Surface-Structured Molecular Sensor for the Optical Detection of Acidity

Langmuir 2008, 24, 2963-2966

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Surface-Structured Molecular Sensor for the Optical Detection of Acidity Alberto Martı´nez-Otero,† Emilia Evangelio,‡ Ramon Alibe´s,§ Jose´ Luis Bourdelande,§ Daniel Ruiz-Molina,‡,| Fe´lix Busque´,*,§ and Jordi Hernando*,§ Institut Catala` de Nanotecnologia, Esfera UAB, 08193 Cerdanyola del Valle` s, Spain, Institut de Cie` ncia de Materials de Barcelona (CSIC), 08193 Cerdanyola del Valle` s, Spain, Departament de Quı´mica, UniVersitat Auto` noma de Barcelona, 08193 Cerdanyola del Valle` s, Spain, and Department of Chemistry, Centro de InVestigacio´ n en Nanociencia y Nanotecnologı´a, 08193 Cerdanyola del Valle` s, Spain ReceiVed December 30, 2007. In Final Form: February 6, 2008 In this letter, we report on the development of a surface molecular sensor for the detection of acidity. Lithographically controlled wetting deposition has been applied to form the nanostructure of a new fluorescent compound with three protonation states featuring different optical properties on a glass substrate. Atomic force microscopy demonstrates the functionalization of the surface with ordered arrays of the sensor molecules. The fluorescence properties of the resulting nanopattern at different pH values have been investigated by confocal fluorescene microsopy, thus revealing the fast, sensitive, reversible response of the prepared nanosensor to gas flows of varying acidity.

Fluorescent pH sensors are analytical tools of increasing interest in various scientific areas, industry, clinical analysis, and environmental protection owing to their high sensitivity and selectivity, ease of integration, and relatively low cost.1 In recent years, there has been considerable research directed toward their miniaturization down to the nanoscale. Miniaturization is an essential tool not only for the rapid and effective sensing of very small volume samples but also for the benefits of sensor integration. So far, the incorporation of optical sensors into fiber optics,2 nanoparticles,3 and micro- and nanofluidic networks4 has been reported. Covalent bonding, including attachment to membranes5 or copolymerization,6 has been the chemical route for the manipulation and integration of the dyes in most of the previously described approaches. Attachment through the formation of selfassembled monolayers has also been used.7 In spite of being successful, such grafting procedures involve several chemical steps for the modification of the indicator and/or the supporting matrix. To overcome this drawback, different approaches such as the use of a nanosensor based on a glass pipet have recently been described.8 However, the finding of a simpler means for the nanostructuration of optochemical sensors still remains a * Corresponding authors. E-mail: [email protected], jordi.hernando@ uab.es. † Institut Catala ` de Nanotecnologia. ‡ Institut de Cie ` ncia de Materials de Barcelona. § Universitat Auto ` noma de Barcelona. | Centro de Investigacio ´ n en Nanociencia y Nanotecnologı´a. (1) (a) de Silva, A. P.; de Silva, S. S. K.; Goonesekera, N. C. W.; Gunaratne, H. Q. N.; Lynch, P. L.; Nesbitt, K. R.; Patuwathavithana, S. T.; Ramyalai, N. L. D. J. Am. Chem. Soc. 2007, 129, 3050. (b) Lin, J. Trends Anal. Chem. 2000, 19, 541. (2) Vo-Dinh, T.; Kasili, P. Anal. Bioanal. Chem. 2005, 382, 918. (3) Bishnoi, S. W.; Rozell, C. J.; Levin, C. S.; Gheith, M. K.; Johnson, D. H.; Halas, N. J. Nano Lett. 2006, 6, 1687. (4) Mela, P.; Onclin, S.; Goedbloed, M. H.; Levi, S.; Garcı´a-Parajo, M. F.; van Hulst, N. F.; Ravoo, B. J.; Reinhoudt, D. N.; van den Berg, A. Lab Chip 2005, 5, 163. (5) Kooronczi, I.; Reichert, J.; Ache, H.; Krause, C.; Werner, T.; Wolfbeis, O. Sens. Actuators, B 2001, 74, 47. (6) Tan, W. H.; Shi, Z.-Y.; Smith, S.; Bimbaum, D.; Kopelman, R. Science 1992, 258, 778. (7) (a) Crego-Calama, M.; Reinhoudt, D. N. AdV. Mater. 2001, 13, 1171. (b) Basabe-Desmonts, L.; Beld, J.; Zimmerman, R. S.; Hernando, J.; Mela, P.; Garcı´aParajo, M. F.; van Hulst, N. F.; van den Berg, A.; Reinhoudt, D. N.; CregoCalama, M. J. Am. Chem. Soc. 2004, 126, 7293. (c) Rudzinski, C. M.; Young, A. M.; Nocera, D. G. J. Am. Chem. Soc. 2002, 124, 1723.

