Metal incorporated molecularly imprinted polymer-based electrochemical sensor for enantio-selective analysis of pyroglutamic acid isomers

Sensors and Actuators B 186 (2013) 407–416

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Metal incorporated molecularly imprinted polymer-based electrochemical sensor for enantio-selective analysis of pyroglutamic acid isomers Bhim Bali Prasad ∗ , Indu Pandey Analytical Division, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India

a r t i c l e

i n f o

Article history: Received 10 April 2013 Received in revised form 8 June 2013 Accepted 11 June 2013 Available online 20 June 2013 Keywords: Molecularly imprinted metallo-polymer d- and l-pyroglutamic acid Electro-polymerization Pencil graphite electrode Enantio-selective analysis

a b s t r a c t This paper reports a new class of pencil graphite electrode duly modified with a molecularly imprinted metallo-polymer for enantio-selective sensing of d-/l-pyroglutamic acid, in aqueous and real samples. Herein, specific binding sites of d- and l-isomers were created in their respective three-dimensional motifs of highly conducting imprinted film. This was developed through the electropolymerization of copper(II)–5-methyl-thiophene–2-carboxylic acid complex, in the presence of analyte (template:monomer molar ratio 1:2). The detection of isomers could be feasible at operating conditions (pH, deposition potential, deposition time, etc.) of differential pulse anodic stripping voltammetry [aqueous sample, linear range 2.8–170.0 ng mL−1 , limit of detection, 0.77 ng mL−1 (S/N = 3)]. The proposed sensor was also validated with dilute real samples (urine, cerebrospinal fluid and blood plasma). Although several chronic diseases (metabolic acidosis, 5-oxoprolinuria, etc.) are known to be manifested at hyper-concentrations of l-pyroglutamic acid (d-isomer is biologically inactive), the sample dilution by several fold, which mitigates biological matrix effect, is necessarily required. This warrants a highly sensitive probe of analysis within the linear quantitation range 1.3–180.0 ng mL−1 , without any crossreactivity and false-positives. The endogenous concentrations of real samples could simply be obtained by multiplying the concentration of dilute sample with respective dilution factor. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Metal ion coordination plays important role in biological recognition systems, since many pharmaceutics demonstrate biological activities when present in the form of metal complexes. Metal ion mediated molecular imprinting has drawn considerable attention [1–7] since its first report by Fujii et al. [8]. Accordingly, the metal ion is employed as an assembled pivot for assembling both the functional monomer and the template. In such complex imprinted polymers (CIPs), metal ion mediated imprinting may impart more stability in water and protic solvents owing to inherent covalent co-ordinations, in comparison to the traditional approach. Also, there is a possibility to alter affinity as well as selectivity by replacing the metal with other metal ions. The intrinsic characteristics of water compatibility of CIPs have been explored in many fields like catalysis [1], separation [2], molecular recognition [3], sensors [4,6,7], and solid-phase micro extraction [5]. Despite the fact that we have already been able to fabricate for the first time a CIP-based electrochemical sensor for the enantio-selective recognition [7].

∗ Corresponding author. Tel.: +91 9451954449; fax: +91 54222368127. E-mail address: [email protected] (B.B. Prasad). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.06.041

This warrants more improvement in the process development of molecularly imprinted polymer (MIP) film. In our earlier work, the MIP film was obtained via cumbersome free radical polymerization using surface “grafting from” approach. Alternatively, an electrochemically grown MIP directly on the solid surface could be more facile to obtain highly sensitive ultra-thin layer (nano-film). MIP involves the formation of cavities in a synthetic polymer matrix that are complementary to a template (analyte) molecule, in terms of functional and structural characters. Metal-containing conducting polymers, i.e. metallo-polymers are unique systems in which metal groups could be tethered, coupled, or incorporated [9]. Of these, the polymer, which has the metal group directly incorporated into the conjugated backbone, is known to influence greatly on the conducting property of the film. This type of system is three-dimensional in presence of metal [Cu(II) containing octahedral geometry] that might exhibit a wide range of physical and electronic properties of n-type semi-conductors, involving typically a hopping mechanism of electro-conductivity [10]. Thus, metallo-polymers are those materials that combine some of the redox properties of the conducting polymer and some of the metal ion. A large range of transition-metal-containing materials, particularly thiophene derivatives, have been electropolymerized to grow thin conducting films easily on a variety

