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Conductive imprinted electrochemical sensor for epinephrine sensitive detection and double recognition
Accepted Manuscript Conductive imprinted electrochemical sensor for epinephrine sensitive detection and double recognition
Fen Liu, Xianwen Kan PII: DOI: Reference:
S1572-6657(19)30065-7 https://doi.org/10.1016/j.jelechem.2019.01.050 JEAC 12880
To appear in:
Journal of Electroanalytical Chemistry
Received date: Revised date: Accepted date:
16 October 2018 14 January 2019 21 January 2019
Please cite this article as: F. Liu and X. Kan, Conductive imprinted electrochemical sensor for epinephrine sensitive detection and double recognition, Journal of Electroanalytical Chemistry, https://doi.org/10.1016/j.jelechem.2019.01.050
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ACCEPTED MANUSCRIPT Conductive imprinted electrochemical sensor for epinephrine sensitive detection and double recognition
Fen Liu, Xianwen Kan*
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College of Chemistry and Materials Science, Anhui Normal University, Wuhu
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241000, P.R. China; The Key Laboratory of Functional Molecular Solids, Ministry
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of Education; Anhui Laboratory of Molecule-Based Materials, Anhui Key
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Laboratory of Chemo-Biosensing.
*Corresponding author: Xianwen Kan
E-mail: [email protected]; Tel: +86-553-3937135; Fax: +86-553-3869303.
ACCEPTED MANUSCRIPT Abstract Epinephrine (EP), an important derivative of a neurotransmitter in the mammalian central nervous system and monitoring the concentration of EP is significant in biological and chemical researches. In this work, a molecularly imprinted polymer (MIP)/gold nanoparticles (AuNPs) composite (MIP/AuNPs) was
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modified on glassy carbon electrode (GCE) surface to fabricate a novel
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electrochemical sensor for EP detection. The modified AuNPs increased the surface
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area of the electrode and improved the sensitivity of the sensor. 3-thiophene boronic acid (3-TBA), as a monomer of conductive polymer, was employed to
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electropolymerize MIP in the presence of EP. Scanning electron microscope, cyclic
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voltammetry, and electrochemical impedance spectroscopy characterization results demonstrated that the prepared sensor possessed sensitive detection and selective
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recognition abilities toward EP. Unlike the other MIP based electrochemical sensors,
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conductive poly(3-TBA) matrix, as well as the conductive AuNPs can enhance the sensitivity of the sensor. Under the optimal conditions, the sensor can sensitively
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detect EP with a linear range and limit of detection of 9.0 × 10-8 ～ 1.0 × 10-4 mol/L
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and 7.6 × 10-8 mol/L, respectively. And the sensor showed double recognition ability to EP due to the following reasons: first, size and shape complementarity between the formed imprinted sites and the template molecules; second, the reversible covalent interaction between boronic acid of 3-TBA and cis-diol of EP. Thus, the sensor can recognize EP from its analogues. And the sensor has been applied to analyze EP in injection samples with satisfactory results.
