Molecular Engineering of Fluorescent Penicillins for Molecularly

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Anal. Chem. 2006, 78, 2019-2027

Molecular Engineering of Fluorescent Penicillins for Molecularly Imprinted Polymer Assays Elena Benito-Pen˜a,† Marı´a C. Moreno-Bondi,*,† Santiago Aparicio,‡ Guillermo Orellana,*,‡ Josefine Cederfur,§ and Maria Kempe§

Department of Analytical Chemistry and Department of Organic Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040 Madrid, Spain, and Department of Experimental Medical Science, Biomedical Center B12, Lund University, SE-22184 Lund, Sweden

The interaction of seven novel fluorescent labeled β-lactams with a library of six polymer materials molecularly imprinted (MI) with penicillin G (PenG) has been evaluated using both radioactive and fluorescence competitive assays. The highly fluorescent competitors (emission quantum yields of 0.4-0.95) have been molecularly engineered to contain pyrene or dansyl labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers. Pyrenemethylacetamidopenicillanic acid (PAAP) is the tagged antibiotic that provides the highest selectivity when competing with PenG for the specific binding sites in a MI polymer prepared with methacrylic acid and trimethylolpropane trimethacrylate (10:15 molar ratio) in acetonitrile in the presence of PenG. Molecular modeling shows that recognition of the fluorescent analogues of PenG by the MI material is due to a combination of size and shape selectivity and demonstrates how critical the choice of label and tether chain is. PAAP has been applied to the development of a fluorescence competitive assay for PenG analysis with a dynamic range of 3-890 µM in 99:1 acetonitrile-water solution. Competitive binding studies demonstrate various degrees of cross-reactivity for some antibiotics derived from 6-aminopenicillanic acid, particularly amoxicillin, ampicillin, and penicillin V (but not oxacillin, cloxacillin, dicloxacillin, or nafcillin). Other antibiotics, such as chloramphenicol, tetracycline, or cephapirin, do not compete with PAAP for binding to the imprinted polymer. The MI assay has successfully been tested for PenG analysis in a pharmaceutical formulation. Biological recognition elements such as antibodies, enzymes, and nucleic acids have traditionally played a key role in the development of highly sensitive and selective analytical methods based on different transduction mechanisms. However, the limited operational and storage stability of these biomolecules, along with the difficulties associated with their preparation and isolation, are among the drawbacks that have limited so far commercialization of many biosensors.1-3 * To whom correspondence should be addressed. E-mail: mcmbondi@ quim.ucm.es; [email protected]. † Department of Analytical Chemistry, Complutense University of Madrid. ‡ Department of Organic Chemistry, Complutense University of Madrid. § Lund University. 10.1021/ac051939b CCC: $33.50 Published on Web 01/28/2006

© 2006 American Chemical Society

Molecular imprinting allows the design and synthesis of artificial materials containing receptor sites for a large variety of target species.4 In this technique, the analyte molecule (template) is allowed to form a noncovalent or covalent complex with a functional monomer that is copolymerized with a cross-linker to yield a three-dimensional network structure.5,6 After removal of the template, the polymer material will bear cavities with complementary size, geometry, and arrangement of functional groups to the target analyte. Molecularly imprinted polymers (MIPs) may eventually become an attractive alternative to antibodies as selective recognition elements for chemical sensing: they are not affected by harsh conditions during the measurement process, can be used both in organic and in aqueous solvents facilitating determination of antigens under nonphysiological conditions, and, in principle, can be produced for recognizing species for which there are no antibodies available. Moreover, the cost of obtaining MIPs is lower and their preparation and storage easier than those of antibodies. The application of MIPs in optical sensing has adopted so far three formats. The first one is based on selective binding of a spectroscopically quantifiable template to the polymer material;7-10 when the analyte does not display optical properties for the spectroscopic analysis it can be determined using a labeled template or analogue derivative in a displacement or competitive assay.11-18 According to a second method, rebinding of the template to the MIP induces a variation of the optical properties (1) Cram, D. J. Nature 1992, 356, 29-36. (2) Lehn, J. M. Angew. Chem., Int. Ed. Engl. 1988, 27, 89-112. (3) Ramamurthy, R.; Schanze, K. S. Optical Sensors and Switches; Marcel Dekker: New York, 2001. (4) Sellergren, B., Ed. Molecularly Imprinted Polymers. Man Made Mimics of Antibodies and Their Applications in Analytical Chemistry; Elsevier: Amsterdam, 2001. (5) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (6) Mayes, A. G.; Mosbach, K. Trends Anal. Chem. 1997, 16, 321-331. (7) Dı´az-Garcı´a, M. E.; Badia, R. Moleculaly Imprinted Polymers for Optical Sensing Devices. In Optical Sensors. Industrial, Environmental and Diagnostic Applications; Narayanaswamy, R., Wolfbeis, O. S., Eds.; Springer: Berlı´n, 2004. (8) Dickert, F. L.; Besenbo ¨ck, H.; Tortschanoff, M. Adv. Mater. 1998, 10, 149151. (9) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495-2504. (10) McNiven, S.; Karube, I. Toward Optical Sensors for Biologically Active Molecules. In Molecularly Imprinted Polymers. Man Made Mimics of Antibodies and Their Applications in Analytical Chemistry; Sellergren, B., Ed.; Elsevier: Amsterdam, 2001. (11) Piletsky, S. A.; Piletskaya, E. V.; Elskaya, A. V.; Levi R.; Karube, I. Anal. Lett. 1997, 30, 445-455.