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of substrates for applications in organic and molecular electronics [10,11]. The high polarizability of sulfur atoms in thiophene rings may lead to stabilization in the conjugated chain for an excellent charge transport. There always exists a possibility of strong interaction between the sulfur atom of thiophene ring and metal ion. This could be, however, not possible while oxidizing thiophene at a potential that gives an activated species, generally a radical cation, for the subsequent dimerization. The dimer so obtained can further undergo polymerization through the metal ion mediation involving associated terminal functionalities, such as amine, imine, and carboxylic acid as ligands for the metal ion complexation [12,13]. In the same meaning, we are interested in this work to develop a Cu(II) mediated MIP-modified electrode using 5-methyl-2-thiophene carboxylic acid (5-MTCA) as a monomer and pyroglutamic acid (PGA) as a template. To the best of our knowledge, such MIPs based on conducting metallo-polymer are not yet reported. PGA is essential amino acid which is electro-inactive species in aqueous solution. Of the two isomers of PGA, l-PGA is bioactive to improve blood circulation in the brain, whereas d-PGA remains inactive. In human genetic disorder, such as acetaminophen-induced metabolic disorder, a high level of pyroglutamic acid is secreted in the urine that is known as 5-oxoprolinuria. Overproduction of PGA also leads to increased PGA in plasma (normal concentration 2.5 × 103 ng mL−1 , higher concentration 284.0 × 103 ng mL−1 ), cerebrospinal fluid (normal concentration 5.3 × 103 ng mL−1 , higher concentration 169.1 × 103 ng mL−1 ) and massive urinary excretion (normal concentration 1.03 × 103 ng mL−1 , higher concentration 813.3 × 103 ng mL−1 ) [14–16] to manifest severe metabolic acidosis, hemolytic anemia and central nervous systems dysfunction [17]. Chromatographic methods have exclusively been reported for enantioselective recognition [15]; others chromatographic work are, however, only limited to l-PGA quantifications [18–21]. A single MIP selective for l-PGA is also reported [22] but no analytical aspects have been investigated. In view of the possible complications from real matrices, hyper-concentrations of l-PGA are difficult to be estimated and therefore, a reliable, rapid, selective and sensitive method of analysis for l-PGA in dilute sample is sought for. 2. Experimental 2.1. Chemicals and reagents All chemicals were of analytical reagent grade, and used without further purification. Demineralized triple distilled water (TDW) (conducting range 0.06–0.07 × 10−6 S cm−1 ) was used throughout the experiment. d- and l-PGA, 5-MTCA, CuSO4 , and all interferents (d-PGA, d-/l-proline, d-/l-glutamic acid, d-/l-aspartic acid, d-/l-methionine, l-cysteine, l-histidine, ascorbic acid, dopamine, glutathione and l-tryptophan) were purchased from Aldrich (Steinheim, Germany), Loba Chemie (Mumbai, India) and Spectrochem Pvt. Ltd. (Mumbai, India). Solvents, acetonitrile and triethylamine, were purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). Stock solution (0.5 mg mL−1 ) was prepared by dissolving 50 mg d- or lPGA in 100 mL water. All working solutions of the test analyte were prepared by an appropriate dilution of the stock solution with water. Standard phosphate buffer solution was prepared by dissolving 0.1290 g sodium dihydrogen phosphate and 7.3350 g KCl in 100 mL water; desired pH values of these solutions were adjusted with the addition of a few drops of 0.1 M HCl or 0.1 M NaOH solution. The human blood plasma, cerebrospinal fluid (CSF), and urine were obtained from the Institute of Medical Sciences, Banaras Hindu University, and stored for a week in a refrigerator at −4 ◦ C, before use. These biological samples were found to be spoiled after a week. Pencil rods (2B grade, 0.5 mm diameter, 5.0 cm length) were purchased from Hi Par, Camlin Ltd. (Mumbai, India).

2.2. Apparatus Differential pulse anodic striping voltammetry (DPASV) and cyclic voltammetry (CV), were performed using a three electrode cell assembly consisted of MIP-modified pencil graphite electrode (PGE), a platinum wire, and Ag/AgCl (saturated KCl) as working, counter, and reference electrodes, respectively. The instrument used was a polarographic analyzer/stripping voltammometer [model 264 A, EG & G Princeton Applied Research (PAR), USA] in conjunction with an electrode assembly (PAR model 303 A) and X-Y chart recorder (PAR model RE 0089). For IR characterization, a section of MIP-modified layer was scrapped out with a razor-blade from the surfaces of a bunch of MIP-modified electrodes. FT-IR (KBr) spectra were recorded on Varian 3100 FT/IR (USA) spectrometer. Morphological images of modified PGEs, were recorded using scanning electron microscope (SEM) (JEOL, JSM, Netherlands, model 840 A). UV–visible analysis was performed on Varian Cary 100 Bio UV–visible spectrophotometer (USA). Atomic force microscopy (AFM) experiments were performed on a dimension 3100 scanning electron microscopy (Vecco Instruments Inc., USA), in the tapping mode. X-ray photoelectron spectroscopy (XPS) measurements have been carried out for MIP-adduct, MIP, and poly [5-MTCA] polymer, using a Perkin Elmer 1257 model operating at an average base pressure of ∼5 × 10−8 torr at 300 K with a nonmonochromatized Al K␣ line at 1486.6 eV, and a hemispherical sector analyzer capable of 25 meV resolution. All experiments were performed at 25 ± 1 ◦ C. 2.3. Electrode preparation The pencil graphite rod was first pretreated with 6 M HNO3 for 15 min, followed by water-washings. After smoothing its surface by soft cotton, this was inserted into a Teflon tube. Any micro gaps all around in between the graphite rod and the tubing were sealed properly with the help of an epoxy spray. The tip of the pencil rod at one end was gently rubbed with an emery paper (No. 400) to level the pencil surface along the tube orifice. Electrical contact was made by soldering a metallic wire to the exposed reverse side of the pencil rod. The PGE was preferred as a working electrode because of its low background current, wide potential window, chemical inertness, and low cost as compared to other solid electrodes (Pt, Au, Pd, Ag, etc.) [23]. 2.4. Fabrication of MIP-modified PGE MIP, i.e. PGA imprinted poly[5-MTCA–Cu(II)] film, was obtained by the electro-polymerization of 5-MTCA (0.1 mM), in the presence of Cu(II) and l-PGA (template), directly over the tip of the PGE. For electropolymerization, the prepolymer solution [CuSO4 (0.05 mM), 0.1 mM 5-MTCA, d- or l-PGA (0.05 mM), 1 mL each] was taken with 7.0 mL of supporting electrolyte (phosphate buffer, pH = 5.0) in a voltammetric cell consisted of three electrodes PGE, Ag/AgCl (saturated KCl), and platinum wire as working, reference and auxiliary electrodes, respectively. Note that all Cu(II) ions were self-assembled in prepolymer mixture and as such there were no possibility to get any Cu(II) to complex with phosphate buffer. After nitrogen purging for 15 min, electro-polymerization was carried out under CV scans (n = 5) from −2.0 V to +2.0 V at the scan rate of 50 mV s−1 . The tentative mechanism of electropolymerization is shown in Fig. S1. The l-PGA–Cu(II)–poly-5MTCA modified electrode was characterized between −2.0 and +2.0 V in the pH region of 1.0–9.0. Electropolymerization CVs scan was performed at optimized pH 5.0 of phosphate buffer solution. Any pH higher or lower than 5.0 always led to an unsymmetrical broad CV peak. Accordingly, the 5-MTCA underwent electro-oxidation [9] and dimerization [24], followed by the deprotonation of terminal

