Flow injection chemiluminescence determination of epinephrine using epinephrine-imprinted polymer as recognition material

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1 Analytica Chimica Acta 489 (2003) Flow injection chemiluminescence determination of epinephrine using epinephrine-imprinted polymer as recognition material Jianxiu Du, Lihua Shen, Jiuru Lu Department of Chemistry, School of Chemistry and Materials Science, Shaanxi Normal University, Xi an , PR China Received 21 January 2003; received in revised form 2 June 2003; accepted 3 June 2003 Abstract A flow injection chemiluminescence (CL) method using epinephrine-imprinted polymer as recognition material was investigated for the determination of epinephrine. The analyte, epinephrine, was selectively and temporarily adsorbed on the epinephrine-imprinted polymer, which was packed into a flow-cell placed in front of the window of a photomultiplier tube. Afterwards, the CL reagent, the emerging stream of luminol (presence of potassium ferrocyanide) and potassium ferricyanide flowed through the flow-cell and reacted with epinephrine adsorbed on the polymer to produce strong CL. The CL intensity responded linearly to the logarithm of the concentration of epinephrine within to mol/l range with a detection limit of mol/l. The R.S.D. for mol/l of epinephrine solution was less than 5% (n = 7). Interference experiments demonstrated that the application of molecular imprinted polymer (MIP) to CL analysis could be improved the selectivity of the method greatly. Tests of the recovery for known amount of epinephrine in serum were carried out and satisfactory results were obtained. At the same time, the binding characteristic of the polymer to epinephrine was evaluated by the batch method and the dynamic method Elsevier B.V. All rights reserved. Keywords: Epinephrine; Chemiluminescence; Flow injection; Molecularly imprinted polymer 1. Introduction In the past two decades, chemiluminescence (CL) analysis has been frequently used to the analysis of a wide variety of important inorganic and organic compounds due to its numerous advantages such as low detection limit and wide linear dynamic range, which both can generally be achieved with simple and relatively inexpensive instrumentation [1,2]. Unfortu- Corresponding author. Tel.: ; fax: address: xzb4@snnu.edu.cn (J. Lu). nately, the relatively low selectivity of the CL method itself limits its direct application to the analysis of analyte in complicated sample. Great efforts were made to overcome this shortcoming and the most common way is to use a separation technique, such as liquid chromatography [3,4] and capillary electrophoresis [5,6], prior to CL detection. Most of these methods not only required relatively complicated and expensive instruments, but also suffered from the incompatibility of separation conditions and CL detection conditions in many applications. Molecular imprinted polymer (MIP) has been known as a new synthetic material capable of /$ see front matter 2003 Elsevier B.V. All rights reserved. doi: /s (03) 转载

2 184 J. Du et al. / Analytica Chimica Acta 489 (2003) molecular recognition [7,8]. Usually molecularly imprinted polymer is prepared by copolymerization of functional monomer with cross-linker in the presence of template molecules to produce three-dimensional network polymers. Removal of the templates molecule results in a functional polymeric matrix with recognition sites complementary in size, shape and functionality to the template molecule. The attractive features of MIP, such as mechanical strength, ease of preparation and stability in harsh environments allows it to be used as chromatographic stationary phase [9], solid phase extraction matrices [10,11] artificial receptors for use in drug assays [12] and recognition elements in sensors [13,14]. More recently, the molecule recognition function of MIP was used by Lin and Yamada to develop CL-based sensing systems [15,16]. Epinephrine, a component of neural transmission media, has an important effect on the transmission of nerve impulses. Many phenomena are related to the concentration of epinephrine in blood as well as urine. The normal amount of epinephrine for healthy person in serum is about nmol/l level and in excreted is roughly 8.0 ± 62 g per day. Therefore, it is very important to develop sensitive