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of molecular probes incorporated in the polymer backbone.19-25 Finally, release of spectroscopically active species after a catalytic reaction originated in a specific MIP cavity can also be employed for sensing purposes.26,27 Competitive binding assays using MIPs as recognition elements in place of antibodies are commonly referred to as a molecularly imprinted assay (MIA). Most of the labeled analytes described in the literature for MIA are radioactive derivatives but fluorescent tags are also known.28-31 However, it is common that the analyte cannot be easily derivatized with a fluorescent label or the labeled derivative is not able to bind specifically to the polymer in competition with the unlabeled analyte.14,18 The latter situation is due to the fact that the MIP cavities are complementary to the template and are not able to accommodate the labeled analogue. This problem has also been found when using fluorescent derivatives for the assay unrelated to the analyte structure.11,13,15 Penicillins and cefalosporins belong to the so-called “classical” β-lactam antibiotics (BLAs) due to characteristic four-membered amide moiety they contain.32 The molecular basis of their bactericide action is extremely complex, yet the essentials are currently well understood. BLAs are lethal to bacteria but innocuous to mammals due to the fact that their toxicity arises from interference with the cell wall biosynthesis, a structure that eukaryotic cells do not possess. The screening for these drugs in dairy products and other foodstuffs has been traditionally carried out using immunological, microbiological, receptor, or enzymatic assays; confirmatory analyses are based mainly on HPLC-MS techniques.33,34 (12) Piletsky, S. A.; Piletskaya, E. V.; Yano, K.; Kugimiya, A.; Elgersma, A. V.; Levi, R.; Kahlow, U.; Takeuchi, T.; Karube, I. Anal. Lett. 1996, 29, 157170. (13) Piletsky, S. A.; Terpetschnig, E.; Anderson, H. S.; Nichols, I. A.; Wolfbeis, O. S. Fresenius J. Anal. Chem. 1999, 364, 512-516. (14) Haupt, K.; Mayes, A. G.; Mosbach, K. Anal. Chem. 1998, 70, 3936-3939. (15) Levi, R.; McNiven, S.; Piletsky, S. A.; Cheong, S. H.; Yano, K.; Karube, I. Anal. Chem. 1997, 69, 2017-2021. (16) McNiven, S.; Kato, M.; Levi, R.; Yano, K.; Karube, I. Anal. Chim. Acta 1998, 365, 69-74. (17) Suarez-Rodriguez, J. L.; Diaz-Garcia, M. E. Biosens. Bioelectron. 2001, 16, 955-961. (18) Rachkov, A.; McNiven, S.; El’skaya, A.; Yano, K.; Karube, I. Anal. Chim. Acta 2000, 405, 23-29. (19) Cooper, M. E.; Hoag, B. P.; Gin, D. L. Polym. Prepr. 1997, 38, 209-210. (20) Rathbone, D. L.; Ge, Y. Anal. Chim. Acta 2001, 435, 129-136. (21) Turkewitsch, P.; Wandelt, B.; Darling, G. D.; Powell, W. S. Anal. Chem. 1998, 70, 2025-2030. (22) Liao, Y.; Wang, W.; Wang, B. Bioorg. Chem. 1999, 27, 463-476. (23) Matsui, J.; Tachibana, Y.; Takeuchi, T. Anal. Commun. 1998, 35, 225227. (24) Wang, W.; Gao, S.; Wang, B. Org. Lett. 1999, 8, 1209-1212. (25) Jenkins, A. L.; Uy, O. M.; Murray, G. M. Anal. Chem. 1999, 71, 373-378. (26) Wolfbeis, O. S.; Terpetschnig, E.; Piletsky S. A.; Pringsheim E. Fluorescence techniques for probing molecular interactions in imprinted polymers. In Applied fluorescence in chemistry, biology and medicine; Rettig W., Strehmel B., Schrader S., Seifert H., Eds.; Springer-Verlag: Berlin, 1999. (27) Chow, C. F.; Lam, M. H. W.; Leung, M. K. P. Anal. Chim. Acta 2002, 466, 17-30. (28) Piletsky, S. A.; Turner, A. P. F. New materials based on imprinted polymers and their application in optical sensors. In Optical biosensors: Present and future; Ligler, F. S., Rowe Taitt, C. A., Eds.; Elsevier: Amsterdam, 2002. (29) Vlatakis, G.; Andersson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645-647. (30) Andersson, L. I. J. Chromatogr., B 2000, 739, 163-173. (31) Al-Kindy, S.; Badia, R.; Sua´rez-Rodrı´guez, J. L.; Dı´az Garcı´a M. E. Crit. Rev. Anal. Chem. 2000, 30, 291-309. (32) Analytical Profiles of Drug Substances; Academic Press: New York, 19721988; Vols. 1-17,

