdr, 0.1 mM 2, 0.1 mM 3, 0.2 mM 4, in 0.02 mM TFA (pH=4.0) and 0.1 M (TBA)ClO4, in acetonitrile:toluene (4:1, v:v). The potential scan rate was 20 mV/s.

electrochemically polymerized rather than that of pristine HAdr in this experiment. Although the anodic peak of HAdr electro-oxidation appeared at a relatively low potential, it decreased and was positively shifted in each consecutive potential cycle (Fig. 2). Apparently, the more advanced the electropolymerization, the thicker the MIP-HAdr film. Hence, HAdr was electro-oxidized to a lesser extent. This was because the growing MIP-HAdr film played a role of a self-barrier of increasing resistance preventing electro-oxidation of HAdr. Therefore, the anodic peak of electro-oxidation of HAdr vanished after ten potential cycles (curve 3 in Fig. 2). The increase of resistance of the MIP-HAdr film with the increase of the film thickness, controlled by the number of potential cycles, was estimated by the EIS measurements of the complex impedance, Z, in the presence of the K4Fe(CN)6 redox probe. This impedance is generally expressed as ′

Z ¼ Z −jZ

″

ð1Þ

1.5

4

1.0

I / μA

By molecular modeling, we herein selected functional monomers forming a feasible 3-D structure for imprinting of HAdr. For that, first, structures of complexes of the protonated amine group and two hydroxy groups of HAdr with several recognition sites, anchored to bis(2,2′-bithienyl)methane (Fig. A1 in Supporting Information), were optimized by calculating the free energy difference, ΔG, between the formation energy of the complex and the sum of formation energies of the individual moieties. It appeared that the ΔG gain for the complex involving the protonated amine group of HAdr and the benzo-[18-crown-6] substituent was much higher than those of the benzo-[15-crown-5], benzoic acid, or hydroxy substituent. Moreover, this gain for complexation of two hydroxy groups of HAdr was higher for the benzoic acid substituent than for the 3,4-dihydroxy benzene substituent. Therefore, 2 and 3 were selected for complexation of HAdr in our subsequent experimental studies. In this complexation (Scheme 1b), oxygen atoms 17, 18, and 23 of the benzo-[18-crown-6] substituent of 2 form hydrogen bonds with hydrogen atoms 96, 97, and 103 of the protonated secondary amine and hydroxy groups of HAdr while the hydrogen atom 88 and the oxygen atom 89 of two different hydroxy substituents of the aromatic ring of HAdr form hydrogen bonds with the oxygen atom 148 and hydrogen atom 150 of the carboxy substituent of 3, respectively. That way, multi-point recognition of HAdr was afforded. Subsequently, changes of enthalpy and entropy for the structure-optimized complex were calculated (Table 1).

8

1 2

0.5

3 0.0 0.5

0.6

0.7

0.8

0.9

E / V (vs. Ag/AgCl) Fig. 2. Potentiodynamic curves for deposition of the MIP-HAdr film from the solution of 0.1 mM HAdr, 0.1 mM 2, 0.1 mM 3, 0.2 mM 4, 0.02 mM TFA (pH = 4.0), and 0.1 M (TBA)ClO4 in acetonitrile:toluene (4:1, v:v) recorded at the 1-mm diameter Pt disk working electrode during the (1) first, (2) fifth, (3) tenth, and (4) two-hundredth potential cycle for the 0.50 to 0.95 V potential range. The potential scan rate was 20 mV/s.

Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003

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where Z′ and Z″ are its real and imaginary parts, respectively, and j is imaginary unit. Apparently, the MIP-HAdr film growing on the Pt electrode increasingly hindered electro-oxidation of Fe(CN)64− to Fe(CN)63− due to the increase of the charge transfer resistance, Rct, determined from diameter of the semicircle of the Z″ vs. Z′ plot (Fig. 3) [38]. Resistance of the 1000-cycle MIP-HAdr film, used in some further experiments (see Section 3.5.1, below), was Rct ≅ 2.3 (±0.1) kΩ. This relatively high resistance additionally indicates that conductivity of the 1000-cycle MIP-HAdr film coating the Pt electrode is sufficiently low for the CI measurements (see Section 3.5.3, below).

