Electrochemical sensor for folic acid based on a hyperbranched molecularly imprinted polymer-immobilized sol–gel-modified pencil graphite electrode

Sensors and Actuators B 146 (2010) 321–330

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Electrochemical sensor for folic acid based on a hyperbranched molecularly imprinted polymer-immobilized sol–gel-modified pencil graphite electrode Bhim Bali Prasad ∗ , Rashmi Madhuri, Mahavir Prasad Tiwari, Piyush Sindhu Sharma Analytical Division, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, UP, India

a r t i c l e

i n f o

Article history: Received 7 December 2009 Received in revised form 29 January 2010 Accepted 6 February 2010 Available online 13 February 2010 Keywords: Hyperbranched (dendrimer-like) polymer Molecularly imprinted polymer Trifunctional monomer Differential pulse cathodic stripping voltammtery Folic acid

a b s t r a c t Contrary to the ‘substructure imprinting approach’ of larger molecule like folic acid, which often leads molecular recognition for both folic acid and structural analogues containing pteridine and glutamic acid substructures, a molecularly imprinted polymer capable of binding specifically folic acid has been prepared by stoichiometric imprinting process (template/monomer molar ratio, 1:3) creating multiple binding sites within the cross-linked hyperbranched polymer. Dendrimer-like chains were obtained by an ‘initiator-fragment incorporation radical polymerization’ technique involving a new trifunctional monomer, 2,4,6-trisacrylamido-1,3,5-triazine. An electrochemical sensor was developed for the selective and quantitative recognition of folic acid, using a preanodized sol–gel coated pencil graphite (grade 2B) electrode with imprinted polymer immobilized to its exterior surface. During preconcentration step at +0.8 V (with respect to Ag/AgCl), the analyte recapture at pH 2.5 in aqueous environment simultaneously involved mixed hydrophobically driven hydrogen bondings and ionic interactions with pteridine substructure and purely hydrogen bonding interactions with glutamic acid residue of folic acid. The encapsulated analyte was instantly oxidized and then cathodically stripped off responding differential pulse cathodic stripping voltammetric signal. The folic acid was selectively detected with a limit of detection of 0.002 ␮g mL−1 (3, RSD ≤3.0%), without any cross-reactivities and real matrix complications. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Several chronic diseases (for example, gigantocytic anemia, leucopoenia, mentality devolution, psychosis, heart attack, and stroke), especially those concerned with malformation during pregnancy and carcinogenic processes, are related to the deficiency of folic acid (FA) (Fig. 1) [1] which is a water-soluble vitamin. Since FA is detected in biological fluids at very low concentration, i.e. 0.003 ␮g mL−1 (for pancreatic cancerous patients) [2], a highly specific and sensitive assay is called for. Available methods for this purpose are generally based on spectrophotometry [3], chromatography [4], fluorescence [5] or phosphorescence detection [6], together with bioassay [7]. Among the different methods, electrochemical methods are found to be very promising [8–10]. However, FA quantification range in many electroanalyses was practically very high and thus not suitable for ultratrace analysis. Literature survey revealed only two molecularly imprinted polymers (MIPs) reported so far. These involved a substructure imprinting approach [11] and a combination of substructure and

∗ Corresponding author. Tel.: +91 9451954449; fax: +91 542 22368127. E-mail address: [email protected] (B.B. Prasad). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.02.025

inhibitors imprinting approach [12]. Notably, both approaches are apparently lacking FA specificity among its structural and substructural analogues. The actual imprinting of FA is a difficult task because of its sensitiveness to temperature, UV radiation, and other extreme conditions [13]. For this reason we have sought to address the problem through low-temperature preparation of a hybrid MIP–sol–gel material capable of preconcentrating template in its oxidized form at a preanodized (+0.8 V vs. Ag/AgCl) pencil graphite electrode (PGE), in the presence of dispersed carbon powder in its coating layer, followed by differential pulse cathodic stripping voltammetric (DPCSV) transduction. This was made feasible by designing a new trifunctional monomer derived from, triaminotriazine (2,4,6-trisacrylamido-1,3,5-triazine, hereafter abbreviated TAT). TAT has a compact, symmetric and rigid structure (Fig. 1) that can give rise to multiple non-bonding interactions in the well-defined three-directional preferences [14]. This can be able to simultaneously target pteridine (PT) ring as well as dicarboxylate glutamic acid (Glu) moiety of FA in onepot synthesis of MIP preferably via a low-temperature radical polymerization (in lieu of atom-transfer radical and photo iniferter mediated polymerization techniques). The newly designed monomer, TAT, combines trigonal polymerization functionalities, comprising trisacrylamide functions, each of which can indepen-

