Author’s Accepted Manuscript Pyrrole-phenylboronic acid: A novel monomer for dopamine recognition and detection based on imprinted electrochemical sensor Min Zhong, Ying Teng, Shufen Pang, Liqin Yan, Xianwen Kan www.elsevier.com/locate/bios
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S0956-5663(14)00679-4 http://dx.doi.org/10.1016/j.bios.2014.08.083 BIOS7074
To appear in: Biosensors and Bioelectronic Received date: 2 August 2014 Revised date: 26 August 2014 Accepted date: 28 August 2014 Cite this article as: Min Zhong, Ying Teng, Shufen Pang, Liqin Yan and Xianwen Kan, Pyrrole-phenylboronic acid: A novel monomer for dopamine recognition and detection based on imprinted electrochemical sensor, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2014.08.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pyrrole-phenylboronic Acid: A Novel Monomer for Dopamine Recognition and Detection based on Imprinted Electrochemical Sensor
Min Zhong, Ying Teng, Shufen Pang, Liqin Yan, Xianwen Kan*
College of Chemistry and Materials Science, Anhui Key Laboratory of Chemo-Biosensing, Anhui Key Laboratory of Functional Molecular Solids, Anhui Normal University, Wuhu 241000, P.R. China
*Corresponding author: Xianwen Kan, E-mail: [email protected]; Tel: +86-553-3937135; Fax: +86-553-3869303.
Abstract A molecular imprinting polymer (MIP) based electrochemical sensor was successfully prepared for dopamine (DA) recognition and detection using pyrrole-phenylboronic acid (py-PBA) as a novel electropolymerized monomer. py-PBA could form cyclic boronic ester bond with DA, thus endowing a double recognition capacity of the sensor to DA in the combination of the imprinted effect of MIP. Compared with the sensor prepared using pyrrole or phenylboronic acid as electropolymerized monomer, the present sensor exhibited a remarkable high imprinted factor to DA. The influence factors including pH value, the mole ratio between monomer and template molecule, electropolymerization scan rate, and scan
cycles of electropolymerization process were investigated and optimized. Under the optimal conditions, the sensor could recognize DA from its analogues and monosaccharides. A linear ranging from 5.0 × 10-8 to 1.0 × 10-5 mol/L for the detection of DA was obtained with a detection limit of 3.3× 10-8 mol/L (S/N=3). The sensor has been applied to analyze DA in injection samples with satisfactory results.
Keywords: pyrrole-phenylboronic acid, electrochemical sensor, dopamine, molecular imprinting polymer, double recognition
1 Introduction Molecular imprinting, a promising technique, has been used to synthesize molecular imprinting polymer (MIP) to recognize template molecule (Günter Wulff, 1995; Cheong et al., 1997; Haupt and Mosbach, 2000). The advantages that MIP possesses over biopolymers are low cost, ease of preparation and stability over a great range of temperatures (Mosbach and Ramström, 1996; Hwang and Lee, 2002; James et al., 1996). The general procedure for synthesis MIP involves: First, the assembly of functional monomers around a template molecule in solution to form a complex by covalent or non-covalent bond. Next, the polymerization of above complex with cross-linker and initiator under photo or thermal condition. Third, the removal of the embedded template molecules in the polymer by certain extraction method to afford MIP. The removal of template molecules leaves microcavities with a three dimensional structure complementary in both shape and chemical functionality to
template molecules (Cheong et al., 1997), which is now capable of rebinding the template molecule with a very high specificity (Cheong et al., 1997; Okutucu and Onal, 2011; Xu et al., 2013; Bossi et al., 2007; Li et al., 2006). Most imprinting polymers synthesis reported were based on the non-covalent bond between functional monomer and template molecule, which has been developed primarily by Mosbach and co-workers (Mosbach and Ramström, 1996). This approach is flexible concerning the extraction and rebinding of template molecule. The covalent imprinting is superior in preventing leakage of template molecules during polymerization because of the formation of stable covalent bonds between functional groups of the template and those of monomer (Wang et al., 2009; Boonpangrak et al., 2006; Hwang and Lee, 2002; Ikegami et al., 2004). In order to remove the template, the covalent bonds connecting the template to the polymer should be cleaved (Hwang and Lee, 2002), which could form again when the polymer is incubated into the template molecule solution under the suitable condition. This reversible reaction between template and monomer could provide much more precise rebinding sites for template molecule, resulting the high rebinding and recognition capacities of MIP (Anderson et al., 1993; Meyer et al., 2007; Subrahmanyam et al., 2001; Mayes and Whitcombe, 2005). It is well known that boronic acids can covalently interact with cis-diol-containing molecules to form stable cyclic esters in an alkaline aqueous solution while the boronate esters dissociate when the environmental pH is switched to acidic (James et al., 1996; Rajkumar et al., 2007; Springsteen et al., 2001). Since the discovery of this
reversible interaction between PBA and saccharide in 1954, many PBA-based diol sensors have been developed (Nishiyabu et al., 2011; Okutucu and Onal, 2011; Liu et al., 2001). Recently, much attention has been devoted to the construction of electrochemical and optical sensors based on the interaction of cis-diol compounds and boronic acid (Dechtrirat et al., 2014; Liu et al., 2013; Bull et al., 2012). Polymerisable
boronic acid derivates also have been designed as monomers to form MIP through covalent approach by dehydrocondensation reaction with carbohydrates (Rajkumar et al., 2007). These boronic acid based MIP not only could selectively bind cis-diol compounds from general compounds, but also could recognize template molecule from its analogues, which also are cis-diol compounds (Friggeri et al., 2001; Lin et al., 2013; Bi et al., 2013). Among these boronic acid derivates, phenylboronic acid (PBA) has been extensively used due to its high operational stability and polymerisable property (D'hooge et al., 2008; Mcculloch et al., 2003; Matsumoto et al., 2010). Herein, another kind of phenylboronic acid derivate, pyrrole-phenylboronic acid (py-PBA) was synthesized as a polymerisable monomer for the MIP based electrochemical sensor preparation. The formation of cyclic boronic ester bond between boronic acid and DA, as well as the imprinted cavities of MIP endowed the prepared sensor double recognition capacities. The results of electrochemical experiments demonstrated that the prepared sensors exhibited a much higher imprinted factor than that of the sensor prepared by using py or PBA as polymerized monomer. The sensor could recognize template molecule from its analogues with a good selectivity and sensitively detect DA with a wide linear range and a low
detection limit. Meanwhile, the sensor was used to detect DA in real samples with satisfactory results.
