Anal. Chem. 1999, 71, 4559-4563
Molecularly Imprinted Sensor Layers for the Detection of Polycyclic Aromatic Hydrocarbons in Water Franz L. Dickert* and Matthias Tortschanoff
Institute of Analytical Chemistry, Vienna University, Wa¨hringer Strasse 38, A-1090 Vienna, Austria Wolf E. Bulst and Gerhard Fischerauer
Siemens Corporate Research and Development, Otto Hahn Ring 6, D-81739 Munich, Germany
Molecularly imprinted polyurethanes are presented as sensitive coatings for the detection of polycyclic aromatic hydrocarbons in water. These sensor layers were combined with fluorescence and mass-sensitive transducers. Imprinting based on van der Waals interactions allows detection of these analytes even without any pronounced functionality. The geometry of the imprint molecule determines the selectivity of the sensor layer. In varying the size of template molecules from anthracene up to 1,12benzoperylene, selectivity is tuned to a distinct analyte. The enrichment factor of up to approximately 107 renders detection down to the ppt range possible with hardly any matrix effect by humic acids. In recent years, chemical sensors1 are of growing importance in environmental analysis, especially in monitoring air quality. In liquid media, sensors are common for the selective detection of ions2 but there are only a few applications for the important task of monitoring small organic pollutants in water.3 Supramolecular recognition based on molecular cavities is used successfully in numerous sensor applications.4 However, time-consuming syntheses are necessary and the adaption to a special analyte is difficult. Molecular imprinting5-7 is a promising tool for the development of sensitive coatings for chemical sensors in a reduced time scale and has met with growing interest.8-10 Using noncovalent imprinting, there is no restriction to the choice of the analyte. Even small molecules without any functionality can be detected with high selectivity. (1) Cammann, K.; Lemke, U.; Rohen, R.; Sander, J.; Wilken, H.; Winter B. Angew. Chem., Int. Ed. Engl. 1991, 30, 516-537. (2) Bailey, P. L. Analysis with Ion-Selective Electrodes; Heyden: London, 1980. (3) Janata, J.; Josowicz, M. Anal. Chem. 1998, 70, 179R-208R. (4) Dickert, F. L.; Haunschild, A.; Kuschow, V.; Reif, M.; Stathopulos, H. Anal. Chem. 1996, 68, 1058-1061. (5) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1833. (6) Dickert, F. L.; Thierer S. Adv. Mater. 1996, 8, 978-990. (7) Mayes, A. G.; Mosbach, K. Trends Anal. Chem. 1997, 16, 321-332. (8) Remcho, V. T.; Tan, Z. J. Anal. Chem. 1999, 71, 248A-255A. (9) Malitesta, C.; Losito, I.; Zambonin, P. G. Anal. Chem. 1999, 71, 13661370. (10) Takeuchi, T.; Fukuma, D.; Matsui, J. Anal. Chem. 1999, 71, 285-290. 10.1021/ac990513s CCC: $18.00 Published on Web 09/08/1999
© 1999 American Chemical Society
The principle of molecular imprinting is schematically shown in Figure 1 for polyurethanes, which are used in the following investigations. A highly cross-linked polymer is synthesized around a template molecule. When the template molecule is removed, a geometrically adapted polymer skeleton with fitting cavities and diffusion pathways for analyte inclusion is left behind. Furthermore, polymers are favorable for application in liquid media because of their insolubility and long-term stability. Polycyclic aromatic hydrocarbons (PAHs) are important analytes11-13 in environmental control because of their toxic and carcinogenic properties and their permanent formation in incomplete combustion processes, e.g., in diesel engines.14 Polyurethanes are used to have a hydrophilic polymer for the application in water to guarantee sufficient wetting. To provide an optimized interaction via π-π-bonds for the PAHs, aromatic monomeric components were selected. EXPERIMENTAL SECTION Chemicals. Bisphenol A (2,2-bis(4-hydroxyphenyl)propane), phloroglucinol, and p,p′-diisocyanatodiphenylmethane containing 30% of the respective triisocyanate (mixture of isomers) were dissolved in THF to form a stoichiometric solution. The freshly prepared mixture of the monomers containing 0.5-5% of the imprint PAH was applied to the quartz plates or the microelectronic devices, respectively. The polymerization was performed under ambient conditions (25 °C in air), at higher temperatures up to 70 °C, and in a saturated THF atmosphere. The polycondensation is complete within several hours, forming clear and transparent coatings on the quartz substrate. The high amount of 30% cross-linking monomer components, namely, phloroglucinol and the triisocyanate, results in rigid sensor layers insoluble in water and organic solvents. The removal of the imprint PAHs was performed using toluene. (11) Kumke, U. M.; Lo ¨hmannro ¨ben H.-G.; Roch, Th. J. Fluoresc. 1995, 5, 139152. (12) Inman, S. M.; Thibado, P.; Theriault, G. A.; Lieberman, S. H. Anal. Chim. Acta 1990, 239, 45-51. (13) Panne, U.; Lewitzka, F.; Niessner, R. Analysis 1992, 20, 533-542. (14) Miguel, A. H.; Kirchstetter, T. W.; Harley, R. A.; Hering, S. V. Environm. Sci. Technol. 1998, 32, 450-455.