5

a

3.3. Ellipsometric determination of thickness of the MIP-HAdr film Thickness of the MIP-HAdr film was determined by spectroscopic ellipsometry. To this end, experimental optical parameters (Table A1 in Supporting Information) of the MIP-HAdr film, deposited by potentiodynamic electropolymerization on a gold coated glass slide in the course of 1000 potential cycles, were acquired by measuring polarization of the light scattered. These parameters were then fitted with those of the amorphous phase (Forouhi–Bloomer) model. The fitting resulted in thickness of the film, l = 30 (±3) nm.

CN / a.u.

b

c

3.4. Extraction of the adrenaline template from the MIP-HAdr film The Adr template was extracted from the MIP-HAdr film before determination of HAdr. For this liquid–solid extraction, 0.01 M NaOH was selected by taking advantage of the HAdr deprotonation in basic solutions. The completeness of the extraction was confirmed by the XPS measurements of the high-resolution N 1s spectrum because HAdr was the only source of nitrogen in the HAdr-templated MIP film. Apparently, the N 1s peak of the secondary protonated amine of HAdr at 401.5 eV (Fig. 4b) disappeared after the extraction (Fig. 4c). Another N 1s peak at 399.6 eV (Fig. 4b and c), still remaining after the extraction, was most likely due to the presence of some nitrogen containing contaminant. This peak was also present in the XPS spectrum of the NIP film (Fig. 4a).

2.0

Rct / kΩ

2.0

1.5 1.0

Z'' / kΩ

0.5 0.0

1.5

6

0

200

400

600

800 1000

NC 1.0

1 0.5

0.0

0.0

0.5

1.0

402

400

398

396

BE / eV Fig. 4. The XPS high resolution spectra of N 1s showing the number of counts, CN, vs. binding energy, BE, for the (a) NIP film and the MIP-HAdr film (b) before and (c) after extraction of 1 with 0.01 M NaOH. The MIP-HAdr film was deposited by potentiodynamic electropolymerization in the course of 1000 potential cycles, as described in the caption of Fig. 2.

3.5. Determination of adrenaline using different transduction schemes For determination of Adr with the MIP-HAdr recognition film, three different signal transductions were used, including DPV as well as PM, and CI under the batch analysis as well as FIA conditions, respectively.

3.0

2.5

404

1.5

2.0

2.5

3.0

Z' / kΩ Fig. 3. The electrochemical impedance spectroscopy (EIS) imaginary impedance, Z″, against real impedance, Z′, plots and the charge transfer resistance, Rct, for MIP-HAdr films of different thicknesses expressed in terms of the number of potential cycles of deposition, NC, (Inset), for 0.1 M K4Fe(CN)6 in 0.1 M KNO3 at 0.30 V vs. Ag/AgCl, for (1) the 1-mm bare Pt disk electrode as well as for the electrode coated by the MIP-HAdr film grown by (2) 50, (3) 100, (4) 200, (5) 600, and (6) 1000 potential cycles. Frequency was scanned in the range of 70 kHz to 0.1 Hz.

3.5.1. Differential pulse voltammetry (DPV) under batch analysis conditions For HAdr determination under batch analysis condition, an indirect DPV procedure was adopted. This procedure exploits hindrance of the Fe(CN)64−/Fe(CN)63− electro-oxidation and the resulting decrease of its DPV peak due to the presence of an analyte in an MIP film coating the working electrode [39]. Apparently, the DPV peak current of the K4Fe(CN)6 electro-oxidation was relatively high for the ~ 30-nm thick MIP-HAdr film after extraction of HAdr (curve 1 in Fig. 5a) because the imprinted cavities were left vacant. Therefore, Fe(CN)64− readily penetrated the film for electro-oxidation to Fe(CN)63− at the electrode substrate. Subsequently, the HAdr-free film was immersed for 10 min in the HAdr solutions of different concentrations, which were 0.5 nM in HCl (pH = 7.0) and 0.1 M in Fe(CN)64−. In effect, the DPV peak for Fe(CN)64− was lower the higher the HAdr concentration in solution (curves 2 through 7 in Fig. 5a) because more imprinted cavities were then occupied by HAdr molecules. Therefore, Fe(CN)64− could not freely permeate through the film. For determination of the dependence of sensitivity and the linear dynamic concentration range on the MIP-HAdr film thickness, four

Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003

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10 7

a

1

1

6

(IDPV,e - IDPV, s) / μA

8

6

4

2 3 4

4 3 2

7

2

5

5 1

0

0.1

0.2

0.3

0

0.4

35

b

2'

20 15

3'

10

4'

5 0

0

40

60

80

100

Fig. 6. DPV calibration plots expressed as the dependence of the difference of the DPV peak current for the HAdr-extracted and then HAdr-soaked MIP-HAdr film, IDPV,e, and for the HAdr-extracted MIP-HAdr film, IDPV,s on solute concentration, cHAdr, (IDPV,e − IDPV,s)/μA = 2.469(±0.089) + 0.043(±0.002)cHAdr/nM at the ~30-nm thick MIP-HAdr film coated 1-mm diameter Pt disk electrode, after removing the HAdr template with 0.01 M NaOH for 2 h at 60 °C, and then soaking with solutes of (1) HAdr, (2) catechol, (3) L-DOPA, and (4) ascorbic acid in 0.1 M K4Fe(CN)6 in 0.1 M KNO3 as well as for (5) the ~30-nm thick NIP film coated 1-mm diameter Pt disk electrode soaked in solutions of different concentrations of HAdr in 0.1 M K4Fe(CN)6 in 0.1 M KNO3.

1'

30 25

20

cL / nM

E / V (vs. Ag/AgCl)

20

40

60

80

100

cHAdr / nM Fig. 5. (a) DPV curves, for 0.1 M K4Fe(CN)6 and 0.1 M KNO3 at the ~30-nm thick MIP-HAdr film coated 1-mm diameter Pt disk electrode, for (1) HAdr-extracted with 0.01 M NaOH, and then (2) soaked in the 1, (3) 10, (4) 30, (5) 50, (6) 70, and (7) 100 nM HAdr solution of 0.5 nM HCl (pH = 7.0). (b) DPV calibration plots for HAdr (expressed as the dependence of the difference of the DPV peak current for the HAdr-extracted and then HAdr-soaked MIP-HAdr film, IDPV,e, and for the HAdr-extracted MIP-HAdr film, IDPV,s) on the HAdr concentration, cHAdr, deposited in the course of (1′) 150, (2′) 300, (3′) 600, and (4′) 1000 potential cycles.

HAdr-templated MIP-HAdr films were deposited on the Pt electrodes in the course of 150, 300, 600, and 1000 potential cycles. Then, the HAdr template was extracted with 0.01 M NaOH and the films immersed for 10 min in 0.5 nM HCl (pH = 7.0) solutions of HAdr of different concentrations (Fig. 5b). As expected, the higher the number of cycles, NC, the lower was the anodic DPV peak current. That was because the thicker the MIP-HAdr film the higher was its resistance. Moreover, the shapes of the calibration plots changed from nearly exponential (curves 1′, 2′, and 3′ in Fig. 5b) for thin films to linear (curve 4′ in Fig. 5b) for a thicker film because of higher number of cavities and higher resistance of the latter. Apparently, the linear range was wider (1 to 2, 1 to 5, 1 to 10, and 1 to 100 nM HAdr) and sensitivity lower (2.23, 0.90, 0.28, and 0.043 μA/nM) the thicker (prepared by 150, 300, 600, and 1000 potential cycles, respectively) the MIP-HAdr film. For the thickest MIP-HAdr film, i.e., that of l ≅ 30(±3) nm deposited in the course of 1000 potential cycles (see Section 3.3, above), the regression equation of the calibration plot (curve 1 in Fig. 6) and the correlation coefficient of the calibration plot were (IDPV,e − IDPV,s)/ μA = 2.47(±0.09) + 0.04(±0.00)cHAdr/nM and 0.99, respectively. Accuracy of this measurement was ~ 60 nA. The cHAdr is the molar concentration of HAdr while IDPV,e and IDPV,s are the DPV peak current for the MIP-HAdr film with the HAdr template being extracted, and

