Optical sensor materials for the detection of amines in organic solvents

Analytica Chimica Acta 565 (2006) 42–47

Optical sensor materials for the detection of amines in organic solvents Anja Gr¨afe a , Karsten Haupt b , Gerhard J. Mohr a,∗ a

Institute of Physical Chemistry, Friedrich-Schiller University Jena, Lessing St. 10, D-07743 Jena, Germany b Universit´ e de Technologie de Compi`egne, UMR CNRS 6022, France Received 8 November 2005; received in revised form 6 February 2006; accepted 9 February 2006 Available online 28 February 2006

Abstract A new optical polymer-based sensor was developed, which is able to recognize amines in organic solvents with high sensitivity. Thin polymer membranes were prepared and investigated, which contain a chromogenic functional dye (reactand) that shows a significant colour change during a reversible chemical reaction with the analyte. For that purpose the azo dye 4-trifluoroacetyl-4 -[N-(methacryloxyethyl)-N(ethyl)amino]-azobenzene (CR-465) was synthesized, which contains a trifluoroacetyl moiety (receptor for interaction with amines) and in addition, a polymerizable methacrylate group. The methacrylate group links the dye covalently to the polymer matrix and the receptor recognizes the analyte via covalent binding. For immobilisation of the dye cross-linked methacrylate polymers with different composition were used. The highly cross-linked polymer network was stable against most organic solvents and exhibited enhanced stability against mechanical strain compared to plasticized PVC. The sensitivity of the reaction between the analyte and the dye was tailored by the choice of the solvent in which the analysis of the sensor layer was performed, with equilibrium constants for 1-butylamine ranging from 80 to 2000 M−1 in chloroform and DMSO, respectively. In toluene as the solvent, sensor layers typically exhibited equilibrium constants of 100 M−1 for 1butylamine, 1300 M−1 for 1,4-diaminobutane and 20,000 M−1 for tris-(2-aminoethyl)amine. We have also investigated the cross-linked sensor layers with respect to molecular imprinting and did not find any enhancement in selectivity through imprinting in the presence of different analyte molecules. © 2006 Elsevier B.V. All rights reserved. Keywords: Trifluoroacetyl dyes; Amines; Molecular imprinting

1. Introduction Optical sensors for electrically neutral analytes such as amines frequently use unspecific interactions (e.g. hydrogen bonding) with the indicator dyes to generate signal changes [1,2]. The disadvantages of this strategy are the lack of sensitivity and signal magnitude. To obtain sensors with higher sensitivity we developed chromogenic chemosensor molecules (reactands), which can perform reversible chemical reactions with neutral analytes [3–5]. The formation of a covalent bond causes a shift of the absorbance maximum of the dye and allows the quantification of neutral analytes both in solution and in polymer sensor membranes (Fig. 1). For the preparation of stable sensor layers, covalent immobilisation of dyes to the polymer matrix is generally preferred

∗

Corresponding author. Tel.: +49 3641 948379; fax: +49 3641 948302. E-mail address: [email protected] (G.J. Mohr).

0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.02.034

over physical entrapment. Due to covalent immobilisation of the dye in a polymer matrix, migration, crystallisation or decomposition is avoided and the indicator cannot leach into the sample [6]. Generally, methacrylates are used as monomer materials, because they are commercially available and easily synthesized and polymerized. Furthermore, they can be easily introduced into the functional chemosensor dye, providing a polymerizable derivate of this dye. In the present work, a polymer-based sensor for the detection of amines via trifluoracetyl azo dyes is described with special emphasis on the stability against organic solvents and on how to improve the sensitivity of the dye to the analyte. In this work the predominantly investigated analytes are primary aliphatic amines [7], such as 1-butylamine (BA), 1,4diaminobutane (DB) and tris-(2-aminoethyl)amine (TA). They are of interest because they are used for preparation of fertilizers (e.g. urea [8]), pharmaceuticals [9], surfactants, biological buffer substances and colorants [10] and thus they can be found as pollutants in industrial and manufacturing areas. Furthermore,

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trifluoroacetyl-azobenzene (ETHT 4001) was obtained from Fluka (prod. no. 18596). The solvents for membrane preparation were of HPLC grade and stored over a molecular sieve. Amine solutions were prepared by first dissolving the appropriate amount of the respective amine in the desired solvent and afterwards by dilution series. 2.3. Synthesis of 4-trifluoroacetyl-4 -[N-(2-hydroxyethyl)N-(ethyl)amino]-azobenzene

