Molecular imprinted polymer based electrochemical sensor for selective detection of paraben

Journal Pre-proof Molecular Imprinted Polymer Based Electrochemical Sensor for Selective Detection of Paraben ˘ Beyhan Buse Yucebas ¨ ¸ , Yesim Tugce Yaman, Gulcin Bolat, Erdogan ¨ ur, Ozg ¨ Lokman Uzun, Serdar Abaci

PII:

S0925-4005(19)31567-9

DOI:

https://doi.org/10.1016/j.snb.2019.127368

Reference:

SNB 127368

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

5 July 2019

Revised Date:

22 October 2019

Accepted Date:

30 October 2019

¨ ur Please cite this article as: Yucebas ¨ ¸ BB, Yaman YT, Bolat G, Ozg ¨ E, Uzun L, Abaci S, Molecular Imprinted Polymer Based Electrochemical Sensor for Selective Detection of Paraben, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127368

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Molecular Imprinted Polymer Based Electrochemical Sensor for Selective Detection of Paraben

Beyhan Buse Yücebaş1, Yesim Tugce Yaman2,3†, Gulcin Bolat3†, Erdoğan Özgür*1,2, Lokman Uzun*1,2, Serdar Abaci2,3

Biochemistry Division, Department of Chemistry, Hacettepe University, Ankara, Turkey

2

Advanced Technologies Application and Research Center, Hacettepe University, Ankara, Turkey

3

Analytical Chemistry Division, Department of Chemistry, Hacettepe University, Ankara, Turkey

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*Corresponding Author:

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Erdogan Ozgur (PhD),

Phone: +90–312-297 7353

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*Corresponding Author:

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E-mail: [email protected]

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Fax: +90–312-297 7354

Lokman UZUN (PhD),

Fax: +90–312-299 2163

Phone: +90–312-297 7337

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E-mail: [email protected]

† These

authors contributed equally.

GraphicalAbstract

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Highlights  Electrochemical detection of paraben was performed via molecular imprinting. Electrochemical molecular imprinted sensor platform offers rapid, sensitive and

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

selective electrochemical detection. 

Plastic antibody based biomimetic sensor platform offers high physical, chemical stability with long shelf-life.

Proposed sensing platform was successfully applied for cosmetic cream samples.

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

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Abstract

A new, simple, rapid, sensitive and selective disposable sensor platform was developed for

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the electrochemical detection of paraben based on molecular imprinting technique. Highly selective and specific recognition was established for the analyte through incorporating amino

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acid based polymerizable functional monomer. Paraben-imprinted poly-(2-hydroxyethyl methacrylate-N-methacryloyl-L-phenylalanine) (PHEMA-MAPA) nanofilm on a screenprinted gold electrode surface in the presence of polyvinyl alcohol (PVA) was synthesized by the molecular imprinting technique. Characterization of the fabricated electrode surfaces were

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performed with cyclic voltammetry (CV) and atomic force microscopy (AFM). The electrochemical behavior of paraben was investigated using CV and electrochemical detection studies were carried out with square wave voltammetry (SWV). Under optimal conditions, the linear working range was found to be 1-30 μM with a low detection limit as 0.706 µM. The obtained recovery values proved that the developed method can be successfully applied to

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cosmetic samples. The fabricated disposable sensor system can be used successfully in the determination of other important analytes in the future due to its good sensor performance.

Keywords: molecular imprinted polymer, paraben, electrochemical sensor.

1. Introduction Endocrine disrupting chemicals (EDCs) change the development and functioning of the

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endocrine system by acting as hormones or by blocking their duties when taken into the body. EDCs may have negative effects on biological processes such as growth, gender development, reproduction, insulin synthesis and metabolic rate etc. [1]. Parabens are para-

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hydroxybenzoic acid derivatives including methyl paraben (MP), ethyl paraben (EP), butyl paraben (BP), propyl paraben (PP) and are classified as EDCs [2,3]. Parabens are often

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preferred as protective chemicals in cosmetic and pharmaceutical industries due to their low

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price as well as their effective antimicrobial effect as they prolong the shelf life of the products by preventing the growth of harmful bacteria and fungus [4,5]. Moreover, paraben derivatives are frequently used in light drinks, fish, fruit products, gelatine jam, olives,

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pickles, wines and in bakery products (cakes, bread crumbs, filling materials, etc.). Although parabens have low toxicity, some studies have reported that they may disrupt the endocrine system by affecting the function of male and female reproductive systems and increasing the

