Accepted Manuscript Title: A strategy for improving the sensitivity of molecularly imprinted electrochemical sensors based on catalytic copper deposition Author: Jianping Li Yijuan Shao Weiling Yin Yun Zhang PII: DOI: Reference:

S0003-2670(14)00185-8 http://dx.doi.org/doi:10.1016/j.aca.2014.02.003 ACA 233095

To appear in:

Analytica Chimica Acta

Received date: Revised date: Accepted date:

6-11-2013 28-1-2014 1-2-2014

Please cite this article as: Jianping LiYijuan ShaoWeiling YinYun Zhang A strategy for improving the sensitivity of molecularly imprinted electrochemical sensors based on catalytic copper deposition (2014), http://dx.doi.org/10.1016/j.aca.2014.02.003 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 proof before it is published in its final 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.

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A strategy for improving the sensitivity of molecularly

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imprinted electrochemical sensors based on catalytic copper

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deposition

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Jianping Li∗, Yijuan Shao, Weiling Yin, Yun Zhang

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(College of Chemistry and Bioengineering, Guilin University of Technology, Guilin

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541004)

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∗

Corresponding author. Tel.:+86 773 2903121. E-mail address: [email protected] (J. P. Li)

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1 Abstract: A novel method to improve the sensitivity of molecularly imprinted polymer sensors

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was developed. Oxytetracycline (OTC), which was selected as the template molecule, was first

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rebound to the imprinted cavities. Gold nanoparticles were then labeled with the amino groups of

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OTC molecules via electrostatic adsorption and non-covalent interactions. Copper ions were

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catalytically reduced by the gold nanoparticles, and copper was deposited onto the electrode. The

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deposited copper was electrochemically dissolved, and its oxidative currents were recorded by

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differential pulse voltammetry (DPV). OTC could be determined indirectly within the

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concentration range of 3.0 × 10-10–1.5 × 10-7 mol/L with a detection limit of 6.8 × 10-11 mol/L.

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Key words: molecularly imprinted polymer sensor, catalytic copper deposition, gold nanoparticles,

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oxytetracycline, differential pulse voltammetry

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1. Introduction Given the high selectivity of many molecular imprinting [1,2] and electrochemical detection

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[3] techniques, increased attention has been paid to the design and application of molecularly

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imprinted electrochemical sensors (MIECSs) [4–6]. MIECSs have been extensively studied and

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developed in many fields [7–9]. However, improvement of the detection sensitivity of these

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sensors remains an important undertaking to meet requirements for trace analysis.

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Numerous methods for increasing the detection sensitivity of MIECS, such as

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enzyme-catalyzed amplification [10–12] and doping of molecularly imprinted polymer (MIP)

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membranes with functionalized nano-materials [13–15], have been studied. However, enzyme

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utilization requires a specific temperature and pH [16], and heterogeneous doped mixtures lead to

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MIP membranes with poor mechanical properties that affect the elution operation and reduce

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sensor life [12]. Gold nanoparticles can act as catalysts for metal ion deposition on nanoparticle

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surfaces [17–19]; thus, they have been used to improve the detection sensitivity of biosensors

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utilizing electrochemical methods because of their unique physical and chemical properties. To the

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best of our knowledge, gold nanoparticles have yet to be applied to improve MIP sensor sensitivity

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by metal deposition.

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In the current paper, an MIECS is achieved by exploiting the amplification effects of gold nanoparticles catalyzing

copper deposition. Oxytetracycline

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(OTC),

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broad-spectrum

antimicrobial drug, was selected as the template molecule. OTC determination is important because drug residues in the food chain may endanger human health [20]. Methods for analyzing ultra-trace amounts of OTC are necessary. High-performance liquid chromatography (HPLC) is mainly employed for OTC residue detection; however, this method is complex and expensive [21, 22]. In the present study, a highly sensitive method for trace OTC detection using the MIECS is

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proposed. After the rebinding reaction, gold nanoparticles are labeled onto OTC molecules

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re-adsorbed in the polymer cavities by the electrostatic attraction between gold nanoparticles and

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amino-group on OTC [23], and copper is deposited onto the electrode by the catalysis of gold

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nanoparticles in a copper deposition solution. The derived copper is electrochemically dissolved,

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and its oxidative currents are recorded using differential pulse voltammetry (DPV). The sensitivity

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of the sensor is significantly increased because of the catalytic copper deposition. A schematic of

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the synthesis of the MIECS is shown in Fig. 1.

