Application of non-specific fluorescent dyes for monitoring enantio ...

Fresenius J Anal Chem (1999) 364 : 512–516

© Springer-Verlag 1999

O R I G I N A L PA P E R

Sergey A. Piletsky · Ewald Terpetschnig · Håkan S. Andersson · Ian A. Nicholls · Otto S. Wolfbeis

Application of non-specific fluorescent dyes for monitoring enantio-selective ligand binding to molecularly imprinted polymers

Received: 24 November 1998 / Revised: 29 January 1999 / Accepted: 2 February 1999

Abstract The displacement of non-specific dyes from molecularly imprinted polymer (MIP) chromatographic stationary phases has been used for the detection and quantification of ligand-polymer binding events. A blank polymer and an L-phenylalaninamide-imprinted polymer were prepared using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as a crosslinker. The MIP is first loaded with dye, and a solution of the dye in the eluent is passed through the MIP. If analyte is injected into the dye solution in the eluent, part of the dye is competitively replaced by the analyte from the MIP. Specifically, the competitive displacement of rhodamine B by amino acids and phenylalaninamide (PheNH2), respectively, has been studied under polar and hydrophobic elution conditions. Enantioselective binding of Phe and Phe-NH2 to the imprinted polymer was shown to occur in the micromolar concentration range. It is proposed that the displacement of non-specific dyes from MIPs be used for the development of multisensors based upon these highly specific and stable materials, which provide promising alternatives to the use of biological macromolecules in sensor technology.

Introduction Biosensors based on electrochemical or optical transducers offer great potential for application in clinical analysis, environmental monitoring, and biotechnology, pharmaceutical and food industries, where they provide a powerful and often considerably less expensive alternative to traditional, well-established analytical techniques [1]. Microorganisms, enzymes, antibodies, nucleic acid oligomers as well

S. A. Piletsky · E. Terpetschnig · O. S. Wolfbeis (쾷) University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, D-93040 Regensburg, Germany H. S. Andersson · I. A. Nicholls University of Kalmar, Institute of Natural Sciences, S-39129 Kalmar, Sweden

as synthetic receptors can be used as the molecular recognition biomaterials, and provide the basis for the often high ligand-selectivity of biosensors [2]. The inherent instability of many biological materials, however, can limit the lifespan and area of application of the biosensors. Artificial receptor systems based on MIPs offer a promising alternative to their natural counterparts. Since their initial development [3], MIPs have been used in displacement chromatography [4], as substitutes for enzymes [5] and antibodies in affinity chromatography [6], and for membrane separation processes [7, 8]. Several applications of MIPs in electrochemical and optical sensor technology have also been described [9–12]. MIP-based sensors offer several advantages over traditional biosensors, due to their relative ease of preparation and high stability. Sensor response and the procedures required for sensor regeneration, however, are generally slow (30–45 min) because of the lengthy washing procedure required [13]. To enable the practical application of MIPs in sensor technology, improvements in polymer preparation and sensor design and performance must be achieved. Although many chromatographic and batch binding applications of MIPs have previously been described in detail, these require either a UV-active or a radio-labelled ligand [14]. More recently, we have reported on recognition analysis using fluorescence detection [15]. Another approach consists in the displacement of the labelled template from a column with an MIP by free template [16]. Nonetheless, these methods are inherently limited in terms of the range of substances, that may be studied. The later studies have suggested the use of displacement chromatography, i.e. the competition between a ligand and a free dye for a binding site. Such a method is envisaged as being useful for the evaluation of ligand-polymer affinities in cases where the material is either not amenable to simple spectroscopic analysis, or not readily available in a radiolabelled form. This is of practical importance for the development of MIP-based sensors for use in network arrays (multisensor arrays). In such a system, an analyte added to an MIP array should displace the dye in proportion to its affinity for the polymer.

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Here we present a study on a further extension of an idea reported recently [16], and demonstrate that selective displacement of a non-specific dye from an MIP using various analytes (the template and template-related substances) is possible.