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Pencil rods (2H, HB, H and 2B), 0.5 mm in diameter and 5 cm in length, were purchased from Hi Par, Camlin Ltd. (Mumbai, India). All voltammetric measurements were performed on EG & G Princeton Applied Research 264A potentiostat model 303A electrode assembly and RE0089 X-Y chart recorder. The threeelectrode cell consisted of modified pencil graphite, a saturated Ag/AgCl and a platinum wire as working, reference, and auxiliary electrodes, respectively. Chronocoulometry measurements were carried out with an electrochemical analyzer (CH instruments USA model 1200A). For IR and NMR spectral analyses, Varian 3100 FTIR (USA) and JEOL AL 300 FT NMR (Japan) were used, respectively. Exeter Analytical Inc. ‘Model CE-400 Elemental Analyzer’, (Mexico), was used for elemental analysis. The surface of coating layers was evaluated using scanning electron microscopy (SEM) (JEOL, JSM, Netherland, Model 840A). Atomic Force Microscopy (AFM) experiments were performed using a Dimension 3100 Scanning Probe Microscope (Vecco Instruments Inc., USA) in the tapping mode. Fig. 1. Structures of trifunctional monomer (TAT) and template (FA).

dently be polymerized, in the presence of a large number of initiator (2,2 -azoisobutyronitrile, AIBN) fragments to give rise a hyperbranched (dendrimer-like) polymer following an initiatorfragment incorporation radical polymerization (IFIRP) mechanism [15]. Such system could be exploited for stoichiometric molecular imprinting to create many binding sites for template rebinding, contrary to ‘monomolecular-imprinting approach’ earlier known for a single binding site created within the cross-linked polymer [16]. In the monomolecular-imprinting approach a single molecule of template was taken as a core toward the development of crosslinked dendrimer, while in the present instance monomer was used as a core for developing hyperbranched (dendrimer-like) receptor system in the presence of several molecules of template. Although PGE has widely been used for the stripping analysis [17], modified PGEs are seldom known [18–20]. In the present work PGE was used for the sol–gel/MIP modification because of its larger electrode active surface area, high electrochemical activity, good mechanical stability, low cost, low technology, low background current, and moreover wide potential window [21]. For coating MIP film, the spin coating method was found suitable in controlling the thickness and permeability of the coating layer [22]. In such case, the addition of carbon powder into the sol–gel texture enabled the film to behave as ‘molecular wire’ connecting top MIP-layer and electrode surface to facilitate electrical conductivity and effective transduction of binding event [23]. 2. Experimental 2.1. Materials and methods Melamine (mel), acryloyl chloride (AC), AIBN, and carbon powder (1–2 ␮m in diameter) were purchased from Loba Chemie (Mumbai, India), and Spectrochem Pvt. Ltd. (Mumbai, India). Dimethylsulfoxide (DMSO), acetonitrile (ACN), triethylamine (TEA), dimethylformamide (DMF), and ethanol were AR grade solvents purchased from Spectrochem Pvt. Ltd. (Mumbai, India), and used as such. Ethylene glycol dimethacrylate (EGDMA), tetraethyl orthosilicate (TEOS), FA and its interferents were AR grade provided by Fluka (Steinheim, Germany). The stock solution (500 ␮g mL−1 ) of FA was prepared by deionized triple-distilled water (conducting range 6–7 × 10−8 S cm−1 ) containing 100 ␮L of 0.1 M NaOH. Working standards of FA were daily prepared by diluting the stock solution with water. Human blood serum samples were procured from a local pathology laboratory and stored in a refrigerator at ∼4 ◦ C, before use.

