Anal. Chem. 1997, 69, 2017-2021
Optical Detection of Chloramphenicol Using Molecularly Imprinted Polymers Raphael Levi, Scott McNiven, Sergey A. Piletsky,† Soo-Hwan Cheong, Kazuyoshi Yano, and Isao Karube*
Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153, Japan
A practical optical sensing system for the determination of chloramphenicol (CAP), utilizing molecularly imprinted polymers (MIPs) and HPLC, has been developed. The method is based on competitive displacement of a chloramphenicol-methyl red (CAP-MR) dye conjugate from specific binding cavities in an imprinted polymer by the analyte. The best of these polymers was obtained using (diethylamino)ethyl methacrylate as functional monomer at a monomer:template ratio of 2:1. HPLC with a mobile phase containing CAP-MR was used as the detection system, and injection of CAP and, to a lesser degree, thiamphenicol resulted in proportional displacement of the conjugate, which was detected at 460 nm. The detection system showed a linear response over a range of 3-1000 µg/mL and effectively detected CAP extracted from serum. This system offers a tailor-made, selective, and rapid method for CAP detection, is able to discriminate between similar molecules, and is effective below and above the therapeutic range (10-20 µg/mL serum, potentially toxic above 25 µg/mL). This technique is quite general and should enable the use of MIPs in a wide variety of applications involving the detection of families of molecules which possess a distinct arrangement of functional groups. Biosensors, which are extensively used in medical and environmental monitoring,1,2 utilize various biological molecules such as antibodies, enzymes, etc. capable of recognizing a specific target molecule. The development of a useful biosensor depends on the availability of a receptor molecule that interacts specifically with the target molecule. In many cases, it is not easy to find a natural candidate that possesses the desired properties. There may be difficulties in purifying the molecule, obtaining it in sufficient amounts, instability, or other factors that impede the practical use of such molecules. The molecular imprinting technique,3,4 which combines the advantages of tailor-made sorbents5 and physical durability,6 is one way to solve this problem. This technique has been extensively used in the production of molecularly imprinted † Present address: Institute of Molecular Biology and Genetics, Academy of Sciences of Ukraine, 252143 Kiev, Zabolotnogo 150, Ukraine. (1) Chen, C.-Y.; Karube, I. Curr. Opin. Biotechnol. 1992, 3, 31-39. (2) Karube, I.; Nomura, Y.; Arikawa, Y. Trends Anal. Chem. 1995, 14, 295299. (3) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9-14. (4) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (5) Wulff, G.; Minarik, M. In Chromatographic Chiral Separation; Zief, M., Crane, L. Y., Eds.; Marcel-Dekker: New York, 1988; pp 15-52. (6) Kriz, D.; Mosbach, K. Anal. Chim. Acta 1995, 300, 71-75.
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© 1997 American Chemical Society
polymers (MIPs) with specific binding sites for a wide variety of molecules.3,4 A monomer chosen especially for its ability to interact with the functional groups of the template molecule, a cross-linker, and a target molecule are dissolved in a solvent that serves as a porogen, and together they are polymerized to form a rigid macroporous polymer. Removal of the target molecules leaves binding cavities within the polymer matrix with the momomer’s functional groups positioned at precise locations, with the overall structural characteristics of the cavity contouring the template molecule as a result of the imprinting process. The main use of this method was for the enantioseparation of racemic mixtures,7-9 but attempts to utilize it in the development of synthetic enzymes for catalysis10 and as receptor mimics11 have been reported. The main obstacle to the implementation of MIPs as part of practical sensors is the development of a suitable detection system. Several MIP-based sensing systems have been proposed, including sensors utilizing field effect devices,12 conductometric measurements,13 amperometric measurements,6 fluorescence measurements,14,15 and the displacement of radioactive analogs from MIP antibody mimics.16 Nevertheless, each has its shortcomings: a relatively high noise level and the possibility of interference from other electroactive molecules in the case of electrochemical measurements, possible interference from extraneous fluorescent molecules in the case of fluorescence measurements, and the problem of waste disposal in the radioactive systems. Chloramphenicol (CAP) is an effective antibiotic with broad spectrum activity which is used in both human and veterinary medicine. Despite toxic effects such as bone marrow suppression, aplastic anemia, and the “gray baby syndrome” that have restrained its use, it is a potent drug for the treatment of lifethreatening infections such as childhood meningitis and typhoid (7) Sellergren, B.; Lepisto, M.; Mosbach, K. J. Am. Chem. Soc. 1988, 110, 58535860. (8) Andersson, L. I.; Mosbach, K. J. Chromatog. 