for the HAdr-extracted MIP-HAdr film soaked with the HAdr analyte, respectively. At the signal-to-noise ratio, S/N = 3, detectability was appreciably high, LOD = 2.0 nM. The ~ 30-nm thick MIP-HAdr film was used to compare the DPV chemosensor selectivity with respect to HAdr and common interfering compounds, such as catechol, L-DOPA, and ascorbic acid (curves 2, 3, and 4, respectively, in Fig. 6). The sensitivity with respect to HAdr, 0.04 (± 0.00) μA/nM, was nearly twice those to catechol, 0.02 (±0.00) μA/nM, and L-DOPA 0.03 (± 0.00) μA/nM, and thrice that to ascorbic acid 0.02 (±0.00) μA/nM (Fig. 6). Moreover, the sensitivity of the ~ 30-nm thick NIP film to HAdr (curve 5 in Fig. 6) was 0.002 (±0.001) μA/nM. Hence, the imprinting factor, calculated from the ratio of the sensitivity to HAdr of MIP and NIP, was advantageously as high as, IF ≅ 20 (±3). Despite this relatively high IF value, the selectivity reached may be insufficient for adrenaline determination in real samples containing excessive amounts of these interferents. Then, either they should be determined independently using other procedures or separated prior to adrenaline determination with the chemosensors devised herein. For testing chemosensor repeatability, the HAdr-extracted DPV chemosensor was immersed in 100 nM HAdr solution (pH = 7.0) for 10 min. The determined relative standard deviation (n = 5) of the HAdr quantization did not exceed 3% indicating high repeatability. 3.5.2. Piezoelectric microgravimetry (PM) under flow-injection analysis conditions The optimized procedure of preparation of the MIP-HAdr film for the DPV chemosensor (see Section 3.3, above) was also used for deposition of a ~ 30-nm MIP-HAdr film on the Au film electrode of the 10-MHz QCR for PM measurements. Thin MIP films of bis(2,2′-bithienyl)methanes are rather rigid; visco-elastic effects negligibly contribute to the measured resonant frequency changes [25,26]. For rigid films, the resonant frequency change, Δf, is opposite to the mass change, Δm, as the Sauerbrey relation, Eq. (2), [40] predicts.

Δf ¼ −

2f 0 2 Δm  1=2 Aac μ q ρq

ð2Þ

Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003

T.-P. Huynh et al. / Bioelectrochemistry xxx (2012) xxx–xxx

In this equation, f0 is the fundamental frequency of the resonator (10 MHz), Aac is the acoustically active area of the resonator (1.96 × 10 −5 m 2), μq is the shear modulus of quartz (2.95 × 10 10 Pa), and ρq is the quartz density (2648 kg m −3). After each injection of the HAdr solution of different concentration, the resonant frequency decreased (Fig. 7) because ingress of HAdr to the film increased its mass. Moreover, this resonant frequency decrease was proportional to the increase of the HAdr concentration. Then, the frequency increased to reach the baseline as HAdr was eluted from the film with excess of the carrier solution. In effect, a peak-shaped detection signal was formed, advantageously. Apparently, the HAdr binding in the film was fully reversible. The linear dynamic concentration range was 1 to 10 μM obeying the regression equation of Δf/Hz = − 6.7(± 0.1) − 1.1(± 0.0)cHAdr/μM with the correlation coefficient of 0.99. Accuracy of this measurement was ~ 0.3 Hz. At S/N = 3, detectability of the PM chemosensor, LOD = 0.50 μM, was much poorer than that of the DPV chemosensor (see Section 3.5.1, above) with the 1.1(± 0.0) Hz/μM sensitivity.

5

a 1

3

5

Moreover, for a complex formed between the imprinted cavity of the HAdr-template free MIP-HAdr and a ligand, L, i.e., the analyte or interferent molecule MIPHAdr þ L⇄ðMIPHAdrÞL

ð3Þ

values of the stability constant, Ks, KS ¼

½ðMIP−HAdrÞ−L ½ðMIP−HAdrÞ½L

ð4Þ

and different interferents were determined for the PM chemosensor (Table A2 in Supporting Information) using the literature procedure [25,41]. In this procedure, the dependence of the (MIP-HAdr)-L concentration, [(MIP-HAdr)- L], on time is expressed as d½ðMIPHAdrÞ−L=dt ¼ −Bðdf =dt Þ ¼ ka cL ð f max −f Þ−kd f :