2.2. Synthesis of functional monomer (TAT) Mel (76.9 mmol) was dissolved in 50 mL of DMF. To this AC (246 mmol) was gradually added and the reaction mixture was stirred for 12 h. After filtration of the monomer, TAT (yield 90%) was obtained. This was washed with hot (35 ◦ C) DMF–water (10 mL, 1:1, v/v) to remove residual precursors, if any. The compound was characterized by FT-IR, 1 H NMR, and elemental analysis: CHN analysis (Found: C, 49.9; H, 4.2; N, 29.2. C12 H12 N6 O3 requires C, 49.9; H, 4.1; N, 29.3%). 1 H NMR, ıppm, d6 DMSO: 7.8 (s, 1H, –NH), 5.5 (s, 1H, –CH), 5.2 (s, 1H, –CH2 ), and 5.0 (s, 1H, –CH2 ). FT-IR (KBr), max /cm−1 : 3343 (–NH stretch), 3130 ( CH stretch), 1683 (amide I), 1499 (amide II), 1342 (–C–N– stretch), 1004 (–CH out of plane bend) and 774 (–CH2 rocking vibration in alkene). 2.3. Synthesis of MIP For the preparation of MIP–FA adduct (template/functional monomer molar ratio, 1:3), 1 mmol of functional monomer (TAT, 10 mL DMSO), 0.33 mmol of template (FA, 10 mL DMSO) and 0.9 mmol of initiator (almost equivalent to functional monomer) (AIBN, 4 mL DMSO) were mixed together in a glass tube. After 10–15 min, 20 mmol cross-linker (EGDMA, 4 mL) was added to this mixture and purged with N2 gas for 10 min. The glass tube was sealed and placed in an oven at 50 ◦ C for 4 h. The produced polymer was grounded and sieved to particle size 10–15 ␮m. Removal of template was made by washing the polymer in ACN–TEA (4:1, v/v), until no voltammetric signal was observed. Non-imprinted polymer (NIP) was obtained under the same conditions, but in the absence of template. Furthermore, since the initiator used in MIP preparation is in large amount (fixed), the chain propagation could only proceed through an IFIRP mechanism, instead of conventional one. Consequently a hyperbranched (dendrimer-like) polymer (Scheme 1A), was systematically formed around trifunctional monomer, with initiator fragments (R) incorporated as terminal groups (Scheme 1B). This is apparently feasible because of monodisperse (Mn = 37,212; Mw = 39,129; Mw /Mn = 1.05, estimated by gel-permeation chromatography) characteristic of MIP–FA adduct, where TAT monomer is a core molecule to lead a hyperbranched polymer of high thermal stability (m.p. > 300 ◦ C, by thermogravimetric analysis). 2.4. Electrode preparation and voltammetric procedure A pencil rod (2B) was first pretreated by dipping in 6 M HNO3 for 15 min, washing with water, and subsequently smoothening the surface by soft cotton. This was inserted into a teflon tube where the tip of the pencil rod at one end was gently rubbed with an emery

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paper (No. 400) to level the pencil surface along the tube orifice. Electrical contact was obtained by soldering a metallic wire to the exposed reverse side of the pencil rod. A separate sol–gel was prepared by mixing 1.0 mL of TEOS, 1.0 mL of ethanol, 0.5 mL of water, and 50 ␮L of 0.1 M HCl. After 35 min stirring, a clear sol–gel solution was obtained which left to gel for another 45 min to reach to ‘syneresis’ stage (a stage between ageing and drying of gel). To prepare the MIP–FA adduct/sol–gelmodified PGE, 1.0 mL of sol–gel viscous solution, an optimized amount of carbon powder (50 mg) and 300 ␮L of MIP–FA adduct (15 × 103 ␮g mL−1 in DMSO) were mixed together by mechanical stirring for 10 min, and 5.0 ␮L of this mixture was placed on PGE surface for spin coating at 2600 rpm for 2 min. During modification of electrode, the purpose of using the carbon powder was to enhance the conductivity and less shrinkage of the film. By

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incorporating carbon powder in the initial cocktail of sol–gel and MIP–adduct solutions, it was possible to obtain viscous gel with carbon powder percolating in the silicate network. The electrode was kept overnight for drying in a desiccator. Template was completely extracted from the doped film by immersion of the modified electrode in 1 mL mixture solution of ACN–TEA (4:1, v/v) for 30 min to obtain MIP–sol–gel-modified PGE; the complete template removal was ensured till no DPCSV signal was noticed. The NIP–sol–gelmodified PGEs were also prepared following the same procedure. The modified electrode was immersed in KCl–HCl mixture (10.04 mL, pH 2.5) for 120 s at +0.8 V vs. Ag/AgCl to record DPCSV and cyclic voltammetry (CV) blank runs. An aliquot of a freshly prepared solution of FA (1–100 ␮g mL−1 ) was added in the cell for analyte accumulation in 120 s at +0.8 V vs. Ag/AgCl under ambient conditions. Both CV and DPCSV runs were recorded under cathodic

Scheme 1. (A) Schematic representation of the preparation of the MIP–sol–gel coated PGE. The symbol (䊉) represents silane binding as Si–O–C–OH at various carbonyl centers of MIP. (B) Recognition mechanism of template (FA) bindings within MIP cavities via non-covalent interactions showing ‘multi-molecular imprinting’ in hyperbranched (dendrimer-like) polymer.

