Molecular imprinting polymer electrosensor based on gold nanoparticles for theophylline recognition and determination
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- Kan, X., Liu, T., Zhou, H. et al. Microchim Acta (2010) 171: 423. doi:10.1007/s00604-010-0455-5
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An electrochemical sensor for theophylline (ThPh) was prepared by electropolymerizing o-phenylenediamine on a glassy carbon electrode in the presence of ThPh via cyclic voltammetry, followed by deposition of gold nanoparticles using a potentiostatic method. The effects of pH, ratio between template molecule and monomer, number of cycles for electropolymerization, and of the solution for extraction were optimized. The current of the electro-active model system hexacyanoferrate(III) and hexacyanoferrate(IV) decreased linearly with successive addition of ThPh in the concentration range between 4.0 × 10−7 ~ 1.5 × 10−5 mol·L−1 and 2.4 × 10−4 ~ 3.4 × 10−3 mol·L−1, with a detection limit of 1.0 × 10−7 mol·L−1. The sensor has an excellent recognition capability for ThPh compared to structurally related molecules, can be regenerated and is stable.
KeywordsMolecular imprinted polymerElectrochemical sensorAu nanoparticlesTheophyllineRecognition
Theophylline (1, 3-dimethyl-1H-purine-2, 6-dione, ThPh), a xanthine derivative with diuretic, cardiac stimulant, and smooth muscle relaxant activities, has been widely used for the treatment of asthma and bronchospasm in adult [1, 2]. However, overdose of this kind of drugs would cause side effect, such as tremor tachycardia. Thus, the pharmaceutical preparations and their therapeutic drug monitoring are necessary for ThPh. The methods reported for the determination of ThPh include Surface plasmon resonance , gas chromatography-mass spectrometry , Liquid chromatographic , high-performance liquid chromatography [6–9], liquid chromatography-mass spectrometry , and solid phase extraction  and so on. The above mentioned motheds suffered disadvantages of long procedures and more other interferences of the similar structure molecules except as reported by Dove JW. etc. . Different substrate materials such as ITO film [12, 13] and carboxylic multi-wall carbon nanotubes [14, 15] were also utilized to determination of ThPh. As an alternative, electrochemical sensors have been used to analyze inorganic ions, drugs, proteins, DNA, and other biomolecules due to their advantages of easy preparation, sensitive determination, and time saving [16–19]. The electrochemical methods for determination of ThPh by square-wave voltammetry in tea and drug formulation at a Nafion®/lead-ruthenium oxide pyrochlore modified electrode had been reported . As well known, ThPh is coexisted with caffeine and other purin alkaloids in real samples . Therefore, many efforts have been employed to improve the selectivity of the modified electrodes.
One of the most promising materials in the field of artificial molecular recognition systems would be molecular imprinting polymer (MIP), which has increasingly attracted considerable attention in recent years. MIP is synthesized by copolymerizing functional and cross monomer in the presence of template molecule. Subsequent extraction of template molecules reveals binding sites in the polymer that are complementary in size and shape to the template molecule, resulting in the prepared MIP with the capabilities of specific rebinding and recognition to template molecule [22–26]. Therefore, as recognition elements, MIP has been strongly developed in wide fields, such as liquid chromatography, capillary electrochromatography, solid phase extraction, drug controlled release, and electrochemical sensor [27–33]. The recognition of ThPh also has been achieved by using molecular imprinted technique [14 15]. But these MIPs could not be used to determine the concentration of ThPh at the same time. The molecular imprinting technique and electrochemical sensor has been combined to develop the novel electrochemical sensor to achieve the determination and recognition simultaneously [34–38].