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Some fluorescent-labeled BLAs have been described in the literature and could be applied for optical sensing purposes. For instance, penicillin bearing a fluorescein moiety allowed the identification and detection of less than 10 fmol of penicillinbinding proteins.35 Some o-phthaldialdehyde,36 fluorescamine,37 or 9-isothiocyanateacridine38 derivatives of BLAs have been produced for chromatographic analysis of the antibiotics. BLAs containing luminescent Ru(II) polypyridyl complexes were synthesized by Liang et al. to quantify the activity of β-lactamases.39 However, the rather small number of fluorescent-labeled BLAs described so far is due to the difficulties of their preparation (low solubility of the starting materials, instability of the β-lactam ring, inadequate purification procedures, etc.). Actually, many of them were generated in situ and never isolated or purified. We report in this paper the development of an imprinted-based competitive assay for penicillin G (PenG) analysis based on novel highly fluorescent derivatives of the β-lactam antibiotics.40 The target antibiotic has been labeled with pyrene or dansyl moieties according to the following criteria essential to the sought application: availability and low price of precursors, ease of preparation, high emission quantum yield, and resemblance to the analyte. Several PenG-imprinted polymers from a library developed by Cederfur et al.41 have been selected for the study. The interaction of the BLAs with the MIPs was evaluated, in a first instance, using ligand-binding assays with radiolabeled PenG. From these experiments, the fluorescent BLA showing the highest competition toward PenG was chosen and applied for the development of a sensitive binding assay for the antibiotic analysis. The figures of merit of the competitive method and the cross-reactivity to other antibiotics have also been evaluated. EXPERIMENTAL SECTION Reagents and Chemicals. Penicillin G potassium salt (PenG), anhydrous amoxicillin (AMOX), ampicillin trihydrate (AMPI), penicillin V potassium salt (PenV), oxacillin sodium salt (OXA), cloxacillin sodium salt (CLOX), dicloxacillin sodium salt (DICLOX), nafcillin sodium salt (NAFCI), norfloxacin (NOR), oxytetracycline sodium salt (OXY), doxycycline hydrochloride (DOXY), chloramphenicol (CLF), erythromycin (ERY), and cephapirin sodium salt (CEPH) were purchased from Sigma-Aldrich (St. Louis, MO). Tetracycline hydrochloride (TCY), 6-aminopenicillanic acid (6-APA), 4-aminophenylsulfone (dapsone, DDS), methacrylic acid (MAA), 2-hydroxyethyl methacrylate (HEMA), methacrylamide (MAM), ethylene glycol dimethacrylate (EDMA), (33) Knecht, B.G.; Strasser, A.; Dietrich, R.; Martlbauer, E.; Niessner, R.; Weller, M.G. Anal. Chem. 2004, 76, 646-654. (34) Di Corcia, A.; Nazzari, M. J. Chromatogr., A 2002, 974, 53-89. (35) (a) Lakaye, B.; Damblon, C.; Jamin, M.; Galleni, M.; Lepage, S.; Joris, B.; Marchand-Brynaert J.; Frydrych, C.; Fre`re J.-M. Biochem. J. 1994, 300, 141-145. (b) Galleni. M.; Lakaye. B.; Lepage. S.; Jamin. M.; Thamm. I.; Joris. B.; Fre`re. J.-M. Biochem. J. 1993, 291, 19-21. (36) Rogers, M. E.; Adlar, M. W.; Saunders G.; Holt G. J. Chromatogr. 1984, 297, 385-391. (37) Nakagawa H.; Nishiyama K.; Higashitani T.; Ishikawa S.; Fukui Y. Yakugaku Zasshi 1985, 105, 1096-1099. (38) Sinsheimer J. E.; Hong D. D.; Burkhalter J. H. J. Pharm. Sci. 1969, 58, 1041-1042. (39) Liang, P.; Dong, L.; Martin, M. T. J. Am. Chem. Soc. 1996, 118, 91989199. (40) Orellana, G.; Aparicio, S.; Moreno-Bondi, M. C.; Benito-Pen ˜a, E. Spanish Patent 2,197,811, 2004. (41) Cederfur, J.; Pei, Y.; Zihui, M.; Kempe, M. J. Comb. Chem. 2003, 5, 67-72.

Figure 1. Chemical structures of the novel fluorescent derivatives of 6-aminopenicillanic acid.

trimethylolpropane trimethacrylate (TRIM), and 1,1′-azobis(cyclohexanecarbonitrile) (ABCHC) were from Sigma-Aldrich. tertButyloxycarbonyl phenylalanine (Boc-L-Phe-OH) was from Fluka (Buchs, Switzerland). Monomers and cross-linkers were purified as described previously.41 2,2′-Azobisisobutyronitrile (AIBN) from Acros (Geel, Belgium) was recrystallized from methanol before use. Scintillation cocktail Ecoscint-A was from National Diagnostics (Hessle Hull, England). [Phenyl-4n-3H]benzylpenicillin was from Amersham Pharmacia (Uppsala, Sweden). Methanol (HPLC gradient grade) was from SDS (Peypin, France), and acetonitrile (p.a. grade) was purchased from Merck (Darmstadt, Germany). Water was purified using a Millipore purification system (MilliQ). Acetic and trifluoroacetic acids were from Scharlab (Barcelona, Spain) and Fluka, respectively. Clavulanic acid lithium salt (CLAV) was provided by Dr. Johannes van Rhijn (Rikilt, Wageningen, The Netherlands). MIP Synthesis. The polymer materials were prepared following the procedure described previously.41 Briefly, 1 mmol of PenG was weighed into a screw-cap borosilicate glass tube and suspended by sonication in a portion of the porogen solvent. Enough MAA to dissolve the antibiotic template was added. The remaining porogen and monomer were then incorporated, followed by addition of the cross-linker and the initiator. The prepolymerization mixture was cooled in ice and purged with a stream of nitrogen gas for 10 min. Copolymerization was carried out by irradiation at 350 nm for 24 h at 4 °C in a Rayonet RMR600 photochemical minireactor (Branford, CT). The bulk polymers were ground in a Retsch ZM 100 ultracentrifugal mill (Haan, Germany). The ground particles were wet sieved in water, and size fractions of 50-100 µm were selected for all the experiments. The composition of the synthesized polymers used in the competitive assays with the fluorescent BLAs is shown in Table 1. Control

