Multiwalled carbon nanotubes-ceramic electrode modified with substrate-selective imprinted polymer for ultra-trace detection of bovine serum albumin

Biosensors and Bioelectronics 39 (2013) 236–243

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Multiwalled carbon nanotubes-ceramic electrode modified with substrate-selective imprinted polymer for ultra-trace detection of bovine serum albumin Bhim Bali Prasad n, Amrita Prasad, Mahavir Prasad Tiwari Analytical division, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 June 2012 Received in revised form 23 July 2012 Accepted 25 July 2012 Available online 17 August 2012

This study describes the synthesis of a new class of substrate-selective molecularly imprinted polymer. This involved tetraethylene glycol 3-morpholin propionate acrylate (functional monomer) and bovine serum albumin (template) for polymerization in aqueous condition, using ‘‘surface grafting-from’’ approach directly on a vinyl exposed multiwalled carbon nanotubes-ceramic electrode. The analyte recapture at pH 6.8 in aqueous environment simultaneously involved hydrophobically driven hydrogen bonds and ionic interactions between negatively charged bovine serum albumin and positively charged imprinted nanofilm. The selectively encapsulated bovine serum albumin first gets reduced at  0.9 V and then oxidized within the cavity, without getting stripped off, to respond a differential pulse voltammetry signal. The limit of detection [0.42 ng mL  1 (3s, RSD r 1.02%)] obtained was free from any cross-reactivity and matrix complications in aqueous, pharmaceutical, serum, and liquid milk samples. The proposed sensor can be used as a practical sensor for ultra-trace analysis of bovine serum albumin in clinical settings. & 2012 Elsevier B.V. All rights reserved.

Keywords: Molecularly imprinted polymer Porogen-water Bovine serum albumin Multiwalled carbon nanotubes-ceramic electrode Differential pulse voltammetry

1. Introduction Bovine serum albumin (BSA) is one of serum albumins that attract many biochemical applications. It is a major component of bovine plasma (5 g/100 mL) and plasma accounts for about 40% of the body pool of albumin (Hilger et al., 1996). It is used as a stabilizing agent in enzymatic reactions and as a carrier protein in many vaccines and medicines (Balen, 2002). On exposure to BSA due to consumption of bovine milk and meat, its affects are similar as that of a prime allergen (Restani et al., 2004). Although great effort has been made to reduce exposure to BSA in pharmaceutics to eliminate the threat of bovine spongiforum encephalopathy, least attentions have been paid to comprehend the human immune response owing to the lack of fool-proof immunological methods for the direct evaluation of either BSA or anti-BSA antibodies. A number of anti-rabies vaccines [Semple Vaccine (ARV), Purified Vero Cell Rabies Vaccine (PVRV-Verorab and Abhayrab), and Purified Chick Embryo Cell Vaccine (PCECRabipur)] have BSA content in ppb (ng mL  1) level. On account of some known complications of neurological accidents and allergic reactions (Chakravaty, 2001), it becomes imperative for quality testing of each batch of these vaccines beyond the statuary

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Corresponding author. Tel.: þ91 9451954449; fax: þ91 5422268127. E-mail address: [email protected] (B.B. Prasad).

0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.07.080

obligations. BSA content should be less than 50 ng mL  1 per human dose according to WHO standards (Deshmukh et al., 2004). Several diseases like a rare kidney disease called membranous naphropathy (Debiec et al., 2011), bovine spongiforum encephalopathy (Deshmukh et al., 2004), insulin dependent diabetes mellitus (Persaud and Barranco-Mendoza, 2004), and crutzfield–jacob (Brown, 2005), are known that may be developed due to anticipated BSA exposure to human. Therefore, the detection of BSA has become a wide area of research in immunology and bio-analytical studies. Extensive investigations have been reported for the determination of BSA that include fluorimetry (Sun et al., 2008), quartz crystal microgravimetry (Lin et al., 2004), reverse-phase high performance liquid chromatography (RP-HPLC) (Hamidi and Zarei, 2009), direct electroanalysis (Chiku et al., 2008a), and biosensor detection (Zhang et al., 2012). However, some of these methodologies needed extensive sample pretreatment, while others suffered from low selectivity, poor sensitivity, and high instrumentation. Electrochemical detection of higher non-metal proteins (e.g., albumin) is reportedly very less. This is due to the apparent complexity and strong adsorption of protein on the electrode surface which may lead signal depression to be unpredictable and irreproducible. Many molecularly imprinted polymers (MIPs) have been developed for BSA (Kryscioa and Peppasa, 2012; Ran et al., 2012; Gai et al., 2011; Zhang et al., 2010), without revealing analytical aspects. Furthermore, the imprinting

