chemosensors Article Dummy Molecularly Imprinted Polymers Using DNP as a Template Molecule for Explosive Sensing and Nitroaromatic Compound Discrimination Anna Herrera-Chacon, Andreu Gonzalez-Calabuig and Manel del Valle * Sensors and Biosensors Group, Department of Chemistry, Universitat Autònoma de Barcelona, Edifici Cn, Bellaterra, 08193 Barcelona, Spain; anna.herrera@uab.cat (A.H.-C.); andreugc27@gmail.com (A.G.-C.) * Correspondence: manel.delvalle@uab.cat Abstract: This work reports a rapid, simple and low-cost voltammetric sensor based on a dummy molecularly imprinted polymer (MIP) that uses 2,4-dinitrophenol (DNP) as a template for the quantification of 2,4,6-trinitrotoluene (TNT) and DNP, and the identification of related substances. Once the polymer was synthesised by thermal precipitation polymerisation, it was integrated onto a graphite epoxy composite (GEC) electrode via sol–gel immobilisation. Scanning electron microscopy (SEM) was performed in order to characterise the polymer and the sensor surface. Responses towards DNP and TNT were evaluated, displaying a linear response range of 1.5 to 8.0 µmol L−1 for DNP and 1.3 to 6.5 µmol L− 1 for TNT; the estimated limits of detection were 0.59 µmol L−1 and 0.29 µmol L−1 , for DNP and TNT, respectively. Chemometric tools, in particular principal component analysis   (PCA), demonstrated the possibilities of the MIP-modified electrodes in nitroaromatic and potential interfering species discrimination with multiple potential applications in the environmental field. Citation: Herrera-Chacon, A.; Gonzalez-Calabuig, A.; del Valle, M. Dummy Molecularly Imprinted Keywords: molecularly imprinted polymers; dummy template; voltammetric detection; 2,4,6- Polymers Using DNP as a Template trinitrotoluene; nitroaromatic compounds Molecule for Explosive Sensing and Nitroaromatic Compound Discrimination. Chemosensors 2021, 9, 255. https://doi.org/10.3390/ 1. Introduction chemosensors9090255 There is an increasing interest in analytical methods for determining, quantifying and discriminating pollutant compounds in different matrices. Desirably, the analytical re- Academic Editor: Pi-Guey Su sponse should be fast, portable, reliable, cost-effective, with low-power consumption, easy to manipulate, and able to be used in indoor and outdoor spaces for on-site measurements. Received: 4 August 2021 The field of sensors and biosensors tackles this challenging demand, improving sensing Accepted: 6 September 2021 abilities year by year. A key element in the performance of sensing devices is its recognition Published: 8 September 2021 capability. Molecularly imprinted polymers (MIPs) can be introduced in biosensing as the necessary recognition element, in this case derived from a synthetic approach. As a Publisher’s Note: MDPI stays neutral result, the sensor field can improve response features in a simple and inexpensive manner with regard to jurisdictional claims in published maps and institutional affil- by incorporating these new recognition elements, which may constitute an alternative to iations. antibodies, for example. MIPs acting as artificial receptors have demonstrated their applicability in several fields; among these, we can note the food industry [1,2], environmental monitoring [3,4], clinical diagnosis [5,6] and forensic field [7]. MIPs are perfect candidates to improve the performance of current analytical methods due to their versatility, functionality, and Copyright: © 2021 by the authors. ability to sample pre-concentration [8], separation [9] and/or purification [10]; alternatively, Licensee MDPI, Basel, Switzerland. they may be used as a recognition element for the development of different biosensing This article is an open access article distributed under the terms and strategies [11,12] and also in drug delivery [13]. conditions of the Creative Commons Recently, the use of dummy MIPs has been described, i.e., when MIPs are synthesised Attribution (CC BY) license (https:// towards an analogue molecule of the target analyte, which cannot be used as a template creativecommons.org/licenses/by/ during the synthesis of the polymer. This happens when the analyte is (1) expensive, (2) 4.0/). has a poor solubility or by-products are generated that hinder the imprinting, or (3) is Chemosensors 2021, 9, 255. https://doi.org/10.3390/chemosensors9090255 https://www.mdpi.com/journal/chemosensors Chemosensors 2021, 9, 255 2 of 15 considered a hazard. For example, the use of dummy MIPs has been reported for detecting fluoroquinolones in fish samples [14], for detecting bisphenol A in river water samples [15], and for providing an alternative to acrylamide removal in foodstuffs [16]. Precisely, the use of dummy MIPs is a choice of interest when aiming for the detection of explosive compounds [17], such as nitroaromatic species [18]. A habitual task in forensic laboratories is to detect or confirm the presence of explosives [19] or explosive residues [20]. There is also a need to quantify explosives at trace level after a detonation [21,22], or in contaminated underground water reservoirs [23]. Nitroaromatic species are excellent candidates for electrochemical sensing due to inherent electroactivity, given their characteristic electron-transfer reduction reactions and their electron-acceptor properties [24,25]. For all the above reasons, their electrochemical sensing ability has become a promising alternative to address the growing security needs of society. Specifically, TNT, a common nitroaromatic explosive, complies with the above- mentioned characteristics, making it a candidate to employ the dummy MIP strategy [26]. The following Table 1 presents a survey of recently published methods for determining TNT and related substances, focusing on sensor based