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409

Scheme 1. Schematic representation of MIP-modified PGE fabrication.

carboxylic groups (pH = 5.0, pKa = 3.5) leading Cu(II)-mediated complexation to eventually form a uniform coating of MIP-adduct film at PGE surface. The modified surface was washed with water to remove any residual monomer left with the polymer matrix. Finally, the MIP-coated PGE was obtained after the complete removal of embedded l-PGA moieties from the MIP-adduct film using an eluent, acetonitrile and triethylamine (1:1) mixture, for 60 min. Herein, triethylamine effectively disrupted Cu(II)–l-PGA coordination bonds to form a basic salt of l-PGA that was readily soluble in acetonitrile. This led to the creation of selective molecular cavities in the film (Scheme 1). The complete removal was ensured by XPS measurement which revealed absence of nitrogen atom peak corresponding to PGA (see Section 3.3). A non-imprinted polymer (NIP)-based PGE (omitting only template) and another PGE modified with only poly[5-MTCA], were also prepared for the comparative studies. 2.5. Voltammetric measurements MIP-modified PGE was immersed in the voltammetric cell containing 10.0 mL phosphate buffer (pH 1.0). An aliquot of a freshly prepared solution of d- or l-PGA was introduced in to the cell for analyte accumulation at −0.4 V vs Ag/AgCl for 90 s, with magnetic stirring. All CV runs were scanned under anodic stripping mode within the potential window −0.2 to +0.6 V (respective to Ag/AgCl), at different scan rates (10–200 mV s−1 ). DPASV runs were recorded from −0.2 to +0.4 V at 10 mV s−1 with 25 mV pulse amplitude. Since dissolved oxygen present in the cell did not affect stripping current, any de-aeration of the cell content was not necessary. The limit of detection (LOD) was calculated as three times the standard deviation for the blank measurement in the absence of test analyte divided by the slope of the calibration plot between analyte concentration and DPASV current [25]. Voltammetric measurements were also performed using the NIP-modified PGE, under identical operating conditions. 3. Results and discussion 3.1. Development of MIP film by electropolymerization Enantioselective imprinting mechanism of PGA is proposed in Scheme 2. The softer pencil lead (2B) is appropriate for modification, because it is thicker containing more graphite contents than

the harder ones (2H, H and HB) [26]. This was advantageous to hold MIP film onto PGE surface through the deep penetration of MIP particles in its micro-pores. In Fig. 1A, CV runs did not show any oxidation or reduction peaks of poly[5-MTCA] in the potential range from −2.0 and +2.0 V, revealing its electro-inactivity in the phosphate buffer solution [27]. Herein, the appearance of a very feeble cathodic peak at about 0.0 V is interesting. This could be ascribed by the buildup of adsorbed impurities or the formation of oxide film that were reduced during repetitive reverse CV scans at activated pre-anodized electrode surfaces. However, in case with modified PGE where 5-MTCA was electro-polymerized (n = 5, CV scans) exclusively in presence of copper, a pair of oxidation and reduction peaks were observed at about +0.3 V and −0.5 V, vs Ag/AgCl (saturated KCl), respectively (Fig. 1B). The difference between oxidation and reduction potentials (Ep = 800 mV) and anodic–cathodic peaks current ratio (Ipa /Ipc = 1.0) suggest a quasireversible one-electron oxidation–reduction of 5-MTCA–Cu(II) film [28]. However, in the presence of analyte, 5-MTCA–Cu(II) complex assumed octahedral conformation inhibiting its electrontransfer behavior and inducing an electro-catalytic action to convert PGA into an electro-active species. Fig. 1C depicts electropolymerization (n = 5, CV scans) of 5-MTCA on PGE surface in the presence of Cu(II) and d-/l-PGA. In this case, a large oxidation peak at −0.1 V, accompanying virtually an ill-defined broad reduction peak (presumably due to electro-reduction of the oxidation product) on the reverse scan, is observed (PGA oxidation is irreversible). The larger magnitude of current in Fig. 1C might be attributed to the exclusive oxidation of the analyte, under “electro-catalytic effect” of complexed monomer. The significant decrease of analyte oxidation peak with increasing number of scans (Fig. 1C) and its negative shift by ∼200 mV might be due to the Cu(II)-mediated complexation of PGA with two molecules of 5-MTCA. Such complexation during the course of electro-polymerization was stabilized to the extent that the electro-activity of complexed metal was totally restricted. The proposed complexation apparently proceeded through the amino and carbonyl groups of PGA involving Cu(II) bridge, which shouldered two electron-rich poly[5-MTCA] chains keeping well apart from each other, under electrostatic repulsive forces (Scheme 2). The decrease in CV current (Fig. 1C) upon repetitive CV scans indicates the continual growth of polymer film at the electrode surface; and thereby, a hindrance in electron transport is observed. Therefore, the thickness of polymer film should be controlled by optimizing the number of CV scans for the

