Electrochemical detection of epinephrine using a biomimic made up of hemin modified molecularly imprinted microspheres

Abstract

In this study, a highly sensitive and selective molecularly imprinted polymer (MIP) was synthesized using a functional monomer, 2,4,6-trisacrylamido-1,3,5-triazine, and its application for electrochemical detection of epinephrine (EP) was demonstrated. This particular monomer was selected based on the interaction energies computed for the formation of a pre-polymer complex using computational studies. Furthermore, EP imprinted microspheres were prepared by precipitation polymerization using hemin as the catalytic centre in order to mimic the active site of an enzyme namely peroxidase. Molecularly imprinted and non-imprinted microspheres were characterized using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). An electrochemical sensor for EP detection was fabricated by modifying a gold disc electrode with molecular imprinted microspheres stabilized by a chitosan/Nafion mixture. A linear concentration range from 5 × 10⁻⁸ M to 40 × 10⁻⁶ M with a very low detection limit of 1.2 × 10⁻⁸ M (S/N = 3) is determined for the proposed sensor. Our results clearly demonstrate an efficient sensing capability of imprinted polymer with good reproducibility, stability and higher selectivity for EP detection over its other structural analogues and potential interferents. Essentially, the proposed electrochemical sensor follows a cascade reaction mechanism since it consists of two catalytic sites that aid in EP detection. The analytical applicability of this sensor towards the determination of EP is demonstrated using human blood serum and injection samples.

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1. Introduction

Epinephrine (4-[(1R)-1-hydroxy-2-(methylamino)ethyl]benzene-1,2-diol) [EP] is an important neurotransmitter in the mammalian central nervous system and acts as a chemical mediator for transferring nerve impulses to different organs, thereby controlling its performance in biological reactions and nervous chemical processes.¹ Numerous diseases, such as contraction of smooth muscles, blood pressure, glycogenolysis in the liver and muscle, and lipolysis in adipose tissue, are related to changes in concentration of EP in the living mammalian system. EP has also been used as a common emergency healthcare medicine.² Enzyme mimics that possess the structure and activity close to that of natural enzymes can be prepared by the molecular imprinting technique.³ In general, this technique involves the creation of selective recognition sites in synthetic polymers and the principle is similar to that of enzyme specificity proposed by Fischer as early as 1894.⁴ Preparation of molecularly imprinted polymers (MIPs) involves the formation of self-assembly between a functional monomer and the analyte (template), followed by co-polymerization of a cross linker via either thermal or UV radiation. Extraction of the pre-formed template leads to the formation of cavities or patterns that are complementary to the shape and size of the template present within the polymer matrix.⁵ In the last two decades, MIPs became increasingly attractive in many fields of chemistry and biology due to their versatile applications and catalytic properties, particularly for sensor applications.^6–8 These new type of enzyme mimics have a lot of advantages, such as moderate cost, ease of preparation and long time stability, compared to the traditional mimics of natural antibodies and enzymes.⁹

Although synthesis of MIPs seems quite easy, screening of the best functional monomer having more binding interactions with the selective template makes the imprinting a tedious and time consuming technique.¹⁰ Combinatorial chemistry and molecular modeling are the two most promising tools for identifying a suitable monomer to develop MIPs with enhanced recognition properties.¹¹ In general, combinatorial methods are more rapid than traditional approaches;¹² however, they are expensive since many conditions are required for necessary evaluation and identification. Combinatorial screening is not just a predictive tool but involves laborious experimental verification procedures.¹³ Computer aided study of MIPs is a rational and fast method to improve their recognition properties. Ab initio calculations based on quantum mechanics at different levels, such as Hartree–Fock,¹⁴ Moller–Plesset¹⁵ or density functional theory (DFT),¹⁶ have been employed to rationally design the desired MIPs. Among the various methods, DFT provides a high level accuracy information with reasonable and affordable computational costs.¹⁷

Several methods have been developed to date to determine EP in pharmaceutical and clinical samples. Conventional methods, namely, high performance liquid chromatography (HPLC),¹⁸ chemiluminescence,¹⁹ fluorimetry,²⁰ spectrophotometry,²¹ capillary electrophoresis²² and flow injection analysis,²³ have been reported. Recently, electroanalytical techniques became popular for the determination of environmental and biological compounds due to their ease of detection, sensitivity, stability, accuracy, lower cost and simplicity.^24–26 Electrochemical detection of EP on bare surfaces (unmodified electrodes) has some fundamental problems, mainly arising from high overpotential associated with the detection of analyte and sluggishness of the electrode kinetics. Various strategies, including electrochemically pretreated glassy carbon electrode (GCE),²⁷ polymer modified GCE and self-assembled monolayer modified Au electrodes,^28,29 were used for the analysis.