challenge.9 As an alternative approach, herein we report on the use of the well-known lithographically controlled wetting (LCW) technique, which is a stamp-assisted methodology allowing for controlled surface nanopatterning with resolution on the order of hundreds of nanometers and feature sizes on the order of tens of nanometers.10 LCW nanostructuration of molecular semiconductors,10 single-molecule magnets,11 DNA molecules12 and organic field-effect transistors13 have so far been reported, thus revealing the versatility of this deposition technique. In this work, we apply LCW to demonstrate the concept of ordered molecular arrays on a surface as a pH sensor. With this aim, a new fluorescent compound with three protonation states featuring different optical properties has been prepared, and its acidity-sensing capabilities after deposition onto a glass substrate by LCW have been investigated. As a molecule of choice, we have synthesized new compound 1 starting from commercially available 5-bromovanillin (Supporting Information). The interest in this molecule is twofold. First, pyridine and 6-bromocatechol groups exhibit distinct acidbase activity at different pH intervals (pKa ) 5.2 for the pyridinium ion in water14 and pKa ) 9.2 and 13.0 for pure catechol in water15). This allows for the reversible interconversion of compound 1 between three different protonation states: the cationic (c1), neutral (n1), and anionic (a1) forms (Scheme 1).16 Second, π-electron delocalization along these two groups leads to fluorescence emission in the visible region of the spectrum with acid-base sensitivity. Figure 1 shows the absorption and fluorescence emission spectra obtained for c1, n1, and a1 in acetonitrile. The three states possess different absorption and (8) Piper, D. J.; Clarke, R. W.; Korchev, Y. E.; Ying, L.; Klenerman, D. J. Am. Chem. Soc. 2006, 128, 16462. (9) Wolfbeis, O. S. J. Mater. Chem. 2005, 15, 2657. (10) Cavallini, M.; Biscarini, F. Nano Lett. 2003, 3, 1269. (11) Cavallini, M.; Biscarini, F.; Gomez-Segura, J.; Ruiz, D.; Veciana, J. Nano Lett. 2003, 3, 1527. (12) Bystrenova, E.; Facchini, M.; Cavallini, M.; Cacace, M. G.; Biscarini, F. Angew. Chem., Int. Ed. 2006, 43, 4779. (13) Cavallini, M.; Stoliar, P.; Moulin, J. F.; Surin, M.; Lecle`re, P.; Lazzaroni, R.; Breiby, D. W.; Andreasen, J. W.; Nielsen, M. M.; Sonar, P.; Grimsdale, A. C.; Mu¨llen, K.; Biscarini, F. Nano Lett. 2005, 5, 2422. (14) Espinosa, S.; Bosch, E.; Rose´s, M. J. Chromatogr., A 2002, 964, 55. (15) Motekaitis, R. J.; Martell, A. E. Inorg. Chem. 1984, 23, 18. (16) Further deprotonation of a1 yields a chemically unstable double anionic form, which is most likely due to a polymerization process. This fact prevents the occurrence of a fourth state capable of sensing very high pH values.

10.1021/la704072z CCC: $40.75 © 2008 American Chemical Society Published on Web 03/04/2008

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Scheme 1. Molecular Structures of the Cationic (c1), Neutral (n1), and Anionic (a1) States of 1

emission properties, making compound 1 a suitable reversible fluorescence sensor to operate over a wide range of pH values. The nanostructuration of compound 1 onto a glass substrate in the form of parallel stripes has been achieved by means of the LCW technique. With this aim, a solution of a1 in acetonitrile (ca1 ) 1 × 10-4 M) was drop-casted onto an ∼120-µm-thick glass coverslide, and a stamp with an imprinted pattern of parallel lines (350 nm wide and 1.4 µm pitch) was immediately placed on top. Once the stamp was detached, a molecular positive replica of the imprinted pattern on the surface was obtained, as confirmed by atomic force microscopy (AFM) and confocal fluorescence microscopy. AFM images show the formation of stripes onto the substrate consisting of droplets aligned along the stretching direction (see Figure 2a). An average height of 9 ( 3 nm, width of 383 ( 59 nm, and distance between lines of 1005 ( 46 nm have been retrieved from our AFM measurements for the imprinted nanopattern. Such patterning is effective across at least a 50 × 50 µm2 area over different regions of the substrate, confirming the possibility of long-range nanostructuration (Supporting Information). In contrast to what has been observed upon the nanopatterning of 1 onto graphite, AFM images do not reveal clear crystalline features for the nanostructures of a1