1990, 516, 313-322. (9) Kempe, M.; Mosbach, K. Anal. Lett. 1991, 24, 1137-1145. (10) Leonhardt, A.; Mosbach, K. React. Polym. 1987, 6, 285-290. (11) Andersson, L. A.; Mu ¨ ller, R.; Vlatakis, G.; Mosbach, K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4788-4792. (12) Hedborg, E.; Winquist, F.; Lundstrom, I.; Andersson, L. I.; Mosbach, K. Sens. Actuators A 1993, 37, 796-799. (13) Piletsky, S. A.; Piletskaya, E. V.; Elgersma, A. V.; Yano, K.; Parhometz, Y. P.; El’skaya, A. V.; Karube, I. Biosens. Bioelectron. 1995, 10, 959-964. (14) Kriz, D.; Ramstro ¨m, O.; Svensson, A.; Mosbach, K. Anal. Chem. 1995, 67, 2142-2144. (15) Piletsky, S. A.; Piletskaya, E. V.; Yano, K.; Kugimiya, A.; Elgersma, A. V.; Levi, R.; Kahlow, U.; Takeuchi, T.; Karube, I.; Panasyuk, T. I.; El’skaya, A. V. Anal. Lett. 1996, 29, 157-170. (16) Vlatakis, G.; Andersson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645-647.
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fever.17 In view of the toxic effects, the use of CAP was banned in food-producing animals. Thus, the monitoring of CAP levels in patients’ blood and its detection in food products are highly important. Currently used methods for the detection of CAP include chromatographic,18 microbiological,19 enzymatic,20 immunological,21 and other assays which are often elaborate and laborious. Hence, new, fast, and easy methods for CAP determination and detection are sought. With this aim, a sensor for CAP using a molecularly imprinted polymer as the recognition element has been devised. Furthermore, while CAP is a worthy candidate for the development of a new sensor, it also serves as a model system for investigations of the utility of imprinted polymers as part of a detection system. In this article we describe the preparation and characteristics of a CAP-imprinted polymer and propose a new method for CAP detection based on the optical detection of a CAP-dye conjugate displaced from the MIP by CAP molecules in the sample solution. This method has proven to be fast, reproducible, linear over a broad range of sample concentrations, and insensitive to a large excess of its diacetyl derivative and was successfully applied in the detection of CAP in CAP-containing serum samples EXPERIMENTAL SECTION Materials. All compounds were obtained from commercial sources and used as received except for ethylene glycol dimethacrylate (EGDMA), which was distilled prior to use. All other chemicals and solvents were of analytical or HPLC grade. The synthesis and characterization of the chloramphenicolmethyl red (CAP-MR) conjugate is available as Supporting Information. Preparation of Imprinted Polymers. CAP (1 mmol) was dissolved in tetrahydrofuran (5 mL), and the required amounts of monomer, either methacrylic acid (MAA) or (diethylamino)ethyl methacrylate (DEAEM), cross-linker (EGDMA, 5 mL), and 2,2′-azobis(isobutyronitrile) (1%) were added, dissolved, and polymerized at 60 °C for 48 h. The resulting polymer was crushed, ground, thoroughly washed with hot DMF and boiling ethanol, and sieved in ethanol. Finally, polymer particles sized 25-63 µm were slurry packed into 10 cm stainless steel HPLC columns. Nonspecific polymers were prepared in the same way, except that the mixtures did not contain CAP. High-Performance Liquid Chromatography Studies. The interactions of the different polymers with CAP and its analogs were analyzed using a Gilson HPLC system. The samples were dissolved in acetonitrile, and 10 µL of a 0.1 mg/mL solution was injected for analysis. The capacity factor was calculated as k′ ) (t - tv)/tv, where t is the retention time of the sample and tv is the retention time of the acetone void marker. The separation factor R ) kx′/ky′ is used to evaluate the ability of a polymer to separate the template from other molecules. We have defined an imprinting factor I ) ks′/kn′, where ks′ and kn′ refer to the capacity factors of the specific (imprinted) and nonspecific (nonimprinted) polymers. Thus, k′ represents the affinity of the analyte for the polymer, while I is a measure of the efficacy of the imprinting process. (17) Shalit, I.; Marks, M. I. Drugs 1984, 28, 281-191. (18) Berry, D. J. J. Chromatogr. 1987, 385, 337-341. (19) de Louvois, J. J. Antimicrob. Chemother. 1982, 9, 253-65. (20) Lietman, P. S.; White, T. J.; Shaw, W. V. Antimicrob. Agents Chemother. 1976, 10, 347-353. (21) Dalbey, M.; Gano, C.; Izutsu, A.; Collins, C.; Jaklitsch, A.; Hu, M.; Fischer, M. Clin. Chem. 1985, 31, 933.
