Detection of antibiotics in food: Extraction of fluoroquinolones by DNA

3 downloads 0 Views 152KB Size Report
Feb 28, 2007 - Abstract The ability of DNA to extract fluoroquinolones from model solutions and real probes of food was demon- strated and investigated ...
Anal Bioanal Chem (2007) 388:253–258 DOI 10.1007/s00216-007-1191-5

ORIGINAL PAPER

Detection of antibiotics in food: Extraction of fluoroquinolones by DNA Limin Cao & Hong Lin & Vladimir M. Mirsky

Received: 22 November 2006 / Revised: 8 January 2007 / Accepted: 6 February 2007 / Published online: 28 February 2007 # Springer-Verlag 2007

Abstract The ability of DNA to extract fluoroquinolones from model solutions and real probes of food was demonstrated and investigated quantitatively. The interaction between fluoroquinolones and different types of DNA was studied by equilibrium dialysis. The first application of this direct approach allowed us to determine binding constants and binding stoichiometries in different conditions. The binding of enrofloxacin to heat-denatured DNA (d-DNA) from herring sperm is pH- and magnesium-dependent; the highest fraction of bound drugs was found at pH 7 and a magnesium concentration of 0.5–1 mM. Results for three types of DNA: d-DNA, double-stranded DNA and singlestranded DNA were compared. The unwound DNA showed almost doubled binding constants and stoichiometries, thus indicating preferable interaction of enrofloxacin with singlestrand regions of DNA. The binding of other fluoroquinolones (lomefloxacin, ciprofloxacin, norfloxacin, danofloxacin and sarafloxacin) with d-DNA is very similar to that of enrofloxacin: the binding constants are in the range from 0.94×105 to 2.40×105 M−1, and the stoichiometries range from 4.1 to 6.9 fluoroquinolone molecules per 100 DNA bases. The binding properties were quantitatively the same for extraction of fluoroquinolones from model aqueous solutions and from liquid food (milk). The results indicate the efficiency of DNA for selective extraction of fluoroquinolones from real samples for further analysis. This selective binding also allows us to consider DNA as a L. Cao : H. Lin Aquatic Products Safety Laboratory, Ocean University of China, Qingdao 266003, China V. M. Mirsky (*) Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany e-mail: [email protected]

natural receptor for development of analytical techniques for fluoroquinolones. Keywords Fluoroquinolone . DNA . Extraction . Binding constant . Binding stoichiometry

Introduction Fluoroquinolones are a group of broad-spectrum antibacterial agents which are widely used in human medicine and in veterinary treatment [1, 2]. Hygiene rules of the EC, the USA and most other countries restrict the maximal residue limit of total fluoroquinolones in food. Such analysis is currently performed mainly by high-performance liquid chromatography with preliminary nonselective extraction by hydrophobic solvents [3–5]; this technique is accurate and sensitive but is time-consuming and expensive. Alternative analysis techniques are mainly based on immunoassays [6–8], but the high specificity of antibodies makes difficult the determination of the total amount of fluoroquinolones; therefore, it is important to find a receptor for fluoroquinolones with about similar affinity to different types of this group of antibiotics. Such receptors could be used for selective extraction of fluoroquinolones from samples, thus providing a possibility to use a simpler separation and analysis technique. In the best case, these receptors could even replace antibodies in assays for fast screening of food or other biological samples. In this and a following paper we demonstrate that DNA can be used as such a broadly selective receptor for fluoroquinolones. Fluoroquinolones inhibit the strand-passage activity of topoisomerases by stabilizing the formation of a cleavedDNA complex [9–11]. The fluoroquinolones–DNA–gyrase complex was studied in numerous publications [12–17].

DO01191; No of Pages 5

254

Anal Bioanal Chem (2007) 388:253–258 O

O

F

COOH

O

F

COOH

COOH

F

N

N

N

N

N

N

N

NH

F

C2H5

N CH3

F

Danofloxacin

O

O

O F

F

COOH

F

COOH

N

N

N

Lomefloxacin

Sarafloxacin

N

N

N N

NH

NH

COOH

C2H5

CH3 Norfloxacin Fig. 1 Structures of the fluoroquinolones studied

Enrofloxacin

Ciprofloxacin

The first indications of fluoroquinolones binding to DNA were obtained from fluorescence quenching [18–20] and electrochemical [21, 22] studies. Both techniques are indirect; they produce different values of the binding constants and do not give any information on the binding stoichiometry. In the work described here, the extraction of fluoroquinolones by DNA was demonstrated directly. The binding was characterized quantitatively, and the values of the binding constants and the stoichiometries as well as the effects of pH and magnesium were evaluated. The results demonstrate that DNA can be used for selective extraction of fluoroquinolones. The binding efficiency is not changed when the extraction is performed from a very complicated matrix (milk). In our next papers we will demonstrate an application of DNA as a broadly selective receptor of fluoroquinolones in biosensors.

