Letter pubs.acs.org/acssensors
Chemosensor for Selective Determination of 2,4,6-Trinitrophenol Using a Custom Designed Imprinted Polymer Recognition Unit Cross-Linked to a Fluorophore Transducer Tan-Phat Huynh,*,†,# Agnieszka Wojnarowicz,†,# Anna Kelm,† Piotr Woznicki,† Pawel Borowicz,† Alina Majka,† Francis D’Souza,*,‡ and Wlodzimierz Kutner*,†,§ †
Department of Physical Chemistry of Supramolecular Complexes, Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland ‡ Department of Chemistry, University of North Texas, Denton, Texas 76203-5017, United States § Faculty of Mathematics and Natural Sciences, School of Sciences, Cardinal Stefan Wyszynski University in Warsaw, Wóycickiego 1/3, 01-815 Warsaw, Poland S Supporting Information *
ABSTRACT: A 3-D molecularly imprinted polymer (MIP) film comprising a unit for recognition of 2,4,6-trinitrophenol (TNP) embedded with a fluorophore for signal transduction and quantification is newly fabricated and shown to be selective and sensitive to the target TNP analyte in solution. The limit of detection of this chemosensor reached a level of subnanogram per liter of TNP concentration. Moreover, this MIP film was fabricated by just one-step electropolymerization from a prepolymerization solution; therefore, the procedure is readily extendable for selective determination of other nitroaromatic explosives.
KEYWORDS: molecularly imprinted polymer, chemosensor, 2,4,6-trinitrophenol, fluorescence, electropolymerization molecule.15,16 Therefore, MIP materials are being continuously exploited in applications ranging from building chemosensors, drug delivery systems, and separation methods.17 Herein, a derivatized thiophene based MIP was devised for TNP chemosensing. Advantageously, this MIP was easy to prepare by electropolymerization18 revealing appreciable conductivity. The present study manifests a useful property of this MIP material, i.e., fluorescence, which was exploited as an extremely sensitive signal transduction platform. This new fluorescence MIP-based chemosensor was successfully used for TNP determination in solution at trace levels with high selectivity and reproducibility. For MIP preparation, the bis(2,2′-bithienyl)-(4aminophenyl)methane (NH2-S4)19 functional monomer and 3,3′-bis[2,2′-bis(2,2′-bithiophene-5-yl)]thianaphthene crosslinking monomer (CLM)20,21 (Scheme 1 and Appendix 1S) were employed. This MIP served as both the TNP recognition unit and transducer of the fluorescence signal. For TNP recognition, we have previously demonstrated the NH2-S4 functional monomer to be a successful candidate.19
2,4,6-Trinitrophenol (TNP), known as picric acid, is an explosive and highly toxic substance.1 Apart from its detection in battlefields, its ingestion can cause severe poisoning in humans, including headache, nausea, vomiting, abdominal pain, and destruction of erythrocytes. The lethal dose (oral) in rabbits is 120 mg/kg.2 Therefore, chemical sensors for selective TNP determination are in high demand. There are several ways to detect TNP at trace levels including voltammetric, HPLC chromatographic, gravimetric, and spectroscopic techniques.3 Among the latter, optical techniques based on fluorescence spectroscopy have become among the most convenient ways of TNP determination.4−11 This is because of a wide variety of available fluorophores, relatively simple instrumentation needs, abundant chemical information, and, prevailingly, high sensing detectability. The importance of supramolecular materials, in part, is attributed to fluorophores forming noncovalent complexes, which upon molecular engineering of the fluorophores could directly be used in building sensitive and selective chemosensors.12−14 Among supramolecular materials, molecularly imprinted polymers (MIPs) provide enhanced detectability and selectivity because of complementarity in their molecularly imprinted cavity size, shape, and orientation of recognition sites to those of the binding sites of the template or analyte © XXXX American Chemical Society
Received: January 26, 2016 Accepted: April 11, 2016
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DOI: 10.1021/acssensors.6b00055 ACS Sens. XXXX, XXX, XXX−XXX