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Figure 1. Structures of the various substances used in this work.
CAP-MR Displacement Measurements. CAP-MR (0.20.6 µg/mL) dissolved in acetonitrile was used as the mobile phase, and the column was equilibrated until a steady base line was achieved. Samples containing CAP, thiamphenicol (TAM), or chloramphenicol-diacetate (CAP-DA) were prepared in MeCN, and 20 µL was injected for analysis. In addition, various concentrations of CAP were prepared in bovine serum (ICN Biomedicals, Tokyo, Japan) and extracted using ethyl acetate as described by Yamato et al.22 After complete evaporation of the ethyl acetate, the residue was dissolved in 300 µL of acetonitrile and analyzed. The signals were monitored at 460 nm, and the area of the resulting peak due to CAP-MR was recorded. Each sample was tested at least in triplicate, and the standard error of the mean (SEM) was determined. Each measurement was run for 5 min in order to ensure that the system returned to a constant absorbance before the injection of the next sample. RESULTS AND DISCUSSION Several CAP-imprinted polymers were prepared using the monomers DEAEM (at monomer:template ratios of 1:1, 2:1, and 3:1) or MAA (at monomer:template ratios of 2:1 and 8:1) and tested for their ability to interact with CAP. The only significant results were obtained with the polymer prepared with DEAEM at a monomer:template ratio of 2:1, so all further investigations were carried out using this polymer. To have a better understanding of the nature of the interactions between the specific binding cavity of the MIP and its target molecule and to determine the ability of this polymer to recognize CAP, analogs of CAP were chromatographed. These analogs were (Figure 1) chloramphenicol diacetate (CAP-DA), in which the two hydroxyl groups were acetylated; thiamphenicol (TAM), in which the nitro group was substituted with a methylsulfonyl group; and chloramphenicol-methyl red (CAP-MR), where the dye methyl red was substituted for the dichloroacetyl moiety. These molecules were eluted using acetonitrile at various flow rates and the calculated values of k′, I, and R are presented in Table 1. The (22) Yamato, S.; Sugihara, H.; Shimada, K. Chem Pharm Bull (Tokyo) 1990, 38, 2290-2292.
Table 1. Influence of the Flow Rate on k′, I, and r Values of the Various Analytes As Determined by HPLC Analysis Using a CAP-Imprinted or Nonimprinted Polymer as the Stationary Phase and Acetonitrile as the Mobile Phase analyte, flow rate 2 mL/min CAP CAP-DA TAM CAP-MR 3 mL/min CAP CAP-DA TAM CAP-MR 4 mL/min CAP CAP-DA TAM CAP-MR 5 mL/min CAP CAP-DA TAM CAP-MR
k n′
ks′
I
1.75 0.00 1.14 0.83
17.4 0.14 9.09 5.32
9.92
1.54 0.00 0.99 0.77
15.4 0.15 8.40 4.75
10.0
1.40 0.00 0.84 0.70
14.4 0.13 7.33 4.51
10.3
1.15 0.00 0.65 0.49
12.0 0.16 7.06 3.94
10.4
7.97 6.40
8.48 6.16
8.72 6.44
10.9 8.04
R 1.00 124 1.91 3.26 1.00 102 1.83 3.24 1.00 110 1.96 3.19 1.0 74.9 1.70 3.04
ks′ values indicate a high affinity of TAM and CAP-MR for the specific polymer, but the original CAP template molecule was much more strongly retained. From these results, it is evident that the interactions between the CAP molecule and the binding cavity are mainly due to CAP’s two hydroxyl groups. Acetylation of these groups, as with CAP-DA, greatly diminished the interaction. Nevertheless, the particular structure of CAP also contributed to the interactions since the substitution of the nitro group (in TAM) and the dichloroacetyl group (in CAP-MR) resulted in significant decreases in the affinity for the polymer. Even for CAP-DA, some level of recognition, though very low, was demonstrated by the specific polymer, while no recognition at all was observed with the nonspecific polymer. The interactions most certainly involve hydrogen bonds, and the addition of as little as 1% ethanol, which is known to interfere with hydrogen