Bio-Medical Materials and Engineering 24 (2014) 1085–1091 DOI 10.3233/BME-130907 IOS Press
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A molecule-imprinted polyaniline membrane modified on carbon fiber for detection of glycine1 Hongjuan Zeng a, b*, Deshun Wang a , Junsheng Yu b a
School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Jianshe Road, Chengdu 610054, PR China b School of Optoelectronic Information, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, PR China
Abstract. A layer of L-glycine-molecule-imprinted polyaniline (LMIP-PANI) polymer film has been modified on a carbon fiber electrode for the determination of L-glycine standard samples and L-glycine in cerebrospinal fluid of wistar mice. It has been found that a linear relationship exists between current and concentration for the glycine standard samples in the range of 0–12 μM by using the LMIP-PANI-modified carbon fiber electrode as a sensor. However, there is no any relationship between current and concentration for the carbon fiber electrode modified with no-glycine-molecule-imprinted polyaniline (NIP-PANI). The MIP-PANI- and NIP-PANI-modified carbon fiber films have been characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and electrochemistry methods. The investigation shows that the MIP-PANI-imprinted carbon fiber electrode will have a potential application in in-situ monitoring neurotransmitter due to its easy fabrication, low cost, bio-compatibility and flexibility. Keywords: Glycine, polyaniline, imprint, film, cerebrospinal fluid
1. Introduction Glycine, one of the free amino acids, is widely distributed in the nervous system and plays an important role as neurotransimitters [1, 2]. It probably is the third major inhibitory neurotransmitter in brain. Glycine helps cells to convert many potentially harmful substances such as phenolic materials including benzoic acid (or sodium benzoate) into harmless ones. It is also important in the control of gluconeogenesis, the manufacture of blood sugar from protein in the liver. Furthermore, glycine can serve as a basic nitrogen source and is useful in the synthesis of haemoglobin, glutathione, DNA and RNA. As glycine plays a key role in the development and quality of our skeletal muscles, tissues and structural integrity, reliable measurement of glycine in trace amount is necessary. Detection methods for glycine include precolumn derivatization [3, 4], HPLC chromatography [5–7], chemiluminescene [8, 9], fluorimetriic methods [10, 11] and capillary electrophoresis [12, 13]. 1
This work was supported by National Natural Science Foundation of China (Grant No. 61071026). * Corresponding author. [email protected] 0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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Cleaning-up and concentration before quantitative evaluation are usually required. In some cases, chemical derivation is even required. Some of these methods are time-consuming. Recently, electrodes modified with conductive polymers as molecular recognition elements in molecule-imprinted polymer (MIP) systems are an emerging research direction [14, 15]. MIPs possessing surface cavities complementary in shape to the template can be controlled by electropolymerization procedures [16]. Electrochemical sensors using MIPs for the determination of different analytes have been prepared with appropriate signal transductions, including amperometry, voltammetry, quartz microbalance, and potentiometry [17]. To our knowledge, the carbon fiber electrode modified with an L-glycine molecule-imprinted PANI (MIP-PANI) polymer membrane for the determination of neurotransmitter has not been explored by far. In this paper, we suggest a novel, convenient and effective carbon fiber electrode modified with MIP-PANI polymer membrane for the determination of L-glycine. The MIP-PANI and no-glycine-molecule-imprinted polyaniline (NIP-PANI) films have been characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and electrochemistry methods. 2. Experimental 2.1. Chemical reagent Aniline (98%), Carbon fiber =7 μm (Xinfeng Carbon Fiber Co. Ltd., China); L-glycine (Kelong Chemical Reagent Factory, China); 0.05 mol/L PBS buffer solution (Na2HPO4·12H2O 9.465 g/L, KH2PO4 6.803 g/L) (pH=6.0). Double distilled water was used throughout the experiment except as otherwise noted. The standard solution was prepared by dissolving the corresponding L-glycine in double distilled water. An aqueous solution of 210-3 M L-glycine (purchased from Sigma Co.) was used as the standard solution to prepare a desired series of different concentrated solutions by diluting it with distilled water. The collected samples of wistar mice’s cerebrospinal fluid were also prepared by the above procedure. 2.2. The preparation of glycine imprinted and non-imprinted PANI polymers modified electrodes A novel self-made electrode is shown in Fig. 1.