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ACS Sensors Scheme 1. Structural Formulas of the Bis(2,2′-bithienyl)-(4aminophenyl)methane (NH2-S4) Functional Monomer, 3,3′-Bis[2,2′-bithiophene-5-yl]thianaphthene (CLM) CrossLinking Monomer, and 2,4,6-Trinitrophenol (TNP) Analyte
Moreover, this monomer is a fluorophore. However, its applicability for fluorescence sensing is limited because of its rather short wavelength (∼430 nm) of emission. Importantly, a polymer should be fluorescent and reusable for sensing applications. Therefore, the CLM indicated above was chosen herein to simultaneously serve as the fluorophore core because of its ∼505 nm visible-range emission in toluene (Figure 1S, Supporting Information). Surprisingly, results of fluorescence titration of CLM with TNP (Figure 2S) demonstrated that this complex was stable with the stability constant, K1 ≈ 5000 M−1, at the 1:1 mol ratio of TNP:CLM.22 This value is higher than that of the TNP-(NH2-S4) complex (K1 = 3000 M−1) reported earlier.19 Presumably, both CLM and NH2-S4 contribute to TNP complexation via a combination of π−π stacking, charge transfer, and hydrogen bonding interactions. For that, first, fluorescence of the CLM polymer film alone was measured. This film was deposited on the indium-tin oxide (ITO) glass slide electrode by potentiodynamic electropolymerization from the 0.6 mM CLM and 0.1 M (TBA)ClO4 mixed solvent solution of the acetonitrile-to-toluene volume ratio of 95:5. The role of toluene was to dissolve CLM completely. The anodic peak of CLM electro-oxidation appeared at 1.10 V (Figure 3Sa). When excited at 350 nm, broad emission covering the 500−750 nm range with peak maxima in the 600 nm region was observed (Figure 3Sb). The red-shifted emission compared to that of the monomer is mainly attributed to extended conjugation as a result of polymerization of CLM. Next, a fluorescence MIP-TNP chemosensor was prepared for TNP determination. For that, an MIP-TNP film was deposited on the ITO glass slide by potentiodynamic electropolymerization from the 0.3 mM TNP, 0.3 mM NH2S4, 0.6 mM CLM, and 0.1 M (TBA)ClO4 solution of the acetonitrile-to-toluene volume ratio of 95:5. A relatively high CLM concentration afforded extensive cross-linking of the functional monomer molecules to form a rigid 3-D polymer matrix as well as to provide fluorescence (see above). The anodic peak of bis(2,2′-bithienyl)methane electro-oxidation appeared at ∼0.95 V in the first potential cycle (curve 1 in Figure 1a).23 This peak increased in two subsequent cycles, most likely because surface area of the polymer film increased during electropolymerization as the surface roughness increased (see below). As a result, the prepolymerization complex formed in solution was transferred to the MIP-TNP films (Scheme 1S) deposited on the ITO electrodes. This successful complex transfer was confirmed by the polarization-modulation infrared reflection−absorption spectroscopy (PM-IRRAS) measurements. For that, the spectrum of the TNP drop-coated film was used as a control with respect to its intense stretching vibrational bands related to the
Figure 1. (a) Potentiodynamic curves recorded on ITO glass slides for the 0.3 mM TNP, 0.3 mM NH2-S4, 0.6 mM CLM, and 0.1 M (TBA)ClO4 solution of the acetonitrile-to-toluene volume ratio of 95:5, during 3 potential cycles; cycle numbers are indicated at curves. The potential scan rate was 20 mV/s. Inset in (a): atomic force microscopy (AFM) image of the MIP-TNP film deposited on the ITO glass slide of the (0.5 × 0.5) μm2 surface area. The depth-color scale of the image is from −338 to 252 nm. (b) Normalized fluorescence spectrum of the MIP-TNP film deposited on the ITO glass slide by potentiodynamic electropolymerization. The excitation wavelength was 350 nm. (c) Differential pulse voltammograms, and (d) electrochemical impedance spectra for 1 mM ferrocene in the 0.1 M (TBA)ClO4 solution of acetonitrile, recorded at the MIP-TNP film coated 1-mm-diameter Pt disk electrode (1′ and 1″) before and (2′ and 2″) after TNP extraction, and then (3′ and 3″) after immersing the Pt/(MIP-TNP) electrode in 1 mL of 50 nM TNP, in acetonitrile, for 20 min under magnetic stirring conditions.