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Fig. 1

2. Experimental

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2.1. Apparatus and materials

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Cyclic voltammetry (CV) was performed on a CHI660D electrochemical workstation (Shanghai

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Chenhua Instruments, Shanghai, China) containing a standard three-electrode cell. An

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MIP-modified glassy carbon electrode (GCE, d = 2 mm) was used as the working electrode, a

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platinum wire electrode was used as the auxiliary electrode, and a Ag/AgCl electrode was used as

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the reference electrode. AC impedance spectroscopy was performed on an Autolab 128N

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(Metrohm Company, Switzerland). An S-4800 field emission scanning electron microscope

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(Oxford Company, England) was used to determine salient features of the nanoparticles and

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electrode surface. All measurements were performed at 25 °C.

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OTC was purchased from ACROS, USA. Methylene blue (MB) trihydrate, ascorbic acid,

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copper sulfate, and potassium ferricyanide were purchased from Sinopharm Group Chemical

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Reagent Co., Ltd., China. All chemical reagents applied in this work were of analytical grade and

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used without further purification. The glassware was cleaned and all chemical reagents were

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some improvements. One milliliter of 0.01% HAuCl4 and 100 mL of double distilled water were

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added to a 250 mL round-bottomed flask, and the solution was heated to boiling with vigorous

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stirring. Afterward, 3.5 mL of 1% trisodium citrate was quickly added to the boiling solution. The

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color of the solution turned blue within a few seconds and finally changed to wine-red 1 min later.

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The solution was allowed to boil and stirred for another 10 min before cooling to room

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prepared using double distilled water. A copper-deposition solution containing 0.05 mol/L

ascorbic acid and 0.05 mol/L CuSO4·5H2O was freshly prepared before each use.

2.2. Gold nanoparticle preparation Gold nanoparticles were prepared based on the method described in the literature [24] with

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temperature with stirring to obtain gold nanoparticles.

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2.3. MIP and nMIP preparation Both MIP and nMIP were electropolymerized on the GCE surface. The GCE was polished

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with a piece of chamois leather using 1.0, 0.3, and 0.05 μm alumina in turn before successive

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washing with double distilled water, 50% HNO3, and alcohol. CV was performed for 30 cycles at

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a rate of 50 mV/s in the potential range of -0.6–1.4 V in phosphate buffer solution (PBS, pH = 7.2)

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containing 5×10-4 mol/L OTC and 1.5×10-3 mol/L MB. Cavity–template molecular compounds

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were synthesized by binding OTC with sites on the cavities. The MIECS was then washed with

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deionized water and 50% ethanol for 15 min to remove template molecules and other adsorbents,

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yielding an MIP membrane with imprinting cavities for OTC. An nMIP electrode was produced

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under the same conditions but without the addition of OTC.

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2.4. Experimental procedure

After elution, the electrode was dipped into 10 mL of sample solution containing

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3.0×10−10–1.5×10-7 mol/L OTC for 15 min to rebind the imprinted cavities. Afterward, 10 μL of

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Au colloid was dropped onto the electrode surface, which was then placed at room temperature for

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20 min to form gold nanoparticle-labeled OTC (AuNP-OTC). One hundred and fifty microliters of

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the copper deposition solution was added to the surface of the electrode at room temperature for

15 min in a dark environment to deposit the copper onto the electrode surface. The OTC concentration can be obtained by determining the change in the oxidative current of copper. After each measurement, the renewal of the sensor was carried out by two steps. Firstly, the

electrode was set at 0.4 V in a stirring solution of 0.1 mol/L HNO3 for 30s to dissolve the copper residues on the electrode surface; then, the elution was performed in 50% ethanol for 15 min to

obtained the renewed sensor again.

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2.5. Electroanalytical measurements

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2.5.1. Electrochemical characterization

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CV was employed to study the characteristics of the MIP membrane using 0.01 mol/L

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K3[Fe(CN)6] as a supporting electrolyte over the potential range of -0.2–0.6 V. The scanning rate

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used was 100 mV/s. AC impedance spectroscopy was performed in 0.01 mol/L K3[Fe(CN)6] at a

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potential of 0.168 V, alternating voltage of 5 mV, and frequency range from 100 mHz to 100 kHz.

