Development of highly selective electrochemical impedance sensor for detection of sub-micromolar concentrations of 5-Chloro-2,4-dinitrotoluene

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A highly selective and sensitive impedimetric sensor based on molecular imprinted polymer (MIP) has been developed for the detection of 5-chloro-2,4-dinitrotoluene (5CDNT). Computational simulations were carried out for the best combinations of MIP
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   J. Chem. Sci.  c  Indian Academy of Sciences.DOI 10.1007/s12039-016-1078-0 Developmentofhighlyselectiveelectrochemicalimpedancesensorfordetectionofsub-micromolarconcentrationsof 5-Chloro-2,4-dinitrotoluene K YUGENDER GOUD, SATYANARAYANA M, K KOTESHWARA REDDY andK VENGATAJALABATHY GOBI ∗ Department of Chemistry, National Institute of Technology Warangal, Telangana 506 004, Indiae-mail: MS received 27 January 2016; revised 9 March 2016; accepted 13 March 2016 Abstract.  A highly selective and sensitive impedimetric sensor based on molecular imprinted polymer (MIP)has been developed for the detection of 5-chloro-2,4-dinitrotoluene (5CDNT). Computational simulations werecarried out for the best combinations of MIP precursors by using Hyperchem software. MIP of 5CDNT issynthesizedusingtheoptimizedcombinationsofpolymerprecursorssuchasthefunctionalmonomerandcross-linker. Synthesized MIP and non-imprinted polymer (NIP) were characterized by Fourier transform infraredspectroscopy (FT-IR) and BET adsorption isotherm analysis. The surface area and pore volume of MIP arequite high compared to that of NIP, while the average pore diameter of MIP is lower than that of NIP. However,the synthesized MIP with high surface area and quite good pore diameter for free flow of analytes is suitablefor efficient binding with the analyte 5CDNT. Binding assay studies of MIP by using UV-Visible spectroscopyshowed that the molecular imprinting factor is nearly thrice to that of NIP. Carbon paste electrodes incorporatedwith MIP were employed as a sensor for 5CDNT by applying electrochemical impedance spectroscopy (EIS)as transduction principle. The charge transfer resistance obtained by impedimetric analysis is proportional tothe concentration of 5CDNT over a wide concentration range: 10 nM to 100 µ  M. A linear determination rangeof 1.0 to 100  µ  M was obtained, and the low-detection-limit was found to be 0.1  µ  M. The MIP carbon pasteelectrode has shown very good selectivity towards specific recognition of 5CDNT despite the coexistence of possibleinterferentslike2,4-dinitrotolueneand1,3-dinitrobenzene.ThepresentMIPbasedsensorsystemcouldbe used successfully for direct determination of 5CDNT. Keywords.  5-Chloro-2,4-dinitrotoluene; combinatorial studies; molecular imprinted polymers; ft-irspectroscopy; binding assay; electrochemical impedance spectroscopy. 1.Introduction Detection of explosives, including 2,4,6-trinitrotoluene(TNT), 2,4-dinitrotoluene (DNT) and their derivativesis an important analytical problem for the preventionof terrorism activities, detection of military explosives,detection of leftover landmines and environmental haz-ards from improperly disposed explosives. The directattempt of TNT detection is difficult for laboratory re-searchers due to high explosive nature and lack of com-pound availability. So, the researchers often focus ondetecting DNT and its derivatives. The leftover TNT,DNT in landmines, environment and earth have a lotof possibilities to form a variety of derivatives withhalides. Among the derivatives, 5-chloro-2,4-dinitroto-luene (5CDNT) is one of the most common and major ∗ For correspondenceDedicated to Professor R. Ramaraj on the occasion of his 60 th birthanniversary derivatives of dinitrotoluene. 1,2 5CDNT, which is acommon impurity and derivative of DNT-based explo-sives, exhibits a higher volatility than TNT. Becauseof these facts, it has been considered that the detec-tion of 5CDNT is very important as much as DNT andTNT. 