and select analytical methods for the detection of epinephrine in biological fluids. Although many techniques have been developed for this purpose, high performance liquid chromatography with fluorescence detection and electrochemical is still the most applicable methods [17,18]. In this work, it is found that epinephrine can react with luminol to produce strong CL in alkaline solution in the presence of potassium ferricyanide and potassium ferrocyanide. Further experiments showed that this CL reaction was suitable to develop a flow injection on-line separation and preconcentration CL detection system for epinephrine using epinephrine-imprinted polymer as molecular recognition material. The MIP for epinephrine was prepared using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker in the presence of template molecule of epinephrine; a portion of the resultant polymer particles of size between 100 and 200 m was packed into flow-cell for selective and temporary adsorption of epinephrine; afterward, the emerging stream of luminol solution (presence of potassium ferrocyanide) and potassium ferricyanide solution flowed through the flow-cell, reacted with epinephrine adsorbed on the polymer to generate out strong CL; during the CL reaction, the epinephrine molecules were destroyed leaving the cavities for new determination. The application of MIP to CL analysis improves the selectivity of CL analysis greatly, and makes it possible to become a selective and sensitive method for the determination of analyte in complicated samples directly. The method was applied to the analysis of epinephrine in diluted serum samples. 2. Experimental 2.1. Apparatus The schematic diagram of flow system used in this work is shown in Fig. 1. Reagent solutions, water carrier solution and sample solution were delivered through two peristaltic pumps. PTFE tubing (0.8 mm i.d.) was used to connect all components in the flow system. Injection was made using a six-way injection valve. CL measurements were performed using an IFFL-D flow injection CL analyzer (Xi an Remax Electronic High-Tech Ltd.) fitting up a 1p21 photomultiplier tube (Hamamatsu). Absorbance measurements were taken on a TU-1901 spectrometer (Beijing Currency Instrumental Ltd.). Equilibrium binding experiments were carried out on a HY-2 variable speed reciprocal oscillator (Apparatus Co. Ltd. Changzhou) at room temperature. The data acquisition and treatment was performed with the IFFL-D Flow injection CL data processing software (Xi an Remax Electronic High-Tech Ltd.). a b c d P1 P2 V Fig. 1. Schematic diagram of the chemiluminescence flow system: (a) luminol solution; (b) potassium ferricyanide solution; (c) water carrier; (d) epinephrine solution; (P 1, P 2 ) peristaltic pump; (V) six-way injection valve; (F) flow-cell packed with epinephrine-imprinted polymer; (D) detector; (PC) computer; (W) waste solution. F D PC W

3 J. Du et al. / Analytica Chimica Acta 489 (2003) Reagents EGDMA ware purchased from Sigma (St. Louis, MO). MAA and 2,2 -azobis(2-methylpropinitrile) (AIBN) were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Luminol was kindly offered by Institute of Analytical Science of Shaanxi Normal University (Xi an, China). Other reagents were purchased from Xi an Chemical Reagent Factory (Xi an, China). All reagents used were of analytical reagent grade except for AIBN, which was chemical purity grade. Before of use, EGDMA, MAA, acetonitrile and benzoic alcohol were distilled and AIBN was recrystallized. Doubly distilled water was used throughout the experiments. Stock standard solution of epinephrine ( mol/l) was prepared by dissolving g of epinephrine in 20 ml of 0.1 mol/l acetic acid, and then diluted with water to 100 ml. Working standard solutions of epinephrine were prepared by diluting this stock solution with water. All epinephrine solutions were stored in the refrigerator and protected from light. Luminol solution ( mol/l) was prepared similar to that reported previously [19]. Potassium ferricyande solution ( mol/l) was prepared with water and protected from light Preparation of epinephrine-imprinted polymer MIP for epinephrine was synthesized according to the method described by Liang et al.