ORIGINAL RESEARCH published: 07 May 2021 doi: 10.3389/fchem.2021.621057 Electrochemical Impedance Spectroscopy Microsensor Based on Molecularly Imprinted Chitosan Film Grafted on a 4-Aminophenylacetic Acid (CMA) Modified Gold Electrode, for the Sensitive Detection of Glyphosate Fares Zouaoui 1,2 , Saliha Bourouina-Bacha 2 , Mustapha Bourouina 2,3*, Albert Alcacer 4 , Joan Bausells 4 , Nicole Jaffrezic-Renault 1*, Nadia Zine 1 and Abdelhamid Errachid 1 1 Institut des Sciences Analytiques, Université de Lyon, Villeurbanne, France, 2 Département de Génie des Procédés, Faculté de Technologie, Université de Bejaia, Bejaia, Algeria, 3 Departement de Chimie, Faculté des Sciences Exactes, Université de Edited by: Bejaia, Bejaia, Algeria, 4 Institute of Microelectronics of Barcelona IMB-CNM-CSIC, Autonomous University of Barcelona, Aziz Amine, Barcelona, Spain University of Hassan II Casablanca, Morocco Reviewed by: A novel electrochemical impedance spectroscopy (EIS) microsensor was implemented Hui Xu, for the dosage of traces of glyphosate, in real and synthetic water samples. Molecularly Ludong University, China Abdellatif Ait Lahcen, imprinted chitosan was covalently immobilized on the surface of the microelectrode King Abdullah University of Science previously modified with 4-aminophenylacetic acid (CMA). The characterization of the and Technology, Saudi Arabia resulting microelectrodes was carried out by using cyclic voltammetry measurement (CV), *Correspondence: scanning electron microscopy (SEM), and electrochemical impedance spectrometry Nicole Jaffrezic-Renault nicole.jaffrezic@univ-lyon1.fr (EIS). EIS responses of the CS-MIPs/CMA/Au microsensor toward GLY was Mustapha Bourouina well-proportional to the concentration in the range from 0.31 × 10−9 to 50 × 10−6 bouryas@yahoo.fr mg/mL indicating a good correlation. The detection limit of GLY was 1 fg/mL (S/N = 3). Specialty section: Moreover, this microsensor showed good reproducibility and repeatability, high selectivity, This article was submitted to and can be used for the detection of GLY in river water. Analytical Chemistry, a section of the journal Keywords: microsensor, CMA, MIP, chitosan, Gly, EIS Frontiers in Chemistry Received: 24 October 2020 Accepted: 06 April 2021 INTRODUCTION Published: 07 May 2021 Glyphosate is an herbicide used to destroy what are commonly known as weeds. From the 1970s, Citation: the production and use of glyphosate increased steadily around the world. So far, despite the Zouaoui F, Bourouina-Bacha S, Bourouina M, Alcacer A, Bausells J, risks involved, this herbicide continues to be widely used (Cuhra et al., 2013). Glyphosate works Jaffrezic-Renault N, Zine N and by interrupting the synthesis of aromatic amino acids essential for the functioning of plants Errachid A (2021) Electrochemical (Lopes et al., 2018). Recently, there has been growing concern about the impact of glyphosate on Impedance Spectroscopy living things and the environment (Johansson et al., 2018; Seide et al., 2018). Among the many Microsensor Based on Molecularly effects of this herbicide are toxicity, changes in the activity of antioxidant enzymes, endocrine Imprinted Chitosan Film Grafted on a disruption, damage to lipids, histological damage, etc. (Lopes et al., 2018; Ren et al., 2018; Lorenz 4-Aminophenylacetic Acid (CMA) Modified Gold Electrode, for the et al., 2019). Glyphosate can be found as a contaminant in soil, plants and food products. Sensitive Detection of Glyphosate. GLY has a high solubility in water and its intensive use leads to contamination of surface Front. Chem. 9:621057. and ground water (Ruiz de Arcaute et al., 2018). A certain number of approaches have been doi: 10.3389/fchem.2021.621057 adopted for the detection of glyphosate in various media such as chromatography, photometry, Frontiers in Chemistry | www.frontiersin.org 1 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor mass spectrometry, etc. (Clegg et al., 1999). However, these primary amine. The use of chitosan in the MIP matrix is techniques are long, difficult to implement, expensive, and often increasingly widespread and the number of published works require qualified personnel. Moreover, their application is limited reporting the extent of research in this area is constantly to the laboratory scale. expanding (Karrat et al., 2020; Zouaoui et al., 2020a). Chitosan The challenge for analytical chemistry is to develop alternative combined with the MIP technique have been reported for analytical methods with an emphasis on simplicity, efficiency the preparation of the electrochemical sensors for different and cost. Molecularly imprinted polymers (MIPs) seem to be target molecules including pharmaceutical compounds (Lin a very good