Fig. 1. Recognition of charged analytes via complexation and recognition of neutral analytes via covalent binding to a dye molecule. Apart from an increase in fluorescence upon interaction with the analyte, changes in absorbance can be observed as well.

amines like histamine (HI) and spermidine (SP) were investigated as analytes of interest, because they play an important role in the human organism. 2. Experimental 2.1. Apparatus The absorbance spectra of the dissolved dyes and that of sensor membranes were recorded in a 10 mm quartz cuvette on a Lambda 16 UV–vis spectrometer (Perkin Elmer) at 20 ± 2 ◦ C. Cross-linked polymer membranes were obtained by illuminating monomer mixtures on glass substrates at a wavelength of 312 nm with a BIO-View Transilluminator Model UXDT20SM-8R (Biostep). The equilibrium constants K for the reaction of amines with CR-465 in the sensor membranes were calculated according to Ref. [7], using the following equation: Sx =

KSAR camine + SR 1 + Kcamine

where Sx is the absorbance signal at a defined amine concentration camine , while SAR is the absorbance of the hemiaminal form and SR the absorbance of the trifluoroacetyl form of CR-465. 2.2. Chemicals and reagents Chemicals for synthesis were of analytic reagent grade. For column chromatography silica gel 60 from Fluka was used. For silanisation of the glass supports methacryloxypropyl trimethoxysilane and dry toluene from Fluka were used. For membrane preparation, 2,2-dimethoxy-2-phenyl-acetophenone (DMPAP), methacrylic acid (MA) and trimethylolpropane trimethacrylate (TRIM) were obtained from Aldrich, ethylene glycol dimethacrylate (EDMA), methyl methacrylate (MMA) from Fluka and chloroform (amylene stabilized) from Riedel-deHa¨en. The chromo-reactand 4-N,N-dioctylamino-4 -

First 1.15 g of N-(ethyl)-N-(2-hydroxyethyl)aniline was dissolved in 20 ml of DMF and cooled to 0 ◦ C in an ice bath. A second solution of 1.5 g of 4-trifluoroacetylaniline in 5 ml of DMF was cooled to 0 ◦ C. After addition of 3.8 ml of 6N HCl and subsequently a solution of 0.5 g of sodium nitrite in 5 ml of distilled water, it was added slowly to the first solution, resulting in a colour change from pale yellow to deep red. The reaction mixture was stirred for 5 h at 0 ◦ C and at room temperature over night. The product was extracted by adding 150 ml of dichloromethane and 80 ml of distilled water to the mixture. The organic phase was washed once with a saturated aqueous sodium hydrogen carbonate solution and three times with distilled water. The aqueous solution was extracted once with 100 ml of dichloromethane. Both organic phases were combined, dried over magnesium sulphate and filtered. The resulting red liquid was purified by column chromatography with hexane/ethyl acetate 1:2 (v/v) as the eluent, yielding 1.8 g (73%) of a deep red solid. 1 H NMR (DMSO-d ): δ (ppm) = 8.2 (d), 7.9 (d), 7.8 (d), 6 7.7 (d), 7.6 (d), 6.9 (d), (8H, CH–); 3.5, 3.6 (m, 3H, –OH, –CH2 –O), 3.3 (s, 4H, –CH2 –N); 1.1, 1.2 (m, 3H, –CH3 ); MS: m/z = 365 [M], 334 [M − CH2 OH]. 2.4. Synthesis of 4-trifluoroacetyl-4 -[N(methacryloxyethyl)-N-(ethyl)amino]-azobenzene (CR-465) A solution of 0.12 ml methacryloylchloride in 0.3 ml dry tetrahydrofuran was added dropwise to a cooled (0 ◦ C) solution of 270 mg of 4-trifluoroacetyl-4 -[N-(hydroxyethyl)-N(ethyl)amino]-azobenzene, 1 mg of hydroquinone, 0.3 ml of triethylamine and 5 ml of dry THF. The reaction mixture was heated up to 45 ◦ C for 4 h. Then, a second solution of 0.12 ml methacryloylchloride in 0.3 ml of dry tetrahydrofuran was added at 0 ◦ C, and the reaction mixture was kept at 45 ◦ C for 20 h. After evaporation of THF, the residue was dissolved in 50 ml of dichloromethane, washed twice with saturated aqueous sodium hydrogen carbonate solution and afterwards twice with distilled water. The organic phase was separated, dried over magnesium sulphate, filtered and the solvent was removed under reduced pressure. The remaining red liquid was purified by column chromatography using dichloromethane and subsequently ethyl acetate as eluent, yielding 170 mg (53%) of a deep red oil. DMSO: λmax = 490 nm, ε = 21,600 M−1 cm−1 ; acetonitrile: λmax = 470 nm, ε = 22800 M−1 cm−1 ; chloroform: λmax = 465 nm, ε = 25300 M−1 cm−1 ; toluene: λmax = 460 nm, ε = 22100 M−1 cm−1 . 1 H NMR (CDCl ): δ (ppm) = 8.2 (d), 7.9 (m), 6.8 (dd) (8H, 3 CH–), 6.1 (s), 5.6 (s) (2H, CH2 ), 4.4 (t, 2H, –CH2 –O), 3.8–3.5