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risk of breast cancer in women [6–8]. In addition, MP was found to cause further damage to the skin under ultraviolet rays [9]. Therefore, in order to reduce these possible hazards, the use of parabens in cosmetic products as preservatives is restricted by regulations in several countries, including The European Economic Community (EEC) Directive, up to an authorized concentration of 0.4% (w/w) for each paraben and up to concentration of 0.8% (w/w) for total parabens [10]. The dermal adsorption of personal care products is the most

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obvious way to exposure to parabens (levels of 2400 mg/kg bw-day) in humans [11]. Since paraben derivatives are widely used in different areas and their toxic effects are not fully established, highly selective and sensitive detection of these chemicals still remains important. Spectroscopic and chromatographic methods have been generally used for the determination of paraben derivatives such as high performance liquid chromatography (HPLC) [5,12], liquid chromatography (LC) [13–15], mass spectroscopy (MS) [16–20], and capillary

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electrophoresis [21,22]. There are also electrochemical studies for the determination of various paraben derivatives by employing various nanomaterials on electrode substrates [23– 28]. Electrochemical methods are attractive because they are inexpensive and less time

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consuming when compared with other conventional analytical techniques, besides being suitable for on-site and rapid analysis [29]. One of the most important parameters affecting

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the performance of an electrochemical sensor is the structure of the working electrode. For

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this purpose, a wide variety of working electrodes such as gold, carbon based, platinum, mercury etc. have been developed for the diagnosis of different analytes in the literature [30– 33]. Among these, solid electrodes show some disadvantages such as surface passivation

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issue, need for surface polishing and need to prepare new modified surface after each measurement. Therefore, researchers have eliminated these time consuming disadvantages by using disposable electrodes such as pencil graphite (PGE) [34] and screen-printed electrodes

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(SPEs) [35,36]. The use of single-use screen printed electrodes is becoming more popular in electrochemical determination studies due to the advantages such as low cost, good mechanical stability, easy adsorption of analyte on the surface and simple modification. Molecular imprinting is a promising technique which offers template-assisted formation of selective recognition sites in a synthetic polymeric network capable of mimicking the biorecognition ability of biomolecules, such as amino acids, nucleic acids, enzymes and

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antibodies etc. [37,38]. In this sense, commonly, a template molecule is complexed with functional monomer(s) in a porogen solvent through non-covalent bonds. Subsequently the monomer is polymerized around the template to create specific cavities easily accessible for the template molecule. After the template molecule is removed from the polymeric network, binding sites complementary to the template in size, shape and orientation are created which serves as a functional recognition element for sensing processes. Molecularly imprinted (MIP) polymers have high stability, low cost, high sensitivity and

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selectivity for targeting of molecules [39]. These properties enable the use of molecularly imprinted polymers for electrochemical sensing applications in a broad variety of areas such as life, pharmaceutical and environmental sciences [40–48]. There are only a few studies on

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MIP based electrochemical sensors investigating the selective determination of parabens [49,50] and development of new selective MIP sensors is still a special concern. The

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integration of biomolecules or active sites into a synthetic polymeric structure is one possible

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way to gain mimicking the biorecognition ability of biomolecules. Amino acids are the basis of the functional features and highly selective biorecognition ability of many biostructures. In this regard, electrochemical sensors based molecular imprinting technique is expected to

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improve the sensing properties [50,51]. Hence, in the presented study N-methacryloyl-Lphenylalanine (MAPA) was synthesized and its pre-complex structure with paraben was polymerized on a screen-printed gold electrode (Au-SPE) surface in the presence of 2-

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hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) with polyvinyl alcohol (PVA). The use of PVA during the polymerization process provides the formation of a porous structure, which favors the access of analyte through the electrode surface [52]. Thus, a disposable electrochemical sensor platform functionalized with MIP based structure having parabenic cavities was developed for the highly selective detection of paraben.

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The fabricated single-use electrochemical biosensor platform based molecular imprinted technology for the highly selective detection of paraben by copolymerization of MAPA, HEMA and EGDMA was introduced for the first time. The developed electrode was characterized by spectroscopic and electrochemical methods. The MIP based Au-SPE surfaces showed wide working range, high reproducibility and high selectivity for the electrochemical detection of the paraben. It also served as a practical and fast method that allowed on-site detection. In order to demonstrate applicability of the developed sensor

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platform in real samples, recovery studies were carried out in a cream sample. 2. Materials and Methods 2.1. Materials

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L-Phenylalanine, L-cysteine, PVA, HEMA, EGDMA, ,’-azobisisobutyronitrile (AIBN),