Experimental Materials All chemicals and solvents were of analytical or HPLC grade and used as received: acetic acid, chloroform (Carl Roth, Germany); 4-aminobenzoic acid, L(+)-α-phenylglycine, L-phenylalaninamide (L-Phe-NH2) and D-phenylalaninamide (D-Phe-NH2) (Fluka; Switzerland); L-phenylalanine (L-Phe), L-tryptophane (L-Trp; from Mann Research Labs; Sweden); acetonitrile, D-phenylalanine (D-Phe), ethyleneglycol dimethacrylate, methacrylic acid, methanol (Merck; Germany); 4-nitrophenol (Riedel-de Haën); dansyl-L-Phe (Sigma; Germany). Polymer synthesis A polymer molecularly imprinted with L-phenylalaninamide (the “template”) was prepared by dissolving the template, methacrylic acid and ethyleneglycol dimethacrylate (the crosslinker) in a 50 mL glass test tube (see Table 1) in chloroform. The monomer template mixture was purged thoroughly with nitrogen and sealed before polymerization. Polymerization was initiated by addition of 1% (w/w) azobis(isobutyronitrile) (AIBN) and heating at 80 °C for 18 h. The resulting polymer was crushed and ground. Particles of ~ 60 µm size were collected by sieving and sedimentation from acetone. The polymer powder was washed with an acetic acid/chloroform solution (1 : 9; v/v) and methanol to remove unreacted monomers, template and initiator. A blank (non-imprinted) reference polymer was prepared identically, the polymerization being carried out in the absence of the template.

tion factors (α) were calculated from the relationship α = κ′D/κ′L, where κ′D and κ′L are the capacity factors of the D- and L-isomers, respectively. Displacement experiments Displacement experiments were carried out as follows. A 10 µM solution of a dye in the eluent was run through the column at flow rate of 1 mL min–1. Saturation was assumed to have occurred when no change was detected in the concentration of dye eluted from the column (1–2 h). Rhodamine B was detected at 551 nm and 4-nitrophenol at 380 nm. Aliquots (100 µL) of analyte solution (1– 500 µM in the eluent containing the dye) were injected, and the peak areas (arising from dye displacement) were calculated (Shimadzu C-R3 A chromatographic integrator). System re-equilibration, i. e the time to re-load the MIP with dye, generally required around 20 min.

Results and discussion L-Phe-NH2 was molecularly imprinted in a (methacrylic acid)(ethyleneglycol dimethacrylate) copolymer, and a nonimprinted polymer was prepared similarly. The ligand recognition of this MIP system is based primarily upon electrostatic interactions which have previously been carefully investigated and characterized [17]. A schematic representation of the binding site of an MIP imprinted with L-Phe-NH2 is given in Fig. 1. The blank polymer is assumed to possess a random disposition of the carboxy groups, forming non-specific binding sites. Solvents varying in polarity from water to acetonitrile and methanol, and several dyes (dansyl-L-Phe, 4-nitrophenol and rhodamine B) were used for displacement chromatography. Due to the low solubility of the amino acids

High-performance liquid chromatography Polymer particles (1.0 g) were packed using acetone into stainless steel HPLC columns (4.6 × 100 mm) at 300 × 105 Pa, using an air driven fluid pump (Haskel Engineering Supply Co., UK). Columns were linked to an HPLC and washed on-line with the acetic acid/chloroform mixture until a stable baseline was obtained. Chromatographic experiments were performed using a Knauer (Germany) HPLC system. To determine retention times, analyte solutions (200 µM) were prepared using the eluent (without dye) as the solvent, and 100 µL were injected for each analysis. Analyses were run at a flow rate of 1 mL min–1 with detection at 257 nm. All chromatographic data presented represent the results of at least triplicate experiments. Capacity factors (κ′) were determined from the relationship κ′= (t–to)/to, where t is the retention time of a given species and to is the retention time of void marker (acetone). Effective enantio-separa-

Table 1 Composition of cocktails for making the moleculary imprinted polymer P1 and the non-imprinted polymer K1 Poly- Methacrylic EDMA, Chloroform, mer acid, mmol mmol (g) mL (g)

L-Phe-NH2, AIBN, mmol (g) g

P1 K1

4 (0.71) –

16 (1.38) 16 (1.38)

65.6 (13) 14 65.6 (13) 14

0.33 0.33

Fig. 1 Schematic representation of L-phenylalaninamide interacting with the specific binding site of the imprinted polymer