bonding,23 to the acetonitrile mobile phase led to a dramatic decline in the ks′ value of CAP (from 17.4 to 4.05 at 2 mL/min). An interesting observation was that increasing the flow rate led to an increase of the I values, indicating a decrease in the nonspecific interactions between the analyte and the polymers, accompanied by a decrease in the values of k′ and R. Thus, the flow rate should be chosen according to the intended use of the MIP, either reducing nonspecific interactions (higher flow rate) or increasing the ability of the polymer to separate different molecules (slower flow rate). Since we were interested in practical applications, we decided to use a shorter (5 cm) column in order to reduce the time needed for the analysis of each sample. It was important to allow the system to return to equilibrium before the injection of the next sample, and using a short column reduced this time also. Furthermore, although the short column had somewhat lower k′ and I values, a sufficient degree of interaction was obtained, with approximately the same R values as the longer column. (23) Whitcombe, M. J.; Esther Rodrigez, M.; Villar, P.; Vulfson, E. N. J. Am. Chem. Soc. 1995, 117, 7105-7111.
Figure 2. Influence of (a) CAP-MR concentration in the MeCN mobile phase and (b) the flow rate on the area of the peak produced by injection of 20 µL of 20 µg/mL CAP onto a 50 mm × 4.6 mm i.d. column containing CAP-imprinted polymer. Each sample was analyzed at least three times, and the error bars represent the SEM values.
The influence of the CAP-MR concentration in the mobile phase and of the flow rate on the area of the peak produced was investigated in order to determine the optimal conditions for the system. A sample containing 20 µg/mL CAP was analyzed with various concentrations (0.1-0.8 µg/mL) of CAP-MR in the acetonitrile mobile phase at a flow rate of 2 mL/min. The results (Figure 2a) indicated that the optimal concentration was 0.4-0.6 µg/mL and that going outside these values reduced the peak area. It seems that, above a certain concentration, the conjugate is competing more effectively with the analyte, thus reducing the area of the peak. A 20 µg/mL CAP sample was then analyzed at different flow rates (1-3 mL/min) with 0.6 µg/mL CAP-MR in the mobile phase. It can be seen (Figure 2b) that lowering the flow rate increases the amount of CAP-MR released from the polymer, due to the longer time available for interaction of CAP with the polymer. However, slower flow rates also resulted in longer analysis times per sample and the peaks were very broad, making the analyses less accurate. Taking all these factors into account and compromising speed with larger peak areas, a flow rate of 2 mL/min and a CAP-MR concentration of 0.6 µg/mL in the mobile phase were used in further investigations of the system. The ability of this system to determine CAP, TAM, and CAPDA was evaluated by measuring the signals produced upon injecting 20 µL of samples with concentrations ranging from 3 to 1000 µg/mL. The results (Figure 3) demonstrate that CAP-DA did not give any appreciable response, even when concentrations of 1000 µg/mL were injected. CAP and TAM, on the other hand, produced signals with linearity throughout the concentration range, with the values for CAP being about 40% higher than those Analytical Chemistry, Vol. 69, No. 11, June 1, 1997
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Figure 3. Displacement of CAP-MR from a CAP-imprinted polymer upon injection of 20 µL of CAP (O), TAM (4), or CAP-DA (0); solutions (3-1000 µg/mL). The inset shows detail over the clinically significant CAP concentrations. The mobile phase was MeCN containing 0.6 µg/mL CAP-MR, flow rate 2 mL/min. Each sample was analyzed at least three times and the error bars represent the SEM values. Correlation coefficients: CAP (O), 0.985; TAM (4), 0.987; inset, 0.995.