1–5 Many methods were reported in the literature for thedetection of DNT and TNT derivatives like fluorescence 6 and chemiluminescence, 3 chromatographic techniques, 7 electrochemical methods, 8–10 quartz crystal microba-lance, 11 electronic noses and sniffers. Among them,electrochemical techniques are very attractive becausethese techniques exhibitadvantages like low cost, ruggedinstrumentation, high sensitivity, high selectivity, easyportability, feasibility for automation and simplicity. 12,13 By considering these benefits, the adaptation of an electro-chemicalimpedimetrictechniquetodetectexplosivecom-pounds is considered rather simple and uncomplicated.Molecular imprinting technology 14–18 is one of the most efficient generic method which introduces  K. Yugender Goud et al. molecular recognition properties into synthetic poly-mers with the use of appropriate templates. Molecularimprinted polymers (MIPs) are used in a wide rangeof applications like chromatographic separations, solidphase extractions, catalysts and sensors. MIPs used asrecognition elements for the detection of biomarkers,pharmaceutical drugs and explosives are reported inthe literature. MIPs have good advantages comparedto other recognition elements, like low cost, long shelf life, high selectivity, sensitivity and inherent stabil-ity under drastic conditions. Preparation methods of MIP comprise the following four stages: functionalmonomer assembly, polymerization, template extrac-tion and rebinding of template.The present investigation involves the developmentof an electrochemical impedimetric sensor based onMIP for the detection of 5CDNT. For the selectionof polymer precursors, we performed combinatorialscreening procedure by using HyperChem softwareover a set of twenty monomers and five cross-linkers.MIP of 5CDNT was prepared with methylacrylate asmonomer and ethylene glycol dimethacrylate as crosslinker in the presence of 5CDNT template. Formationof MIP and non-imprinted polymer (NIP), and templateextraction from the MIP were investigated by Fouriertransform infrared spectroscopy (FT-IR). Binding assaystudieswerecarriedouttoanalysethepreferentialbind-ing of 5CDNT with MIP compared to NIP. Electro-chemical impedance of the fabricated MIP carbon pasteelectrodes was investigated for the detection of 5CDNT,and the sensitivity and selectivity of the electrode in thepresence of the homologues of compounds have beeninvestigated. 2.Experimental 2.1  Reagents 5-Chloro-2,4-dinitrotoluene (5CDNT), 2,4-dinitrotoluene(DNT), ethylene glycol dimethacrylate (EGDMA) andn-eicosane were purchased from Sigma–Aldrich (St.Louis, USA), 1,3-dinitrobenzene (DNB) from ReidelChemicals (India), methylacrylate and graphite finepowder from Loba Chemie (India) and azo-bis-iso-butyronitrile (AIBN) from Spectrochem (Mumbai,India). All other chemicals were of analytical grade andwere used as received. Phosphate buffered saline solu-tion (PBS) of pH 7.4 was prepared by using 5 M NaCl,1M KH 2 PO 4  and 1 M K 2 HPO 4  according to the SigmaAldrichprocedure.Allaqueoussolutionswerepreparedusing double distilled water finally passed through 0.22micron filter cartridge (Whatman).2.2  Combinatorial Screening Combinatorial studies were carried out following theprocedure reported elsewhere 19 for the selection of polymer precursors for MIP preparation. The param-eters were optimized by the simulation studies withMM +  and semi-empirical methods using HyperChem8.0 Professional molecular modelling system. Initially,the possible minimum energy conformations of the mo-nomers and 5CDNT were optimized using the MM + and semi-empirical (PM3) quantum methods. Then, thebinding energy between the functional monomer andthe template (5CDNT) was computed. From these com-putational results, we selected methyl acrylate (MA) asmonomer and ethylene glycol dimethacrylate (EGDMA)as cross linker for the preparation of 5CDNT imprintedpolymer.2.3  Preparation of molecular imprinted polymers 5CDNT MIP was prepared by a procedure similar toWang  et al . 