Fig. 1: Carbon fiber electrodes
A fine copper wire was surrounded by five carbon fibers, and then encapsulated with PVC, with 1 mm-long fibers outside the PVC. After that, the carbon fiber microelectrodes was put in ethanol, HNO3/water 1:1 (V/V) to ultrasonically clean, and subsequently immerged into 0.5 mol/L H2SO4 for chemical activation for 2–3 hours. Thus, constructed carbon fiber microelectrode was ready to be used
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as a working electrode for deposition of the MIP-PANI polymer film at 0.9 V constant potential in a HCl-Tris solution containing 0.20 M aniline and 0.20 M L-glycine. Saturated calomel electrode and platinum wire electrode were selected as reference electrode and counter electrode, respectively. All the electrodes were connected to a CHI 660B electrochemical workstation. The MIP-PANI-modified carbon fiber electrodes were immersed in 0.05 mol/L pH 6.8 PBS cold solution at 4 °C before using. NIP-PANI modified electrodes were prepared according to the same procedures but with the solution only containing 0.20 mM aniline. 2.3. Characterizations Morphologies of the MIP-PANI and NIP-PANI membranes deposited on ITO glasses were observed by SEM (JSM-6100, Japan). XPS detection was performed with an XSAM-800 spectrometer (Krapos, UK) equipped with an Mg Ka X-ray source at 1253.6 eV. The chamber pressure was kept under 5×10-9 Pa during the measurement. Electrochemical properties of the MIP-PANI and NIP-PANI membranes modified on ITO glasses were characterized using a cyclic voltammetric scan through the CHI 660B electrochemical workstation. 2.4. Wistar mice and cerebrospinal fluid Either male or female wistar mice (10–12 weeks of age) weighing 250–280 g were purchased from Sichuan University (China). The mice were housed in stainless steel cages (5–6 rats/cage) for one week prior to the experiment. All animals were allowed free access to water and fed on commercial diet. For further tests, 10–50 μL CSF sample was collected through lumbar puncture. 2.5. Current and HPLC measurements The current responses of the MIP-PANI- and NIP-PANI-modified carbon fiber electrodes were recorded through an open circuit potential measurement at room temperature in L-glycine solutions of different concentrations. The solution of the cell was deoxygenated with nitrogen gas for 5 minutes. Chromatography was carried out with a modular liquid chromatographic unit consisting of a constant flow solvent delivery pump (Model 6000A, Waters Assoc., Milford, Mass., USA), a continuously variable wavelength UV-VIS Spectroflow monitor (Spectro Monitor - III, Laboratory Data Control, Riviera Beach, Fla., USA), a strip chart recorder (Model 56, Perkin-Elmer Corp., Norwalk Ct., USA), and a device (Model Vista 401, Varian Associates, Santa Clara, Ca., USA) to integrate the area under the curves of the eluates. Chromatographic conditions were as follows: Zorbax NH2 column; 0.01M potassium dihydrogen phosphate in acetonitrile/water solution (7/3, V/V) [5]. 3. Results and discussion 3.1. Morphological characterization of MIP-PANI and NIP-PANI membranes Fig. 2 shows typical SEM images of the MIP-PANI and NIP-PANI membranes prepared at 0.9 V constant potential for 100 s on ITO glass.
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Fig. 2 SEM images of ITO glass coated with MIP-PANI and NIP-PANI films: (a) MIP-PANI film, (b) NIP-PANI film, (c) MIP-PANI film after soaked in PBS for 4 hours, (d) NIP-PANI film after soaked in PBS for 4 hours.
It can be seen from the images that spatial distribution of the polymers is homogeneous for both films and their granular structures. But the average grain size of the NIP-PANI film is larger than that of the MIP-PANI (see Fig. 2a and 2b). After soaked in PBS solution for 4 hrs, the mesh-shaped and grain-shaped morphologies can be observed for the MIP-PANI (Fig. 2c) and NIP-PANI (Fig. 2d), respectively. Apparently, for the NIP-PANI film, the morphology has no significant change before and after immerged in PBS solution. 3.2. The XPS characterizations of NIP-PANI and MIP-PANI films 1600
1S
N
E1=399.6eV
2000
1400
1S
E2=399.6eV E1=397.9eV
Intensity
Intensity
N
1200
E2=401.3eV
1500
E3=406.2eV
E3=402.2eV
1000
800
1000 396
400 404 Binding energy (eV)
(a)
408
394
396
398 400 402 404 Binding energy (eV)
406
408
(b)
Fig. 3 N ( ls ) core-level spectra of (a) MIP-PANI films and (b) NIP-PANI films prepared by 0.2 M monomer at pH 4.0 and 0.9 V constant potential. The colorized curves in each spectrum represent the well-resolved Gaussian lineshapes corresponding to non-equivalent nitrogen sites.