[20]. A 1 mmol of epinephrine, 4 mmol of MAA and 20 mmol of EGDMA were added into a glass vial and dissolved in 10 ml mixture of acetonitrile and benzoic alcohol (v/v, 3:2). Then 0.31 mmol of AIBN was added, and the solution were purged with nitrogen for 15 min and polymerized at 65 C for 24 h. The resultant bulk polymers were crushed, ground and sieved to collect the particles of size between 100 and 200 m for the following experiments Preparation of MIP-packed flow-cell A portion of above collected polymer particles (50.0 mg) was packed into a colorless glass tube (4 mm i.d. 5 cm length) and plugged with a small amount of glass wool at both ends. This glass tube was connected into the flow system and placed in front of the window of the photomultiplier. For a new MIP-packed flow-cell, the emerging stream of luminol and potassium ferricyanide was allowed to flow through the flow-cell until a stable blank signal was recorded. Then, the doubly distilled water flowed through the flow-cell to clean the polymer Binding experiments The binding characteristic of the polymer was evaluated by two methods: batch method and dynamic method. For the batch method: the above collected polymer particles (20.0 mg) were mixed with 5.0 ml of various concentration of epinephrine solution in 25 ml conical flask and then oscillating for 12 h on a oscillator at room temperature. After centrifuging at 3000 rpm for 10 min, the concentration of free epinephrine in the supernatant was detected by measuring the absorbance at 280 nm. The amount of epinephrine bound to the polymer was calculated by subtracting the concentration of free epinephrine from the initial epinephrine concentration. The data obtained was used for the Scatchard analysis. For the dynamic method: the epinephrine solution was continuously flowed through a glass tube (4 mm i.d. 5cm length) packed with 50.0 mg of epinephrine-imprinted polymer at constant flow rate. The absorbance of free epinephrine in the efflux solution was detected at 280 nm Separation and preconcentration of epinephrine The procedure for separation and preconcentration detection of epinephrine could be summarized as four steps. Step 1 (preconcentration of epinephrine): In this step, six-way injection valve is in the sampling position and pump 2 is stopped. Pump 1 delivered epinephrine solution to flow through the flow-cell and epinephrine was selectively absorbed on the polymer packed into the flow-cell. Step 2 (removing the other substances except epinephrine): In this step, six-way injection valve is in the sampling position and pump 1 was stopped. Pump 2 was started and pumped continuously water carrier through the polymer to remove the other substances except epinephrine.

4 186 J. Du et al. / Analytica Chimica Acta 489 (2003) Step 3 (chemiluminescence detection): In this step, six-way injection valve is in the injection position and pump 1 is stopped. The emerging stream of luminol solution and potassium ferricyanide solution was driven by water carrier to flow through the flow-cell and reacted with epinephrine molecule adsorbed in the MIP to produce strong CL. Step 4 (cleaning the polymer): In this step, six-way injection valve is in the sampling position and pump 1 is stopped. Water carrier washed the flow-cell continuously to clean the polymer for new determination. 3. Results and discussion In the preliminary studies, the very weak CL emission was observed by mixing epinephrine with an alkaline solution of luminol. In the presence of potassium ferricyanide, this very weak CL emission was enhanced greatly. However, the blank signal was too high to be used to develop a CL method for epinephrine because potassium ferricyanide can also oxidize luminol to give a strong CL in alkaline solution. Since the CL reaction of luminol with potassium ferricyanide could be inhibited by potassium ferrocyanide [21], the blank signal arising from luminol-ferricyanide reaction could be decreased effectively by addition of potassium ferrocyanide into reaction system. The possible mechanism for the present reaction may be that in alkaline solution, epinephrine reduces dissolved oxygen to superoxide radical [22] and the produced superoxide radical oxidizes luminol to give out very weak CL emission [23]; the presence of potassium ferricyanide and potassium ferrocyanide not only catalyze the CL reaction between superoxide radical and luminol [19], but also destroys the reaction equilibrium between epinephrine and