alternative for designing analysis tools because et al., 2015; Song et al., 2019), protein (Fatoni et al., 2014; of its many properties such as selectivity, robustness, physical Xia et al., 2016), sweet (Li et al., 2014), phenolic compounds and chemical resistance, inertia and ease of synthesis and Low (Deng et al., 2013; Li et al., 2013; Chakroun Galai et al., 2020; cost (Haupt and Mosbach, 2000, Qiao et al., 2006). MIPs can Salvo-Comino et al., 2020), organic compounds (Chen et al., selectively recognize a molecule or even a family of analogous 2011), ions (Wu et al., 2020), and pesticides (Zouaoui et al., molecules. Molecular printing consists of creating cavities, which 2020a). are complementary from the structural and functional point of Several sensors are being developed by electrodeposition view of the target molecules within a functional polymer. The of molecularly imprinted chitosan. The application of a synthesis of MIPs is the interaction of the functional monomer negative potential during the electrodeposition of chitosan with template molecules through covalent, semi-covalent, and causes release of hydrogen gas. H2 remains trapped in the non-covalent interactions (Yan and Row, 2006); which leads membrane of the CS which makes it inhomogeneous and to the formation of the pre-polymerization complex. After the affects its sensitivity. The covalent grafting of chitosan allows addition of a crosslinking monomer, a polymer network is eliminating these effects and achieving a more stable response formed around the functional monomer/template complex by (Zouaoui et al., 2020a). copolymerization between the functional monomers and the In this work, covalent modifications of the surfaces of crosslinking monomer, thus allowing the formation of a more gold microelectrodes were explored by the use of a 4- resistant and more stable matrix (Wulff et al., 1983). As a aminophenylacetic acid, more commonly called CMA, to ensure last step, cavities complementary to the target molecules with the covalent immobilization of molecularly imprinted chitosan regard to their size and the presence of specific functional in order to develop microsensors, distinguished to detect traces groups are obtained after the extraction of the template from the of glyphosate in water. polymer. The porous matrices obtained can be used to specifically The process used to deposit CMA resides in the formation recomplex the target molecules present in various media. The of a diazonium salt cation which makes it possible to form selectivity of MIPs is conditioned by the functional monomer a covalent bond between the acid and the gold surface. The involved in the synthesis of molecular printing. The monomer is diazonium cation is an unstable species, easily hydrolyzable at chosen according to its physicochemical properties and its ability room temperature and very reactive (Chira et al., 2017). to interact with the template (Pichon and Haupt, 2006). The diazonium ion can be prepared by a standard The many properties of chitosan (CS) allow it to be a diazotisation procedure, by treatment of aromatic amines very good choice for use in the synthesis of MIPs. It is in with nitrites NO− 2 in the presence of hydrochloric acid (HCl). particular biocompatible, biodegradable, non-toxic and has good After this treatment, the –NH2 group of the aniline derivative antimicrobial and antioxidant activities (Younes and Rinaudo, was converted to a –N+ 2 group leads to the formation of an aryl 2015). In addition, this material has various physical and diazonium. The electrochemical reduction of the diazonium chemical characteristics. It is considered to be the second most cation on a metal cathode by an electron transfer from the abundant natural biopolymer after cellulose. Chitosan is a natural surface of the electrode to the diazonium salt generates an cationic polysaccharide (in dilute acid medium) extracted from intermediate aryl radical after the release of N2 . The aryl radical chitin having as reactive functional groups a primary and attaches to the conductive surface by formation of a covalent secondary hydroxyl group and a primary amino group (Pa and bond between the carbon of the aromatic cycle and the metallic Yu, 2001). element (Corgier et al., 2005). The strong functionality of chitosan through its hydroxyl and The carboxylic function of the acid is then available as primary amine functions gives it a considerable opportunity of an accessible surface of the electrode for grafting chitosan. chemical modification (Aranaz et al., 2010). These modifications CS is functionalized by the CMA via the formation of an were intended in order to enhance the characteristics and internal amide bond. The pair 