Materials and methods Fluoroquinolones (Fig. 1) were purchased from the Veterinary Medicine Supervisory Institute of China (Beijing,

China). Stock solutions (200 μg ml−1) were prepared in 0.01 M NaOH and were diluted by buffers. Milk containing 1.5% fat and 3.4% protein was purchased from Kaufland Warenhandel (Neckarsulm, Germany), spiked with a defined concentration of enrofloxacin and diluted 10 times with 10 mM phosphate buffers (pH 7.0). Double-stranded DNA (ds-DNA; from herring sperm) and single-stranded DNA (ss-DNA; from calf thymus) were from Sigma. Denatured DNA (d-DNA) was prepared from ds-DNA according to the following procedure: 5 mg ml−1 ds-DNA in 10 mM phosphate-buffered saline (PBS; pH 7.0) containing 10 mM NaCl and 1 mM EDTA was heated in boiling water for 15 min and cooled in ice–water under stirring for about 15 min. The concentration of DNA (base) was determined by measuring the absorbance at 260 nm using a molar extinction of 6,600 M −1 cm−1 for ds-DNA and of 8,300 M−1 cm−1 for ss-DNA and d-DNA. The heating and cooling procedure resulted in about 130% increase of absorbance at 260 nm, thus indicating that most of the DNA helix was transferred into single-stranded form. Deionized water additionally purified by a Milli-Q system (Millipore) was used.

Table 1 Excitation and emission wavelengths used for determination of the fluoroquinolones concentrations in dialysate Enrofloxacin pH Excitation (nm) Emission (nm)

4.0 330 443

6.0 330 422

7.0 330 412

8.0 330 412

10.0 330 420

Ciprofloxacin

Sarafloxacin

Norfloxacin

Lomefloxacin

Danofloxacin

7.0 334 414

7.0 334 420

7.0 334 412

7.0 334 412

7.0 340 415

Anal Bioanal Chem (2007) 388:253–258

255

50 45 40 35 30 25 20 15 10 5 4

5

6

7

8

9

10

pH

Fig. 3 Effect of pH on the enrofloxacin binding to denatured DNA (d-DNA). The experiments were performed in 0.02 M acetate buffer (pH 4), 10 mM phosphate buffer (pH 6, 7 and 8) and 0.01 M carbonate buffer (pH 10). The concentration of d-DNA was 1.48 mM, and the initial concentration of enrofloxacin was 2.8 μM. Each point represents the average of three measurements

a single-site binding model giving the following linear dependence [23]: R ¼ Ka ðN  RÞ; cf where cf is the concentration of free fluoroquinolones remaining in the dialysate after dialysis, Ka is the binding constant, and N is the binding stoichiometry.

Results and discussion Binding properties of enrofloxacin relative to denatured DNA were investigated at pH 4–10. As shown in Fig. 3, the pH has a strong influence on the affinity of enrofloxacin to nucleic acids. The highest extraction efficiency was observed at neutral pH (pH 7), while the fraction of bound drug decreased dramatically in acidic or in alkaline conditions (Fig. 3). Enrofloxacin possesses two ionizable functional groups: a carboxylic group (pK1 =5.94) and a basic piperazinyl group (pK2 =8.70). Depending on the pH, it can exist in four forms: cationic, neutral nonionized, zwitterionic and anionic [24]. The overall pK of DNA is about 4.0–4.5; therefore, the highest affinity is observed for the zwitterion of enrofloxacin and negatively charged DNA (Table 2).