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Figure 2. Fluorescence sensor and mass-sensitive devices and their integration into a flow system.
sensors were fabricated on 36° rotY,X-LiTaO3 for two reasons. First, the leaky wave supported by this substrate cut exhibits an almost complete shear horizontal polarization; hence, it does not couple (and lose energy) to compressional waves in the loading liquid. Second, the relative permittivity of LiTaO3 is larger than 40, a value comparable to that of water (80); consequently, the SAW devices operate even in watery solutions which, at higher frequencies, produce a quasi short circuit at the surface of any low-permittivity substrate such as quartz. The flow cells used for mass-sensitive measurements were temperature controlled ((0.01 °C). Fluorescence intensity and frequency were recorded by a PC which also controlled the flow of the pumps. The flow diagram and the sensor devices are shown in Figure 2 schematically. Figure 1. Principle of molecular imprinting with polyurethanes using pyrene as template/analyte.
Measurements. The transducer was a quartz substrate in the case of optical measurements and a QMB or SAW device in the case of mass-sensitive detection. The fluorescence measurements were performed with a Perkin-Elmer LS50B fluorescence spectrometer. Excitation was perpendicular to the sensor layer on the quartz sheet in a thickness of 1.2 mm. Emission was observed at the narrow face of the planar waveguide as indicated in Figure 2 in the upper left. The QMB consists of a 10-MHz AT-cut quartz with gold electrodes (5.5 mm in diameter), operating in the thickness shear mode. The device was placed in a self-constructed flow cell with a sample volume of 140 µL. The quartz was driven by an oscillator circuit built up with the integrated amplifier OPA660.15 The frequency was measured with a HP 53131A frequency counter. The SAW devices were designed from a LiTaO3 substrate and gold electrodes, operating at a resonance frequency of 428 MHz. The sensor response was determined by a network analyzer HP 8752C or by using an oscillator circuit combined with a HP 53131A frequency counter. The two-port SAW resonators we used as liquid (15) Auge, J.; Hauptmann, P.; Hartmann, J.; Ro¨sler, S.; Lucklum, R. Sens. Actuators B 1995, 24-25, 43-48.
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RESULTS AND DISCUSSION The freshly prepared sensor layers were investigated by fluorescence, UV, and FT-IR spectroscopy to characterize the imprinting process. Generally a higher amount of imprint molecules should increase the number of cavities and therefore enhance sensitivity. However, at concentrations above 5%, the formation of excimers16 is observed since the PAH molecules are incorporated as clusters. In the case of pyrene, the fluorescence intensity at the emission wavelength of 373 nm decreases above 5% and a broad emission maximum arises at 470 nm. According to the concentration in the washing solution and UV bands, more than 80% of the template was removed by toluene. However, the fluorescence of the sensor layers was reduced to less than 1% of the initial intensity. The strong interaction between the remaining imprint molecules and the polymer initiates a radiationless energy transfer, which is favorable because there is no background intensity to be subtracted from the PAH spectra. To test the selective inclusion of analytes from aqueous samples, a polymer was imprinted with PAHs of different size and shape from naphthalene up to perylene. The characteristic selectivity pattern found for the detection of pyrene is presented in Figure 3. Furthermore, a polymer without imprint molecules (16) Yip, W. T.; Levy, D. H. J. Phys. Chem. 1996, 100, 11539-11545.
Figure 4. Sensor response of polyurethanes imprinted with anthracene (9) and chrysene (light shading) to different analyte PAHs sorted by size.