2 Experimental 2.1 Chemicals Commercially available reagents and solvents were used without further purification. 3-aminophenylboronic acid monohydrate (APBA) and ethanol-d6 were ordered from Aldrich. 2, 5-Dimethoxytetrahydrofuran, glucose (Glu), fructose (Fru), and mannose (Man) were obtained from Aladdin (Aladdin Industrial Inc., China). Dopamine (DA, ≥98.5%), ascorbic acid (AA, >98%), uric acid (UA, >99%), epinephrine hydrochloride (EP, >98%), and pyrrole were purchased from Fluka (Fluka Chemie AG, Switzerland). All other reagents were of at least analytical-reagent grade. And double-distilled deionized water was used for all solutions. 2.2 Apparatus
Electrochemical experiments, such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed on CHI 660C workstation (ChenHua Instruments Co., Shanghai, China) with a conventional three-electrode system. A bare or modified glassy carbon electrode (GCE) was served as a working electrode. A saturated calomel electrode and a platinum wire electrode were used as a reference electrode and a counter electrode, respectively. Field emission scanning electron microscope (FE-SEM) images were obtained on an S-4800 field emission scanning electron microanalyser (Hitachi, Japan). 1H NMR (300 MHz) spectrum was recorded
at 300 MHz NMR spectrometer in C2D5OD. All chemical shifts (δ) are given in ppm relative to TMS (δ = 0 ppm) as internal standard. 2.3 Synthesis and characterization of 3-(1H-pyrrol-1-yl)phenylboronic acid (py-PBA) py-PBA was synthesized according to Boyacı’s work with a simple modification (Aytaç et al., 2011). 3-Aminophenylboronic acid monohydrate (100 mg, 0.646 mmol) was
dissolved
in
10
mL
acetonitrile.
With
the
addition
of
2,
5-Dimethoxytetrahydrofuran (0.167 mL, 1.292 mmol) into above solution under stirring, acetic acid (0.018 mL, 0.323 mmol) was also added to the solution. The resulting mixture was refluxed for 6 h. The reaction was monitored by thin-layer chromatograpgy. After completion of the reaction, the fulvous mixture was passed through a small plug of silica, eluting with 20 mL CH2Cl2, and the solvent was removed under vacuum. The obtained brownish powder was py-PBA. 1H NMR (CD3OD, 300 MHz): δ (ppm): 6.262 (bs, 2H, pyr-H), 7.154 (s,2H, BOH), 7.417-7.465 ᧤m,1H, Ar-H᧥᧨7.634᧤d,1H,Ar-H᧥᧨7.829᧤bs,1H,Ar-H᧥. 2.4 Fabrication of MIP modified GCE (MIP/GCE) The clean GCE was immersed into 5 mL phosphate buffer solution (PBS, 0.05 mol/L, pH=8.0) containing 1.12 mg synthesized py-PBA and 100 μL 0.05 mol/L DA. Then cyclic voltammetry (CV) was performed from -0.2 V to +1.2 V for 20 cycles at a scan rate of 50 mV/s, obtaining polymer film modified electrode. Subsequently, the embedded DA was extracted by immersing above electrode into 0.5 mol/L H2SO4 aqueous solution, subsequently with the electrochemical scanning between 0 - 1.5 V
for several cycles, getting MIP modified electrode (MIP/GCE). The procedure of preparation of MIP/GCE was depicted in Fig. 1A. As a control, non-molecular imprinting polymer modified electrode (NIP/GCE) was prepared and treated in exactly the same way except for the omitting of DA in the electropolymerization process. 2.5 Electrochemical properties measurements Electrochemical measurements to characterize the prepared modified electrodes were carried out in 0.05 mol/L PBS (pH=7.0) by using CV and DPV methods. AA, UA, and EP were selected as coexisted or structural similar compounds to evaluate the recognition capacity of the prepared sensor.
3 Results and discussion 3.1 Characterization of MIP/GCE The morphological structure of MIP/GCE was tested by SEM, as shown in Fig. 1B and Fig. 1C. It could be seen that a layer of rough film coated onto the surface of GCE (Fig. 1C), which should be attributed to the electropolymerization of polymer film in the presence of DA. One mmol/L [Fe(CN)6]3−/[Fe(CN)6]4− was chosen as an electro-active probe to investigate the extraction of template molecule. As shown in Fig. 1D, no redox peaks of probe could be found on MIP/GCE before the extraction of DA because embedded DA molecules blocked the arrival of probe to surface of the electrode. With the removal of DA, an obvious redox peaks of probe appeared on MIP/GCE, which could
be explained that the extraction of DA molecules left imprinted cavities for probe reaching electrode surface. However, CV curves of NIP/GCE before and after the extraction both had no redox peaks of the probe, which could be ascribed to no imprinted cavities were created during the electropolymerization process. 3.2 Polymerized monomer for MIP/GCE Although the synthesis of py-PBA has been reported, it is a novel monomer in the field of MIP. Py or PBA has been applied individually in the MIP preparation since py could be electrpolymerized to form MIP on electrode surface and PBA could selectively bind cis-diol compounds (Choong et al., 2009; Mazzotta et al., 2008; Tomsho and Benkovic, 2012). In order to choose an efficient monomer, py, PBA, and synthesized py-PBA were employed as different monomer to prepare three sensors and the specific rebinding properties were investigated. From Fig. 2, sensor prepared using py as monomer showed the specific adsorption since the current of DA on NIP/GCE was really low, implying that the Ppy film could efficiently decrease the non-specific adsorption of DA. The sensor prepared using PBA as monomer showed the highest current of DA compared with other sensors, which could be explained that the existed boronic acid group of PBA in MIP film could rebind lots of DA molecules. Similarly, the boronic acid group in NIP also could covalent bind DA molecules to show a high current response. Although the current response of DA on MIP/GCE decreased slightly, a really low current response of DA on NIP/GCE could be found when py-PBA was used as the monomer to fabricate the sensor, In this case, boronic acid groups in both polymer films could covalent bind DA molecule. As speculated
above, the py group in monomer could decrease the non-specific adsorption, leading to a lower binding on NIP/GCE. The imprinted factors (IF, IF=ႤiMIPs/ႤiNIPs) have been calculated and compared on the three kinds of sensors, which were 5.1, 2.1, and 18.4, corresponding to the sensors prepared using py, PBA, and py-PBA as monomer, respectively. These results fully illustrated the advantage of py-PBA as a polymerized monomer.
