Anal. Chem. 1999, 71, 1366-1370
Molecularly Imprinted Electrosynthesized Polymers: New Materials for Biomimetic Sensors Cosimino Malitesta, Ilario Losito, and Pier Giorgio Zambonin*
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Dipartimento di Chimica, Universita` degli Studi, Via Orabona 4, I-70125 Bari, Italy
The preparation and characterization of electrosynthesized poly(o-phenylenediamine) (PPD) imprinted by glucose (iPPD) is reported as the first case of an electrosynthesized polymer molecularly imprinted by a neutral template. The material is employed as the recognition element of a QCM biomimetic sensor for glucose. Scatchard analysis of the relevant calibration curve offers information on the equilibrium and binding sites involved in glucose detection. XPS comparison of PPD and iPPD supports the occurrence of a templating effect. On this basis, molecular imprinting electropolymerization is proposed as a possible strategy for the preparation of new materials with molecular recognition properties to be applied in biomimetic sensors. The development of highly selective chemical sensors for complex matrixes of medical, environmental, and industrial interest has been the object of great research efforts in the last years. The application of biomolecules such as enzymes or antibodies and supramolecular assemblies such as membranes or even cells and tissues has been widely investigated. In particular, their extraordinary molecular recognition capabilities have been successfully exploited in the realization of the sensing element for a number of biosensors (see, for example, ref 1 for a review). Recently, the use of artificial materials with comparable recognition properties has been proposed for designing biomimetic sensors. This approach could overcome some limitations of biosensors such as those due to the availability or the cost of biocomponents for a particular analyte. Moreover, artificial materials could offer the possibility to overcome some of the problems encountered when biocomponents are employed, such as instability (due, for example, to inhibition or denaturation), particularly in adverse operative conditions (e.g., high temperatures, harsh chemical environments, or extreme pH values). Probably the most promising materials in the field of artificially generated molecular recognition are molecularly imprinted polymers2 (MIPs). Their synthesis, first reported more than 25 years ago,3 is based on the chemical polymerization of a functional monomer and a cross-linking agent in the presence of a molecule used as (1) Sethi, R. S. Biosens. Bioelectron. 1994, 9, 243 and references therein. (2) Andersson, L. I. Anal. Chem. 1996, 68, 111 and references therein. (3) Wulff, G.; Sharhan, A. Angew. Chem., Int. Ed. Engl. 1972, 11, 341.
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a template, often interacting with them by relatively weak interactions (e.g., hydrogen bonds). At the end of polymerization and after removal of the print molecule, a cross-linked (rigid) polymer is obtained, containing sites with a high affinity for the template, due both to their shape and to the arrangement of the functional groups of the monomer units around the print molecule. This property has already been exploited successfully in several analytical applications, including chromatographic separations of diastereoisomers or enantiomers by using columns packed with an imprinted polymer,4,5 ligand-binding assays2,6 (where MIPs mimic the role of antibodies in immunoassays), and selective sample enrichment by solid-phase extraction.7 Only a few applications of MIPs in biomimetic sensors (based on either optical or electrochemical transduction) have been reported.8 In the present work, the first successful application of electropolymerization as a general strategy for the preparation of molecularly imprinted electrosynthesized polymers (MIEPs) is reported. Electropolymerization has been already proposed as a procedure for imprinting polymers to be used in a nitrate-selective potentiometric sensor.9 The nitrate template was not removed from the polymer after the synthesis. The inability of other anions to replace nitrate in the polymer conferred high selectivity to the sensor response. However, the approach was limited to charged polymers and templates. In addition, the preservation of recognition sites upon removal of the template has not been considered, so that the possibility of using those polymers in chemical sensors