Figure 1. Absorption and fluorescence (normalized to the absorption at λexc ) 310 nm) spectra of c1, n1, and a1 in acetonitrile (c1 ) 1 × 10-5 M). Fluorescence quantum yields of these species are Φc1 ) 0.07, Φn1 ) 0.05, and Φa1 ) 0.22. To ensure full conversion to the desired state of 1, the pH of the medium was varied by the addition of acid or base: pH 8.5 (c1), 14.5 (n1), and 24.0 (a1). These pH values are provided as sspH values, which have been measured in acetonitrile with pH standarization in the same solvent (Supporting Information).

Figure 2. (a) Topographic AFM image of a 10 × 10 µm2 area of a sample prepared by LCW deposition of a1 onto a glass coverslide from an acetonitrile solution (ca1 ) 1 × 10-4 M). (b) Fluorescence image of a 10 × 10 µm2 area of the same sample (λexc ) 532 nm, power density ≈ 0.25 kW cm-2, pixel rate ) 1 kHz).

prepared on glass substrates. Together with the fast response of the resulting nanopattern to acidity changes (see below), this suggests that the structure of the deposited droplets of 1 is amorphous. Importantly, fluorescence images of the samples prepared by LCW demonstrate that the imprinted nanostripes do fluoresce, thus preserving the emissive character found for compound 1 in solution (Figure 2b). However, wider stripes are observed in fluorescence (fwhm ≈ 850 nm) with respect to AFM measurements. This is due to the lower lateral resolution of confocal fluorescence microscopy, which causes the dimensions of the features observed to be a convolution of their actual size with that of the diffraction-limited spot of the excitation light (∼250 nm under our experimental conditions). To explore the sensing capabilities of 1 on the nanoscale in view of future integration into opto-chemical devices, the fluorescence response of the prepared nanostructured surfaces upon exposure to atmospheres of different acidity has been studied. The experimental conditions applied are such that photodegradation of 1 is minimized and only emission from the c1 and, primarily, a1 states of the sensor is detected by using λexc ) 532 nm and λdet > 550 nm. Thus, we have observed that chromophore photobleaching under our excitation conditions occurs only for long irradiation times (on the order of tens of seconds), which are orders of magnitude larger than those required to acquire the fluorescence images in Figures 2 and 3 (illumination time per pixel ) 1 ms). We ascribe this behavior to the low absorption of 1 at λexc ) 532 nm (e.g., a1,532 ) 50 L mol-1 cm-1 in acetonitrile). However, we have also observed that the optical

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Figure 3. (a-d) Consecutive fluorescence images of a 14 × 14 µm2 area of a sample prepared by LCW deposition of a1 onto a glass coverslide from an acetonitrile solution (ca1 ) 1 × 10-4 M): (a) initially, (b) after exposure to an acidic gas flow (HCl/N2), (c) after exposure to a basic gas flow (triethylamine/N2), and (d) after exposure to air. All four images were obtained with the same instrument settings (λexc ) 532 nm, power density ≈ 0.25 kW cm-2, pixel rate ) 1 kHz). (e) Mean intensity cross sections of the fluorescence images. The profiles plotted are obtained by averaging the signal within the region confined inside the white rectangle in image a.

spectra of 1 broaden when deposited on glass (Supporting Information). Accordingly, larger absorptivity values at λexc ) 532 are expected for the nanostructured molecules with respect to solution, thus allowing for fluorescence detection from the nanopatterned surface under such excitation conditions. Figure 3a-d shows consecutive fluorescence images on the same area of an imprinted nanopattern of 1 that has been sequentially exposed to atmospheres of different acidity. Figure 3e plots a detailed comparison of the fluorescence intensity profiles retrieved from such images. Clear, measurable changes in the emission arising from microscopic areas of the sample occur upon varying the acidity of the environment. This demonstrates that protonation and deprotonation processes also affect the photophysical properties of 1 in the solid state. Figure 3a depicts the initial fluorescence image registered for a freshly nanostructured surface equilibrated in air (pH ∼7 on the aqueous scale).17 A fluorescence intensity rate of ∼35 kcount s-1 is measured in this case. This emission must stem from the a1 state, which is the predominant species in the original acetonitrile solution used for the deposition of 1. When the patterned surface is exposed to an acid atmosphere (HCl/N2 gas flow), a noteworthy decrease in the fluorescence emission is detected (Figure 3b). This arises from the protonation of a1 subsequently to yield n1 and c1, which are species that appear to be nonfluorescent or only slightly fluorescent, respectively, under our experimental conditions. The remaining emission intensity of ∼20 kcount s-1 after prolonged exposure to the acid atmosphere is therefore ascribed to the complete interconversion of a1 to c1. A pH value of ∼1 (on the aqueous scale) has been measured upon exposure of a pH indicator to the same environment. Therefore, we indeed expect such complete conversion to the c1 state to occur under our experimental conditions because the pKa of the pyridinium group in water is ∼5.14 In a subsequent step, the acid-base equilibrium is shifted back (i.e., c1 is interconverted to a1) after exposure to a basic atmosphere (triethylamine/N2 gas flow). A pH value of ∼12 (on (17) In contrast to the measurements in solution, the pH values of the different atmospheres to which the nanopatterned surface was exposed are given on the aqueous scale (i.e., measured in water with standardization in the same solvent).