3.3 Optimization of conditions for MIP/GCE preparation Different influencing factors including pH value, the mole ratio between monomer and template molecule, electropolymerization scan rate, and scan cycles of electropolymerization process were investigated to fabricate an efficient sensor. The change of current response of DA on each electrode was calculated by subtracting the current response recorded in the absence of DA from current response caused in the presence of 110-4 mol/L DA. pH value in the polymerization process is an important factor for the sensor preparation. The change of current of DA was shown in Fig. 3A at pH 5, 6, 7, 8, 9. The stronger binding capacity of the sensor could be seen when the pH value of the polymerization solution shifted form 5 to 8. It could be attribute to that a tetrahedral anion structure of phenylboronic acid and its derivatives can efficiently react with cis-diols to form five- or six-membered cyclic boronate esters in an alkaline aqueous solution (Liu et al., 2011; He et al., 2011; Martin et al., 2011). However, the binding capacity decreased at pH 9. This phenomenon could be explained that DA would
self-polymerize distinctly at this pH value (Jiang et al., 2011; Ma et al., 2011), which influence the formation of boronate ester. With the selecting of py-BPA as the monomer, the mole ratio between monomer and template molecule should be investigated because this ratio would influence the amount of the imprinted cavities in MIP, which should affect the rebinding and recognition capacities of the sensor. The MIP were prepared in solutions of a constant DA concentration (110-3 mol/L) and of varying py-PBA concentrations in the range of 110-3~1.610-3 mol/L to obtain four sensor with the different ratio of 1:1, 1:1.2, 1:1.4, and 1:1.6. As shown in Fig. 3B, the sensor prepared with the ratio between template and monomer molecule of 1:1.2 displayed the highest net current of DA. DA molecules probably could not be captured during the electropolymerization process when the monomer was used at a lower concentration. On the other hand, higher concentration of py-PBA used would cause the deeply embedded of DA molecules, which could not be extracted easily (Kan et al., 2012). Therefore, the optimize ratio between DA and py-PBA was selected as 1:1.2 in the subsequent experiments. Electropolymerization scan cycles and rate are both the important factors for the formation of MIP film (Rezaei et al., 2014; Xu et al., 2014), which should influence the thickness and compactness of the polymers, respectively. The thickness of the polymer film would increase with the increase of scan cycles of electropolymerization, which would also affect the sensitivity of sensor. The number of scan cycles was varied from 10 to 30 in this research to determine the optimal film thickness (Fig. 3C). Polymer films that were formed less than 20 scan cycles were found to be unstable.
Higher cycles lead to form the thicker sensing film with less accessible imprinted sites. The current response changes of DA on MIP/GCE implied that the optimum polymerization cycle was to be
20. The effect of scan rate in the
electropolymerization process was shown in Fig. 4D. The MIP film produced at a slower scan rate was found to form a tight film to decrease the accessibility of template molecule to imprinted sites. However, a faster scan rate formed a loose and rough film with a low recognition capacity. The current response of DA on the MIP/GCE was found to increase with an increase of scan rate up to 50 mV/s and decrease as the scan rate increased above that value. Thus, the optimum polymerization scan rate was found to be 50 mV/s. 3.3 Recognition capacity of MIP/GCE The selectivity of MIP/GCE was investigated using AA, UA, and EP as the structural analogues or coexisted compounds (Mao et al., 2012; Maouche et al., 2012). Fig. 4A showed the current responses of above four compounds on MIP/GCE and NIP/GCE. Compared with NIP/GCE, the current response caused by DA on MIP/GCE was much higher because of the presence of imprinted cavities. The current of EP on MIP/GCE showed no obvious increase in comparison to the other compounds, whereas the current of DA on MIP/GCE was at least 6 times than that of other compounds. The selectivity experiments were also carried out by incubating MIP/GCE or NIP/GCE in the mixed solution of DA and an analogue, whose concentration was the same as or 10 times of the concentration of DA. The mixed solution of DA and 100 times concentration of AA was also used to investigate the
selectivity of the sensor since the concentration level of AA in bodies is much higher than DA. The ratios of current responses on MIP/GCE were calculated by current recorded in DA and an analogue solution (As) dividing current recorded in DA solution (A0). It could be found in Fig. 4B that the current of the mixed compounds of DA and AA or UA on MIP/GCEshowed less than 10% of current increase than that of only DA in the solution, indicating no obvious interference of AA and UA and the good selectivity of the prepared sensor. When the same concentration of EP coexited with DA, no obvious increase of current response could be observed, indicating the slightly binding with EP. However, a current increase of about 25% was found when the concentration of EP was 10 times as DA. This phenomenon was not surprising due to the very similarity of the structures of DA and EP, as well as the covalent binding between phenolic hydroxyl groups of EP and the boronic acids of PBA. The specificity of MIP/GCE to monosaccharides, such as Glu, Fru, and Man, also has been investigated, since they are cis-diol-containing molecules. Although these monosaccharides have no redox active on MIP/GCE in the chosen potential window, the addition of each monosaccharide in DA solution should influence the adsorption of DA on MIP/GCE, which would change the current response of DA on MIP/GCE. Therefore, the current responses were recorded by incubating MIP/GCE in the mixed solution of DA and a monosaccharide, whose concentration was the same or 10 times as the concentration of DA. The ratios of current responses on MIP/GCE were also calculated by current recorded in DA and a monosaccharide solution (As) dividing current recorded in DA solution (A0). It could be found in Fig. 4C that current of DA
slightly changed with the addition of monosaccharide. These monosaccharides have no similar molecular structure of DA, they could not be adsorbed well in the imprinted cavities of MIP, although phenolic hydroxyl groups of monosaccharide could covalent bind boronic acid groups of PBA. This result also demonstrated that the prepared sensor possessed good recognition capacity to DA.