514 Table 2 Retention times (RT; min) of the analytes on imprinted (P1) and blank (K1) polymers, respectively, using methanol. The analyte concentration was 200 µM in a 100-µL injection sample Analyte

RT with methanol

L-Phe-NH2 D-Phe-NH2 L-Phe D-Phe L-phenylglycine L-Trp Aminobenzoic acid Rhodamine B Acetone

Imprint P1

Blank K1

10.02 8.91 2.09 2.03 1.7 3.12 – 2.33 1.79

7.89 7.9 1.4 1.4 1.34 1.46 – 1.76 1.38

Table 3 Dissociation constants (Kd) and number of the binding sites (Bt) obtained for MIP and blank polymer from frontal analysis in methanol. The retention time of the void marker is 1 min Polymer

P1 K1

L-Phe-NH2

Rhodamine B

Kd, µM

Bt, mmol/g

Kd, µM

Bt, mmol/g

59.3 83.2

1.8 1.1

133 108

2.7 1.9

in hydrophobic solvents, most of the measurements were carried out in methanol. The recognition characteristics of the polymers were evaluated – using the template and structurally related substances – in terms of retention times and by frontal analysis (Table 2 and 3), which permits determination of the binding site populations and the dissociation constant for the ligand bound to the MIP. As expected, L- Phe-NH2 showed a higher affinity for the MIP than the blank polymer. The MIP demonstrated enantioselectivity (α = 1.29) in experiments using acetonitrile/acetic acid while the blank polymer did not at all. In the case of methanol, the enantioselectivity of the MIP was lower (α = 1.18), which is attributed to the adverse influence of the more polar solvent methanol on the strength of the electrostatic interactions responsible for molecular recognition. Again, no specificity was observed in the case of blank polymer. The specificity of the MIP for L- and D-Phe in methanol is quite pronounced (α = 1.25), but unfortunately the difference in the retention times for the L- and D-enantiomers is very small. Rhodamine B displays strong absorption and fluorescence in the visible and contains tertiary amino groups capable of interacting with the carboxy moieties of the polymer. Dansylated Phe requires UV excitation and can be expected to strongly interact through hydrogen and electrostatic bonding with the cavities of the MIP. This is true to some extent for the rather small (and non-fluorescent) 4-nitrophenol. Initial experiments were carried out with 4nitrophenol. When a solution of L- or D-Phe-NH2 in the eluent containing dye was injected onto a column with a constant flow of eluent containing 10 µM 4-nitrophenol, displacement peaks were observed which were rather

broad, thus making quantification difficult in case of template concentrations of 0.25 mM. In general, L-Phe-NH2 generated displacement peaks 1.3 to 2 times larger than D-Phe-NH2, depending on the solvent used. In case of dansyl-L-Phe, no displacement of dye by template was observed in the solvents studied, i.e water, methanol and acetonitrile. This result may be explained by the steric requirement of dansyl-L-Phe relative to the template, and by its relatively high hydrophobicity, leading to less efficient competition for the polar carboxy residues present in the binding sites. Rhodamine B yielded the best results when displacement experiments were performed in either acetonitrile containing 0.5% (v/v) acetic acid or water/methanol. No displacement was observed in the case of water or water/acetonitrile mixtures, again probably because of too high a level of hydrophobic interaction between dye and polymer thus preventing competition between dye and template. If, however, experiments were carried out in methanol (where the solubility of amino acids is better than in acetonitrile and non-specific interactions between dye and polymer are less pronounced than in water), the areas of the displacement peaks in blank polymers were proportional to the analyte retention time (Fig. 2). In the case of the MIP this dependence was only observed at analyte concentrations of > 100 µM, though at analyte concentrations of < 100 µM the dependence of the displacement peak area of the D- and L-forms of the template were different (Fig. 3). The difference in the sensor response to L- and D-enantiomers (DL/DD, the ratio of displacement dye peaks areas for L- and D-enantiomers) varied from 1.3 to 1.7, depending on analyte concentration. The role of solvent polarity on the specificity of the displacement process was examined by eluting with acetonitrile (0.5% acetic acid). Because amino acids are not soluble in this composition, 4-aminobenzoic acid was used as a non-specific marker. High selectivity was ob-

