Thin Film Optical Sensors Employing Polyelectrolyte Assembly

subsequently assembled with a polycation employing electrostatic layer-by-layer ... deficient analytes such as ferric ions, mercury, and 2,4-dinitroto...
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Thin Film Optical Sensors Employing Polyelectrolyte Assembly Soo-Hyoung Lee,† J. Kumar,‡ and S. K. Tripathy*,† Center for Advanced Materials, Department of Chemistry and Physics, University of MassachusettssLowell, Lowell, Massachusetts 01854 Received August 17, 2000. In Final Form: October 6, 2000 This work describes the development of thin film optical sensors for pH, metal ions (ferric and mercury), and 2,4-dinitrotoluene detection. To fabricate the pH sensor, a fluorescent molecule, 1-hydroxypyren3,6,8-trisulfonate, was assembled with a polycation by an electrostatic layer-by-layer assembly technique. The fluorescent indicator molecule exhibits distinct and well-defined emission peaks for protonated and deprotonated forms. The relative peak positions and intensity of fluorescence of the protonated and deprotonated forms change in response to pH variations. For metal ion (ferric and mercury) and 2,4dinitrotoluene sensing, the indicator molecules were covalently incorporated into poly(acrylic acid) and subsequently assembled with a polycation employing electrostatic layer-by-layer assembly. The sensor is based on the fluorescence quenching of indicator molecules by electron transfer from indicator to electrondeficient analytes such as ferric ions, mercury, and 2,4-dinitrotoluene. Fluorescence intensities decreased with increasing concentration of analytes. Quenching behavior follows Stern-Volmer bimolecular quenching kinetics. Linear increase in absorbance, film thickness, and emission intensity was observed as a function of number of bilayers deposited in all these films.

Introduction Optical-sensing techniques have recently attracted considerable attention and have been widely used for quantitative measurements of various analytes such as H+,1-4 carbon dioxide,5-7 oxygen,8-12 metal ions,13-15 ammonia,16 amine,17 urea,18 glucose,19-21 humidity,22 and penicillin 23,24 in environmental, industrial, clinical, medical, and biological applications. Optical sensors offer many † ‡

Department of Chemistry. Department of Physics.

(1) Lin, H.-J.; Szmacinski, H.; Lakowicz, J. R. Anal. Biochem. 1999, 269, 162. (2) Nivens, D. A.; Zhang, Y.; Angel, S. M. Anal. Chim. Acta 1998, 376, 235. (3) Munkholm, C.; Rarkinson, D.-R.; Walt, D. R. J. Am. Chem. Soc. 1990, 112, 2608. (4) Offenbacher, H.; Woflbeis, O. S.; Furlinger, E. Sens. Actuators 1986, 9, 73. (5) Tabacco, M. B.; Uttamlal, M.; McAllister, M.; Walt, D. R. Anal. Chem. 1999, 71, 154. (6) Marazuela, M. D.; Moreno-Bondi, M. C.; Orellana, G. Appl. Spectrosc. 1998, 52, 1314. (7) He, X.; Rechnitz, G. A. Anal. Chem. 1995, 67, 2264. (8) Choi, M. M. F.; Xiao, D. Anal. Chim. Acta 1999, 387, 197. (9) Gouin, J. F.; Baros, F.; Birot, D.; Andre, J. C. Sens. Actuators, B 1997, 38-39, 401. (10) Xu, W.; Schmidt, R.; Whaley, M.; Demas, J. N.; DeGraff, B. A.; Karikari, E. K.; Farmer, B. L. Anal. Chem. 1995, 67, 3172. (11) Papkovsky, D. B. Sens. Actuators, B 1995, 29, 213. (12) Chung, K. E.; Lan, E. H.; Davidson, M. S.; Dunn, B. S.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1995, 67, 1505. (13) Ji, J.; Rosenzweig, Z. Anal. Chim. Acta 1999, 397, 93. (14) Shortreed, M. R.; Dourado, S.; Kopelman, R. Sens. Actuators, B 1995, 38-39, 8. (15) Madden, J. E.; Cardwell, T. J.; Cattrall, R. W.; Deady, L. W. Anal. Chim. Acta 1996, 319, 129. (16) Wolfbeis, O. S.; Posch, H. E. Anal. Chim. Acta 1986, 185, 321. (17) Charlesworth, J. M.; McDonald, C. A. Sens. Actuators, B 1992, 8, 137. (18) Wolfbeis, O. S.; Klimant, I.; Werner, T.; Huber, C.; Kosch, U.; Krause, C.; Neurauter, G.; Durkop, A. Sens. Actuators, B 1998, 51, 17. (19) Resenzweig, Z.; Kopelman, R. Anal. Chem. 1996, 68, 1408. (20) Resenzweig, Z.; Kopelman, R. Sens. Actuators, B 1996, 35-36, 475. (21) Muller, C.; Hitzmann, B.; Schuber, F.; Scheper, T. Sens. Actuators, B 1997, 40, 71. (22) Sadaoka, Y.; Matsuguchi, M.; Sakai, Y.; Murata, Y-u. Sens. Actuators, B 1992, 7, 443.