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Scheme 1. (Continued).

stripping mode from +0.1 and −0.2 V and terminated at −1.1 and −0.95 V, respectively (scan rate 10 mV s−1 , pulse amplitude 25 mV, equilibration time 15 s). In stripping method the analyte is first deposited onto electrode surface, usually from a stirred solution. After an accurately measured deposition time the electrolysis is discontinued, the stirring stopped, and the deposited analyte is determined by a stripping voltammetric step. In cathodic stripping method the electrode behaves as an anode during the deposition (preconcentration) and as a cathode during stripping. Because the material is deposited onto a much smaller solid-phase volume than the bulk solution volume, the analyte is often concentrated by factors of 100 to more than 1000 in the preconcentration step. Therefore a major advantage of stripping analysis is the capability for electrochemical preconcentration of the analyte prior to the voltammetric measurement. Since oxygen did not influence the FA oxidation, deaeration of the cell content in this work was not required. The limit of detection (LOD) was calculated as three times the standard deviation for the blank measurement in the absence of FA divided by the slope of the calibration plot [24]. The above voltammetric procedure was also performed using the NIP-modified PGE under identical operating conditions. The sol–gel layer in between PGE surface and MIP is essentially required to avoid any delamination as well as shrinkage of the MIP film. The purpose of using sol–gel at syneresis stage was to avoid any encapsulation of receptor and to favor surface immobilization of MIP over sol–gel so that binding sites could be fully accessible at the restricted access media for template recapture. As shown in Scheme 1A, the physical sorption of sol–gel into dense pores of PGE produced a first microphase interface. The softer pencil lead (2B) is

appropriate for sol–gel modification at first microphase because it is thicker containing more graphite contents than the harder ones (2H, H and HB). The charging current of bare PGEs, where pencil hardness varies from 2H (hardest) to 2B (softest), was measured in cathodic and anodic scans of CV runs (stripping mode) from +0.1 to −1.1 V vs. Ag/AgCl, at scan rate of 10 mV s−1 . A significant decrease in charging current was observed in the order: 2H > HB > H > 2B Interestingly, pencil leads with hardness grade H and 2B (modified or non-modified) revealed approximately 0.05 ␮A charging current, respectively, in the potential window (−0.5 to −0.95 V) where test analyte responded voltammetric peak. However, optimized analyte accumulation potential and deposition time were realized higher (+1.2 V, 180 s) for modified PGE (H) than those for modified PGE (2B); and moreover, the first one was responsive, with restricted and non-quantifiable current, for only higher concentration range of analyte. Therefore, the PGE with 2B grade was used throughout this work. 3. Results and discussion 3.1. Characterization of coating layer FT-IR (KBr) spectra of template (FA), MIP–FA adduct, MIP, sol–gel, MIP–FA adduct immobilized sol–gel, and MIP–sol–gel were comparatively studied (see Supporting Information, SI 1) which showed following downward absorption band shiftings:

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Fig. 2. SEM images (magnification 6000×): (A) bare PGE (2B grade), (B) sol–gel-modified PGE, (C) MIP–FA adduct immobilized sol–gel-modified PGE, and (D) MIP–sol–gelmodified PGE.

• MIP –NH stretching vibration shifted from 3350 to 2994 cm−1 ; hydroxyl (–OH) stretching band of Glu moiety from 3544 to 3400 cm−1 ; –NH group of PT ring shifted from 3300 to 3185 cm−1 on account of hydrophobic driving force, acting upon binding in DMSO or rebinding process in aqueous medium, which overcome solvent influences, if any, against the formation of stable hydrogen bond. The downward shifting of template bands (Supporting Information SI 1) occurred due to non-covalent interactions, i.e. hydrophobically driven hydrogen bonding between MIP and template. Accordingly, as shown in Scheme 1B, FA binds to the site by hydrogen bonds between PT ring (N-5 and –NH2 ) and mel [–NH (b)], respectively, in combination with a salt bridge between the PT ring (N-3 and C4 –O) and mel [N-a and –NH (c)] as a consequence of mutual polarization of receptor and guest on analyte rebinding in aqueous medium (Scheme 1B). • Aromatic ring characteristic vibrations of FA (phenyl and PT residues) at 1484 and 1410 cm−1 are shifted to 1424 and 1396 cm−1 , respectively on FA binding with MIP under ␲–␲ electron donor–acceptor interactions. Reportedly PT ring, a planar N-heterocyclic, would parallel the planar ␲ system of mel to produce a complex by ␲–␲ electron donor–acceptor interaction [25].