dissolved oxygen. epinephrine + O 2 (aq) + OH superoxide radical superoxide radical + luminol K 3 Fe(CN) 6 -K 4 Fe(CN) 6 excited state of 3-aminophthalate ion Excited state of 3-aminophthalate ion 3-aminophthalate ion + hν Q/[epinephrine]/ml Q/ mol Fig. 2. Scatchard plots to estimate the binding nature of epinephrine-imprinted polymer. Q is the amount of epinephrine to 20.0 mg of epinephrine-imprinted polymer Binding characteristic of epinephrine-imprinted polymer In this work, the binding characteristic of epinephrine-imprinted polymer was estimated by two methods: batch method and dynamic method. For the batch method, the equilibrium binding experiments were carried out by varying the concentration of epinephrine from 0.5 to 5.0 mmol/l in the presence of 20.0 mg of above collected polymer particles. The obtained data were plotted according to the Scatchard equation [24] to estimate the binding parameters of epinephrine-imprinted polymer. As shown in Fig. 2, the Scatchard plot was not linear with at whole epinephrine concentration range indicating that the binding sites in the epinephrine-imprinted polymer are non-uniform in respect to the affinity for epinephrine. However, it is observed that two distinct sections within the plot can be regarded as straight lines. This revealed that two classes of binding sites were in epinephrine-imprinted polymer. The equilibrium dissociation constant K d1 and the apparent maximum amount Q max1 of the higher affinity binding sites can be calculated to be 4.66 mmol/l and mol/g of dry polymer from the slope and intercept of its Scatchard plot. By the same treatment, K d2 and Q max2 of the lower affinity binding sites were calculated to be 1.67 mmol/l and 71.3 mol/g. For the dynamic method, the epinephrine solution at different concentrations was continuously flowed through a glass tube (4 mm i.d. 5 cm length) packed

5 J. Du et al. / Analytica Chimica Acta 489 (2003) with epinephrine-imprinted polymer (50.0 mg) at the flow rate of 1.5 ml/min. The absorbance of epinephrine in the efflux solution was detected every 3 ml. It was observed that the absorbance of epinephrine in the efflux solution was increasing with the increase in the time of epinephrine flowing through the polymer. Finally, it reached a constant value. The time for absorbance reaching constant were different for different concentrations of epinephrine solution at same flow rate. The lower concentration of epinephrine solution, the longer time was needed. The equilibrium time for , and mol/l epinephrine solution at the flow rate of 1.5 ml/min were about 10, 5 and 4 min, respectively Optimization of CL reaction conditions When the MIP-packed flow-cell replaced by a spiral glass tube (1 mm i.d. 15 cm length), the schematic diagram of the flow system showed in Fig. 1 was used to optimize the reagents concentrations for the CL determination of epinephrine. The effect of luminol concentration was examined in the range of to mol/l. The CL intensity increased with raising the concentration of luminol up to mol/l, above this concentration, the CL intensity decreased. The effect of sodium hydroxide concentration was examined over mol/l ranges. The maximum CL intensity was obtained when 0.5 mol/l sodium hydroxide was used. The effect of potassium ferricyanide concentration was examined in the range of to mol/l. The CL intensity increased as the concentration of potassium ferricyanide increasing, The CL intensity reached up to maximum value when mol/l of potassium ferricyanide was used. Higher concentration of potassium ferricyanide lowered the CL intensity. In the absence of potassium ferrocyanide, the blank signal was very high because potassium ferricyanide can oxidize luminol to give a strong CL in alkaline solution. Sherlin and Neufeld have ever reported that the CL reaction of luminol with potassium ferricyanide could be inhibited by potassium ferrocyanide [21]. Therefore, the blank signal could be reduced by addition of potassium ferrocyanide into reaction system. The effect of potassium ferrocyanide concentration was also examined in the range of mol/l when potassium ferricyanide concentration was fixed at mol/l. The experimental results showed that 0.25 mol/l potassium ferrocyanide could decrease the blank signal efficiently and give the maximum CL intensity Optimization of separation and