ACCEPTED MANUSCRIPT Keywords: gold nanoparticles, conductive molecularly imprinted polymer, modified
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electrode, 3-thiophene boronic acid, epinephrine
ACCEPTED MANUSCRIPT 1. Introduction Molecular imprinting is a synthesis technique for artificial material preparation with specific recognition ability toward template molecule [1-3]. Prepared molecularly imprinted polymer (MIP) has lots of imprinted cavities with complementary sizes,
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shapes, and functional groups with template molecules, which make it can recognize
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template molecule from its analogues [4]. Thus, MIP has been applied in many fields [8, 9],
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including solid phase extraction [5-7], similar enzyme catalysis
chromatographic separation [10-15], and chemical sensing [16-21]. Due to the
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advantages of simplicity, sensitivity, and stability, electrochemical method has been
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combined with MIP for template molecule sensitive and selective detection [22-24]. In recent years, nanomaterials, such as nobel metal nanoparticles based nanocomposites
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(gold nano urchins/graphene oxide [25], platinum nanoparticles/carbon nitride
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nanotubes [26], silver nanoparticles/polyoxometalate functionalized reduced graphene oxide [27]) and quantum dots based nanocomposites (boron nitride quantum dots [28], nitride nanotubes/graphene quantum
dots
[29],
graphene quantum
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carbon
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dots/two-dimensional hexagonal boron nitride nanosheets [30]), have been modified on electrode for imprinted electrochemical sensors preparation to improve the sensitivity of the sensors. Besides the single template recognition and detection, dual templates have been involved into the imprinted polymer synthesis, in which dual templates can been simultaneously selectively detected [31]. Computational approach has also been used to select adequate functional monomers for imprinted electrochemical sensor preparation [32]. The results of these work demonstrated that
ACCEPTED MANUSCRIPT the MIP based electrochemical sensors have endowed the selectivity of the sensors besides the sensitivity for template molecules detection. Most MIP based electrochemical sensors were prepared by electropolymerizing functional monomer in the presence of template molecule. And the most interaction
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between functional monomer and template is non-covalent bond due to the flexible
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concerning the extraction and rebinding of template molecule. Compared with
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non-covalent interaction, covalent imprinting is superior in preventing leakage of template molecules during polymerization process because of the formation of stable
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covalent bonds between the template and monomer [33-35]. Thus, the precise
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imprinting sites facilitate the recognition ability of the prepared MIP [36-38]. Reversible covalent interaction has been employed for MIP preparation, which
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results in a rather homogenous population of binding sites with minimizing the
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existence of non-specific sites. However, the covalent interaction based MIP has been restricted since it’s difficult to design an appropriate template-monomer reversible
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covalent interaction under mild condition. It is worth mentioning that boronic acid
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derivatives exhibit high affinity to cis-diol containing compounds (CDCCs) to form cyclic esters in mild basic or neutral aqueous solution, while the cyclic esters dissociate once switching the medium to acidic solution [39, 40]. Thus, boronic acid based MIP has been investigated, which also has been applied for CDCCs electrochemical sensing [41-43]. And if a monomer possesses two functions of affinity and imprinted sites toward template molecules, it should simplify the procedure of MIP preparation process.
ACCEPTED MANUSCRIPT Thiophene, as a monomer of conductive polymer, has received a significant amount of attention in many fields, such as supercapacitor materials [44-46] and electrochemical sensing interface [47]. Also, thiophene has been used as functional monomer for MIP preparation [48]. To restrict the non-specific adsorption of MIP, the
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conductivity of MIP based electrochemical sensors is usually unsatisfying, which
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limits its sensitivity for template detection in turn. Thus, thiophene is seldom used as
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functional monomer for MIP based electrochemical sensor preparation due to the conductivity of poly(thiopnene) [49], which would provide a sensing interface for
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template electrochemical redox. As the consequences, the non-specific adsorption can
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not be well restrained and the selectivity of the sensor should be affected. It’s well-known that boronic acid formed cyclic esters with cis-diol containing
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compounds through covalent interaction in neutral or basic solution. Epinephrine (EP),
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a cis-diol compound, is an important derivative of a neurotransmitter in the mammalian central nervous system, which can lead to glycogenolysis in the liver and
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skeletal muscle, the mobilization of free fatty acid, the increase of plasma lactate and the
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improved force and rate of heart contraction [50, 51]. Numerous diseases are related to the concentration of EP, such as Parkinson’s disease, Alzheimer’s disease, hypertension and multiple sclerosis [52-54]. Thus, monitoring the concentration of EP is significant in biological and chemical researches. Poly(thiophene) matrix based MIP can form imprinted sites with complementary size and shape with EP [55, 56]. To achieve the sensitivity and selectivity, 3-thiophene boronic acid (3-TBA) was chosen as a novel functional monomer for MIP based electrochemical sensor
ACCEPTED MANUSCRIPT preparation in this work. Thus, the prepared electrochemical sensor can selectively detect EP due to the dual recognition ability. Besides, the modified gold nanoparticles (AuNPs) and the conductive poly(3-TBA) matrix would improve the sensitivity of the
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sensor.