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Scheme 2. Schematic illustration of enantioselective imprinting and rebinding of d-PGA and l-PGA.

development of maximum current response, upon analyte binding. For details on the optimization of polymerization conditions, vide Supplementary data section S.1. Accordingly, the optimized CV cycles n = 5, scan rate 50 mV s−1 , and template–Cu(II)–monomer ratio 1:1:2, were adjudged as best parameters for obtaining the maximum current response (Figs. S2 and S3). Stoichiometry of Cu(II)–l-PGA (1:1) complex was studied by UV–vis spectroscopy and the Job’s method of continuous variation. The preferential

use of Cu(II) amongst other divalent ions (Ni(II), Co(II), Zn(II)) was based on relatively high stability constant (ˇ = 0.85) of Cu(II)–l-PGA (1:1) complex (vide Supplementary Data section S.1). 3.2. Spectral characterizations of imprinted films FT-IR spectra (Fig. S4) of monomer (curve A), template (curve B), MIP-adduct (curve C), and MIP (curve D) are comparatively studied

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411

Fig. 1. CVs scans for polymer modification of PGE: (A) poly[5-MTCA], (B) poly[5-MTCA–Cu(II)], and (C) poly[5-MTCA–Cu(II)–l-PGA]. Electropolymerization conditions; 0.05 mM l-PGA, 0.1 mM 5-MTCA, 0.01 M phosphate buffer (supporting electrolyte pH 5.0), no. of CV scans 5, potential range −2.0 to +2.0 V vs Ag/AgCl, scan rate 50 mV s−1 .

to support the binding mechanism suggested in Scheme 2, In the presence instance, the coordination (ionic) bonds are considered as a key factor to promote a stable analyte binding in the aqueous medium [29]. Consequently, the major bands due to monomer [curve A, OH (3034 cm−1 ), C O (1727 cm−1 )] and due to template [curve B, NH (3431.8 cm−1 ), C O (1725 cm−1 )] are shifted downward [2826.36 cm−1 (OH), 3318.1 cm−1 ( NH), 1660 cm−1

( C O, monomer), 1640 cm−1 ( C O)], on complexing with Cu(II) in MIP-adduct (curve C). It could be interesting to note that IR bands corresponding l-PGA are disappeared after its extraction from the MIP-adduct (curve D); whereas bands due to the monomer [complexed with Cu(II)] remained unaffected. Based on this, one may conclude that template and monomer were mediated by the metal ion for complexation. It should be further noted that the CH3

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Fig. 2. SEM and AFM images of MIP-adduct modified PGE (A and C) and MIP modified PGE (B and D), respectively.

stretching vibration occurring at −2950 cm−1 (curve A) of 5-MTCA is vanished and CH CH (1580 cm−1 , in curves C and D) vibration band is appeared. This indicates CH3 conversion into vinyl group, consequent upon electro-polymerization. Interestingly, template bands, which were found to be disappeared (curve D) on the template retrieval, reinstated at their original positions after analyte rebinding in the aqueous medium (cf, curve C). 3.3. Surface characterization SEM image of the surface of MIP-adduct modified electrode revealed a uniformly grown electropolymerized compact film (Fig. 2A). However, MIP-modified PGE surface (Fig. 2B) appeared to be consisted of conglomerate beads having porous texture, after template extraction, which might increase surface area to benefit electrochemical reactivity. Unlike the SEM which provides twodimensional projection or a two-dimensional image of a surface, the AFM provides a true three-dimensional surface profile. In this work, polymeric coatings as reflected by AFM image offered a topographic contrast direct height measurement. Accordingly, an elevated layer with the surface height of 14.8 nm, having root mean square (RMS) roughness (Rq ) 1.12 nm and arithmetic mean roughness (Ra 1.02 nm), is obtained for bare PGE surface (Fig. 2C). When a MIP layer was electrochemically assembled on the bare PGE surface, the total film height is increased up to 18.4 nm (Fig. 2D) with Rq 2.03 and Ra 2.57 nm. An average estimate of the thickness of MIP film can be calculated using Eq. (1) [30]: z(x, y) = s(x, y) + t + z(x, y)

(1)

where z(x,y) is the surface height (18.4 nm) of MIP/PGE, s(x,y) is the surface height (14.8 nm) of bare PGE, t is the average thickness, and

z(x,y) is the inherent roughness (RMS roughness, Rq = 2.03 nm) of MIP film. Accordingly, the mean thickness (t) of imprinted polymer film on the exposed tip of PGE could be obtained as 1.57 nm. This value is approximately equal to that (1.58 nm) derived from the total charge density of CV curve (Fig. 1C) recorded during electropolymerization; for details vide Supplementary Data section S.1. XPS (Fig. S5, A–C) was used to investigate the surface composition of poly[5-MTCA], MIP-adduct, and MIP-coated PGE surfaces, respectively. It also indicated the presence of Cu(II) in MIPadduct and MIP films. Fig. S5A corresponding to the poly[5-MTCA] film indicates sharp peaks of C1s, O1s, and S2p. Upon electropolymerization of 5-MTCA in the presence of Cu(II) and PGA, all signals corresponding to poly[5-MTCA] are retained with Fig. S5B. However, in this case, additional signals are emerged related to Cu(II) with binding energies (BE) 935.5 eV (for Cu 2p3/2) and 950.5 eV (for Cu 2p1/2) as well as N1s with BE 399.2 eV (for PGA). The Cu(II) incorporation in the film is indicated by the shift of oxygen BE from 531.5 to 526.0 eV consequent upon complexation of COOH group of 5-MTCA with Cu(II). Notably, thiophene sulfur atom was not participated in such complexation, because S2p BE (164 eV) remained unaltered during the course of electro-polymerization. Thus, it could be concluded that electropolymerization in this work exclusively involved the formation of Cu(II) O bonds to obtain incorporated metal ions in the polymer backbone (metallopolymer). Further, the complete retrieval of the template from MIP-adduct is ensured by the conspicuous absence of N1s peak of the template (Fig. S5C) in the polymer motif. The fitted (deconvoluted) O1s, N1s and C1s XPS spectra of MIPadduct are shown in Fig. S6(A–C). Fig. S6A depicts two peaks at 531.0 and 532.5 eV, corresponding to C O and C O, respectively. Fig. S6B displays only one peak at 399.2 for N1s. Fig. S6C is