advantages over other sensing techniques.6,8,9,25,26 Usually optical sensing does not consume analytes, no reference is required, and the signal is insensitive to sample flow rate, stirring speed, and exterior electrical interferences. In addition, optical sensors have the potential for miniaturization, remote sensing, and easy installation when optical fibers are used.9,13,23,27 Suitable fluorescence indicators that are sensitive to analyte concentrations and exhibit changes in fluorescence intensity are used as molecular recognition materials in common optical sensors.8 Both organic and inorganic polymers have been used as solid supports for the indicator. It is known that the choice of solid supports and the immobilization of indicator into the supports have significant effects on the performance of the optical sensors in terms of selectivity, sensitivity, response time, and stability.28,29 The indicators are immobilized by physical or chemical procedures onto the polymeric materials.28 The physical procedures used for immobilization include adsorption,30-32 dissolution,33,34 entrapment in a porous network,35,36 and ion exchange.37 These methods are simple (23) Fuh, M.-R. S.; Burgess, L. W.; Christian, G. D. Anal. Chem. 1988, 60, 433. (24) Kulp, T. J.; Camins, I.; Angel, S. M.; Munkholm, C.; Walt, D. R. Anal. Chem. 1987, 59, 2849. (25) Brook, T. E.; Narayanaswamy, R. Sens. Actuators, B 1997, 3839, 195. (26) Gabor, G.; Chadha, S.; Walt, D. R. Anal. Chim. Acta 1995, 313, 131. (27) Watkins, A. N.; Wenner, B. R.; Jordan, J. D.; Xu, W.; Demas, J. N.; Bright, F. V. Appl. Spectrosc. 1998, 52, 750. (28) Brook, T. E.; Narayanaswamy, R. Sens. Actuators, B 1998, 51, 77. (29) Xavier, M. P.; Garcia-Fresnadillo, D.; Moreno-Bondi, M. C.; Orellana, G. Anal. Chem. 1998, 70, 5184. (30) Demas, J. N.; DeGraff, B. A.; Xu, W. Anal. Chem. 1995, 67, 1377. (31) Carraway. E. R.; Demas, J. N.; DeGraff, B. A. Langmuir 1991, 7, 2991. (32) Xu, W.; Kneas, K. A.; Demas, J. N.; DeGraff, B. A. Anal. Chem. 1996, 68, 2605. (33) Klimant, I.; Wolfbeis, O. S. Anal. Chem. 1995, 67, 3160. (34) Mills, A.; Lepre, A.; Theobald, B. R.; Slade, E.; Murrer, B. A. Anal. Chem. 1997, 69, 2842.