1-ethyl-3- (3-dimethylaminopropyl) morphology of the CS or to give it new properties (Wang carbodiimide and N-hydroxysuccinimide (EDC/NHS) makes et al., 2016). Many studies have looked at the modification possible the preceding amide bond, thanks to its carboxylic acid of chitosan (Martins et al., 2004; Pereira et al., 2013). function. It consists of the formation of an ester intermediate Chitosan is biofunctionalized according to a very common between NHS and the carboxylic function. The intermediate thus technique resulting in the formation of a bond of the primary formed reacts more easily with the primary amine of chitosan amine of chitosan with a carboxylic acid (Chung et al., which improves the reaction kinetics. Better adhesion of the 2002). Nucleophilic reactions on electrophilic carbons such as chitosan will thus make it possible to obtain a homogeneous aldehydes, ketones and carboxylic acids are thus made possible functionalized chitosan membrane on the surface of the sensor by the non-binding nucleophilic doublet possessed by the (Corgier et al., 2005). Frontiers in Chemistry | www.frontiersin.org 2 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor MATERIALS AND METHODS regeneration, and analysis of the real sample. With the responses of the four microsensors we made the error bars. Reagents A pH- meter: Mettler Toledo FE20/EL20 was used for the Chitosan (CS, degree of deacetylation 80.0–95%), acetic measure of solution pH. A FEI Quanta FEG 250 SEM (University acid (purity 99.7%), methanol (purity 99.9%), 1-ethyl- of Lyon 1), France, was used to obtain the Scanning Electron 3-(3-dimethylaminopropyl)carbodiimide (EDC), 4- Microscopy (SEM) images. aminophenylacetic acid (CMA), sodium nitrite (NaNO2 ), hydrochloric acid (37%, HCl), Pure ethanol (99.8%), Sulfuric acid CS-MIPs/CMA/Au Sensor Fabrication (purity 95%), sodium hydroxide (NaOH) and glyphosate were Diazonium Grafting Onto Gold Microelectrodes obtained from Sigma Aldrich, France. N-hydroxysuccinimide Preparation of Diazonium Salt (CMA) (NHS) was purchased from Acros Organics, France. All 3 mM CMA solution was prepared with 20 mM of HCl (20 mM) interferents: gluphosinate-ammonium (GLU), chlorpyrifos and 20 mM of NaNO2 (20 mM) in an aqueous solution (Figure 1 (CHL), and phosmet (PHO) were supplied by Sigma Aldrich, step. 1). Then, the mixture was stored at 4 ◦ C until the appearance France. The experimental runs were performed at room of a pinkish hue (about 5 min) (El Alami El Hassani et al., 2019). temperature with analytical grade reagents. Electrodeposition of CMA The Au microelectrodes were washed beforehand for 10 min with Apparatus ethanol and deionized water under ultrasound. The final step The data of electrochemistry were obtained through a is their exposure for 30 min to UV/ozone. 5 cyclic voltammetry multichannel potentiostat analyzer (Biologic-EC-Lab VMP3). (CV) scans between −1.2 and 0.0 V at a scanning rate of 50 mV/s The tests were performed with a transducer supplied by the were carried out for the electrodeposition of the CMA solution National Center for Microelectronics (CNM), CSIC, Barcelona. (Figure 1 step. 2). A cathodic peak is observed; the decrease It’s made up of a plate of four bare gold working microelectrodes in maximum intensity with each successive sweep (Figure 2) is (WE) (s = 0.64 mm²), one counter microelectrode (CE) (s = probably due to the obstruction of the working electrode surface. 0.13 mm²), and two Ag/AgCl reference microelectrodes (RE) After electrodeposition, the microelectrodes undergo rinsing (s = 1.37 mm²) (see transducer in Figure 1). The use of the with deionized water and drying under a stream of nitrogen. four working microelectrodes allowed us to go faster in the experimental part. With the multichannel potentiostat it was Activation of the Carboxylic Function possible to prepare four microsensors at the same time and to use Solution of 400 mM of 1-ethyl-3- (3 dimethylaminopropyl) them simultaneously either for detection, interference studies, carbodiimide (EDC) and 100 mM of N-Hydroxysuccinimide FIGURE 1 | (A) Transducer holding an array of four bare-gold working microelectrodes (WE) s = 0.64 mm², one counter microelectrode (CE) s = 0.13 mm², and two Ag/AgCl reference microelectrodes (RE) s = 1.37 mm². (B) Preparation of CS-MIPs/CMA/Au and its recognition for GLY. Frontiers in Chemistry | www.frontiersin.org 3 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor (NHS) was prepared in absolute ethanol. The microelectrodes To eliminate the molecule template, a second incubation functionalized by the CMA were incubated in this solution for 1 h of about 30 min was made in acetic acid/methanol solution to activate the carboxylic function (Figure 1 step. 