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dimethyl sulfoxide (DMSO), ethyl acetate, 1,4-dioxane, sodium hydroxide (NaOH) and 1Hbenzotriazole were supplied by Sigma Chemical Co (USA). Paraben, methyl paraben (MP),

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ethyl paraben (EP), butyl paraben (BP) and propyl paraben (PP) were purchased from Fluka Chemie AG (Buchs, Switzerland). Potassium dihydrogen phosphate (KH2PO4, ≥99.0%),

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potassium chloride (KCl, ≥99.0%), dipotassium hydrogen phosphate (K2HPO4, ≥99.0%), potassium ferricyanide (K4[Fe(CN)6], ≥99.0%) and potassium ferrocyanide (K3[Fe(CN)6], ≥98.5%) were supplied from Sigma-Aldrich. Other chemicals were supplied from Merck A.G. (Darmstadt, Germany).

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2.2. Instruments

All electrochemical studies were performed with a Gamry Interface 1000 model potentiostat/galvanostat. Surface characterization was obtained using Nanomagnetics Instruments (Oxford, UK) brand atomic force microscope. pH measurements were carried out

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with a Hannah model pH meter. Attenuated total reflection Fourier Transform Infrared Spectrophotometer (ATR FT-IR) were carried out with Nicolet™ iS50 FTIR Spectrometer. 2.3. Synthesis of N-methacryloyl-(L)-phenylalanine (MAPA), N-methacryloyl-(L)-cysteine (MAC) MAPA and MAC were synthesized as previously reported [46]. Firstly, 5.52 mmol of amino acid (L-phenylalanine or L-cysteine) was dissolved in 1.0 M NaOH. Benzotriazole methacrylate (MA-Bt) in 1,4-dioxane was added to the amino acid solution and the reaction

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occurred at room temperature for 30 min. Then, the 1,4-dioxane was evaporated under vacuum. The residue was extracted with ethyl acetate to remove unreacted chemicals. The solution was acidified to pH 6-7 and then the water was evaporated to obtain MAPA or MAC.

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2.4. Design of the paraben imprinted electrochemical sensor

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2.4.1. MAC coating on the screen-printed gold electrodes

Au-SPEs were used as substrates for the modification which were commercial and purchased

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from Metrohm DropSens (Spain). The gold surfaces of the SPE electrodes were cleaned with 1.0 M sulphuric acidic solution (1.0 M H2SO4) electrochemically and electrodes were kept in

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a vacuum oven (200 mmHg, 25°C) for 2 h. Then, 100 µL of MAC (5 mg/mL) was drop casted onto the gold surfaces and waited for 6 hours in order to generate a self-assembled monolayer containing polymerizable functional groups of methacrylate to facilitate further surface modifications [53]. Unbound MAC was removed by DI water and MAC modified electrodes

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were kept in vacuum oven (200 mmHg, 25°C). 2.4.2. Polymerization of paraben imprinted polymeric film on electrode surface The stock monomer solution containing MAPA-paraben complex (stoichiometric molar ratio was 1:1), HEMA and EGDMA was prepared for the formation of paraben imprinted porous polymeric film. PVA (25 mg/mL) aqueous stock solution was prepared to obtain a porous structure. AIBN (2 mg/mL) dissolved in DMSO was used as initiator. The polymer solution 7

was prepared by mixing 100 μL of stock monomer solution with 20 μL of PVA (25 mg/mL in DI) stock solution and 40 μL of AIBN solution. The polymer solution of 3.0 µL was dropped onto modified surface and bulk polymerization was performed using UV lamp (100 W, 365 nm) for 30 min. After the synthesis of polymeric film, pore-maker PVA was removed with DI water at 25°C for 1 h in shaking incubator. 2.5. Characterization studies Microscopic characterization of the sensor surfaces was carried out by atomic force

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microscopy (AFM) in dynamic mode in air atmosphere with an oscillation resonance frequency at 341.30 kHz. The vibration amplitude was applied at 1xVRMS and free vibration

get a view of a 2 µ × 2 µm and 4 µm × 4 µm areas.

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amplitude at 2xVRMS. Surfaces were scanned at a 1 µm/s and 256 × 256 pixels resolution to

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Electrochemical characterization studies were performed in 5 mM Fe(CN)63-/4- 0.1 M KCl solution. Cyclic voltammograms were recorded in the range from -0.5 V to 1.0 V (vs.

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Ag/AgCl) with scan rate 100 mVs-1.