10.1021/la0011836 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/28/2000

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but suffer from the problem of insolubility of indicator in the polymeric support, which results in leaching-out of the indicator. The chemical procedure involves the formation of covalent bonds between the indicator and support materials. Sensors with covalently immobilized indicators have the advantage of not suffering from indicator leaching-out but require complicated chemical reactions.38,39 In this paper, the electrostatic layer-by-layer (ELBL) assembly technique is used as a novel and easy way to immobilize the fluorescence indicator into the polymer for fabricating optical chemical sensors. The ELBL assembly technique is a powerful approach to create welldefined ultrathin film structures40,41 Alternate adsorption of anionic and cationic polyelectrolytes on a charged substrate by sequential dipping of the substrate into aqueous polyelectrolyte solutions yields a thin film which is both durable and reproducible in terms of film thickness and has a well-organized bilayer structure, high thermal stability, and high uniformity. The electrostatic assembly process has recently been found suitable for the assembly of a number of photonic materials including purple membrane fragments,42,43 photoprocessable materials for holographic storage,44 and acentric choromophore assembly for nonlinear optics.45,46 The pyrene derivative, 1-hydroxypyrene-3,6,8-trisulfonate (HPTS) was chosen as an indicator because HPTS has many attractive features as a fluorescent indicator. It has large Stokes shifts, high quantum yield, high absorbance, and excellent photostability and is nontoxic.4 Optical chemical sensors were fabricated by a layer-bylayer assembly technique using poly(allylamine hydrochloride) (PAH) as a polycation and mixture (PAA&HPTS) of HPTS and poly(acrylic acid) (PAA) or polymer (PAAHPTS) that HPTS is covalently attached into PAA as a polyanion. Recently Mohwald and co-workers have reported the mechanism of adsorption and desorption behavior from the sequential deposition of PAH and an analogous anionic pyrene dye, 1,3,6,8-pyrenetetrasulfonic acid (4-PSA).47 They have provided extensive structural details of the multilayered films of 4-PSA and PAH. In light of this we have not included morphological and structural details in this paper instead focusing on the optical, sensing, and stability aspects of the robust films. After deposition layered PAH/PAA films were crosslinked via heat-induced amide bond formation to make a (35) Di Marco, G.; Lanza, M.; Campagna, S. Adv. Mater. 1995, 7, 468. (36) McDonagh, C.; MacCraith, B. D.; McEvoy, A. K. Anal. Chem. 1998, 70, 45. (37) Zhujun, Z.; Seitz, W. R. Anal. Chim. Acta 1984, 160, 47. (38) Zhujun, Z.; Zhang, Y.; Wangbai, M.; Russell, R.; Shakhsher, Z. M.; Grant, C. L.; Seitz, W. R. Anal. Chem. 1989, 61, 202. (39) Lobnik, A.; Oehme, I.; Murkovic, I.; Wolfbeis, O. S. Anal. Chim. Acta 1998, 367, 159. (40) Lvov, Y.; Decher, G.; Sukhorukov, G.Macromolecules 1993, 26, 5396. (41) Decher, G.; Lvov, Y.; Schmitt, J. Thin Solid Films 1994, 244, 772. (42) He, J.-A.; Samuelson, L.; Li, L.; Kumar, J.; Tripathy, S. K. Langmuir 1998, 14, 1674. (43) He, J.-A.; Samuelson, L.; Li, L.; Kumar, J.; Tripathy, S. K. Adv. Mater. 1999, 11, 435. (44) He, J.-A.; Bian, S.; Li, L.; Kumar, J.; Tripathy, S. K. Appl. Phys. Lett. 2000, 76, 3233. (45) Lee, S.-H.; B Balasubramanian, S.; Kim, D. Y.; Viswanathan, N. K.; Bian, S.; Kumar, J.; Tripathy, S. K. Macromolecules 2000, 33, 6534. (46) Wang, X.; Balasubramanian, S.; Li, L.; Jiang X.; Sandaman, D. J.; Rubner, M. F.; Kumar, J.; Tripathy, S. K. Macromol. Rapid Commum. 1997, 18, 451. (47) Tedeschi, C.; Caruso, F.; Mohwald, H.; Kirstein, S. J. Am. Chem. Soc. 2000, 122, 5841.

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stable optical sensor.48 In the pH sensor, the relative peak positions and intensity of fluorescence of the protonated and deprotonated fluorescent indicator molecule change in response to pH variations. Other sensors, metal ion sensors (ferric and mercury ions) and a nitro compound (2,4-dinitrotoluene, DNT) sensor are based on fluorescence quenching of indicator molecules by electron transfer from indicator molecules to analytes (electron acceptor). Quenching-based fluorescence sensors utilize SternVolmer bimolecular quenching kinetics given by49

F + hv f F*

(1)

F* f F + hv or ∆

k1

(2)