3.4 Analytical performance of the electrochemical sensor In order to obtain an analytical curve of MIP/GCE for DA, the sensitive DPV measurements were performed under the optimized conditions on the sensor Fig. 5 showed the DPV curves recorded for MIP/GCE to the successive addition of DA in PBS solution. And the dependence of the peak currents on the concentration of DA was shown in the insert of Fig. 5. It could be found that the peak current of DA increased with the increase of its concentration. A linear relationship between the peak current and the concentration of DA was obtained range from 5.0× 10-8 to 1.0 × 10-5 mol/L, the linear regression equation being I (μA)= 2.935+ +0.664c (μmol/L), with a correlation coefficient of 0.996. The detection limit was calculated to be 3.3 × 10-8 mol/L based on the 3 of the blank signals. Thus, the DA response on MIP/GCE presented a higher sensitivity and a wider linear detection range. In order to evaluate the validity of the MIP/GCE method, it was used to determine DA in two kinds of dopamine hydrochloride injections, which were obtained from the first affiliated hospital of Wannan Medical College. As summarized in Table 1, the amount of DA in the injection detect by the present sensor kept the almost value with
the instruction. In order to perform the recovery test, the samples were spiked with 4.0 × 10-6 mol/L, 6.0× 10-6 mol/L, and 8.0× 10-6 mol/L DA. The results showed that the recoveries from the samples were excellent, and varied from 91.5% to 105.2%. The reproducibility of the sensor was investigated in 1.0 × 10-5 mol/L DA. After the first electrochemical determination of DA, the sensor was CV scanned in 0.5 M H2SO4 to remove the bound DA from the MIP film. The sensor was then incubated in the same concentration of DA solution for the next electrochemical measurement. The current intensity of DA decreased to about 88.9% of the initial value after being used more than 6 binding/detection/extraction cycles with a relative standard deviation of 4.3% of peak currents. The stability of the MIPs/GCE was also tested in 1.0 ×10−5 mol/L DA. The current response of the imprinted sensor decreased to 89.4% after storing for 15 days at 4 oC. The results demonstrated that the prepared electrochemical sensor had an excellent regeneration property and stability, which provided a new class of polymer modified electrodes for sensor applications.
4. Conclusions In this work, py-PBA was synthesized as a novel electropolymerized monomer for the preparation of MIP based electrochemical sensor. Due to the inherent molecular recognition capacity of MIP and the affinity binding ability of boronic acid toward cis-diol compound, the prepared sensor exhibited a double recognition capacity to DA. And the sensor could sensitively detect DA with a wide linear range. These results indicated that py-PBA is a promising polymerized monomer for MIP preparation with
high affinity and recognition toward cis-diols containing compounds.
Acknowledgements We greatly appreciate the support of the National Natural Science Foundation of China for young program (21005002), Anhui Provincial Natural Science Foundation for Young Program (11040606Q35), Anhui University Provincial Natural Science Foundation Key program (KJ2010A138).
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Figure Captions Fig. 1 Schematic illustrations of the fabrication procedure for the MIP/GCE (A), SEM images of bare GCE (B) and MIP/GCE (C), and CV curves of MIP/GCE (D) , which was characterized using 1 mmol/L [Fe(CN)6]3−/[Fe(CN)6]4−. Fig. 2 IF (IF=ႤiMIPs/ႤiNIPs) of the sensors prepared using py, PBA, or py-PBA as polymerized monomer.
Fig. 3 Effect factors of pH value (A), the mole ratio between monomer and template molecule (B), electropolymerization scan cycles (C), and scan rate (D) of electropolymerization process on MIP/GCE preparation. Fig. 4 Selectivity of MIP/GCE. Current responses of DA, AA, EP, or UA on MIP/GCE and NIP/GCE (A), the ratios of current responses on MIP/GCE calculated by current recorded in DA and an analogue solution dividing current recorded in DA solution (B), and the ratios of current responses on MIP/GCE calculated by current recorded in DA and a monosaccharide solution dividing current recorded in DA solution (C). Fig. 5 DPV of MIP/GCE in the presence of DA (a to g are 5.0× 10-8 to 1.0 × 10-5 mol/L). Inset: the calibration plot of the concentration of DA versus peak current. Table 1Application of the sensor to determine DA in injection samples spiked with different amounts of DA. Theoretical Actual The number Added Founda Recovery value value -6 -6 (%) of samples (10 mol/L) (10 mol/L) (10-6 mol/L) (10-6 mol/L) a
b a
2.0 2.0 2.0 2.0 2.0 2.0
2.03 2.21 1.99 2.06 2.04 2.10
Average value of three determinations.