Table 1. Composition of the Molecularly Imprinted (template: 1 mmol of penicillin G) and Control (template: 1 mmol of Boc-L-Phe-OH) Polymer Libraries41

MIP

functional monomer(s) (mmol)a

cross-linker (mmol)b

initiator (mmol)c

solvent (mL)

1 2 3 4 5 6

MAA (10) MAA (10) MAA (14) MAA-MAM (10:6) MAA-HEMA (10:6) MAA-HEMA (14:4)

TRIM (15) TRIM (40) EDMA (70) TRIM (48) TRIM (28) EDMA (90)

AIBN (0.6) ABCHC (0.5) AIBN (0.84) ABCHC (0.64) AIBN (0.4) AIBN (1.1)

acetonitrile (7.5) acetonitrile (8.0) acetonitrile (14) acetonitrile (33.6) acetonitrile (13.6) acetonitrile (25.5)

a MAA, methacrylic acid; MAM, methacrylamide; HEMA, 2-hydroxyethyl methacrylate. b TRIM, trimethylolpropane trimethacrylate; EDMA, ethylene glycol dimethacrylate. c AIBN, 2,2′-azobisisobutyronitrile; ABCHC, 1,1′-azobis(cyclohexanecarbonitrile).

polymers (CP) were prepared for each MIP composition using Boc-L-Phe-OH instead of PenG as the template. Thorough extraction of the template and fluorescent impurities from the polymers was carried out using the following procedure: the ground polymers were suspended on a shaking table in 1:4 methanol-acetic acid solution (3 × 1 h + 1 × 16 h), methanol (4 × 25 min + 1 × 16 h), acetonitrile (3 × 2 h + 1 × 120 h), and methanol (4 × 1 h + 1 × 72 h). The granules were then extracted with acetonitrile in a Soxhlet apparatus for 48 h, shaken with 1:1 acetonitrile-water for 48 h, and Soxhlet-extracted again with acetonitrile for 48 h. Finally, the MIP powder was dried in a vacuum oven (40 °C, 48 h). The fluorescence of the washing solvent in the 350-450-nm region was checked after each successive cleaning step until no signal could be distinguished from that of the blank solvent. Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

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Synthesis of Pyrene- and Dansyl-Labeled BLAs. β-Lactam antibiotics containing a pyrene moiety (Figure 1) were prepared from 6-APA or the corresponding BLA and the succinimidyl esters (obtained from the commercial acids and N-hydroxysuccinimide) of pyrenebutyric or pyreneacetic acids (Aldrich) in acetonewater-NaHCO3.40 The following pyrene derivatives were synthesized: [2S,5R,6R]3,3-dimethyl-7-oxo-6-[(pyren-1-ylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (PAAP); [2S,5R,6R]-3,3-dimethyl-7-oxo-6-[(4-pyren-1-ylbutanoyl]amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (PBAP); [2S,5R,6R]-6-{[(2R)-2-amino-2(4-hydroxyphenyl)ethanoyl]amino}-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (PAAX); [2S,5R,6R]3,3-dimethyl-7-oxo-6-({(2R)-2-phenyl-2-[(pyren-1-ylacetyl)amino]ethanoyl}amino)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (PAAM); [2S,5R,6R]-3,3-dimethyl-7-oxo-6-({(2R)-2-phenyl-2-[(pyren1-ylbutanoyl)amino]ethanoyl}amino)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (PBAM). Dansyl chloride was substituted for the succinimidyl esters in order to obtain the dansyl-labeled substrates, namely: [2S,5R,6R]6-{[(5-(dimethylamino)-1-naphthyl]sulfonyl}amino-2-phenylethanoyl]amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (DAM); [2S,5R,6R]-6-{[(5-(dimethylamino)-1-naphthyl]sulfonyl}amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (DAP). After stirring the mixture overnight, the acetone was removed under reduced pressure and the aqueous mixture was subjected to repetitive extraction with diethyl ether in order to remove the unreacted succinimidyl ester. Then the aqueous phase was brought to pH 2.5 with 10% phosphoric acid and extracted several times with diethyl ether. The combined extracts were dried over anhydrous magnesium sulfate. After removing the drying agent, the organic solvent was eliminated by rotavaporation to yield the target compound. The purity and structure of the labeled substrates was confirmed by elemental analysis and FT-IR, 1H NMR, and 13C NMR spectroscopy, as well as mass spectrometry with electrospray ionization (ESI-MS) and has been reported elsewhere.40 Apparatus. Steady-state photoluminescence spectra have been measured with a photon-counting Fluoromax 2 (Horiba-Jobin Yvon) spectrofluorometer, the sample cell of which was thermostated at 25.0 °C. Instrument control and data processing were performed with the manufacturer’s original software (Datamax). The excitation and emission wavelengths were set at 327 and 376 nm, respectively. Fluorescence quantum yields (Φem) were calculated by the Parker and Rees method42 that relates the emission quantum yield of an unknown (u) to that of a standard (std) fluorophore, the emission spectra of which have been recorded under the same conditions in optically diluted solution (A < 0.1):

(Φem)u ) (Φem)std

( )

Astd Su nu Au Sstd nstd

2

(1)

where, A is the absorbance of the solution at the excitation wavelength, S is the area under the fluorescence curve, and n is (42) Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587-600.