Fig. 3 shows the XPS spectra for N1s core level of the MIP-PANI and NIP-PANI films. The signal of N 1s in both films can be fitted with three peaks with their maxima at 397.9 eV, 399.6 eV, and >400 eV, respectively. The first one at 397.9 eV can be attributed to the neutral species corresponding to imine groups; the second one at 399.6 eV is probably originated from the neutral species such as amine groups [18]; while the last one >400 eV can be assigned to positively-charged N atoms. This behavior is similar to those observed in copolymers of aniline, aminobenzoic and 2-aminoterephthalic acids [19]. It is known that the ratio between charged and neural species (N+/N) is related to the amount of charge carriers (and, therefore, to the conductivity). Table 1 shows the values of the N+/N
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ratio for the MIP-PANI and NIP-PANI films obtained by integrating the area of the XPS peaks. In the case of the MIP film, the N+/N (%) is 64.2, higher than that of NIP-PANI film (32); which suggests that the MIP-PANI film will be more conductive than the NIP-PANI film. Table 1 +
The relative percent of N /N determined by the integrated intensities of the XPS N(Is) lines Film MIP-PANI NIP-PANI
A1 (397.9 ev) -12676.7
A2 (399.6 eV) -12961.4 -17845.9
A3 (401.3 eV) -16213.7
A4 (402.2 eV) -12099.2
A5 (406.2 eV) -15861
¦A -37737 -49920
N+/N (%) 32.06 64.25
3.3. The electrochemical response of NIP-PANI and MIP-PANI films The electroactivity and electrochemical behaviour of the NIP-PANI and MIP-PANI films have also been analyzed in order to explore the use of these materials as electrodes. The NIP-PANI and MIP-PANI films were deposited on clean ITO glasses. Fig. 4 shows the electrochemical response of the NIP-PANI and MIP-PANI films in 25 mM concentrated phosphate buffers and nal ionic strength maintained at 60 mM. At pH 6.8, the current of the MIP-PANI film is about 40 times of that of the NIP-PANI film. It shows that the excellent conductive of the MIP film. In addition, a pair of redox peaks (Ea =70 mV and Ec = -370 mV) with ΔEp=440 mV can be seen for the MIP-PANI film. And a pair of redox peaks (Ea=500 mV and Ec= -550 mV) with ΔEp=1050 mV can be obtained for the NIP-PANI film. It implies that H+ ions can move much faster in the MIP-PANI film than in the NIP-PANI film. 200 MIP NIP
0 4 NIP
2 current/μA
current/μA
100
0 -2 -4
-100
-6 -8 -1.0
-0.8
-0.4
0.0 potential/V
-0.5
0.0 potential/V
0.4
0.5
1.0
0.8
Fig. 4 Cyclic voltammogram of MIP-PANI and NIP-PANI films in pH 6.8 PBS buffers.
Fig. 5 The open circuit-time curves of MIP-PANI modified carbon fiber and NIP-PANI modified carbon fiber in pH 6.8 PBS buffers with addition of 2.1×10-6 M glycine at a fixed time (100 s).
3.4. current measurements and analytical application All of the current response of the MIP-PANI carbon fibers electrodes were investigated and compared with that of the NIP-PANI-modified carbon fibers by using the method of open circuit potential with time. Prior to measurement of the current, the polymer films were conditioned in PBS (pH=6.8) until reached a constant value. The length of time required was recorded as a function of time. Potential measurements were obtained by mildly stirring PBS buffers (pH=7.0) while adding
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glycine. Fig. 5 displays a typical response obtained in standard glycine solutions. The response to glycine was significantly higher for the MIP-PANI film than for the NIP-PANI film. This confirms that the imprinting process creates a microenvironment for the recognition of glycine due to the presence of specific molecular interaction sites at the interfaces between the membranes and electrolytes where the exchange reaction between the glycine ions and the dopant (phosphate ions used in the preconditioning step) occurs. The insert graph shows a linear relationship between current and the concentration of glycine standard samples in the range from 0 to 12 μM for the carbon fiber electrode, but no relationship for the NIP-PANI modified carbon fiber electrode can be obtained. Table 2 Results of glycine concentration of cerebrospinal fluid of wistar mice analysis (n=5) Methods
HPLC (μM) 1.23 1.19 1.25 glycine concentration of cerebrospinal fluid of wistar mice/μM
Sample 1 Sample 2 Sample 3
MIP-PANI modified carbon fiber electrode (μM) 1.25 1.23 1.28
RSD (%) 1.65 3.4 2.5
1.30 Error Bars
1.25
1.20 1
2 samples
3
Fig.6 The error bars of glycine concentration of cerebrospinal fluid in different wistar mice.