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All measurements were performed at room temperature.

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Electrochemical measurement of copper was performed in 10 mL of 0.1 mol/L HNO3 using

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DPV from -0.5 V to 0.5 V with a scanning rate of 50 mV. The oxidation peak current of copper

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was recorded at 0.08 V.

3. Results and discussion

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3.1. MB electropolymerization

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MB is an electroactive compound that has been observed to have the ability to form polymers

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under a sufficiently positive potential [25–27]. Therefore, MB was selected as a functional

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monomer to synthesize poly(MB). The electropolymerization of MB on the GCE is an irreversible

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process. As the number of cycles increased, the irreversible oxidation peak at 1.2 V continuously

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decreased (Fig. 2). When the number of cycles was increased to 30, the peak currents became very

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peak of copper, which obviously showed a good relationship with the OTC concentrations for

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quantitative analysis, was obtained at a potential of 0.08 V. Figure 3 shows the oxidative currents

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of deposited copper obtained with (curve “c”) and without (curve “b”) AuNP-OTC. In the

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presence of OTC, the oxidation peak at 0.08 V was much higher than that obtained without OTC.

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This difference demonstrates that gold nanoparticles act as catalysts during copper deposition. The

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small, which indicates that a low conductive MIP film had formed on the electrode surface. Fig. 2

3.2. Voltammetric response of copper The dissolving of deposited copper was measured by DPV in 0.1 mol/L HNO3. The oxidation

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non-specific adsorption of gold NPs on nMIP is demonstrated in curve “a,” which shows no peak

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at all. These results indicate that gold nanoparticles are indispensable in the copper deposition

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process.

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3.3. Characterization of MIP formation on the electrode

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3.3.1. Characterization by CV and AC impedance measurements

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OTC showed no oxidation or reduction peaks in the selected potential range; thus,

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K3[Fe(CN)6] was used as an electron probe between the MIP-modified electrode and the solution

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to determine the binding of OTC to MIP. The cavities can be used as electron transfer channels,

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which may be obstructed during molecule recognition and bring about a change in the probe ion

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currents. Figure 4A shows the current changes of the MIP-modified GCE during the binding and

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elution of OTC template molecules. The decrease in oxidation-reduction peak current of the

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MIP-GCE from curve “a” to curve “b” illustrates the production of a film covering the GCE

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surface. When OTC was removed (curve “c”), a peak current may be seen. Curve “c” to curve “d”

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shows a peak current reduction because of the rebinding of OTC to the cavities in the MIP film.

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Since less probes could access to the surface of the electrode through the cavities in MIP, the peak

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current decreased.

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AC impedance changes in the MIP electrode are shown in Fig. 4B The change trends

observed are similar to changes in the CVs, which demonstrates the ability of the MIP membrane

to recognize template molecules.

In contrast, the redox peak currents of K3[Fe(CN)6] decreased dramatically from “a” to “b” on

the nMIP electrode (Fig. 4C), while, curve “b” to curve “c” shows a negligible change after

elution because there were no specific cavities in the nMIP film polymerized on the electrode in the absence of template OTC. Fig. 4

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3.3.2. Characterization by SEM and energy-dispersive X-ray spectroscopy (EDS)

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SEM was performed to describe the electrode surface before and after copper deposition.

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Figure 5A shows the AuNPs prior to copper deposition, and Fig. 5B shows the AuNPs after copper

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deposition. The AuNPs volume increased significantly after copper deposition, which indicates

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that copper is indeed deposited onto the AuNPs surface. Fig. 5

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EDS was performed to check the deposited copper particles by using an indium tin oxide (ITO)

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electrode in the preparing of the MIP. Figure 6A shows the EDS spectrum of the electrode surface

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before copper deposition, and Fig. 6B shows the EDS spectrum of the electrode surface after copper deposition. Copper deposition on the ITO electrode is evident.

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3.4. Optimization of the rebinding time, catalytic deposition time, and supporting

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electrolyte for electrochemical measurement

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Rebinding was performed in 10 mL of 1.5 × 10-7 mol/L OTC. CV was performed to check the

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currents change and the voltammograms are shown as Fig. 7A. Peak currents gradually increased

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for up to 15 min and then remained stable with further increases in rebinding time, probably

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because the rebinding for the molecule to the cavity achieved a dynamic balance when the time

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came to 15 min. Thus, 15 min was selected as an appropriate rebinding time.