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solvents (porogens) for MIP development routinely being used were organic solvents that might unfold the native BSA, even at pre-polymerization stage. Insofar as MIP-based sensors (Chen et al., 2012; Yu et al., 2010) dealing ultra-trace analysis of BSA are concerned, there were some false-positive contributions with considerable amount of non-specific interferences and crossreactivity. Although the recent seminal work of surface imprinted chitosan coated multiwalled carbon nanotubes (MWCNTs)-based biosensors (Chen et al., 2012) revealed a high level of sensitivity, unfortunately it was found stable only at 4 1C for 15 day. Thus, this cannot be recommended as a practical sensor for in-field and clinical settings. MIPs are synthetic antibody mimics, formed by cross-linking of organic (or inorganic) polymers in the presence of an analyte (template), which yield recognitive polymer networks with specific binding pockets for the biomolecules. Considering the incompatibility between the protein and organic solvents and moreover, its unfolding (denaturation) in harsh chemical conditions, the substrate (whole protein) imprinting in biological benign conditions, maintaining the physiological status of protein, is rather challenging. Protein imprinting in neutral aqueous condition might be an alternative that has, however, been drawn limited attention till date, primarily because of water competition toward hydrogen bonding interactions (Lin et al., 2009; Yang et al., 2011). We are of the opinion that the concerted effects of hydrophobically driven hydrogen bonding and electrostatic interactions might rescue the situation and a stable MIP-template adduct formation in aqueous condition could be feasible for the substrate-selective imprinting of entire protein molecule, without its denaturation in the experimental conditions. Besides, surface imprinting can also be imbibed in such work to avoid the protein entrapment in the polymer matrix and to facilitate the analyte mass-transfer without any impediment or blocking effect (Turner et al., 2006). We have adopted this approach for the first time for BSA imprinting where imprinted chains were grown via free radical polymerization, directly on the surface of a vinyl exposed MWCNTs–ceramic electrode (MWCNTs-CE). For MIP development in aqueous medium, both monomer and cross-linker ought to be soluble in water. In the present work, a typical monomer, tetraethylene glycol-3morpholine propionate acrylate (TEGMPA), and a cross-linker, diacryloyl urea (DAU), were synthesized, which are watersoluble. TEGMPA is consisted of two kinds of functional group: one is the acrylate, which could be polymerized by free radical chain growth process, linking the TEGMPA in the polymer network and the other the 30 amine which readily abstracts a proton from water in neutral condition. Nevertheless, taken an excess amount of TEGMPA, may lead to the generation of corresponding free radical, after reaction with ammonium persulphate (APS, initiator). This species may serve as a co-initiator (Wu et al., 2006; Yu et al., 2009) to expedite the polymerization process. The objective of such fabrication is to develop a water-compatible, highly sensitive, and selective tool for the detection of BSA in real samples.

2. Experimental 2.1. Reagents All chemicals were of analytical reagent grade and used without further purification. Acryloyl chloride (AC), urea, BSA, and APS, were purchased from Loba Chemie (Mumbai, India). 3-(trimethoxysilyl)propyl methacrylate (TMPM), tetraethylene glycol diacrylate (TEGDA), morpholine, MWCNTs (internal diameter 2–6 nm, outer diameter 10–15 nm, length 0.1–10 mm, and purity 490%), and interferents were obtained from CDH (Delhi, India) and Aldrich (Steinheim, Germany). Phosphate buffer