F* + Q f F + Q*

k2

(3)

where F is fluorophore, Q is a quencher, k1 is the rate constant for unimolecular decay of the excited-state F*, and k2 is the rate constant for bimolecular quenching processes by Q that can deactivate the excited state. On photoexcitaion, fluorophore electron is excited from the highest occupied energy band (the π-band) to the lowest unoccupied energy band (the π*-band) leaving a “hole” which is the empty state in the π-band (eq 1). When the excited electron recombines with the hole, a photon is emitted (photoluminescence, eq 2). Relatively, when the excited electron of fluorophore transfers to a quencher (nearby cationic electron acceptor) and the electron and the hole are separated, the luminescence is quenched (eq 3). This kinetic scheme gives the well-known Stern-Vomer equation

I0/I ) 1 + Ksv[Q] where, I0 is the fluorescence intensity with absence of quencher, I is the intensity when quencher is present, Ksv is the Stern-Vomer constant, and [Q] is the concentration of quencher. A linear calibration curve results for a plot I0/I versus [Q]. Our approach has several advantages over a conventional spin coating method: it is simple and inexpensive, the thickness of the sensor layer is easily controlled at the molecular level, and the cross-linked, nylon-like sensor material exhibits long-term stability. Further, it may be possible to fabricate and multiplex multiple-sensing devices by employing different sensing layers or functionalities. Detailed investigations on multilayer fabrication and sensing ability of assembled film are reported. Experimental Section Materials. Poly(acryloyl chloride) (PAC) used in the synthesis of fluorescent molecule functionalized polymer was acquired from Polyscience. All other chemicals were purchased from Aldrich and used without further purification. A buffer solution covering the pH range 1-13 was composed of 0.2 M boric acid, 0.05 M citric acid, and 0.1 M sodium phosphate in deionized (DI) water.50 Citric acid-sodium citrate buffer solution of pH 4.4 was prepared using 0.1 M citric acid and 0.1 M sodium citrate.50 Microscope glass slides used as substrates for multilayer fabrication were purchased from VWR Scientific. The reagent used for cleaning the glass slides, Chem-solv cleaner, was obtained from Mallinckrodt. Water from a Milli-Q system was used in the multilayer (48) Harris, J. J.; DeRose, P. M.; Bruening, M. L. J. Am. Chem. Soc. 1999, 121, 1978. (49) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (50) Perrin, D. D.; Dempsey, B. Buffers for pH and Metal Ion Control; John Wiley & Sons: New York, 1974; p 132.

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fabrication process. The resistivity of the water used was higher than 18.2 MΩ cm with a total organic content of less than 10 ppb. Synthesis of Polymer (PAA-HPTS): A solution of HPTS (0.724 g, 1.38 mmol) in 150 mL of anhydrous N,N′-dimethylformamide (DMF) was added to a solution of PAC (5 g, 13.8 mmol, MW)10 000 g/mol, 25% in dioxane) in 150 mL of anhydrous DMF while purging with dry nitrogen gas. Two milliliters of anhydrous pyridine was added, and the solution was stirred at 25 °C for 24 h. The solution was concentrated under reduced pressure to remove most of the solvent and poured into water under agitation while adding a few drops of hydrochloric acid to precipitate the polymer. The precipitated PAAHPTS was collected by filtration and washed with excess water until pH of the wash solution was neutral. The solid was dried under vacuum at 25 °C for 24 h. Fabrication of Sensor Layers. Microscope glass slides (25 × 75 mm) with negatively charged surfaces were used as substrates for the sensor-layer fabrication. Treatment with 1% Chem-solv solution in DI water under ultrasonication for 3 h generates negative charges on the slide surfaces due to partial hydrolysis.51 Subsequently, the slides were rinsed with DI water in an ultrasonicator for 30 min. Multilayers for optical sensors were fabricated using two different polyanions. For a multilayerbased pH sensor, a 10 mM solution of PAA (MW ) 50 000 g/mol) and a 10 mM solution of HPTS were prepared in citric acidsodium citrate buffer. Forty milliliters of PAA and 10 mL of HPTS solution were mixed (PAA&HPTS) and used as the polyanion. For other multilayer-based sensors, a 10 mM solution of PAAHPTS in citric acid-sodium citrate buffer was prepared and used as the polyanion. Commercially available PAH was used as a polycation in the fabrication of pH as well as metal ion sensors. All solutions were filtered through Gelman 0.45 µm membrane filters before use. The deposition process was carried out in two steps (one dipping cycle). In the first step, glass slides were immersed in 10 mM PAH solution in citric acid-sodium citrate buffer for 10 min at room temperature and subsequently washed with citric acidsodium citrate buffer for 5 min. After the deposition and washing steps, the slides were dried with a stream of nitrogen gas. In the second step, the substrates with a single layer of PAH were immersed into the polyanion, PAA-HPTS or PAA&HPTS, solution for 10 min followed by the washing and drying procedures. This dipping cycle was repeated many times in order to build thick multilayer films. After the deposition process, dry multilayer films were heat treated at 130 °C for 2 h in a nitrogen atmosphere to cross-link the films. Characterization and Sensor Activity. UV-visible absorption spectra were recorded using a GBC UV/VIS 916 spectrophotometer. The IR spectra were recorded on a PerkinElmer 1760X FT-IR spectrometer. Thickness of the assembled films was obtained using an ellipsometer (AutoEL-III, Rudolph Research, USA) and a Dektak profilometer. Sensing abilities of multilayer films were characterized using fluorescence emission spectra obtained from a fluorescence spectrofluorometer (SLMAMINCO, model 8100). The glass slides containing sensor layers were placed in a 1-cm quartz cuvette (Sigma) which is filled with various pH buffer solutions, metal salt, or DNT solutions of different concentration (analytes). The cuvette was placed in the sample holder of the spectrofluorimeter, and emission spectral data were obtained in the region between 400 and 600 nm.