procedures. Table 1. Summary of recent studies dedicated to the detection of TNT, detailing the strategy and technique employed, linear range and limit of detection. Linear Range (mol LOD Sensing Platform Interferents Technique Reference L−1 ) (mol L−1 ) Molecularly imprinted Trimesic acid, isopthalic acid Cyclic 0.1 × 10−9 –10.0 × polydopamine films onto and 50.0 × 10−12 [27] Voltammetry 10−9 gold electrodes 4-nitrophenol Phenol, Aniline, Carbon paste electrodes Square-Wave 5.0 × 10−9 –1.0 × para-Nitrophenol, 1.5 × 10−9 [28] modified with MIP particles Voltammetry 10−6 Benzoic acid and Nitrobenzene Gold nanoparticles/poly Paracetamol-caffeine-based (carbazole-aniline) analgesic drug, acetylsalicylic Square-Wave 4.4 × 10−7 –4.4 × 1.1 × 10−7 [29] film-modified glassy carbon acid (aspirin), sweetener, Voltammetry 10−6 electrode and sugar Alkanethiols self-assembled Trinitrobenzene, dinitrotoluene Differential Pulse 4.0 × 10−8 –3.2 × on AuNPs modified glassy 1.3 × 10−8 [30] and dinitrobenzene Voltammetry 10−6 carbon Dummy molecularly 2,4-dinitrophenol, imprinted polymers with 0.8 × 10−6 –30.0 × 4-nitrophenol, Fluorescence 0.28 × 10−6 [18] capped CdTe quantum 10−6 phenol, and dinitrotoluene dots Nitrobenzene, p-Nitrotoluene, Dinitrotoluene, Trinitrophenol, cyclotrimethylenetrinitramine, Naphthalene based UV–Vis 5 × 10−9 – cyclotetra- 1.5 × 10−9 [31] fluorescent probe spectroscopy 1 × 10−6 methylenetetranitramine and Hexanitrohexaazaisowurtzi- tane Amine functionalised Nitrobenzene, dinitrotoluene 4.4 × 10−11 –4.0 × Fluorescence 4.3 × 10−11 [32] nanoparticles and trinitrophenol 10−8 Nitrobenzene, dinitrotoluene 2.2 × 10−10 –3.1 × Fluorescent paper Fluorescence 1.4 × 10−10 [33] and trinitrophenol 10−8 MIP functionalised carbon Paracetamol, serotonin, Differential pulse The present graphite epoxy composite 1.5 × 10−6 –8 × 10−6 0.29 × 10−6 tryptamine voltammetry work electrodes The presence of TNT and its degradation products in bodies of water has been consid- ered by environmental agencies as a major concern. For example, monitoring, clean-up and adsorption experimental methods are needed, especially in the case of explosive-related compounds, where the legally tolerated levels in ground waters are in the microgram per litre range [34]. Apart from facilities dedicated to the manufacturing, processing and Chemosensors 2021, 9, 255 3 of 15 storage of munitions, TNT can be released into the environment through spills, weapon firing, leaching from inadequately sealed impoundments, or zone demilitarization. TNT has been classified as a probable human carcinogen by the United States Environmental Protection Agency (USEPA) [35]. According to USEPA, TNT in drinking water poses a considerable risk of inducing cancer at concentrations above 0.44 µmol L−1 [36]. Thus, there is a need to develop affordable and reliable methods to determine TNT levels, which can be deployed on-field, especially in countries where the access to more sophisticated techniques, e.g., high-performance liquid chromatography (HPLC) or solid-phase extrac- tion and gas chromatography/mass spectrometry (GC/MS), may not be accessible. In the same direction, the electrochemical sensor and polymer synthesis proposed here is easy to conduct in non-specialist laboratories and undemanding to reproduce, due to the cheap and readily available reagents and laboratory apparatus, which is an advantage in cases of resource-limited facilities. Electrochemical sensors for the detection of nitroaromatic compounds, such as TNT, have been described in the literature in studies that use screen-printed sensors [37,38]; additionally, voltammetric sensors modified with cobalt phtalocyanine [39], carbon fi- bres [28], carbon nanotubes [40], mesoporous carbon [41] and gold nanoparticles and poly(carbazole-aniline) have been specially electropolymerized to imprint nitroaromatics and nitramines [29]. These electrochemical sensors have special advantages, including their ability to be miniaturised which enables them to be used in portable equipment, making on-field detection in difficult scenarios possible. In the same line, these electrochemical sensors may be also involved in the detection of analytes in water samples without the need for sample pre-treatment, which translates to faster measurements due to the absence of extractions with organic solvents or demanding procedures to clean up and pre-treat the samples. Although several approaches have been recently reported, see Table 1, with extensive linear ranges and low limits of detection, we think that there has been a persistent, urgent need for a simple, cheap, and robust sensor which is able to detect and quantify TNT and DNP and to identify related substances in a fast and reliable manner in on-field situations. In this study, the abovementioned purpose was accomplished by a molecularly im- printed polymer for TNT using the TNT analogue 2,4-dinitrophenol (DNP) as a dummy template. Obtained polymers in the form of microbeads were next integrated into graphite epoxy composite (GEC) electrodes in a fast, simple and versatile process, converting in- sulating polymers in a biomimetic recognition element able to be employed as a reusable voltammetric sensor. Responses of these developed sensors towards TNT and DNP were studied, and the selectivity was evaluated with respect to additional nitroaromatic species which could be interferents in real samples, such as: 1-nitrobenzene (1-NB), 4-nitrotoluene (4-NT), 1,3-dinitrobenzene (1-DNB), 2-nitrotoluene (NT), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2-DNT). At the same time, many of these mono-nitro and di- nitroaromatic compounds may be degradation products of energetic materials, which envisages their use in environmental applications [42–44]. The schematic plot of the imprinting process is depicted in Figure 1. The dummy molecularly imprinting approach is shown by replacing the DNP imprinting motif by the TNT as the final analyte. Chemosensors 2021, 9, x FOR PEER REVIEW 4 of 15 Chemosensors 2021, 9, 255 The schematic plot of the imprinting process is depicted in Figure 1. The dummy 4 of 15 molecularly imprinting approach is shown by replacing the DNP imprinting motif by the TNT as the final analyte. Figure 1. Schematic procedure of polymer imprinting synthesis process employing 2,4-dinitrophenol (DNP) and its Figure 1. Schematic procedure of polymer imprinting synthesis process employing 2,4-dinitrophenol (DNP) and its im- immobilization onto the surface sensor. mobilization onto the surface sensor. 