Attenuated total reflection Fourier Transform Infrared Spectrophotometer (ATR FT-IR)

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spectra of molecular imprinted polymer based electrochemical sensor surface (before and after extraction of paraben) were carried out with Thermo Fisher Nicolet is50 model in a reflectance mode.

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2.6. Electrochemical procedures

All electrochemical studies were performed with three-electrode system. Screen printed gold electrode (Au-SPE) was used as a working electrode; platinum wire was used as the counter electrode and silver chloride (Ag/AgCl in 3 M KCl) was used as the reference electrode. Electrochemical behavior of paraben was studied by using CV in the range from -0.4 V to 1.0 V (vs. Ag/AgCl) with scan rate 100 mVs-1. Square wave voltammograms were recorded from 0.5 to 0.9 V (vs. Ag/AgCl) with 50 Hz (frequency) and 50 mV (pulse size) for the 8

electrochemical detection of paraben. Limit of detection (LOD) and limit of quantification (LOQ) were calculated from 3 Sb/m and 10 Sb/m (Sb: standard deviation of blank solution, m: slope of calibration range). 2.6. Real sample preparation In order to demonstrate the applicability of the developed sensor system, a cream sample was selected as a real sample which is one of the cosmetic products containing paraben derivatives. 0.1 g of the cream sample was weighed and dissolved in 2.0 mL of ethanol. Then,

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this sample was allowed to stand for 20 minutes at a stirring speed of 120 rpm on a magnetic stirrer until the solution got a complete homogeneity. The homogenous sample was passed through a filter and the stock solution was prepared and diluting to 100 mL with phosphate

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buffered saline (PBS; pH: 7.0). Recovery tests were carried out by using the standard addition method.

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3. Results and Discussion

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3.1. Characterization studies of MIP/Au-SPE

Surface morphology of the bare Au-SPE surface, MAC coated SPE before imprinting process and paraben imprinted porous polymeric film structures were examined by atomic force

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microscopy (AFM). The morphological changes on SPEs were clearly observed after MAC coating and MIP functionalization (Figure 1). Although SPE surface had almost planar properties (Figure 1a), the formation of MAC film on SPE increased the roughness (Figure

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1b). After paraben imprinted polymeric film coated onto MAC/Au-SPE produced a highly uniform porous structure (Figure 1c).

It was clear that the presence of PVA during

polymerization process caused a porous structure to the polymeric film whereas the pore size ranged from 40 to 400 nm. ---- Please insert Figure 1----

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The electrochemical characterization of MIP based sensor platform was carried out using a redox probe, Fe(CN)63-/4-, which possess a well-known electrochemical behaviour in order to clarify the electrochemical behavior of MIP/Au-SPE. Cyclic voltammograms were recorded to evaluate all changes on the electrode surface at each step as shown in Figure 2. Bare AuSPE showed a well-defined voltammogram, corresponding to the reduction/oxidation peak currents of the redox pair which were approximately equal and reversible. The highest peak current values for the bare surface indicated a faster electron-transfer than the modified

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surfaces (Figure 2.a). ---- Please insert Figure 2----

The Au-SPE surface modified with MAC to form a self-assembled layer containing

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polymerizable functional groups of methacrylate, showed lower anodic/cathodic peak currents of redox probes (Figure 2.b) as an indication of the limitation of the electron transfer in the

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reduction/oxidation reactions of the Fe(CN)6-3 /-4 ion pair due to successful coating of gold

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surface with MAC layer. The surface conductivity was more restricted at non imprinted (NIP) Au-SPE than MAC/Au-SPE (Figure 2.c). According to this result, it can be understood that deposition of this new organic layer hindered the transfer of electrons on the surface

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demonstrating that NIP polymerization step was obtained successfully. After modification with paraben imprinted porous polymeric film (MIP/Au-SPE), peak current values decreased obviously as shown in Figure 2.d and peak potential values shifted to more negative values

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leading to an increased peak to peak separation (ΔEp). This situation indicated that the peak currents decreased due to the presence of the electrical insulating organic layers (MAC, paraben and polymer) on the surface. After the template (paraben) molecules were removed from the surface (denoted as MIP(removed)/Au-SPE), the redox peak currents increased compared to MIP (non-removed)/Au-SPE surface (Figure 2.e). Based on the electrochemical