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(m, 4H, –CH2 –N), 1.6 (m, 3H, –CH3 ), 1.3, 1.2 (t, 3H, –CH3 ); MS: m/z = 433 [M], 334 [M − CH2 –O–C( O)–C( CH2 )–CH3 ]. 2.5. Silanisation of the glass supports The glass supports were obtained by cutting 76 mm × 26 mm × 1 mm microscope slides into pieces of 9.5 mm × 26 mm. The resulting cuvette-size glass supports were first washed at 80 ◦ C with a mixture of 20 ml of conc. NH3 and 100 ml of H2 O, then with a mixture of 20 ml of 30% H2 O2 and 100 ml of H2 O, and finally with a mixture of 15 ml of conc. HCl, 15 ml of 30% H2 O2 and 90 ml of H2 O. The glass supports were purged two times with water and two times with acetone and afterwards dried in hot air. Immediately after washing the slides twice with dry toluene, the silanisation process was performed by placing the glass substrates into a solution of 3 ml of methacryloxypropyl trimethoxysilane and 0.3 ml of triethylamine in 150 ml of dry toluene. The supports were incubated 17 h in a closed container at room temperature. Then they were washed two times with dry toluene and dried under a stream of nitrogen. The silanised supports were stored dry and in darkness at 4 ◦ C. 2.6. Cocktail preparation For preparation of the monomer solution, 11.8 mg of the initiator (DMPAP) was weighed in a 2 ml plastic cone and dissolved in 350 ␮l solvent, if no other amount is noted in Table 1. Afterwards the (co)monomers and cross-linker (shown in Table 1) were added to this solution and thoroughly mixed. A defined amount of this monomer solution (one-hundredth of the total volume of the monomer solution) and 3.5 ␮l solvent were added to the vial containing 0.43 mg (1 ␮mol) of the dye CR-465. For preparation of molecularly imprinted membranes the amount of 3.5 ␮l plain solvent was replaced by a solution of the template molecule. 2.7. Preparation of the membranes Three microliters of the monomer cocktail were pipetted onto the activated glass plate at room temperature. The agent used for

silanisation of the glass surface contains methacrylate groups that bind the monomers in the cocktail covalently to the glass surface. The solution was immediately covered with a non-activated cover glass plate and a weight of 500 g was placed onto the cover plate. This procedure guarantees thin polymer layers, prevents evaporation of solvents and protects the polymerisation process against oxygen. The glass support with the monomer cocktail was then illuminated for 10 min at 312 nm using a Biostep Transilluminator. Finally, the glass support was placed over night into the same solvent that was used for monomer cocktail preparation (see also Table 1) and then the cover glass slide was removed by ultrasound. 2.8. Membrane characterisation Various thin polymer membranes, consisting of different types and amounts of monomers and solvents were prepared. Table 1 shows the amounts of monomers and cross-linkers used for membrane preparation. The amine solutions were prepared in the same solvent as used for preparation of the membranes. Solutions with different concentrations of the desired analyte were obtained by dilution series. The concentration for selectivity and sensitivity measurements covered a range from 1 × 10−1 to 1 × 10−6 M. To measure the optical properties of the membranes, the sensor layers on the glass supports were placed in a cuvette with the appropriate solvent and examined with an UV/vis-spectrometer against different types and amounts of amines (Fig. 2). 3. Results and discussion 3.1. Optical properties of the indicator dye The synthesized dye CR-465 shows an absorbance maximum at a wavelength of 465 nm for the trifluoroacetyl form and at 420 nm for the hemiaminal form in chloroform as the solvent (Figs. 3 and 4). The maximum of the absorbance spectrum exhibits a significant dependence on the type of solvent and a positive solvatochromism is observed [11].