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consisted of four peaks at 284.5, 285.8, 286.9, and 287.6 eV which can be attributed to C C, C N, C O, and C O groups, respectively. Providing the polymer surface X moles of 5-MTCA and Y moles of PGA, the following equation can be used to estimate the ratio of 5-MTCA to PGA in MIP-adduct [31]: 8 − C/N X = Y 2C/N − 6

3.5. Chronocoulometry Diffusion coefficient (D) could be determined chronocoulometrically involving charge (Q) versus square root of time (t1/2 ) plot of the Anson equation [35]: Q = 2nFAC(Dt)1/2 −1/2 + Qads + Qdl

(2)

Qads = nF 0 A

where C and N represent the XPS corrected peak areas of carbon and nitrogen. The ratio of peak areas C/N as shown in Fig. S6 is 4:1. Thus, an actual 5-MTCA and PGA molar ratio (X/Y) is obtained as 2:1, which is in good accordance to that obtained from the stoichiometry discussed in the Supplementary Data section S.1.

O

O

(6)

where A is the electrochemical surface area vide Supplementary Data Section S.2), C is the concentration of analyte (0.85 ng mL−1 ), Qdl is the double layer charge, Qads the faradic oxidative charge, and  0 is the surface coverage. The slope 4.015 × 10−6 ␮C s−1 of Q vs t1/2 plot revealed an estimate of D as 3.0 × 10−7 cm2 s−1 . The intercept of this plot tantamounts to the total charge [(Qdl + Qads ) = 9.088 × 10−6 C]. The value of Qdl could be obtained using the similar plot made in the absence of analyte. On subtraction of Qdl from the total charge, Qads is obtained to be 9.033 × 10−6 C. The value of  0 can be obtained in terms of the number of electrons (n) from the following empirical equation defining Nerstian adsorbate layer [36]:

MIP film is a hybrid material carrying both organic and inorganic moieties that candidly serve as an active mediator for the electro-catalytic oxidation of l-PGA. Fig. 3A presents CV runs of l-PGA (concentration 129.0 ng mL−1 ) on the l-PGA imprinted MIPmodified PGE (electrode 1). These runs were recorded within the potential ranging from −0.2 to +0.6 V (respective to Ag/AgCl) in phosphate buffer (pH 1.0), at different scan rates (10–200 mV s−1 ), after analyte accumulation in MIP cavities at −0.4 V for 90 s. Since, no peak is observed in run 1 (blank), this implies that Cu(II) ion, when incorporated within the polymer matrix, is not oxidized/reduced in the potential range studied. However, with the presence of l-PGA, an anodic peak at about 0.1 V is obtained, notwithstanding the fact that l-PGA is electro-inactive in water. On reverse scan, the broader current between −0.1 V and −0.2 V could be ascribed to the reduction of 3,4-dihydropyrrole-2-one (oxidation product). The reason could be the “electro-catalytic action” of 5-MTCA–Cu(II) that turned l-PGA to be electro-active. In the metallo-polymer, the polymer moiety acts as “synthetic metal/organic metal” having semiconductor characteristics [32] and Cu(II) acts as a electro-catalyst. Under such condition, l-PGA decarboxylation occurred followed by its stripping from the MIPcavity at the electrode surface. Consequently, the analyte after rebinding disturbed the steady state of charge mediation along the polymer chain and provoked an oxidative current. The anodic shift of CV peak potential with scan rate (Fig. 3A) indicates the involvement of proton in electro-oxidation of l-PGA. The enhanced electro-conductivity in the present instance can be better comprehended in terms of the p-type semiconductor behavior of complexed metal in the polymer chain. Accordingly, Cu(II) ion being an electron acceptor might inject positive hole to l-PGA [33] to induce electro-oxidation in accordance to the following known electrode process [34]:

H N

(5)

(36.9 × 10−3 cm2 ,

3.4. Electrochemical behavior

O

413



Ipa =

n2 F 2 4RT



 0 Av

(7)

The values of n and  0 are obtained as ∼2.0 and 1.26 × 10−9 mol cm−2 , respectively, on the basis of Eqs. (6) and (7). In the present instance,  0 reflects the total surface coverage of analyte (0.46 × 10−10 mol or 2.8 × 1013 molecules) specifically bound to MIP cavities (each molecule per cavity). The apparent decrease in the diffuse distance, owing to metal ion mediation in between the redox center and the surface of electrode, could be considered as a prime factor for the faster diffusion of analyte molecules into MIP cavities. It should be noted that d-PGA imprinted MIP-modified PGE had similar behavior in terms of electrodics as that with l-PGA imprinted PGE. 3.6. Analytical applications DPASV is superior to CV in terms of the sensitivity of the measurement. As is evident from Fig. 3(B-C), the proposed dor l-PGA imprinted sensor did not reveal any response in blank (supporting electrolyte) solution (run 1). l-PGA imprinted sensor (electrode 1), however, was responsive (Fig. 3B) exclusively for l-PGA either alone (run 5) or in racemic mixture (run 6) but not for d-PGA (run 3). This revealed no change in the current even after spiking with d-PGA (run 4). In the similar tune, D-PGA imprinted sensor (electrode 2) was found to be