preconcentration conditions Using the schematic diagram showed in Fig. 1, a series of experiments was conducted to optimize the experimental conditions of separation and preconcentration of epinephrine. The preconcentration time for epinephrine is one of most important parameters in the experiments. It is relevant to the concentration of epinephrine, the binding capacity of the polymer and the flow rate. When the amount of the polymer was 50.0 mg and the flow rate was fixed at 1.5 ml/min, the relation of the CL intensity with the preconcentration time was examined. It was observed that the CL intensity was increasing with the increase in preconcentration time within the examined range (5 20 min) for mol/l epinephrine solution. Finally, 10 min was selected. The dynamic binding experiments showed under this preconcentration time, lower than mol/l epinephrine solution can be effectively adsorbed by the polymer. The washing time in step 2 is a critical parameter for the selective determination of epinephrine. A longer washing time is helpful to remove the other substances expect for epinephrine, whereas, too long washing time would result in the loss of epinephrine, thereby decrease the sensitivity of detection. The effect of the washing time was examined in the 0 5 min when the flow rate of water carrier was fixed at 1.5 ml/min. The experiments showed that when the washing time was 3 min, the other substances can be effectively removed and the CL intensity varied slightly. So, 3 min was selected as the suitable time. The reagent volume, the volume of the emerging stream of luminol solution and potassium ferricyanide solution is another key parameter in the experiments. If the volume is too small, the epinephrine adsorbed on the polymer cannot react completely in one determination. However, if the volume is too larger, a long cleaning time is needed to remove the excessive reagent and a long analytical time is required. The

6 188 J. Du et al. / Analytica Chimica Acta 489 (2003) Table 1 Calibration data for the determination of epinephrine Signal s R.S.D. (%) Net signal Reagent blank mol/l mol/l mol/l mol/l mol/l effect of different reagent volume ( l) was compared. The experimental results showed that the optimum reagent volume was 600 l. The effect of the cleaning time for the polymer was also examined within 0 5 min. It was observed that 3 min is enough to clean the polymer, giving a good repeatability when the flow rate of water carrier was fixed 1.5 ml/min Analytical performance Under the selected experimental conditions, the CL intensity responded linearly with the logarithm of the concentration of epinephrine in the range of to mol/l with a slope of (n=5, r 2 =0.996). The data for calibration was listed in Table 1. The possible explanation for the CL intensity responding linearly with log[epinephrine] may be that the present CL reaction is not same as the usual CL reaction performing in the solution, it actually occurs in the porous structure of MIP. For the different concentration of epinephrine, the rate of diffusion of CL reagents in the porous structure of MIP may be different, this causes the different rate of CL reaction and results in the CL intensity responding linearly with log[epinephrine]. The standard deviations for mol/l of epinephrine solution was less than 5% (n=7). When the concentration of epinephrine was mol/l, the column with packed of 50.0 mg of epinephrine-imprinted polymer could be used more than 100 times. The detection limit (3σ) was mol/l. While under the same conditions, the detection limit for epinephrine without preconcentration step is mol/l. As can be seen, the detection limit for epinephrine using MIP as preconcentration matrix is 1 order of magnitude lower than that obtained without the preconcentration Table 2 Tolerable ratio of interfering species to epinephrine with and without MIP Specie With MIP Without MIP Glucose Galactose Lactose Glycin Creatine Uric acid Ascorbic acid 50 1 Glutathione Norepinephrine Dopamine step. This indicates that MIP can be used as concentration matrix in the CL analysis and it can decrease the detection limit of the method Selectivity Under the optimized conditions and using the manifold depicted in Fig. 1, the interference of some reducing species normally existed in serum was investigated by analyzing a standard