Results and Discussion Multilayer Fabrications. Since three sulfonate groups of HPTS can be easily ionized and make anionic charges in a broad pH range, ELBL assembly is possible between HPTS and a polycation. PAA&HPTS as a polyanion was assembled with PAH as a polycation employing the ELBL assembly technique. The deposition process was monitored using UV-visible absorption spectroscopy. Absorbance of a single bilayer formed was measured as a function of dipping time and is shown in Figure 1. The absorbance saturates in a dipping time of about 9 min. A dipping time (51) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 224.

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Figure 1. UV absorbance as a function of deposition time for a single bilayer film of PAH/PAA&HPTS.

Figure 2. UV absorption spectra of a multilayer film of PAH/ PAA&HPTS as a function of number of dipping cycles.

of 10 min was used for each deposition step for all multilayer fabrication processes. Figure 2 shows UV spectra of assembled films with various dipping cycles. Maximum UV absorbance occurs at 410 nm. A linear increase in absorbance as a function of the number of dipping cycles is shown in the inset. Emission spectra were measured from multilayer films. Emission maximum from these films occurs at 480 nm, and the measured fluorescence intensities increased linearly with the number of dipping cycles (Figure 3). A linear increase in the thickness was also observed from the deposited films with the number of dipping cycles. The thickness of a bilayer obtained in each dipping cycle was 310 Å. Alternatively, multilayer films were fabricated using a different polyanion (PAA-HPTS). PAA-HPTS was synthesized by covalent functionalization of HPTS into PAA. A synthetic scheme is shown in Figure 4. HPTS-functionalized PAA was produced by the reaction between the hydroxyl group of HPTS and the carboxyl chloride group of PAC. To calculate the extent of functionalization, PAAHPTS solution and PAA solution with different concentrations of HPTS in water were prepared and UV

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Figure 3. Fluorescence emission spectra of a multilayer film of PAH/PAA&HPTS as a function of number of dipping cycles. Figure 5. UV absorption (A, B) and fluorescence emission (C, D) spectra of HPTS and PAA-HPTS.

Figure 6. UV absorbance as a function of deposition time for a single bilayer film of PAH/PAA-HPTS.

Figure 4. Synthesis scheme for polymer PAA-HPTS.

absorbances were recorded. From the calibration curve of UV absorbance versus concentration of HPTS, the amount of HPTS functionalization in PAA-HPTS was determined to be 5%. Figure 5 shows UV absorption (A, B) and fluorescence emission (C, D) spectra of HPTS and the polymer PAA-HPTS. Almost similar absorbance and emission maxima were obtained at 405 and 495 nm, respectively.

