Author’s Accepted Manuscript Electrochemical sensor based on molecularly imprinted film at Au nanoparticles-carbon nanotubes modified electrode for determination of cholesterol Jian Ji, Zhihui Zhou, Xiaolian Zhao, Jiadi Sun, Xiulan Sun www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(14)00956-7 http://dx.doi.org/10.1016/j.bios.2014.12.014 BIOS7330
To appear in: Biosensors and Bioelectronic Received date: 8 October 2014 Revised date: 2 December 2014 Accepted date: 2 December 2014 Cite this article as: Jian Ji, Zhihui Zhou, Xiaolian Zhao, Jiadi Sun and Xiulan Sun, Electrochemical sensor based on molecularly imprinted film at Au nanoparticles-carbon nanotubes modified electrode for determination of c h o l e s t e r o l , Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2014.12.014 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.
Electrochemical sensor based on molecularly imprinted film at Au nanoparticles-carbon nanotubes modified electrode for determination of cholesterol Jian Ji1, Zhihui Zhou2, Xiaolian Zhao2, Jiadi Sun1, Xiulan Sun1* 1 State Key Laboratory of Food Science and Technology，School of Food Science of Jiangnan University, Synergetic Innovation Center Of Food Safety and Nutrition, Wuxi, Jiangsu 214122, China 2 Wuxi Jinkun Bio-Technology Co., Ltd. Wuxi, Jiangsu 214122, China
Corresponding author: Xiulan Sun (e-mail: [email protected]) First author: Jian Ji (e-mail: [email protected])
Abstract: A novel electrochemical sensor for cholesterol (CHO) detection based on molecularly imprinted polymer (MIP) membranes on a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWNTs) and Au nanoparticles (AuNPs) was constructed. p-Aminothiophenol (P-ATP) and CHO were assembled on the surface of the modified GCE by the formation of Au-S bonds and hydrogen-bonding interactions, and polymer membranes were formed by electropolymerization in a polymer solution containing p-ATP, HAuCl4, tetrabutylammonium perchlorate (TBAP) and the template molecule CHO. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were used to monitor the electropolymerization process and its optimization, which was further characterized by scanning electron microscopy (SEM). The linear response range of the MIP sensor was between 1 × 10-13 and 1 × 10-9 mol L−1, and the limit of detection (LOD) were 3.3×10−14 mol L−1. The proposed system has the potential for application in clinical diagnostics of cholesterol with high-speed real-time detection capability, low sample consumption, high sensitivity, low interference and good stability. Keywords Cholesterol, Moleculary imprinted polymer, Electrochemical sensor, Multiwalled
carbon nanotubes; Au nanoparticles.
Introduction Recently there has been an intense interest in molecularly imprinted technology. Molecularly imprinted polymers (MIP) are prepared by creating a three-dimensional polymeric matrix around a template molecule (Spégel et al. 2002). After the removal of the template, the resulting imprinted cavitiy with complementary shape and functional groups remain. The MIP technique has been demonstrated as one of the most promising techniques in the sensor field for its low cost, simplicity, reliability, and wide choice of templates and functional monomers (Fuchs et al. 2012; Haupt and Mosbach 2000). The technique has been widely explored in the field of analytical chemistry, especially in studies on stationary phases for HPLC (Moein et al. 2011), capillary electrochromatography (Huang et al. 2011), enzyme-linked sorbents for chemiluminescence (Jing et al. 2011) and colorimetric detection (Sergeyeva et al. 2014). Owing to the complementarity in binding sites and shape, the created nanocavities can exhibit good selectivity and act as artificial antibodies toward the imprinted molecules, including a large and diverse set of important metal ions (Güney and Cebeci 2010), organic molecules (Sun et al. 2014) and bioorganic molecules (Bossi et al. 2012). Furthermore, MIP electrochemical sensors have attracted considerable attention and have been widely utilized to detect different molecules ranging from small hazardous molecules to biomacromolecules (Kan et al. 2012; Li et al. 2012; Malitesta et al. 2012; Wang et al. 2014). At the same time, these reports pointed out that the fabrication of MIP on electrode surfaces was a critical factor (Afkhami et al. 2013). Hitherto, several methods have been developed for fabricating MIP on electrode surfaces, including in-situ polymerization by electrochemical (Pernites et al. 2011), optical (Ton et al. 2013) and thermal technologies (Turner et al. 2010), and to coat electrode surfaces with MIP suspensions containing glutinous agents (Yang et al. 2014). To achieve a high sensitivity, nanomaterials were used with MIP (Kong et al. 2013; Lian et al. 2012).