The MIP-PNAI-modified carbon fiber was used to detect glycine concentration of cerebrospinal fluid of wistar mouse and compared with HPLC method [20]. The results are listed in Table 2. The relative standard derivations (RSD) by using the MIP-PANI-modified carbon fiber electrode are all less than 5%. The error bars of the samples are given in Fig. 6. The results are satisfactory. 4. Conclusions L-glycine-molecule-imprinted PANI and NIP-PANI films have been successfully synthesized through electrochemical polymerization in aqueous media. Both of them have been characterized by SEM, XPS and electrochemistry methods. A mesh-shaped structure, higher N+/N percent, and faster electron transfer have been found for the MIP-PANI films. The MIP-PANI-modified carbon fiber electrode was endowed higher specic recognition and selectivity to template L-glycine when it was in glycine standard solutions and real samples, cerebrospinal fluid of wistar mice. The MIP-PANImodified carbon fiber electrode may have a potential application in in-situ monitoring neurotransmitter due to its easy fabrication, low cost, biocompatibility, and flexibility.
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5. Acknowledgement The authors gratefully acknowledge the financial support of this work by National Foundation of China (Grant No. 61071026). References [1]
Martinez, M., Frank, A., Diez-Tejedor, E., and Hernanz, A. Amino acid concentrations in cerebrospinal fluid and serum in Alzheimer's disease and vascular dementia. J. Neural Transm., 6 (1993)1-9. [2] Albin, R.L. and Greenamyre, J.T. Alternative excitotoxic hypotheses. Neurology, 42 (1992)733-738. [3] Bruckner, H., Haasmann, S., Langer, M., Westhauser, T., Wittner, R., and Godel, H. Liquid chromatographic determination of D- and L-amino acids by derivatization with o-phthaldialdehyde and chiral thiols. J. Chromatogr. A, 666 (1994) 259-273. [4] Masoumeh H., Leila Y., Hamid A. A kinetic spectrophotometric method for simultaneous determination of glycine and lysine by artificial neural networks. Anal. Biochem., 365 (2007)74-81. [5] Koziol, T. R., Jacob, J. T., Achari, R. G. Ion-pair liquid chromatographic assay of decongestants and antihistamines. J Pharm Sci. 68(1979)1135-1138. [6] Marrubini G, Caccialanza G, Massolini G. Determination of glycine and threonine in topical dermatological preparations. J Pharm Biomed Anal., 47 (2008)716-722 [7] Uchikura, K., Chem. Pharm. Bull. Determination of aromatic and branched-chain amino acids in plasma by HPLC with electrogenerated Ru(bpy)3(3+) chemiluminescence detection. 51 (2003)1092-1094. [8] Zhou, G.J. and Chen, H.Y. Flow injection chemiluminescence determination of amino acids by oxidation with N-bromosuccinimide. Anal. Sci., 18 (2002) 693-696. [9] Yokota, K., Saito, K., Yamazaki, S., and Muromatsu, A. new detection method of -, -, and -amino acids coupled with an online photochemical oxidation and tris(2,2’-bipyridine)-ruthenium(iii) chemiluminescence, Anal. Lett., 35 (2002) 185-194. [10] Swanepoel, E., de Villiers, M.M., and du Preez, J.L. Fluorimetric method of analysis for D-norpseudoephedrine hydrochloride, glycine and L-glutamic acid by reversed-phase high-performance liquid chromatography. J. Chromatogr. A., 1–2 (1996) 287-291. [11] Cha, K.W., Park, C., and Park, K.W. Spectrofluorimetric Determination of Oxalate Based on Its Ternary Complex between Eu3+ and Thenoyltrifluoroacetone, Bull. Korean Chem. Soc., 23 (2002) 1456-1458. [12] Veledo, M.T., de Frutos, M., and Diez-Massa, J.C. Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection, J. Chromatography A., 1079 (2005) 335–343. [13] Miao-Jen Lu, Tai-Chia Chiu,Po-Ling Chang, Hsin-Tsung Ho, Huan-Tsung Chang. Determination of glycine, glutamine, glutamate, and -aminobutyric acid in cerebrospinal fluids by capillary electrophoresis with light-emitting diode-induced fluorescence detection, Analytica Chimica Acta., 538 (2005)143–150. [14] Hutchis, R.S., Bachas, L.G. Nitrate-selective Electrode Developed by Electrochemically Mediated Imprinting/Doping of Polypyrrole, Anal. Chem., 67 (1995) 1654 -1660. [15] Blanco-López, M. C., Lobo-Castanón, M. J., Miranda-Ordieres, A. J., Tunón -Blanco, P. Electrochemical sensors based on molecularly imprinted polymers, TrAC Trends Anal. Chem., 23 (2004)36-48. [16] P.-Y. Chen, P.-C. Nien, C.-T. Wu,T.-H. Wu, C.-W. Lin, K.