3). Then, the (1:1, v/v). The incubation time of 30 min is considered to devices were carefully rinsed with pure ethanol and dried using be an optimal time for maximum extraction of GLY from nitrogen (El Alami El Hassani et al., 2019). the CS film. Because for an incubation time >30 min, EIS plot and CV voltammogram can be superimposed on those Grafting of CS on Activated CMA for an extraction time of 30 min. Knowing that initially the Preparation of GLY/CS Mixture GLY (anionic) is adsorbed on active sites of chitosan (cationic To prepare the CS solution, 1 g of CS powder was dissolved biopolymer) by electrostatic interaction at pH = 5, it is desorbed in a volume of 100 mL of acetic acid 0.1 M and ultrasonicated in AcAC/methanol (1:1; v:v) solution at pH <4.5 for the most for 6 h at room temperature. 1 mg/mL of GLY was prepared in part. While the chitosan remains adherent and fixed by covalent CS solution and stirring of the suspension for 2 h is followed bonds to the surface of the functionalized microelectrode. by an adjustment of the pH to 5 with a solution of ammonia So, a microsensor constructed with CS molecularly imprinted hydroxide. Chitosan used has a medium molecular weight (≈250 film modified with CMA was applied for a specific recognition of kDa). The monomer/template ratio is 1/1, that means that GLY. The process is shown in Figure 1 (step. 4 and 5). in the mixture consists of one glyphosate molecule for each The steps in making the unprinted polymer sensor (CS- glycosamine monomer. The molecular weight of chitosan being NIP/CMA/Au) are identical to those for the CS-MIP/CMA/Au 250 kDa which correspond to 1,470 glycosamine monomers per microelectrodes, except that GLY was absent from the entire polymer chain, the mixture contains one molecule of glyphosate process. Storage at ambient temperature of the sensors was for one glycosamine monomer. carried out for further applications. Experimental Conditions CS-MIPs Deposition A multichannel potentiosat (Biologic-EC-Lab VMP3) was used GLY/CS mixture was deposited by drop-casting on the for the characterization by the techniques of cyclic voltammetry functionalized microelectrodes surface for 24 h. The deposition (CV) and electrochemical impedance spectroscopy (EIS) of all was followed by incubation during 1 h of the GLY-CS/CMA/Au the modifications made to the working microelectrodes. All in 0.5 M H2 SO4 as the cross-linker to remove residues of the measurements were performed in 5 mM ferro-ferricyanide chitosan. Chitosan is soluble in all mineral acids except sulfuric ([Fe(CN)6 ]3−/4− ) and phosphate buffer saline solution (PBS). acid. In this case, sulfuric acid is used as a crosslinking agent For the cyclic voltammetric measurements, the potential was during the formation of the biopolymer. The deposited CS film scanned between 0 and 0.45 V at a scan rate of 80 mV/s. EIS data remains stable; moreover the reproducibility results confirm this are obtained for an initial potential of 0.2 V and a frequency range (see section About the Sensor Regeneration and Reproducibility). varying from 300 mHz to 100 kHz. For detection step, after an During the crosslinking, ionic bonds are formed between the immersion in aqueous GLY solutions of concentrations varying sulfate ions of sulfuric acid and the ammonium ions of chitosan. between 0.31 × 10−9 and 50 × 10−6 mg/mL for a period of The mechanism of crosslinking of chitosan with sulfuric acid is 30 min, CS-MIPs/CMA/Au electrodes were characterized by EIS. presented in Supplementary Figure 1. RESULTS AND DISCUSSION Characterization of the Surface Modifications Electrochemical impedance spectroscopy (EIS) has emerged as an alternative to conventional analysis techniques to study the modified surfaces. EIS provides information on the kinetics of molecular phenomena at the interface, as well as on the coverage rates and the effectiveness of surface modifications (Proud and Müller, 1993). By modeling the electrochemical response by equivalent electrical circuits, it is possible to relate the physical and chemical properties of the electrode material to the data from the EIS curves (Janata, 2002). The equivalent circuit shown in Figure 3C was used to fit the Nyquist plots a, b, c, and d of Figure 3A, and the equivalent circuit shown in Figure 5B was used to fit the Nyquist plots e of Figure 3A. From the examination of Figure 3A, a, it emerges that the sensor has a high charge transfer rate for [Fe(CN)6]3−/4− (Rct = FIGURE 2 | Cyclic voltammogram of electrodeposition of CMA on the 783 Ohm). An obvious increase of the charge transfer resistance microelectrode surface. value was observed with the electrodeposition of CMA on the Frontiers in Chemistry | www.frontiersin.org 4 