Carbon nanotubes (CNTs) are promising for electrochemical sensing due to their large surface areas, high electrical conductivities and excellent electron transfer rates (Lota et al. 2011). CNTs have been widely used as sensors in various electroanalyticaland biosensing applications (Yáñez-Sedeño et al. 2010). Au nanoparticles (AuNPs) have been intensively studied on the fabrication of electrodes due to their large specific surface area, good biocompatibility, and high conductivity (Li et al. 2010b). Self-assembled monolayers (SAMs) can be induced by the strong chemisorption between the substrate and organic molecule (Fuller et al. 2010). SAMs have excellent stability and integration with biomolecular electronic devices and have been widely used in sensors. Cholesterol (CHO) is an essential lipid with several biological functions in organisms. Hypercholesterolemia is a major cause of the early development of coronary heart and peripheral atherosclerotic diseases (Stitziel et al. 2013). The determination of cholesterol levels in food and blood has been increasingly important for clinical analysis/diagnosis because of an alarming rise reported in the rate of clinical disorders due to abnormal levels of blood cholesterol. CHO has been detected by various methods, such as HPLC (Kotani et al. 2011), gas chromatography (Garcí a ‐ Llatas et al. 2012), cholesterol biosensors (Safavi and Farjami 2011) and electrophoretic chips (Kaminikado et al. 2011). Therefore, it is highly desirable for analytical research and clinical applications to establish a sensitive, selective and quick method to detect trace CHO in biological samples. To accurately determine CHO levels in individual laboratories, a sensitive and selective analytical methodology that requires less expensive apparatus and direct analytical determination without complicated derivatization procedures is needed. In this study, a novel sensor for CHO determination based on MIP and co-polymerized technology was constructed. A molecularly imprinted film was fabricated on the electrode surface, and CHO was linked to the cavities constructed by binding sites of molecularly imprinted film. It was hypothesized that the combination of surface molecular self-assembly by co-polymerization of poly-aminothiophenol (p-ATP) and AuNPs on GCE modified with multi-walled carbon nanotubes (MWNTs)
would maximize the amount of effective imprinted sites and would enhance its conductivity. 2. Experimental 2.1. Instruments and reagents The morphology of the molecularly imprinted polymers (MIP) on the electrode was observed using a scanning electron microscope (Hitachi S-4800, Japan). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were conducted using a CHI660e
workstation
(Chenhua,
Shanghai,
China),
using
a
conventional
three-electrode system in a solution containing 2.5×10−3 mol L-1 Fe(CN)63−/4− consisting of 0.0824 g K3Fe(CN)6, 0.1164 g K4Fe(CN)6·3H2O, and 0.1 M KCl. A glassy carbon electrode (GCE, Φ 3 mm) was used as the working electrode, a platinum wire was used as the auxiliary electrode and a saturated calomel electrode was used as the reference electrode. Multi-walled carbon nanotubes (MWNTs, CVD method, purity >95%, diameter 50 nm, length 10-20 µm) was obtained from Nanjing Xianfeng Nanomaterials Technology Co. Ltd. (Nanjing, China). P-aminothiophenol (p-ATP, 97 %), tetrabutylammonium perchlorate (TBAP, 99 %)，chitosan (CS, minimum 90 % deacetylated) and tetrachloroaurate (III) acid (HAuCl4) were purchased from Sigma-Aldrich China, Inc. Cholesterol (CHO, 99 %) was purchased from Sigma-Aldrich Co. LLC. (Mainland, China). All other reagents were Chromatography analytical grade and prepared with ultrapure water (18.2 MΩ.cm) from a Milipore Mili-Q system. Standard solutions of CHO were prepared in ethanol. 2.2 Fabrication of AuNPs-MWNTs modified GCE Before electrode modification, a bare GCE was immersed in a fresh piranha solution of H2SO4/30 % H2O2 (3:1, v/v) for 15 min, and then rished with redistilled water. The GCE was polished to a mirror-finished with slurry of alumina (0.30 μm and 0.05 μm), washed with ultrapure water, and then ultrasonicated in ethanol and ultrapure water for 2 min, respectively. The MWNTs modified GCE was prepared as reported previously (Yang et al. 2013). -COOH was grafted on the surface of MWNTs to enhance the dispersion and
stability of MWNTs. MWNTs-COOH (5 mg) and CS-acetic acid solution (1.0 wt %, 10 mL) were mixed in a glass tube with the help of ultrasonic treatment to form a homogeneous MWNTs-CS suspension solution (0.5 mg mL−1), which palyed an important role in immobilization of the MWNTs on electrode. Then, the GCE was coated with 10.0 μL of the resulting MWNT-CS composite and allowed to dry at room temperature for 30 min. The obtained MWNTs modified GCE was then dipped in 2.43 mmol L−1 aqueous HAuCl4 solution with 0.1 mol L−1 H2SO4 and treated at -0.2 V constant potential for 200 s. After rinsing several times with ultrapure water and absolute ethanol, and then dried under nitrogen flow for 1 min at room temperature (Sun et al. 2013). 2.3 p-ATP and CHO self-assembly on the AuNPs-MWNTs/GCE The p-ATP modified electrode was prepared according to the reported methods (Li et al. 2010a). The prepared AuNPs-MWNTs/GCE was immersed in an ethanol solution containing 5 mM p-ATP for 24 h at room temperature, removed, and then washed with ethanol and redistilled water to remove physically absorbed p-ATP. p-ATP has the functional agent –SH, which could connect with AuNPs based with S-Au bond. Then, the p-ATP modified AuNPs-MWNTs/GCE was immersed in an ethanol solution with 1 mmol L−1 CHO for 4 h. The electrode was removed, rinsed with ethanol and redistilled water to remove physically absorbed CHO, and then dried under nitrogen gas at ambient temperature. 2.4 Fabrication of imprinted MIP-AuNPs-MWNTs /GCE The p-ATP and CHO modified AuNPs-MWNTs/GCE was immersed in an ethanol solution containing 1×10−2 mol L-1 p-ATP, 5×10−2 mol L-1 TBAP, 1×10−2 mol L-1 CHO and 0.4 g L−1 HAuCl4 (Wang et al. 2011). The co-polymerization was performed by the application of five CV cycles in an ice bath with a potential range from -0.3 to 1.2 V (scan rate 50 mV s−1). After electropolymerization, the composite membrane modified electrode was immersed in an ethanol/water (4:1, v/v) solution containing 0.5 mol L−1 HCl to remove the CHO template molecule. The imprinted electrode was then rinsed with ultrapure water, and finally dried under nitrogen for further use.