N

Cu(II)

+ CO2 + 2e- + 2H+

pyroglutamic acid

OH

3,4-Dihydro-pyrrol-2-one

The, CV runs so obtained bear linear relationships between peak current (Ip ) and scan rate (v1/2 ) and in between peak potential (Ep ) and v, as described below: Ip (␮A) = (5.89 ± 1.83) + (0.75 ± 6.61)v1/2 ,

R2 = 0.997

Ep (V) = (0.25 ± 0.004) + (0.107 ± 0.003) log v,

R2 = 0.997

(3) (4)

Accordingly, the electron process in this study was basically a diffusion-controlled phenomenon.

responsive for D-PGA either alone or in racemic mixture, and not responsive for l-PGA at any concentration studied (Fig. 3C). Based on these observations, the development of Cu(II) ion mediated MIP template interactions and spatial arrangements of MIP cavities for both d- and l-PGA are suggested in Scheme 2. Accordingly, enantioselective binding of both isomers was feasible owing to their distinct 3D spatial arrangements generating corresponding imprints (chiral cavities) in the polymer network. Herein, functional constraints were imposed toward rebinding of d-and l-PGA,

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Fig. 3. (A) CV runs (anodic stripping) of l-PGA (129.0 ng mL−1 ) on MIP-modified PGE (electrode 1) at different scan rates: (1) blank, 10, (2) 10, (3) 20, (4) 50, (5) 100, and (6) 200 mV s−1 ; (B) DPASV response on MIP-PGE (electrode 1, imprinted for l-PGA) for different concentrations of PGA isomers in aqueous solutions: (1) blank, (3) d-PGA (12.0 ng mL−1 ), (4) d-PGA (20.0 ng mL−1 ), (5) l-PGA (12.5 ng mL−1 ), and (6) l-PGA and d-PGA mixture (each 26.0 ng mL−1 ); (C) DPASV response on MIP-modified PGE (electrode 2, imprinted for d-PGA) for different concentrations of PGA isomers in aqueous solutions: (1) blank, (3) l-PGA (12.0 ng mL−1 ), (4) l-PGA (20.0 ng mL−1 ), (5) d-PGA (15.0 ng mL−1 ), and (6) d- and l-PGA mixture (each 24.0 ng mL−1 ). DPASV response of different concentrations of PGA isomers in urine sample (D), blood plasma (E), and CSF (F) on electrode 1: (1) blank, (3) d-PGA (10 ng mL−1 ), (4) d-PGA (15.0 ng mL−1 ), (5) l-PGA (10.0 ng mL−1 ), and (6) l-PGA and d-PGA mixture (each 29.7 ng mL−1 ). DPASV response of 150 ng mL−1 PGA on NIP-modified PGE (run 2) in aqueous and real samples (urine, blood plasma and CSF; all containing l-PGA) as shown in B–F, respectively. [Optimized operating conditions: Eacc −0.4 V vs Ag/AgCl, tacc 90 s, phosphate buffer pH 1.0].

primarily due to the difference in configuration of carboxylic groups of PGA isomers that reinforced requisite enantioselectivity in MIP cavities (carboxylic groups in the molecular structure of PGA are shown above and below the plane in the case of d-and l-isomers, respectively, in Scheme 2). The l-PGA analysis was validated on electrode 1 in the complicated matrices of real sample (Fig. 3D–F). Optimum operating conditions for both d- and l-PGA imprinted sensors are equivalent. Both d- and l-imprinted sensors revealed almost equal current and quantitative (100%) recovery of their respective template (analyte) of known concentration (Table S1). Fortuitously, the respective NIP-modified PGE sensors responded test analyte insignificantly <0.5 ␮A not detectable in DPASV curve drawings even at concentration as high as 150.0 ng mL−1 , both in aqueous as well as real samples [Fig. 3(B–F), run 2]. For the quantitative measurement, a series of different concentrations of l-PGA in aqueous and real samples, were investigated with electrode 1 at the optimized operating conditions. The corresponding results are summarized in Table 1 and respective calibration plots are shown in Fig. S7. [For details on optimization of analytical parameters, vide Supplementary Data section S.3.] The real samples were necessarily diluted to several folds so as to move the measurement to the linear range of concentration (1.3–180.0 ng mL−1 ). Sample dilutions otherwise also helped to mitigate the matrix complications and false-positive (non-specific) contributions. Any pretreatment

of biological samples such as ultra-filtration, deproteinization, or centrifugation have to be deliberately avoided as these may lead inaccuracies in results. Insofar as the generation of calibration curves (Fig. S7) is concerned, a series of biological samples were first diluted and the original concentration of test analyte was obtained by the proposed sensor employing the “method of standard addition” for each sample. Latter, the diluted sample was spiked with different amounts of authentic analyte to obtain various samples of known concentrations (the term ‘concentration’ means original amount plus spiked amount of test analyte in a certain volume of diluted samples) for DPASV measurements. However, the endogenous (original) concentration of supplied samples (undiluted) can only be obtained simply multiplying the result of diluted (unspiked) sample by the respective dilution factor (Table 1). Different techniques used for l-PGA determination were compared with the present work (Table S2). The proposed sensor is relatively simple and cost-effective involving less instrumentation for enantio-selective analysis of PGA isomers, in aqueous and real samples (LODs = 0.7–0.9, S/N = 3). The major limitation of this sensor is its time consuming (60 min) regeneration process involving the method of template retrieval. Apparently, MIP-based HPLC column would also require equivalent amount of time for the regeneration, and unfortunately routine HPLC method [2] was found to be relatively less sensitive, laborious and highly expensive than this work

415

[Table S2]. In view of the fact that no enantioselective analysis of PGA in biological fluids has been reported by HPLC till date, the proposed sensor is quite suitable for enantioselective analysis of PGA in clinical settings, despite the time consuming regeneration process involved in consecutive DPASV measurements.