The deposition process was monitored using UV-visible absorption spectroscopy. Absorbance of a single bilayer formed was measured as a function of dipping time and shown in Figure 6. The absorbance was saturated in about 9 min. A dipping time of 10 min in each deposition step was used for all multilayer fabrication processes. UV spectra of assembled films from PAH and PAA-HPTS up to 16 dipping cycles are shown in Figure 7. Linear increase in absorbance with dipping cycles was observed. Maximum UV absorbance occurs at 410 nm. Figure 8 shows fluorescence emission spectra of these multilayer films. Emission maximum from these films occurs at 485 nm, and the measured fluorescence intensities increased linearly with number of dipping cycles. From thickness measurements of multilayers with dipping cycles, the film thickness was also observed to increase linearly with the number of dipping cycles. The thickness of 260 Å was obtained for each dipping cycle. The linear behavior of UV absorption, fluorescence emission, and film thickness with dipping cycles indicates the uniformity of the adsorption process.

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Figure 7. UV absorption spectra of a multilayer film of PAH/ PAA-HPTS as a function of number of dipping cycles.

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Figure 9. FTIR spectra of multilayer films before heating (A) and after heating at 130 °C (B).

Figure 8. Fluorescence emission spectra of a multilayer film of PAH/PAA-HPTS as a function of number of dipping cycles.

Harris et al. have demonstrated that cross-linked multilayer films of PAH and PAA via heating are stable over a wide pH range unlike other polyelectrolyte multilayer films.48 In this case, heat-induced amide bond formation between PAH and PAA occurs with the formation of very stable films which do not degrade even under severe pH conditions. The multilayer film of PAH with PAA-HPTS (20 dipping cycles) was heated at 130 °C for 2 h under nitrogen. FTIR spectra shown in Figure 9 indicate amide bond formation between amine groups of PAH and carboxylate groups of PAA. Before heating, peaks due to -COOH (1709 cm-1) and -COO- asymmetric (1563 cm-1) and symmetric (1401 cm-1) stretches are dominant and shown in spectrum A. After heating at 130 °C the carboxylate (COOH and COO-) are no longer present and amide peaks appear at 1542 cm-1 (Amide I) and 1668 cm-1 (Amide II) as seen in spectrum B. The same results are obtained on the PAH/PAA&HPTS multilayer film. Figure 10 is the photograph of fluorescence emission from cross-linked PAH/PAA&HPTS (A) and PAH/PAAHPTS (B) multilayer films (20 dipping cycles) with shining of 360 nm UV radiation from a lamp. There is strong fluorescence quenching in high concentration of HPTS (self-quenching)52 and hence no fluorescence in the solid state. However, fluorescence of HPTS indicator is not (52) Stokes, G. G. Philos. Trans. R. Soc. London, A 1852, 142, 463.

Figure 10. Photograph of fluorescence emission of PAH/ PAA&HPTS (A) and PAH/PAA-HPTS (B) multilayer films.

quenched in the ELBL assembled film because a polymer, PAA or PAH, provides fluorophore with the appropriate spacer avoiding self-quenching. In fact the ELBL assembly technique can be utilized to adsorb the optimum concentration and separation of the fluorophores in the thin film optical sensors. Sensing Ability of Multilayer. HPTS is a fluorescent pH indicator dye and was first reported by Wolfbeis and co-workers.53 They measured pH values in the near neutral range using HPTS and found HPTS to be an almost ideal pH indicator. A detailed study of the properties of the indicator have been described by a number of researchers.4,7,37,53-57 HPTS can exist in both acid (protonated) and (53) Wolfbeis, O. S.; Furlinger, E.; Kroneis, H.; Marsoner, H. Fresenius’ Z. Anal. Chem. 1983, 314, 119. (54) Wolfbeis, O. S.; Weis, L. J. Anal. Chem. 1988, 60, 2028. (55) Zhujun, Z.; Seitz, W. R. Anal. Chim. Acta 1984, 160, 305. (56) Luo, S.; Walt, D. R. Anal. Chem. 1989, 61, 174.

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Figure 11. Fluorescence emission spectra of multilayer film of PAH/PAA&HPTS as a function of pH.

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Figure 12. Fluorescence intensities in the multilayer film of PAH/PAA& HPTS as a function of pH: protonated (416 nm, A) and deprotonated (493 nm, B) forms of indicators.

base (deprotonated) forms, and the fluorescence intensity and spectrum depend on the following acid-base equilibrium.