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the aqueous scale) is retrieved for such an environment by means of a pH indicator. Because the first pKa of catechol in water is ∼9,15 full recovery of the a1 species must therefore occur. As expected, this process is accompanied by a large increase in emission (Figure 3c). Noticeably, a fluorescence rate of ∼70 kcount s-1 is measured in this case, which represents a 2-fold rise in the emission intensity with respect to that initially found in the freshly nanostructured surface (∼35 kcount s-1). The lower emission of the fresh surface is ascribed to the partial conversion of a1 to nonfluorescent n1 molecules as a result of its interaction with protonated sites on the glass surface18 and/or partial evaporation of the neutral form of the countercation (ammonia) after equilibration in air (pH ∼7 on the aqueous scale). In fact, by assuming a pKa value of ∼9 for the n1-a1 pair in water, we expect the corresponding acid-base equilibrium to be predominantly shifted to the neutral form at pH ∼7. This fact is confirmed once the triethylamine/N2 gas flow is stopped and the sample is allowed to equilibrate in air. The partial evaporation of triethylamine molecules leads to a decrease in the fluorescence intensity down to the original value of ∼35 kcount s-1 (Figure 3d). Importantly, Figure 3d closely resembles the fluorescence image registered at zero time (Figure 3a; freshly nanostructured surface), evidencing the reversibility of the sensing process and the fair robustness of the nanostructured surface upon exposure to successive acidic and basic atmospheres. Indeed, an AFM inspection of the patterned nanostrucutures demonstrates that their morphology does not suffer significant changes upon a few complete acidification/basification cycles. However, care has to be taken when larger exposures to acidic and basic gas flows are performed. In that case, progressive structural changes could affect the imprinted nanopattern, which might be ascribed to both dewetting of the sensor molecules and the deposition of ammonium salts arising from succesive acidic (HCl) and basic (triethylamine, NEt3) gas flows. Together with robustness and reversibility, future application of LCW surface nanopatterning to sensor miniaturization requires the resulting devices to show optimal properties in terms of response time and sensitivity. In the case of nanometer-sized stripes of 1 imprinted by LCW in this work, the fluorescence changes observed upon variation of the surrounding atmosphere are instantaneous and constant. In fact, they are found to occur faster than the time required to change the acidity of the surrounding atmosphere (less than 1 to 2 s). Therefore, we conclude that LCW deposition allows for the preparation of sensing platforms with response times as fast as those achieved in solution and by means of other sensor miniaturization techniques.2,4,8 Because the concentrations of acid and base used in our experiments are orders of magnitude larger than the number of deposited sensor molecules, this reveals a very fast diffusion of the gas through the LCW-patterned nanostructures and points toward the absence of crystallinity in the ordered arrays of sensor molecules prepared. Conversely, we have observed much slower sensing times (on the order of tens of seconds) for micrometersized polycrystalline particles of either c1 or a1 placed directly onto glass coverslides. Even more importantly, sensor nanostructuration by means of LCW also leads to large sensitivities due to the contrast between functionalized and nonfunctionalized areas. This behavior can be uncovered by comparing the fluorescence response of the LCW nanopatterned surface with that from a continuous thin film prepared by drop-casting from an acetonitrile solution of (18) (a) Zang, L.; Liu, R.; Holman, M. W.; Nguyen, K. T.; Adams, D. M. J. Am. Chem. Soc. 2002, 124, 10640. (b) Al-Kaysi, R. O.; Bourdelande, J. L.; Gallardo, I.; Guirado, G.; Hernando, J. Chem.sEur. J. 2007, 13, 7066.