Keeping this in mind, in the present study, an electrochemical detection of EP is demonstrated using hemin modified microspheres of MIP. A functional monomer, namely, 2,4,6-trisacrylamido-1,3,5-triazine (TAT) derived from triamino triazine, has been selected based on the computational studies and is being used for the preparation of EP imprinted polymer. This particular MIP has a compact, symmetric and rigid structure (ESI; Fig. S1†) that can facilitate multiple non-bonding interactions in the well-defined three dimensional preferences.³⁰ Preparation of MIP using a precipitation polymerization method involves the use of TAT as a functional monomer and chlorohemin as a co-monomer having catalytic activity for specific recognition of the template and replacing the natural enzyme chloroperoxidase.^31,32 Chlorohemin is covalently linked to the polymer during polymerization and incorporation of chlorohemin leads to the formation of active sites into MIP that are further utilized for the catalytic reduction of hydrogen peroxide (H₂O₂). Hemin in the MIPs can be oxidized by H₂O₂ and then be re-reduced by EP. The latter reaction involves the formation of EP–quinone, which is electroactive and can be reduced further, leading to an overall cascade reaction for determining EP. MIP is immobilized on the Au electrode surface along with chitosan due to its effective transduction, membrane forming ability, biocompatibility and detection in aqueous systems with a longer period of stability.³³ After MIP immobilization, a thin layer of Nafion is coated over the surface to stabilize MIP because of its chemical and mechanical stability, along with the cationic selectivity and higher conductivity.³⁴ Differential pulse voltammogram (DPV) assays were performed for the detection and quantification of EP in the present study. The creation of binding sites in EP MIP provides a better enhancement in sensitivity of the proposed sensor. Various important parameters, such as the amount of MIP to be coated, concentration of H₂O₂ and pH of the electrolytic medium, which influences the sensitivity and selectivity of the electrochemical sensor, were optimized. The fabricated electrochemical biosensor showed excellent performance characteristics for the determination of EP. Furthermore, the feasibility of the proposed sensor for detecting EP in human blood serum and injection samples is demonstrated for practical applications.

2. Experimental section

2.1. Chemicals

Epinephrine (EP), melamine (Mel), 2,2′-azoisobutyronitrile (AIBN) and ferri-protoporphyrin IX (hemin) were purchased from Sigma-Aldrich. Ethylene glycol dimethacrylate (EGDMA), acryloyl chloride (AC) and acetonitrile (HPLC grade) were obtained from Merck chemicals. Similarly (−)-isoproterenol hydrochloride (IP), 3,4-dihydroxy-L-phenylamine (HPA), 3,4-dihydroxyhydrocinnamic acid (HCA), L-ascorbic acid (LAA) and 1,2-dihydroxy naphthalene (DN) were procured from Sigma-Aldrich. All these chemicals were of analytical grade and used without any further purification. All the aqueous solutions were prepared using Millipore water having a resistivity of 18.2 MΩ cm obtained from the Milli-Q system (Millipore Inc.).

2.2. Hardware and software

A personal computer, namely, an Intel Pentium 4, running with the Red Hat Linux operating system with a 2.0 GHz CPU, 2 GB RAM having 200 GB hard disc was used for the computational studies involving simulation of functional monomers. This system was used to execute the software package Gaussian 03. Avogadro 1.0.1 was employed as a graphical user interface for Gaussian.

2.3. Geometry optimization and energy calculations

Three-dimensional structures of the template, monomer and monomer–template complexes were built with the aid of Avogadro program. The virtual library of functional monomers essentially consists of acidic, basic and neutral monomers. The molecular geometries were optimized using DFT calculations at the B3LYP/6-31G(d) level implemented in Gaussian 03 and the single point energies were calculated at the B3LYP/6-31+G(d,p) level of theory.³⁵ The interaction energy (ΔE) values of the pre-polymer complexes were calculated using eqn (1) as follows:

ΔE = E_{(template–monomer)} − E_template − ∑E_monomer, (1)

where E_{(template–monomer)} is the energy of template–monomer complex, E_(template) is the energy of EP and E_(monomer) is the energy associated with a functional monomer. The basis set superposition error (BSSE), which often substantially affects the calculated stabilization energy values, was corrected by means of the counterpoise method.³⁶

2.4. Synthesis of the functional monomer

The selected functional monomer, TAT, was synthesized using a procedure reported elsewhere.³⁰ Melamine (Mel, 20 mmol) was dissolved in 20 mL of dimethyl formamide (DMF). AC (65 mmol) was gradually added to Mel in DMF and the reaction mixture was stirred for about 10 hours. Then, the resultant mixture was washed with hot (35 °C) DMF–water (10 mL; 1 : 1 v/v) to remove the residual precursors. The product obtained was then characterized by FTIR, ¹H NMR, and elemental analysis: CHN analysis (found: C – 49%; H – 4.5%; N – 30.1% and the formula C₁₂H₁₂N₆O₃ requires C – 49.9%; H – 4.1%; N – 29.3%). ¹H NMR, δ ppm d₆ DMSO, 7.75 (s, 1H, –NH), 3.45 (s, 1H, –CH) and 2.50 (s, 1H, –CH₂). FTIR (KBr), cm⁻¹: 1681 (amide I), 1487 (amide II), 3329 (–NH stretching; broad), 3118 (CH stretching), 1338 (–C–N– stretching), 1004 (–CH bending) and 772 (–CH₂ rocking vibration of alkene). These studies clearly reveal the successful synthesis of the TAT functional monomer.