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2. Experimental
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2.1 Chemicals
3-Thiophene boronic acid (3-TBA), thiophene (Th), and epinephrine (EP) were
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purchased from Aladdin Reagent Co. Ltd. Dopamine (DA), ascorbic acid (AA), and
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uric acid (UA) were ordered from Sigma-Aldrich Reagent Co. Ltd. All other reagents were of analytical grade and used without further purification. All aqueous solutions
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2.2 Apparatus
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purification system.
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were prepared with ultrapure water (18.25 MΩ cm) from a Millipore water
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Electrochemical experiments, such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS), were performed on CHI 660C workstation (ChenHua Instruments Co., Shanghai, China) with a conventional three-electrode system. A bare or modified glassy carbon electrode (GCE) was served as a working electrode. A saturated calomel electrode and a platinum wire electrode were used as a reference electrode and a counter-electrode, respectively. Field emission scanning electron microscope
ACCEPTED MANUSCRIPT (FE-SEM) images were obtained on an S-4800 field emission scanning electron microanalyzer (Hitachi, Japan).
2.3 Fabrication of gold nanoparticles modified GCE (AuNPs/GCE)
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Prior to fabricating gold nanoparticles (AuNPs) modified electrode, a bare GCE
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was successively polished with 1.0, 0.3, and 0.05 μm α-Al2O3 powder. Then the clean
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electrode was immersed into 4 mmol/L HAuCl4 and treated by the use of a constant
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potential of − 0.2 V for 500 s, obtaining the AuNPs modified GCE (AuNPs/GCE).
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2.4 Fabrication of MIP modified AuNPs/GCE (MIP/AuNPs/GCE) The clean AuNPs/GCE was immersed into 5.0 mL phosphate buffer solution (PBS,
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0.1 mol/L, pH 6.0) containing 400 μL 0.05 mol/L 3-TBA and 100 μL 0.05 mol/L EP
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[57]. Then cyclic voltammetry (CV) was performed from - 0.2 V to + 1.2 V for 20 cycles at a scan rate of 50 mV/s, obtaining polymer film modified electrode.
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Subsequently, the embedded EP was extracted by immersing the modified electrode
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into 0.05 mol/L HCl aqueous solution and was electrochemically scanned between 0.0 V – 1.5 V for several cycles, getting MIP modified AuNPs/GCE (MIP/AuNPs/GCE). As a control, non-molecular imprinting polymer (NIP) modified AuNPs/GCE (NIP/AuNPs/GCE) was prepared in exactly the same way except for the omitting of EP in the electropolymerization process. And the MIP and NIP film prepared using thiophene (Th) as functional monomer was also modified on AuNPs/GCE in the same way, which were labeled as MIP(Th)/AuNPs/GCE and NIP(Th)/AuNPs/GCE,
ACCEPTED MANUSCRIPT respectively. The procedures for the construction of the MIP/AuNPs/GCE and MIP(Th)/AuNPs/GCE were illustrated in Scheme 1. Also, MIP/GCE and NIP/GCE were prepared by direct electropolymerization of MIP and NIP on GCE, respectively.
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2.5 Electrochemical properties measurements
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Electrochemical characterization of the prepared modified electrodes were
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carried out in 5.0 mmol/L [Fe(CN)6]3-/[Fe(CN)6]4- using CV and EIS methods. Electrochemical detection of EP was performed in 0.1 mol/L PBS (pH 7.0) by using
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CV and DPV approaches. AA, UA, and DA were selected as coexisted or structural
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similar compounds to evaluate the recognition capacity of the prepared sensor.
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2.6 Regeneration of MIP/AuNPs/GCE
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After the detection of 5.0 × 10-5 mol/L EP, MIP/AuNPs/GCE was immersed in 0.05 M HCl and electrochemically scanned between 0.0 V – 1.5 V for several cycles
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to remove adsorbed EP molecules. The eluted sensor was then immersed in the same
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concentration of EP for detection.