2. Materials and Methods 2. Materials and Methods 2.1. Chemicals and Reagents 2.1. Chemicals and Reagents Tetramethyl orthosilicate (TEOS), hydrochloric acid, sodium hydroxide, 1,3- Tetramethyl dinitrobenzene orthosilicate (DNB), (TEOS),(NT), 2-nitrotoluene hydrochloric acid, sodium 2,4-dinitritoluene (DNT), hydroxide, 1,3-dinitro- 2,6-dinitrotoluene benzene and (DNB), 2-nitrotoluene 2,4-dinitrophenol (DNP) were (NT), 2,4-dinitritoluene purchased from Merck(DNT), 2,6-dinitrotoluene (Darmstadt, and 2,4- Germany). Ethylene dinitrophenol (EGDMA), dimethacrylate (DNP) were purchased methacrylic from acid Merck (MAA), (Darmstadt, (NB) 1-nitrobenzene Germany). Ethylene di- and 4-nitrotoluene methacrylate (NT) were supplied(EGDMA), methacrylic by Acros Organicsacid (MAA), (Geel, 1-nitrobenzene Belgium). (NB) and 4-nitrotoluene 2,4,6-trinitrotoluene (TNT) was (NT) were obtained fromsupplied by Acros(Teddington, LGC standards Organics (Geel, Belgium). Middlesex, 2,4,6-trinitrotoluene UK). Methanol (MeOH), (TNT) was ethanol obtained (EtOH) and from LGC acetic standards acid (Teddington, (HAc) were acquiredMiddlesex, from Scharlab UK).(Barcelona, Methanol (MeOH), Spain) and ethanol 2,2- (EtOH) and acetic acid (HAc) were azobis(2,4-dimethylvaleronitrile) acquired (AIVN) from Scharlab was purchased from(Barcelona, Wako ChemicalsSpain) GmbH and 2,2- azobis(2,4-dimethylvaleronitrile) (Neuss, Germany). Graphite powder (AIVN) was purchased (particle size < 50 µm) fromwasWako Chemicals received from GmbH BDH (BDH (Neuss,Laboratory Germany). Supplies, GraphitePoole, powder UK) and Resineco (particle size < 50 Epoxy µm) was Kitreceived resin was supplied from BDH (BDH by Resineco LaboratoryGreen Composites Supplies, Poole,(Barcelona, Spain). All UK) and Resineco reagents Epoxy used were Kit resin of analytical was supplied reagent by Resineco grade, Greenand all solutions Composites were made (Barcelona, up using Spain). MilliQ water All reagents fromof used were MilliQ System analytical (Millipore, reagent grade, Billerica, MA, USA). The final experimental samples were prepared in and all solutions were made up using MilliQ water from MilliQ System (Millipore, Biller- phosphate buffer (100 ica, mmol MA, USA).L−1 KCl, The50final mmol L−1 K2 HPOsamples experimental 4 ·2H2 O,were 50 mmol L−1 KH prepared PO4 , pH 7.0). in 2phosphate buffer (100 mmol L KCl, 50 mmol L K2HPO4·2H2O, 50 mmol L KH2PO4, pH 7.0). −1 −1 −1 2.2. Equipment and Software 2.2. Imprinted polymers Equipment and Softwarewere synthesized in a water bath controlled with a Huber CC1 thermoregulation Imprinted polymerspump (Huber were GmbH,inOffenburg, synthesized Germany). a water bath Polymer controlled with a beads Huberand CC1 polymer-modified electrodes were characterized by scanning electron microscopy thermoregulation pump (Huber GmbH, Offenburg, Germany). Polymer beads and (SEM) poly- and FT-IR, employing mer-modified a MERLIN electrodes FE-SEM and by were characterized an scanning IR Spectrophotometer Tensor 27, electron microscopy Bruker, (SEM) and respectively. The resulting microscopy images were treated with Fiji package software and Image J software [45] (Zeiss GmbH, Jena, Germany). The electrochemical cell for voltammetric measurements employed a commercial 52–61 combined Ag/AgCl reference and counter platinum electrode (Crison Instruments, Barcelona, Spain) and was connected Chemosensors 2021, 9, 255 5 of 15 to an AUTOLAB PGSTAT30 (Ecochemie, Utrecht, The Netherlands) controlled with the GPES Multichannel 4.7 software package. Electrochemical data were plotted using Origin 8.0, whereas PCAs were calculated by the authors using techniques in MATLAB 2016b (MathWorks, Natick, MA, USA). 2.3. Polymer Synthesis Quantities of 0.5 mmol of DNP and 1.05 mmol of MAA were transferred into a 250 mL round-bottomed flask and dissolved with 40 mL of EtOH. The solution was gently stirred in an ice bath for 30 min. Afterwards, 10.2 mmol of EGDMA and 0.10 mmol of AIVN were added to the solution and the obtained mixture was purged with a flow of dry nitrogen for 15 min. Polymerisation, in bead forms, was initiated in a water bath at 60 ◦ C with magnetic stirring; the obtained polymer was collected after an overnight reaction. After this, the MIP was dried overnight at room temperature, the polymer was transferred into a Soxhlet system, and the template was extracted using a mixture of methanol:acetic acid (9:1) over 72 h. Non-imprinted polymers (NIPs) were prepared following the same methodology, but without the addition of the template molecule. 2.4. Sensor Preparation The so-called graphite epoxy composite (GEC) electrode was prepared by soldering a 5 mm diameter copper disc into an electrical connector. Then, the connector was placed into a 6 mm inner diameter PVC tube. Afterwards, a mixture of epoxy resin and graphite was prepared in order to allocate the conductive graphite particles into a robust matrix. Once the epoxy graphite resin was added, the sensor was cured for 48 h in an oven at 65 ◦ C. The resulting sensors had a sturdy surface which could be regenerated after a light polishing with sandpaper [46,47] and easily functionalised with several recognition elements [48,49]. Once the bare sensor was prepared, the MIPs were incorporated into the sensor. Once discarded, GEC sensors could be regenerated and reused by repeating the polishing procedure. 