We prepared a control electrode following the same procedure but without a template molecule. The control electrode was treated using the same procedure as for the imprinted electrode to ensure that any effects observed were only due to the imprinting features and not the subsequent treatments undergone by the electrode. 2.5 Electrochemical measurements CV and DPV measurements were carried out to evaluate the current response of the modified electrodes immersed in a solution of 2.5 mmol L−1 [Fe(CN)6]3-/4- with 0.1 mol L−1 KCl as the supporting electrolyte over an applied potential range from -0.2 to +0.6 V. All the measurements were carried out at ambient temperature. CV of the MIP film were performed by scanning the potential between -0.3 and +1.2 V at a scan rate of 50 mV s−1. 3. Result and discussion 3.1 Preparation and characterization of the MIP-modified electrodes Basic functional monomers with amino groups are usually used for acidic template containing hydroxy groups since very stable complexes could be formed between them through stronger ionic interactions. It is of obvious importance that the functional monomers strongly interact with the template and form stable host-guest complexes before polymerization. In the CHO imprinting process, p-ATP acted as a functional monomer due to its amino groups, which can interact with the hydroxy groups of CHO simultaneously, AuNPs acted as crosslinkers to form polymeric networks through Au-S. The whole preparation process for developing the molecularly imprinted sensor is shown in Scheme 1. This process can be summarized in four steps: 1) Self-assembly of p-ATP onto the surface of AuNPs-MWNTs/GCE, exposing an array of amino groups to the solution; 2) Hydrogen bonding adsorption of CHO molecules onto the surface of the p-ATP modified electrode; 3) Co-polymerization of p-ATP-AuNPs onto the surface of the modified electrode and 4) Removal of the template CHO molecules from MIP-AuNPs-MWNTs/GCE. A large number of tailor-made cavities formed on the surface of the modified electrode. Before co-polymerization, the GCE was modified with MWNTs and AuNPs. CV of the modified electrodes in 2.5 mmol L−1 [Fe(CN)6]3-/4- and 0.1 mol L−1 KCl are
shown in Fig. 1(A). The bare GCE showed a couple of redox peaks. When the electrode surface was covered with the MWNTs-CS composite, the redox peak current in the CV curve increased, indicating that the incorporation of MWNTs improved the current response of K3[Fe(CN)6] on the MWNTs/GCE. The strategy of electrodeposition of AuNPs onto the surface ofMWNTs/GCEwas not only to enhance the electrochemical signal but also to plant a site for p-ATP binding, which was the functional monomer of the MIP sensor. The effect of the scan rate on the redox reaction of AuNPs-MWNTs/GCE was investigated using CV. Fig. 1(B) shows the CV curves for AuNPs-MWNTs/GCE at scan rates from 50 to 150 mV s-1. Both the anodic and cathodic peakcurrents (Ipa and Ipc, respectively) were linearly related to the square scan rate (Fig. 1(C)), with the linear regression equations Ipa (μA) = 39.47 + 16.40 v 1/2 (mV s−1, R2 = 0.999) and Ipc (μA) = -32.47 – 17.18 v1/2 (mV s−1, R2 = 0.998). The results indicated that the process was predominantly
diffusion-controlled
(Laviron
1979),
which
suggested
that
AuNPs-MWNT played a role in increasing the electron-transfer rate (the comparison of bare GCE, MWNTs/GCE and AuNPs-MWNTs/GCE is shown in Fig. S1). CV for the MIP film were recorded in 2.5×10−3 mol L-1 [Fe(CN)6]3−/4− and 0.1 mol L−1 KCl, andwere used to confirm whether or not CHO was embedded in the MIP film. During the procedure, [Fe(CN)6]3−/4− was used as a mediator between the imprinted electrodes and the substrate solutions. Fig. 1(D) shows the relationship between peak current and surface modification conditions of AuNPs-MWNTs/GCE. The bare electrode showed a couple of redox peaks with current value of approximately 500 μA, seen in Fig. 1(E) curve a. After assembly with p-ATP, a decrease in the ΔIp (200 μA) was observed, as shown in curve b. Compared to the current response of AuNPs-MWNTs/GCE, almost no redox peaks were observed, as shown in curve c, which indicated that the MIP film had formed on the AuNPs-MWNTs/GCE surface. After removing CHO (curve d), a decrease in ΔIp (150 μA) was observed. A possible reason was that after CHO removal the cavities enhanced the diffusion of [Fe(CN)6]3-/4- through the MIP film and promoted the redox reaction of [Fe(CN)6]3-/4- on the sensor (the cyclic voltammograms of the assembly
process of only AuNPs modifying GCE are shown in Fig. S2). The one-step co-polymerization method was improved by conducting CV in a 5 mL ethanol solution containing 1×10−2 mol L-1 p-ATP, 5×10−2 mol L-1 TBAP, 1×10−2 mol L-1 CHO and 0.4 g L−1 HAuCl4. Fig. 1(F) shows the electrochemical process used to form a MIP film on AuNPs-MWNTs/GCE. The MIP film was deposited by repetitively sweeping the potential from −0.3 to 1.2 V at a scan rate of 50 mV s −1. An irreversible oxidation process appeared during the first cycle and disappeared during the second cycle. HAuCl4 was reduced to AuNPs and absorbed onto the electrode surface. Two oxidation peaks were clearly observed at potentials of approximately 0.32 V and 0.68 V are possibly the polymerization reaction of p-ATP (Wang et al. 2011), in the first scan, which indicated that a compact polymeric film was formed and bound to the electrode surface. The decrease in peak current seemed to be related to the continuous formation of p-ATP-AuNPs composite membranes that led to the suppression of the voltammetric response (the contact angle experiment of MIP-GCE is shown in Fig. S3 and the morphology of the modified electrode’s surface observed using SEM is shown in Fig. S4). 3.2 Characterization and optimization of the electrochemical behavior of the MIP sensor After electropolymerization, the composite membrane modified electrode was immersed in an ethanol:water (4:1) solution containing 0.5 mol L−1 HCl to remove the template. As shown in Fig. 2(A) and Fig. 2(B), the current gradually increased and reached a maximum at approximately 10 min and then remained stable for 10 min, which meant that the template was washed out after 10 min in this solution. The kinetic adsorption of CHO onto the MIP sensor is shown in Fig. 2(C) and Fig. 2(D). The amount of CHO adsorbed onto the MIP sensor increased with increasing adsorption times. It was also found that CHO was quickly absorbed by the MIP sensor and the adsorption reachedkinetic equilibrium within 150 s. The kinetic curve observed was typical of most rebinding processes and revealed the rapid dynamic adsorption of CHO onto the MIP-modified GCE. The elution time of MIP-GCE after adsorption of 10 ng mL-1 CHO ethanol