2.5. Preparation of EP imprinted and non-imprinted microspheres

Hemin modified EP imprinted polymer (MIP) was prepared using a non-covalent immobilization protocol (ESI; Fig. S2†). Accordingly, the pre-polymerization complex was prepared by mixing 0.5 mmol EP (template), 2 mmol TAT (functional monomer) and 5 mL mixture DMSO and acetonitrile (3 : 2 v/v) in a 15 mL glass vial. The contents were shaken well to allow the formation of self-assembly by host–guest chemistry. Subsequently, 5 mmol EGDMA (cross linker), 0.02 mmol hemin and 0.1 g AIBN were sequentially added. The mixture was then sonicated and purged with nitrogen gas for 10 minutes to create an inert atmosphere. The vial was completely sealed and kept in a water bath at 60 °C for 15 hours to complete the polymerization reaction. Then, the polymer was washed with excess methanol to remove any residual precursors. The resultant polymer was observed to be microspheres and was collected on a nylon filter (0.27 μm pore size); washed repeatedly with methanol–acetic acid (9 : 1 v/v) mixture to remove the template. The reference or non-imprinted polymer (NIP) was also prepared in the same manner, following the above-mentioned procedure but in the absence of template, EP.

2.6. Preparation of MIP modified electrode

Initially, a bare gold disc electrode was cleaned by polishing with alumina slurry and ultrasonically cleaned with de-ionized water, absolute ethanol and Millipore water each for 5 minutes. Then, the electrode was cleaned with a hot mixture of “piranha” solution (1 : 3 mixture of 30% H₂O₂ and conc. sulphuric acid), rinsed with ultra-pure water and dried. Followed by polishing, the electrode was subjected to a potential cyclic sweeping between −0.4 V and 1.6 V in 0.5 M H₂SO₄ until a constant cyclic voltammogram representing the electrochemical characteristics of a bare gold electrode (gold oxide formation and stripping peaks) was obtained. Initially, 5 μL of 1% chitosan in 0.8% acetic acid was coated onto the pre-cleaned gold disc electrode and dried for an hour. About 1 mg of EP imprinted microspheres dispersed in 1 mL of 0.1 M phosphate buffer solution (PBS) (pH = 7.0) was sonicated for 10 minutes. 10 μL of this well dispersed MIP (1 mg mL⁻¹) in PBS (pH – 7.0) solution was drop casted onto the chitosan modified Au disc electrode and dried in air. Finally, 5 μL of 0.5% Nafion in acetonitrile solution was coated onto the electrode to obtain chitosan/MIP/Nafion modified Au surface and this electrode was explored further for the detection of EP. This modified electrode was stored at 4 °C when not in use. Analytical applicability of the proposed sensor was investigated for EP detection using human blood serum samples and injection solutions as model systems. EP hydrochloride injection samples were purchased from a local pharmacy with proper approval and permission. Human blood serum samples were obtained from a local pathology clinic with proper permission. All these experiments were performed in compliance with the relevant laws and institutional guidelines and the institutional committees have approved these experiments. It can also be noted that informed consent was obtained for these experimentations wherein human blood serum samples were employed for real sample analysis.

2.7. Electroanalytical measurements

All electrochemical measurements were performed using 0.1 M PBS (pH = 7.0) as the supporting electrolyte, which is degassed with nitrogen for 10 minutes to avoid the interference from the reduction of oxygen present in the aqueous electrolyte during the measurements. The cyclic voltammograms were obtained by scanning the potential from −0.3 V to +0.8 V vs. SCE at a fixed scan rate of 100 mV s⁻¹. The current measurements were performed using DPV in a potential range between +0.0 V and +0.6 V and to record DPVs, a step potential of 8 mV, a modulation amplitude of 50 mV and a scan rate of 16 mV s⁻¹ were employed. Electrochemical impedance spectroscopy (EIS) experiments were recorded at a half-wave peak potential of the redox mixture consisting of 1 mM [Fe(CN)₆]^3−/4− solution along with 100 mM KCl as a supporting electrolyte using frequency ranging from 100 kHz to 0.1 Hz and 5 mV as the alternating current amplitude.