9 8 Fluorescence intensity

55

Bound efrofloxacin (%)

Dialysis tubes with a molecular weight cutoff of 12,000–14,000 were from Carl Roth. Aliquots of 10 ml fluoroquinolones in buffers were dialyzed against 1 ml DNA in PBS for 22–24 h at room temperature under permanent stirring. It was proved that this time is enough to reach a steady-state concentration of dialysate. Control experiments were performed in the same conditions, except that 1 ml PBS was used instead of DNA. After dialysis, the fluorescence intensities of fluoroquinolones in dialysate (outer solution) were determined according to the conditions shown in Table 1, and the results from controls and test samples were recorded as F0 and F1, respectively. In the concentration range used, the fluorescence vs. concentration dependence was found to be linear, and the native fluorescence of the buffers was found to be less than 1% of that from the fluoroquinolones; therefore, the percentage of bound fluoroquinolones was calculated directly from the difference between F0 and F1 as (F1−F0)/ F0. The dialysis experiments for milk samples were carried out according to the same procedure as for enrofloxacin; however, because of significant native fluorescence of this probe under the experimental conditions, the percentages of the bound drugs were calculated from the calibration plot (Fig. 2). Binding constants and binding stoichiometry were calculated from a number of dialysis experiments with fixed DNA concentrations and varied fluoroquinolones concentrations. The data obtained were analyzed in Scatchard plots, as the dependence of the binding ratio (molar quantity of the bound antibiotics to the molar quantity of DNA bases) R vs. R/cf. The data were fitted by

7 6 5

Table 2 Electrical charge of DNA and enrofloxacin at different pH

4

pH 0.0

0.5

1.0

1.5

2.0

Enrofloxacin concentration (µM) Fig. 2 Dependence of the fluorescence of milk samples on the concentration of enrofloxacin added. Each point represents the average of three measurements of one probe. The line obtained by linear regression was used for calibration

DNA Carboxyl group of enrofloxacin Piperazinyl group of enrofloxacin

4

6

7

8

10

0 0 +

− 0 +

− − +

− − 0

− − 0

256

Anal Bioanal Chem (2007) 388:253–258

2.4 2.2 -1

Ka (10 , M )

2.0

5

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0

1

2

3

4

5

6

2+

Mg concentration (mM)

Fig. 4 The effect of Mg2+ concentration on the binding constants of d-DNA and enrofloxacin at pH 7. The d-DNA concentration was 0.38 mM

This may indicate the role of dipole–dipole interaction in the binding. Probably, a carboxy group of the fluorochinolone molecule interacts with a positive charge of the protonated DNA base, while the positive charge of enrofloxacin interacts with a phosphate group of DNA. The involvement of the DNA base in the binding also explains the higher affinity of unwound DNA to this antibiotic. The presence of Mg2+ strongly increases the binding of enrofloxacin to DNA. The highest binding efficiency was observed at concentrations of 0.5–1.0 mM (Fig. 4). The optimal concentration was independent of enrofloxacin concentration. Nonmonotonous dependence indicates that at least two different processes are involved in the magnesium effect. The first process should favor the binding of

7 6

Fluorescence intensity

1- 3.37 mM 2- 2.31 mM 3- 1.48 mM 4- 0.99 mM 5- 0.33 mM 6- Control

6 5

5

4

4

3 2

3

1

2

enrofloxacin to DNA; an understanding of the chemical nature of this effect demands further investigation. The second process should hinder the binding. This can be explained by electrostatic shielding, taking into account the local increase of enrofloxacin concentration in strongly the nonhomogeneous electric field caused by phosphate groups of DNA (the Debye length in the experimental conditions was comparable with the dipole length of zwitterionic enrofloxacin; therefore the antibiotic molecules migrate towards the maximal electrical field). An increase of the ionic strength decreases the electric field near DNA, resulting in a decrease of the local enrofloxacin concentration near DNA and of the apparent binding constant. The dependence of the DNA concentration on the binding was investigated at fixed enrofloxacin concentration (2.5 μM) and various concentrations of d-DNA (0.33– 3.37 mM). The fraction of the bound drugs increases gradually from 20 to 58% with increase of d-DNA concentration (Fig. 5). ss-DNA from calf thymus, ds-DNA and d-DNA from herring sperm were dialyzed against different concentrations of enrofloxacin in the presence of 1 mM Mg2+ at pH 7; the data obtained were linearized by presentation in the Scatchard plot (Fig. 6). The binding constants Ka for ss-DNA, d-DNA and ds-DNA were found to be 1.73 (±0.25)×105, 2.19(±0.20)×105 and 1.31(±0.014)×105 M−1, respectively. The results demonstrate that ss-DNA binds enrofloxacin more effectively than ds-DNA. The binding stoichiometries (referring to the number of enrofloxacin molecules per 100 DNA bases) for these types of DNA were estimated to be 4.46±0.74, 4.11±0.44 and 2.21±0.03, respectively. This indicates that the binding