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Figure 2. Fluorescence excitation (λem ) 376 nm) and emission (λex ) 345 nm) spectra of PAAP (250 nM) in acetonitrile.

the solvent refractive index, in the case where the standard and the unknown have to be measured in different solvents. Purified pyrene (Aldrich, +99%, sublimated twice; Φem ) 0.62 in O2-free acetonitrile)43 and 5-(dimethylamino)-1-naphthalene sulfonic acid hydrate (Aldrich; Φem ) 0.36 in 0.1 M NaHCO3)44 have been used as reference fluorophores. Oxygen was removed from the solutions by argon purging for a minimum of 30 min before the measurements. Radioligand Competitive Assays. The competition between PenG and each fluorescent BLA for binding to the MIPs was evaluated using radiolabeled PenG-based assays. For this purpose, 0.1 mg of each MIP was incubated in microcentrifuge tubes for 16 h on a shaking table, with 1 pmol of [phenyl-4n-3H]benzylpenicillin and 1 mL of a solution of the tested BLA (1 nM-1 mM) in 99:1 acetonitrile-water (MIPs 1, 2, and 4) or in 98:2 acetonitrilewater (MIPs 3, 5, and 6). After the incubation time, the slurries were centrifuged (5 min at 14000 rpm) and 500 µL of the supernatant was added to 10 mL of the scintillation cocktail. Radioactivity was measured with a Rackbeta 1219 liquid scintillation counter (LKB Wallac, Turku, Finland). Binding of PAAP to the MIPs/CPs. To calculate the optimum amount of polymer to be used in the competitive assays, a constant amount of PAAP (250 nM) was incubated with increasing concentrations of MIP or CP (0-10 mg/mL). After centrifugation (5 min, 14 000 rpm), the fluorescence of the supernatant was measured with the instrumental setup described above. Fluorescence Competitive Assays. MIP particles (5 mg) were mixed with 500 pmol of PAAP and varying amounts of the corresponding competitive analytes (final concentration 1 nM890 µM) in the suspension solvent (99:1 acetonitrile-water) up to a final volume of 2 mL. The mixture was incubated for 7 h at room temperature in polypropylene tubes on a shaking table. All the solutions were prepared in triplicate, and each sample was analyzed three times (n ) 9). The fluorescence of the supernatant (43) Karpovich D. S.; Blanchard G. J. J. Phys. Chem. 1995, 99, 3951-3958. (44) Himel C. M.; Mayer R. T. Anal. Chem. 1970, 42, 130-132.

was measured using a flow injection system. The 99:1 acetonitrilewater carrier was flowed at 6 mL min-1 with a syringe pump (Kloehn, Las Vegas, NV). Sample injections were performed via a six-way rotary valve, fitted with a 200-µL loop in line with a flowthrough fluorescence cell (100 µL, Hellma, Germany) placed in the fluorometer sample holder. Fluorescence intensity measurements were taken with the instrumental setup described above. The amount of bound PAAP to the MIP or CP was plotted as a function of log[PenG]. The experimental signals were normalized using eq 2,

normalized signal ) B/B0

(2)

Table 2. Emission Features of the Novel Fluorescent β-Lactam Antibiotics in O2-Free Acetonitrile Solution at 25.0 °C (λExc ) 327 nm; n ) 3) labeled BLAa

λemmax (nm)

Φem

PAAP PAAM PAAX PBAP PBAM DAP DAM

376 376 374 374 375 520 522

0.89 ( 0.07 0.94 ( 0.07 0.90 ( 0.07 0.75 ( 0.06 0.77 ( 0.06 0.39 ( 0.02 0.43 ( 0.02

a See Figure 1 and text for the structure and names of the fluorescent β-lactams.

where B is the fluorescence intensity measured in the presence of increasing PenG concentrations and B0 is the signal in the absence of antibiotic. The experimental (sigmoidal) data plot was fitted to a four-parameter logistic function:45

normalized signal ) Bmin +

Bmax - Bmin b

1 + 10(log[PenG]-logEC50)

(3)

where Bmax is the asymptotic fluorescence maximum intensity (emission in the absence of analyte), log EC50 stands for the logarithm of analyte concentration at the inflection point, and Bmin is the asymptotic minimum intensity. Interference Species Study. Several antibiotics commonly used in veterinary practice, both structurally related to PenG (AMOX, AMPI, PenV, OXA, CLOX, DICLOX, NAFCI, CEPH) and nonrelated (NOR, DOXY, CLF, ERY, DDS, TCY, OXY), have been tested for cross-sensitivity in the MIP competitive assay. Clavulanic acid, usually found in pharmaceutical formulations of AMOX, and pyrene were also tested for interference. The degree of competition between each antibiotic and PAAP for the MIP sites was evaluated from binding assays according to the procedure described above. The specificity of each assay was calculated by measuring cross-reactivity as the ratio of the EC50 value for PenG to that obtained for the potential competitor. Binding Kinetics of PAAP to the MIP or CP. Different batches of MIP or CP particles (2.5 mg mL-1) were incubated between 0 and 18 h in polypropylene tubes at room temperature on a shaking table with 250 nM PAAP in the absence and in the presence of 426 µM PenG in 99:1 acetonitrile-water by volume. After centrifugation, the fluorescence of the supernatant was measured with the instrumental setup described above, and the percentage of PAAP bound to the MIP/CP was plotted as a function of the incubation time to determine the binding kinetics. All solutions were prepared in triplicate, and each sample was analyzed three times (n ) 9). The experimental data were analyzed using Prism 4.0 software (Graphpad, San Diego, CA). Molecular Modeling. The 3-D chemical structures of the labeled BLAs have been modeled using molecular mechanics followed by energy minimization by molecular dynamics (MOPAC, AM1 force field) using Chem3D Ultra 7.0 software (CambridgeSoft, MA). Analysis of a Commercial Pharmaceutical Formulation. The fluorescence MIP assay has been applied to the analysis of (45) Motulsky, H.; Christopoulus, A. Fitting dose response curves; GraphPad Software Inc., 2002.