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor FIGURE 3 | (A) EIS of bare Au (a), CS-NIPs/CMA/Au (b), CMA/Au (c),CS-MIPs/CMA/Au before extraction of template (d), CS-MIPs/CMA/Au after extraction of template (e), in 5 mM [Fe(CN)6 ]3−/4− and PBS, initial potential E = 0.2 V. Highest Freq = 100 khz, Lowest Freq = 300 mHz. (B) VC of bare Au (a), CS-NIPs/CMA/Au (b),CMA/Au (c),CS-MIPs/CMA/Au before extraction of template (d), CS-MIPs/CMA/Au after extraction of template (e), in 5 mM [Fe(CN)6 ]3−/4− and PBS, from 0 to 0.45 V at a scan rate of 80 mV/s. (C) Randles’ circuit, where Rs is the electrolyte resistance, CPE is the constant phase elements, Rct is the charge transfer resistance and Zw is the Warburg impedance. gold microelectrode surface (Rct = 26,269 Ohm) (Figure 3A, c). peaks is lower after deposition of the CS which is a non- Impedance becomes higher value after the chemical deposition of conductor polymer (Figure 3B, d). It was observed that redox chitosan (Rct = 95,452 Ohm) (Figure 3A, d). peaks of NIPs film (Figure 3B, b) are higher than MIPs film which From the comparison of the diagrams obtained with the NIP is thicker. Due to extraction of template from the MIPs matrix film (Figure 3A, b), the MIP film (Figure 3A, d) has a larger (Figure 3B, e), it was observed a significant increase in redox Re (Z), and therefore, a greater thickness than that of the NIP peak, this peak is bigger comparing to NIPs which is related to film. Indeed, the NIP, the chitosan chains are positioned in a the presence of imprinted hole promoting the charge transfer. flat manner on the CMA. Logically, the thickness of the NIP membrane equals the width of the glucosamine in the chitosan. Surface Features For the MIP, the presence of glyphosate gives a film with The surface morphologies investigations of CMA/Au, CS- curvatures which modifies the morphology of the film, in MIPs/CMA/Au and CS-NIP/CMA/Au (Figure 4) were carried particular its thickness in comparison with the NIP film. If the out by scanning electron microscopy (SEM). It can be seen film is thicker, its resistance to electron transfer is higher. Also, it from the completely different morphologies of the surfaces of is possible that the presence of glyphosate in the MIP film makes the bare electrodes (Figure 4a) and that of the modified with it more resistive in comparison with the NIP film (see diagram of CMA (Figure 4b), that the operation of depositing the CMA the Supplementary Figure 2). film was well-accomplished. After the chemical deposition of The removal of the pesticide template from the MIPs was the chitosan onto CMA layer, another morphology modification lead to significant decrease in impedance (Figure 3A, e) and the has appeared on the surface of the microelectrode which formation of two well-defined loops has been observed (for the confirms the deposition of the CS (Figure 4c). Therefore, SEM first semicircle R2 = 11,775 Ohm, for the second semicircle R3 = does not allow seeing the difference between the morphology 53,728 Ohm). Moreover, charge transfer resistance of NIPs (Rct = of MIPs (Figure 4b) and NIPs films (Figure 4c); because the 59,890 Ohm) is higher than MIPs film after removing template, diameters of the cavities on the chitosan layer have the same due to the presence of imprinted cavities on MIPs layer, which order of magnitude with the GLY molecules; in the order of a favors the electron transfer. few nanometers. This result is also proved by the cyclic voltammetry (CV) curves obtained in a ferro-ferricyanide solution (E varying from 0 to 0.45 V and scan rate of 80 mV/s). Like the results Electrochemical Responses of MIPs to of the EIS, those of the CV (Figure 3B) correspond to the Glyphosate same characteristics of the electrodes. It can be seen from the From the EIS graphs in Figure 5A, we can see a very CV (Figure 3B), after electrodeposition of CMA (Figure 3B, c) remarkable variation in response amplitudes with increasing GLY the decrease of intensity current of redox peaks was recorded concentration, which is an indicator of the relationship between compared with bare gold surface (Figure 3B, a). Then, redox the latter and the impedance of the GLY sensor. Frontiers in Chemistry | www.frontiersin.org 5 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor FIGURE 4 | SEM images. (a) Bare gold, (b) CMA/Au, (c) CS-MIPs/CMA/Au, (d) CS-NIPs/CMA/Au. FIGURE 5 | (A) EIS of CS-MIPs/CMA/Au in 5 mM [Fe(CN)6]3−/4− and PBS. Before pre-concentration and after incubation in different concentrations of GLY. (B) Schematic of equivalent electrical circuit. TABLE 1 | Estimated values of the parameters fitted by equivalent electrical circuit model R1 + Q2 /R2 + Q3 /R3 . [GLY].109 mg/mL R1 (Ohm) R2 (Ohm) Q2 . 