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solution (PBS, pH 6.8, ionic strength 0.1 M) was used as a supporting electrolyte. Standard stock solution of BSA (500 mg mL  1) was prepared using deionized triple-distilled water (conducting range (0.06–0.07)  10  6 S cm  1). Human blood serum samples were collected from a local pathology laboratory and stored in a refrigerator at 4 1C, before use. The pharmaceutical sample analyzed was Rabipur (Rabies vaccine). 2.2. Apparatus All voltammetric measurements were carried out with a polarographic analyzer/stripping voltammometer [model 264A, EG & G Princeton Applied Research (PAR), USA] in conjunction with an electrode assembly (PAR model 303A) and a X–Y chart recorder (PAR model RE 0089). A conventional three-electrode system was adopted where platinum wire was used as an auxiliary electrode, saturated Ag/AgCl electrode as a reference electrode, and MIP-modified MWCNTs-CE as a working electrode. Chronocoulometry measurements were performed on an electrochemical analyzer (CH instruments USA, model 1200A). FT-IR and NMR spectroscopic measurements were performed by Varian 3100 FT/IR (USA) and JEOL AL 300 FT-NMR (Japan), respectively. Morphological studies of bare and MIP-modified electrode surface were made using scanning electron microscope (SEM) [JEOL, JSM, Netherlands, Model 840A]. All experiments were carried out at 2571 1C. 2.3. Synthesis of TEGMPA Synthesis of TEGMPA is similar to that of ethylene glycol 3-morpholine propionate acrylate (Yu et al., 2009). For this a mixture of 1.3 mL of morpholine (15 mmol) dissolved in 7.5 mL methanol was added drop-wise in TEGDA (1.36 mL, 15 mmol) at 0–5 1C, under magnetic stirring in nitrogen atmosphere. FT-IR was used to monitor the completion of the reaction. When the N–H peak at 3515 cm  1 was disappeared, methanol was removed by rotatory evaporation. The product so obtained was identified by 1H NMR as follows: 1H NMR (D2O): d 6.1 (1H), d 5.8 (1H), d 6.4 (1H), d 3.7 (4H), d (4H), d 3.6 (4H), d 4.2 (4H), d 3.5 (8H), d 2.6–2.8 (4H). 2.4. Synthesis of cross-linker (1,3-diacryloyl urea, DAU) Preparation and characterization of cross-linker, DAU, are described elsewhere (Prasad et al., 2010). In brief, to alkaline solution of urea (1.8 g urea/15 mL 1.0 M NaOH) 4.87 mL AC was added drop-wise and heated for 20 min at 80 1C. A crude white product was separated out (the completion of reaction was indicated by the disappearance of the pungent smell of AC) and this was re-crystallized with ethanol. 2.5. Electrode preparation MWCNTs render biocompatibility to the ceramic electrode, besides inculcating electro-conductivity. Furthermore, CNTs are homogeneously dispersed in ceramic (sol–gel) matrix to improve stability as compared to pest or composite electrode. MWCNTs-CE surface is more amenable to ‘‘surface grafting from’’ approach for the growth of a nanometer thin MIP film (Prasad et al., 2011). Also, this electrode is reportedly best amongst other modified electrodes (carbon CE, carbon CE modified with MWCNTs) in terms of providing lower charging current and hence better signal/noise ratio (Prasad et al., 2011). For MWCNTs-CE preparation in this work, 1.0 mL ethanol, 1.0 mL TMPM, 0.5 mL water, and 10.0 mL of 0.1 M HCl were mixed together for 30 min to obtain sol–gel, followed by a hand on mixing with 100 mg MWCNTs. The suitable amount of this homogenized mixtures was filled in a glass tube (outer diameter 0.4 cm,

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length 3.0 cm) under physical pressure, and then left to dry for 48– 72 h at room temperature. Electrical contact was achieved by inserting a copper wire through the top via open tip of the glass tube. The bottom tip was first smoothened and polished with an emery paper to obtain a bright surface and then rinsed with water. As many as three vinyl exposed MWCNTs-CEs were obtained from the entire reaction mixture. In order to optimize the concentration of double bonds at the surface of electrode, four additional MWCNTs-CE were prepared using MWCNTs (100 mg) and the varying amount of sol–gel (50, 100, 150, and 200 mL). The amount of double bonds at the vinyl exposed MWCNTs-CE surface was determined by a catalytic bromine addition reaction between Br2 (KBrO3/KBr in acidic condition) and the prevalent double bonds catalyzed by HgCl2. The excess Br2 was evaluated by the standard method of iodometric titration. This led the determination of actual amount of Br2 consumed by double bonds that is equivalent to the concentration of vinylic double bonds at the electrode surface [For details vide Supplementary Data Section (S.1)]. 2.6. Grafting of MIP on the exposed vinyl groups of MWCNTs-CE For grafting MIP on to the vinyl exposed MWCNTs-CE (Scheme 1), the pre-polymerization mixture [template (BSA, 0.001 mmol/2 mL TDW), monomer (TEGMPA, 0.02 mmol/1.0 mL TDW), cross-linker (DAU, 0.2 mmol/1.0 mL TDW), and APS (10 mL, 20% w/v)] was mixed with 15.0 mg of MWCNT-COOH. The whole content was purged with N2 gas for 10 min, and 10 mL of this solution was spin coated on the MWCNTs-CE surface at 1500 rpm for 20 s. The electrode was kept in a UV chamber at 38 1C for 3 h (with intermittent heating at 10 min interval) to initiate free radical polymerization. Note that continuous exposure of electrode in UV chamber was avoided just to safeguard against denaturation of protein. Similar procedure was also followed to prepare the non-imprinted (control) polymer i.e., NIP-grafted electrodes in the absence of template (BSA). Template molecules were retrieved from the polymeric (MIP-template adduct) film by stirring the modified electrode into 0.1 M NaOH for 30 min. The complete template removal was ensured until no voltammetric response of the template was noticed. 2.7. Voltammetric procedure For electrochemical measurement, the analyte was first accumulated with stirring for 60 s (accumulation time) in an open circuit

and then after a negative potential of 0.9 V vs. Ag/AgCl was imposed. After 15 s equilibration time, differential pulse voltammetric (DPV) run was scanned from 0.7 V to þ0.7 V vs. Ag/AgCl using pulse amplitude of 25 mV, pulse width 50 ms, and scan rate 10 mV s  1. Cyclic voltammograms (CVs) were also recorded in the potential window  0.7 V to þ0.7 V at different scan rates (10–200 mV s  1). Since oxygen did not influence the voltammetry of analyte, any deaeration of the cell content was not required. All DPV runs for each concentration of test analyte were quantified using the method of standard addition.