Figure 3. Cross-reactivities of the fluorescent β-lactams vs PenG in the presence of the prepared MIPs in competitive binding assays. Solvent: 99:1 acetonitrile-water for MIPs 1, 2, and 4; 98:2 acetonitrile-water for MIPs 3, 5, and 6. The concentration of radioactivelabeled PenG is 1 nM in all cases. (Cross-reactivity values expressed in % collected in Table S1, Supporting Information.)

a pharmaceutical formulation used in veterinary practice. Erbacilina Enzima´tica 4+5 (Industrial Veterinaria, Barcelona, Spain) was kindly provided by Dr. Casilda Rodrı´guez from the Veterinary Faculty (UCM, Madrid, Spain). The sample composition per dosage is 1 000 000 IU of PenG potassium salt (626.6 mg) and 3 000 000 IU of PenG procaine salt (3000 mg). Following the manufacturer’s recommendations, the lyophilized samples were reconstituted with 20 mL of purified water and sonicated during 10 min (nominal value 347 mM PenG). A 20-µL aliquot of the solution was diluted to 50 mL with acetonitrile and water to a final 99:1 ratio (v/v, acetonitrile-water). For the recovery studies, samples were prepared by spiking 20-µL aliquots of the reconstituted solution with 20 and 40 µL, respectively, of an aqueous standard containing PenG potassium and PenG procaine salts (total PenG concentration 349.6 mM) in the same ratio as in the pharmaceutical sample. The solutions were made up to 50 mL with acetonitrile and water to a final 99:1 ratio (v/v, acetonitrilewater). The samples were analyzed as described previously. Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

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Figure 4. Energy minimized (AM-1) structures of PenG, PAAP, PBAP, and DAP. For the sake of comparison, the orientation of the fused β-lactam and thiolane rings has been maintained in all the structures.

RESULTS AND DISCUSSION MIP Library. A MIP library targeted for PenG was synthesized from various functional monomers and cross-linkers.41 The MIP library, and a corresponding control polymer library prepared with Boc-Phe-OH as the template, were screened for binding to PenG. Binding studies in acetonitrile-water mixtures showed higher affinity with lower amounts of water. To maintain selectivity for PenG, assays were performed in acetonitrile containing 1-2% water. Among the best MIP candidates found during the screening,41 six MIPs were selected for further studies. These MIPs represent different combinations of functional monomers and cross-linkers and was therefore expected to show some diversity in cross-reaction profiles against fluorescent PenG analogues. Spectral Characteristics of the Labeled BLAs. All the spectroscopic data show that the pyrene and dansyl moieties keep their features after incorporation with the BLA structures. As an example, the excitation and emission spectra in acetonitrile of the 2024

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PAAP pyreneacetic analogue of PenG are depicted in Figure 2 and display the typical vibronic structure of pyrene absorption and fluorescence bands. The emission maximums and fluorescence quantum yields (Φem) of the novel labeled β-lactams in deaerated acetonitrile are collected in Table 2. The measured Φem values of the synthesized pyrene- and dansyl-labeled BLAs are equal or higher than 0.75 and 0.40, respectively. Interestingly, the pyreneacetic derivatives display stronger fluorescence (Φem ∼ 0.90) than their pyrenebutyric analogues (Φem ∼ 0.75), and both of them show higher Φem values than pyrene itself under the same conditions (Φem ) 0.62)43 used as a reference. Taking into account the negligible perturbation of pyrene electronic features by alkyl substituents at the 1-position,46 and that there is no correlation between the emission quantum (46) Yao, C.; Kraatz, H.-B.; Steer, R. P. Photochem. Photobiol. Sci. 2005, 4, 191199.