with a different transducer cannot be evaluated. During the time of this work, attempts to imprint of electrosynthesized polypyrrole by charged and neutral species were reported.10 The work was aimed at improving selectivity and sensitivity of film electrodes based on that polymer. Little success was obtained in the case of neutral species, perhaps due to the choice of overoxidizing the polymer after the imprinting procedure. (4) Karube, J.; Kato, T.; Takeuchi, T.; Suzuki, M.; Yokoyama, K.; Tamiya, E.; Karube, I. Anal. Chem. 1993, 65, 2223. (5) Kempe, M. Anal. Chem. 1996, 68, 1948. (6) Vlatakis, G.; Anderson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645. (7) Rashid, B. A.; Briggs, R. J.; Hay, J. N.; Stevenson, D. Anal. Commun. 1997, 34, 303. (8) Kriz, D.; Ramstro¨m, O.; Mosbach, K. Anal. Chem. 1997, 69, 345A. (9) Hutchins, R. S.; Bachas, L. G. Anal. Chem. 1995, 67, 1654. (10) Spurlock, L. D.; Jaramillo, A.; Praserthdam, A.; Lewis, J.; Braither-Toth, A. Anal. Chim. Acta 1996, 336, 37. 10.1021/ac980674g CCC: $18.00
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Many peculiar features of the electrosynthetic approach could be very helpful both in improving the molecular imprinting polymerization procedure itself and in extending the application of MIPs as sensing elements for chemical sensors with various transduction mechanisms. By this methodology, polymeric films can be easily grown adherent to conducting electrodes of any shape and size and with a thickness controlled by the amount of circulated charge. In the perspective of sensor development, this feature gives the possibility of creating, in a very simple way, a direct communication between the polymer and the surface of the transducer, provided the latter is conductive. In the same direction, miniaturization, one of the major goals of chemical sensor technology, could also be easily realized. On the other hand, the structural versatility offered by the several monomers and the relevant electrosynthesized polymers investigated so far (see, for example, ref 11), with their variety of functional groups, is a promising starting point to turn imprinting polymerization into an easy procedure for producing biomimetic sensors for several analytes of different classes. In this work, poly(o-phenylenediamine) (PPD), already used in our laboratory as an enzyme-entrapping membrane for biosensors,12,13 has been chosen to check the feasibility of a molecular imprinting electropolymerization, using a neutral template such as glucose. A (glucose) biomimetic sensor was produced by this approach. PPD seemed suitable for the imprinting procedure because it grows compact (rigid) and exhibits functional groups of various natures12,14,15 (hydrophilic, hydrophobic, basic, etc.). In addition, the relevant films are thin,12 a desirable feature in relation to the response time of the sensor.8 A quartz crystal microbalance (QCM) has been selected as the transducer of the biomimetic sensor, as already done in many bioaffinity sensors (see, for example, ref 16). A direct communication between the imprinted polymer and the transducer has been obtained by using one of the platinum electrodes of the piezoelectric quartz crystal as the working electrode during the polymerization stage. Also, QCM represented a useful tool during the development of the material. Analytical performances of the QCM biomimetic sensor based on glucose-imprinted PPD (iPPD) will be reported in this work, together with the results of control tests performed both on bare crystals and with not-imprinted PPD films. The results show that the presence of the template in the synthesis medium plays a fundamental role in determining the molecular recognition capabilities of the imprinted polymer. Moreover, results relevant to the chemical characterization of iPPD as well as to the investigation of interactions involved in analyte detection process will be presented. (11) Scrosati, B., Ed. Applications of electroactive polymers; Chapman and Hall: London, 1993. (12) Malitesta, C.; Palmisano, F.; Torsi, L.; Zambonin, P. G. Anal. Chem. 1990, 62, 2735. (13) Palmisano, F.; Guerrieri, A.; Quinto, M.; Zambonin, P. G. Anal. Chem. 1995, 67, 1005. (14) Centonze, D.; Malitesta, C.; Palmisano, F.; Zambonin, P. G. Electroanalysis 1994, 6, 423. (15) Losito, I.; De Giglio, E.; Malitesta, C.; Sabbatini, L.; Zambonin, P. G. Spectrosc. Eur. 1997, 9/3, 24. (16) Raman Suri, C.; Mishra, G. C. Biosens. Bioelectron. 1996, 11, 1199.