2.5. MIP Modification of Electrodes by Sol–Gel Entrapment The polymers employed herein had an insulator nature; therefore, the proposed strategy was to create a conductive paste which incorporated insulating polymer beads and graphite particles. The polymer beads were allocated into the surface electrode by drop-casting via sol–gel immobilisation. The employed sol–gel was prepared with 0.5 mL of TEOS, 0.5 mL of EtOH, 0.25 mL of H2 O and 25 µL of HCl 0.1 mol L−1 which were vigorously stirred for 45 min and then rested for 35 min in order to achieve the optimal polymerisation conditions. Subsequently, 0.2 mL of the rested solution and 7 mg of graphite were added to 40 µL of a 15 mg mL−1 polymer (MIP or NIP) suspension in EtOH. This mixture was stirred for 10 min at 1400 rpm. Then, the surface was prepared by spin-coating 10 µL of the polymer solution using a homemade spin-coater. Polymerisation was ended with overnight drying of the electrodes at 4 ◦ C [46,47]. 2.6. Characterisation by Scanning Electron Microscopy In order to prepare the sample for SEM studies, MIP and NIP beads were sprinkled into different aluminium stubs that contained carbon-tape on the surface. Then, the conductive carbon-tapes were metallised, employing a Au–Pd alloy (80:20) for 4 min, which added a 15–20 nm layer onto the sample, which enabled visualisation of the synthesised polymer beads with an adequate contrast. Five SEM images were developed for each kind of polymer (MIPs and NIPs) in order to count at least 500 particles in order to obtain a histogram of both polymers (Figures 2 and S1). Microscopy studies for the electrodes were performed by placing them on the stage and fixing them with carbon tape without employing any kind of metallisation. Chemosensors 2021, 9, 255 6 of 15 Figure 2. Characterisation of the polymers for MIP (A,C) and NIP (B,D): scanning electron microscopy (SEM) with secondary electron (A,B) and size distribution of the particles (C,D). 2.7. Electrochemical Measurements All the electrochemical measurements were performed using the differential pulse voltammetry (DPV) technique. The potential scan window was performed between 0 V and −1.2 V with a scan rate of 100 mV/s, a step potential of 5 mV and a modulation amplitude of 50 mV. No stirring was employed during the measurements. A cleaning step was performed between measurements, applying a potential of +1.4 V for 45 s in NaOH 0.1 mol L−1 . According to previous studies in our laboratories, immersion of the electrode into the solution for at least 5 min is desirable to preconcentrate the analyte and to obtain a perceptible signal [2,50]. All stock standard solutions were prepared at 5000 mg L−1 in acetonitrile (ACN). All the electrochemical measurements were treated with baseline correction (Figure S5, supplementary data) and all the analytical response calculations were performed for the first peak reduction, around −0.45 V, as suggested in the literature for TNT [25]. The first peak was considered for quantification of all the aforementioned nitro-derived species. 3. Results and Discussion 3.1. MIP Physical Characterisation Standard acrylic MIP synthesis was adopted in order to obtain the recognition material in beads form. Once the polymers were synthesised and the template was removed, SEM was performed in order to check the morphology of the abovementioned polymers before electrode immobilisation. Obtained MIPs beads showed a non-regular, highly cross-linked material with a spherical shape. Their average size and standard deviation were diameters of 0.416 ± 0.116 µm and 0.721 ± 0.247 µm for MIPs and NIPs, respectively, (Figure 2). Chemosensors 2021,9,9,255 Chemosensors2021, x FOR PEER REVIEW 77 of of 15 15 As As can can be be observed, observed, thethe MIPs MIPs presented presented aa slightly slightly smaller smaller particle particle size size in in comparison comparison with with NIPs. NIPs. This This might might suggest suggest that that during during the the synthesis synthesis there there was was more more reactivity reactivity due due to to the the imprinting, imprinting, which which might might bebe due due the the template template presence. presence. One One ofofthe thekeykeysteps stepsofof thethe presented presented protocol is the protocol immobilisation is the immobilisationontoonto the electrode, the elec- as trode, as can be seen in Figure 3. Materials were deposited by a drop-casting,spin-coating can be seen in Figure 3. Materials were deposited by a drop-casting, spin-coating technique technique viavia sol–gel sol–gel immobilisation, immobilisation, as as mentioned mentioned below,below, presenting presenting aa homogeneous homogeneous dispersion dispersion onon the the whole whole surface surface ofof the the sensors. sensors. OnOn the the other other hand, hand, Figure Figure 33 also also shows shows that the surface that the surfaceof ofthe thebare bareelectrode electrode was was totally totally different different fromfrom the the polymer-modified polymer-modified sen- sensors. As can be seen, the surface modification is self-evident; the sors. As can be seen, the surface modification is self-evident; the roughness after sol–gelroughness after sol–gel deposition deposition is very noticeable is very noticeable when comparing when comparing unmodified unmodified sensors sensors (Figure 3C)(Figure 3C) with mod- with modified sensors (Figure 3A,B). There are no clear differences