ð5Þ

B = −[A (μq ρq) 1/2 / 2 f02 Mw V] being the proportionality factor obtained from Eq. (2). Mw is the molecular weight of the (MIP-HAdr)- L complex, V is the volume of the MIP film, and fmax is the maximum change of the resonant frequency for a particular L concentration. The stability constant is determined from the ratio of the complex association, ka, and dissociation rate constant, kd. K s ¼ ka =kd

10 μM

7.5

7

ð6Þ

Integration of Eq. (5) against time as well as substitution

0

f eq ¼ ka cL f max =ðka cL þ kd Þ -5

ð7Þ

Δf / Hz

result in f eq ¼ f max ½1− expðkobs t Þ

-10

ð8Þ

where -15

kobs ¼ ka cL þ kd :

-20 0

20

40

60

80

100

120

t / min

b

-18

Δf / Hz

-15

-12

-9

-6

0

2

4

6

8

10

cHAdr / M Fig. 7. (a) The resonant frequency change with time for HAdr, recorded at the ~30-nm thick MIP-HAdr film coated 10-MHz QCR after extraction of the HAdr template with 0.01 M NaOH for 2 h at 60 °C, and then injection of the 200-μL samples of aqueous solutions of HAdr of different concentrations as well as (b) the calibration plot for HAdr, Δf/Hz = −6.72(±0.11) − 1.08(±0.02)cHAdr/μM.

ð9Þ

Here, feq is the equilibrium frequency at time t (200 s) and kobs is the apparent association rate constant. Values of kobs were derived from Eq. (7) by fitting them to the experimental data. Values of ka were determined from slopes and those of kd from intercepts of the linear regression plots of kobs vs. analyte or interferent concentration, cL, according to Eq. (9) (Fig. A2 in Supporting Information). The mass transport effects were accounted for with the K4Fe(CN)6 redox probe, as described previously [27]. Apparently, the order of changes of the determined Ks values is consistent with that of the sensitivity of the DPV chemosensor (see Section 3.5.1, above), i.e., Ks for the complex of MIP-HAdr with HAdr, 913 (±77) M−1, is almost twice those with catechol, 441 (±57) M−1, and L-DOPA, 470 (±46) M−1, and nearly thrice that with ascorbic acid 348 (±78) M−1. 3.5.3. Capacitive impedometry (CI) under flow-injection analysis conditions The HAdr analyte was also determined under FIA conditions using the CI transduction. In this determination, the change of capacity of the electrical double layer, Cdl, was measured. For constant and sufficiently low frequency, f = 1 / 2πω (where ω is angular frequency), this capacity can easily be determined for the electrode of the area A from the measured imaginary component of impedance, Z″, by adopting a simple Randles equivalent circuit [42–44]. ″

Z ¼

1 ωC dl A

ð10Þ

Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003

8

T.-P. Huynh et al. / Bioelectrochemistry xxx (2012) xxx–xxx

The determined herein double-layer capacity was higher the higher the concentration of HAdr in consecutively injected samples (Fig. 8). Most likely, this capacity increase originated from the increase of capacity of the compact part of the double layer. This was because nearly no contribution of capacity of the diffuse layer part to the overall capacity was expected at a relatively high concentration of the supporting electrolyte of specifically non-adsorbing ions (0.1 M KF) used. Therefore, the Helmholtz model of the compact layer is applicable here with its double-layer capacity dependent on electric permittivity, ε, and the compact layer thickness, d, according to Eq. (11)

C dl ¼

ð11Þ

where ε0 is electric permittivity of free space. Apparently, the permittivity increase due to the HAdr ingress to the MIP-HAdr film was responsible for the measured capacity increase. This capacity was higher the higher the HAdr concentration in solution (Fig. 8). The linear dynamic concentration range extended from 1 to 22 μM HAdr with the calibration plot described by the regression equation of Cdl/F m −2 = 5.867(±0.001) × 10 −2 + 2.44(±0.02) × 10 −5 cHAdr/ μM with the correlation coefficient of 0.99. Accuracy of this measurement was ~ 7 × 10 −5 F m −2. At S/N = 3, detectability and sensitivity were LOD = 1.5 μM and 2.4 × 10 −8 F m −2 μM −1, respectively.