yield of pyrene and the solvent polarity,43 such a result seems difficult to rationalize. Binding of the Labeled β-Lactams to the MIPs. It has been discussed in the literature13 that one limiting factor for the application of fluorescent measurements in MIA is often the poor recognition of the labeled guest by the binding sites in the MIP. To evaluate the interaction of the tagged BLAs with the PenGimprinted polymers, the cross-reactivity of the former in competitive binding assays was measured against radioactive-labeled PenG. As discussed previously,14 this type of assay is extremely sensitive and providea valuable information for optimizing the fluorescence experiments. The measured cross-reactivity for the different labeled β-lactams in 99:1 acetonitrile-water mixture is depicted in Figure 3. The dansyl derivatives (DAP, DAM) turned out to be poor competitors of PenG for the binding sites of all the six imprinted polymers tested. However, MIP 1 selectively recognized the pyrene derivative PAAP, while PBAP also bound to MIP 6 but to a lower extent than PAAP. This differential binding is probably a consequence of the higher conformational freedom of PBAP due to the longer spacer between the fluorescent and the penicillanic acid moieties it features. The labeled ampicillin PAAM was only recognized by MIP 6. The energy-minimized 3-D structures of PenG and PAAP (Figure 4) show a similar pocketlike compact conformation that seems to be the key for an efficient recognition of the arylsubstituted β-lactams by MIP 1. In this way, the methylene group of the benzyl moiety plays a crucial role toward a good fit of the guest into the host binding site. Neither DAP, DAM, nor PBAP adopts such a folded conformation (Figure 4). This is a clear example of the shape selectivity that underlies molecular recognition by imprinted polymer materials. The size of the guest molecule also plays an important role: PAAM does have the single methylene group of the benzyl moiety (Figure 1) but it is not recognized by the PenG-imprinted MIP. From those competition experiments, and the fact that MIP 6 showed unspecific recognition of β-lactam antibiotics PenG, PenV, AMOX, and AMPI while MIP 1 was selective to PenG (radiolabeled competitive assays, data not shown), the MIP 1/PAAP pair was selected for the fluorescent competitive binding assay development. Optimization of a Fluorescent Competitive Binding Assay for PenG. The titration curve of PAAP (250 nM in 99:1 per volume acetonitrile-water) binding to MIP 1 or CP 1 is shown in Figure 5. It can be observed that binding of the fluorescent probe to the control polymer was very low in all cases. To select the optimum MIP concentration for PenG analysis, the binding of a fixed amount of PAAP to increasing amounts of imprinted polymer was evaluated in the presence of a constant concentration of PenG.47 As shown in Figure 5, PAAP competes with PenG for the binding sites in the polymer, as the labeled BLA is partially displaced from the MIP in the presence of the template molecule. The largest difference in the fraction of PAAP bound to the polymer, in the presence and in the absence of the analyte, (47) Ekins, R. P. Immunoassay Design and Optimisation. In Principles and Practice of Immunoassay, 2nd ed.; Price, C. P., Newman D. J., Eds.; McMillan: London, U.K., 1997; p 199.

Figure 5. Binding assay (n ) 6; 7-h incubation) of PAAP (250 nM) in 99:1 acetonitrile-water in the presence of increasing concentrations of the MIP 1 (b) and control polymer 1 (1). The binding assay in the presence of a constant concentration of PenG (426 µM) has also been included (O).

Figure 6. Binding kinetics (n ) 6) of 250 nM PAAP to MIP 1 (b), control polymer 1 (1), and MIP 1 in the presence of 426 µM PenG (O) in 99:1 acetonitrile-water (2.5 mg mL-1 polymer in all cases).

corresponds to a MIP slurry of 2.5 mg mL-1. This value was therefore selected for subsequent development of the competitive assay. The extent of the PAAP binding to MIP 1 as a function of the incubation time is depicted in Figure 6. The kinetics of fluorescent β-lactam binding to the imprinted polymer slows down in the presence of PenG due to effective competition. Nevertheless, it levels off after 7 h so that this contact time was selected for all subsequent competitive assays. Competitive calibration curves were prepared using 99:1 acetonitrile-water and PenG standards with concentrations ranging between 0.0013 and 890 µM. PAAP displacement from MIP 1 by PenG is depicted in Figure 7. Higher levels of PenG could not be tested due to the solubility limit of these species in the solvent mixture. The presence of higher amounts of water in the solution strongly decreases selective binding of PAAP to the polymer (data not shown). This feature limits the application of the MIP assay to direct PenG analysis in aqueous samples. The experimental data have successfully been fit to eq 3 (n ) 9, r ) 0.990). The measured EC50 value is 0.053 mM, and the limit of detection Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

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Table 3. Analytical Characteristics of the Competitive Fluorescence Binding Assays and Cross-Reactivities Related to PenG When the MIP Assay Is Conducted for an Antibiotic Concentration Ranging from 0.0005 to 333 µg mL-1 (0.0005-167 µg mL-1 for AMOX and AMPI) in 99:1 Acetonitrile-Watera antibioticb

Figure 7. Dose-response curve (n ) 9) for PenG (from 0.0013 to 890 µM) in 99:1 acetonitrile-water for MIP 1 in the presence of 250 nM labeled β-lactam PAAP (b), for control polymer 1 in the presence of 250 nM labeled β-lactam PAAP (O), and for MIP 1 in the presence of 250 nM pyrene (1) (2.5 mg mL-1 polymer in all cases).

(LOD), calculated as the analyte concentration for which PAAP binding to MIP is inhibited by 10%, is 0.32 µM. The assay dynamic interval, i.e., the analyte concentration that produces a normalized signal in the 20-80% range, is theoretically between 3 and 2800 µM according to eq 3, although the maximum assayed concentration has been 890 µM. The use of lower PAAP concentrations (e.g., 125 nM) for the competitive assay did not produce a significant decrease of the EC50 values, but the signal-to-noise ratio was much better when the competition was carried out using 250 nM PAAP. Employing unsubstituted pyrene instead of PAAP as fluorescent competitor did not yield competition (Figure 7). This result stresses that molecular recognition is indeed driven by the penicillin moiety of the probe. When the assay was carried out in the presence of the control polymer instead of MIP 1, there was no competition either (Figure 7). On the contrary, the amount of PAAP bound to the CP slightly increased with the concentration of PenG. This result might be explained by considering that the interaction of PenG with the carboxylic groups randomly distributed in the control polymer increases the overall hydrophobicity of the polymer surface and favors nonspecific interactions with the hydrophobic moiety of the label. The specificity of the developed fluorescence MIA was evaluated by measuring the cross-reactivity (i.e., the ratio of the EC50 value measured for PenG to that of the potential competitor) of general antibiotics structurally related to PenG and other nonrelated antibiotics that are commonly used in veterinary practice. The results are collected in Table 3. AMOX, AMPI, 6-APA, and PenV proved to be the strongest competitors. The latter, an antibiotic used for human treatment but not in veterinary practice, afforded the maximum cross-reactivity. All these compounds have structures similar to PenG, and AMOX, AMPI, and PenV also contain the angled benzylmethylene moiety. This finding supports the idea that such a molecular shape plays a key role in the recognition process. Other antibiotics such as those belonging to the isoxazolpenicillin family showed significantly lower cross-reactivity in the 2026 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