3. Results and discussion 3.1 Characterization of MIP/AuNPs/GCE The morphologies of AuNPs/GCE and MIP/AuNPs/GCE were characterized by SEM, as shown in Fig. 1A and B. It can be found that a layer of nanoparticles coated onto the electrode surface, which can improve the surface area of the electrode and
ACCEPTED MANUSCRIPT facilitate the electronic transfer. With the electropolymerization of MIP, an obvious layer of film can be observed on the surface of AuNPs, indicating the preparation of MIP/AuNPs/GCE. [Fe(CN)6]3-/[Fe(CN)6]4- is a commonly used probe for the characterization of
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MIP based electrochemical sensor preparation procee, which was also chosen to
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investigate the prepared process of MIP/AuNPs/GCE. The reaction between boronic
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acid and cis-diol containing compound can produce boronate ester [58], [59], which causes the conversion from sp2 to sp3 hybridization of boron atom [60]. Thus,
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the electrostatic repulsion between boronate moieties and [Fe(CN)6]3-/[Fe(CN)6]4-
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couple should block the arrival of [Fe(CN)6]3-/[Fe(CN)6]4- to sensor surface [61]. As shown in Fig. 2A, before the extraction of EP, a pair of weak redox peak of be
found
on
MIP/AuNPs/GCE
(Fig.2Aa)
or
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[Fe(CN)6]3-/[Fe(CN)6]4- can
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NIP/AuNPs/GCE (Fig. 2Ac), although the polymer matrix (poly(3-TBA) or poly(thiphene)) was conductive. Compared with NIP/AuNPs/GCE (Fig. 2Ad), a
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remarkably increased peak current of probe can be found on MIP/AuNPs/GCE (Fig.
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2Ab) after the removal of template molecules. It may implied that the cavities were left by the extraction of template molecules, forming channels for the probe to pass through the cavities. EIS is a valuable way for the characterization of the sensor preparation process. Fig. 2B shows the Nyquist diagrams of the modified electrode fabricated at each step in the presence of [Fe(CN)6]3-/[Fe(CN)6]4-. Compared with bare GCE (Fig. 2Ba), the slope of the line part of AuNPs/GCE (Fig. 2Bb) dramatically increased, indicating
ACCEPTED MANUSCRIPT that AuNPs had good electric conducting property and could enhance the electron transfer rate [62, 63]. Before the extraction, MIP/AuNPs/GCE (Fig. 2Bc) exhibited an obvious interfacial resistance because the polymer film decreased the electron transfer rate. A remarkable decrease of the interfacial resistance can be
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observed on MIP/AuNPs/GCE after the removal of template molecules (Fig. 2Bd),
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which implied that the formed imprinted sites enhanced the diffusion rate of
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[Fe(CN)6]3-/[Fe(CN)6]4- through the MIP film and made it easier for electronic transfer. With the addition of EP, the impedance increased (Fig. 2Be), which can be
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ascribed to the block of the channels for probe to reach the electrode surface by the
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adsorbed EP molecules. Thus, the impedance and CV changes of the modified process, as well as the SEM characterization demonstrated the successful preparation of the
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sensor.
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Fig. 3 exhibits the UV-vis spectra of Th (light blue), TBA (red), EP (green), Th-EP (blue), and TBA-EP (black) aqueous solution. A characteristic absorption peak
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at 290 nm for EP can be observed. When EP was mixed with Th, no obvious shift of
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adsorption peak can be found, indicating no interaction between EP and Th. Although TBA has no adsorption like Th, the adsorption peak of TBA-EP was red-shifted from 290 nm to 296 nm, compared with individual EP. The results implied the obvious interaction between TBA and EP [64].