-C. Ho. Fabrication of a molecularly imprinted polymer sensor by self-assembling monolayer/mediator system, Anal. Chim. Acta., 643 (2009)38-44. [17] Prasad, K., Prathish, K.P., Gladis, J.M., Naidu, G.R.K., Rao, T.P. Molecularly imprinted polymer (biomimetic) based potentiometric sensor for atrazine, Sens. Actuators B., 123 (2007)65-70. [18] Golczak, S., Kanciurzewska,A., Fahlman, M., Langer, K., Langer, J., J. Comparative XPS surface study of polyaniline thin Solid State Ionics,179 (2008)2234–2239. [19] J. Arias-Pardilla, H.J. Salavagione, C. Barbero, E. Morallón, J.L. Vázquez. Study of the chemical copolymerization of 2-aminoterephthalic acid and aniline: Synthesis and copolymer properties, Eur. Pol. J. 42 (2006) 1521-1532. [20] ZHANG Lu-rong, JIANG Guo-rong. Quantitative analysis of Asp, Glu and Gly in CFS by RP-HPLC, Chinese J. of Hospital Phar., 24 (2004) 689-691.
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A molecule-imprinted polyaniline membrane modified on carbon fiber for detection of glycine1 Hongjuan Zeng a, b*, Deshun Wang a , Junsheng Yu b a
School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Jianshe Road, Chengdu 610054, PR China b School of Optoelectronic Information, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, PR China
Abstract. A layer of L-glycine-molecule-imprinted polyaniline (LMIP-PANI) polymer film has been modified on a carbon fiber electrode for the determination of L-glycine standard samples and L-glycine in cerebrospinal fluid of wistar mice. It has been found that a linear relationship exists between current and concentration for the glycine standard samples in the range of 0–12 μM by using the LMIP-PANI-modified carbon fiber electrode as a sensor. However, there is no any relationship between current and concentration for the carbon fiber electrode modified with no-glycine-molecule-imprinted polyaniline (NIP-PANI). The MIP-PANI- and NIP-PANI-modified carbon fiber films have been characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and electrochemistry methods. The investigation shows that the MIP-PANI-imprinted carbon fiber electrode will have a potential application in in-situ monitoring neurotransmitter due to its easy fabrication, low cost, bio-compatibility and flexibility. Keywords: Glycine, polyaniline, imprint, film, cerebrospinal fluid
1. Introduction Glycine, one of the free amino acids, is widely distributed in the nervous system and plays an important role as neurotransimitters [1, 2]. It probably is the third major inhibitory neurotransmitter in brain. Glycine helps cells to convert many potentially harmful substances such as phenolic materials including benzoic acid (or sodium benzoate) into harmless ones. It is also important in the control of gluconeogenesis, the manufacture of blood sugar from protein in the liver. Furthermore, glycine can serve as a basic nitrogen source and is useful in the synthesis of haemoglobin, glutathione, DNA and RNA. As glycine plays a key role in the development and quality of our skeletal muscles, tissues and structural integrity, reliable measurement of glycine in trace amount is necessary. Detection methods for glycine include precolumn derivatization [3, 4], HPLC chromatography [5–7], chemiluminescene [8, 9], fluorimetriic methods [10, 11] and capillary electrophoresis [12, 13]. 1
This work was supported by National Natural Science Foundation of China (Grant No. 61071026). * Corresponding author. [email protected] 0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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Cleaning-up and concentration before quantitative evaluation are usually required. In some cases, chemical derivation is even required. Some of these methods are time-consuming. Recently, electrodes modified with conductive polymers as molecular recognition elements in molecule-imprinted polymer (MIP) systems are an emerging research direction [14, 15]. MIPs possessing surface cavities complementary in shape to the template can be controlled by electropolymerization procedures [16]. Electrochemical sensors using MIPs for the determination of different analytes have been prepared with appropriate signal transductions, including amperometry, voltammetry, quartz microbalance, and potentiometry [17]. To our knowledge, the carbon fiber electrode modified with an L-glycine molecule-imprinted PANI (MIP-PANI) polymer membrane for the determination of neurotransmitter has not been explored by far. In this paper, we suggest a novel, convenient and effective carbon fiber electrode modified with MIP-PANI polymer membrane for the determination of L-glycine. The MIP-PANI and no-glycine-molecule-imprinted polyaniline (NIP-PANI) films have been characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and electrochemistry methods. 