20 by the polymerization of methylacry-late monomer and ethylene glycol dimethacrylate crosslinker in presence of the template 5CDNT. Methylacry-late (MA; 0.5 g), EGDMA (5.7 g) and 5CDNT (0.3 g)were dissolved in 20 mL chloroform, and AIBN (0.3 g)was added to the mixture. 21 The resultant mixture washeated for 24 h at 60–70 ◦ C. A solid polymer wasformed which was centrifuged, dried and collected inpetri dish. For the extraction of the template molecule,the polymer matrix was treated with methanol : aceticacid mixture (80:20) for 6 h by Soxhlet extractionmethod. The resultant imprinted polymer of 5CDNTwas labelled as 5CDNT-MA-MIP. For control exper-iments, a non-imprinted polymer (NIP) was preparedsimilarly, in the absence of 5CDNT, and the resultantpolymer matrix was treated with methanol:acetic acidmixture (80:20), similar to MIP. The resultant NIP waslabelled as 5CDNT-MA-NIP.2.4  Carbon paste electrode preparation Carbon paste electrodes were prepared by mixing dif-ferent weight proportions (1:1:1, 1:2:1 and 2:1:1) of MIP, graphite powder and n-eicosane, and the optimumweight proportion was chosen based on the redox activ-ity of potassium ferricyanide at the carbon paste elec-trodes. Carbon paste electrode (CPE) incorporated withMIP was prepared 22,23 by mixing the optimized combi-nation of 0.5 g MIP, 1.0 g graphite powder and 0.5 gn-eicosane. The resultant carbon paste was filled into ahollow Teflon tube of 10 mm diameter. The other endof the Teflon tube was connected to a copper wire for   MIP based Impedimetric Sensor for 5CDNT  electrical contact. The exposed electrode surface waspolished on a butter paper to get a smooth and freshsurface. For every set of experiments, a fresh electrodesurface was exposed by polishing.2.5  FT-IR, UV-Visible, BET and electrochemicalimpedance experiments The prepared MIP, NIP and template-extracted MIPpolymers were characterized by using FT-IR spec-trophotometer (Perkin Elmer, model100) employingKBr pellet method in the frequency range of 400 –4000 cm − 1 with a resolution of 2 cm − 1 . The resultswere analyzed by using Spectrum software. Bindingassay studies of MIP and NIP were carried out by usingUV-Visible spectrophotometer (Perkin Elmer Lambda25). From the difference in absorbance values, theamount of 5CDNT absorbed onto MIP or NIP wascalculated and the imprinting factors of MIP and NIPwere calculated. The specific surface area, pore volumeand pore diameter of the imprinted and non-imprintedpolymers were determined by using a MicromeriticsASAP 2020 V3.00H analyzer, and the obtained resultswere analyzed by the Brunauer-Emmett-Teller (BET)method. A sample of MIP (300 mg) was degassedat 150 ◦ C for 24 h under nitrogen flow prior to themeasurements. The dried degassed polymer was usedfor the experiment. The nitrogen adsorption/desorptionisotherms were recorded, and Barrett-Joyner-Halenda(BJH)methodwasappliedtoanalyzetheporesize,porevolume and pore diameter.Electrochemical impedance measurements were per-formed on electrochemical workstation (Model IM6e),Zahner-Eletrik GmbH, Germany. The frequency rangeof 10 mHz to 100 KHz was used, and the results wereanalyzed by using Thales 3.08 USB software. Threeelectrode system was used for impedance analysis,where the carbon paste electrode acts as working elec-trode, Pt spiral wire as counter electrode and Ag | AgCl(3 N KCl) as reference electrode. The experiments werecarried out at the open circuit potential with small exci-tation amplitude of 10 mV peak-to-peak. All the exper-iments were carried out in aqueous phosphate buffersolution (PBS) of pH 7.4. Before the experiments, theexperimental solution was purged with nitrogen gas for15 min to eliminate the dissolved oxygen. 