presence of the −NO2 group at 875, 1157, 1248, 1365, and 1428 cm−1 of TNP (Figure 4Sa). Expectedly, the −NO2 bands at 876, 1158, 1178, 1202, 1227, 1377, and 1431 cm−1 were also present in the spectrum of the MIP-TNP film (Figure 4Sb). The assigned calculated normal modes are indicated with vertical arrows in the spectra shown in Figure 4S. Topography of the MIP-TNP film was examined by the atomic force microscopy (AFM) imaging (Figure 1a, inset). Apparently, the TNP-templated MIP-TNP film deposited on the ITO glass slide was composed of ∼10-nm-diameter distinguishable grains. This film was inhomogeneous; the determined film roughness was high, equaling 14.4(±0.9) nm.24 At 350 nm excitation, three fluorescence bands were observed for the MIP-TNP film coated ITO glass slide (Figure 1b). Predictably, the first short-wavelength (∼420 nm) weak band confirmed the presence of nonpolymerized monomers in the film.19,24 The intermediate (∼600 nm) most intense band was assigned to the MIP-TNP polymer film. Additionally, a long-wavelength (∼710 nm) weak band also appeared. This band was seen only for the MIP-TNP film deposited on the ITO glass slide (Figure 2b) but was absent when a Au substrate was used (data not shown) instead. Presumably, this band was due to heterogeneous electropolymerization governed by the ITO substrate, which caused formation of another polymer phase of higher conjugation.25 Quantum yield of the MIP-TNP polymer film obtained from the integrating sphere measurements using two different methods (Appendix 1S) was 3.1 × 10−3 (with estimated maximum error of 50%). B
DOI: 10.1021/acssensors.6b00055 ACS Sens. XXXX, XXX, XXX−XXX
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For constructing calibration plots, relative fluorescence, ΔI = I/I0, was used where I0 and I are the fluorescence intensity of the MIP-TNP film before and after wetting, respectively, with a drop of the acetonitrile solution of TNP. The linear dynamic concentration range of fluorescence at 600 nm extended from at least 0.2 to 20.4 ng L−1 (curve 1 in Figure 2b) obeying the linear regression equation of ΔI = 0.973 ± (0.030) + 0.060(±0.003) cTNP /(ng L−1). The sensitivity and correlation coefficient was 60(±3) × 10−3 ng−1 L and 0.98, respectively. At the signal-to-noise ratio of 3, the limit of detection was appreciable, equaling 0.2 ng L−1. To confirm the imprinting, a nonimprinted polymer (NIP, a polymer without imprinted molecular cavities) film was used for the TNP determination in a control experiment (curve 2 in Figure 2b). In view of no imprinted cavities, the TNP binding by NIP was much weaker than that by the TNP-extracted MIPTNP, as manifested by only minor increase of relative fluorescence with the TNP concentration increase in solution. By calculating the ratio of slopes of the calibration curves for the MIP-TNP and NIP film, we arrived to the apparent imprinting factor, which appreciably amounted ∼6.9. Moreover, the present chemosensor was selective with respect to the most typical interferences, i.e., 2,4,6-trinitrotoluene (TNT), and phenol. That is, the sensitivity to TNP was five times that to TNT, 12(±1) × 10−3 ng−1 L (curve 3 in Figure 2b) and six times that to phenol, 10(±1) × 10−3 ng−1 L (curve 4 in Figure 2b). For phenol, the fluorescence of the MIP film was quenched after deposition the first drop of the phenol solution, and then saturated with subsequent drops.
Figure 2. (a) Steady-state fluorescence spectra of the TNP-extracted MIP-TNP film, deposited on the ITO-coated glass slide, for different TNP titrant concentrations; excitation at 350 nm. The TNP concentration was increased stepwise in the range of 0.46 to 22.45 ng L−1, as indicated with the vertical arrow. (b) The calibration plots of relative fluorescence at the peak maxima for (1) TNP, (3) TNT, and (4) phenol constructed for measurements under the drop-wetting conditions as well as for (2) TNP and the NIP film used as a control.