Table 1 Amounts of the (co)monomer and cross-linker molecules used for membrane preparation and type of template molecule used for preparation of imprinted polymers Membranes

Solvent for monomer cocktail (␮l)

Monomer (␮l)

Cross-linker (␮l)

Template molecule

Molar ratio dye:template

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13

350 Chloroform 350 Toluene 350 Acetonitrile 350 DMSO 350 Chloroform 350 Toluene 700 Toluene 700 Toluene 700 Toluene 350 DMSO 350 DMSO 350 Toluene 350 Toluene

43 MMA 43 MMA 43 MMA 43 MMA 43 MMA 40 MMA + 1.7 MA 43 MMA 43 MMA 43 MMA 43 MMA 43 MMA 39 MMA + 3.4 MA 41 MMA + 1.7 MA

377 EDMA 377 EDMA 377 EDMA 377 EDMA 430 TRIM 377 EDMA 377 EDMA 377 EDMA 377 EDMA 377 EDMA 377 EDMA 377 EDMA 377 EDMA

TA DB BA HI SP TA BA

3:1 2:1 1:1 1:1 1:1 1:1 1:1

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Fig. 3. Synthesis of the functional dye CR-465. Table 2 Equilibrium constants of polymer membranes M1, M2, M3, M4 and a 15 ␮M solution of ETHT 4001 for 1-butylamine Fig. 2. Polymer membrane M1 (containing the dye CR-465) in chloroform (left cuvette) and in 0.1 M 1-butylamine solution in chloroform (right cuvette).

3.2. Sensitivity and selectivity of sensor membranes based on CR-465 toward amines The reaction of the polymer membranes with amines is fast and this rapid response to the analyte is necessary for application in sensor layers. The sensitivity of the membrane towards amines is calculated using the equilibrium constants Kequ. . In principle two effects on sensitivity are observed, the solvent-dependent sensitivity and the dependence on the type of analyte molecule. Membranes M1, M2, M3 and M4 show different sensitivity towards 1-butylamine depending on the type of solvent. Membranes examined in DMSO show by far the highest sensitivity, followed by membranes evaluated in toluene and acetonitrile, which provide comparable sensitivity. The lowest sensitivity is obtained using chloroform as the solvent for the analyte. Table 2 gives an overview about the equilibrium constants of the membranes for 1-butylamine in different solvents. As a ref-

Membranes

Solvent

Kequ. (M−1 )

M1 M2 M3 M4

Chloroform Toluene Acetonitrile DMSO

80 100 150 2000

ETHT 4001 solved in

Kequ. (M−1 )

Chloroform Toluene Acetonitrile DMSO

3.5 60 280 30000

erence, the equilibrium constants of ETHT 4001 to 1-butylamine in the same solvent are shown as well. In the case of DMSO and acetonitrile the reaction with the dye in solution is more efficient than the reaction with the polymer membranes. On the other hand, in toluene and chloroform the membranes react more sensitively than dissolved ETHT 4001. Generally, the order in sensitivity is the same for both

Fig. 4. Reaction of 1-butylamine with the trifluoroacetyl group of immobilised CR-465 causing a colour change from red to yellow.