3.ResultsandDiscussion 3.1  Computational studies Molecular dynamics simulation studies 24,25 were ex-ploited to select the best monomer and cross-linkercombination for MIP. The molecular dynamic (MD)simulations were performed at 298 K for the calculationof interaction energy of constructed molecular systems.In the first step, 2-D chemical structures of the func-tional monomers (a virtual library of 20 monomers),template, and cross linking agents were prepared usingHyperChem software. Using the molecular builderoption, the 2-D structures were converted to 3-D struc-tures. Then, geometric optimization was carried out byusing Molecular Mechanics (MM + ) and semi-empirical(SE) methods to obtain minimum energy structures. Inthis analysis, we have chosen Polak-Ribiere algorithm,which is a conjugate gradient method used specificallyfor aromatic and conjugated organic compounds.Using conformation optimization, the interactionenergy or binding score (  E) of 5CDNT–monomer– Table1.  Geometrical optimization of template-monomer cross linker interactions in vacuum by molecular mechanics andsemi-empirical method. BondEnergyBindingScore  (  E ) Template-MonomerCrosslinker  (kcal/mol)5-Chloro-2,4-DNT + Methyl acrylate DVB  − 5307.68  − 0.56 EGDMA  − 5943.86  − 3.69 MBAA  − 5234.92  − 1.21DAPZ  − 5956.44  − 1.625-Chloro-2,4 DNT + Diethylaminoethylmethacrylate DVB  − 7144.88 0.54EGDMA  − 7788.76  − 2.91MBAA  − 7882.84  − 2.05DAPZ  − 7794.72  − 1.605-Chloro-2,4 DNT + Itaconic acid DVB  − 5697.94 0.16EGDMA  − 6342.29  − 3.67MBAA  − 5627.32  − 2.63DAPZ  − 6348.02  − 2.22DVB – divinylbenzene; MBAA – N,N ′ -methylenebisacrylamide.  K. Yugender Goud et al. cross linker complexes was calculated. The  E valueswere calculated using the following equation:  E = E ( template − monomer ) − E ( template ) −  E ( monomer )  (1)  E  =  E ( template − monomer − cross linker ) − E ( template ) −  E ( monomer ) −  E ( cross linker )  (2) Figure1.  FT-IR spectra of 5CDNT-MA-MIP before (a)and after template extraction (b) and of 5CDNT-MA-NIP (c). The most stable template-monomer-cross linker com-plex was selected based on the interaction energy(table 1), and the 5CDNT template-methyl acrylate- EGDMA combination has been selected. Consequently,the MIP for 5CDNT has been synthesized accordingly.3.2  FT-IR study The infrared spectral properties of the synthesizedMIP and NIP of 5CDNT were studied by FTIR spec-trophotometry. FTIR spectra of 5CDNT MIP, NIP andtemplate-extracted MIP are shown in figure 1. Thevibrational peak of nitro group assigned to 5CDNT at ∼ 1520 cm − 1 is present in the FTIR spectrum of MIP(figure 1 (a)) along with various vibrational peaks of the MIP matrix, while that of nitro group is not presentin the FTIR spectra of both NIP and template-extractedMIP (figure 1 (b, c)). The presence or absence of thevibrational peak of nitro group, respectively, confirmedthe capture and removal of the template molecule inMIP and template-extracted MIP (figure 1 (a, b)). Thevibrational frequency of nitro group of the 5CDNT tem-plate in MIP matrix ( ∼ 1520 cm − 1 ) is slightly higherthan that of free 5CDNT molecule, indicating theexistence of substantial weak interactions between (a)    A   b  s  o  r   b      a   n  c  e Wav elen g th / nm  0.1 mM 0.075 mM 0.05 mM 0.025 mM 0.0125 mM Wav elen g th / nm  0.1 mM 0.075 mM 0.05 mM 0.025 mM 0.0125 mM    A   b  s  o  r   b      a   n  c  e (b) 2202402602 8 03003202202402602 8 03003202202402602 8 03003200. 8 (c) Wav elen g th / nm    A   b  s  o  r   b      a   n  c  e  0.1 mM 0.075 mM 0.05 mM 0.025 mM 0.0125 mM Figure2.  UV–Visible spectra of aqueous 5CDNT solution of different concentrations before (a) and after adsorption withMIP (b) or NIP (c). 