Electrochemical method based on MIP for ThPh recognition and determination has been reported [39–41]. In order to further improve the recognition property of sensor, herein, we reported a new MIP electrochemical sensor combined with Au-nanoparticles to improve the current response. In situ electropolymerization method was employed by using ThPh and o-phenylenediamine (o-PD) as template molecule and functional monomer, respectively. Au nanoparticles (AuNPs) were deposited on the above polymers film surface. The prepared electrochemical sensor was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectrum (EIS), and differential pulse voltammetry (DPV). As expected, the electrochemical sensor exhibited high recognition capacity toward ThPh, as well as broader linear range and lower detection limit.
o-Phenylenediamine (o-PD) was purchased from Shanghai Chemical Reagent Company, China (http://www.reagent.com), and purified by sublimation before use. Theophylline (ThPh) and theophylline-7-acetic acid (ThPh-7) were purchased from Alfa Aesar (http://www.alfa.com). Guanine (GUAN) and adenine (ADEN) were obtained from Sanland-chem International Inc. HAuCl4·4H2O was obtained from Aaladdin Reagent Co., Ltd.(http://www.aladdin-reagent.com). All other reagents were of analytical grade and used without further purified. All solutions were prepared with triple distilled water.
All electrochemical measurements were performed on a CHI 660A electrochemical workstation (Chenhua Instruments Co., Shanghai, China) with a standard three-electrode configuration. A modified glassy carbon electrode (GCE) was used as the working electrode (3.0 mm diameter), a saturated calomel electrode (SCE) was used as the reference electrode and a platinum electrode was employed as the auxiliary electrode. The actual pH values were determined with a pH/Ion Analyser model pHS-3CT (Da Pu Instrument Co., Ltd. Shanghai, China). Scanning electron microscopy (SEM) was performed with Hitachi S-4800 SEM (operated at 10 kV).
Preparation of Au-nanoparticles and imprinted modified electrode
Results and discussion
Characterization of the imprinted modified electrode
Optimization of conditions for imprinted modified electrode preparation
In order to construct an efficient sensor, different influencing factors including pH value of electropolymerization solution, scan cycles of electropolymerization process, template molecule/monomer ratio, and extraction solution were investigated.
The effect of electropolymerization solution (0.1 mol·L−1 HAc-NaAc buffer) pH over the range of 4.0 ~ 6.7 on the current intensity of [Fe(CN)6]3−/[Fe(CN)6]4− on MIP/GCE was investigated. A maximum response was observed at about pH 5.2, indicating the formation of maximum imprinted cavities under this condition. Therefore, the electropolymerization was carried out at pH 5.2 HAc-NaAc solution.
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 40 in this research to determine the optimal film thickness. 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 curves of [Fe(CN)6]3−/[Fe(CN)6]4− on MIP/GCE implied that the optimum polymerization cycles was to be 20.
To determine the effect of the ratio between ThPh and o-PD to the response of MIP/GCE, the MIP films were electropolymerized in solutions of constant o-PD (5 × 10−3 mol·L−1) concentration and varying ThPh concentrations in the range of 5–20 × 10−3 mol·L−1. After extracting the ThPh, the response of [Fe(CN)6]3−/[Fe(CN)6]4− to the modified electrode increased with the increase of the ratio of ThPh to o-PD from 1:1 to 3:1. However, when the ratio was increased to 4, the current response was almost negligible, which was possibly attributed to unstable MIP film on the electrode surface by using too little o-PD. Thus, the optimum ThPh/o-PD ratio was chosen as 3.
The removal of the template from the MIP is necessary to release the imprinted sites, which would selectively rebind the template molecule . Citric-phosphate buffer (0.1 mol·L−1; pH 2.2, 3.0), HAc-NaAc buffer (0.2 mol·L−1; pH 3.0, 3.5, 4.0, 4.5), H2SO4 (0.5 mol·L−1), and ethanol were respectively applied to remove ThPh. The results indicated that ThPh could be removed almost completely by ethanol within 5 h, and the AuNPs/MIP/GCE could be regenerated for several times by this method. So soaking in ethanol for 5 h was chosen as the best condition for template removal.