3. Results and discussion 3.1. Evaluation of graft efficiency and its effect on surface imprinting The concentration of the double bonds per unit area of electrode surface (E, mol/cm2) is used to evaluate the graft efficiency, and this can be defined as E¼

ðV b V s ÞC 2A

ð1Þ

where Vb and Vs, are the volumes (L) of Na2S2O3 standard solution consumed in the blank and sample experiments (in the absence and presence of electrode), respectively, C is the concentration of Na2S2O3 standard solution (mol/L), and A is the area of MWCNTsCE (cm2). From the above equation, graft efficiencies were calculated for four different electrodes obtained using varying amount of TMPM as shown in Fig. S1.A. This depicted an increase in graft efficiency with the increase of TMPM. Interestingly, of all electrodes prepared with different amounts (50 mL, 100 mL, 150 mL, 200 mL) of TMPM and duly modified with MIP, MIP-modified MWCNTs-CE carrying 150 mL of TMPM at preliminary layer responded a highest current (Fig. S1.B) for BSA (7.59 ng mL  1). At the surface of this MIP-modified MWCNTsCE, optimized vinyl double bonds by the virtue of TMPM grafting might have induced the selective and high occurrence of imprinting polymerization, leading to form thin MIP layer consisted of maximum number of recognition sites. Any amount lower than 150 mL TMPM and thereby the less amount of vinyl groups led to the low efficiency of imprinting polymerization, at the surface of electrode. On the contrary, vinyl groups were so crowded at MWCNTs-CE surface that the excessive amount of TMPM (4150 mL) always resulted in a low occurrence of imprinting polymerization, due to

Scheme 1. Schematic representation of MIP-modified MWCNTs-CE fabrication.

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the possible steric hinderance or self-polymerization of surface vinyl groups. Hence, MWCNTs (100 mg) mixed with 150 mL of TMPM might be considered to be the best optimized composition to obtain MWCNTs-CE for polymer chain growth with efficient imprinting. 3.2. Polymer characteristics Vinyl exposed MWCNTs-CE inherited a unique characteristics provided by the sol–gel matrix with excellent biocompatibility and CNTs with attractive electrochemical features. Herein, TMPM is sandwiched between MWCNTs and MIP; TMPM molecules on the one hand interacted with the hydrophobic side walls of nanotubes while on the other side covalently anchored the imprinted polymer network (Scheme 1). The parts of nanotubes shielded by a chain of silicate particles, however, may not be electrochemically accessible. Nevertheless, the exposed (unshielded) parts of the nanotubes still remain intact and are readily accessible for solution species to serve as nano-electrodes (Gong et al., 2005). The ratio of template, monomer, and cross-linker needs to be optimized since the recognition ability of imprinted polymer is primarily dependent on both the print molecule and functional precursors of the polymer. Different template to monomer molar ratios (1:10, 1:20, 1:40, 1:60, and 1:80) were tested on MWCNTs-CEs; the optimized template-monomer stoichiometry of 1:20 yielded a maximum DPV response for a known concentration (9.95 ng mL  1) of BSA (Fig. S2.A). There are reportedly 18 net negative charges on each BSA molecule (Carter and Ho, 1994) in neutral medium. Consequently, functional monomers carrying the equivalent amount of positive charges are minimally required for charge compensation to ensure a stable self assembly of BSATEGMPA complex in pre-polymer mixture. Accordingly, this complex with 1:20 stoichiometry, [BSA.TEGMPA20]þþ, eventually led the production of a cationic polymer motif, [BSA.TEGMPA20]2n þ , for substrate imprinting on MWCNTs-CE surface. Any amount of TEGMPA less than 0.02 mmol revealed instability of the system responding lower current due to the increased heterogeneity in the structure. Furthermore, too many functional monomers excess than requisite for self assembly may lead non-specific analyte binding. The crosslinker amount could be an aided factor toward the stability of protein–monomer complex. In this work, the maximum development of DPV current response occurred when the cross-linker (DAU) amount 0.2 mmol was used with 1:20 complex. This is due to the improved stabilization of binding sites. The further increase of crosslinker should be avoided, since this may impede the template diffusion across the MIP motif, as was evinced by the declined response of BSA encapsulation (Fig. S2.B). In the polymerization, APS served as an initiator while monomer acted as an accelerator of the polymerization process in the capacity of co-initiator on abstraction of proton and generation of initiating free radicals thereof, as shown in Fig. S3 (Yu et al., 2009). Polymerization conditions have drastic impact on protein imprinting. The concerted effect of initiator and co-initiator in the form of two generated free radicals (Fig. S3) triggered the polymerization kinetics at low polymerization temperature that helped avoiding protein denaturation on exposure to UV light. An optimum of 38 1C polymerization temperature for 3 h, on intermittent exposure to UV light for 10 min interval, resulted in electrode modification for optimum DPV current response (Fig. S2.C). Exceeding temperature above 38 1C (Fig S2.D) might have induced conformational changes in BSA molecule (Takeda et al., 1989) that resulted in decreased DPV current. Also, at temperature lower than 38 1C, the polymerization process required longer time for complete reaction.