2. Experimental 2.1. Chemical reagent Aniline (98%), Carbon fiber =7 μm (Xinfeng Carbon Fiber Co. Ltd., China); L-glycine (Kelong Chemical Reagent Factory, China); 0.05 mol/L PBS buffer solution (Na2HPO4·12H2O 9.465 g/L, KH2PO4 6.803 g/L) (pH=6.0). Double distilled water was used throughout the experiment except as otherwise noted. The standard solution was prepared by dissolving the corresponding L-glycine in double distilled water. An aqueous solution of 210-3 M L-glycine (purchased from Sigma Co.) was used as the standard solution to prepare a desired series of different concentrated solutions by diluting it with distilled water. The collected samples of wistar mice’s cerebrospinal fluid were also prepared by the above procedure. 2.2. The preparation of glycine imprinted and non-imprinted PANI polymers modified electrodes A novel self-made electrode is shown in Fig. 1.
Fig. 1: Carbon fiber electrodes
A fine copper wire was surrounded by five carbon fibers, and then encapsulated with PVC, with 1 mm-long fibers outside the PVC. After that, the carbon fiber microelectrodes was put in ethanol, HNO3/water 1:1 (V/V) to ultrasonically clean, and subsequently immerged into 0.5 mol/L H2SO4 for chemical activation for 2–3 hours. Thus, constructed carbon fiber microelectrode was ready to be used
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as a working electrode for deposition of the MIP-PANI polymer film at 0.9 V constant potential in a HCl-Tris solution containing 0.20 M aniline and 0.20 M L-glycine. Saturated calomel electrode and platinum wire electrode were selected as reference electrode and counter electrode, respectively. All the electrodes were connected to a CHI 660B electrochemical workstation. The MIP-PANI-modified carbon fiber electrodes were immersed in 0.05 mol/L pH 6.8 PBS cold solution at 4 °C before using. NIP-PANI modified electrodes were prepared according to the same procedures but with the solution only containing 0.20 mM aniline. 2.3. Characterizations Morphologies of the MIP-PANI and NIP-PANI membranes deposited on ITO glasses were observed by SEM (JSM-6100, Japan). XPS detection was performed with an XSAM-800 spectrometer (Krapos, UK) equipped with an Mg Ka X-ray source at 1253.6 eV. The chamber pressure was kept under 5×10-9 Pa during the measurement. Electrochemical properties of the MIP-PANI and NIP-PANI membranes modified on ITO glasses were characterized using a cyclic voltammetric scan through the CHI 660B electrochemical workstation. 2.4. Wistar mice and cerebrospinal fluid Either male or female wistar mice (10–12 weeks of age) weighing 250–280 g were purchased from Sichuan University (China). The mice were housed in stainless steel cages (5–6 rats/cage) for one week prior to the experiment. All animals were allowed free access to water and fed on commercial diet. For further tests, 10–50 μL CSF sample was collected through lumbar puncture. 2.5. Current and HPLC measurements The current responses of the MIP-PANI- and NIP-PANI-modified carbon fiber electrodes were recorded through an open circuit potential measurement at room temperature in L-glycine solutions of different concentrations. The solution of the cell was deoxygenated with nitrogen gas for 5 minutes. Chromatography was carried out with a modular liquid chromatographic unit consisting of a constant flow solvent delivery pump (Model 6000A, Waters Assoc., Milford, Mass., USA), a continuously variable wavelength UV-VIS Spectroflow monitor (Spectro Monitor - III, Laboratory Data Control, Riviera Beach, Fla., USA), a strip chart recorder (Model 56, Perkin-Elmer Corp., Norwalk Ct., USA), and a device (Model Vista 401, Varian Associates, Santa Clara, Ca., USA) to integrate the area under the curves of the eluates. Chromatographic conditions were as follows: Zorbax NH2 column; 0.01M potassium dihydrogen phosphate in acetonitrile/water solution (7/3, V/V) [5]. 3. Results and discussion 3.1. Morphological characterization of MIP-PANI and NIP-PANI membranes Fig. 2 shows typical SEM images of the MIP-PANI and NIP-PANI membranes prepared at 0.9 V constant potential for 100 s on ITO glass.