solution was analyzed, as shown in Fig. 2 (E). Only 3 min was needed to remove the CHO molecule from the MIP film, which was much shorterthan the first template removal after electropolymerization. The elution solution was optimized as in Fig. 2(F), which indicated that the MIP-GCE after adsorption of 10 ng mL-1 CHO immersed in an ethanol solution could not be cleaned out completely, even with the elution time extending to 60 min. Remarkably the added 0.5 mol L−1 HCl could remove the CHO template from the MIP film after a short amount of time, as shown in Fig. 2 (F) curve a. From these results, an adsorption time of 150 s and an elution time of 3 min were selected for all subsequent assays. 3.3 Electrochemical detection of CHO For CHO detection, MIP-AuNPs-MWNTs/GCE was immersed in different concentrations of 5 mL samples CHO solution at room temperature in an open chamber (from 1×10−14 to 1×10−8 mol L−1). When CHO adhered to the modified electrode it showed higher charge-transfer impedance and this increase represented the combined effects of a reduction in the DPV current peak value, as shown in Fig. 3(A). This showed that when CHO was rebound, a compact film appeared on the surface of the electrode which hindered electron transfer from the [Fe(CN)6]3−/4− ion pair to the electrode surface. The formed MIP-AuNPs-MWNTs complex membrane resulted in a decrease in the electrochemical reaction of the [Fe(CN)6]3−/4− probe. The function of MWNTs in the complex membrane was analyzed as shown in Fig. 3(A). Compared with the MIP-AuNPs-MWNTs membrane, the extent of current fluctuation of MIP-AuNPs influenced by the concentration of CHO was much weaker, which indicated that the MWNTs played a significance role in the MIP sensor. As shown in Fig. 3(B), the MIP sensor equipped with MWNTs had a large-scale, sensitive, and credible detection range, and the limit of detection (LOD) was much better than that of the MIP sensor equipped without MWNTs (the MIP and NIP sensors’ response to the incubation concentration of CHO are shown in Fig. S5). Linear relationships existed between the peak current and the log of CHO concentration from 1×10 −13 to 1×10−9 mol L−1 (Ip = − 5.033 logC + 16.885 (R2 = 0.995)). The LOD of the CHO MIP sensor was 3.3×10−14 mol L−1, which were more sensitive than most available CHO
MIP sensor detection methods (Table 1). 3.4 Selectivity, reproducibility and stability of the MIP sensor An excellent sensor not only possesses good sensitivity, but also has good selectivity. To determine the selectivity of the molecularly imprinted sensor four compounds were investigated: cholic acid, deoxycholic acid, ascorbic acid, uric acid and 7-dehydrocholesterol as the control. Fig. S6 shows different current response signals for the proposed sensing system after the addition of 1×10−9 mol L-1 cholic acid, deoxycholic acid,ascorbic acid, uric acid and 7-dehydrocholesterol under the same experimental conditions. To evaluate the reproducibility and repeatability of the MIP sensor, experiments were carried out in a 1×10-10 mol L-1 CHO solution. The MIP sensor was regenerated with a peak current relative standard deviation (RSD) of 3.54 % using four different GCE electrodes. Excellent reproducibility was obtained with a relative standard deviation of 2.72 % after 20 washes and measurements, therefore the MIP sensor had good reversibility. Moreover, the stability of the sensor was evaluated,as stability is a key factor for CHO detection. The MIP sensor was stored in an ethanol:water (4:1) solution containing 0.5 mol L−1 HCl at room temperature for 10 days, and the peak current did not significantly change. After one month, it decreased to 91.68 %. All measurements indicated that the MIP sensor possessed excellent stability. 4. Conclusions In this work, a MIP film electrochemical sensor was used to indirectly detect CHO. It was constructed and developed by co-polymerization of p-ATP and HAuCl4 in the presence of template CHO molecules using CV. CHO molecules were absorbed by hydrogen bonding with p-ATP onto the surface of the AuNPs-MWNTs-modified GCE, which greatly increased the amount of imprinted sites. The doped nanoparticles enhanced the sensitivity of the MIP sensor, especially for MWNTs, whose irreplaceable function was discussed in the paper. The lowest detectable concentration of CHO was 3.3×10−14 mol L−1 and the linear detection range extended to 1×10−9 mol L−1. Furthermore, the fabrication of MIP-AuNPs-MWNT-modified GCEs was simple and controllable. These results demonstrated that the electrochemical sensor
significantly improved the sensitivity and selectivity of CHO analysis with good repeatability. But its application in clinical analysis/diagnosis or food analysis is a long process needed intensive study. Acknowledgements This work was supported by the National Research Program (No.201203069-1), Jiangsu Tech-enterprise technology innovation fund, enterprise project of high-tech business incubator (NO. BC2011034). References Afkhami, A., Ghaedi, H., Madrakian, T., Ahmadi, M., Mahmood-Kashani, H., 2013. Biosensors and Bioelectronics 44, 34-40. Aghaei, A., Milani Hosseini, M.R., Najafi, M., 2010. Electrochimica Acta 55(5), 1503-1508. Bossi, A.M., Sharma, P.S., Montana, L., Zoccatelli, G., Laub, O., Levi, R., 2012. Analytical chemistry 84(9), 4036-4041. Chou, L., Liu, C.-C., 2005. Sensors and actuators B: Chemical 110(2), 204-208. Fuchs, Y., Soppera, O., Haupt, K., 2012. Analytica Chimica Acta 717, 7-20. Fuller, R.M., Gao, K., Li, L., 2010. Google Patents. Güney, O., Cebeci, F.Ç., 2010. Journal of Applied Polymer Science 117(4), 2373-2379. García‐Llatas, G., Vidal, C., Cilla, A., Barberá, R., Lagarda, M.J., 2012. European Journal of Lipid Science and Technology 114(5), 520-526. Gong, J.-L., Gong, F.-C., Zeng, G.-M., Shen, G.-L., Yu, R.-Q., 2003. Talanta 61(4), 447-453. Haupt, K., Mosbach, K., 2000. Chemical Reviews 100(7), 2495-2504. Huang, B.-Y., Chen, Y.-C., Wang, G.-R., Liu, C.-Y., 2011. Journal of chromatography A 1218(6), 849-855. Jing, T., Xia, H., Guan, Q., Lu, W., Dai, Q., Niu, J., Lim, J.-M., Hao, Q., Lee, Y.-I., Zhou, Y., 2011. Analytica Chimica Acta 692(1), 73-79. Kaminikado, K., Ikeda, R., Idegami, K., Nagatani, N., Mun'delanji, C.V., Saito, M., Tamiya, E., 2011. Analyst 136(9), 1826-1830.