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The impact of the copper deposition time on the peak current was studied by dipping the

electrode into 1×10-7 mol/L OTC solution for 15 min, labeling with gold nanoparticles by electrostatic forces for 20 min, and then immersing in the copper deposition solution. Copper was deposited on the AuNPs surface to form a core-shell structure. The OTC concentration may be

obtained by measurement of the stripping current of deposited copper. The oxidation peak slowly

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increased as the copper deposition time increased, which can be explained by the increasing

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amount of copper deposited onto the gold nanoparticle surface with increasing time. However,

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long deposition times may also give rise to unstable peak currents and increasing background

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signals. The proposed method shows an optimal signal-to-noise ratio when the reaction time is 15

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min (Fig. 7B).

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Fig. 7

2 Experiments were performed in substrates of 0.01 mol/L H2SO4, HCl, and HNO3, and the

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results illustrate that the well-defined and high copper oxidation peak may be observed in 0.01

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mol/L HNO3. HNO3 concentrations between 0.01 and 0.5 mol/L were then selected for the

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detections. Considering the peak heights and repeatability of results, 0.1 mol/L HNO3 was finally

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selected as a supporting electrolyte.

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3.5. Calibration Curve

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Figure 8 shows the relationship between the oxidation peaks obtained and the OTC

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concentration. The currents increased with increasing OTC concentration because of the

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increasing number of imprinting cavities on the MIP membrane occupied by OTC molecules. A

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standard curve of the OTC concentration was established according to the oxidation peaks (Inset

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Fig. 8). The linear relationship of the oxidation peak (∆I) and OTC concentration (c) is represented

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by the equation (1) with a linear correlation of 0.9987 for OTC concentrations ranging from

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3.0×10−10 mol/L to 1.5×10-7 mol/L.

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ΔI (10-6 A) = 0.0143×c (10-10 mol/L)

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The detection limit of the sensor was 6.8×10−11 mol/L, as calculated according to the equation

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(1)

(2):

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(2) Fig. 8

Table 1 showed a comparison of analytical parameters such as the determination ranges and

detection limits of the proposed molecularly imprinted sensor with other sensors or even methods reported for OTC determination. The sensor clearly exhibited a low detection limit, indicating that it is one of the most sensitive method compared with most reported methods. Table 1

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3.6. Reproducibility, stability, and selectivity of the MIP electrode

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The reproducibility of the MIECS was studied by measuring a solution of 5.0×10-8 mol/L

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OTC seven times using one sensor. The relative standard deviation (RSD) obtained was 3.8%,

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which indicates good reproducibility. The sensor-to-sensor reproducibility was tested by

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determining responses to 5.0×10-8 mol/L OTC using seven different sensors. The RSD obtained

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was 3.2%, which again proves that the sensor exhibits good reproducibility.

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Stability is a very significant indicator that describes sensor quality. Sensor stability was

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evaluated by determining 5.0×10-8 mol/L OTC. No significant difference in concentrations

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determined was found even after 2 wk. After 3 wk, the peak current decreased by approximately

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12%. One month later, the peak current decreased by approximately 19%.

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The influence of possible interfering substances present in milk samples on the detection of

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OTC was studied. The allowable error was defined as the maximum concentration of different

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interfering substances that causes a relative error of 5% in the analytical signal. Results revealed

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that 1000-fold molar excesses of various substances, such as Ca2+, Mg2+, and Zn2+, VC, VB1, VB2,

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and VB3 in 6.0×10-8 mol/L OTC show no interference effects on the OTC signal. Lactose, glucose,

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and urea showed no significant effects even at 5000-fold molar excess concentrations. However,

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doxycycline, chlortetracycline, and tetracycline influenced the OTC signal at 230-fold, 350-fold,

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400-fold, and 380-fold molar excess concentrations, respectively. These results confirm that the

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sensor has high selectivity.