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characterization studies results, the developed sensor platform was suitable for the detection of paraben. Also, ATR FT-IR analysis was carried out to show the removal of paraben from the surface of molecular imprinted polymer based electrochemical sensor. Hence, MIP/Au-SPE and MIP (removed)/Au-SPE surfaces were analyzed. IR spectra and spectral subtraction of MIP/AuSPE and MIP (removed)/Au-SPE surfaces given in Figure S1a-b. In the IR spectra of MIP (removed)/Au-SPE surface, the absence of the benzene C-H stretching vibrations were

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observed in the 3100-3000 cm-1 region, in-plane C-H bending vibrations in the 1600-1000 cmregion and especially C-H bending in the 810-830 cm-1 region (1,4-disubstituted benzene)

indicated the removal of paraben. ATR FT-IR results indicated that the intensity of the peak

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absorbance of the O–H functional group (in the 3200-3600 cm-1 region) and carboxylic acid C=O stretching vibrations (1710 cm-1) decreased by removal paraben. The absence of those

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peaks indicates breaking the secondary interactions via aromatic ring in its structure and

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carboxylic acid groups between paraben and MAPA and emphasized that the template molecules, paraben was successfully removed from the polymer structure (Figure S1a, b). 3.2. Electrochemical behavior of paraben on MIP/Au-SPE

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Cyclic voltammetry method is generally used to reveal the behavior of the analyte in electrochemical assays. Therefore, cyclic voltammograms were recorded at bare and modified electrodes to understand the electrochemical behavior of the parabens at different surfaces

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(Figure 3). Firstly, when the anodic potential was scanned at 10 µM paraben adsorbed on MIP (removed) /Au-SPE, a single and a well-defined oxidation peak which is characteristic for all parabens was recorded at 0.734 V (vs. Ag/AgCl) with peak current of 3.075 µA (Figure 3.a). In the reverse scan, no reduction peak was observed due to irreversible nature of the reaction process as previously reported [54,55]. ---- Please insert Figure 3----

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However, in the absence of paraben in PBS (pH 7.0), no peak corresponding to paraben oxidation was observed as was expected (Figure 3.b). On the other hand, for bare Au-SPE, the presence of the irreversible oxidation peak appeared in lower magnitude (0.087 µA) at 0.8 V (vs. Ag/AgCl) (inset Figure 3.a) and the CV obtained in blank PBS did not show this oxidation peak (inset Figure 3.b). Thus, it can be deduced that oxidation peak current of paraben increased as 35-folds at imprinted polymer modified working electrode. Besides, oxidation peak potential shifted to zero which showed that the oxidation reaction of the

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paraben was facilitated by the modification of the sensor generating specific recognition sites for the target molecule, paraben [56]. The integration of MAPA into the polymeric structure enables mimicking of biorecognition ability of biomolecules through the secondary

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interactions via aromatic ring in its structure and carboxylic acid groups. Based on these results, the MIP/Au-SPE electrode served as a suitable microsurface for the sensitive

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detection of paraben. Scan rate study is also an important in order to obtain data on the

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oxidation/reduction mechanism of electroactive species. It is possible to understand whether the oxidation/reduction reaction mechanism of the analyte is controlled by adsorption or diffusion process. Therefore, CV measurements were recorded by increasing scan rate

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linearly in the range of 75 to 300 mVs-1 as given in Figure S2. As shown in voltammograms measured at different scan rates; oxidation peak current values increased with scan rate. The linear relationship between peak current-scan rates was given in Equation1; 𝐦𝐕

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𝐈(µ𝐀) = 𝟎. 𝟎𝟏𝟓

𝐬

– 𝟎. 𝟖𝟓𝟏

(𝐑𝟐 = 𝟎. 𝟗𝟗𝟐)

(Eq.1)

This linearity indicated that the paraben oxidation process was adsorption controlled at MIP/Au-SPE as expected which also confirmed/proved the positive contribution of MIP surface via capturing the analyte into polymeric network by pseudo-biorecognition process. 3.3. Optimization studies

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Experimental parameters were investigated and optimized to improve the performance of the developed MIP based sensor. One of the most critical step affecting the performance of the molecular imprinted polymers is to identify the optimal adsorption and removal (desorption) time of analyte from the surface (Figure 4a-b). After paraben was removed from the MIP surface, the effect of adsorption/contact time was studied by immersing the electrode in paraben-containing solutions at different time intervals. As can be seen from the Figure 4a, the anodic peak current of the analyte increased up to 60 min adsorption time. However, the

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surface reached saturation after this time and there was no significant increase in peak current. Hence, optimum and efficient adsorption time was determined as 60 min and kept constant during further studies. Removal time of paraben from the imprinted polymer surface was