Emission spectra shown in Figure 11 were measured in cross-linked multilayer films of PAH/PAA&HPTS at various pH values using 365 nm excitation. Two emission peaks appeared in acidic condition. These peaks are centered at 416 and 493 nm and result from the protonated and deprotonated forms of the indicator, respectively.4,37,56 This ratiometric sensor is insensitive to system instabilities such as variation of source intensity, fluorescence quenching, photobleaching, and indicator leachingout.5,14,37,56 Fluorescence intensities were decreased at 416 nm and increased at 493 nm with increasing pH. Figure 12 shows experimental values for fluorescence intensities versus pH in protonated (416 nm, A) and deprotonated (493 nm, B) forms of indicators. Figure 13 shows the calibration curve which plots the ratio of fluorescence intensities of two forms with pH. From quenching theory, the electron-deficient metal cations such as Fe3+ or Hg2+, a nitro aromatic such as DNT, and dicationic electron acceptor, N,N′-dimethyl-4,4′bipyridinium (methyl viologen, MV2+) can play a role as quenchers for electron transfer quenching based sensors. Metal ion sensors using the quenching of fluorescence by metal ions are reported by several workers.13-15,58-60 Seitz et al. show that a membrane containing pyrene fluores(57) Wolfbeis, O. S.; Kovacs, B.; Goswami, K.; Klainer, S. M. Mikrochim. Acta 1998, 129, 181. (58) Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1997, 119, 3443. (59) Kim, J.; Wu, X.; Herman, M. R.; Dordick, J. S. Anal. Chim. Acta 1998, 370, 251. (60) Birch, D. J. S.; Suhling, K.; Holmes, A. S.; Salthammer, T.; Imhof, R. E. Pure. Appl. Chem. 1993, 65, 1687.

Figure 13. The ratio of fluorescence intensities (493 nm/416 nm) of a multilayer film of PAH/PAA&HPTS as a function of pH.

cence indicator was efficiently quenched by organic nitro compounds such as DNT and TNT.61 The solution of PAA-HPTS was tested with several metal ions such as Ba2+, Ca2+, Fe3+, K+, Zn2+, Hg2+, Cd2+, and Pb2+. Only additions of Fe3+ and Hg2+, which are electron-deficient metal cations, have a significant effect on the fluorescence intensity showing efficient quenching. Figure 14 shows fluorescence intensities of PAA-HPTS solution in water with addition of Fe3+. Fluorescence intensities decreased with ferric ion concentration by quenching. Fluorescence spectra as a function of different concentrations of ferric ion were measured in a multilayer film of PAH/PAA-HPTS. Fluorescence intensity decreased with ferric ion concentration (Figure 15). Similar behaviors (61) Jian, C.; Seitz, W. R. Anal. Chim. Acta 1990, 237, 265.

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Figure 14. Fluorescence emission spectra of PAA-HPTS solution as a function of ferric ion concentration. The inset shows relative fluorescence intensities (I/I0) at 498 nm with ferric ion concentration.

Figure 16. Fluorescence emission spectra of a multilayer film of PAH/PAA-HPTS as a function of mercury ion concentration. The inset shows relative fluorescence intensities (I/I0) at 490 nm with mercury ion concentration.

Figure 15. Fluorescence emission spectra of a multilayer film of PAH/PAA-HPTS as a function of ferric ion concentration. The inset shows relative fluorescence intensities (I/I0) at 490 nm with ferric ion concentration.

Figure 17. Fluorescence emission spectra of a multilayer film of PAH/PAA-HPTS as a function of DNT concentration. The inset shows relative fluorescence intensities (I/I0) at 490 nm with DNT concentration.

were observed in the multilayer films with addition of Hg2+ and DNT as shown in Figures 16 and 17. Each inset picture shows relative fluorescence intensity (I/I0) decreases with analyte concentration. We believe that this fluorescence intensity drop is the quenching effect due to complex formation (HPTS indicator(-)/quencher(+)), followed by ultrafast electron transfer from excitations on multilayer films to quencher (analytes, electron acceptor) and the degree of quenching depends on the amount of analyte. Whitten and co-workers62 and Heeger et al.63 have reported the fluorescence of an entire polyanionic conjugated polymer, polyphenylene vinylene (PPV), was quenched by strong electron acceptor, biotin-methyl viologen (B-MV), which combines a methyl viologen linked to a biotin molecule, in aqueous solutions. Fluorescence-based sensing through avidin-biotin complexation in thin films assembled by the Langmuir Blodgett process has been investigated by us earlier.64,65 Chen et