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binding mechanism (Fig. 1) in aqueous medium. BSA is a large guest molecule with several exposed reactive functionalities, at its surface, for binding with the host MIP. Such bindings are suggested on the basis of downward shifts of IR bands of participating key groups. [For details, vide Supplementary Data Section S.2]. An insight into the surface morphology of the modified surface of MWCNTs-CE was feasible through SEM images (Fig. 2). The bare MWCNTs-CE showed distinctly visible nanotubes (Fig. 2A). Surface of MIP-BSA adduct-modified MWCNTs-CE is relatively compact (Fig. 2B). Upon template retrieval from this, the MIPmodified electrode (Fig. 2C) revealed rather a rough surface and thereby a high surface area of film, which is beneficial for the adsorption of proteins. Fig. 2D displays the side view of MIP modified MWCNTs-CE reflecting 68.1 nm thickness of coating layer [vide Supplementary Data Section S.3]. 3.4. Electrochemical behavior Fig. 3A shows CV runs of 1.99 ng mL  1 of BSA, recorded within the potential window  0.7 V to þ 0.7 V (vs. Ag/AgCl), after analyte accumulation for 60 s and subsequently exposing the MIP-modified MWCNTs-CE sensor at  0.9 V for 15 s equilibration time. The bare (unmodified) electrode, however, responded only at a higher scan rate ( Z100 mV s  1) for the higher concentration of BSA (Z20.0 ng mL  1), with broader and ill defined features (Fig. 3A, inset). The accumulation of analyte was favored owing to strong electrostatic interaction between positively charged MIP film and negatively charged BSA molecule, irrespective of negative accumulation potential r  0.9 V imposed. The polarity on the modified MWCNTs-CE could drastically be altered on applying more negative potential ( 4 0.9 V) which restricted analyte binding. BSA electrochemistry is reportedly confined with the minimum of three redox active amino acid residues [Cystein (CYS),

3.3. Spectral and surface characterization FT-IR (KBr) spectra (Fig. S4) of template, monomer, MIP, and MIP-adduct are comparatively examined to support the proposed

Fig. 1. Suggested binding mechanism of BSA via multiple point electrostatic and hydrogen bonding interactions (BSA: monomer molar ratio 1:20; for the sake of brevity, only a few monomeric units are shown) with the MIP motif.

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Fig. 2. SEM images of (A) unmodified MWCNTs-CE, (B) MIP-adduct modified MWCNTs-CE, (C) MIP modified MWCNTs-CE and (D) side view of modified MWCNTs-CE.

tyrosine, and tryptophan (TRP)] (Chiku et al., 2008b). Besides, BSA redox reaction is also expected to take place on the sulphur double bonds in typical cases (Shao et al., 2005; Stankovich and Bard, 1978). In the present instance, the electro-active groups of CYS, tyrosine and TRP are bound through H-bondings within the MIP cavities (Fig. 1), and thus not free to take part in redox process in bound condition. Notably, BSA molecules are not stripped off the cavities owing to the strong electrostatic attraction between positively charged electrode and negatively charged template molecules on anodic scan. Since MIP particles are covalently linked with MWCNTs-CE, an apprehension of leaching of MIP adduct is ruled out during anodic scan. Accordingly, only option left for BSA oxidation is solely dependent upon the disulphide bonds. Disulphide bonds exposed at the surface of BSA were reduced initially at accumulation stage, given negative potential for the reduction. This process may thus be written as follows (Shao et al., 2005):

 Accumulation stage: (R–S ¼S–R)solution-(R–S ¼S–R)adsorbed

 Cathodic reduction stage: (R–S¼S–R)adsorbed þ2H þ þ2e  -(R–SH–SH–R)adsorbed (at  0.9 V)

 Anodic oxidation stage: (R–SH–SH–R)adsorbed-(R–S¼S–R)adsorbed þ2H þ þ2e 

The quasi-reversibility nature of the peaks is suggested by the corresponding DEp range, varying from 0.075 V to 0.300 V with the increase of scan rates (10–200 mV s  1), and the value of Ipa/Ipc

greater than unity. The broad peak width at all scan rates indicates the strong adsorption of both reduced and oxidized species; pre and post-adsorption peaks are concealed within the drawn-out peaks (feeble pre-adsorption peaks, however, seen at high scan rates). The quasi-reversibility for this process was also confirmed from the different slopes of Ipa vs. n1/2 and Ipc vs. n1/2 profiles as shown below: Ipa ¼ ð2:89 7 0:21Þ n1=2 þ ð7:497 1:84Þ,

R2 ¼ 0:98

ð2Þ

Ipc ¼ ð1:89 70:15Þ n1=2 þ ð4:93 7 1:34Þ,

R2 ¼ 0:98

ð3Þ

Under these conditions of quasi-reversibility, it may be possible to study the kinetics of the electrode reaction. Accordingly, the separation of peak potential, DEp, should be a measure of the standard rate constant (K0) for electron transfer process. These DEp values were introduced in the working curve described by Nicholson (1965) for obtaining the transfer parameter, c, and then the value of K0 was estimated according to the following equation (Pad and Leddy, 1995).