ified sensors (Figure 3A,B). There are no clear differences between MIPs and NIPs: bothbetween MIPs and NIPs: present both present asurface a modified modified surface where the where the immobilized immobilized polymer beads polymer can beads be seen can be seen alongside alongside the sol–gelthe and sol–gel and graphite. graphite. Figure 3. Figure 3. Scanning Scanningelectron electronmicroscopy microscopy(SEM) (SEM)images with images secondary with electron secondary detector electron for the detector for MIP-sensor (A) and the MIP-sensor (A) NIP- and sensor (B) and GEC electrode (C). NIP-sensor (B) and GEC electrode (C). An FT-IR An FT-IR assay assay waswas performed performed to to observe observe thethe main main chemical chemical bonds. bonds. The The FT-IR FT-IR was was performed in triplicates of MIP and NIP samples. MIP and performed in triplicates of MIP and NIP samples. MIP and NIP polymers showed very NIP polymers showed very similarFT-IR similar FT-IRspectra spectrawith with little little or ornono significant significant differences differences (Figure (Figure S2). S2). Thus, Thus, no differ- no differences ences in the in the chemical chemical bondsbonds were wereobservedobserved betweenbetween the different the different synthesis synthesis methodolo- methodologies. gies.main The The main bands bands observed observed are the are the broadbroadbandband of hydroxyl of hydroxyl groups groups around around 3430cm 3430 −−1 cm 1 from the from the MMA MMA units; units; the the stretching stretching and and bending bending of of methyl methyl groups groups from from the the MMAMMA and and the EGDMA the EGDMA at atthe theband bandaround around 2950–2980 2950–2980 cmcm −1 1450–1470 −1 and and 1450–1470 the−carbonyl cm−1; cm 1 ; the carbonyl stretch- ing at 1720atcm stretching −1; the 1720 −1 ; the C–O–C cmC–O–C stretching at 1145 at stretching and11451250andcm1250 −1; and −1 ; rocking cmthe and the of the C–H rocking of stretching the at 750 cm at. The C–H stretching −1 750 peak cm at− 1 1636peak . The cm is −1 atassociated 1636 cm with− 1 C=C bonds, is associated which with C=C indicates bonds, which indicates polymerized an incompletely an incompletely polymerized monomer. Probably, monomer. EGDMA Probably, EGDMA was not totally was not crosslinked. totallyIncrosslinked. the case of MIPs, in which DNP could be retained during the synthesis, the nitro groups In the case of showed MIPs, in a strong which band aroundDNP1550 could be1350 and retained cm−1during the synthesis, , corresponding the nitro to asymmetric groups showed a strong band around 1550 and 1350 cm −1 , corresponding−1to asymmetric and symmetric stretching, respectively. The absence of a band at 1550 cm confirmed the and symmetric absence of DNPstretching, respectively. from the polymer. The absence The band at 1390 of cma−1band couldatbe 1550 cm−to related 1 confirmed the asym- the absence of DNP −1 could be related to the metric bending of thefrom –OHthe polymer. groups of theThe band atacid carboxylic 1390 of cm the MMA units. asymmetric bending The obtained of the –OH chemical bondinggroups of thewith agreed carboxylic acid of the the expected MMAproduced polymer units. from The obtained free radical chemical The polymerisation. bondingslightagreed with in differences thethe expected wavenumber polymer andproduced from peak intensity free could radical polymerisation. be associated with theThe slight structure chemical differences andin space the wavenumber distributionand duepeak intensity to differences could be associated in the synthesis. with the chemical structure and space distribution due to differences in the synthesis. 3.2. Electrochemical Response 3.2. Electrochemical Response Prior to the evaluation of the electrochemical response, the pH and enrichment times Prior to the evaluation of the electrochemical response, the pH and enrichment times between template sample and sensor were studied and optimised. Stock solutions of 10 between−1 template sample and sensor were studied and optimised. Stock solutions of µmol L for TNT and DNP were employed. The samples were measured with a contact 10 µmol L−1 for TNT and DNP were employed. The samples were measured with a time from 0 to 100 min, and the resultant plots and fittings are shown in Figure S3, in the contact time from 0 to 100 min, and the resultant plots and fittings are shown in Figure S3, supplementary information. The evaluated pH ranged from 5 to 8 (Figure S4). pH 7 was in the supplementary information. The evaluated pH ranged from 5 to 8 (Figure S4). pH 7 chosen because TNT and DNP presented maximum peak intensities at this neutral value. Chemosensors 2021, 9, x FOR PEER REVIEW 8 of 15 Chemosensors 2021, 9, 255 8 of 15 In order to achieve a balance between the increase in signal and speed, plus for simplicity was chosen because TNT and DNP presented maximum peak intensities at this neutral of the method, a contact time of 5 min was chosen, which enabled reaching more than value. In order to achieve a balance between the increase in signal and speed, plus for two-thirds of the final response with a reduced analysis time. simplicity of the method, a contact time of 5 min was chosen, which enabled reaching more In order to study the repeatability of the manufactured sensors, 10 µmol L−1 samples than two-thirds of the final response with a reduced analysis time. of TNT and DNP were measured 15 times with three DNP-MIP-sensors and three DNP- In order to study the repeatability of the manufactured sensors, 10 µmol L−1 samples NIP-sensors on the same day. The obtained repeatability values for DNP-MIPs and DNP- of TNT and DNP were measured 15 times with three DNP-MIP-sensors and three DNP-NIP- NIPS sensors when measuring TNT were 3.97% and 