for TAM at the same concentrations, showing that CAP competed more effectively with the bound CAP-MR than did TAM. The SEM for these results was less than 10% for all of the CAP and most of the TAM samples, but at the lowest TAM concentrations (3-10 µg/mL), it was as high as 18.7%. It seems that the lower detection limit of this system is around 3 µg/mL (∼60 ng analyte), and the main targets in the further development of this system are improving its sensitivity and precision at the lower concentrations. Nevertheless, the proposed method is adequate for detection below and above the recommended therapeutic range (10-20 µg/mL serum, with potential toxicity above 25 µg/ mL serum).17 TAM, which is almost identical in structure to CAP and, more importantly, possesses the same two free hydroxyl groups (Figure 1), was also efficiently detected by our system, although we aimed to develop a CAP-specific sensor. Nevertheless, in the case of a sensor developed for clinical purposes, when it is known precisely which drugs are involved, it can be used with confidence. Furthermore, in designing a sensor to interact with functionalities that are characteristic of a family of molecules, a single sensor could be used for the detection of any one of several different molecules. Naturally, a calibration curve should be prepared for each analyte, since the differing interactions of each substance with the MIP result in differing displacements of CAP-MR. To estimate the efficiency of the displacement of CAP-MR by CAP under these conditions, a calibration curve for CAP-MR was prepared, and the areas of the peaks obtained upon injection of CAP-MR (in the presence of CAP-MR in the eluent) were compared to the values obtained with CAP under the same conditions. It was found that the peaks generated by the injection of CAP amounted to approximately only 10% of those obtained with the same concentration of CAP-MR. This implied that only a small portion of the CAP molecules actually displaced CAPMR from the polymer. This is not surprising given the overwhelming excess of CAP-MR in the system. Possible interference in the system was investigated by mixing various amounts of CAP-DA with 100 µg/mL CAP and comparing the results with those obtained with the CAP solution only. As 2020 Analytical Chemistry, Vol. 69, No. 11, June 1, 1997
Figure 4. Peak area of the CAP-MR displaced upon injection of 100 µg/mL CAP mixed with various amounts of CAP-DA. The polymer was packed into a 5 cm column, and the mobile phase was MeCN containing 0.6 µg/mL CAP-MR, flow rate 2 mL/min. Each sample was analyzed at least three times, and the error bars represent the SEM values. The cross-hatched region corresponds to the range of the peak areas produced by 100 µg/mL CAP solution.
Figure 5. Detection of various CAP concentrations in serum, extracted in duplicate. CAP was extracted from the serum using ethyl acetate and dissolved in MeCN, and 20 µL was injected for analysis. The mobile phase was MeCN containing 0.6 µg/mL CAP-MR, flow rate 2 mL/min. Each point represents the mean of three measurements, and the SEM was approximately 5% , r2 ) 0.990.
can be seen in Figure 4, the values obtained with mixtures of analytes were within experimental error of those of the CAP solution alone, even when CAP-DA was in 10-fold excess. To assess the ability of this system to detect CAP in real samples, serum samples containing various concentrations of CAP were prepared. Each serum sample was divided in half, extracted separately and analyzed at least three times. Our system was able to detect CAP concentrations as low as 5 µg/mL (see Figure 5), and the response was linear over the range examined (correlation coefficient, 0.990), with SEM values lower than 10%, demonstrating the reproducibility and reliability of both the detection and the extraction methods. To summarize, we present here a detection system for chloramphenicol which is based on the competitive displacement of a CAP-dye conjugate from a CAP-imprinted polymer. This system rapidly and effectively detected CAP and, to a lesser degree, TAM with good reproducibility. CAP-DA, which is structurally similar to CAP yet lacks the appropriate functional groups, was not detected and was shown not to interfere with CAP detection, even in high concentrations. Our sensor system is capable of detecting chloramphenicol in serum samples both
above and below the clinically useful range. These results herald a new method of constructing tailor-made MIP-based sensors.
ACKNOWLEDGMENT This project was financially supported by the Ministry of Education, Science, Sports and Culture of Japan: Large-Scale Research Projects under the New Program in Grants-in-Aid for Scientific Research. R.L, S.M, S.A.P., and S.-H.C are grateful to the Japan Society for the Promotion of Science for postdoctoral fellowships. We thank Elena V. Piletskaya for technical assistance.
SUPPORTING INFORMATION AVAILABLE Synthesis and characterization of the CAP-MR conjugate (4 pages). Ordering information is given on any current masthead page.
Received for review September 25, 1996. Accepted March 4, 1997.X AC960983B X
Abstract published in Advance ACS Abstracts, April 15, 1997.
Analytical Chemistry, Vol. 69, No. 11, June 1, 1997
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