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Table 3 Calculated equilibrium constants for the reaction of the investigated membranes with different analyte solutions Membrane Template molecule

Solvent Kequ. (M−1 )

M2 M7 M8 M9 M10 M11 M12 M13

Toluene Toluene Toluene Toluene DMSO DMSO Toluene Toluene

– TA DB BA HI SP TA BA

BA

DB

TA

SP

HI

100 150 150 120 – – 120 140

1300 1500 1000 1300 – – 1600 2000

20000 20000 10000 20000 – – 14000 15000

– – – 17000 10000 – –

– – – 2000 – – –

dye solutions and membranes, i.e. highest Kequ. is observed in DMSO followed by acetonitrile, toluene and chloroform. This clearly indicates that the polymer matrix already defines a certain microenvironment and polarity for the dye. The dye inside the polymer matrix is less affected by the added organic solvent than the dye dissolved in the pure solvent. The second effect on sensitivity results from the type of analyte molecule. Independently from the solvent the membranes show an increase in sensitivity with higher number of functional groups at one analyte molecule. As shown in Table 3 the polymer membrane M2 is more sensitive to tris-(2-aminoethyl)amine, having three functional groups than to 1,4-diaminobutane (with only two amino groups). The lowest sensitivity is observed for 1-butylamine as the analyte. As seen in Table 3, the higher sensitivity for analyte molecules with more than one amino group does not only result from a higher number of amino groups per molecule, because the values of Kequ. increase about a factor of 10 for each additional amino group. The higher sensitivity can be explained by an effect observed from Zimmerman and coworkers, where the diamine is expected to provide a more stable adduct due to formation of a hydrogen bonded ring shown in Fig. 5 [12]. In contrast the observed sensitivities for 1-propylamine, 1butylamine and 1-hexylamine were all similar. Consequently, different lipophilicity of primary aliphatic amines did not affect the sensitivity and selectivity in the recognition process. The membrane M5 was also examined towards its reaction with secondary and tertiary amines, as well as to its reaction with 1-pentanol (Fig. 6). The reaction rate with diethylamine and triethylamine is significantly slower than with primary amines, and no shift in the absorbance maximum could be observed within 5 min of reaction time [5,7].

Fig. 5. Formation of a cyclic reaction product from 1,4-diaminobutane (R H) and the trifluoroacetyl group of the reactand.

Fig. 6. Absorbance spectra of membrane M5 upon exposure to 0.1 M triethylamine (TEA), 0.1 M diethylamine (DEA) and 0.1 M 1-pentanol solutions (solvent: chloroform).

3.3. Reversibility and stability After measurement of a concentration series of different amines, the membrane was purged with the plain solvent to remove most of the analyte (>90 %) which was visible from the regeneration of the initial spectrum of the trifluoroacetyl form. This measurement and cleaning procedure could be repeated with the same membrane three to four times, before the colour of the membrane remained orange (i.e. in the hemiaminal form). The membranes M4 and M6 could be used for more than four measurement cycles. The stability of the polymer membranes towards organic solvents is high. Because of the covalent binding of the polymer to the glass surface, the cross-linked polymer network is not soluble in any solvent and resists minor mechanical strain. However, the polymer seems to shrink with time, because the reversibility of the hemiaminal formation gradually decreases after more than four measurement cycles. 3.4. Attempting to affect selectivity via molecular imprinting A major focus of our work was dedicated to enhance the selectivity in the recognition of a desired amine. For that purpose the technique of molecular imprinting [13,14] was used. Before polymerisation, the chromogenic reactand with the polymerizable methacrylate group is brought to reaction with the desired amine (template molecule) in a suitable solvent. To this covalent pre-organized complex, the other monomers and cross-linking molecules are added. The organized complex in the monomer cocktail is then stabilized by the light-induced polymerisation process. After polymerisation, the amine is removed from the polymer by solvent extraction (in the present case >90% of the template were removed according to the ratio in absorbance of the trifluoroacetyl and hemiaminal form), leaving a cavity in the polymer matrix that corresponds in shape and functionality to this amine. This imprint inside the polymer matrix should then selectively recognize the desired amine [15]. The quantity of template used for membrane preparation was adjusted relative to the amount of the functional dye in order