4.61%, respectively. For DNP, the sensors on the same day. The obtained repeatability values for DNP-MIPs and DNP-NIPS DNP-MIP and DNP-NIP sensors had repeatability values of 9.14% and 7.34%, respec- sensors when measuring TNT were 3.97% and 4.61%, respectively. For DNP, the DNP-MIP tively. and DNP-NIP sensors had repeatability values of 9.14% and 7.34%, respectively. The reproducibility was studied for three DNP-MIP sensors: 10 µmol L−1 samples of The reproducibility was studied for three DNP-MIP sensors: 10 µmol L−1 samples of TNT and DNP were measured five times, obtaining values of 6.5% for TNT and 5.5% for TNT and DNP were measured five times, obtaining values of 6.5% for TNT and 5.5% for DNP. DNP-MIP DNP. DNP-MIP sensors’ sensors’ repeatability repeatability values values against against DNP DNP showed showed slightly slightly better better results results than TNT, which is explained by the dummy imprinting performed for DNP, than TNT, which is explained by the dummy imprinting performed for DNP, as can as can be be seen in seen in Figure Figure 4. 4. Figure 4. Repeatability measurements of MIP and NIP for (A) DNP and (B) TNT (n = 3). Figure 4. Repeatability measurements of MIP and NIP for (A) DNP and (B) TNT (n = 3). The obtained voltammograms for DNP and TNT are presented in Figure 5. DNP has a The obtained voltammograms for DNP and TNT are presented in Figure 5. DNP has double reduction peak between −0.4 V and −0.6 V, whereas TNT exhibits three reduction a double reduction peak between −0.4 V and −0.6 V, whereas TNT exhibits three reduction peaks in the same region. The three peaks that TNT presents are due the reduction in the peaks in the three nitro same The groups. region. firstThe nitrothree peaksisthat reduction TNT presents produced around are −0.5due the usually V, and reduction in the presents three nitro groups. The first nitro reduction is produced around −0.5 V, a sharper and more defined peak; the second nitro group is reduced around −0.65 V; and and usually pre- sents a sharper and more defined peak; the second nitro group is reduced the last nitro group is around −0.75 V when measured with MIP-functionalised electrodes. around −0.65 V;appears It and thethat lastthe nitro MIPgroup is around electrode shifted−0.75 theseVreduction when measured with MIP-functionalised peaks to more negative potentials electrodes. when It appears compared thatNIP with the the MIP electrode electrode, whichshifted these peaks presented reduction at −peaks 0.35 V,to−more 0.55 Vnega- and −0.65 V. In the case of DNP, the two nitro reduction peaks appeared at aroundat− tive potentials when compared with the NIP electrode, which presented peaks −0.35 0.52 V, V −0.55−V and andV−0.65 0.68 V. In for MIP andtheNIP, caserespectively, of DNP, the with two nitro reduction no apparent peaks shift appeared at around in potential. −0.52These V andsubstances −0.68 V forwere MIP and NIP,inrespectively, studied withrange a concentration no apparent of 0.55 shift to 19inµmol potential. L−1 for DNP and from 0.45 to 10 µmol L for TNT. − 1 3.3. Calibration Curves An MIP sensor, NIP sensor and bare electrode (GEC) were used to compare the voltam- metric responses for DNP and TNT in detail, as depicted in Figure 6. Three sensors of each type were used to build the calibration curves on different days. The obtained voltammo- grams were treated with baseline correction and the first peak was chosen to determine the peak height. To illustrate the processing, the baseline-corrected voltammograms are plotted in Figure S5, in the supplementary information. Chemosensors Chemosensors 2021,2021, 9, x FOR PEER REVIEW 9, 255 9 of 1515 9 of Figure Figure 5. Voltammetric 5. Voltammetric responses responses from from 0.55 0.55 to 19 to 19 µmol L−L1−1ofofDNP µmol DNPand andfrom from0.45 0.45to to 15 15 µmol L− µmol L −11 TNT for the prepared TNT for the prepared sensors. (A) Voltammetric response vs. DNP measured with the MIP sensor. (B) Voltammetric response sensors (each colour represents an increasing concentration of the corresponding compound). (A) Voltammetric vs. DNPresponse measuredvs. with the NIP sensor. (C) Voltammetric response of DNP measured with the GEC sensor. (D) Voltammetric response vs. DNP measured with the MIP sensor. (B) Voltammetric response vs. DNP measured with the NIP sensor. (C) Voltammetric TNT measured with the MIP sensor. (E) Voltammetric response of TNT measured with the NIP sensor. (F) Voltammetric response of DNP measured with the GEC sensor. (D) Voltammetric response vs. TNT measured with the MIP sensor. response vs. TNT measured with the GEC sensor. (E)Chemosensors Voltammetric response 2021, 9, of REVIEW x FOR PEER TNT measured with the NIP sensor. (F) Voltammetric response vs. TNT measured 10 with of 15 the GEC sensor. These substances were studied in a concentration range of 0.55 to 19 µmol L−1 for DNP and from 0.45 to 10 µmol L−1 for TNT. 3.3. Calibration Curves An MIP sensor, NIP sensor and bare electrode (GEC) were used to compare the volt- ammetric responses for DNP and TNT in detail, as depicted in Figure 6. Three sensors of each type were used to build the calibration curves on different days. The obtained volt- ammograms were treated with baseline correction and the first peak was chosen to deter- mine the peak height. To illustrate the processing, the baseline-corrected voltammograms are plotted in Figure S5, in the supplementary information. Calibration curves from 0.550.55 to to 1919 −1 for DNP (A) and from 0.45 to 15 µmol −1 Figure 6. Figure 6. Calibration curves from µmolLL−1 for µmol DNP (A) and from 0.45 to 15 µmol L−1 forLTNTfor(B)TNT (B) for the for the three three different sensorsensor different typestypes usedused in this study. in this Linear study. Linearranges rangesfrom 1.6to from 