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optimized in acetic acid:water mixture (1% HAc:H2O) (1:9, V:V) solution by recording square wave voltammograms in PBS (pH: 7.0) solution (Figure 4b). It was observed that the

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peak currents of the paraben decreased with removal time and the oxidation peak was

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completely disappeared at the end of 60 min demonstrating that paraben was successfully removed from the electrode surface. Therefore, optimum removal time was performed as 60 min for the next studies. Furthermore, the effect of the pH was optimized by recording the

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oxidation current in the paraben solutions having different pH values in the range of 4.0-8.0. As seen in the Figure 4c, the peak current increased up to pH 7.0; then decreased when it was adjusted to 8.0. The reason of this behaviour may depend on the solubility variation of

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paraben due to medium pH as well as change in polarization of side groups of template molecule (paraben) and functional monomer (MAPA). Therefore, the optimal and possible interactions between template and functional monomer inhibited after medium pH (7.0) which resulted in a significant decrease at pH 8.0. As a result, the optimum pH value was determined as 7.0 and kept constant during further studies. ---- Please insert Figure 4----

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3.4. Analytical performance MIP/Au-SPE Square wave voltammetry (SWV) method was utilized to reveal the working linear range of the developed sensor. Square wave voltammograms were recorded to determine the amount of paraben adsorbed onto the specific cavities. The measured current values were plotted against the concentration in order to obtain calibration curve. As shown in Figure 5, the obtained oxidation peak currents for paraben were linear at the concentration range from 1 µM to 30 µM. After the 30 µM concentration of the analyte, the peak current increased

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slightly as an indication of saturation of the sensing layer. For this reason, 30 µM was accepted as upper limit for the calibration curve. ---- Please insert Figure 5----

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Limit of detection (LOD) and limit of quantitation (LOQ) were calculated as 0.706 µM and 2.35 µM, respectively. Many sensor systems were developed for single/multiple detection of

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paraben derivatives in the literature (Table 1). The obtained linear range and LOD value were

[24,25,51,55,57–59]. ---- Please insert Table 1----

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comparable with the previously proposed electrochemical sensors for paraben derivatives

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For the reproducibility study, which is an important parameter in the analytical performance evaluation, four different MIP surfaces were prepared independently in the same way and SW voltammograms recorded and compared. The relative standard deviation (RSD) value was

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found as 2.45% (n=4) which revealed a reproducible fabrication of MIP surfaces for paraben detection. Finally, repeatability of the signals was evaluated by using the same sample for four consecutive measurements at a single day and RSD value was calculated as 3.15%. The results revealed the reproducibility of the sensor surface as well as having repeatable and reusable detection performance. Selectivity studies

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The selectivity of the developed sensor for paraben was investigated using paraben derivatives as competitors. Competitive adsorption studies were carried out using ethyl paraben (EP), methyl paraben (MP), propyl paraben (PP) and butyl paraben (BP) molecules. Two different approaches were followed for selectivity studies. In the first approach; both MIP and NIP coated electrodes were interacted with each competitor molecule as a singular solution and peak current values were measured (Figure 6). The selectivity and relative selectivity coefficients were calculated by using these current values.

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---- Please insert Figure 6---The selectivity and the relative selectivity coefficients were defined as k = current valuetemplate/ current valuecompetitor and k´ = kMIP / kNIP. k and k´ values were summarized in Table 2,

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respectively. The current values of the paraben imprinted MIP/Au-SPE against MP, EP, PP and BP were 0.95 µA, 0.68 µA, 0.57 µA and 0.51 µA, respectively. The current value for

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paraben at the same concentration was 13.64 µA. According to these values, paraben

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imprinted SPE was 14.35, 20.11, 23.84 and 26.74 times more selective for paraben than MP, EP, PP and BP, respectively. These results indicated that the spatial and chemical structure of the paraben molecules was successfully transferred to the polymeric structure.

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---- Please insert Table 2----

In the second approach; binary mixtures of the target molecule paraben with other competitive molecules were prepared and the interference values of competitive molecules

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were examined (Figure 7). It was found that MIP/Au-SPE was selective to paraben in the presence of other competitor molecules. ---- Please insert Figure 7---3.5. Real sample analysis

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In order to show the applicability of the sensor in real sample, standard addition method was utilized. As a real example, cream was chosen from cosmetic products and recovery study was carried out [49,50]. The results of the recovery experiments were summarized in Table 3. ---- Please insert Table 3---As given in the Table 3; recovery values were found as 91.9% and 116.6%. Also, the calculated RSD values were found to be very low as 2.35% and 5.52% with high accuracy. These results proved that the developed sensor can be applied successfully in real samples.