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to form a stoichiometric complex. For amines with only one amino group the ratio between template and dye is one and for templates with more amino groups, this ratio decreases. Working with an excess of template molecule shifts the equilibrium of the reversible chemical reaction to the side of the hemiaminal form. In most cases an excess of amine was necessary to render the hemiaminal formation complete. In a first approach we used a polymer composition containing 80 mol.% of EDMA, 16 mol.% of MMA and 4 mol.% of the dye to prepare imprinted membranes. Table 3 gives an overview about the calculated equilibrium constants of the membranes M7, M8 and M9 investigated with different solutions containing 1-butylamine, 1,4-diaminobutane and tris-(2aminoethyl)amine. Additionally, the membranes M10 and M11 were investigated towards their reaction with histamine and spermidine. Because no enhanced selectivity towards the imprint amine was observed (see Kequ. in Table 3), type and amount of solvent were modified. However, the membranes did not show any solvent-dependent change in selectivity towards the analyte. Therefore, in addition to the covalent recognition, also non-covalent types of binding inside the polymer were tested. For that purpose different amounts of methacrylic acid were used for preparation of the polymer. The carboxylate group of the methacrylic acid and the amino group of the template were intended to form a hydrogen bond that should also hold the template molecule in position. The resulting binding site was supposed to exhibit three sites for recognition, one aminespecific trifluoroacetyl group and two unspecific carboxylate groups [12]. A polymer composition containing 0.8–1.6 mol.% of MA gave membranes with good sensor performance due to appropriate thickness, homogeneity and stability. The membranes M12 and M13 with the methacrylic acid copolymer were examined towards their selectivity (Table 3), but again, they did not exhibit any enhanced selectivity towards solutions containing either tris-(2-aminoethyl)amine or 1-butylamine. 4. Conclusion The cross-linked polymer sensor membranes perform fast reversible chemical reactions with solutions of primary aliphatic amines in most organic solvents. They are stable against organic solvents and mechanical strain, which is necessary for application in industrial waste solutions or reactors. The equilibrium constants vary, depending on the solvent and analyte molecule, between 80 M−1 (e.g. for BA in chloroform) and 20,000 M−1 (e.g. for TA in toluene). A change in selectivity due to the size or

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polarity of the analyte could not be observed. The reaction rate of the membranes with secondary and tertiary amines as well as with alcohols is slower than the rate with primary aliphatic amines. An enhancement in selectivity of polymer membranes for selected primary amines via molecular imprinting could not be achieved. Neither changing the polymer composition, nor the solvent or the type of imprint molecule caused any increase in selectivity. The binding sites inside the polymer were probably not specific enough, because the template molecules were too small and too flexible to form a rigid arrangement during the polymerisation process. However for molecular imprinting, templates with a more functional and rigid structure are expected to cause a higher specificity in the recognition process. For example, larger analytes such as steroids or antibiotics that have an inelastic ring structure and different functional groups are supposed to be better templates for molecular imprinting. We are currently investigating these templates in our laboratory in order to provide enhanced selectivity in analyte recognition. Acknowledgement This work was supported by the Heisenberg Fellowship MO 1062/1-1 and research grant MO 1062/2-1 of Deutsche Forschungsgemeinschaft. This support is most gratefully acknowledged. References [1] S.C. Zimmerman, N.G. Lemcoff, Chem. Commun. (2004) 5. [2] K. Haupt, Chem. Commun. (2003) 171. [3] G.J. Mohr, D. Cittero, C. Demuth, M. Fehlmann, L. Jenny, C. Lohse, A. Moradian, T. Nezel, M. Rothmaier, U.E. Spichiger, J. Mater. Chem. 9 (1999) 2259. [4] G.J. Mohr, U.E. Spichiger, W. Jona, H. Langhals, Anal. Chem. 72 (2000) 1084. [5] G.J. Mohr, Sens. Actuators B 107 (2005) 2. [6] G.J. Mohr, M. Wenzel, F. Lehmann, P. Czerney, Anal. Bioanal. Chem. 374 (2002) 399. [7] G.J. Mohr, C. Demuth, U.E. Spichiger, Anal. Chem. 70 (1998) 3868. [8] M.Z. Liu, F.L. Zhan, L. Wu, et al., J. Polym. Mater. 21 (2004) 213. [9] M.D. Berry, J. Neurochem. 90 (2004) 257. [10] Clayden, Greeves, Warren, Wothers, Org. Chem. (2001) 572f. [11] G.J. Mohr, N. Tirelli, C. Lohse, U.E. Spichiger-Keller, Adv. Mater. 10 (1998) 1353. [12] E. Mertz, J.B. Beil, S.C. Zimmerman, Org. Lett. 5 (2003) 3127. [13] K. Haupt, Analyst 126 (2001) 747. [14] S.A. Piletsky, S. Alcock, A.P.F. Turner, Trends Biotechnol. 19 (2001) 9. [15] G. Wulff, Angew. Chem. (International Edition) 34 (1995) 1812.