107 (F.sn-1 ) n2 R3 (Ohm) Q3 . 106 (F.sn-1 ) n3 X2 Blank 147.0 11,775 3.09 0.86 53,728 8.84 0.74 0.042 0.31 142.4 14,633 2.49 0.88 45,421 8.52 0.72 0.013 6.25 135.1 19,369 2.22 0.88 40,029 7.72 0.73 0.014 125 134.8 21,937 2.16 0.88 34,817 7.21 0.73 0.013 2,500 132.6 23,910 2.03 0.87 30,561 6.45 0.73 0.013 50, 000 131.6 28,397 2.83 0.87 28,673 5.73 0.77 0.011 Frontiers in Chemistry | www.frontiersin.org 6 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor The EIS data were fitted by equivalent electrical circuit model given in Table 1. The results reported by the fitting reveals that (EEC). The optimal fit was done using (R1 + Q2 /R2 + Q3 /R3 ) n2 and n3 are <1, which confirmed the roughness and the EEC, which consists of three components associated in series heterogeneity of the microelectrode surface. As these values are (Figure 5B). The first component R1 expresses the electrolyte invariant with the increase in the applied potential, the stability resistance. The parallel elements Q2 and R2 , which are related of the electrode surface is confirmed. to the first Nyquist semi-circle, correspond respectively to the At high frequencies, the Nyquist plot shows a first semi-circle, constant phase element (CPE) and the charge transfer resistance. while the second is observed at lower frequencies. This element is For to the second Nyquist semi-circle, the parallel elements Q3 comprised of contributions from three different processes which and R3, correspond respectively to CPE and charge transfer are electron transfer process, diffusion control and adsorption of resistance. This electrical circuit was reported to characterize ionic species (Terbouche et al., 2016; Tanujit and Asokan, 2019). porous structures. CPE1 and CPE2 are interpreted as the non- The diminution of R3 charge transfer resistance is probably ideal capacitances which are caused by the surface geometry, related to the adsorption of [Fe(CN)6 ]3−/4− on the CMA roughness, and porosity. The relation between these constant microelectrode, or may be attributed to the electrostatic phase elements Q and the impedance Z is given by: ZCPE = interaction between this negative ions and membranes modified 1/Q(jω)n ; where j is the imaginary number, ω is the angular surface. This decrease is due to the accumulation of ions on the frequency and n is correction factor (0 < n < 1). If the value surface, which increases the active surface area of the electrode. n tends to 0, CPE becomes less capacitive (Ebdelli et al., 2011; These behaviors are allowed by the morphology and surface Gaied et al., 2014; Aymen et al., 2015). The selection of the porosity of the CS-MIPs/CMA/Au electrodes. equivalent circuit was dependent upon the fitting of the Nyquist On another side, the resistance R2 increases due to re- plot to produce the smallest error, expressed as the standard occupancy of the complementary sites which are located on the deviation (X 2 ). membrane of the biopolymer. These complexations make the The estimated values of the parameters after adjusting the surface of the electrode more resistant to charge transfer. model of the equivalent circuit with the impedance spectra are If R2 and R are the load transfer resistances of the sensor corresponding respectively to a given and zero concentration of GLY, the ratio |R–R2 |/R2 = 1R/R, therefore makes it possible to measure the relative variation of the charge transfer resistance of the electrode. Therefore, 1R/R is related to the evolution of the GLY content of the solution. The exploitation of the data of the EIS diagrams made it possible to find a linear relation between this parameter and the logarithm of the concentration of GLY. The linear range is from 0.31 × 10−9 up to 50 × 10−6 mg/mL as shown in Figure 6. The calibration equation is given below (with a coefficient of determination = 0.998): 1R/R = 0.206log[GLY] − 0.188, This new sensor has a detection limit (LOD) of 10−12 mg/mL. The estimation of the LOD is carried out thanks to the commonly used equation: 3S/m (Shrivastava and Gupta, 2011), S and m are respectively the residual standard deviation and the slope of the calibration line. The high sensitivity of these MIP/Au/CMA sensors allows them to be used successfully for the detection of very low concentrations of GLY. Indeed, the LOD obtained is FIGURE 6 | Sensitivity of CS-MIPs/CMA/Au (1R/R = 0.206 log [GLY] – 0.188, R² = 0.999) and CS-MIPs/Au (1R/R = 0.087 log [GLY] +0.673; R² = 0.991). 