C¼

K 0 ðDoxi =Dred Þa=2 ðpDoxi nFv=RTÞ1=2

ð4Þ

To estimate K0 from Eq. (4), the diffusion coefficient (D) (assuming Dox ¼Dred ¼D) was obtained from the chronocoulometry experiment. According to the integrated Cottrell equation (Bard and Faulker, 2001), the relationship between Q and t1/2 (Anson plots) can be described as follows: Q ¼ 2nFACD1=2 t 1=2 p1=2 þ Q dl þ Q ads

ð5Þ

Q ads ¼ nFaG0

ð6Þ

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Fig. 3. (A) CV runs of BSA (1.99 ng mL  1) at different scan rates: (a) blank, 10, (b) 10, (c) 20, (d) 50, (e) 100, and (f) 200 mV s  1 at MIP modified MWCNTs-CE and unmodified MWCNTs-CE (inset). (B) DPV responses on MIP modified MWCNTs-CE run a for blank, runs c and d for BSA (1.99 and 6.99 ng mL  1) in aqueous medium, runs (f) and (g) for BSA (2.89 and 4.91 ng mL  1) in serum, run (i) for BSA (4.48 ng mL  1) in vaccine, run (k) for BSA (5.25 ng mL  1) in milk, runs (b), (e), (h), and (j) for BSA (20.0 ng mL  1) at NIP modified MWCNTs-CE in aqueous, serum, pharmaceutical, and milk samples, respectively.

where A is area of electrode (0.0827 cm2), C is the concentration (1.99 ng mL  1) of BSA, Qdl is the double layer charge, Qads is the faradic oxidative charge, and G0 is the surface coverage. For bare and modified electrodes, Qdl and total charge (Qdl þQads) were estimated from the respective intercepts of the Anson plots (Q and t1/2) in the absence and presence of BSA. Accordingly, Qads was calculated as 8.79  10  6. Surface coverage can be obtained in terms of number of electron ‘n’ by the equation defining Nerstian adsorbent layer (Hassen et al., 2007): "

# n2 F 2 0 Ip ¼ G An 4RT

ð7Þ

Accordingly, n and G0 were obtained to be 1.95 and 5.59  10  10 mol cm  2, respectively. This reflects total surface coverage of specifically bound analyte (4.6  10  11 mol or 2.77  1013 molecules) to MIP cavities (each molecule per cavity). The slope of the Anson plot (0.346  10  3 mC s  1) revealed an estimate of 2.55  10  2 cm2 s  1 for D. Substituting the D value (Dox ¼Dred) the K0 values at different scan rates were calculated from Eq. (4) for different values of c [1.75 (10 mV s  1), 0.17 (20 mV s  1), 0.11 (50 mV s  1)] as 0.437, 0.060, and 0.061 cm s  1 (mean K0 ¼0.186 cm s  1). The decrease in K0 represents sluggish kinetics of electron-transport for BSA oxidation with the increase of scan rate, under the adsorbed state of analyte in the domain of molecular cavities of imprinted polymer.

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Insofar as the sensitivity of the measurement is concerned, the DPV technique is better than CV at the scan rate, 10 mV s  1. This is because of the fact that an approximately 3.5-fold higher current is obtained in the sufficient time scale of measurement, using pulse amplitude 25 mV and pulse width 25 ms. DPV runs (Fig. 3B) showed symmetrical peaks for BSA detection in aqueous and real samples (serum, milk, and vaccine) with the MIP modified electrodes, without any matrix complication. NIP-modified electrodes did not respond to BSA in aqueous and pharmaceutical samples. However, some nonspecific adsorption of BSA could be seen on NIP-modified electrode in serum samples which was easily removed simply by water washing (n¼ 3, 0.5 mL). The reproducible regeneration of modified electrodes for the next use was feasible adapting the method of template retrieval (vide Section 2.6). Accordingly, the used electrode was regenerated by dipping in 0.1 M NaOH solution for 30 min, under stirred condition. The overall renewal of the modified electrode was confirmed by DPV until no template residue was left in the modified film to respond any voltammetric current. Any deformation of MIP cavities was ruled out after regeneration, as the renewed sensor always responded quantitative (100%) response of BSA in aqueous medium. In this work, any single modified electrode could be used for as many as 60 consecutive runs, with quantitative recoveries, after regeneration by the method of template extraction (Fig. S5). Furthermore, the reproducibility on a single electrode, which was renewed after each run, was examined by obtaining multiple DPV runs (Fig. 3B, run c) for BSA (1.99 ng mL  1) in aqueous medium. Insofar as electrode to electrode variation is concerned, a parallel measurement for BSA (2.89 ng mL  1) in blood serum on three modified electrodes, prepared in the same ways in different batches, responded quantitative recoveries with RSD 1.5% (Fig. 3B, run f). This indicates precision of the result as well as reproducibility in MIP sensor development. Notably, the multiple runs (Fig. 3B, runs c and f) both in aqueous and blood serum samples obtained with the regenerated MIP-sensor, show requisite ruggedness of the sensor without revealing any medium effect and false-positives. 3.5. Analyte adsorption behavior The Langmuir equation (Eq. (8)) provides a relationship between the concentration (C) of BSA solution, and the amount of BSA adsorbed on the surface (! 0) (Smiechowski et al., 2006): C