The presence of AIBN fragments (R), on account of IFIRP mechanism, within the entire MIP network (Scheme 1B), was indicated by a characteristic –C N stretching peak at 2200 cm−1 which was unaltered after immobilization on sol–gel layer. Other prominent FT-IR features of pure sol–gel [SiO4 internal vibrations (1075 cm−1 ), Si–O–Si bridge (481 cm−1 ), Si–OH stretching (937 cm−1 )] remained intact in all the curves corresponding to MIP–FA adduct immobilized sol–gel and MIP–sol–gel. A number of such Si–O–C linkages could be feasible at sol–gel/polymer interface due to SiO− attack of sol–gel matrix at various >C O sites of MIP, as revealed by the disappearance of band corresponding to all >C O groups of MIP occurring at 1700 cm−1 and appearance of a typical Si–O–C stretch-

ing vibrations at 1170 cm−1 . Hydrophobically induced hydrogen bondings in this instance are also substantiated by downfield peak shiftings in 1 H NMR spectra (DMSO-d6 , 300 MHz, TMS, ıppm ) (see Supporting Information, SI 2): 7.8 (MIP amide protons) shifted to 8.5, 10.5 [Glu (protons –COOH)] shifted to 11.0, 5.0 [PT (protons –OH)] shifted to 5.48 after FA rebinding with MIP. Interestingly, these shifted peaks both in IR and NMR resumed their original positions after the removal of FA. An extraneous peak for –CH3 protons of AIBN fragments in MIP–FA adduct/MIP occurred at 1.5 ppm and no peak corresponding to vinylic double bond of monomer was noticed after polymerization. This suggests the incorporation of corresponding isobutyronitrile (R) AIBN fragments in polymer resulting in hyperbranched (dendrimer-like) polymer [26]. Hyperbranched dendritic polymerization in this work was further supported by an additional experiment showing encapsulation and transport properties of malachite green as a dye probe into the void spaces of dendritic structure [15]. For this the solid MIP (10 mg) was added into an aqueous solution of malachite green (0.001 wt.%, 2 mL) and shaken well. All dye molecules were adsorbed to the polymer surface changing water phase to be completely colorless. Interestingly no such color sorption was observed when a linear polymer, polyvinyl chloride (mol. wt. 48000), was used, in lieu of MIP. This indicates encapsulation and phase-transfer (solid ↔ liquid) properties of MIP often observed in the case of hyperbranched network. It may be added that solid → liquid phasetransfer of dye color was relatively slow in the case of NIP as compared to that of MIP presumably because of strong non-specific color retention. 3.2. Surface morphology The bare PGE (2B grade), whose morphology is shown in Fig. 2A, represents an uniform and smooth surface. Fig. 2B corresponds to the sol–gel stationary phase film created on the PGE surface. Here,

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Fig. 3. AFM images (tapping mode) of MIP–FA adduct immobilized sol–gel film: (A) before template extraction and (B) after template extraction.

the SEM image shows homogeneously dispersed colloidal particles of sol with enhanced surface area in the presence of carbon powder. The MIP–FA adduct layer on sol–gel-modified PGE surface revealed that the exterior coating was porous with microvoids and compact owing to the carbon dispersion without any cracks (Fig. 2C). Interestingly, upon template removal from this film delivered larger pores (Fig. 2D) with vertical tethering of MIP dendrimer chains on the lateral surface. The appearance of such pores channelized from the top to bottom within the polymer film ‘grafted to’ sol–gel surface amidst carbon powder, with increased contact area and apertures of different depths, favored high wettability and electrical conductivity all along the entire film thickness. Surface morphology was further examined from the AFM images recorded under tapping mode for MIP–FA adduct immobilized sol–gel (Fig. 3A) and MIP–sol–gel (Fig. 3B). The rootmean-square roughness of MIP–FA adduct immobilized sol–gel and MIP–sol–gel is obtained to be 0.8 and 1.8 nm, respectively. This indicates that adduct has more compact and smooth surface than the MIP film. The arithmetic mean roughness for the above-modified surfaces is 0.7 and 1.6 nm, respectively [27]. In this case the space between the cavities shows elevated bumps of polymer material (Fig. 3B), after the template extraction. While the decrease in roughness is obvious upon analyte binding, the elevation in the image (50 nm) reflects the fact that the polymer matrix is ‘pushed’ aside by the species on template stamp, thus filling in the gaps between the individual template molecules [28].

3.3. Electrochemical behavior of the MIP-hybrid PGE sensor Both bare and modified electrodes have not responded any current, in blank (Fig. 4, inset, ‘a’ and dotted curve). Thus the hydrogen ion reduction within the potential window (+0.1 to −1.1 V) at pH 2.5, was absent either with bare or modified electrodes. At preanodized electrode the entrapped FA molecules in MIP cavity were irreversibly oxidized at C9 –C10 mimicking biological oxidation process [29], as shown below:

The oxidized FA subsequently stripped off on cathodic scan following an electrochemically reversible reduction (2e− /2H+ ) to PT ring, as reported elsewhere [30]. The cathodic stripping CV peak (at −0.55 V vs. Ag/AgCl at 10 mV s−1 ) was broader in shape (Fig. 4, inset) reflecting a sluggish reduction behavior owing to hydrophobically driven hydrogen bonding of oxidized FA in the MIP cavity. This wave shifted slightly toward more negative poten-