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Table: Table 1 Comparison with other published MIP methods for the determination of CHO NO.
Method
Linear range
LOD
Shelf life
(mol L-1)
(mol L-1)
(days)
Reference
1
2-MBI-GE
1×10−6 to 3×10−5
0.42×10−6
18
(Aghaei et al. 2010)
2
GC/PB/CS-SiO2-COX-MWCNTs
4×10−6 to7×10−4
1×10−6
50
(Tan et al. 2005)
3
PMBI-GE
up to 2×10−5
0.7×10−6
PII: DOI: Reference:
S0956-5663(14)00956-7 http://dx.doi.org/10.1016/j.bios.2014.12.014 BIOS7330
To appear in: Biosensors and Bioelectronic Received date: 8 October 2014 Revised date: 2 December 2014 Accepted date: 2 December 2014 Cite this article as: Jian Ji, Zhihui Zhou, Xiaolian Zhao, Jiadi Sun and Xiulan Sun, Electrochemical sensor based on molecularly imprinted film at Au nanoparticles-carbon nanotubes modified electrode for determination of c h o l e s t e r o l , Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2014.12.014 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.
Electrochemical sensor based on molecularly imprinted film at Au nanoparticles-carbon nanotubes modified electrode for determination of cholesterol Jian Ji1, Zhihui Zhou2, Xiaolian Zhao2, Jiadi Sun1, Xiulan Sun1* 1 State Key Laboratory of Food Science and Technology，School of Food Science of Jiangnan University, Synergetic Innovation Center Of Food Safety and Nutrition, Wuxi, Jiangsu 214122, China 2 Wuxi Jinkun Bio-Technology Co., Ltd. Wuxi, Jiangsu 214122, China
Corresponding author: Xiulan Sun (e-mail: [email protected]) First author: Jian Ji (e-mail: [email protected])
Abstract: A novel electrochemical sensor for cholesterol (CHO) detection based on molecularly imprinted polymer (MIP) membranes on a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWNTs) and Au nanoparticles (AuNPs) was constructed. p-Aminothiophenol (P-ATP) and CHO were assembled on the surface of the modified GCE by the formation of Au-S bonds and hydrogen-bonding interactions, and polymer membranes were formed by electropolymerization in a polymer solution containing p-ATP, HAuCl4, tetrabutylammonium perchlorate (TBAP) and the template molecule CHO. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were used to monitor the electropolymerization process and its optimization, which was further characterized by scanning electron microscopy (SEM). The linear response range of the MIP sensor was between 1 × 10-13 and 1 × 10-9 mol L−1, and the limit of detection (LOD) were 3.3×10−14 mol L−1. The proposed system has the potential for application in clinical diagnostics of cholesterol with high-speed real-time detection capability, low sample consumption, high sensitivity, low interference and good stability. Keywords Cholesterol, Moleculary imprinted polymer, Electrochemical sensor, Multiwalled
carbon nanotubes; Au nanoparticles.
Introduction Recently there has been an intense interest in molecularly imprinted technology. Molecularly imprinted polymers (MIP) are prepared by creating a three-dimensional polymeric matrix around a template molecule (Spégel et al. 2002). After the removal of the template, the resulting imprinted cavitiy with complementary shape and functional groups remain. The MIP technique has been demonstrated as one of the most promising techniques in the sensor field for its low cost, simplicity, reliability, and wide choice of templates and functional monomers (Fuchs et al. 2012; Haupt and Mosbach 2000). The technique has been widely explored in the field of analytical chemistry, especially in studies on stationary phases for HPLC (Moein et al. 2011), capillary electrochromatography (Huang et al. 2011), enzyme-linked sorbents for chemiluminescence (Jing et al. 2011) and colorimetric detection (Sergeyeva et al. 2014). Owing to the complementarity in binding sites and shape, the created nanocavities can exhibit good selectivity and act as artificial antibodies toward the imprinted molecules, including a large and diverse set of important metal ions (Güney and Cebeci 2010), organic molecules (Sun et al. 2014) and bioorganic molecules (Bossi et al. 2012). Furthermore, MIP electrochemical sensors have attracted considerable attention and have been widely utilized to detect different molecules ranging from small hazardous molecules to biomacromolecules (Kan et al. 2012; Li et al. 2012; Malitesta et al. 2012; Wang et al. 2014). At the same time, these reports pointed out that the fabrication of MIP on electrode surfaces was a critical factor (Afkhami et al. 2013). Hitherto, several methods have been developed for fabricating MIP on electrode surfaces, including in-situ polymerization by electrochemical (Pernites et al. 2011), optical (Ton et al. 2013) and thermal technologies (Turner et al. 2010), and to coat electrode surfaces with MIP suspensions containing glutinous agents (Yang et al. 2014). To achieve a high sensitivity, nanomaterials were used with MIP (Kong et al. 2013; Lian et al. 2012).