1/108 times lower compared to the EQS value of pesticides equal to 0.1 µg/mL. TABLE 2 | A comparison of analytical parameters of the proposed electrode with other MIP electrochemical sensors. Electrode Technique Linear range mg/mL Limit of detection mg/mL Sensitivity References Ppy-MIP/ HAuCl4 -PB/ITO DPV 400 × 10−6 −1,200 × 10−6 92 × 10−6 / Xu et al., 2017 Ppy-MIP/Au DPV 5 × 10 −6 −800 × 10 −6 0,27 × 10−6 12,8 Zhang et al., 2017 Ppy-MIP/Au SWV 1,7 × 10−11 −1,7 × 10−2 1,7 × 10−10 / Mazouz et al., 2017 CS-MIP/Au EIS 0,31 × 10−9 −50 × 10−6 10−12 0,087 Zouaoui et al., 2020b CS-MIP/CMA/Au EIS 0,31 × 10−9 −50 × 10−6 10−12 0,206 This work Ppy-MIP/HAuCl4 -PB/ITO, Molecularly imprinted Polypyrrole/Composite of urchin-like gold nanoparticles and Prussian Blue/Indium-Tin Oxide glass; Ppy-MIP/Au, Molecularly imprinted Polypyrrole/Gold electrode; CS-MIP/Au, Molecularly imprinted Chitosan/Gold microelectrode; DPV, Differential Pulse Voltammetry; SWV, Square Wave Voltammetry. Frontiers in Chemistry | www.frontiersin.org 7 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor The analytical performance of the CS-MIPs/CMA/Au sensor of GLY. As seen in Figure 7, 1R/R of the CS-MIPs/CMA/Au was compared to other MIPs electrochemical sensors used for were stronger compared with 1R/R of the CS-NIPs/CMA/Au GLY detection reported in the literature (Table 2). To the best of indicating that the proposed sensor had excellent specificity, our knowledge, this sensor had the best analytical characteristics because the fixation of GLY molecule by the CS-NIPs/CMA/Au than most previously reported sensors. microsensor is very low which confirms the success and efficiency of the GLY’s impression. CMA Effect on Sensor Sensitivity The sensitivity of the sensor based on CS-MIPs modified with About the Sensor Regeneration and CMA (CS-MIPs/CMA/Au) was compared with the sensitivity of the sensor based only on chitosan membrane (CS-MIPs/Au) Reproducibility Three working microelectrodes, prepared according to the (Zouaoui et al., 2020b). As shown in Figure 6, the sensitivity of same method, are used to study the reproducibility of the CS-MIPs/CMA/Au is 2.37 times higher than CS-MIPs/Au. For CS-MIPs/CMA/Au microsensor. Acceptable reproducibility is applications in the sensor field, the electrodeposition of chitosan obtained with a relative standard deviation of 1.46%. (CS-MIPs/Au) on a conductive surface by pH gradient causes the degassing of H2 . These molecules are blocked inside the membrane, which makes it non-homogeneous. “The grafting of chitosan onto the diazonium salt by the formation of strong interactions probably leads to more homogeneous membranes and to the resolution of interface problems.” Interference The selective sensing of the designed microsensor of the GLY was tested using interfering pesticides; this includes phosmet (PHO), gluphosinate-ammonium (GLU), and chlorpyrifos (CHL). EIS analyzes are performed using the same experimental process as that for GLY detection. The detection tests of PHO, GLU, and CHL were performed in the same concentration range between 0.31 × 10−9 and 50 × 10−6 mg/mL (Figure 7). For the different interferences, the response of CS-MIPs/CMA/Au sensor were lower compared with GLY detection indicating that the microsensor displays the best selectivity to GLY. Non-imprinted microsensor (CS-NIPs/CMA/Au) was used to study the effectiveness of the imprinting in the presence FIGURE 8 | Regeneration of the CS-MIPs/CMA/Au microsensor. FIGURE 7 | Selectivity of CS-MIPs/CMA/Au microsensor (black) response of CS-MIPs/CMA/Au to GLY, (red) response of CS-NIPs/CMA/Au to GLY, (blue) response of CS-MIPs/CMA/Au to GLU, (pink) response of CS-MIPs/CMA/Au FIGURE 9 | EIS of CS-MIPs/CMA/Au before and after incubation in Rhone to CHL, and (green) response of CS-MIPs/CMA/Au to PHO. river water. Frontiers in Chemistry | www.frontiersin.org 8 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor To study the regeneration of the CS-MIPs/CMA/Au has been registered with good correlation coefficient equals to microsensor, EIS repetitive measurements were carried 0.997 (Figure 10B). The form of regression equation y = ax out using the same imprinted microsensor which was re- (1R/R = 0.20log[GLY]) is an indication of the absence of immersed in solutions containing 125 pg/mL of GLY. After GLY in the river water considered. In addition, the sensitivity each measurement, the CS-MIPs/CMA/Au microsensor of this designed microsensor in the real and buffer samples were was incubated for 5 min in CH3 COOH/ CH3 OH solution very close, which indicated that the CS-MIPs/CMA/Au exhibited (1:1, v/v) to eliminate attached GLY molecules. As high reliability. shown in Figure 8, the results of second and the third repeat measurements showed weak relative variation of 1R/R compared with the first cycle (3.08 and 12.75%, CONCLUSION respectively). For the fourth repeat, the variation was more significant (50.75%), indicating that the sensor can only be This work is a new investigation around the design