al.’s work62 describes complex formation and electron transfer between PPV and B-MV in solution. They also show this quenching could be recovered when the PPV/ B-MV complex formations were broken by formation of other stronger biotin (B-MV)/avidin complex by addition of avidin solution. In our approach, fluorescence spectra of the multilayer films (PAH/PAA-HPTS) in the solid state were measured with different concentrations of MV2+. Fluorescence intensity decreased with MV2+ concentration showing effective quenching occurs (Figure 18) in the film. The quenching on the multilayer film should be recovered if we use a biotin-methyl viologen/avidin system, which opens up the possibility for making reversible optical thin film sensors based on this approach. We are further investigating the reversible sensing ability on these multilayer films. The data obtained by performing a Stern-Volmer analysis in each sensor are shown in Figure 19. Linear plots between concentration of quencher and I0/I in the

(62) Chen, L.; McBranch, D. W.; Wang, H.-L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287. (63) Heeger, P. S.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12219.

(64) Samuelson, L. A.; Miller, P.; Galotti, D. M.; Marx, K. A.; Kumar, J.; Tripathy, S. K.; Kaplan, D. L. Langmuir 1992, 8, 604. (65) Samuelson, L. A.; Miller, P.; Galotti, D. M.; Marx, K. A.; Kumar, J.; Tripathy, S. K.; Kaplan, D. L. Thin Solid Films 1992, 210/211, 796.

Thin Film Optical Sensors

Langmuir, Vol. 16, No. 26, 2000 10489

was covalently attached to the polymer. The multilayer film of PAH/PAA-HPTS could be tested with different pH solutions but showed less sensitivity as a pH sensor in comparison with PAH/PAA&HPTS. This difference in sensitivity may be due to the absence of a hydroxyl group, which plays an important role in the pH sensing, in PAA-HPTS. The two kinds of multilayer films, PAH/ PAA&HPTS and PAH/PAA-HPTS, fabricated and used as pH and metal ion/DNT sensors, respectively, also attest to the many variations possible in this type of sensor material design. When analyte solutions containing mixtures of competing species are used, the issue of sensor selectivity becomes important. This is under present exploration. The electrostatic multilayer assembly approach presents the opportunity to include multiple compatible fluorescent dyes, and the question of selectivity may be addressed as a differential response to individual analytes. Figure 18. Fluorescence emission spectra of a multilayer film of PAH/PAA-HPTS as a function of MV2+ concentration. The inset shows relative fluorescence intensities (I/I0) at 490 nm with MV2+ concentration.

Figure 19. Stern-Volmer plots of multilayer films of PAH/ PAA-HPTS as a function of different quencher concentrations.

multilayer films are obtained showing a Stern-Volmer relationship. Stern-Volmer constants (Ksv) were calculated from slopes of each plot. Multilayer films of PAH/PAA-HPTS were found to be more stable than those of PAH/PAA&HPTS because HPTS

Conclusions We have successfully developed thin film optical sensors for pH, metal ions (ferric and mercury), and DNT detection by an ELBL assembly technique. A broad general strategy to form robust sensors based on thermo-cross-linked films is established. UV absorbance, film thickness, and emission intensity increased linearly with increase in the number of bilayers deposited in all multilayer films. As a pH sensor, the emission peak positions and relative intensity of fluorescence in the protonated and deprotonated forms of fluorescence indicator changed when the pH was changed from 1 to 13, and the relative change could be used to determine pH of a specific analyte. A calibration curve was obtained for pH in terms of the ratio of fluorescence intensities of the protonated and deprotonated forms. As metal ion and DNT sensors, multilayer films were efficiently quenched by electron-deficient analytes and fluorescence intensities decreased with increasing concentration of analytes. A Stern-Volmer bimolecular quenching relationship was found to hold when I0/I was used to determine analyte concentration. Methyl viologen is a strong quencher, and fluorescence of multilayer films was efficiently quenched by formation of a HPTS indicator and methyl viologen complex. A reversible optical thin film sensor can be realized by breaking of indicator/quencher complexation and fluorescence recovery. Acknowledgment. Financial support from ONR is gratefully acknowledged. Extensive discussions with the MURI team and Dr. Srinivasan Balasubramanian are acknowledged. LA0011836