This useful fluorescence property of the MIP film was then exploited for transduction of the TNP recognition signal. For that, first, the TNP template was extracted from the TNPtemplated MIP-TNP film with methanol for 4 h under magnetic stirring conditions.19 The differential pulse voltammetry (DPV) measurements served for indirect tracing the presence of the TNP residue in the MIP-TNP film after TNP template extraction. For such a tracing, a ferrocene redox probe is used.23 In our DPV experiments, the TNP molecules occupied imprinted cavities of the MIP-TNP film apparently affording conductivity manifested by a pronounced ferrocene DPV peak at 0.30 V vs Ag/AgCl (curve 1′ in Figure 1c). After extraction of the TNP template, however, this peak vanished (curve 2′ in Figure 1c). This was because the imprinted cavities were then emptied and the film conductivity decreased, as evidenced by a huge semicircle in the electrochemical impedance spectroscopy (EIS) (curve 2″ in Figure 1d) corresponding to the ferrocene charge transfer resistance. Then, the TNP-extracted MIP-TNP film was immersed in 50 nM TNP for 20 min under magnetic stirring conditions. As a result, the DPV peak increased (curve 3′ in Figure 1c) because the TNP molecules entered the MIP cavities and increased its conductivity, as evidenced by the decrease of the semicircle in the EIS spectrum (curve 3″ in Figure 1d). For analytical measurements, modulation of fluorescence intensity, after consecutive film wetting with drops of the acetonitrile solutions of TNP of different concentrations, was monitored. As shown in Figure 2a, this experiment revealed the emergence of a new fluorescence peak at ∼670 nm whose intensity increased with the increase of the TNP concentration. Generally, interaction of nitroaromatic compounds with fluorophores in solution results in fluorescence quenching mainly due to the occurrence of photoinduced electron transfer.25 Additionally, although MIP film swelling26 and, in few instances, aggregation27 could increase the fluorescence, the significantly red-shifted peak appearing here suggests its origin to be different from emission of the film in the absence of TNP. It is likely that charge transfer (CT) type interactions between TNP and CLM within the polymer film could be responsible for this behavior.25 If that was the case, the moderately rigid MIP film could facilitate CT type interactions resulting in CT emission that was not possible to observe for the complex in solution (Figure 2Sa) because of dissociation of this complex (as a result of its moderate stability). Further studies are in progress to unravel the mechanistic details of this phenomenon in our laboratories.
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CONCLUSION As a proof of concept, the MIP-TNP film is demonstrated to be a promising chemosensor material due to its emission in the visible range and high TNP detectability (at the subnanogram per liter TNP concentration). Importantly, the CLM played a triple role of the functional and cross-linking monomer as well as the fluorophore in this chemosensor. The CLM cross-linked the functional monomer to form 3-D MIP cavities while its fluorescent property allowed signal transduction and quantification. The presently developed approach of TNP chemosensing can readily be extended for selective determination of other nitroaromatic explosives; this research is currently underway.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00055. Instrumentation, procedures, polymerization as well as fluorescence of CLM and FTIR (PDF)
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AUTHOR INFORMATION
Corresponding Authors
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[email protected]. Author Contributions #
These authors equally contributed to this work.
Notes
The authors declare no competing financial interest. C
DOI: 10.1021/acssensors.6b00055 ACS Sens. XXXX, XXX, XXX−XXX
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ACKNOWLEDGMENTS We thank Dr. Marta Sosnowska and Prof. Tiziana Benincori for syntheses of NH2-S4 and CLM, respectively. The present research was financially supported by the European Regional Development Fund through Project ERDF (Grant No. POIG.01.01.02-00-008/08 2007-2013 to W.K.), the European Union 7.FP (Grant No. REGPOT-CT-2011-285949-NOBLESSE to W.K.), the Foundation for Polish Science (MPD/ 2009/1/styp19 to T.P.H.), the National Science Center of Poland (Grant No. 2014/15/B/NZ7/01011 to W.K.), and the US National Science Foundation (Grant No. 1401188 to F.D.). Access to the AFM instrumentation was funded by the Foundation for Polish Science under the FOCUS Program No. FG 3/2010 Grant.
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