G0

¼

1 C þ Bads Gmax Gmax

ð8Þ

where Bads is the adsorption coefficient and ! max represents the maximum amount of protein that can adsorb on the surface. Thus, a linear equation C/G0 ¼(0.003470.0003)  1011Cþ(0.046670.0029) (R2 ¼0.984), for the plot of C/G0 vs. C is obtained. The intercept (equivalent to slope/Bads) of this equation suggests an estimate of adsorption coefficient (Bads) to be 7.29  109 L mol  1. The Gibbs free energy of adsorption, DGads can be estimated using equation (Wright et al., 2004):   1 DGads exp ð9Þ Bads ¼ 55:5 RT where 55.5 represents the molar concentration of water (mol L  1) which was used as the solvent. The large negative value of DGads ( 66.23 KJ mol  1) indicates spontaneous analyte adsorption on the MIP surface. The value of DG, in the present case, is higher to that reported earlier for proteins (Smiechowski et al., 2006). 3.6. Optimization of analytical parameters As observed in CV measurement, the extent of analyte accumulation was negligibly affected by the negative polarization of modified MWCNTs-CE and accumulation was effective even at an

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open circuit. The maximum development of DPV current occurred given accumulation time for 60 s, followed by saturation in binding sites. However, the modified electrode needed its activation, before voltammetric measurement. The optimum activation potential in this work was 0.9 V; any potential less or higher than this might have tremendous effect on the stability of the proposed electrostatic model of template-adduct film coated on MWCNTs-CE (Fig. S6.A and B). As far as pH of the working solution is concerned, the DPV current for BSA (5.95 ng mL  1) was observed maximally at pH 6.8 (Fig. S6.C). The observed decreased current response in acidic or basic pH/6.8S may be accorded with the aggregation of BSA which causes defolding both at low and high pH (Terashima et al., 2002). The DPV anodic peak was found to be shifted positively with pH in accordance with the linear equation: Ep (V)¼ (0.05570.004) pHþ( 0.28870.025), R2 ¼0.98, the slope of which corroborates an equal number of protons and electrons (2e  /2H þ ) in the electrode process. 3.7. Analytical determination Under optimized operating DPV conditions, the estimation of BSA was carried out in aqueous, human blood serum, pharmaceutics, and liquid milk. The corresponding results are depicted as linear calibration equations between peak current (Ip, mA) and concentration (C, ng mL  1) along with respective recoveries and LODs in Table 1. For all samples studied, a complete saturation of binding sites in the concentration range, 20.02–30.91 ng mL  1, was attained.

Notably, the blood serum samples were diluted as many as 1000fold so as to move the detection within the range of detection limits and also to mitigate the matrix effect to the larger extent. Any pretreatment such as deproteinization and/or ultrafiltration of serum has deliberately been avoided as this may lead inaccuracies in the final results. Instead, the dilution was found to be very effective against matrix effect, and the sample behavior almost approximated to that of aqueous solution. As a matter of fact, the slopes of calibration equations of all the real samples studied were very close to that of aqueous sample. The proposed sensor is compared with a recently reported surface imprinted chitosan coated MWCNTs based biosensor (Chen et al., 2012), by means of Student’s t-test. Herein, tcal (1.6) is found to be less than ttab (4.3), at the confidence level of 95% (R2 ¼0.99). This revealed that although a similar order of precision in the final result is realized within the concentration range (1.99–9.84 ng mL  1) by both methods, the latter one, despite being highly sensitive than ours, is not suited for in-field investigation owing to its instability at room temperature; and moreover, the obtained data could be regarded equivocal as a consequence of heavy matrix effect and cross-reactivity. On the other hand, the proposed sensor is relatively cost-effective, portable, and easy-to-use with high sensitivity for disease diagnosis in clinical settings, without any false-positive or cross-reactivity. It is worth to compare the proposed MIP sensor with other known sensors for BSA determination (Table 2). Accordingly, the detectibility, i.e., LOD and sensitivity, i.e., limit of quantitation (LOQ) of most of the techniques are inferior to this work. The proposed sensor has high stability