1.6 to8.0 8.0µmol µmol − 1 L Lfor DNP −1 for DNP and 1.3 and from from 1.3µmol to 6.5 for L−1 for to 6.5L µmol −1 TNT are added TNT are added in (C,D). in (C,D). In order to obtain LOD values (S/N = 3), the regression lines were fitted with the first five concentration values of the calibration curves, from 1.6 to 8.0 µmol L−1 for DNP and 1.3 to 6.5 µmol L−1 for TNT, and the linear portions of the calibration curve were used for the calculation. Detailed parameters of the calibration curves are shown in Table 2. Chemosensors 2021, 9, 255 10 of 15 In order to obtain LOD values (S/N = 3), the regression lines were fitted with the first five concentration values of the calibration curves, from 1.6 to 8.0 µmol L−1 for DNP and 1.3 to 6.5 µmol L−1 for TNT, and the linear portions of the calibration curve were used for the calculation. Detailed parameters of the calibration curves are shown in Table 2. Table 2. Summary of calibration results in the linear concentration region from 1.6 to 8.0 µmol L−1 towards DNP and from 1.3 to 6.5 µmol L−1 for TNT. MIP NIP DNP TNT DNP TNT Sensitivity 1.84 56.6 1.33 4.41 (µA µmol−1 L) Intercept (µA) 2.31 5.72 3.44 11.9 R2 0.99 0.98 0.99 0.99 LOD (µmol− 1 L) 0.59 0.29 1.38 0.95 LOQ (µmol−1 L) 1.79 0.88 4.18 2.87 As per the results obtained, it may seem that the affinity of the MIP is better for TNT than for DNP. These results may be explained in view of the electrostatic attraction between a bulk polymer and the number of nitro groups borne by each compound: TNT has three groups whereas DNP only has two. A quick assessment of specificity was performed, verifying the interference effect of three related phenolic compounds, acetaminophen, tryptamine and serotonin, in mixtures with TNT, to check any significant alteration of the voltammetric signals and/or the adsorption abilities of the MIP materials. In all assayed cases (Figure S6, supplementary info), the TNT signals were only minimally affected, demonstrating the accomplished selectivity with the MIP modified devices. 3.4. Specificity versus Other Nitroaromatic Compounds Other mono-nitro and di-nitro aromatic compounds available in the laboratory were measured five times at the same concentration in order to evaluate the specificity of the imprinted sensor. Examined compounds, in addition to DNP and TNT, were 2,4- dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), 2-nitrotoluene (2-NT), 1,3- dinitrobenzene (1,3-DNB), 4-nitrotoluene (4-NT) and 1-nitrobezene (1-NB). Fixed con- centrations of 10 µmol L−1 of the corresponding compounds were employed to evaluate the differences in specificity. As can be seen in Figure 7, current intensities are clearly higher for the MIP sensor when compared with the NIP and GEC sensors for all the evaluated compounds. This higher current may be attributed to the enrichment of electroactive species on the surface electrode due to the imprinted sites. However, when we examined the specificity, it was evident that TNT exhibited the highest current, followed by the group formed by 2-NT, 2,4-DNT, 1,3-DNB and 2,6-DNT, and finally, in the last group, we observed 4-NT, 1-NB and DNP. These different groups can be explained again by the number of nitro groups present in the molecule. It may seem that a larger number of nitro groups allows a higher concentration on the polymer surface and an increase in the signal intensity. 10 µmol L−1 of the corresponding compounds were employed to evaluate the differences in specificity. As can be seen in Figure 7, current intensities are clearly higher for the MIP sensor when compared with the NIP and GEC sensors for all the evaluated compounds. This Chemosensors 2021, 9, 255 higher current may be attributed to the enrichment of electroactive species on the surface 11 of 15 electrode due to the imprinted sites. Figure Figure 7. Comparison of voltammetric 7. Comparison response of voltammetric of 10 µmol response of 10Lµmol L−1 nitroaromatic −1 nitroaromatic compounds for eachfor each compounds sensor and different sensor nitroaromatic and different species.species. nitroaromatic As an However, additional when feature the we examined thatspecificity, may be derived it wasfrom evidentthese assays, that TNT we foreseethe exhibited possible highestuses in discriminating current, followed by the thegroup different formed nitroaromatic compounds by 2-NT, 2,4-DNT, in aand 1,3-DNB type of intelligent 2,6-DNT, sensor, and finally, infor thewhich an exploratory last group, we observedapplication wasand 4-NT, 1-NB attempted. Multivariate DNP. These differentanalysis groups of the can be complete explainedvoltammogram employing again by the number a principal of nitro component groups present analysis in the (PCA) molecule. wasseem It may performed in order to evaluate whether the voltammetric signals obtained were that a larger number of nitro groups allows a higher concentration on the polymer surface promising for future studies involving classification and an increase in the signal intensity. and/or quantification of the substances. Five samples for each compound at 10 µmol L −1 were measured with the MIP, NIP and GEC sensors (see As an additional feature that may be derived from these assays, we foresee possible uses incharacteristic discriminating voltammogram profiles in the the different nitroaromatic supplementary compounds information, in a type Figure of intelligent sen-S8). In sor, forFigure which8,anthe scores obtained exploratory after the application wasPCA transformation attempted. are plotted, Multivariate analysisand as can of the com-be seen, plete voltammogram employing a principal component analysis (PCA) was performed in in the each different compound formed its own cluster in the case of MIP [A], whereas case of the NIP sensor [B] and GEC