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a1 (ca1 ) 1 × 10-4 M). Thus, when switching from an acidic (HCl/N2 gas flow, pH ∼1 on the aqueous scale) to a basic (triethylamine/N2 gas flow, pH ∼12 on the aqueous scale) atmosphere, a fluorescence intensity increase (Fbasic/Facid) equal to 6.0 is determined for the LCW nanometer-sized imprinted stripes by confocal fluorescence microscopy. To retrieve the Fbasic/Facid value, the average emission signal measured for the fluorescent stripes has been corrected for the background signal detected for the nonfunctionalized areas of the surface. Importantly, such a correction procedure cannot be applied to continuous thin film samples. This leads to the determination of significantly smaller changes in emission intensity (Fbasic/Facid ) 4.1) when the same measurement conditions are applied to thin films of 1 on glass, thus clearly demonstrating the larger pH sensitivity provided by the LCW-imprinted sensor. In fact, by exploiting the simultaneous detection of functionalized and nonfunctionalized areas, small nanopatterned domains of 1 composed of only ∼10-50 molecules could ultimately be exploited as pH nanosensors according to the photochemical properties of this species in solution. A large change in the fluorescence emission arising from the nanostructured sensor has also been observed when switching from a neutral (pH ∼7 on the aqueous scale) to a basic atmosphere (pH ∼12 on the aqueous scale): Fbasic/Fneutral ) 3.1. Because the pKa value of catechol in water is ∼9,15 this pH interval covers the acid-base equilibrium between the n1 and a1 species, which are nonfluorescent (off) and fluorescent (on) under our experimental conditions, respectively. Assuming a typical value of ∼2 pH units for the sensing dynamic range of the off-on n1-a1 pair,1a our measurements indicate that the prepared nanopatterned surface yields a very sensitive response to small pH changes within the pKa ( 1 interval of this system (pH ∼8-10). Thus, taking the fluorescence intensities measured at the edges of this pH range (pH ∼7 and 12), we determine that the nanopatterned sensing surface undergoes changes in fluorescence intensity of ∼100% per pH unit relative to the emission at pH ∼7. In principle, similar sensitivities could also be obtained for the pH range covered by the c1-n1 pair (pH ∼4-6 on the aqueous scale) by properly choosing the excitation/detection conditions so that the system behaves as a fluorescent off-on sensor (for instance, by selectively and efficiently detecting one of these two states). Therefore, because of the capability of the sensor molecules developed in this work to switch between different states with distinct fluorescence properties, the use of sophisticated experimental schemes such as dual-wavelength detection could be

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envisaged in order to attain specific pH sensing over large pH ranges (pH ∼4-10 on the aqueous scale). Whereas the response time and sensitivity of the surface nanopatterned pH sensor developed in this work are similar to those reached by other miniaturization approaches,2,4,8 several particular features arise from the use of LCW nanostructuration. Similarly to nanosensors based on glass pipettes,8 LCW deposition does not involve the covalent bonding of the active molecules to a substrate, which provides a simple, rapid methodology for sensor miniaturization that, more interestingly, can be reproduced on several surfaces of technological or industrial interest. In case of LCW deposition, however, this prevents the use of the resulting patterned platform under liquid environments. Nevertheless, the LCW nanostructuration approach is not restricted to charged molecules but can be applied to any type of sensor systems, in contrast to the methodology reported in ref 8. Moreover, it allows fast sensing of gas samples, which is a behavior that is scarcely reported for fluorescent sensors.19 In conclusion, a new fluorescent compound with acidity sensing capability and its deposition onto a glass substrate by the LCW technique have been presented. Confocal fluorescence microscopy demonstrates that the emission arising from the resulting nanostructured surface can be modulated by gas flows of different acidities, which can therefore be envisaged as a potential fluorescent pH sensor. Interestingly, sensor patterning leads to larger sensitivity to pH variations with respect to thin film sensing devices. Together with the reproducibility and fast response times encountered, this demonstrates the potential of the LCW deposition approach to attain sensor miniaturization. Acknowledgment. We acknowledge the financial support of the Ministerio de Educacio´n y Ciencia (MEC) through projects MAT2006-13765-C02-01, CTQ2004-02539, and CTQ200601040. F.B. and J.H. thank the MCyT-ERDF for their Ramon y Cajal contracts, and A.M.-O. and E.E. thank the Generalitat de Catalunya and MEC for their predoctoral grants, respectively. Supporting Information Available: Detailed experimental procedures on the synthesis, spectroscopic characterization in solution, and topographic and spectroscopic characterization of the nanopattern of 1. This material is available free of charge via the Internet at http://pubs.acs.org. LA704072Z (19) Nastasi, F.; Puntoriero, F.; Palmeri, N.; Cavallaro, S.; Campagna, S.; Lanza, S. Chem. Commun. 2007, 4697.