solution of mol/l epinephrine. The normal concentration ranges for these reducing species in blood are 2 14 mg/l for ascorbic acid, mg/l for creatine, mg/l for glutathione, mg/l for glucose, 3 28 mg/l for galactose, mg/l for uric acid [25]. The tolerable limit of an interfering species was taken as a relative error less than 5%. The results obtained are shown in Table 2. At the same time, the interference of these species to epinephrine under the same conditions without MIP was also carried out. The results obtained are also shown in Table 2. As can be seen for Table 2, with MIP, the tolerable ratio of all interfering species to epinephrine was enhanced. These results showed that MIP can be used as molecular recognition material in the CL analysis and improve the selectivity of the CL method. In the presence of MIP, the tolerable limits for most reducing species are higher than their normal level in blood except for glutathione and glucose. However, for the 1000-fold diluted samples, the concentrations for glutathione and glucose are lower than their interference concentrations. It should be indicated that 100-fold is the highest tolerable ratio that we examined. The

7 J. Du et al. / Analytica Chimica Acta 489 (2003) Table 3 Results of recovery tests on serum samples Sample Concentration (mol/l) Found a (mol/l) a Average of three measurements. Recovery (%) actual tolerable ratios for some reducing species are possible higher than 100-fold Application Blood samples were obtained from the hospital of Shaanxi Normal University and centrifuged at 3000 r/min for 30 min. The supernatant was transferred into the test tube and used as serum sample. A known amount of epinephrine standard solution was added to 0.1 ml of serum sample and then the mixture was diluted to 100 ml with doubly distilled water for analysis. The results of the recovery test are showed in Table 3. As can be seen from Table 3, the recoveries of added epinephrine can be quantitative and t-test assumed that there is no significant difference between recoveries and 100% at confidence level of 95%. 4. Conclusion In this work, the epinephrine-imprinted polymer was used as molecule recognition material in the CL analysis. The characteristic of the selective binding function of epinephrine-imprinted polymer to epinephrine molecule enables the method to have the advantage of selectivity, make it possible to be applied to analysis of epinephrine in complicated sample directly. The application of the method was validated by testing the recovery of known amount of epinephrine in serum samples. Acknowledgements The authors gratefully acknowledge financial support from National Natural Science Foundation of China (Grant No ) and Natural Science Foundation of Shaanxi Province. References [1] K. Robards, P.J. Worsfold, Anal. Chim. Acta 266 (1992) 147. [2] M.G. Sanders, P.J. Worsfold, J. Biolumin. Chemilumin. 11 (1996) 61. [3] A. Dapkevicius, T.A. Beek, H.A.G. Niederlander, A. Groot, Anal. Chem. 71 (1999) 736. [4] B. Gammelgaard, O. Joens, B. Nielsen, Analyst 117 (1992) 637. [5] A.M. Garcia-Campana, W.R.G. Baeyens, Y. Zhao, Anal. Chem. 69 (1997) 83A. [6] X. Huang, Z. Fang, Anal. Chim. Acta 414 (2000) 1. [7] G. Wulff, Angew. Chem. Int. Ed. Engl. 34 (1995) [8] K. Mosbach, O. Ramstrom, Biotechnology 14 (1996) 163. [9] V.T. Remcho, Z.J. Tan, Anal. Chem. 71 (1999) 248A. [10] N. Masque, R.M. Marce, F. Borrull, Trends Anal. Chem. 20 (2001) 477. [11] D. Sterenson, Trends Anal. Chem. 18 (1999) 154. [12] G. Vlatakis, I.I. Andersson, R. Muller, K. Mosbach, Nature 361 (1993) 645. [13] D. Kriz, O. Ramstrom, K. Mosbach, Anal. Chem. 69 (1997) 345A. [14] K. Haupt, K. Mosbach, Chem. Rev. 100 (2000) [15] J. Lin, M. Yamada, Anal. Chem. 72 (2000) [16] J. Lin, M. Yamada, Analyst (2001) 810. [17] B. Kagedal, D.S. Goldstein, J. Chromatogr. 429 (1988) 177. [18] R.P.H. Nikolajsen, A.M. Hansen, Anal. Chem. Acta 449 (2001) 1. [19] J. Du, Y. Li, J. Lu, Anal. Lett. 34 (2001) [20] C. Liang, H. Peng, A. Zhou, L. Nie, S. Yao, Anal. Chim. Acta 415 (2000) 135. [21] P.B. Sherlin, H.A. Neufeld, J. Org. Chem. 35 (1990) [22] M. Sun, S. Zigman, Anal. Biochem. 90 (1978) 81. [23] R.E. Bensinger, C.M. Johnson, Anal. Biochem. 116 (1984) 142. [24] R.L. Veazey, T.A. Nieman, Anal. Chem. 51 (1979) [25] J. Matsui, Y. Miyoshi, O. Doblhoff-Dier, T. Takeuchi, Anal. Chem. 67 (1995) 4404.