Determination of theophylline
Comparison with other methods for the determination of ThPh
1.0 × 10−4 ~ 4.8 × 10−2 mol·L−1
2.0 × 10−4 mol·L−1
Surface plasmon resonance
0 ~ 3.3 × 10−2 mol·L−1
2.2 × 10−3 mol·L−1
Gas chromatography-mass spectrometry
1.1 × 10−6 ~ 5.6 × 10−5 mol·L−1
Reversed-phase high-performance liquid chromatographic
2.8 × 10−6 ~ 5.6 × 10−5 mol·L−1
1.4 × 10−6 mol·L−1
Ratio-spectra derivative spectrophotometry
1.1 × 10−4 ~ 1.0 × 10−3 mol·L−1
4.1 × 10−6 mol·L−1
2.7 × 10−6 mol·L−1
High-performance liquid chromatographic
1.1 × 10−5 ~ 2.8 × 10−5 mol·L−1
5.6 × 10−7 mol·L−1
5.0 × 10−5 ~ 1.7 × 10−4 mol·L−1
2.8 × 10−5 ~ 8.4 × 10−4 mol·L−1
3.3 × 10−6 mol·L−1
Liquid chromatography–mass spectrometry
4.25 × 10−5 mol·L−1
Solid phase extraction (MIP)
1.1 × 10−5 ~ 1.1 × 10−4 mol·L−1
5.6 × 10−6 mol·L−1
1 × 10−6 ~ 1 × 10−2 mol·L−1
5.5 × 10−7 mol·L−1
1.0 × 10−7 ~ 1.0 × 10−4 mol·L−1
1.0 × 10−7 mol·L−1
electrochemical sensor based on MIP
2.0 × 10−5 ~ 7.5 × 10−5 mol·L−1
3.0 × 10−9 mol·L−1
0 ~ 1.5 × 10−5 mol·L−1
1.0 × 10−6 mol·L−1
4.0 × 10−7 ~ 1.5 × 10−5 mol·L−1
1.0 × 10−7 mol·L−1
2.4 × 10−4 ~ 3.4 × 10−3 mol·L−1
Recognition of imprinted modified electrode
The special selectivity test of AuNPs/MIP/GCE was carried out by using Caffeine (CAFF), ThPh -7-acetic acid (ThPh -7), guanine (GUAN), and adenine (ADEN) as comparative compounds. It was also performed by DPV in [Fe(CN)6]3−/[Fe(CN)6]4−.
Regeneration and stability
After the first electrochemical determination of ThPh, AuNPs-MIP/GCE was immersed in ethanol to remove ThPh bounding in the polymer. AuNPs-MIP/GCE was then incubated in [Fe(CN)6]3−/[Fe(CN)6]4− solution containing the same concentration of ThPh for the next electrochemical measurement. The current of [Fe(CN)6]3−/[Fe(CN)6]4− decreased to about 95% of the original value after being used more than 10 binding/detection/extraction cycles. After a 15-day storage period at 4 °C in dry condition, the sensor retained 85% of its initial current response, which was similar to the result reported by Wang . The results demonstrated that the prepared electrochemical sensor had excellent regeneration property and stability, which maybe provides a new class of polymer modified electrodes for sensor applications.
Analytical result and recovery
7.2 × 10−6
4.3 × 10−6
10.6 × 10−6
The present study describes the development of molecular imprinting polymer electrochemical sensor for special recognition and determination of ThPh. Sensor was fabricated by electropolymerization of o-PD in the presence of ThPh on GCE surface and electrodeposition Au NPs on MIP modified electrode surface successively. The prepared sensor not only exhibited a special recognition capacity to ThPh, but also had a high sensitivity for ThPh determining. Peak current of [Fe(CN)6]3−/[Fe(CN)6]4− varied linearly with the concentration of ThPh in the range of 4.0 × 10−7 ~ 1.5 × 10−5 mol·L−1 and 2.4 × 10−4 ~ 3.4 × 10−3 mol·L−1, and the detection limit reached 1.0 × 10−7 mol·L−1, which may be enhanced by the deposited AuNPs and could be used as a basis for ThPh determination.
We greatly appreciate the support of the National Natural Science Foundation of China for General program (20675001) and young program (21005002), Anhui University Provincial Natural Science Foundation Key program (KJ2010A138), Dr Start-up Fundation of Anhui Normal University (160-750834).