4.0 6.0 8.0 4.0 6.0 8.0
3.75 6.31 8.05 3.78 5.60 7.32
93.8 105.2 100.6 94.5 93.3 91.5
Highlights
► A novel polymerized monomer, py-PBA, was synthesized to prepare MIP/GCE. ► The present sensor possessed a high imprinted factor. ► Double recognition of DA due to the specific adsorption of MIP and covalent binding between DA and PBA. ► The sensor exhibited sensitive detection capacity to DA.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
PII: DOI: Reference:
S0956-5663(14)00679-4 http://dx.doi.org/10.1016/j.bios.2014.08.083 BIOS7074
To appear in: Biosensors and Bioelectronic Received date: 2 August 2014 Revised date: 26 August 2014 Accepted date: 28 August 2014 Cite this article as: Min Zhong, Ying Teng, Shufen Pang, Liqin Yan and Xianwen Kan, Pyrrole-phenylboronic acid: A novel monomer for dopamine recognition and detection based on imprinted electrochemical sensor, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2014.08.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pyrrole-phenylboronic Acid: A Novel Monomer for Dopamine Recognition and Detection based on Imprinted Electrochemical Sensor
Min Zhong, Ying Teng, Shufen Pang, Liqin Yan, Xianwen Kan*
College of Chemistry and Materials Science, Anhui Key Laboratory of Chemo-Biosensing, Anhui Key Laboratory of Functional Molecular Solids, Anhui Normal University, Wuhu 241000, P.R. China
*Corresponding author: Xianwen Kan, E-mail: [email protected]; Tel: +86-553-3937135; Fax: +86-553-3869303.
Abstract A molecular imprinting polymer (MIP) based electrochemical sensor was successfully prepared for dopamine (DA) recognition and detection using pyrrole-phenylboronic acid (py-PBA) as a novel electropolymerized monomer. py-PBA could form cyclic boronic ester bond with DA, thus endowing a double recognition capacity of the sensor to DA in the combination of the imprinted effect of MIP. Compared with the sensor prepared using pyrrole or phenylboronic acid as electropolymerized monomer, the present sensor exhibited a remarkable high imprinted factor to DA. The influence factors including pH value, the mole ratio between monomer and template molecule, electropolymerization scan rate, and scan
cycles of electropolymerization process were investigated and optimized. Under the optimal conditions, the sensor could recognize DA from its analogues and monosaccharides. A linear ranging from 5.0 × 10-8 to 1.0 × 10-5 mol/L for the detection of DA was obtained with a detection limit of 3.3× 10-8 mol/L (S/N=3). The sensor has been applied to analyze DA in injection samples with satisfactory results.
Keywords: pyrrole-phenylboronic acid, electrochemical sensor, dopamine, molecular imprinting polymer, double recognition
1 Introduction Molecular imprinting, a promising technique, has been used to synthesize molecular imprinting polymer (MIP) to recognize template molecule (Günter Wulff, 1995; Cheong et al., 1997; Haupt and Mosbach, 2000). The advantages that MIP possesses over biopolymers are low cost, ease of preparation and stability over a great range of temperatures (Mosbach and Ramström, 1996; Hwang and Lee, 2002; James et al., 1996). The general procedure for synthesis MIP involves: First, the assembly of functional monomers around a template molecule in solution to form a complex by covalent or non-covalent bond. Next, the polymerization of above complex with cross-linker and initiator under photo or thermal condition. Third, the removal of the embedded template molecules in the polymer by certain extraction method to afford MIP. The removal of template molecules leaves microcavities with a three dimensional structure complementary in both shape and chemical functionality to
template molecules (Cheong et al., 1997), which is now capable of rebinding the template molecule with a very high specificity (Cheong et al., 1997; Okutucu and Onal, 2011; Xu et al., 2013; Bossi et al., 2007; Li et al., 2006). Most imprinting polymers synthesis reported were based on the non-covalent bond between functional monomer and template molecule, which has been developed primarily by Mosbach and co-workers (Mosbach and Ramström, 1996). This approach is flexible concerning the extraction and rebinding of template molecule. The covalent imprinting is superior in preventing leakage of template molecules during polymerization because of the formation of stable covalent bonds between functional groups of the template and those of monomer (Wang et al., 2009; Boonpangrak et al., 2006; Hwang and Lee, 2002; Ikegami et al., 2004). In order to remove the template, the covalent bonds connecting the template to the polymer should be cleaved (Hwang and Lee, 2002), which could form again when the polymer is incubated into the template molecule solution under the suitable condition. This reversible reaction between template and monomer could provide much more precise rebinding sites for template molecule, resulting the high rebinding and recognition capacities of MIP (Anderson et al., 1993; Meyer et al., 2007; Subrahmanyam et al., 2001; Mayes and Whitcombe, 2005). It is well known that boronic acids can covalently interact with cis-diol-containing molecules to form stable cyclic esters in an alkaline aqueous solution while the boronate esters dissociate when the environmental pH is switched to acidic (James et al., 1996; Rajkumar et al., 2007; Springsteen et al., 2001). Since the discovery of this
reversible interaction between PBA and saccharide in 1954, many PBA-based diol sensors have been developed (Nishiyabu et al., 2011; Okutucu and Onal, 2011; Liu et al., 2001). Recently, much attention has been devoted to the construction of electrochemical and optical sensors based on the interaction of cis-diol compounds and boronic acid (Dechtrirat et al., 2014; Liu et al., 2013; Bull et al., 2012). Polymerisable
boronic acid derivates also have been designed as monomers to form MIP through covalent approach by dehydrocondensation reaction with carbohydrates (Rajkumar et al., 2007). These boronic acid based MIP not only could selectively bind cis-diol compounds from general compounds, but also could recognize template molecule from its analogues, which also are cis-diol compounds (Friggeri et al., 2001; Lin et al., 2013; Bi et al., 2013). Among these boronic acid derivates, phenylboronic acid (PBA) has been extensively used due to its high operational stability and polymerisable property (D'hooge et al., 2008; Mcculloch et al., 2003; Matsumoto et al., 2010). Herein, another kind of phenylboronic acid derivate, pyrrole-phenylboronic acid (py-PBA) was synthesized as a polymerisable monomer for the MIP based electrochemical sensor preparation. The formation of cyclic boronic ester bond between boronic acid and DA, as well as the imprinted cavities of MIP endowed the prepared sensor double recognition capacities. The results of electrochemical experiments demonstrated that the prepared sensors exhibited a much higher imprinted factor than that of the sensor prepared by using py or PBA as polymerized monomer. The sensor could recognize template molecule from its analogues with a good selectivity and sensitively detect DA with a wide linear range and a low
detection limit. Meanwhile, the sensor was used to detect DA in real samples with satisfactory results.