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Fig. 2 SEM images of ITO glass coated with MIP-PANI and NIP-PANI films: (a) MIP-PANI film, (b) NIP-PANI film, (c) MIP-PANI film after soaked in PBS for 4 hours, (d) NIP-PANI film after soaked in PBS for 4 hours.
It can be seen from the images that spatial distribution of the polymers is homogeneous for both films and their granular structures. But the average grain size of the NIP-PANI film is larger than that of the MIP-PANI (see Fig. 2a and 2b). After soaked in PBS solution for 4 hrs, the mesh-shaped and grain-shaped morphologies can be observed for the MIP-PANI (Fig. 2c) and NIP-PANI (Fig. 2d), respectively. Apparently, for the NIP-PANI film, the morphology has no significant change before and after immerged in PBS solution. 3.2. The XPS characterizations of NIP-PANI and MIP-PANI films 1600
1S
N
E1=399.6eV
2000
1400
1S
E2=399.6eV E1=397.9eV
Intensity
Intensity
N
1200
E2=401.3eV
1500
E3=406.2eV
E3=402.2eV
1000
800
1000 396
400 404 Binding energy (eV)
(a)
408
394
396
398 400 402 404 Binding energy (eV)
406
408
(b)
Fig. 3 N ( ls ) core-level spectra of (a) MIP-PANI films and (b) NIP-PANI films prepared by 0.2 M monomer at pH 4.0 and 0.9 V constant potential. The colorized curves in each spectrum represent the well-resolved Gaussian lineshapes corresponding to non-equivalent nitrogen sites.
Fig. 3 shows the XPS spectra for N1s core level of the MIP-PANI and NIP-PANI films. The signal of N 1s in both films can be fitted with three peaks with their maxima at 397.9 eV, 399.6 eV, and >400 eV, respectively. The first one at 397.9 eV can be attributed to the neutral species corresponding to imine groups; the second one at 399.6 eV is probably originated from the neutral species such as amine groups [18]; while the last one >400 eV can be assigned to positively-charged N atoms. This behavior is similar to those observed in copolymers of aniline, aminobenzoic and 2-aminoterephthalic acids [19]. It is known that the ratio between charged and neural species (N+/N) is related to the amount of charge carriers (and, therefore, to the conductivity). Table 1 shows the values of the N+/N
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ratio for the MIP-PANI and NIP-PANI films obtained by integrating the area of the XPS peaks. In the case of the MIP film, the N+/N (%) is 64.2, higher than that of NIP-PANI film (32); which suggests that the MIP-PANI film will be more conductive than the NIP-PANI film. Table 1 +
The relative percent of N /N determined by the integrated intensities of the XPS N(Is) lines Film MIP-PANI NIP-PANI
A1 (397.9 ev) -12676.7
A2 (399.6 eV) -12961.4 -17845.9
A3 (401.3 eV) -16213.7
A4 (402.2 eV) -12099.2
A5 (406.2 eV) -15861
¦A -37737 -49920
N+/N (%) 32.06 64.25
3.3. The electrochemical response of NIP-PANI and MIP-PANI films The electroactivity and electrochemical behaviour of the NIP-PANI and MIP-PANI films have also been analyzed in order to explore the use of these materials as electrodes. The NIP-PANI and MIP-PANI films were deposited on clean ITO glasses. Fig. 4 shows the electrochemical response of the NIP-PANI and MIP-PANI films in 25 mM concentrated phosphate buffers and nal ionic strength maintained at 60 mM. At pH 6.8, the current of the MIP-PANI film is about 40 times of that of the NIP-PANI film. It shows that the excellent conductive of the MIP film. In addition, a pair of redox peaks (Ea =70 mV and Ec = -370 mV) with ΔEp=440 mV can be seen for the MIP-PANI film. And a pair of redox peaks (Ea=500 mV and Ec= -550 mV) with ΔEp=1050 mV can be obtained for the NIP-PANI film. It implies that H+ ions can move much faster in the MIP-PANI film than in the NIP-PANI film. 200 MIP NIP
0 4 NIP
2 current/μA
current/μA
100
0 -2 -4
-100
-6 -8 -1.0
-0.8
-0.4
0.0 potential/V
-0.5
0.0 potential/V
0.4
0.5
1.0
0.8
Fig. 4 Cyclic voltammogram of MIP-PANI and NIP-PANI films in pH 6.8 PBS buffers.