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4. Conclusion In this study, highly-selective cavities specific to paraben were formed on single-use screen printed electrodes via molecular imprinting technology. Thus, a simple, fast, economic,

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sensitive and selective electrode platform was fabricated for the electrochemical detection of paraben. The characterization of the bare and modified Au-SPEs surface was performed by

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AFM and electrochemical measurements. Under optimum conditions, the LOD and LOQ

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values were calculated as 0.706 µM and 2.35 µM, respectively. As a result of reproducibility study, RSD value was found to be 2.45% (n=4) indicating that the obtained results had good precision. The developed sensor was successfully applied in cosmetic samples with standard

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addition method with high accuracy. Recovery values were obtained in the range of 91119.6%. It was shown that the developed sensor can be used for selective and sensitive determination for µM levels of paraben content in cosmetic samples. The designed sensor

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platform can also be applied used for different analytes in a wide range of applications such as food control, cosmetics and environmental analysis.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Acknowledgement The authors gratefully acknowledge the financial support for this work was provided by the

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by Research Council of Hacettepe University (Project number FHD-2017-13834).

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24

Biographies Beyhan Buse Yücebaş received BS degree from department of chemistry and MSc degree in chemistry from Hacettepe University, Turkey.

Yesim Tugce Yaman is currently a PhD student at the Surface Electrochemistry Research Group and in 2016 she received her master's degree in Analytical Chemistry from Hacettepe University. She earned a bachelor's degree (2013) from Hacettepe University Department of Food Engineering. Her research interests are development of novel electrochemical surfaces using different methodologies for detection important analytes such as cancer cells, DNA,

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drugs and endocrine disruptors etc.

Dr. Gulcin Bolat is a research assistant at Hacettepe University, Department of Chemistry, Turkey. She obtained her bachelor’s degree in Hacettepe University, Faculty of Engineering at Department of Chemical Engineering in 2008. She earned her PhD (2016) and master's

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degree (2011) in Analytical Chemistry from Hacettepe University with Prof. Serdar Abaci. Her main research interests are synthesis and characterization of conducting polymers,

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ultramicroelectrodes and electrochemical sensors for drugs, vitamins, proteins, DNA and

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pesticides.

Erdoğan Özgür received BS degree from department of chemistry education (2008) and MSc and PhD degrees in chemistry from Hacettepe University, Turkey (2011 and 2016). His

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research interests are molecular imprinted polymeric micro/nano materials and their applications in biosensors.

Prof. Serdar Abaci is the head of Surface Electrochemistry Research Group at Hacettepe University in Ankara, Turkey. After obtaining his PhD (2002) from Hacettepe University on

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the synthesis and applications of PbO2 electrodes, Prof. Abaci worked as a postdoc researcher at Auburn University, USA. He has authored 42 research papers and works as a reviewer in roughly 10 journals. Prof. Abaci is an expert in the field of electrochemistry and he has a wide teaching

statement

includes

chemistry,

analytical

chemistry,

electrochemistry,

electroanalytical chemistry, surface analysis methods, surface chemistry. His current main research interest is the development of novel electrochemical surfaces using various materials including nanomaterials and conducting polymers. At the same time, different types of sensors such as ultramicroelectrodes, biosensors and thin films have been constructed for the 25

detection of various analytes. His contributions in the field of nanotechnology, material characterization and microfabrication have enhanced the power of electrochemistry more.

Lokman Uzun is Professor at the Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey where he also received his PhD in 2008. He is the author of more than 100 articles in peer-review journals with a h-index value of 29 and one of Associate Editors of Advanced Materials Letters. He recently awarded by an European Union Marie-Curie fellowship with the Biosensors and Bioelectronics Centre, Linköping University, Sweden for two years. His research interest is mainly in materials science, surface

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modification, affinity interaction, polymer science, especially molecularly imprinted polymers and their applications in biosensors, bioseparation, food safety, and the environmental

Jo

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sciences.

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Captions of the Figures Figure 1. AFM images of bare SPE surface (a), MAC coated SPE surface (b), and paraben imprinted porous polymeric film modified SPE surface (c). Figure 2. Cyclic voltammograms of bare Au-SPE (a), MAC modified Au-SPE (b), NIP AuSPE (c), MIP (non-removed) Au-SPE (d), MIP (removed) Au-SPE (e) in 5 mM Fe(CN)63-/4in 0.1 M KCl. Figure 3. Cyclic voltammograms of bare Au-SPE (a), MIP (template removed) / Au-SPE (b)

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in 10 µM paraben solution in PBS (pH: 7.0) with a scan rate of 100 mVs-1. Inset: Cyclic voltammograms of bare Au-SPE (a) and MIP (template removed)/Au-SPE (b) in absence of paraben.