2 Experimental 2.1 Chemicals Commercially available reagents and solvents were used without further purification. 3-aminophenylboronic acid monohydrate (APBA) and ethanol-d6 were ordered from Aldrich. 2, 5-Dimethoxytetrahydrofuran, glucose (Glu), fructose (Fru), and mannose (Man) were obtained from Aladdin (Aladdin Industrial Inc., China). Dopamine (DA, ≥98.5%), ascorbic acid (AA, >98%), uric acid (UA, >99%), epinephrine hydrochloride (EP, >98%), and pyrrole were purchased from Fluka (Fluka Chemie AG, Switzerland). All other reagents were of at least analytical-reagent grade. And double-distilled deionized water was used for all solutions. 2.2 Apparatus
Electrochemical experiments, such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed on CHI 660C workstation (ChenHua Instruments Co., Shanghai, China) with a conventional three-electrode system. A bare or modified glassy carbon electrode (GCE) was served as a working electrode. A saturated calomel electrode and a platinum wire electrode were used as a reference electrode and a counter electrode, respectively. Field emission scanning electron microscope (FE-SEM) images were obtained on an S-4800 field emission scanning electron microanalyser (Hitachi, Japan). 1H NMR (300 MHz) spectrum was recorded
at 300 MHz NMR spectrometer in C2D5OD. All chemical shifts (δ) are given in ppm relative to TMS (δ = 0 ppm) as internal standard. 2.3 Synthesis and characterization of 3-(1H-pyrrol-1-yl)phenylboronic acid (py-PBA) py-PBA was synthesized according to Boyacı’s work with a simple modification (Aytaç et al., 2011). 3-Aminophenylboronic acid monohydrate (100 mg, 0.646 mmol) was
dissolved
in
10
mL
acetonitrile.
With
the
addition
of
2,
5-Dimethoxytetrahydrofuran (0.167 mL, 1.292 mmol) into above solution under stirring, acetic acid (0.018 mL, 0.323 mmol) was also added to the solution. The resulting mixture was refluxed for 6 h. The reaction was monitored by thin-layer chromatograpgy. After completion of the reaction, the fulvous mixture was passed through a small plug of silica, eluting with 20 mL CH2Cl2, and the solvent was removed under vacuum. The obtained brownish powder was py-PBA. 1H NMR (CD3OD, 300 MHz): δ (ppm): 6.262 (bs, 2H, pyr-H), 7.154 (s,2H, BOH), 7.417-7.465 ᧤m,1H, Ar-H᧥᧨7.634᧤d,1H,Ar-H᧥᧨7.829᧤bs,1H,Ar-H᧥. 2.4 Fabrication of MIP modified GCE (MIP/GCE) The clean GCE was immersed into 5 mL phosphate buffer solution (PBS, 0.05 mol/L, pH=8.0) containing 1.12 mg synthesized py-PBA and 100 μL 0.05 mol/L DA. Then cyclic voltammetry (CV) was performed from -0.2 V to +1.2 V for 20 cycles at a scan rate of 50 mV/s, obtaining polymer film modified electrode. Subsequently, the embedded DA was extracted by immersing above electrode into 0.5 mol/L H2SO4 aqueous solution, subsequently with the electrochemical scanning between 0 - 1.5 V
for several cycles, getting MIP modified electrode (MIP/GCE). The procedure of preparation of MIP/GCE was depicted in Fig. 1A. As a control, non-molecular imprinting polymer modified electrode (NIP/GCE) was prepared and treated in exactly the same way except for the omitting of DA in the electropolymerization process. 2.5 Electrochemical properties measurements Electrochemical measurements to characterize the prepared modified electrodes were carried out in 0.05 mol/L PBS (pH=7.0) by using CV and DPV methods. AA, UA, and EP were selected as coexisted or structural similar compounds to evaluate the recognition capacity of the prepared sensor.
3 Results and discussion 3.1 Characterization of MIP/GCE The morphological structure of MIP/GCE was tested by SEM, as shown in Fig. 1B and Fig. 1C. It could be seen that a layer of rough film coated onto the surface of GCE (Fig. 1C), which should be attributed to the electropolymerization of polymer film in the presence of DA. One mmol/L [Fe(CN)6]3−/[Fe(CN)6]4− was chosen as an electro-active probe to investigate the extraction of template molecule. As shown in Fig. 1D, no redox peaks of probe could be found on MIP/GCE before the extraction of DA because embedded DA molecules blocked the arrival of probe to surface of the electrode. With the removal of DA, an obvious redox peaks of probe appeared on MIP/GCE, which could
be explained that the extraction of DA molecules left imprinted cavities for probe reaching electrode surface. However, CV curves of NIP/GCE before and after the extraction both had no redox peaks of the probe, which could be ascribed to no imprinted cavities were created during the electropolymerization process. 3.2 Polymerized monomer for MIP/GCE Although the synthesis of py-PBA has been reported, it is a novel monomer in the field of MIP. Py or PBA has been applied individually in the MIP preparation since py could be electrpolymerized to form MIP on electrode surface and PBA could selectively bind cis-diol compounds (Choong et al., 2009; Mazzotta et al., 2008; Tomsho and Benkovic, 2012). In order to choose an efficient monomer, py, PBA, and synthesized py-PBA were employed as different monomer to prepare three sensors and the specific rebinding properties were investigated. From Fig. 2, sensor prepared using py as monomer showed the specific adsorption since the current of DA on NIP/GCE was really low, implying that the Ppy film could efficiently decrease the non-specific adsorption of DA. The sensor prepared using PBA as monomer showed the highest current of DA compared with other sensors, which could be explained that the existed boronic acid group of PBA in MIP film could rebind lots of DA molecules. Similarly, the boronic acid group in NIP also could covalent bind DA molecules to show a high current response. Although the current response of DA on MIP/GCE decreased slightly, a really low current response of DA on NIP/GCE could be found when py-PBA was used as the monomer to fabricate the sensor, In this case, boronic acid groups in both polymer films could covalent bind DA molecule. As speculated
above, the py group in monomer could decrease the non-specific adsorption, leading to a lower binding on NIP/GCE. The imprinted factors (IF, IF=ႤiMIPs/ႤiNIPs) have been calculated and compared on the three kinds of sensors, which were 5.1, 2.1, and 18.4, corresponding to the sensors prepared using py, PBA, and py-PBA as monomer, respectively. These results fully illustrated the advantage of py-PBA as a polymerized monomer.