60

Bound enrofloxacin (%)

2.6

50 40 30 20 10

1 0

0 380

a

400

420

440

460

480

500

520

0.0

540

Wavelength (nm)

Fig. 5 Fluorescence emission spectra of enrofloxacin recorded in 0.01 M phosphate buffer (pH 7.0, at 1exc =330 nm, initial concentration 2.8 μM) after dialysis against d-DNA (a) and dependence of the

b

0.5

1.0

1.5

2.0

2.5

3.0

3.5

d-DNA concentration (base, mM)

bound enrofloxacin on d-DNA concentration (b). The amount of bound enrofloxacin was calculated from the fluorescence at 412 nm

Anal Bioanal Chem (2007) 388:253–258

257

7000

Table 3 Binding constants and binding stoichiometries for interaction of fluoroquinolones with denatured DNA evaluated from Scatchard plots

6000

Fluoroquinolone

Binding constant (105, M−1)

Binding stoichimetry (the number of fluoroquinolone molecules per 100 DNA bases)

Enrofloxacin Ciprofloxacin Norfloxacin Sarafloxacin Danofloxacin Lomefloxacin

2.19±0.20 1.90±0.09 1.13±0.09 1.04±0.04 2.40±0.26 0.94±0.07

4.11±0.44 4.91±0.25 6.57±0.57 6.86±0.27 6.24±0.76 4.83±0.38

R/C f

8000

5000 4000 3000 2000

a

1000 0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

R

Dialysis was performed in 0.01 M phosphate buffer (pH 7) in the presence of 1 mM Mg2+

9000 8000

constants are slightly different, which may be caused by different sequences of these biopolymers. The binding of five other fluroquinolones to d-DNA was also investigated, and the data obtained are summarized in Table 3. The values of Ka varied from 0.94×105 to 2.40× 105 M−1; the calculated stoichiometries were in the range from 4.1 to 6.9 per 100 DNA bases. The observed variations of the binding characteristics between different types of fluoroquinolones are very small; therefore, DNA really had an affinity to fluoroquinolones with broad selectivity. Let us compare the extraction of fluoroquinolones by DNA and by antibodies. The values obtained for binding constants for DNA are approximately 3 orders of mag-

7000

R/Cf

6000 5000 4000 3000 2000 1000 0.00

0.01

0.02

b

0.03

0.04

R 3200 2800

R/C f

2400 2000

30 1600 1200

c

0.003

0.006

0.009

0.012

0.015

0.018

R

Fig. 6 Scatchard plots for enrofloxacin binding to single-stranded DNA (a), d-DNA (b) and double-stranded DNA (c). The measurements were performed in the presence of 1 mM Mg2+ at pH 7.0. Each point represents the average of three measurements

constant and the number of binding sites of ds-DNA are about a half those of the denatured forms; therefore, the formation of ds-DNA from ss-DNA decreases the number of binding sites for enrofloxacin nearly twofold. Assuming that the mechanisms of the fluoroquinolone binding to ss-DNA and d-DNA are the same, this observation may indicate that some groups of DNA bases involved in the hydrogen bond forming between the base pairs participate in the binding of fluoroquinolones. The stoichiometries of ss-DNA and d-DNA are similar, but the binding

Extracted enrofloxacin (%)

25

800

20 15 10

from milk

5

from phosphate buffer

0 0

1

2

3

4

5

6

Enrofloxacin concentration ( µM) Fig. 7 The enrofloxacin–DNA binding efficiency in milk samples and in phosphate buffers (0.01 M, pH 7.0). The concentration of dDNA was 0.38 mM. The measurements were performed in the presence of 1 mM Mg2+. Each data point represents the average of three measurements