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3.7. Sample testing

Milk samples purchased from a supermarket were subjected to OTC determination using the

developed sensor. Sample pretreatment was performed according to the reported procedure [28]. Protein in samples was precipitated using McIlvaine buffer solution before assay. Water sample was taken from Lijiang River in Guilin, and it was treated by a microporous membrane. The

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MIECS was dipped into the sample for 15 min to operate rebinding. As no OTC was found in the

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samples, standard addition method was performed by which OTC solutions of certain

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concentrations were added into the samples. The recoveries obtained ranged from 98.6% to

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103.0% (Table 2), which agreed with that obtained by chromatographic method. The RSDs

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obtained were less that 3.0%, which indicates the stability and practical utility of our developed

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sensor.

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Table 2

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4. Conclusions

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In this paper, an MIP electrochemical sensor was developed based on the signal amplification

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of catalytically deposited copper and applied in the detection of OTC. A low detection limit for

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OTC was determined. Good selectivity was obtained because of the reorganization of molecularly

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imprinted cavities. The sensor exhibited long-term stability, enabled convenient preparation, and

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showed good analytical performance.

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Acknowledgements

The authors gratefully acknowledge the financial support of the National Natural Science

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Foundation of China (No. 21375031 and No. 21165007).

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Figures

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Fig. 1. Schematic of the proposed procedure for constructing a molecularly imprinted polymer

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sensor and determining OTC.

5 Fig. 2. Cyclic voltammograms of the electropolymerization of methylene blue at the GCE. OTC:

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5×10-4 mol/L; MB: 1.5×10-3 mol/L.

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Fig. 3. DPV-stripping curves of the molecularly imprinted polymer (MIP): (a) DPV-stripping curve on nMIP; (b) DPV-stripping curve on MIP without OTC; (c) DPV-stripping curve on MIP.

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Fig. 4. (A) CVs on MIP , (B) AC impedances of the molecularly imprinted polymer (MIP)

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electrode: (a) Bare GCE electrode; (b) MIP electrode; (c) MIP electrode after OTC removal; (d)

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MIP electrode after OTC rebinding and (C) CVs on nMIP.

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Fig. 5. SEM images of AuNPs (A) before and (B) after copper deposition.

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Fig. 6. EDS images of the ITO electrode (A) before and (B) after copper deposition. Fig. 7. Effects of time of (A) rebinding and (B) catalytically deposited copper on response signals:

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(A) 1.5 × 10-7 mol/L OTC; (B) 1 × 10-7 mol/L OTC: (a) the oxidation current of copper deposition

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catalyzed by Au nanoparticles; (b) the oxidation current of copper deposition without Au

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nanoparticles (background); (c) the difference between a and b.

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Ac ce p

19

Fig. 8. DPV responses after rebinding of the molecularly imprinted polymer electrode with different OTC concentration: a-0, b-3×10-10, c-6×10-10, d-9×10-10, e-1.2×10-9, f-1.5×10-9, g-3.0×10-9, h-5.0×10-9, i-7.0×10-9, j-9.0×10-9, k-1.2×10-8, l-1.8×10-8, m-2.4×10-8, n-3.0×10-8, o-4.0×10-8, p-5.0×10-8, q-7.0×10-8, r-9.0×10-8, s-1.1×10-7, t-1.3×10-7, u-1.5×10-7 mol/L OTC,

respectively.