3.3 Optimization of conditions for MIP/GCE preparation Different influencing factors including pH value, the mole ratio between monomer and template molecule, electropolymerization scan rate, and scan cycles of electropolymerization process were investigated to fabricate an efficient sensor. The change of current response of DA on each electrode was calculated by subtracting the current response recorded in the absence of DA from current response caused in the presence of 110-4 mol/L DA. pH value in the polymerization process is an important factor for the sensor preparation. The change of current of DA was shown in Fig. 3A at pH 5, 6, 7, 8, 9. The stronger binding capacity of the sensor could be seen when the pH value of the polymerization solution shifted form 5 to 8. It could be attribute to that a tetrahedral anion structure of phenylboronic acid and its derivatives can efficiently react with cis-diols to form five- or six-membered cyclic boronate esters in an alkaline aqueous solution (Liu et al., 2011; He et al., 2011; Martin et al., 2011). However, the binding capacity decreased at pH 9. This phenomenon could be explained that DA would
self-polymerize distinctly at this pH value (Jiang et al., 2011; Ma et al., 2011), which influence the formation of boronate ester. With the selecting of py-BPA as the monomer, the mole ratio between monomer and template molecule should be investigated because this ratio would influence the amount of the imprinted cavities in MIP, which should affect the rebinding and recognition capacities of the sensor. The MIP were prepared in solutions of a constant DA concentration (110-3 mol/L) and of varying py-PBA concentrations in the range of 110-3~1.610-3 mol/L to obtain four sensor with the different ratio of 1:1, 1:1.2, 1:1.4, and 1:1.6. As shown in Fig. 3B, the sensor prepared with the ratio between template and monomer molecule of 1:1.2 displayed the highest net current of DA. DA molecules probably could not be captured during the electropolymerization process when the monomer was used at a lower concentration. On the other hand, higher concentration of py-PBA used would cause the deeply embedded of DA molecules, which could not be extracted easily (Kan et al., 2012). Therefore, the optimize ratio between DA and py-PBA was selected as 1:1.2 in the subsequent experiments. Electropolymerization scan cycles and rate are both the important factors for the formation of MIP film (Rezaei et al., 2014; Xu et al., 2014), which should influence the thickness and compactness of the polymers, respectively. The thickness of the polymer film would increase with the increase of scan cycles of electropolymerization, which would also affect the sensitivity of sensor. The number of scan cycles was varied from 10 to 30 in this research to determine the optimal film thickness (Fig. 3C). Polymer films that were formed less than 20 scan cycles were found to be unstable.
Higher cycles lead to form the thicker sensing film with less accessible imprinted sites. The current response changes of DA on MIP/GCE implied that the optimum polymerization cycle was to be
20. The effect of scan rate in the
electropolymerization process was shown in Fig. 4D. The MIP film produced at a slower scan rate was found to form a tight film to decrease the accessibility of template molecule to imprinted sites. However, a faster scan rate formed a loose and rough film with a low recognition capacity. The current response of DA on the MIP/GCE was found to increase with an increase of scan rate up to 50 mV/s and decrease as the scan rate increased above that value. Thus, the optimum polymerization scan rate was found to be 50 mV/s. 3.3 Recognition capacity of MIP/GCE The selectivity of MIP/GCE was investigated using AA, UA, and EP as the structural analogues or coexisted compounds (Mao et al., 2012; Maouche et al., 2012). Fig. 4A showed the current responses of above four compounds on MIP/GCE and NIP/GCE. Compared with NIP/GCE, the current response caused by DA on MIP/GCE was much higher because of the presence of imprinted cavities. The current of EP on MIP/GCE showed no obvious increase in comparison to the other compounds, whereas the current of DA on MIP/GCE was at least 6 times than that of other compounds. The selectivity experiments were also carried out by incubating MIP/GCE or NIP/GCE in the mixed solution of DA and an analogue, whose concentration was the same as or 10 times of the concentration of DA. The mixed solution of DA and 100 times concentration of AA was also used to investigate the
selectivity of the sensor since the concentration level of AA in bodies is much higher than DA. The ratios of current responses on MIP/GCE were calculated by current recorded in DA and an analogue solution (As) dividing current recorded in DA solution (A0). It could be found in Fig. 4B that the current of the mixed compounds of DA and AA or UA on MIP/GCEshowed less than 10% of current increase than that of only DA in the solution, indicating no obvious interference of AA and UA and the good selectivity of the prepared sensor. When the same concentration of EP coexited with DA, no obvious increase of current response could be observed, indicating the slightly binding with EP. However, a current increase of about 25% was found when the concentration of EP was 10 times as DA. This phenomenon was not surprising due to the very similarity of the structures of DA and EP, as well as the covalent binding between phenolic hydroxyl groups of EP and the boronic acids of PBA. The specificity of MIP/GCE to monosaccharides, such as Glu, Fru, and Man, also has been investigated, since they are cis-diol-containing molecules. Although these monosaccharides have no redox active on MIP/GCE in the chosen potential window, the addition of each monosaccharide in DA solution should influence the adsorption of DA on MIP/GCE, which would change the current response of DA on MIP/GCE. Therefore, the current responses were recorded by incubating MIP/GCE in the mixed solution of DA and a monosaccharide, whose concentration was the same or 10 times as the concentration of DA. The ratios of current responses on MIP/GCE were also calculated by current recorded in DA and a monosaccharide solution (As) dividing current recorded in DA solution (A0). It could be found in Fig. 4C that current of DA
slightly changed with the addition of monosaccharide. These monosaccharides have no similar molecular structure of DA, they could not be adsorbed well in the imprinted cavities of MIP, although phenolic hydroxyl groups of monosaccharide could covalent bind boronic acid groups of PBA. This result also demonstrated that the prepared sensor possessed good recognition capacity to DA.