Figure 4. Optimization of adsorption time (a), desorption time of paraben (b), and pH (c).

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Figure 5. Square wave voltammograms of the paraben solution at different concentrations (a)

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(a. 0, b. 1, c. 5, d. 10, e. 15, f. 20, g. 30 µM paraben), inset: linear calibration graph. Figure 6. Selectivity study for each competitor molecule at different concentrations. MIP

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coated (a) and NIP coated (b) SPEs. a. Paraben (P):30 µM, b1. P + MP:30 µM + 30 µM, b2. P + MP:30 0µM + 300 µM, b3. P + MP:3000µM + 3000 µM, c1. P + EP: 30 µM + 30 µM,

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c2. P + EP: 300 µM + 300 µM, c3. P + EP: 3000 µM + 3000 µM, d1. P + PP: 30 µM + 30 µM, d2. P + PP: 300 µM + 300 µM, d3. P + PP: 3000 µM + 3000 µM, e1. P + BP: 30 µM + 30 µM, e2. P + BP: 300 µM + 300 µM, e3. P + BP: 3000 µM + 3000 µM. Figure 7. Peak current values for binary mixtures of the target molecule paraben with other

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competitive molecules. MIP coated SPE (a) and NIP coated SPE (b). (a, b1, b2, b3, c1, c2, c3, d1, d2, d3, e1, e2 and e3 were same at Figure 6) Figures

Figure 1

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Figure 3

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Figure 4

29

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Tables Table 1. Comparison of the developed sensor performance with the literature. Target

Sensing system Mode

Detection range (M)

LOD (M)

MP EP PP BP EP

PMAA/GCE

SWV

SWV

4 × 10−7 4 × 10−7 2 × 10−7 2 × 10−7 3.5 × 10− 10

[41]

CF and trimetallic nanoparticles Hemoglobin and multiwalled carbon nanotube GCE

2 × 10−5 - 1 × 10−4 2 × 10−5 - 1 × 10−4 5 × 10−6 - 1 × 10−4 5 × 10−6 - 8 × 10−5 1 × 10− 9 - 1 × 10− 7

DPV, CV

0.1 x10-6 - 13x10-6

25 x10-9

[44]

CV

5 x10-6 - 50 x10-6

BP BP

GCE/MWCNT- AdSV NAF CV, EIS In2O3/GCE

10 x10-6 - 100 x10-6 0.14 x10-6 - 2.14 x106

CF

DPV

0.1 x10-6

[23]

0.20 x10-6

[45]

8 x10-4

[46]

1.20 x10-6 - 36.62 x10-6 20– 180 x10-6 1 x10-4 - 5 x10-5

10 x10-6

[47]

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PP

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BP

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MP

Ref.

BDDE Cas, CV 1.97 x10-6 [48] 3.8 x10-9 [24] C60NRs– NH– CV, EIS Ph–GCE Paraben PHEMASWV, 1 x10-6 - 30 x10-6 0.706 x10-6 This study MAPA) CV nanofilm Abbreviations: Poly(methacrylic acid) (PMAA); Glassy Carbon Electrode (GCE); Carbon nanofibers (CF); Multi-Wall Carbon Nanotubes (MWCNTs); Nafion (NAF); Boron-Doped Diamond Electrode (BDDE); Fullerene Nanorods (C60NRs).

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EP EP

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Table 2. The selectivity and relative selectivity coefficients Paraben Derivatives

k (MIP)

k (NIP)

k’

Singular

MP

14.35

1.013

14.16

solution

EP

20.11

1.013

19.85

PP

23.84

1.033

23.07

BP

26.74

1.049

25.47

Paraben/MP 1,24

1.013

1.22

mixture

Paraben/EP

1,27

1.031

1.23

Paraben/PP

1.39

1.031

1.34

Paraben/BP

1.58

1.049

1.51

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Binary

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Table 3. Recovery results for cream sample Sample Measured (µM) Cream

-

Added (µM)

Found (µM)

%RSD

%Recovery

1

0.76 ± 0.44

2.35

91.88

20

24.1 ± 3.30

5.52

119.60

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(95% confidence interval, N=3, t=4.30)

36