258

nitude less than those for antibodies. Taking into account the binding stoichiometry and the molecular weight of one DNA base, one can estimate that one fluoroquinolone molecule can be bound by a DNA fragment with a molecular weight of about 5,000. Assuming that the binding stoichiometry for antibodies is 1, the corresponding weight of this “fragment” for antibodies is equal to their molecular weight (about 250,000); therefore, the extraction capacity of DNA is much higher than that for antibodies. Finally, the possible application of DNA for extraction of enrofloxacin was tested with real samples. Dialysis of the commercial milk samples without added enrofloxacin against d-DNA in PBS showed that there is no significant fluoroquinolones residue (or other substances which have similar fluorescence and which also bind DNA) in the milk; therefore, the changes of fluorescence in the milk samples enriched by enrofloxacin occur mainly owing to enrofloxacin–DNA binding. Despite the expected effect of magnesium and other ions in milk, no significant extraction of enrofloxacin by DNA is observed without addition of Mg2+. Probably, polyvalent metals of the milk probes tested are present in the form of strong complexes with other compounds. In the presence of 1 mM Mg2+, the enrofloxacin– DNA binding efficiency in milk samples was found to be very similar to that in the phosphate buffers (Fig. 7). The results demonstrated that d-DNA can be used for effective extraction of enrofloxacin from complicated biological samples.

Conclusion Extraction of fluoroquinolones by DNA was demonstrated for the first time; binding constants and binding stoichiometries for different types of fluoroquinolones were measured. An application of this direct technique demonstrated that fluoroquinolones have higher affinity to DNA than was earlier suggested from indirect data. The same affinity properties were obtained in experiments on extraction of fluoroquinolones from real samples. The results suggest that DNA can be used as a broadly specific receptor for the development of a fast and simple assay of fluoroquinolones. Relatively high binding constants and the possibility to control the affinity by variation of pH and magnesium concentration allow us to suggest applications of DNA as an

Anal Bioanal Chem (2007) 388:253–258

extraction phase, or as a receptor in affinity chromatography and in biosensors. Acknowledgements This work was supported by National Natural Science Funding of China (no. 30400336) and the German Academic Exchange Service (DAAD). The authors are thankful to O. S. Wolfbeis and A. Karasyov for helpful discussions.

References 1. Higgins G, Fluit C, Schmitz (2003) J Curr Drug Targets 4:181– 190 2. Brown A (1996) J Vet Pharmacol Ther 19:1–14 3. Deng D, Yang X, Chen L (2000) Chin J Vet Drugs 34:53–58 4. Hernandez-Arteseros JA, Barbosa J, Compano R (2002) J Chromatogr A 945:1–24 5. Hotzapple K, Buckley A, Stanker H (2001) J Chromatogr B 7541–7549 6. Bucknall S, Silverlight J, Coldham N, Thorne L, Jackman R (2003) Food Addit Contam 20:221–228 7. Holtzapple K, Buckley A, Stanker H (1997) J Agric Food Chem 45:1984–1990 8. Duan J, Yuan ZH (2001) J Agric Food Chem 49:1087–1089 9. Barnard FM, Maxwell A (2005) Antimicrob Agents Chemother 45:1994–2000 10. Bhanot SK, Singh M, Chatterjee NR (2001) Curr Pharm Des 7:311–335 11. Drlica K, Zhao X (1997) Microbiol Mol Biol Rev 61:377–392 12. Shen LL, Baranowski J, Pernet AG (1989) Biochemistry 28:3879–3885 13. Shen LL, Mitscher LA, Sharma PD, O’Donnell TJ, Chu DTW (1989) Biochemistry 28:3886–3894 14. Palu’ G, Valisena S, Ciarrocchi G, Gatto B, Palumbo M (1992) Proc Natl Acad Sci USA 89:9671–9675 15. Fan J-Y, Sun D, Yu H, Kerwin SM, Hurley LH (1995) J Med Chem 38:408–424 16. Critchlow SE, Maxwell A (1996) Biochemistry 35:7387–7393 17. Cabral MJH, Jackson AP, Smith CV, Shikotra N, Maxwell A, Liddington RC (1997) Nature 388:903–906 18. Sissi C, Andreolli M, Cecchetti V (1998) Bioorg Med Chem 6:1555–1561 19. Sortino S, Condorelli G (2002) New J Chem 26:250–258 20. Hwangbo HJ, Yun BH, Cha JS (2003) Eur J Pharm Sci 18:197– 203 21. Nawaz H, Rauf S, Akhtar K, Khalid AM (2006) Anal Biochem 354:28–34 22. Radi A, El Ries MA, Kandil S (2003) Anal Chim Acta 495:61–67 23. Yu S, Wang H, Zhu N, Ye R (1999) In: Introduction to immunology. China Higher Education Press, Beijing, and Springer, Heidelberg, pp 150–151 24. Lizondo M, Pons M, Gallardo M, Estelrich J (1997) J Pharm Biomed Anal 15:1845–1849