3.4 Analytical performance of the electrochemical sensor In order to obtain an analytical curve of MIP/GCE for DA, the sensitive DPV measurements were performed under the optimized conditions on the sensor Fig. 5 showed the DPV curves recorded for MIP/GCE to the successive addition of DA in PBS solution. And the dependence of the peak currents on the concentration of DA was shown in the insert of Fig. 5. It could be found that the peak current of DA increased with the increase of its concentration. A linear relationship between the peak current and the concentration of DA was obtained range from 5.0× 10-8 to 1.0 × 10-5 mol/L, the linear regression equation being I (μA)= 2.935+ +0.664c (μmol/L), with a correlation coefficient of 0.996. The detection limit was calculated to be 3.3 × 10-8 mol/L based on the 3 of the blank signals. Thus, the DA response on MIP/GCE presented a higher sensitivity and a wider linear detection range. In order to evaluate the validity of the MIP/GCE method, it was used to determine DA in two kinds of dopamine hydrochloride injections, which were obtained from the first affiliated hospital of Wannan Medical College. As summarized in Table 1, the amount of DA in the injection detect by the present sensor kept the almost value with
the instruction. In order to perform the recovery test, the samples were spiked with 4.0 × 10-6 mol/L, 6.0× 10-6 mol/L, and 8.0× 10-6 mol/L DA. The results showed that the recoveries from the samples were excellent, and varied from 91.5% to 105.2%. The reproducibility of the sensor was investigated in 1.0 × 10-5 mol/L DA. After the first electrochemical determination of DA, the sensor was CV scanned in 0.5 M H2SO4 to remove the bound DA from the MIP film. The sensor was then incubated in the same concentration of DA solution for the next electrochemical measurement. The current intensity of DA decreased to about 88.9% of the initial value after being used more than 6 binding/detection/extraction cycles with a relative standard deviation of 4.3% of peak currents. The stability of the MIPs/GCE was also tested in 1.0 ×10−5 mol/L DA. The current response of the imprinted sensor decreased to 89.4% after storing for 15 days at 4 oC. The results demonstrated that the prepared electrochemical sensor had an excellent regeneration property and stability, which provided a new class of polymer modified electrodes for sensor applications.
4. Conclusions In this work, py-PBA was synthesized as a novel electropolymerized monomer for the preparation of MIP based electrochemical sensor. Due to the inherent molecular recognition capacity of MIP and the affinity binding ability of boronic acid toward cis-diol compound, the prepared sensor exhibited a double recognition capacity to DA. And the sensor could sensitively detect DA with a wide linear range. These results indicated that py-PBA is a promising polymerized monomer for MIP preparation with
high affinity and recognition toward cis-diols containing compounds.
Acknowledgements We greatly appreciate the support of the National Natural Science Foundation of China for young program (21005002), Anhui Provincial Natural Science Foundation for Young Program (11040606Q35), Anhui University Provincial Natural Science Foundation Key program (KJ2010A138).
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Figure Captions Fig. 1 Schematic illustrations of the fabrication procedure for the MIP/GCE (A), SEM images of bare GCE (B) and MIP/GCE (C), and CV curves of MIP/GCE (D) , which was characterized using 1 mmol/L [Fe(CN)6]3−/[Fe(CN)6]4−. Fig. 2 IF (IF=ႤiMIPs/ႤiNIPs) of the sensors prepared using py, PBA, or py-PBA as polymerized monomer.
Fig. 3 Effect factors of pH value (A), the mole ratio between monomer and template molecule (B), electropolymerization scan cycles (C), and scan rate (D) of electropolymerization process on MIP/GCE preparation. Fig. 4 Selectivity of MIP/GCE. Current responses of DA, AA, EP, or UA on MIP/GCE and NIP/GCE (A), the ratios of current responses on MIP/GCE calculated by current recorded in DA and an analogue solution dividing current recorded in DA solution (B), and the ratios of current responses on MIP/GCE calculated by current recorded in DA and a monosaccharide solution dividing current recorded in DA solution (C). Fig. 5 DPV of MIP/GCE in the presence of DA (a to g are 5.0× 10-8 to 1.0 × 10-5 mol/L). Inset: the calibration plot of the concentration of DA versus peak current. Table 1Application of the sensor to determine DA in injection samples spiked with different amounts of DA. Theoretical Actual The number Added Founda Recovery value value -6 -6 (%) of samples (10 mol/L) (10 mol/L) (10-6 mol/L) (10-6 mol/L) a
b a
2.0 2.0 2.0 2.0 2.0 2.0
2.03 2.21 1.99 2.06 2.04 2.10
Average value of three determinations.
4.0 6.0 8.0 4.0 6.0 8.0
3.75 6.31 8.05 3.78 5.60 7.32
93.8 105.2 100.6 94.5 93.3 91.5
Highlights
► A novel polymerized monomer, py-PBA, was synthesized to prepare MIP/GCE. ► The present sensor possessed a high imprinted factor. ► Double recognition of DA due to the specific adsorption of MIP and covalent binding between DA and PBA. ► The sensor exhibited sensitive detection capacity to DA.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