2.8. Instrumentation

Electrochemical studies using voltammetric and electrochemical impedance spectroscopy (EIS) measurements were performed using an Eco Chemie, electrochemical work station, model Autolab 302N using GPES software version 4.9 and frequency response analyzer (FRA), software version 2.0. A three electrode system employing a gold disc electrode (BAS Inc.) as a working electrode, saturated calomel electrode (SCE) as a reference electrode and a spiral platinum (Pt) wire as a counter electrode were used in this study. All potentials referred in the text were against SCE. A Toshniwal pH meter was used for measuring pH of the solutions. Surface morphologies of the prepared polymers were analyzed with a help of FESEM (Carl Zeiss, Supra 35 VP, Germany). FTIR spectra of these samples were obtained using a Shimadzu model IR Affinity FTIR spectrophotometer.

3. Results and discussion

3.1. Selection of the functional monomer for the preparation of MIP

A virtual library of 15 functional monomers was analyzed to select the most suitable functional monomer. The interaction energy values between EP and each of these functional monomers were calculated and presented in Table 1. The interaction energy of such a pre-polymer complex is directly related to its stability.³⁷ From the computational studies, TAT is identified to be the most promising functional monomer capable of forming stronger interactions with the template, EP. This may be due to the formation of multiple interactions in well-defined three dimensional preferences with the functional monomer, TAT.³⁸ This particular monomer is used further to prepare the molecularly imprinted polymer (MIP) using EP as a template.

Table 1 Interaction energy values obtained for the formation of pre-polymer complexes between EP and various functional monomers employed for the computational study

S. no.	Pre-polymer complex	Interaction energy^a ΔE (kcal mol^−1)
a BSSE corrected interaction energies.
1	EP–2,4,6-trisacrylamido-1,3,5-triazine	−43.9507
2	EP–p-vinyl benzoic acid	−37.9957
3	EP–urocanic acid	−37.3744
4	EP–trans-3-pyridyl acrylic acid	−36.4834
5	EP–itaconic acid	−35.1531
6	EP–2-(trifluoromethyl) acrylic acid	−31.6578
7	EP–acrylamide	−30.7479
8	EP–methacrylic acid	−30.5848
9	EP–acrylic acid	−30.3965
10	EP–4(5) vinylimidazole	−28.5768
11	EP–4-vinyl pyridine	−27.9242
12	EP–N,N-methylene bisacrylamide	−26.5248
13	EP–acrylonitrile	−26.0855
14	EP–allylamine	−25.0062
15	EP–acrylamido 2-(methyl) 1-propane sulphonic acid	2.324

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3.2. Characterization of EP imprinted polymer

The structure and morphology of EP imprinted MIP were characterized using FTIR and FESEM analyses. FTIR spectra were obtained for synthesized EP imprinted microspheres (MIP), before (B) and after (C) the extraction of EP along with the non-imprinted microspheres (NIP) prepared in the absence of EP (A) and the corresponding spectra are shown in Fig. 1. EP MIP before extraction (Fig. 1B) showed a broad band in the region 2950–3490 cm⁻¹ due to the hydrogen bonding between the –NH group of TAT and –OH group of EP. Shifting of the hydroxyl group of EP and –NH of TAT from 3500 to 3490 cm⁻¹ and 3350 to 2990 cm⁻¹, respectively, is attributed to the formation of hydrogen bonding. This shift essentially arises due to the non-covalent interactions, i.e., hydrophobically driven hydrogen bonding between TAT and EP. On the other hand, non-imprinted polymers (Fig. 1A) do not show any such type of shift in peaks to the lower wavenumber region, suggesting the absence of template, EP. Some common bands were observed for TAT, hemin and EGDMA viz., 1720 cm⁻¹ corresponds to C=O stretching; 1454 cm⁻¹ is attributed to –CH₂ and –CH₃ deformation and 1159 cm⁻¹ due to C–O vibration. It is interesting to notice that a broad band in the region 2950–3300 cm⁻¹ disappeared in the EP extracted MIP spectra (Fig. 1C), indicating a complete removal of EP.

Fig. 1 FTIR spectra of EP imprinted microspheres before and after extraction (B and C) along with the non-imprinted microspheres (A).

Furthermore, FESEM analysis was carried out to investigate the structural and morphological changes that occur in the resultant MIP and for comparison, a similar study using NIP was also performed. Fig. 2 shows the FESEM images of EP imprinted (A) and non-imprinted (B) polymers. FESEM image of MIP (Fig. 2A) clearly reveals the formation of uniformly sized, discrete and nearly monodispersed particles resembling microspheres, whereas the NIP (Fig. 2B) displays irregular and highly agglomerated particles. This indicates the possible ordered cross linker reactions and imprinted effect of MIP on the resultant microspheres.

Fig. 2 FESEM images of EP imprinted (A) and non-imprinted microspheres (B).