14

Page 14 of 25

1 2 3

ip t

4

AuNP labeling

OTC

AuNP

AuNP deposited with cupper

5

M

6 7

Fig. 1

Ac ce p

te

d

8 9

us

an

Cupper deposition

cr

Binding

15

Page 15 of 25

1 2

ip t

3

50

cr

-50

us

I / µA

0

-150

-0.5

an

-100

0.0

0.5

E/V

4

Fig. 2

Ac ce p

te

d

6 7

1.5

M

5

1.0

16

Page 16 of 25

1 2

0

ip t

3

a b c

-5

cr

-15 -20

us

I / µA

-10

-25

-0.4

-0.2

0.0

0.2

0.4

0.6

E/V

4

M

5

Fig. 3

Ac ce p

te

d

6 7

an

-30

17

Page 17 of 25

1000

a c

I / µA

500

b

0

ip t

d -500

-0.2

0.0

0.2

0.4

0.6

2

us

E/V

1 A

b

1500

d

500

M

300

1000 Z'' / ohm

Z'' / ohm

an

2000

200

c

100

0

1000

2000

te

0

d

0 0

Ac ce p

3 4

cr

-1000

a 100

300

3000

4000

5000

6000

Z' / ohm

B 15

a

10

I/μA

1000

c

5 0

b

-5

500

I/μA

200

Z' / ohm

-10

-0.2

0.0

b

0

0.2

0.4

0.6

E/V

c

-500

-1000 -0.2

5

0.0

0.2

0.4

0.6

E/V

6

C

7

Fig. 4

8 18

Page 18 of 25

1 2

M

an

us

cr

ip t

3

4

A

Ac ce p

te

d

5

6 7

B

8

Fig. 5

9 10 19

Page 19 of 25

1 2

4

A

6 7 8 9

Ac ce p

te

d

M

5

an

us

cr

ip t

3

B Fig. 6

20

Page 20 of 25

1 2

ip t

200 160

cr

80

us

I / μA

120

40 0 6

9

12

15

18

an

3

t / min

3

A

M

4

d

5

te

200

I / μA

Ac ce p

150

a

c

100

b

50

0

6

21

0

5

10

15

20

t / min

7

B

8

Fig. 7

9 10

21

Page 21 of 25

1 2 3

0

ip t

a

cr

u

-100

us

200 150

-150

ΔI / μA

I / µA

-50

100

0

0.0

0.2

E/V

0

500

1000

1500

-10

C / 10 mol/L

0.4

0.6

M

-0.2 4

Fig. 8

Ac ce p

te

d

5 6

an

50

-200

22

Page 22 of 25

1 2 3

Table.1 Analytical parameters for OTC in different methods Method

Determination range (mol/L)

Detection limit (mol/L)

Molecularly imprinted 5.0 × 10-9 ~ 1.0 × 10-6 mol/L

2.3 × 10−11 mol/L

Renewable molecularly imprinted sensor

[30]

1.8 × 10−9mol/L.

[31]

-

[32]

2×10-5~1×10-2 mol/L

Potentiometric sensor

4.0 × 10-7~5.0 × 10-2 mol/L

1.0 × 10-7 mol/L

[33]

1× 10-7~ 1× 10-5mol/L

1.0× 10-8 mol/L

[34]

2× 10-7~ 4× 10-6mol/L

4.03× 10-9 mol/L

[21]

2× 10-7~ 7× 10-6mol/L

2.0× 10-8 mol/L

[22]

6.8 × 10-11 mol/L

-

Ac ce p

HPLC

d

HPLC

te

quantum dots as probe

M

Potentiometric sensor

Fluorescence with CdS

This sensor

5

8.0 × 10-9 ~ 6.0 × 10-7 mol/L

2.7 × 10−8 mol/L

an

sensor

3.0 × 10-8 ~ 8.0 × 10-5 mol/L

us

amplification Molecularly imprinted

[29]

cr

sensor with enzyme

Ref.

ip t

4

3.0 × 10-10 ~ 1.5 × 10-7 mol/L

23

Page 23 of 25

1 2

Table 2 Sample analysis results

Water

Total found

RSD

10-8 mol/L

10-8 mol/L

%

3

3.05

2.31

6

6.13

9

8.87

4

4.12

7

7.08

--

--

5

7

14

102.2

1.93

98.6

3.24

103.0

1.97

101.1

te Ac ce p

10

13

2.19

Highlights

9

12

101.7

d

8

11

%

M

6

Recovery

ip t

Added

cr

Milk

Found

us

Sample

an

3 4

● A strategy for improving sensitivity of molecular imprinted electrochemical sensor was proposed.

● Gold nanoparticles were labeled on template molecules for catalytic reduction of copper ions.

15

● The deposited copper was electrochemically oxidized by DPV method, and the

16

stripping current was measured.

17

● The sensitivity was increased significantly due to the large amount of copper.

18 19 24

Page 24 of 25

Graphical Abstract

1 2

ip t

3

AuNP labeling

cr

Binding

OTC

AuNP

AuNP deposited with cupper

an

4

us

Cupper deposition

5

Gold nanoparticals were labeled on the OTC molecules re-adsorbed in cavities, and

7

then copper was deposited on the electrode by the catalysis of gold nanoparticles in

8

copper deposition solution. The derived copper was electrochemically dissolved and

9

the oxidative currents were recorded by DPV method.

d

te

Ac ce p

10

M

6

25

Page 25 of 25