Figure 3. Selectivity pattern of pyrene detection by fluorescence, using polyurethanes imprinted with PAHs of different sizes.
was prepared to investigate the amount of unspecific adsorption. According to Figure 3, the response of the nonimprinted sensor layer is less than 1% compared to the optimum. A bulk effect has been proven, indicating a direct relationship between sensor response and layer height. The artificial molecular recognition system shows clearly a pronounced selectivity pattern even with these small template molecules without functionality. The analyte inclusion is a pure van der Waals interaction between the analyte and the sensor layer. Because there are no specific binding sites, the size and the shape of the cavities determine the strength of interactions and therefore the selectivity of the sensor.17 Access to imprint cavities is hindered for analytes that are significantly larger than the imprint molecule. If the analyte is too small, it will be washed out of the coating since there is no tight fit to the pockets. As shown in Figure 3, the optimum cavity geometry is not generated by the analyte itself in the case of these polyurethane layers. The maximum of sensitivity for the detection of pyrene is achieved when the smaller imprint molecule acenaphthene is used. The cavities are larger than the printing molecules especially when the monomer units have about the same size and shape as the template molecules. Maximum selectivity can be tuned to each analyte of interest, as shown in Figure 4. The sensor effects of two polyurethanes imprinted with anthracene and chrysene, respectively, toward analytes of different size from anthracene to benzoperylene were investigated. The enrichment factor, which represents the relation of the PAH concentration within the sensitive layer compared to the concentration in the aqueous solution, allows a direct comparison of the incorporation into the sensor layer despite different fluorescence intensities of different PAHs. As shown above, an analyte that is slightly larger than the imprint molecule shows a better inclusion into the cavity than the imprint itself. Using anthracene to generate the cavities, maximum sensitivity is achieved for chrysene as analyte. Using the bigger chrysene as imprint molecule, the characteristic sensitivity pattern is shifted (17) Yoshizako, K.; Hosoya, K.; Iwakoshi, Y.; Kimata, K.; Tanaka, N. Anal. Chem. 1998, 70, 386-389.
Figure 5. QMB sensor response of an anthracene- and chryseneimprinted layer to a pulse of 2 µg/L chrysene and perylene, respectively (flow rate, 10 mL/min).
to bigger analytes with an optimum at perylene. Thus, the sensor responses to chrysene and perylene can be optimized by choosing the print molecules anthracene and chrysene, respectively. As shown in Figure 5 the same selectivity pattern as in Figure 4 can be obtained by mass-sensitive measurements with the QMB. Again, imprinting with anthracene favors the incorporation of chrysene in comparison to perylene whereas the opposite is found for chrysene imprinting. Additionally, the sensor responses in Figure 5 validate the data in Figure 4 and prove that the relations between PAH fluorescence intensities and their concentrations are real. The positions of the maximums observed in Figure 4 depend on the conditions of polymerization.18 At elevated temperatures or in more dilute solutions, imprinted polymers are generated that show the highest sensitivity to the template molecule. In parallel to this behavior, the percentage of template removal is reduced to, e.g., 50% at 70 °C. Both temperature and solvent allow a better organization of the polymer around the template because of a higher mobility of the oligomers during the polymerization process. Then the hollows show an optimized size and shape for the template, and this excellent fitting leads to a reduced removal of this molecule because of energetic and geometrical reasons. (18) Sellergren, B.; Kenneth, S. J. J. Chromatogr. 1993, 635, 31-49.