Carbon nanotubes (CNTs) are promising for electrochemical sensing due to their large surface areas, high electrical conductivities and excellent electron transfer rates (Lota et al. 2011). CNTs have been widely used as sensors in various electroanalyticaland biosensing applications (Yáñez-Sedeño et al. 2010). Au nanoparticles (AuNPs) have been intensively studied on the fabrication of electrodes due to their large specific surface area, good biocompatibility, and high conductivity (Li et al. 2010b). Self-assembled monolayers (SAMs) can be induced by the strong chemisorption between the substrate and organic molecule (Fuller et al. 2010). SAMs have excellent stability and integration with biomolecular electronic devices and have been widely used in sensors. Cholesterol (CHO) is an essential lipid with several biological functions in organisms. Hypercholesterolemia is a major cause of the early development of coronary heart and peripheral atherosclerotic diseases (Stitziel et al. 2013). The determination of cholesterol levels in food and blood has been increasingly important for clinical analysis/diagnosis because of an alarming rise reported in the rate of clinical disorders due to abnormal levels of blood cholesterol. CHO has been detected by various methods, such as HPLC (Kotani et al. 2011), gas chromatography (Garcí a ‐ Llatas et al. 2012), cholesterol biosensors (Safavi and Farjami 2011) and electrophoretic chips (Kaminikado et al. 2011). Therefore, it is highly desirable for analytical research and clinical applications to establish a sensitive, selective and quick method to detect trace CHO in biological samples. To accurately determine CHO levels in individual laboratories, a sensitive and selective analytical methodology that requires less expensive apparatus and direct analytical determination without complicated derivatization procedures is needed. In this study, a novel sensor for CHO determination based on MIP and co-polymerized technology was constructed. A molecularly imprinted film was fabricated on the electrode surface, and CHO was linked to the cavities constructed by binding sites of molecularly imprinted film. It was hypothesized that the combination of surface molecular self-assembly by co-polymerization of poly-aminothiophenol (p-ATP) and AuNPs on GCE modified with multi-walled carbon nanotubes (MWNTs)
would maximize the amount of effective imprinted sites and would enhance its conductivity. 2. Experimental 2.1. Instruments and reagents The morphology of the molecularly imprinted polymers (MIP) on the electrode was observed using a scanning electron microscope (Hitachi S-4800, Japan). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were conducted using a CHI660e
workstation
(Chenhua,
Shanghai,
China),
using
a
conventional
three-electrode system in a solution containing 2.5×10−3 mol L-1 Fe(CN)63−/4− consisting of 0.0824 g K3Fe(CN)6, 0.1164 g K4Fe(CN)6·3H2O, and 0.1 M KCl. A glassy carbon electrode (GCE, Φ 3 mm) was used as the working electrode, a platinum wire was used as the auxiliary electrode and a saturated calomel electrode was used as the reference electrode. Multi-walled carbon nanotubes (MWNTs, CVD method, purity >95%, diameter 50 nm, length 10-20 µm) was obtained from Nanjing Xianfeng Nanomaterials Technology Co. Ltd. (Nanjing, China). P-aminothiophenol (p-ATP, 97 %), tetrabutylammonium perchlorate (TBAP, 99 %)，chitosan (CS, minimum 90 % deacetylated) and tetrachloroaurate (III) acid (HAuCl4) were purchased from Sigma-Aldrich China, Inc. Cholesterol (CHO, 99 %) was purchased from Sigma-Aldrich Co. LLC. (Mainland, China). All other reagents were Chromatography analytical grade and prepared with ultrapure water (18.2 MΩ.cm) from a Milipore Mili-Q system. Standard solutions of CHO were prepared in ethanol. 2.2 Fabrication of AuNPs-MWNTs modified GCE Before electrode modification, a bare GCE was immersed in a fresh piranha solution of H2SO4/30 % H2O2 (3:1, v/v) for 15 min, and then rished with redistilled water. The GCE was polished to a mirror-finished with slurry of alumina (0.30 μm and 0.05 μm), washed with ultrapure water, and then ultrasonicated in ethanol and ultrapure water for 2 min, respectively. The MWNTs modified GCE was prepared as reported previously (Yang et al. 2013). -COOH was grafted on the surface of MWNTs to enhance the dispersion and
stability of MWNTs. MWNTs-COOH (5 mg) and CS-acetic acid solution (1.0 wt %, 10 mL) were mixed in a glass tube with the help of ultrasonic treatment to form a homogeneous MWNTs-CS suspension solution (0.5 mg mL−1), which palyed an important role in immobilization of the MWNTs on electrode. Then, the GCE was coated with 10.0 μL of the resulting MWNT-CS composite and allowed to dry at room temperature for 30 min. The obtained MWNTs modified GCE was then dipped in 2.43 mmol L−1 aqueous HAuCl4 solution with 0.1 mol L−1 H2SO4 and treated at -0.2 V constant potential for 200 s. After rinsing several times with ultrapure water and absolute ethanol, and then dried under nitrogen flow for 1 min at room temperature (Sun et al. 2013). 2.3 p-ATP and CHO self-assembly on the AuNPs-MWNTs/GCE The p-ATP modified electrode was prepared according to the reported methods (Li et al. 2010a). The prepared AuNPs-MWNTs/GCE was immersed in an ethanol solution containing 5 mM p-ATP for 24 h at room temperature, removed, and then washed with ethanol and redistilled water to remove physically absorbed p-ATP. p-ATP has the functional agent –SH, which could connect with AuNPs based with S-Au bond. Then, the p-ATP modified AuNPs-MWNTs/GCE was immersed in an ethanol solution with 1 mmol L−1 CHO for 4 h. The electrode was removed, rinsed with ethanol and redistilled water to remove physically absorbed CHO, and then dried under nitrogen gas at ambient temperature. 2.4 Fabrication of imprinted MIP-AuNPs-MWNTs /GCE The p-ATP and CHO modified AuNPs-MWNTs/GCE was immersed in an ethanol solution containing 1×10−2 mol L-1 p-ATP, 5×10−2 mol L-1 TBAP, 1×10−2 mol L-1 CHO and 0.4 g L−1 HAuCl4 (Wang et al. 2011). The co-polymerization was performed by the application of five CV cycles in an ice bath with a potential range from -0.3 to 1.2 V (scan rate 50 mV s−1). After electropolymerization, the composite membrane modified electrode was immersed in an ethanol/water (4:1, v/v) solution containing 0.5 mol L−1 HCl to remove the CHO template molecule. The imprinted electrode was then rinsed with ultrapure water, and finally dried under nitrogen for further use.