EXPERIMENTAL SECTION Materials. o-Phenylenediamine (o-PD) (Aldrich) was purified before use by sublimation under vacuum; all the other chemicals (Aldrich) were used as received. Apparatus. Microgravimetric measurements were performed by using 9-MHz, AT-cut quartz crystals (QA-AM9-PT, Seiko EG&G) with circular platinum electrodes (electrode area, 0.196 cm2) on the opposite faces. All the data reported in this paper refer to new crystals. The crystals were mounted in a Teflon holder (well-type, QA-CL4, Seiko EG&G), which enabled exposure of only one face to the liquid, and their terminals connected to the oscillator unit of a quartz crystal analyzer model QCA917 (Seiko EG&G). The ∆f output of the analyzer was recorded by an y-t recorder (Linseis L250 E). Conductance spectra of quartz resonators were acquired by an impedance analyzer (HP4194A). A potentiostat-galvanostat PAR 273 (EG&G), controlled by the M270 software, was used for electropolymerization of o-PD. The well of the QCA917 Teflon holder was used as an electrochemical cell in a three-electrode configuration, using the Pt electrode of the quartz resonator, a Pt wire, and a Ag/AgCl/KCl saturated electrode as working, counter, and reference electrodes, respectively. Sample Preparation. Polymerization of o-PD was performed by cyclic voltammetry (20 scans) in the range 0.0-0.8 V (scan rate 50 mV/s) from a solution of o-PD 5 mM in acetate buffer (pH 5.2). For imprinted polymerizations, glucose was also added at a concentration 20 mM. After the synthesis, the polymer film, still mounted inside the Teflon holder, was subjected to a washing procedure in triply distilled water, to remove the glucose possibly entrapped in the polymeric matrix. Samples of PPD and iPPD for XPS study of glucose-iPPD interactions were prepared by equilibrating the polymer films with 10 mM glucose in acetate buffer and then immersing them in acetate buffer for a short time to remove loosely bonded analyte molecules. Acetate buffer was used in place of glucose in the corresponding blank experiment. Each XPS sample was dried by a gentle nitrogen flux before the analysis. Calibration of the Quartz Crystal Microbalance for Maximum Admittance Measurements. The QCA917 microbalance provides a second output (resonant admittance intensity, Vout) which can be correlated to the maximum admittance magnitude (Ymax) of the quartz resonator equivalent circuit by a calibration procedure. The approach described in ref 17 was selected for this purpose. Briefly, Vout and Ymax values were measured, when quartz resonators were in contact with different media (air, water, ethanol, and water-ethanol and water-glycerol mixtures), by the microbalance and the impedance analyzer, respectively, and used to draw the calibration curve. Verification of the Imprinting Effect. The interaction between glucose and imprinted PPD was checked by exposing the crystals, unmodified or modified by PPD or iPPD and mounted in the well-type holder, to increasing concentrations of glucose. Small aliquots of stock glucose solution (20 or 500 mM) were (17) Muramatsu, H.; Ye, X.; Suda, M.; Sakuhara, T.; Ataka, T. J. Electroanal. Chem. 1992, 322, 311.
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Figure 1. (a) Repetitive cyclic voltammetry of o-PD 5 mM in acetate buffer (pH 5.2) on a Pt electrode of a QCM. Scan rate, 50 mV/s. Number of scans, 20. Glucose concentration, 20 mM. The current decreases cycle by cycle. (b) Corresponding frequency variation.
successively added by a microsyringe to the acetate buffer contained in the well (total volume 550 µL). The same procedure was performed for the selectivity tests. XPS Experiments. XPS analysis was performed with a Leybold LHS10 spectrometer equipped with a twin anode (Mg KR/Al KR) source. Survey scans (kinetic energy range 0-1500 eV, FRR mode, retarding ratio of 3) and high-resolution spectra (spectral range 50 eV, FAT mode, pass energy of 50 eV) were recorded. High-resolution spectra were analyzed by two software packages, both running on a Compaq Deskpro 386 microcomputer. The curve-fitting procedure has been described elsewhere.18 RESULTS AND DISCUSSION Molecular Imprinting Electropolymerization. A typical cyclic voltammogram recorded during the electropolymerization of o-PD on the electrode of a quartz crystal in the presence of glucose is reported in Figure 1a. No significant differences are observed in comparison with those obtained under the same conditions (pH, monomer concentration) but without the molecular template.12 The result could be in part rationalized, considering that glucose does not show any electroactivity on platinum in the potential window chosen for the polymerization. As a result, the template structure is not electrochemically altered while the polymer is growing around it. As suggested by the remarkable decrease of the current intensity, a nonconducting film is formed, progressively covering the electrode surface and leading to the suppression of the voltammetric response. (18) Malitesta, C.; Losito, I.; Sabbatini, L.; Zambonin, P. G. J. Electron. Spectrosc. Relat. Phenom. 1995, 76, 629.
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Figure 2. (a) QCM response of an iPPD-based sensor to glucose injection. The curve is relevant to the evolution of frequency upon addition of glucose up to 20 mM following the steady value for glucose 10 mM. ∆fss represents the steady-state total value. (b) Calibration curve for the sensor. (c) Calibration curve for a sensor showing saturation at lower concentration.