3.2 The specific adsorption of the sensor
ACCEPTED MANUSCRIPT The specific adsorption is a natural property of MIP, which was investigated by immersing different modified electrodes in 5.0 × 10-5 mol/L EP solution, as shown in Fig. 4A. It’s different from other reported MIP based electrochemical sensors, a pair of redox peaks of EP can be observed when NIP/AuNPs/GCE was used as the
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working electrode (blue and red) due to the conductivity of poly(thiophene) matrix of
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NIP film. Thus, the non-specific adsorbed EP molecules can be redoxed on NIP/GCE
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and NIP/AuNPs/GCE. An increased current of EP was found on MIP/GCE (green), which can be attributed to the specific adsorption of EP into imprinted sites. Owing to
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the good conductivity of AuNPs, MIP/AuNPs/GCE (black) showed much higher
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current of EP than that of MIP/GCE. By contrast, the same experiments were performed on MIP(Th)/AuNPs/GCE and NIP(Th)/AuNPs/GCE. Although the
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NIP(Th)/AuNPs/GCE (rose red) and NIP/AuNPs/GCE (red) showed low peak
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currents of EP, MIP(Th)/AuNPs/GCE (light blue) exhibited a much lower current than that of MIP/AuNPs/GCE. The oxidation current of EP was chosen to calculate
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imprinted factor (IF, IF=ΔiMIP/ΔiNIP). As shown in Fig. 4B, MIP/AuNPs/GCE and
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MIP/GCE exhibit almost the same IF of 2.18 and 2.25, respectively. Compared with MIP/AuNPs/GCE, MIP(Th)/AuNPs/GCE possessed a lower IF of 1.66. These phenomena can be explained that the MIP prepared using 3-TBA as the functional monomer possessed double recognition capacity toward template molecule. Besides the removal of template molecules left imprinted sites with complementary sizes, shapes, and functional groups with template molecules, boronic acid formed cyclic
ACCEPTED MANUSCRIPT esters with EP through covalent interaction, which made the MIP/AuNPs/GCE show a higher specific adsorption capacity toward EP than that of MIP(Th)/AuNPs/GCE. 3.3 Optimization of the conditions for sensor preparation To
obtain
an
ideal
imprinted
sensor,
experimental
conditions
for
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MIP/AuNPs/GCE preparation, such as the pH value of the prepolymerization solution,
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the molar ratio of functional monomer to template molecule, the number of
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electropolymerization cycles and the electropolymerization rate were optimized. The change of current response of EP on each electrode was calculated by subtracting the
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current response recorded in the absence of EP from current response caused in the
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presence of 5.0 × 10-5 mol/L EP.
Prepolymerization solution pH is an important factor for sensor preparation,
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which influences both of the formation of polymer and the interaction between EP
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and 3-TBA. Fig. 5A shows the oxidation current of EP on the sensor prepared under different pH value. It’s unlike the result described in common literatures, in which the
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optimal pH value for cyclic esters formed by boronic acid with cis-diol compound is
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in neutral or basic solution. In the present work, the sensor exhibited the highest current when the sensor was prepared in prepolmerizatblyion solution with the pH of 6.0. This was probably because proton is significantly important in the electropolymerization of thiophene [65]. If the electroploymerization in neutral or base solution, there are no sufficient amounts of protons for the growth of poly(thiophene) chain [66]. Thus, it was speculated that the poly(3-TBA) matrix of
ACCEPTED MANUSCRIPT the present MIP film can not be well formed in neutral or base solution. Based on these, the optimized pH value of electropolymerization solution was chosen as 6.0. The molar ratio of functional monomer to template molecule directly affects the number of imprinted sites in the MIP/AuNPs/GCE. The sensors were prepared by
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electropolymerizing MIP in the electrolyte solution containing varied concentration of
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3-TBA and a consistent concentration of EP (1.0 × 10-3 mol/L ) with the mole ratios
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of 1:1, 2:1, 3:1, 4:1, and 5:1. It can be found in Fig. 5B, with the increase of mole ratio, the current of EP increased, indicating the increase of imprinted sites. However,
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the current of EP decreased when the mole ratio was over 4:1, which probably
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because too many functional monomer decreased the imprinted sites in turn. Thus, the optimal mole ratio between functional monomer and template molecule was chosen as
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4:1.