sensor [C], the samples were grouped clearly but with a lower degree of clustering, or did not group at all in the case of the GEC sensor. It is significative that the intended compounds in this study, i.e., DNP and TNT, exhibited distinct clusters, meaning that there is a clear difference in the response obtained from the template (DNP) and the intended analyte (TNT). The remaining compounds considered as possible interferents appeared in clusters more or less together; however, a separation could be seen inside the main “nitroaromatic cluster”, and each mono-nitro and di-nitro compound tested enabled its identification. The differences in the PCA results for the compared sensors show that the MIP sensor enabled a better discrimination among the tested nitroaromatic compounds (as can be seen in Figure 8). These results shown by this PCA treatment are a promising starting point to apply a combination of electrochemistry and chemometrics, which is commonly known as Electronic Tongue [51], to identify nitrated compounds in the environmental field. compound tested enabled its identification. The differences in the PCA results for the com- pared sensors show that the MIP sensor enabled a better discrimination among the tested nitroaromatic compounds (as can be seen in Figure 8). These results shown by this PCA treatment are a promising starting point to apply a combination of electrochemistry and chemometrics, which is commonly known as Electronic Tongue [51], to identify nitrated Chemosensors 2021, 9, 255 12 of 15 compounds in the environmental field. Figure 8. Figure 8. Score Score plots plots for for each each nitroaromatic nitroaromatic compound compound performed performed with with the the voltammetric voltammetric signals signals of of (A) (A) MIP, (B) NIP MIP, (B) NIP and and (C) GEC sensors for five replicates of each nitroaromatic compound at 10 µmol L−1 after principal component analysis. (C) GEC sensors for five replicates of each nitroaromatic compound at 10 µmol L−1 after principal component analysis. 4. Conclusions The use of DNP as a dummy template in a biomimetic MIP-based sensor for the deter- mination and discrimination of TNT has been proven to offer a suitable electrochemical response for identification and quantification of the latter. Electron microscopy showed how the spatial distribution of the synthesized MIP was homogeneous within the electrodes modified by spin-coating. The optimisation of the electroanalytical system resulted in a lin- ear response range from 1.6 to 8.0 µmol L−1 for DNP and from 1.3 to 6.5 µmol L−1 for TNT. Furthermore, a specificity study was carried out in order to discriminate TNT from other nitroaromatic species. The constructed sensory platform was sensitive to TNT, capable of detecting TNT below the concentrations where it is considered a risk, i.e., 0.44 µmol L−1 , among other mono, di and tri-nitro compounds that can be found and discriminated as derivate species in polluted surface or underground waters. However, more work is still needed to improve the sensitivity of MIP-GEC sensors while retaining a high selectivity; this is needed as well as choosing the simplest way of increasing the enrichment time by one or more orders of magnitude. In future studies, the authors intend to apply this approach to build a sensor array with different imprinted polymers and use chemometrics to extract the maximum amount of information from the obtained electrochemical data in the electronic tongue approach [52]. Obtaining such an electronic tongue will hopefully enable the simultaneous quantification and identification of different nitrobenzene species in complex mixtures with enhanced Chemosensors 2021, 9, 255 13 of 15 sensitivity and selectivity features, with remarkable interest specifically in environmental applications. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/chemosensors9090255/s1, Figure S1: Representative SEM images employed to determine the polymers’ particle sizes for [A] MIP and [B] NIP. Figure S2: Comparison between MIP (red) and NIP (blue) FT-IR spectra. Figure S3: Adsorptive kinetics fitted curves for the different sensor types. Figure S4: [A] pH study for TNT at different pH values; [B] pH study for DNP at different pH vales for the three MIP sensors (n = 3). Figure S5: Voltammetric results with baseline correction responses for the calibration curves. Figure S6: Interferent study at different ratios for TNT versus acetaminophen, serotonin and tryptamine. Figure S7: Schematic representation of different species used into the discrimination study. Figure S8. Differential pulse voltammograms for 10 mol L−1 of [A] NB, [B] NT, [C] 1,3-DNB, [D] 2-NT, [E] 2,6-DNT, [F] 2,4-DNT, [G] 2,4-DNP, and [H] TNT employed in the principal component Analysis. Author Contributions: Conceptualization, M.d.V.; Methodology, M.d.V.; Investigation, A.H.-C., A.G.- C.; A.H.-C. performed the design and synthesis of polymers and the experimental measurements. A.G.-C. performed the calculations for the qualitative modelling. M.d.V.; supervision, M.d.V.; funding acquisition. All authors contributed to the writing, reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: Financial support for this work was provided by the Spanish Ministry of Economy and Innovation, MINECO (Madrid) through project PID2019-107102RB-C21. Manel del Valle thanks the support from program ICREA Academia from Generalitat de Catalunya. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: Anna Herrera-Chacon and Andreu González-Calabuig thank Universtitat Autònoma de Barcelona (UAB) for the PIF fellowship. Conflicts of Interest: The authors declare no conflict of interest. References 1. Liang, C.; Ristic, R.; Jiranek, V.; Jeffery, D.W. 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