Fig. 2 Dependence of the peak area of dye displaced by analytes from blank polymer on analyte concentration. Displacement was detected in methanol containing 10 µM of Rhodamine B. Detection at 551 nm. Displacement peak maxima were observed at 1.7 min for amino acids and at 3 min for the respective amides

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Fig. 3 Dependence of the peak area of dye displaced by various analytes from a polymer imprinted with L-phenylalaninamide on analyte concentration. Same experimental conditions as in Fig. 2

Fig. 4 Dependence of the peak area of dye displaced by analytes from L-phenylalaninamide-imprinted polymer on analyte concentration. Displacement was detected in acetonitrile/acetic acid (0.5%) containing 10 µM of Rhodamine B. Flow rate 1 mL min–1. Detection at 551 nm. Displacement peak maxima were observed at 2.1 min for both polymers (P1 and K1)

served in the experiments on displacement of rhodamine B by Phe-NH2 in acetonitrile with 0.5% of acetic acid (Fig. 4). The difference in response for L- and D-enantiomers (DL/DD) varied from 1.1 to 2.5, depending on analyte concentration. The higher specificity of rhodamine B displacement in acetonitrile in comparison to methanol is due to the fact that imprints formed in chloroform can preserve their structure better in the relatively hydrophobic acetonitrile than in water or methanol, wherein they show different swelling. Additionally, methanol – unlike acetonitrile – is capable of forming hydrogen bonds and thus is able to disrupt the specific interaction between the polymer and template. In the case of blank polymer, no difference in displacement peaks for L-, and D-enantiomers of phenylalaninamide was observed. In order to better understand these observations, and the difference in response of the imprinted and blank

polymers, it is necessary to characterize the nature of the “binding” of both analyte and dye to the polymer. Dissociation constants were determined for L- and D-Phe-NH2 and for rhodamine B by frontal analysis [18] (Table 3). Analysis was carried out only for amides (the templates) and rhodamine B, because of the low solubility of amino acids in methanol. The dissociation constants obtained are indicative for the average specificity of the polymer receptor sites. This analysis revealed the presence of a larger site population and sites of higher affinity for L-Phe-NH2 in the MIP than in the blank polymer. Another important fact is the longer retention time of rhodamine B on the imprinted polymer when compared to the blank, which suggests that this is not so much due to higher affinity sites, but to the larger number of sites available. The results indicate that the blank polymers contain a population of binding sites of relatively low affinity, whereas MIPs possess a range of binding sites of low and high affinity [19]. We assume that at low concentrations the competition between template and dye takes place to a slight extent only. This is supported by the enantioselective displacement observed for the enantiomers of the template, where template molecules interact primarily with specific sites and rhodamine B (because of steric hindrance) interacts mostly with non-specific sites. Because specific binding sites are most suitable for accommodation of templates (and molecules of closely related structure), steric factors play an important role in the displacement process. This is reflected by the fact that binding sites for L-Phe-NH2 can interact with L-Phe, but cannot readily accommodate the more sterically demanding LTrp molecules or D-enantiomers. The high response for L-Phe can be explained by its ability to compete with the dye for the binding with both specific and non-specific sites. No significant difference was observed in the behavior of the MIP and blank polymers in titration experiments with sodium hydroxide (data not shown). This indicates that it is only the spatial disposition of the functional groups that differs between the MIP and blank polymers and that this difference, along with shape complementarity, is responsible for the specificity. Evidently, several factors are essential for characterizing MIP sensors based on dye displacement. The specific displacement of the dye by an analyte from a chromatographic column packed with an MIP represents a novel approach to multisensor technology. In particular, we envisage that a multiple analytical device may be constructed on a chip containing a microfabricated column as described by Regnier [20].

Conclusion Displacement chromatography has been demonstrated to be useful means for monitoring interactions between analyte and a molecularly imprinted polymer. The method, based on the displacement of a non-specific dye by an an-

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alyte, is highly reproducible and relatively sensitive, as shown by the detection of the optical isomers of Phe and Phe-NH2 in the micromolar concentration range. It is suggested that the approach be used for multisensor development, particularly for the determination of non-fluorescent analytes.

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