PenG 6-APA AMOX AMPI PenV OXA CLOX DICLOX NAFCI CEPH, CLAV, TCY, OXY, NOR, DOXY, CLF, ERY, DDS

EC50 (mM)

CRc (%)

LODd (µM)

DRe (mM)

0.053 0.092 0.180 0.140 0.130 0.250 0.930 5.40 1.70 nc

100 57 29 37 40 21 6.0 0.9 3.0 nc

0.32 0.89 16 4.0 0.39 6.2 100 580 270 nc

0.003-0.89f 0.007-0.65 0.030-0.46f 0.015-0.41f 0.004-0.86f 0.025-0.63 0.26-0.70f 0.63-0.65f 0.66-0.73f nc

a Fluorescent competitor: 250 nM PAAP (n ) 3). nc, no competition detected. b Structures shown in Supporting Information (Figure S1). c Cross-reactivity (see text for definition). d LOD, limit of detection. e Dynamic range. f Maximum assayed concentration depending on the particular antibiotic solubility.

Figure 8. Comparison of the binding curves to MIP 1 (n ) 3) obtained using PenG potassium salt (b) or a standard mixture of PenG potassium and PenG procaine salts (1 000 000 IU/3 000 000 IU) (O), in 99:1 acetonitrile-water.

investigated range, e.g., oxacillin (21%), cloxacillin (6%), nafcillin (3%), and dicloxacillin (0.9%). Cephapirin, a β-lactam derived from 7-aminocephalosporanic acid, did not compete either. Other antimicrobials not related structurally to PenG (namely, chloramphenicol, norfloxacin, tetracycline, oxytetracycline, erythromycin, and doxycycline) were also tested, and none of them showed cross-reactivity in the investigated range. Dapsone was a positive interference in the fluorescence assay, as it emits light in the same range as PAAP. Nevertheless, after subtracting the emission of the interference, the molecule displayed no competition with PAAP for the MIP-binding sites. Clavulanic acid, the most clinically important inhibitor of β-lactamases that mediates bacterial resistance to penicillin-type antibiotics, was also tested with negative competition results.

Table 4. PenG Analysis in a Reconstituted Pharmaceutical Formulation Using the Fluorescence Molecularly Imprinted Assay with MIP 1 (n ) 3) nominal valuea (mM)

added (mM)

found (mM)

RSD (%)

recovery (%)

347

0 349 699

318 716 1083

11 6 6

92 102 103

a Nominal value: 50 000 UI mL-1 (Pen G potassium) + 150 000 UI mL-1 (PenG procaine).

Analysis of a Pharmaceutical Formulation. The optimized fluorescent MIA has been applied to the analysis of penicillin G in a commercial pharmaceutical formulation. To this aim, a calibration curve was prepared by codissolving a mixture of PenG potassium and PenG procaine salts in 99:1 acetonitrile-water at same ratio as in the formulation (1 000 000/3 000 000 IU) and compared to that measured for PenG potassium alone. At shown in Figure 8, binding of the PenG salts mixture to MIP 1 is slightly underestimated at high analyte concentration and slightly overestimated at low analyte concentration so that a larger slope of the calibration curve is obtained. Therefore, to avoid this effect in the analysis of the pharmaceutical formulation, the calibration plots were carried out with the mixture of PenG potassium and procaine salts. For validation purposes, a recovery study was performed by spiking the reconstituted pharmaceutical formulation samples with increasing amounts (349 and 699 mM) of a PenG potassiumPenG procaine mixture. As shown in Table 4, recoveries ranging

from 92 to 103% in the assayed range were found, demonstrating the applicability of the MIP assay to the analysis of samples that can be dissolved in organic solvents. CONCLUSIONS Carefully designed fluorescent competitors of target analytes provide high selectivity to molecularly imprinted polymer assays. The spatial arrangement, nature, and size of the fluorescent tag must be tailored to the template structure to achieve an efficient competition for the binding sites. Optimum recognition of PenG by the MIP requires less than 1% water in the organic solvent. This fact limits the application of the assay to the analysis of Pen G in aqueous samples at low concentration (e.g., milk). Further work is in progress to overcome such a problem using MIPs capable of binding penicillin antibiotics in such medium. ACKNOWLEDGMENT This work has been funded by the European Union “Quality of Life and Management of Living Resources” V Framework program (Grant QLK1-CT-1999-00902) and by the Spanish Ministry of Education and Science (Grant BQU-2002-04515-C02). E.B.P. thanks Complutense University for a predoctoral grant. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 28, 2005.

October

30,

2005.

Accepted

AC051939B

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