The mass variation on the electrode during the polymerization, monitored by the QCM, confirms this mechanism. As shown in Figure 1b, a remarkable negative variation of the resonance frequency for the quartz crystal (corresponding to a mass increase) is observed during the beginning of polymerization. In the following stage, the decrease of frequency is smaller and finally it reaches a steady value, confirming that there is no further polymerization. Analytical Performances. The response to 20 mM glucose of a sensor based on iPPD film is shown in Figure 2a. A pronounced decrease of the resonant frequency is observed immediately after each injection, and then a steady value (∆fSS) is reached in ∼17 min, a response time in the low range of those generally reported.9 Similar frequency transients were observed on bare Pt electrode of quartz resonators, while the steady-state response was zero. Addition of buffer aliquots showed that the
transient did not originate from the mechanical perturbation caused by injection. Considering that the admittance varied during the transient on both Pt and iPPD, the phenomenon can be tentatively explained by liquid loading (see below) associated to the elevated transitory variation of density and viscosity in the solution layer upon the addition of the concentrated stock glucose solutions. The sensor showed a shelf life of at least one month. Figure 2b shows the typical trend of the iPPD-based sensor calibration curve. It exhibits a linear range up to ∼20 mM; a response saturation is obtained at ∼100 mM. The occurrence of this saturation condition has been associated in the MIP literature19 with the minor role played by nonspecific binding. In some cases (Figure 2c), saturation at much lower concentrations was observed. These cases were always correlated to a polymeric mass deposited lower (by ∼20%) than in the normal case. The origin of this phenomenon likely involves some role for the Pt surface, since a similar weak imprinting and a lower mass deposited were also observed when quartz resonators were reused after mechanical cleaning of electrode surface by alumina powder. Verification of the Imprinting Effect. Different tests were performed in order to confirm that the observed frequency variations were really both correlated to mass changes in the imprinted polymer and caused by the imprinting procedure. Several effects other than mass variation ∆m can cause the observed frequency variation20 ∆f. Among others, modifications of density and/or viscosity of the liquid in contact with the quartz may result in a frequency variation (liquid loading). Its contribution to ∆f was evaluated by measuring Ymax and employing the equations21
∆f )
2f 2
∆m Fη +( ) ] [ A 4πf F
(1)
(
(2)
xjc66
1/2
Q
)
ηQ Fη 1 1 ) + Ymax jc66C1 πC1 πfcj66FQ
where f is the fundamental resonance, A is the piezoelectrically active area, FQ is the density of quartz, jc66 is its piezoelectrically stiffened elastic constant, ηQ is the effective quartz viscosity, and C1 is the motional capacitance of the equivalent circuit for unperturbed QCM. The other symbols have the usual meaning. Even for the highest glucose concentrations the effect of liquid loading is negligible (0.4%). This finding is furtherly confirmed by the result of the experiment with an unmodified quartz crystal (see above). As far as viscoelastic effects of polymer are concerned, they are likely negligible22 since the polymer films are very thin (∼100 Å); also, Ymax shows only a slight variation (∼10%) for the highest glucose concentration. This is confirmed by the similar values of widths of the conductance spectrum for bare and iPPD-covered crystals. A major role for other factors (e.g., surface roughness) is also excluded on comparing iPPD response with Pt and notimprinted PPD ones. (19) Matsui, J.; Miyoshi, Y.; Doblhoff-Dier, O.; Takeuchi, T. Anal. Chem. 1995, 67, 4404. (20) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1355. (21) Martin, S. J.; Edwards Granstaff, V.; Frye, G. C. Anal. Chem. 1991, 63, 2272. (22) Borjas, R.; Buttry, D. A. J. Electroanal. Chem. 1990, 280, 73.
Figure 3. Scatchard plot relevant to calibration curve in Figure 2b (see text).