3.3. Electrochemical characterization of MIP coated Au electrodes

Electrochemical impedance spectroscopy (EIS) is used for investigating and analyzing the electron transfer characteristics of EP imprinted and non-imprinted polymer modified Au electrodes. Generally, the formation of semicircle in EIS is used for the determination of the charge transfer resistance (R_ct) value. Impedance spectra of various modified electrodes were obtained using 0.1 M KCl containing 5 mmol L⁻¹ [Fe(CN)₆]^3−/4− as a redox probe with a small excitation amplitude of 5 mV peak to peak over the frequency range from 100 kHz to 100 mHz and the respective plots are shown in Fig. 3. In order to determine the R_ct values, the collected impedance data were best fitted to Randle's equivalent circuit. From these Nyquist plots, it is evident that the bare Au surface (Fig. 3a) exhibited a semi-circle with a higher R_ct value of 868 Ω, suggesting a relatively sluggish electrochemical performance of the redox probe on a bare Au electrode.³⁹ Moreover, R_ct values at the Au electrode modified with NIP (Fig. 3b) and MIP before extraction of EP (Fig. 3c) remarkably decreased to 155 Ω and 198 Ω, respectively. It is very interesting to note that after the extraction of the template, the MIP coated Au electrode (Fig. 3d) showed a very low R_ct value of 57 Ω, indicating the formation of cavities due to removal of EP, which facilitates and enhances the electron transfer characteristics at the MIP modified electrode interface.⁴⁰ These results confirm the surface modification using MIP and NIP microspheres and the EP-MIP modified Au electrode shows a facilitated electron transfer process along with good electrical conductivity.

Fig. 3 EIS of bare Au disc electrode (a), NIP (b) and MIP microspheres modified Au electrodes before (c) and after extraction (d) of EP in 5 mmol L⁻¹ [Fe(CN)₆]^3−/4− solution containing 0.1 M KCl as a supporting electrolyte. Inset: equivalent circuit used for fitting the impedance data.

In order to confirm that the changes in R_ct values are due to the modified EP-MIP films, impedance studies were performed at various steps of chitosan and Nafion coated electrodes for the EP-MIP system before and after extraction of EP using the same above-mentioned electrolyte solution consisting of a redox probe along with a supporting electrolyte. Results obtained from these studies (ESI; Fig. S3†) clearly rule out the possibility of electrostatic attraction between chitosan and the redox probe along with the repulsive interaction between Nafion and the redox probe. These studies clearly indicate that the observed changes in R_ct values are mainly due to the process of imprinting and due to the removal of template and EP by extraction.

3.4. Electrochemical detection of EP using MIP modified Au electrodes

EP imprinted MIP modified Au electrodes were explored further for the possible application in electrochemical detection of EP. The mechanism associated with EP sensing relies mainly on the cascade reaction involving redox change of EP at molecularly imprinted microspheres that act as a catalyst. This reaction involves the oxidation of EP to EP–quinone, whereas the hemin unit present within MIP is reduced and furthermore the oxidized form of hemin is regenerated by H₂O₂. The presence of H₂O₂ in the electrolyte aids the conversion of reduced hemin to oxidized hemin and in-turn enhances the EP sensing process. Overall, the proposed electrochemical biosensor has essentially two active sites, namely, MIP and hemin. It is interesting to note that both these active sites play a crucial role and that EP is broken down during the electrochemical detection process in a cascade reaction mechanism. The principle of EP detection is illustrated in Fig. 4. Quantification of EP is determined by monitoring the electrochemical reduction of EP–quinone species on the electrode surface by applying a suitable potential. The mechanism is similar to those proposed for the electrochemical determination of glucose and determination of phenolic compounds using biomimetic catalysts such as dopamine β-monooxygenase, peroxidase and tyrosinase enzymes.⁴¹ This particular mechanism also explains the higher sensitivity of the proposed sensor arising from signal amplification due to the cascade redox reactions.^42,43

Fig. 4 Schematic of the principle associated with the electrochemical detection of EP.