0.062

a Cdl / (F m-2)

Chemosensor transduction

LOD

Ref.

Colorimetry LC–MS–MSa ISFETb DPV SWVc CV PM Amperometry

20 μM 55 nM 1 μM 2, 40, and 250 nM 0.15, 9.4, and 34 nM 1 μM 20 nM 5.2 μM

[3] [4] [5] [6,10,11] [7,9,12] [8] [28] [29]

a

εε0 dA

0.060

0.058

10 μM

0.056

30

50

50

0

75

100

100

150

200

t / min 0.0615

b 0.0610

Cdl / (F m-2)

Table 2 Comparison of LOD of different chemosensors described in literature for determination of adrenaline.

0.0605

b c

LC–MS–MS—liquid chromatography–tandem mass spectrometry. ISFET—ion-sensitive field-effect transistor. SWV—square wave voltammetry.

4. Conclusions Molecular modeling appeared to be an effective tool for approximating structure and stability of the pre-polymerization complex of HAdr and functional monomers selected from a range of bis(2,2′-bithienyl) methane derivatives. The highly negative gain of the free energy of the complex formation proved that the complex (Scheme 1) was sufficiently stable to survive the electropolymerization leading to formation of the MIP-HAdr film. Moreover, the order of changes of the determined values of the stability constant of complex formation, Ks, is consistent with that of the experimental chemosensor sensitivity. Potentiodynamic electropolymerization with the lowest possible positive potential reversal is another novel way of deposition of an MIP film in the presence of an electroactive template, such as HAdr. It takes advantage of a pronounced anodic shift of the electrooxidation potential of the complexed HAdr template with the MIP-HAdr film growth during the electropolymerization. At sufficiently low anodic potential reversal selected, this oxidation is shifted outside this potential advantageously preventing HAdr electro-oxidation even though this procedure requires enormous numbers of potential cycles for electropolymerization. The LOD value for the devised chemosensor featuring a ~ 30-nm thick MIP-HAdr film, with the high imprinting factor of ~ 20, was as low as 2 nM HAdr for the DPV transduction, reached under batch analysis conditions. Moreover, this LOD value is lower than those reported by most of the other published researches using the DPV transduction (Table 2). However, the LOD values of the PM and CI chemosensors, attained under the FIA conditions, were much higher. Nevertheless, these chemosensors are more practical in use because of their easier and less time consuming operation. LOD for the FIA-PM chemosensor was much lower than that for the FIA-CI one. However, the PM experiments are more demanding. Importantly, the HAdr determination under FIA conditions is advantageous from the throughput point of view because it is less chemicals and time consuming than that under the batch conditions.

0.0600

Acknowledgments 0.0595 0.0590

20

40

60

80

100

cHAdr / μM Fig. 8. (a) The electrical double layer capacitance, Cdl, change with time, recorded at the ~30-nm thick MIP-HAdr film coated 10-MHz QCR, after extraction of the HAdr template with 0.01 M NaOH for 2 h at 60 °C, and then injection of the 200-μL samples of the 0.1 KF (pH = 8.3) solutions of different concentrations of HAdr as well as (b) the calibration plot for HAdr, Cdl/F m−2 = 5.867(± 0.001) × 10−2 + 2.44(±0.02) × 10−5 cHAdr/μM.

The financial support of the Foundation for Polish Science (MPD/2009/1/styp19) to TPH, the NanOtechnology, Biomaterials, and aLternative Energy Source for ERA integration (FP7‐REGPOT‐ CT‐2011‐285949-NOBLESSE project) to WK as well as the National Science Foundation of USA (Grant No. 1110942 to FD) is gratefully acknowledged.

Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bioelechem.2012.07.003.

Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003

T.-P. Huynh et al. / Bioelectrochemistry xxx (2012) xxx–xxx

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Please cite this article as: T.-P. Huynh, et al., Molecularly imprinted polymer of bis(2,2′-bithienyl)methanes for selective determination of adrenaline, Bioelectrochemistry (2012), doi:10.1016/j.bioelechem.2012.07.003