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As important factors for MIP preparation, the scan cycles and rate of electropolymerization are related to the thickness and density of the film, respectively.
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When the sensor is prepared under less electropolymerization scan cycle, low current
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of EP can be found due to the formation of less imprinted sites (Fig. 5C). Too many scan cycles would cause the formation of thick film, in which embedded EP molecules is hard to be extracted. The highest current of EP can be observed when the number of electropolymerzation cycle was 20, which was selected as optimized cycle. A dense MIP film would be formed if the sensor was prepared under a low scan rate, which was also difficult for template molecules removal. However, too fast scan rate would cause a loose film, which was probably not stable. Fig. 5D shows that the
ACCEPTED MANUSCRIPT highest current of EP can be obtained when scan rate was 50 mV/s, which was chosen as optimal scan rate.
3.4 Analytical performances of MIP/AuNPs/GCE
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To explore the ability of MIP/AuNPs/GCE for EP quantitative detection, DPV
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method was chosen to examine the relationship between the current response of EP
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and the concentration of EP on MIP/AuNPs/GCE. As shown in Fig. 6A, with the addition of EP, the oxidation peak current of EP continually increased. A linear range
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and a limit of detection (LOD) were 9.0 × 10-8 ～ 1.0 × 10-4 mol/L and 7.6 × 10-8
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mol/L, respectively (Inset of Fig. 6A). And the sensor prepared using thiophene as functional monomer was also used for EP detection with the linear range and LOD of
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4.0 × 10-6 ～ 1.0 × 10-4 mol/L and 3.2 × 10-7 mol/L, respectively (Fig. 6B). It’s
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obvious that MIP/AuNPs/GCE possessed higher sensitivity for EP detection. The results of the reported electrochemical sensors for EP detection were summarized in
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Table S1. Overall, the present sensor showed a wider linear range or a lower LOD
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than that of the other electrochemical sensors, which can be attributed to the conductivity of polymer film and AuNPs, as well as the double specific adsorption of the prepared MIP. Thus, MIP/AuNPs/GCE is a promising candidate of fabricating sensitive biosensors for cis-diol compounds selective detection.
3.5 Reproducibility, stability, and selectivity of MIP/AuNPs/GCE
ACCEPTED MANUSCRIPT The reproducibility of the sensor was investigated by detecting 5.0 × 10 -5 mol/L EP on eight sensors, which were fabricated under the same conditions. A relative standard deviation (RSD) of 4.1% was obtained, implying the good reproducibility of the sensor. When a sensor was used to detect 5.0 × 10-5 mol/L EP for six times with
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subsequent cycles of extraction and measurement operations. The obtained RSD was
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5.9%. And EP kept about 87.2% of the initial current response after being stored for
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fifteen days at room temperature. These results demonstrated that the present sensor possessed good reproducibility, regeneration, and stability, which should facilitate
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itself to be used for real sample determination.
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Selectivity is another essential property of MIP, which was investigated using AA, UA, and DA as the structural analogues or coexisted compounds. Fig. 7A and B the
current
of
EP
or
each
analogue
on
MIP/AuNPs/GCE
and
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show
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MIP(Th)/AuNPs/GCE, respectively. It can be seen that each compound showed its current on both MIP based sensors and NIP based sensors. This was consistent with
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the aforementioned description that poly(thiophene) possesses good conductivity.
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However, the highest IF of 2.18 can be found on MIP/AuNPs/GCE, indicating the good selectivity of the prepared sensor.