EP oxidized product EP–quinone is electrochemically active and could be reduced at the electrode surface to produce an amplified response.⁴⁴ Both cyclic voltammetry (CV) and DPV measurements were employed to evaluate the electrochemical activity of the EP-MIP modified Au electrode towards EP detection. CV studies were carried out in 0.1 M PBS (pH = 7.0) solution at a fixed potential sweep rate of 100 mV s⁻¹ (ESI; Fig. S4†). Formation of redox peaks in CV is attributed to EP and the EP–quinone redox couple, and this correlates very well with the proposed cascade reaction mechanism for EP sensing wherein this redox couple plays a critical role. Interestingly, the change in redox current due to incremental addition of EP is very minimal; making it very difficult to distinguish. In contrast, differential pulse voltammetry (DPV) assays exhibit significant changes in the reduction current, due to elimination of double layer charging current associated with the modified electrodes, which maximizes the signal to noise ratio. These experiments were performed by sweeping the potential between 0.0 V and 0.6 V and monitoring the redox reaction for EP and EP–quinone formation.⁴⁵ The comparative DPV responses of MIP and NIP coated Au disc electrodes obtained after subtraction of background current are displayed in Fig. 5. The reduction current is significantly increased at the MIP modified Au electrode (Fig. 5b) after addition of 10 μmol L⁻¹ EP. On the contrary, the NIP microspheres coated electrode (Fig. 5a) did not show any response in the absence and presence of EP, indicating the absence of active sites for EP detection. In contrast, MIP modified electrodes show peak formation at 380 mV due to the formation of EP–quinone species upon adding EP, indicating the effect of cascade reactions essentially arising from the hemin unit present within the immobilized microspheres that could be oxidized by H₂O₂ and re-reduced by the added analyte, EP. In order to prove that this sensing mechanism works only in the presence of H₂O₂, a few other experiments were also carried out using CV studies. These CVs were recorded using EP-MIP coated Au electrodes in PBS solution without H₂O₂ and along with a fixed concentration of EP (ESI; Fig. S4†). The respective cyclic voltammograms show clearly that the EP-MIP coated Au electrode shows a clear redox peak corresponding to EP and EP–quinone redox reaction only if H₂O₂ is present. This cyclic reaction is particularly responsible for EP detection and is illustrated in Fig. 4.

Fig. 5 Baseline corrected DPV responses of (a) NIP and (b) EP-MIP modified Au disc electrodes in 0.1 mol L⁻¹ PBS (pH – 7.0) containing 200 μmol L⁻¹ H₂O₂ and 10 μmol L⁻¹ EP.

3.5. Optimization of conditions

From the electrochemical studies, it is clear that EP-MIP modified Au electrode could be used for the detection of EP and it is necessary to optimize the conditions at which the proposed electrochemical sensor works best and exhibits good sensor characteristics, namely, sensitivity, limit of detection, linear concentration range and stability. First, the quantity of EP imprinted microspheres to be used for coating onto the working electrode for fabrication of the sensor was analyzed. An insufficient quantity of active sites does not allow for suitable amplification of the analyte response. On the other hand, a higher quantity of MIP microspheres can lead to slow diffusion of analyte to the recognition sites and hence results in an inefficient communication between the binding sites and transduction.⁴⁶ Henceforth, the optimization of various parameters and conditions is absolutely necessary for any sensor. The change in DPV peak current with respect to an increase in reaction time was examined by coating 10, 20, 30 and 50 μg of EP imprinted microspheres onto the working electrode (ESI; Fig. S5A†). It is identified that the sensors prepared with 10, 20 and 30 μg of microspheres showed an increase in peak current during the initial 20 minutes, while the 50 μg microspheres coated sensor exhibited decreased reduction current for 20 minutes and a slight increase after 20 minutes (ESI; Fig. S5A†). The sensor prepared with 10 μg of microspheres was found to display a rapid increase and higher reduction current, suggesting a suitable communication between the reaction product, EP–quinone and the electrode surface. The gradual decrease in analyte response with increasing the quantity of microspheres may be attributed to the increase in resistance of the electrode surface that inhibits the transduction response. Hence, 10 μg of microspheres with an incubation time of 20 minutes was considered as the optimal value.

Similarly, the dependence of sensor response on H₂O₂ concentration in the range of 50–500 μmol L⁻¹ was also studied (ESI; Fig. S5B†) since it plays a vital role in cascading the reaction of peroxidase enzymes, with higher concentrations inhibiting the catalysis.⁴⁷ It was noticed that at a fixed EP concentration of 10 μmol L⁻¹, the variation in reduction current increased with H₂O₂ concentration and reached a maximum at 200 μmol L⁻¹ (ESI; Fig. S5B†). Hence, this concentration was chosen for all the subsequent experiments.

Moreover, the influence of pH on the reduction peak current of EP–quinone was also investigated in the 2–10 pH range. As the pH of the medium was gradually increased, the peak current reached its maximum value at pH 7.0 and at pH values higher than 7.0, the reduction current was found to decrease (ESI; Fig. S5C†). This is due to the negative charge on the EP–quinone species that inhibits the reduction at higher pH values. Therefore, an optimized pH value of 7.0 for the detection medium was selected.

3.6. Evaluation of electrochemical sensor characteristics for EP detection using DPV studies

Finally, an EP-MIP microspheres coated Au disc electrode fabricated under optimized conditions was used as a sensing platform to monitor the changes in reduction current values by following a DPV assay, as shown in Fig. 6. The EP reduction current increases with systematic increasing concentration of EP suggesting an efficient electrocatalytic property of MIP microspheres due to the presence of specific cavities formed during the polymerization. The reaction product EP–quinone is reduced resulting in the formation of a peak and the response was explored via DPV (Fig. 6A). It is evident from the analysis that increasing concentration of EP results in increased reduction current, suggesting the potential applicability of MIP electrodes as a sensor matrix. For quantification, the change in reduction current at the peak value was plotted against the concentration of EP (Fig. 6B). There are two linear ranges from 5.0 × 10⁻⁸ M to 1.1 × 10⁻⁶ M and 1.1 × 10⁻⁶ M to 40 × 10⁻⁶ M with the regression equations Δi (μA) = −0.4588 + 0.2222 [EP] μmol L⁻¹ (R² = 0.9890) and Δi (μA) = −0.7152 + 0.0066 [EP] μmol L⁻¹ (R² = 0.9895).