of a regenerated twice. sensitive and selective sensor for the detection of traces of GLY in synthetic and real water samples. It is based on REAL SAMPLE ANALYSIS the grafting of the MIP molecularly imprinted CS chitosan onto an Au microelectrode modified with 4-aminophenylacetic In order to assess the robustness and the applicability of the acid CMA. Thanks to EIS investigations, the quantification developed microsensor in a real environment, water samples of GLY was carried out to in a linear concentration range retrieved from the Rhone River in Lyon, France, were analyzed. between 0.31 × 10−9 and 50 × 10−6 mg/mL and a limit of CS-MIPs/CMA/Au microsensor was incubated in river water detection of 10−12 mg/mL. samples for 30 min, and then EIS experiments were carried out The sensitivity of CS-MIPs/CMA/Au is higher compared for different GLY concentrations. The EIS diagrams were plotted with CS-MIPs/Au which arouses the interest to use this to deduce electron transfer resistance of the sensor surface. diazonium salt. The developed electrochemical microsensor Respective Nyquist plots before and after the sample analysis are has very good selectivity for GLY in comparison to three almost superimposed, probably indicating the absence of GLY in interferent compounds. The printing efficiency was confirmed the analyzed sample (Figure 9). using non-imprinted microsensor. In addition, the high To confirm the above observation, the GLY was quantified reliability of this biosensor allows it to be used precisely to using the standard additions method. A remarkable variation of detect low traces of GLY in both synthetic and real water impedance spectra was observed with a gradual increase of GLY samples. The ease of implementation, the simplicity and concentration in the river water sample in the interval of 0.31 the miniaturization of this sensor guide research for the × 10−9 -50 × 10−6 mg/mL (Figure 10A). A linear relationship development of economical, precise, and rapid tools for between GLY concentration and the relative transfer resistance environmental analysis. FIGURE 10 | (A) EIS of CS-MIPs/CMA/Au in 5 mM [Fe(CN)6 ]3−/4− and PBS Before pre-concentration and after incubation in river water with different concentrations of GLY. (B) detection of GLY in Rhone river water using standard addition method with the following concentrations (0.031 pg/mL, 6.25 pg/ml, 125 pg/ml, 2.5 ng/ml, and 50 ng/ml) (1R/R = 0.20 log [GLY]; R² = 0.997). Frontiers in Chemistry | www.frontiersin.org 9 May 2021 | Volume 9 | Article 621057 Zouaoui et al. Electrochemical Impedance Spectroscopy Microsensor DATA AVAILABILITY STATEMENT FUNDING The raw data supporting the conclusions of this article will be The authors acknowledge the financial support of CAMPUS made available by the authors, without undue reservation. FRANCE program under grant agreement PROFAS B+ and PHC PROCOPE #40544QH and the EU H2020 AUTHOR CONTRIBUTIONS research and innovation program entitled Kardia Tool grant agreement #768686. FZ carried out the experimental work under the supervision of NJ-R and AE, and wrote the article. JB and AA contributed to the SUPPLEMENTARY MATERIAL design of the sensors. NJ-R supervised and contributed writing and editing. 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Conflict of Interest: The authors declare that the research was conducted in the doi: 10.1080/14786435.2020.1841914 absence of any commercial or financial relationships that could be construed as a Terbouche, A., Lameche, S., Ait-Ramdane-Terbouche, C., Guerniche, D., Lerari, potential conflict of interest. D., and Bachari, K. (2016). A new electrochemical sensor based on carbon paste electrode/Ru(III) complex for determination of nitrite: electrochemical Copyright © 2021 Zouaoui, Bourouina-Bacha, Bourouina, Alcacer, Bausells, impedance and cyclic voltammetry measurements. Measurement 92, 524–533. Jaffrezic-Renault, Zine and Errachid. This is an open-access article distributed doi: 10.1016/j.measurement.2016.06.034 under the terms of the Creative Commons Attribution License (CC BY). The use, Wang, J., Wang, L., Yu, H., Zain-ul-Abdin, S., Chen, Y., Chen, Q., distribution or reproduction in other forums is permitted, provided the original et al. (2016). Recent progress on synthesis, property and application author(s) and the copyright owner(s) are credited and that the original publication of modified chitosan: an overview. Int. J. Biol. Macromol. 88, 333–344. in this journal is cited, in accordance with accepted academic practice. No use, doi: 10.1016/j.ijbiomac.2016.04.002 distribution or reproduction is permitted which does not comply with these terms. Frontiers in Chemistry | www.frontiersin.org 11 May 2021 | Volume 9 | Article 621057