Fig. 5 The open circuit-time curves of MIP-PANI modified carbon fiber and NIP-PANI modified carbon fiber in pH 6.8 PBS buffers with addition of 2.1×10-6 M glycine at a fixed time (100 s).
3.4. current measurements and analytical application All of the current response of the MIP-PANI carbon fibers electrodes were investigated and compared with that of the NIP-PANI-modified carbon fibers by using the method of open circuit potential with time. Prior to measurement of the current, the polymer films were conditioned in PBS (pH=6.8) until reached a constant value. The length of time required was recorded as a function of time. Potential measurements were obtained by mildly stirring PBS buffers (pH=7.0) while adding
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H. Zeng et al. / A molecule-imprinted polyaniline membrane modified on carbon fiber
glycine. Fig. 5 displays a typical response obtained in standard glycine solutions. The response to glycine was significantly higher for the MIP-PANI film than for the NIP-PANI film. This confirms that the imprinting process creates a microenvironment for the recognition of glycine due to the presence of specific molecular interaction sites at the interfaces between the membranes and electrolytes where the exchange reaction between the glycine ions and the dopant (phosphate ions used in the preconditioning step) occurs. The insert graph shows a linear relationship between current and the concentration of glycine standard samples in the range from 0 to 12 μM for the carbon fiber electrode, but no relationship for the NIP-PANI modified carbon fiber electrode can be obtained. Table 2 Results of glycine concentration of cerebrospinal fluid of wistar mice analysis (n=5) Methods
HPLC (μM) 1.23 1.19 1.25 glycine concentration of cerebrospinal fluid of wistar mice/μM
Sample 1 Sample 2 Sample 3
MIP-PANI modified carbon fiber electrode (μM) 1.25 1.23 1.28
RSD (%) 1.65 3.4 2.5
1.30 Error Bars
1.25
1.20 1
2 samples
3
Fig.6 The error bars of glycine concentration of cerebrospinal fluid in different wistar mice.
The MIP-PNAI-modified carbon fiber was used to detect glycine concentration of cerebrospinal fluid of wistar mouse and compared with HPLC method [20]. The results are listed in Table 2. The relative standard derivations (RSD) by using the MIP-PANI-modified carbon fiber electrode are all less than 5%. The error bars of the samples are given in Fig. 6. The results are satisfactory. 4. Conclusions L-glycine-molecule-imprinted PANI and NIP-PANI films have been successfully synthesized through electrochemical polymerization in aqueous media. Both of them have been characterized by SEM, XPS and electrochemistry methods. A mesh-shaped structure, higher N+/N percent, and faster electron transfer have been found for the MIP-PANI films. The MIP-PANI-modified carbon fiber electrode was endowed higher specic recognition and selectivity to template L-glycine when it was in glycine standard solutions and real samples, cerebrospinal fluid of wistar mice. The MIP-PANImodified carbon fiber electrode may have a potential application in in-situ monitoring neurotransmitter due to its easy fabrication, low cost, biocompatibility, and flexibility.
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5. Acknowledgement The authors gratefully acknowledge the financial support of this work by National Foundation of China (Grant No. 61071026). References [1]
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