Fig. 6 (A) DPV curves recorded in 0.1 mol L⁻¹ PBS (pH = 7.0) containing 200 μmol L⁻¹ H₂O₂ at the MIP modified Au electrode for different EP concentrations: (a) blank (0 M), (b) 5.00 × 10⁻⁸ M, (c) 4.03 × 10⁻⁷ M, (d) 7.98 × 10⁻⁷ M, (e) 1.13 × 10⁻⁶ M, (f) 5.84 × 10⁻⁶ M, (g) 10.07 × 10⁻⁶ M, (h) 13.94 × 10⁻⁶ M, (i) 20.75 × 10⁻⁶ M, (j) 26.54 × 10⁻⁶ M, (k) 30.15 × 10⁻⁶ M, (l) 35.93 × 10⁻⁶ M and (m) 40 × 10⁻⁶ M respectively. (B) A plot of variation in reduction current vs. EP concentration. Inset: similar plot shown at lower concentrations of EP.

At concentrations above 40 μmol L⁻¹, the sensor exhibits saturation and follows Michaelis–Menten behaviour with a K_m value of 18.23 μmol L⁻¹. The molecularly imprinted polymers as recognition elements behave similarly to biosensors constructed with the natural enzymes and also possess advantages such as low cost, high stability and ease of preparation.^48,49 The detection limit and linear concentration range values were calculated according to the IUPAC recommendations and those values were found to be 1.2 × 10⁻⁸ M and 5.0 × 10⁻⁸ to 40 × 10⁻⁶ M, respectively. Compared with other sensors described in the literature over the past decade for EP detection, the proposed sensor in this study displayed a significant improvement in detection limit and comparable linear concentration range, as shown in Table 2.^{29,45,50–55} In addition, to investigate the reproducibility and repeatability of the proposed electrochemical sensor, several experiments were performed in PBS (pH = 7.0) solution containing 200 μmol L⁻¹ H₂O₂ with addition of 10 μmol L⁻¹ EP into the electrochemical cell and recording the change in reduction current (Δi) as the sensor response to the target analyte. The EP-MIP electrode exhibited a relative standard deviation (RSD) value of 4.4% of reduction peak current for a series of five sensors prepared in the same manner. Similarly, there was no significant change in the response even after 25 measurements. The current response was observed to retain 95% of its initial value after 20 days. These results clearly demonstrate very good stability and reproducibility of the imprinted sensors.

Table 2 Comparison of the various electrochemical methods reported for EP determination

S. no.	Method	Electroanalytical technique used	Linearity range (M)	Detection limit (M)	Reference
a SAM – self assembled monolayer.b Indium tin oxide.c Graphene/gold nanoparticles/glassy carbon electrode.
1	SAM^a on gold electrode	Square wave adsorptive stripping voltammetry	5.0 × 10^−7 to 1.0 × 10^−5 and 1.0 × 10^−5 to 6.0 × 10^−4	1.0 × 10^−8	29
2	SAM^a on gold nanoparticles	Cyclic voltammetry	1.0 × 10^−7 to 3.2 × 10^−8 and 1.0 × 10^−5 to 2.0 × 10^−4	6.0 × 10^−8	45
3	Ionic liquids/carbon nanotubes based carbon paste electrode	Differential pulse voltammetry	3 × 10^−7 to 450 × 10^−6	9.0 × 10^−8	50
4	MIP in sol–gel on ITO^b electrode	Amperometry	1.0 × 10^−4 to 1.0 × 10^−3	—	51
5	Carbon paste electrode/iron phthalocyanin	Differential pulse voltammetry	1.0 × 10^−4 to 1.0 × 10^−3	5 × 10^−6	52
6	Gr/Au NPs/GCE^c	Cyclic voltammetry	5.0 × 10^−8 to 8.0 × 10^−6	7.0 × 10^−9	53
7	Electropolymerized MIP	Amperometry	3 × 10^−7 to 1.0 × 10^−3	9.0 × 10^−8	54
8	MIP based sensor	Differential pulse adsorptive stripping voltammetry	4.9 × 10^−10 to 3.2 × 10^−8	1.1 × 10^−10	55
9	Hemin modified MIP	Differential pulse voltammetry	5.0 × 10^−8 to 1.1 × 10^−6 and 1.1 × 10^−6 to 40 × 10^−6	1.2 × 10^−8	Present work

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3.7. Selectivity of the EP sensor