Fig. 4. DPCSV response of FA (concentration 0.108 ␮g mL−1 ) with (A) sol–gelmodified PGE, (B) FA-imprinted sol–gel-modified PGE, (C) bare PGE, (D) NIP–sol–gel-modified PGE, and (E) MIP–sol–gel-modified PGE. DPCSV response of FA (␮g mL−1 ): (G) 0.0498, (H) 0.072 (with MIP–sol–gel-modified PGE), (F) 0.072 (with NIP–sol–gel-modified PGE) in human blood serum. DPCSV response of FA (␮g mL−1 ): (J) 0.058, (K) 0.083 (with MIP–sol–gel-modified PGE), (I) 0.083 (with NIP–sol–gel-modified PGE) in multivitamin tablet. Inset figure shows typical cyclic voltammograms of 0.05 ␮g mL−1 aqueous solution of FA at different scan rates (mV s−1 ): (a) 10, blank, (b) 10, (c) 20, (d) 50, (e) 100, and (f) 200 in stripping mode with MIP–sol–gel-modified PGE. The dotted curve represents CV response on bare PGE.

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Table 1 Sample behavior. Range (␮g mL−1 )

LOD (3, ␮g mL−1 )

RSD (%) (n = 3)

0.0021

2.3

98.1–102.4

0.0021

1.7

0.017–0.118

98.3–102.1

0.0022

3.0

0.202–2.750

98.5–101.8

Sample

Regression equation

Correlation coefficient ()

Aqueous sample

IP = (43.1468 ± 0.0275)C + (0.0153 ± 0.0021), n = 9 (lower range) IP = (4.3214 ± 0.0025)C + (−0.0111 ± 0.0064), n = 16 (higher range) IP = (43.8446 ± 0.0992)C + (0.0139 ± 0.0051), n=8 IP = (41.1307 ± 0.0792)C + (0.0256 ± 0.0046), n = 7 (lower range) IP = (4.3524 ± 0.0099)C + (0.0623 ± 0.0167), n = 13 (higher range)

0.99

0.007–0.156

99.3–103.4

0.99

0.198–4.624

97.9–103.6

0.99

0.010–0.077

0.99 0.99

Blood serum sample Multivitamin sample

tial with increasing scan rate owing to the strong stability of MIP–FA adduct which required relatively high energy for cathodic reduction. The reduced product was probably weakly adsorbed at the electrode surface; such adsorption, however, was not favored at lower scan rates because of the sufficient time available for the removal of the reduction product from the electrode surface for re-oxidation. The reduction product progressively dissolved near the electrode surface, weakening its adsorption within a limited time with increasing scan rate, and thereby responded enhanced anodic peak with positive shift. This resulted in quasireversibility [(Epa − Epc ) > 29.5 mV, Ipa /Ipc > 1] with increasing scan rates (>20 mV s−1 ). Quantifications of CV cathodic peaks were difficult owing to its ‘drawn-out’ nature. However, DPCSV peaks (Fig. 4) with MIP–sol–gel-modified PGE are found symmetrical and easily quantifiable. For a comparative study, we have also prepared different kinds of modified electrodes (including bare PGE) using various coatings, viz., sol–gel, sol–gel FA composite (FA-imprinted sol–gel), NIP–sol–gel and MIP–FA adduct (different template/monomer molar ratios, 1:1, 1:2, 1:3, 1:4) immobilized sol–gel, all with embedded carbon powder. Direct MIP coating on bare electrode was not stable as the coating layer was found to be crippled down, after drying. As can be seen in Fig. 4, DPCSV responses with sol–gel and NIP-modified electrodes were found to be non-responsive (curves A and D), while FA-imprinted sol–gel PGE showed somewhat restricted response (curve B). A very drawn-out (curve C) response was noticed with unmodified PGE. MIPs (template/monomers molar ratios 1:1 and 1:4) immobilized sol–gel-modified electrodes showed insignificant response at all concentrations of analyte, whereas MIP (1:2) behaved with irregular response showing a limiting plateau in the current response beyond concentrations 0.04 and 2.0 ␮g mL−1 both in lower and higher concentration regions of analyte solutions, respectively (see Supporting Information, SI 3). However, the activity of MIP (1:3) immobilized sol–gel-modified PGE was found to be significantly enhanced showing specific binding of analyte with linear response in both lower and higher concentrations ranges (see Supporting Information, SI 3). This is because of high symmetricity of DPCSV responses (Fig. 4, curves E, G, H, J, and K) realized with MIP (1:3)–sol–gel-modified PGE, without any matrix or nonspecific complications in aqueous and real samples (blood serum and multivitamin tablet). Interestingly, no signal was observed on non-imprinted control electrodes for FA concentration below 1.5 ␮g mL−1 . This demonstrates the profound influence of the template in the formation of the selective binding sites for FA. Consequently, non-specific contributions to DPCSV current in all samples were absent (Fig. 4, curves D, F and I). Any non-specific contribution with NIP–sol–gel-modified PGE above 1.5 ␮g mL−1 could be mitigated in the present instance by multiple waterwashings (n = 4, 0.2 mL) of both NIP and MIP–sol–gel-modified PGEs. The reproducible regeneration of modified electrodes was possible simply by soaking in ACN–TEA (4:1, v/v) for half an hour,