We prepared a control electrode following the same procedure but without a template molecule. The control electrode was treated using the same procedure as for the imprinted electrode to ensure that any effects observed were only due to the imprinting features and not the subsequent treatments undergone by the electrode. 2.5 Electrochemical measurements CV and DPV measurements were carried out to evaluate the current response of the modified electrodes immersed in a solution of 2.5 mmol L−1 [Fe(CN)6]3-/4- with 0.1 mol L−1 KCl as the supporting electrolyte over an applied potential range from -0.2 to +0.6 V. All the measurements were carried out at ambient temperature. CV of the MIP film were performed by scanning the potential between -0.3 and +1.2 V at a scan rate of 50 mV s−1. 3. Result and discussion 3.1 Preparation and characterization of the MIP-modified electrodes Basic functional monomers with amino groups are usually used for acidic template containing hydroxy groups since very stable complexes could be formed between them through stronger ionic interactions. It is of obvious importance that the functional monomers strongly interact with the template and form stable host-guest complexes before polymerization. In the CHO imprinting process, p-ATP acted as a functional monomer due to its amino groups, which can interact with the hydroxy groups of CHO simultaneously, AuNPs acted as crosslinkers to form polymeric networks through Au-S. The whole preparation process for developing the molecularly imprinted sensor is shown in Scheme 1. This process can be summarized in four steps: 1) Self-assembly of p-ATP onto the surface of AuNPs-MWNTs/GCE, exposing an array of amino groups to the solution; 2) Hydrogen bonding adsorption of CHO molecules onto the surface of the p-ATP modified electrode; 3) Co-polymerization of p-ATP-AuNPs onto the surface of the modified electrode and 4) Removal of the template CHO molecules from MIP-AuNPs-MWNTs/GCE. A large number of tailor-made cavities formed on the surface of the modified electrode. Before co-polymerization, the GCE was modified with MWNTs and AuNPs. CV of the modified electrodes in 2.5 mmol L−1 [Fe(CN)6]3-/4- and 0.1 mol L−1 KCl are
shown in Fig. 1(A). The bare GCE showed a couple of redox peaks. When the electrode surface was covered with the MWNTs-CS composite, the redox peak current in the CV curve increased, indicating that the incorporation of MWNTs improved the current response of K3[Fe(CN)6] on the MWNTs/GCE. The strategy of electrodeposition of AuNPs onto the surface ofMWNTs/GCEwas not only to enhance the electrochemical signal but also to plant a site for p-ATP binding, which was the functional monomer of the MIP sensor. The effect of the scan rate on the redox reaction of AuNPs-MWNTs/GCE was investigated using CV. Fig. 1(B) shows the CV curves for AuNPs-MWNTs/GCE at scan rates from 50 to 150 mV s-1. Both the anodic and cathodic peakcurrents (Ipa and Ipc, respectively) were linearly related to the square scan rate (Fig. 1(C)), with the linear regression equations Ipa (μA) = 39.47 + 16.40 v 1/2 (mV s−1, R2 = 0.999) and Ipc (μA) = -32.47 – 17.18 v1/2 (mV s−1, R2 = 0.998). The results indicated that the process was predominantly
diffusion-controlled
(Laviron
1979),
which
suggested
that
AuNPs-MWNT played a role in increasing the electron-transfer rate (the comparison of bare GCE, MWNTs/GCE and AuNPs-MWNTs/GCE is shown in Fig. S1). CV for the MIP film were recorded in 2.5×10−3 mol L-1 [Fe(CN)6]3−/4− and 0.1 mol L−1 KCl, andwere used to confirm whether or not CHO was embedded in the MIP film. During the procedure, [Fe(CN)6]3−/4− was used as a mediator between the imprinted electrodes and the substrate solutions. Fig. 1(D) shows the relationship between peak current and surface modification conditions of AuNPs-MWNTs/GCE. The bare electrode showed a couple of redox peaks with current value of approximately 500 μA, seen in Fig. 1(E) curve a. After assembly with p-ATP, a decrease in the ΔIp (200 μA) was observed, as shown in curve b. Compared to the current response of AuNPs-MWNTs/GCE, almost no redox peaks were observed, as shown in curve c, which indicated that the MIP film had formed on the AuNPs-MWNTs/GCE surface. After removing CHO (curve d), a decrease in ΔIp (150 μA) was observed. A possible reason was that after CHO removal the cavities enhanced the diffusion of [Fe(CN)6]3-/4- through the MIP film and promoted the redox reaction of [Fe(CN)6]3-/4- on the sensor (the cyclic voltammograms of the assembly
process of only AuNPs modifying GCE are shown in Fig. S2). The one-step co-polymerization method was improved by conducting CV in a 5 mL ethanol solution containing 1×10−2 mol L-1 p-ATP, 5×10−2 mol L-1 TBAP, 1×10−2 mol L-1 CHO and 0.4 g L−1 HAuCl4. Fig. 1(F) shows the electrochemical process used to form a MIP film on AuNPs-MWNTs/GCE. The MIP film was deposited by repetitively sweeping the potential from −0.3 to 1.2 V at a scan rate of 50 mV s −1. An irreversible oxidation process appeared during the first cycle and disappeared during the second cycle. HAuCl4 was reduced to AuNPs and absorbed onto the electrode surface. Two oxidation peaks were clearly observed at potentials of approximately 0.32 V and 0.68 V are possibly the polymerization reaction of p-ATP (Wang et al. 2011), in the first scan, which indicated that a compact polymeric film was formed and bound to the electrode surface. The decrease in peak current seemed to be related to the continuous formation of p-ATP-AuNPs composite membranes that led to the suppression of the voltammetric response (the contact angle experiment of MIP-GCE is shown in Fig. S3 and the morphology of the modified electrode’s surface observed using SEM is shown in Fig. S4). 