The correlation of the response with the imprinting procedure was investigated by testing the glucose response of not-imprinted PPD films, which did not show a significant response to the analyte (less than 5% of typical response at 1 mM). This result emphasizes the role played by the molecular template in determining the recognition capabilities of iPPD. The specificity of the recognition mechanism in glucoseimprinted PPD has been also taken into account by checking its response to substances with a different molecular structure. In particular ascorbic acid, paracetamol, and cysteine, which are common interferences in glucose determination in real matrixes, were considered. No appreciable frequency variation was observed for a 1 mM concentration level, thus confirming the selectivity of recognition. On the other hand, a small response was obtained with fructose, which is more similar to glucose in structure. Glucose-iPPD Interactions. Information on the equilibrium
glucose-iPPD h glucose + iPPD was extracted by Scatchard analysis of the calibration curve, a tool already applied in MIP work.19 This approach employs the equation
B/[F] ) Bmax/KD - B/KD
(3)
where B is the amount of glucose bound to the polymer, as calculated by the frequency (mass) variation upon addition of the analyte, and [F] is the concentration of free glucose (approximated by the analytical concentration of glucose). Bmax represents the apparent maximum number of binding sites, and KD the dissociation constant of the complex glucose-iPPD. A Scatchard plot of data in Figure 2b is reported in Figure 3. The fitting of eq 3 to the high-concentration data produced the binding properties (KD ) 34 mM, Bmax ) 3.7 nmol) of the interaction sites. Comparing the number of monomers present in the deposited film, as evaluated from the frequency variation occurring during the polymerization, with the number of binding sites it is possible to estimate that sites involve about two monomers for each glucose molecule. This low ratio could be partially due to the contemporaneous occurrence of some specific adsorption/permeation. Analytical Chemistry, Vol. 71, No. 7, April 1, 1999
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Figure 5. Fitted O 1s XP spectrum for an iPPD film after interaction with 10 mM glucose (see text).
This finding confirms in an independent manner the occurrence of a templating effect.
Figure 4. Comparison of composition of iPPD and PPD as evaluated by C 1s and N 1s XPS signals.
Low-concentration data seem to involve a second group of binding sites, even if the relevant KD and Bmax values cannot be reliably evaluated. The data of Figure 2c permit us to obtain binding parameters (KD ) 4.6 mM, Bmax ) 0.30 nmol) for this second group of sites, whose interaction with analyte appears stronger. Spectroscopic Characterization of iPPD. XPS was employed to compare the chemical structure of the iPPD with that of notimprinted PPD, which has been already investigated.12,14,15 In the last case, XPS worked in practice as a bulk technique, since it was able to sample over all the film thickness (∼100 Å12). Figure 4 reports results of the comparison: only minor differences can be observed. This finding agrees with the idea that molecular imprinting influences mainly the tridimensional arrangement of the polymer, which keeps its chemical composition. A further comparison has been performed after exposure of the above polymers to 10 mM glucose. Figure 5 shows O 1s spectrum for an iPPD-glucose sample. Two components are fitted to the spectrum profile. The peak (OHBE) at higher binding energy was attributed to groups (N-OH) also present in PPD14,15 and to groups (C-OH) belonging to glucose present in the sites of iPPD. The amount of C-OH groups was evaluated by comparing OHBE/N values for the iPPD-glucose sample and for a blank iPPD sample. The obtained difference value (0.3) is in good agreement with that (0.28) estimated by the QCM response (i.e., mass of glucose in sites) of iPPD to the same glucose concentration and by the mass of deposited monomeric units. A similar behavior was not obtained in the experiments involving not-imprinted PPD.
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CONCLUSIONS For the first time, the feasibility of molecular imprinting electropolymerization by a neutral template (glucose) has been demonstrated, so that the procedure can be proposed as a possible way for MIP preparation. This statement is supported by preliminary results obtained by using other templates (e.g., lactose). The approach offers an easy way to the preparation (and, in perspective, to the miniaturization) of biomimetic sensors based on the imprinted polymers (recognition element) directly grown on the (conducting) transducer (QCM). The assembled sensor showed interesting analytical performances, even if it suffers from protein interferences, as already reported for QCM immunosensors.16 Work using a different transducer (electrical or optical) is underway for resolving this problem. XPS characterization of the imprinted polymer supported the hypothesis that the templating procedure influences mainly the spatial organization of the polymeric framework. Schatchard analysis showed the occurrence of two types of binding sites. Theoretical and experimental studies are in progress for rationalizing the values of the relevant KD’s and for understanding the interactions between monomer/oligomer/polymer and glucose causing the templating effect. Work is planned for extending the methodology to other class of analytes (e.g., pesticides, drugs, metabolites) even employing other electrosynthesized polymers. ACKNOWLEDGMENT This work is part of the Ph.D. thesis of I.L. F. Palmisano and L. Sabbatini are gratefully acknowledged for many helpful discussions. Financial support from MURST (ex 40%) and CNR (“Target Project on Biotechnology“) is acknowledged. Received for review June 22, 1998. Accepted January 5, 1999. AC980674G