Recovery (%)

as is evident from the multiple runs (Fig. 4, curve E). After regeneration of the modified electrode, all measurements revealed a high degree of precision (RSD 2.3%, n = 3, Table 1) with quantitative (100%) recovery in the aqueous sample. Any single electrode could be used after regeneration for as many as 30 consecutive runs with quantitative recoveries. Insofar as sample-to-sample and electrode-to-electrode variations are concerned, the three freshly modified electrodes always responded reproducible current response (Fig. 4, curve H) with three duly spiked blood samples (FA concentration 0.072 ␮g mL−1 , recovery 102%, RSD 1.1%). This confirmed reproducible method of PGE modification, and ruggedness of the electrode against any matrix complications. In the range of pH 1.4–5.5, the cathodic stripping peak potential (Ep ) shifted to positive potential (contrary to usual shift to higher negative value) as the pH increased making Ep vs. pH profile to be perfectly linear [Ep (V) = (−0.745 ± 0.018) + (0.053 ± 0.005) pH ( = 0.97)]. This is consistent with the transfer of 2e− /2H+ during the oxidation–reduction of FA. The DPCSV peak current of FA increased up to pH 2.5, and then progressively decreased when the pH is increased. It is known that at low pH (90 s) always responded diminishing response because of cracks observed in monolayer coating. However, the effect of exposure time of electrode in analyte solution revealed poor partitioning of analyte across film/solution interface until an accumulation time of 120 s was reached, and above this a drastic fall in current response appeared as a consequence of the poor stability of MIP–FA adduct.

Linear range (␮g mL−1 )

FA selectivity Selective in the presence of 200-fold AA, 100-fold UA, 1000-fold Na+ , K+ , NH4 + , Mg2+ , Ca+2 , Cl− , F− , CO3 2− , SO4 2− , glucose, urea, and oxylate Selective in the presence of 100-fold UA, glucose, cysteine, glutamic acid, hypoxanthine, Vit. C, B1 , B2 , and B12 Non-selective in the presence of AA and DA

Selective in the presence of 1000-fold AA, 500-fold UA, 10-fold DA, MTX, FCA, 5-fold AD, and 2-fold URA

In the absence of carbon powder, the modified PGE was found to be totally non-responsive to detect FA. The current response, however, was observed to be increasing with increase of carbon powder content up to 50 mg due to the increase of reactive surface area, and then gradually decreased either owing to the restricted permeability through denser matrix or due to the mechanical deterioration of more brittle film with high carbon content [23]. In the second kind of saturation experiment, the PGE was modified with fixed amount of MIP–FA adduct (with sol–gel) and DPCSV responses were recorded for varying concentration of template analyte (FA). This revealed a complete saturation in the binding site due to optimum uptake up to 4.624 ␮g mL−1 . Surprisingly, an earlier saturation of current in the dilute range of analyte concentrations was observed at 0.156 ␮g mL−1 , when FA concentrations were initially measured from 0.007 to 0.156 ␮g mL−1 in a new set of experiment. Under the optimized conditions, average DPCSV currents for concentrated solutions were found to be reduced by approximately 10-fold with decrease in slope of linear calibration equation between Ip (␮A) and C (␮g mL−1 ) (Table 1). This may be ascribed to the some sort of hindrance in homogeneous charge-transport and mass-transfer (diffusion) from bulk to the electrode owing to the probable intermolecular aggregation through hydrogen bondings between FA molecules [33]. This means the electrocatalytic activity of MIP film is much favored with the sorbed FA (under predominating hydrophobically induced hydrogen bonding in aqueous-rich media in lower concentration range causing an early saturation of FA uptake) vis-à-vis diffused FA (under competition with hydrophobically induced FA rebinding interactions in higher concentration ranges) onto the modified electrode surface. The negative intercept in aqueous solutions of higher FA concentrations is insignificant and within the limit of experimental error. Under the optimized operating conditions one may, however, opt either of the concentration regions, as the situation demands, for the measurement with MIP–sol–gel-modified PGE, on the basis of linear calibration equations and LODs presented in Table 1. 3.6. Cross-reactivity studies Modified PGEs prepared with MIP–sol–gel and NIP–sol–gel were subjected to cross-reactivity with different interferents (see Supporting Information, SI 5), viz., methotrexate (MTX), folinic acid (FCA), dopamine (DA), uracil (URA), ascorbic acid (AA), adenine (AD), uric acid (UA), and their clinically relevant mixture. Accordingly, MIP-modified sol–gel electrode without washing treatment was not responsive for all the interferents when studied individually. The non-specific adsorption with NIP-modified electrode was easily removed from MIP/NIP immobilized sol–gel surface simply by water-washes. Despite the fact that the MIP had a strong