3.2 Characterization and optimization of the electrochemical behavior of the MIP sensor After electropolymerization, the composite membrane modified electrode was immersed in an ethanol:water (4:1) solution containing 0.5 mol L−1 HCl to remove the template. As shown in Fig. 2(A) and Fig. 2(B), the current gradually increased and reached a maximum at approximately 10 min and then remained stable for 10 min, which meant that the template was washed out after 10 min in this solution. The kinetic adsorption of CHO onto the MIP sensor is shown in Fig. 2(C) and Fig. 2(D). The amount of CHO adsorbed onto the MIP sensor increased with increasing adsorption times. It was also found that CHO was quickly absorbed by the MIP sensor and the adsorption reachedkinetic equilibrium within 150 s. The kinetic curve observed was typical of most rebinding processes and revealed the rapid dynamic adsorption of CHO onto the MIP-modified GCE. The elution time of MIP-GCE after adsorption of 10 ng mL-1 CHO ethanol
solution was analyzed, as shown in Fig. 2 (E). Only 3 min was needed to remove the CHO molecule from the MIP film, which was much shorterthan the first template removal after electropolymerization. The elution solution was optimized as in Fig. 2(F), which indicated that the MIP-GCE after adsorption of 10 ng mL-1 CHO immersed in an ethanol solution could not be cleaned out completely, even with the elution time extending to 60 min. Remarkably the added 0.5 mol L−1 HCl could remove the CHO template from the MIP film after a short amount of time, as shown in Fig. 2 (F) curve a. From these results, an adsorption time of 150 s and an elution time of 3 min were selected for all subsequent assays. 3.3 Electrochemical detection of CHO For CHO detection, MIP-AuNPs-MWNTs/GCE was immersed in different concentrations of 5 mL samples CHO solution at room temperature in an open chamber (from 1×10−14 to 1×10−8 mol L−1). When CHO adhered to the modified electrode it showed higher charge-transfer impedance and this increase represented the combined effects of a reduction in the DPV current peak value, as shown in Fig. 3(A). This showed that when CHO was rebound, a compact film appeared on the surface of the electrode which hindered electron transfer from the [Fe(CN)6]3−/4− ion pair to the electrode surface. The formed MIP-AuNPs-MWNTs complex membrane resulted in a decrease in the electrochemical reaction of the [Fe(CN)6]3−/4− probe. The function of MWNTs in the complex membrane was analyzed as shown in Fig. 3(A). Compared with the MIP-AuNPs-MWNTs membrane, the extent of current fluctuation of MIP-AuNPs influenced by the concentration of CHO was much weaker, which indicated that the MWNTs played a significance role in the MIP sensor. As shown in Fig. 3(B), the MIP sensor equipped with MWNTs had a large-scale, sensitive, and credible detection range, and the limit of detection (LOD) was much better than that of the MIP sensor equipped without MWNTs (the MIP and NIP sensors’ response to the incubation concentration of CHO are shown in Fig. S5). Linear relationships existed between the peak current and the log of CHO concentration from 1×10 −13 to 1×10−9 mol L−1 (Ip = − 5.033 logC + 16.885 (R2 = 0.995)). The LOD of the CHO MIP sensor was 3.3×10−14 mol L−1, which were more sensitive than most available CHO
MIP sensor detection methods (Table 1). 3.4 Selectivity, reproducibility and stability of the MIP sensor An excellent sensor not only possesses good sensitivity, but also has good selectivity. To determine the selectivity of the molecularly imprinted sensor four compounds were investigated: cholic acid, deoxycholic acid, ascorbic acid, uric acid and 7-dehydrocholesterol as the control. Fig. S6 shows different current response signals for the proposed sensing system after the addition of 1×10−9 mol L-1 cholic acid, deoxycholic acid,ascorbic acid, uric acid and 7-dehydrocholesterol under the same experimental conditions. To evaluate the reproducibility and repeatability of the MIP sensor, experiments were carried out in a 1×10-10 mol L-1 CHO solution. The MIP sensor was regenerated with a peak current relative standard deviation (RSD) of 3.54 % using four different GCE electrodes. Excellent reproducibility was obtained with a relative standard deviation of 2.72 % after 20 washes and measurements, therefore the MIP sensor had good reversibility. Moreover, the stability of the sensor was evaluated,as stability is a key factor for CHO detection. The MIP sensor was stored in an ethanol:water (4:1) solution containing 0.5 mol L−1 HCl at room temperature for 10 days, and the peak current did not significantly change. After one month, it decreased to 91.68 %. All measurements indicated that the MIP sensor possessed excellent stability. 4. Conclusions In this work, a MIP film electrochemical sensor was used to indirectly detect CHO. It was constructed and developed by co-polymerization of p-ATP and HAuCl4 in the presence of template CHO molecules using CV. CHO molecules were absorbed by hydrogen bonding with p-ATP onto the surface of the AuNPs-MWNTs-modified GCE, which greatly increased the amount of imprinted sites. The doped nanoparticles enhanced the sensitivity of the MIP sensor, especially for MWNTs, whose irreplaceable function was discussed in the paper. The lowest detectable concentration of CHO was 3.3×10−14 mol L−1 and the linear detection range extended to 1×10−9 mol L−1. Furthermore, the fabrication of MIP-AuNPs-MWNT-modified GCEs was simple and controllable. These results demonstrated that the electrochemical sensor
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Table: Table 1 Comparison with other published MIP methods for the determination of CHO NO.
Method
Linear range
LOD
Shelf life
(mol L-1)
(mol L-1)
(days)
Reference
1
2-MBI-GE
1×10−6 to 3×10−5
0.42×10−6
18
(Aghaei et al. 2010)
2
GC/PB/CS-SiO2-COX-MWCNTs
4×10−6 to7×10−4
1×10−6
50
(Tan et al. 2005)
3
PMBI-GE
up to 2×10−5
0.7×10−6