In order to evaluate the selectivity of synthesized MIP, three compounds which are structural analogues to EP viz., (−)-isoproterenol hydrochloride (IP), dihydroxy naphthalene (DN), 3,4-dihydroxy-L-phenylamine (HPA) and two other compounds, namely, 3,4-dihydroxy hydrocinnamic acid (HCA) and L-ascorbic acid (LAA), with different structures but are capable of forming quinone derivatives were considered in this study. DPV experiments were carried out by monitoring the change in reduction current for both MIP and NIP modified Au electrodes toward EP sensing in the presence of these interferences at two different concentrations viz., 10 μM and 20 μM and the corresponding data are displayed in Fig. 7. The EP imprinted microspheres based sensor showed at least five times more current response for EP–quinone than a non-imprinted microspheres based sensor. It is evident from these results that in case of interferents, there is no significant difference in the response exhibited by MIP and NIP microspheres modified electrodes, proving the specific selectivity towards EP for the synthesized MIP microspheres (Fig. 7). It is very interesting to notice that although IP nearly possesses the same structure as EP, the response of the sensor is much lower than that for EP. This suggests that the high selectivity of the sensor mainly arises due to the specific recognition sites and not merely the cavities, which reflect the process of templating during the polymerization in terms of size, shape and arrangement of the functional groups.

Fig. 7 Variation of reduction current at MIP and NIP microspheres modified electrodes towards EP detection and other potential interferents each added at 10 μmol L⁻¹ and 20 μmol L⁻¹ concentrations. Supporting electrolyte: 0.1 mol L⁻¹ PBS (pH = 7.0) containing 200 μmol L⁻¹ H₂O₂.

3.8. Analytical applicability of the proposed EP sensor

The analytical applicability of the proposed method was investigated for the determination of EP in human serum samples and injection solutions. EP hydrochloride injection samples (standard content of EP 1 mg mL⁻¹; 1 mL per injection) were purchased from a local pharmacy with proper approval and permission as mentioned above. Human blood serum samples were obtained from a local pathology clinic with proper permission as stated in the experimental section and it was stored under refrigeration. 50 μL of serum samples were diluted with 9.5 mL of PBS (pH = 7.0) solution to avoid the interferences arising from the serum matrix. The concentration of EP in the samples was varied from 1 μM to 10 μM and were analyzed for EP content using the proposed electrochemical sensor (ESI; Table S1†). The corresponding comparison values obtained from this EP sensor are shown in Table 3. These results clearly indicate that the recovery percentages for both injection and human serum samples are in the range of 96–112% with an RSD value between 1.75 and 5.48. These values demonstrate a highly promising analytical applicability of the proposed biomimetic sensor for electrochemical determination of EP in pharmaceutical and real samples analyses.

Table 3 Recovery studies of EP sensing in human blood serum and injection samples^a

Samples	Added (μmol L^−1)	Found (μmol L^−1)	Recovery (%, n = 3)
a These results are expressed as mean values and the ±RSD values are based on three replicate measurements.
Serum sample	—	ND	—
	1.48	1.67	112.8 (±3.46)
	2.91	3.22	110.6 (±2.25)
	4.41	4.36	98.86 (±2.45)
Injection sample	1.00	0.96	96 (±5.48)
	3.46	3.65	105.5 (±4.35)
	5.88	5.75	97.78 (±1.75)

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4. Conclusions

Rational synthesis of a new sensory MIP material for electrochemical detection of EP is presented, and EP imprinted microspheres are prepared by a precipitation polymerization method. A gold disc electrode was modified with microspheres of MIP for the electrochemical sensing of EP. Under optimized conditions, MIP modified Au electrodes exhibit a higher electrocatalytic activity, better sensitivity and selectivity towards EP detection in comparison to NIP modified electrodes. This confirms the formation of shape specific and site selective cavities during polymerization for the target analyte molecule. Electrochemical interface properties of the bare and MIP/NIP modified electrodes were studied using EIS. MIP modified electrodes showed less charge transfer resistance than with a NIP modified surface. The proposed method was applied for the determination of EP in human serum and pharmaceutical samples and the obtained results were more than satisfactory. This study clearly demonstrates the biomimetic polymers (MIPs) as promising materials to be used as artificial receptors for the electrochemical sensing of EP.

Acknowledgements

Authors are thankful to Dr S. Umadevi, UGC – Asst. Prof., Department of Industrial Chemistry, Alagappa University, Karaikudi for her technical guidance in synthesis of the functional monomer. V. G. acknowledges the funding from the Council of Scientific and Industrial Research (CSIR), India through the 12^th five year plan network project, namely, Molecules to Materials to Devices (M2D) having project number CSC 0134.

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Footnote

† Electronic supplementary information (ESI) available: More results and analysis (Fig. S1 to S5 and Table S1). See DOI: 10.1039/c5ra16636e

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