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preference for PT and Glu substructures of FA showing maximum current response with quantitative (100%) recovery, it could not retain other structural analogues like MTX, FCA and AD which are having either of PT and Glu substructure or both (see Supporting Information, SI 6). This confirmed a substrate-selective (systemspecific) imprinting effect in the present case. A parallel cross-reactivity study (see Supporting Information, SI 6) has been made in the presence of FA since real samples always carry concomitant interferents. In the presence of mixture of interferents taken in their clinically relevant concentration ratios, the characteristic peak current due to FA cathodic stripping was less than 1.6% of the value obtained in the absence of interferents; this shows that the sensor can be used quantitatively in mixtures with these compounds prevalent even at higher concentrations. The linear range, LOD, and the anti-interferents ability for FA at MIP–sol–gel-modified PGE were compared with the recently reported chemically modified electrodes and were given in Table 2. Accordingly, the p-AMT modified GCE [8] had poor limit of quantitation (0.044 ␮g mL−1 ) to detect the normal level (0.0151 ± 0.0045 ␮g mL−1 ) of FA in blood serum with no tolerance for the major interferents, AA and UA, present at higher concentrations. Although the SWCNT-ILPE [9] had shown better sensitivity in terms of the quantitation range, it was not tested for FA determination in human blood serum sample. The MDWCNPE is inferior in all respect for the evaluation of normal level of FA in blood serum. Furthermore, the above electrodes were not examined for crossreactivity with different structural analogues like, MTX and FCA. However, in this work, the MIP–sol–gel-modified PGE is more suitable for the determination of FA even in the presence of 1000-fold AA and 500-fold UA including other clinically relevant interferents and structural analogues. The proposed electrode is also compared with SWCNT-ILPE [9], by means of Student’s t-test, where tcal (1.48) was found less than ttab (2.77) at confidence level 95% ( = 0.99). This revealed a similar order of precision in the final results within the concentration range (0.007–0.156 ␮g mL−1 ) by both methods. The proposed method is, however, rather simpler, more selective, and cost-effective. 4. Conclusions This work has introduced a new concept to prepare MIP for complex target molecule like FA, where its both substructures (PT and Glu) were targeted together under spatial dispositions of dendrimer chains showing a phenomenal imprinting effect in aqueous environment. The proposed approach herein demonstrates a new generation of solid sensors based on conducting MIP–sol–gel hybrid material, which is sensitive and highly suitable for the electrochemical detection of FA at ultratrace level in aqueous and real conditions, without any complication of interference from co-existing complexing matrices of the test solution. Acknowledgements The authors thank the Council of Scientific and Industrial Research, New Delhi for the award of a junior research fellowship (to R.M.). Instrumental facility by the Department of Science and Technology, through project SR/S1/IC-18/2006, is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.snb.2010.02.025.

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India for about one-and-half years 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. Rashmi Madhuri is currently pursuing a PhD at Banaras Hindu University under the supervision of Professor Bhim Bali Prasad. She received her BSc in 2005 and MSc in 2007 from Banaras Hindu University, India. She is recipient of CSIR NET Junior Research Fellowship and BHU research fellowship. Her research interests lie in the field of chemical sensor development, molecularly imprinted biomimetic polymers, and electroanalytical chemistry.

Biographies

Mahavir Prasad Tiwari is a research scholar in Department of Chemistry, Faculty of Science, BHU, India under the supervision of Professor Bhim Bali Prasad. He received his BSc in 2005 and MSc in 2007 from Purvanchal University, India. His research interests lie in the field of solid-phase extraction/microextraction, molecularly imprinted polymers, and electroanalytical chemistry.

Bhim Bali Prasad is currently a professor of analytical chemistry at Banaras Hindu University, India where he has mentored 20 PhD students and published 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.,

Piyush Sindhu Sharma is currently working as a post-doctoral fellow at University of Verona, Italy. He received his BSc and MSc degrees from Banaras Hindu University, India. He obtained his PhD from BHU with Professor Bhim Bali Prasad. His thesis dealt with the use of imprinted polymer materials for biomolecules analyses from complex matrices. His research interests lie in the field of biotechnology and MIPsensors.