1.9

1.4

1.1

0.8

1.7

RSDd (%) (n = 3)

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0.9

0.86

0.75

99.0–100.1 (5.43 × 10 to 5.24 × 10 )

99.7–101.2 (2.52 × 103 to 3.03 × 105 )

99.9–100.5 (1.08 × 103 to 1.28 × 105 )

99.0–101.3

1.8–173.6

1.5–180.0

1.3–153.8

2.8–165.0

e

c

d

IP is DPASV current (␮A) for l-PGA concentration (C, ng mL−1 ). The values in parentheses denote endogenous concentration (ng mL−1 ) range of real samples, obtained after multiplying the experiment results with dilution factors. LOD calculated as three times the standard deviation for the blank measurement in the absence of l-PGA divided by the slope of respective calibration plots. RSD (%) for three sets of LOD data. Aqueous sample studied on d-PGA imprinted polymer modified PGE (electrode 2) at optimized operating conditions. a

b

0.999

0.999

0.999

CSF sample (l-PGA) (diluted to 3016.2-fold) Blood plasma (l-PGA) (diluted to 1682.0-fold) Urine sample (l-PGA) (diluted to 828.3-fold) Aqueous (d-PGA)e

0.999

0.999

Ip = (0.137 ± 0.145) + (0.296 ± 0.001)C n = 11 Ip = (0.231 ± 0.152) + (0.252 ± 0.002)C n=7 Ip = (0.385 ± 0.621) + (0.264 ± 0.007)C n=8 Ip = (0.218 ± 0.248) + (0.303 ± 0.003)C n=8 Ip = (0.125 ± 0.114) + (0.295 ± 0.001)C n = 11 Aqueous (l-PGA)

3

Correlation coefficient () Regression equationa Sample

Table 1 Sample behavior on l-PGA imprinted polymer modified PGE (electrode 1).

0.77

0.77 99.0–101.1 2.8–170.0

5

Recovery (%)b Range (ng mL−1 )

LODc (ng mL−1 , 3)

3.7. Enantioselectivity and interferences Enantiomeric discrimination with quantitative recoveries has been well established for binary mixtures of d- and l-PGA in aqueous samples using electrode 1 and electrode 2 (Table S1). Spatial orientation of organic functionalities of the film enabled enantio-selective binding of PGA isomers with their respective molecular cavity. Many bio-molecules namely, d-PGA, d- and lglutamic acid, d- and l-proline, glutathione, d- and l-aspartic acid, d- and l-methionine, l-tryptophan, l-cysteine and dopamine are some known coexisting interferents with l-PGA in biological fluids. Table S3 reveals the impact of imprinting on electrode 1. The test analyte (l-PGA) responded with highest imprinting factor (IF = 14), when studied either alone or with the mixture of all interferents (clinically relevant molar ratio 1:10) on electrode 1. When interferents were studied alone on electrode 1 in aqueous samples, their IF values are drastically reduced to approximately 1.0. This reflects their negligible interferences to cause as non-specific adsorption, in the present instance. The very small non-specific contributions, as revealed by NIP-modified PGE could easily be mitigated by waterwashings (n = 1, 0.2 mL) of MIP-sensor, after rebinding experiment, both in the case of aqueous and real samples. Note that the similar trend of non-specific adsorption was noted with all biological samples studied and that could be washed away from the sensor, after rebinding experiment. This ensured a fool-proof analysis of l-PGA on electrode 1, without any cross-reactivity and false positives in real samples. 3.8. Stability and reproducibility of the sensor The reproducibility and ruggedness of the proposed sensor were investigated in order to ensure its practicability in clinical settings. As a matter of fact, as many as ten successive measurements of l-PGA (24.0 ng mL−1 , aqueous sample) revealed the quantitative response with RSD about 1.0% [only three multiple DPASV runs are shown in Fig. 3B (curve 6)]. However, any single electrode could be used for ninety consecutive runs (Fig. S8), with quantitative (100%) recoveries, after regenerating the sensor each time by the method of template retrieval. Despite the fact that an one hour being consumed for the electrode regeneration each time, the memory effect of the sensor to PGA remained unaffected till ninetieth consecutive DPASV measurements. This could be attributed to the higher degree of stability of molecular cavities manifested upon metal ion mediated imprinting during electropolymerization. Insofar as electrode-to-electrode reproducibility in real sample (urine) is concerned, three sensors fabricated under identical conditions yielded l-PGA concentration (29.7 ng mL−1 ) with RSD 2.1% (three successive DPASV runs with each electrode are shown in Fig. 3D, curve 6). The stability of the proposed sensor (electrode 1) has been examined by monitoring the response of l-PGA (24.9 ng mL−1 ) at a regular interval of three days, for a period of one month; only about 10% degeneration in the quantitative results was noticed if the same electrode was used beyond a month. The observed enantio-selective ability, easy regeneration, high reproducibility, ruggedness, and above all a month-long stability have made the proposed sensor perfectly efficient for the highly selective and sensitive analysis of PGA isomers at trace level, without any cross-reactivity and interference, in clinical investigations.