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In these specifically adapted sensitive layers, 20% of the resulting cavities can be refilled by analyte molecules. A coefficient of distribution up to approximately 107 is obtained according to the Nernst law. Under conditions of equilibrium, the chemical potential of the analyte (A) must be equal in the liquid aqueous solution (l) and in the solid sensor layer (s) with x the mole fraction of the analyte (eq 1).19
(µ0(A) + RT ln x)l ) (µ0(A) + RT ln x)s
(1)
The expression in eq 2 is obtained for the equilibrium constant K with [CA] the concentration of cavities filled with the analyte,
K ) [CA]/[C]0x(A)
(1)
whereas [C]0 corresponds to the overall cavities and x(A) is the mole fraction of the analyte in the aqueous solution. The amount of incorporated pyrene, i.e., the complex [CA], can directly be determined by mass-sensitive measurements, preferentially with the QMB. The overall number of cavities available result from the saturation behavior of the layers. Fluorescence spectrometry allows us to determine both the PAH concentration in the aqueous solution and that in the coating. The equilibrium constant of a sensor layer imprinted with 3% of pyrene toward pyrene as analyte is 9.4 × 107 (eq 2). This excellent sensitivity renders detection limits with a IUPAC signal/noise ratio of 3:1 in the parts per trilion (ppt) range down to 30 ng/L possible. The fluorescence intensity increases linearly with the pyrene concentration within a wide range up to approximately 40 µg/L.20 At higher concentrations, saturation is observed caused by the limited number of cavities in the polymer. The linear range can be extended by raising the amount of imprint molecules. However, a higher percentage of imprint results in inclusion of template clusters, causing a broadening of the size distribution of the cavities and therefore a reduced selectivity. The equilibrium constants in eq 2 are also true for diluted mixed PAH solutions of some micrograms per liter. Besides increased sensitivity, the separation of the analyte from a complex matrix21 is a further advantage in using selective sensor layers. Often, there are different quenchers that prevent a direct fluorescence measurement of an unknown sample. Common quenchers in environmental samples are humic acids. The quenching effects of humic acids to the fluorescence intensity of an aqueous solution of pyrene measured in a cuvette and using the sensor are compared in Figure 6. A humic acid concentration of 14 mg/L causes a decrease of fluorescence intensity of more than 50% in the case of the solution but less than 10% using a sensitive layer. The sensitive layers presented can also be combined with transducer other principles than fluorescence spectroscopy, as already shown in Figure 5. The advantages of mass-sensitive devices are low costs and ease of miniaturization and integration into microelectronic sensor units. Investigations have also been (19) Dickert, F. L.; Ba¨umler, U.; Stathopulos, H. Anal. Chem. 1997, 69, 10001005. (20) Dickert, F. L.; Besenbo¨ck, H.; Tortschanoff, M. Adv. Mater. 1998, 10, 149151. (21) Muldoon, M. T.; Stanker L. H. Anal. Chem. 1997, 69, 803-808.
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Figure 6. Relative fluorescence decrease caused by the presence of humic acids using the proposed sensor (O) compared to the direct measurement of the aqueous solution (/).
Figure 7. Sensor response of a 428-MHz shear wave resonator to a pulse of 3.5 µg/L pyrene in water (temperature, 25 °C; flow rate, 2 mL/min).
performed with QMBs (Figure 5) yielding no cross sensitivity to quenchers such as humic acids that are not incorporated into the layers. Shear wave resonators might show promising sensitivities because of the high resonance frequency22 of 428 MHz which renders detection of mass changes down to 1 pg possible. Furthermore, the damping of this device at the resonance point only increases by three or four db in going from air to water. The effects produced by temperature and pressure changes can be eliminated by the use of a reference sensor coated with the nonimprinted polymer. The frequency change of a shear wave resonator caused by an aqueous solution of 3.5 µg/L pyrene is shown in Figure 7. The response time is some minutes, and the effect is fully reversible. The layer height on this device was approximately only a quarter of the QMB coatings in Figure 5. The signal-to-noise ratio can further be improved by using an electronic mixer for the compensation of temperature effects. CONCLUSIONS We have shown that molecular imprinting is a great tool in the development of chemical sensors. Insoluble, chemically resistive, and durable sensitive layers are synthesized, perfect for (22) Dickert, F. L.; Forth, P.; Bulst, W.-E.; Fischerauer, G.; Knauer, U. Sens. Actuators B 1998, 46, 120-125.
application in liquid media and tailored to a specific analyte. Even chemically homologous compounds such as PAHs are detected with high selectivity. The preparation is compatible with common production technologies; they can be combined with many kinds of transducers including fiber-optic systems23 or the universal mass-sensitive devices. Further improvement in selectivity is accomplished by interpretation of the whole fluorescence spectrum, or in the case of mass-sensitive detection, several devices
can easily be integrated on a single chip. These sensor arrays in combination with pattern recognition methods24 render the simultaneous detection of many analytes possible.
(23) Wolfbeis O. S., Ed. Fiber Optic Chemical Sensors and Biosensors; CRC Press: Boca Raton, 1991, Vol. I, pp 25-110. (24) Dickert, F. L.; Hayden, O.; Zenkel, M. E. Anal. Chem. 1999, 71, 13381341.
Received for review May 13, 1999. Accepted July 29, 1999.
ACKNOWLEDGMENT Part of this work was supported by the German Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie under Grant 16SV545/0.
AC990513S
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