Table 1 Sample behavior. Sample

Regression equation

Range (ng mL  1)

Recovery (%)

LODa (3r) (ng mL  1)

RSDb (%) (n¼ 3)

Aqueous

IP ¼(7.738 7 0.034) n¼8, R2 ¼ 0.999 Ip ¼(7.850 7 0.033) n¼8, R2 ¼ 0.999 Ip ¼(7.859 7 0.047) n¼6, R2 ¼ 0.999 IP ¼(7.978 7 0.056)

Cþ (1.797 7 0.633),

1.99–30.91

99.2–101.0

0.42

1.02

Cþ (0.7557 0.52),

2.89–27.28

98.3–102.0

0.42

0.48

4.48–20.02

97.6–103.0

0.41

0.60

5.25–24.00

97.6–103

0.40

0.73

Human blood serum (dilution 1000-times) Pharmaceutics (antirabies injection) Liquid milk a b

Cþ (1.114 70.668), 2

Cþ (0.524 70.899), n¼5], R ¼ 0.999

LOD based on the minimal distinguishable signal for lower concentration of analytes. RSD (%) for three set of LOD data.

Table 2 Comparison of different methods for determination of BSA. S. n.

Method

LOD (ng mL  1)

Range (ng mL  1)

Buffer (pH)

Remarks

Reference

1.

Direct electrochemical detection at diamond electrodes Modified Si nanowires sensors MIP based flow injection chemiluminescence sensor MIP as a sorbent for solid phase extraction Highly sensitive detection of protein at Ag nanostructures HPLC—UV detection Fluorimetric determination

190

(50–400)  103

PBS (7.4)

Chiku et al. (2008b)

– 1.5

(24.7–65.0)  103 10–5000

PBS (7.0) –

No real sample analysis, study of denatured BSA, no interferents study No real sample analysis Analysis in liquid milk, milk powder

670

(20–200)  103

PBS (5.5)

Soleimani et al. (2012)

0.05

10  2–10  5

PBS (7.0)

500 182

(1–100)  103 (0–12)  103

0.55

1–1000

0.28

10  1–105

– Britton–robbinson (2.8) Carbonate/ bicarbonate (9.32) PBS (6.8)

Analysis in whey, milk, urine, and serum No analysis in real sample, no interferents study. So lacks selectivity Pharmaceutical vaccines Milk and milk powder, lacks sensitivity, difficult instrumentation No real samples analysis

0.40

1.99–30.91

PBS (6.8)

2. 3. 4. 5. 6. 7. 8. 9.

10.

Cd–Te quatum dots modified ITO electrodes Chitosan coated magnetic nanoparticles modified MWCNTs modified MIP biosensor Present work

Shao et al. (2005) Yu et al. (2010)

Devi et al. (2012) Hamidi and Zarei (2009) Sun et al. (2008) Zhang et al. (2012)

Chen et al. (2012) No interferents study, very low stability (15 day, 4 1C), no in-field analysis – Liquid milk, serum, anti-rabies vaccines, highly stable, cost-effective, can perform in-field analysis

B.B. Prasad et al. / Biosensors and Bioelectronics 39 (2013) 236–243

with a longer endurance [for details, vide Supplementary Data Section S.4]. 3.8. Cross-reactivity studies MIP and NIP modified MWCNTs-CEs were subjected to crossreactivity studies with a number of proteins having different isoelectric points [(insulin (INS), haemoglobin (Hb), human serum albumin (HSA), ovalbumin (OVA), and lysozyme (LYS)], amino acids [Glutamic acid (GA), CYS, TRP, Ascorbic acid AA, histidine (HIS)], and dopamine (DOP), which are known interferents during BSA detection. Barring OVA and HSA, none of the interferents were observed to be responsive on MIP modified electrode, when studied individually or in binary BSA-interferent mixtures (Fig. S7) [For details, vide Supplementary Data Section S.5].

4. Conclusions For the first time, we are reporting MIP-based biomimetic electrochemical sensor for BSA determination in real samples. This responded the quantitative analysis of BSA at ultra-trace level, (LOD, 0.40 ng mL  1, S/N¼3), without any matrix complication and false-positive contribution. For the faster ingress and egress of analyte, the ‘‘surface grafting from’’ approach have successfully been exploited on vinyl exposed MWCNTs-CE for MIP nano-film coating in aqueous condition. The MIP coated MWCNTs-CE may serve as a practical sensor for clinical studies, under environmentally and biologically benign conditions. Herein, BSA molecules were not denatured both during MIP development in aqueous medium and DPV measurement at applied optimum operating conditions.

Acknowledgments Authors thank Council of Scientific and Industrial Research— University Grant Commission (CSIR-UGC), New Delhi for granting junior research fellowship to A. P. and CSIR for a senior research fellowship to M. P. T. Instrumental facilities were procured out of a recent project (SR/S1/IC-30/2010) funded by the Department of Science and Technology, New Delhi.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.07.080.

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