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4. Conclusion In this work, a simple, sensitive, water-compatible, electroconducting, and highly stable metal-ion-mediated MIP film has been obtained by the electro-polymerization method. The PGE sensor modified with MIP film has shown enantioselective feature for the trace-level analysis of d- and l-PGA, in aqueous and real samples. Despite the fact that PGA is an electrochemically inactive substance, the proposed sensor commands a unique electro-catalytic activity, at the behest of Cu(II) incorporated electro-conducting film, for the oxidative stripping of d- and lisomer to respond DPASV current, without any cross-reactivity, interference and matrix complications. Since MIP-based sensor for PGA analysis is, hitherto, not yet reported, this work merits special significance in clinical investigations. Acknowledgment One of the authors (I.P.) is thankful for the financial support received from the Department of Science and Technology, New Delhi (project SR/S1/IC-30/2010). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.06.041. References [1] M. Erdem, R. Say, A. Ersoz, A. Denizli, H. Turk, Biomimicking metal-chelating and surface-imprinted polymers for the degradation of pesticides, Reactive and Functional Polymers 70 (2010) 238–243. [2] H.S. Lee, J. Hong, Chiral and electrokinetic separation of amino acids using polypyrrole coated adsorbents, Journal of Chromatography A 868 (2000) 189–196. [3] L. Qin, X.W. He, W. Zhang, W.Y. Li, Y.K. Zhang, Macroporous thermosensitive imprinted hydrogel for recognition of protein by metal coordinate interaction, Analytical Chemistry 81 (2009) 7206–7216. [4] A. Gultekin, A. Ersoz, D. Hur, N.Y. Sarıozlu, A. Denizlid, R. Say, Gold nanoparticles having dipicolinic acid imprinted nanoshell for Bacillus cereus spores recognition, Applied Surface Science 256 (2009) 142–148. [5] J. Huang, Y. Hu, Y. Hu, Y.G. Li, Development of metal complex imprinted solidphase microextraction fiber for 2,2 -dipyridine recognition in aqueous medium, Talanta 83 (2011) 1721–1729. [6] T.A. Sergeyevaa, O.A. Slinchenko, L.A. Gorbach, V.F. Matyushov, O.O. Brovko, S.A. Piletsky, L.M. Sergeeva, G.V. Elska, Catalytic molecularly imprinted polymer membranes: development of the biomimetic sensor for phenols detection, Analytica Chimica Acta 659 (2010) 274–279. [7] B.B. Prasad, D. Kumar, R. Madhuri, M.P. Tiwari, Metal ion mediated imprinting for electrochemical enantioselective sensing of l-histidine at trace level, Biosensors and Bioelectronics 28 (2011) 117–126. [8] Y. Fujii, K. Kikuchi, K. Matsutani, K. Ota, M. Adachi, M. Syoji, I. Haneishi, Y. Kuwana, Template synthesis of polymer schiff-base cobalt(III) complex and formation of specific cavity for chiral amino-acid, Chemistry Letters 13 (1984) 1487–1490. [9] M.O. Wolf, Transition-metal-polythiophene hybrid materials, Advanced Materials 13 (2001) 545–553. [10] J. Margolis, Conductive Polymers and Plastics, Chapman and Hall, New York, 1989, pp. 2–11. [11] A. Mishra, C.Q. Ma, P. Bäuerle, Functional oligothiophenes: molecular design for multidimensional nanoarchitectures and their applications, Chemical Reviews 109 (2009) 1141–1276. [12] F.A. Cotton, G. Willinson, Advanced Inorg Chem, Wiley, New York, 1988, pp. 35–83. [13] R.C. Mehrotra, R. Bohra, Metal Carboxylates, Academic Press, London, 1983. [14] R.W. Friesen, E.M. Novak, D. Hasman, S.M. Innis, Relationship of dimethylglycine, choline and betain with oxoproline in plasma of pregnant women and their newborn infants, Journal of Nutrition 137 (2007) 2416–2641. [15] A.A. Jackson, C. Persaud, T.S. Meakins, R. Bundy, Urinary excretion of 5l-oxoproline (pyroglutamic acid) is increased in normal adults consuming vegetarian or low protein diets, Journal of Nutrition 126 (1996) 2813–2822. [16] A. Mandal, C. Guo, K.K. Chaudhary, P. Liu, F.S. Yallou, E. Dong, F. Aziat, D.S. Wishart, Multi-platform characterization of the human cerebrospinal fluid metabolome: a comprehensive and quantitative update, Genome Medicine 4 (2012) 1–11.

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Biographies Bhim Bali Prasad is currently a faculty member (professor) at the Banaras Hindu University, India where he has mentored 20 PhD students and published more than 90 research papers in several reputed international and national journals. He received his BSc degree in chemistry in 1972 and MSc degree in 1974 form Banaras Hindu University, India. He obtained his PhD from the department of chemistry, Faculty of Science, BHU, India with Dr. Lal Mohan Mukherjee. He is a recipient of several national awards for his research contribution in analytical chemistry. He also went to Ranbaxy Ltd., India for about one and half year and elaborated a protocol for pharmaceutical analysis, interfaced with several sophisticated instruments. His research interests include environmental chemistry, chromatography, electroanalysis, and detection principle for chemical analysis and development of biomimetic chemical sensor using molecularly imprinted polymers for clinical, pharmaceutical and biological analyses. Indu Pandey is currently pursuing a PhD at Banaras Hindu University under the supervision of Prof. Bhim Bali Prasad. She received her BSc in 2007 and MSc in 2009 from Banaras Hindu University, India. She is recipient of Junior Research Fellowship from the Department of Science and Technology, New Delhi (project SR/S1/IC30/2010). Her research interests lie in the field of chemical sensor development, molecularly imprinted biomimetic polymers, and electro-analytical chemistry.