036912151 8 21    B   i  n       d    i  n      g   c      a     p     a   c   i   t      y    /  m      g     g   -   1 Concentr a tion of 5CDNT / mM (a) 8 8 0.10  (b) Concentr a tion of 5CDNT / mM    I  m      p   r   i  n   t   i  n      g      F     a   c   t  o  r (n = 3) Figure3.  Plots of the binding capacity (a) and imprinting factor (b) of 5CDNT-MA-MIP against the concentration of 5CDNT.   MIP based Impedimetric Sensor for 5CDNT  encapsulated 5CDNT and the surrounding MIP matrix.The results clearly indicate that the complex betweenthe MIP matrix and the template had been formed  via reversible non-covalent interactions.3.3  Binding assay studies by UV-visible spectroscopy Binding assay studies 26–28 of MIP and NIP were carriedout by using UV-Visible spectroscopic analysis. 0.25 gof MIP or NIP was added into aq. 5CDNT solutionsof five different concentrations (0.1, 0.075, 0.05, 0.025,0.0125 mM). These mixtures were incubated for sixhours with continuous shaking at 300 rpm. UV-Visiblespectraof 5CDNT solutionswere taken before the treat-ment with MIP. After the treatment with MIP and NIPfor6h,thesolutionswerecentrifugedandUV-Visspec-tra of the supernatant solutions were recorded. From thedifference in absorbance values, the amount of 5CDNTabsorbed onto MIP or NIP was calculated. The bind-ing capacity and imprinting factor of MIP and NIP werecalculated, similar to Chang  et al . 29 Binding Capacity (mg/g)  =  analyte (mg) absorbed / polymer (g) (3) Table2.  Specific surface area, pore volume and porediameter of MIP and NIP by BET experiments.Specific Total poresurface volume Average porePolymer area (m 2  /g) (cm 3  /g) diameter (Å)5CDNT-MA-MIP 326 0.2416 405CDNT-MA-NIP 150 0.1748 51 # Percentage of error: < 1%. Imprinting factor  =  Binding capacity of MIP / Binding capacity of NIP (4)When analyzing the UV-Visible spectra (figure 2) of 5CDNT before and after absorption onto MIP and NIP,the spectra of 5CDNT solutions treated with MIP showless absorbance values compared to those treated withNIP. This observation indicates that 5CDNT moleculeinteracted with the binding sites of MIP much moreeffectively than that of NIP. The binding capacity of MIP is higher than that of NIP in a wide range of 5CDNT concentrations (figure 3). The experimentswere repeated three times and the resultant imprint-ing factor values were plotted against the concentra-tions of 5CDNT. By decreasing the concentration of 5CDNT, the imprinting factor values of MIP becomesmore prominent; this result indicates that the preparedMIP shows more specificity towards 5CDNT at lowconcentrations.3.4  BET analysis of MIP and NIP In general, an increase in the surface area and averageporevolumeofapolymermatrixindicatesthatthepoly-mer possesses higher accessibility and a better capac-ity for rebinding in its pores. 29 Specific surface area,pore volume and pore diameter of MIP and NIP weremeasured by BET nitrogen adsorption method (table 2).The specific surface area of MIP is relatively large andis nearly twice to that of NIP, and similarly the totalpore volume of MIP also is significantly higher thanthat of NIP. The average pore diameter of NIP is rela-tively higher (51.46 Å), though that of MIP (40.47 Å)is high enough for the free flow of the template 5CDNTmolecules in the MIP matrix. Overall, the observedresultsfromBETanalysisclearlyindicatedthattheMIP 1001k10k100k    I  m      p   e       d     a   n  c  e   /      Ω F req u enc y  / Hz    100 µ M 10 µ M 1 µ M 0.1 µ M 0.01 µ M Bl a nk PBS (a) 10m100m1101001k10k100k10m100m1101001k10k100k101001k10k100k  100 µ M 5CDNT Bl a nk PBS    I  m      p   e       d     a   n  c  e   /      Ω F req u enc y  / Hz (b) Figure4.  (a) Bode plots of EIS analysis of 5CDNT-MA-MIP carbon paste elec-trode and (b) 5CDNT-MA-NIP carbon paste electrode in phosphate buffer (pH 7.4) atdifferent concentrations of 5CDNT ((a) 0, 0.01, 0.1, 1, 10, 100  µ  M, (B) 0, 100  µ  M).
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