3.6 Application of MIP/AuNPs/GCE In order to evaluate the practical application of the sensor, two kinds of EP hydrochloride injections were taken as real samples, which were obtained from the first affiliated hospital of Wannan Medical College. The DPV was used to detect EP
ACCEPTED MANUSCRIPT in the real sample. The results were summarized in Table 1. The test value is close to the labeled standard value of the injection. In order to evaluate the recovery of the sensor, 4.0 × 10−5 mol/L, 6.0 × 10−5 mol/L, and 8.0 × 10−5 mol/L EP were added to the samples, respectively. The obtained recoveries ranged from 90.6% to 103.5%,
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indicating that the prepared sensor was reliable for EP detection in real samples.
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4. Conclusion
In summary, 3-TBA was used as a novel functional monomer for the MIP based
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electrochemical sensor preparation. Thiophene and boronic acid groups endowed the
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sensor with imprinted effect and boronic acid affinity to EP, respectively. Thus, the sensor possessed double recognition capacity, which made it selectively detect EP
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from its analogues. Due to the conductivity of AuNPs and poly(3-TBA), the sensor
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can sensitively detect EP in the concentration range of 9.0 × 10-8 ～ 1.0 × 10-4 mol/L. The results implied that 3-TBA can be expected to become a promising
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functional monomer for cis-diol containing compounds sensitive and selective
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detection with double recognition capacity.
Acknowledgements We greatly appreciate the support of the National Natural Science Foundation of China (21005002, 21575003) and Foundation for Innovation Team of Bioanalytical Chemistry of Anhui Province.
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ACCEPTED MANUSCRIPT Figure Captions Scheme 1 Preparation process of MIP/AuNPs/GCE using 3-TBA and thiophene as functional monomers, respectively. Fig. 1 (A) SEM images of AuNPs/GCE and (B) MIP/AuNPs/GCE.
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Fig. 2 (A) CV curves of MIP/AuNPs/GCE before (a) and after (b) the extraction of
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EP, CV curves of NIP/AuNPs/GCE before (c) and after (d) the extraction of EP in
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[Fe(CN)6]3-/[Fe(CN)6]4-. (B) EIS curves of different modified electrodes in [Fe(CN)6]3-/[Fe(CN)6]4-. GCE (a), AuNPs/GCE (b), MIP/AuNPs/GCE before (c) and
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after (d) the extraction of EP, and MIP/AuNPs/GCE after incubating in 5.0 × 10-5
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mol/L EP solution (e).
Fig. 3 UV-vis spectra of Th (light blue), TBA (red), EP (green), Th-EP (blue), and
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TBA-EP (black) aqueous solution.
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Fig. 4 (A) CV curves of MIP/AuNPs/GCE (black), NIP/AuNPs/GCE (red), MIP/GCE (green), NIP/GCE (blue), MIP(Th)/AuNPs/GCE (light blue), NIP(Th)/AuNPs/GCE
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(rose red) in [Fe(CN)6]3-/[Fe(CN)6]4-. (B) Imprinted factor of MIP/GCE,
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MIP/AuNPs/GCE, MIP(Th)/AuNPs/GCE. Fig. 5 Effect factors for the preparation of MIP/AuNPs/GCE. (A) the mole ratio between monomer and template molecule of the electropolymerization solution, (B) electropolymerization scan cycles (C) and scan rate, (D) pH value. Fig. 6 (A) DPV curves of MIP/AuNPs/GCE with the increase of EP concentration (a～k: 9.0×10-8 ～1×10-4 mol/L) (Inset: the calibration plot of the concentration of EP vs. peak current); (B) DPV curves recorded on MIP(Th)/AuNPs/GCE with the
ACCEPTED MANUSCRIPT increase of EP concentration (a～h: 7.0×10-7 ～ 1×10-4 mol/L) (Inset: the calibration plot of the concentration of EP vs. peak current).
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Fig.7 Selectivity of MIP/AuNPs/GCE (A) and MIP(Th)/AuNPs/GCE (B).
ACCEPTED MANUSCRIPT Table 1 The application of the sensor for EP detection in real samples. Theoretical value
Injection b
mol/L)
(10
mol/L)
Recovery (%)
RSD (%)
2.0
4.0
6.53
108.9
1.5
2.0
6.0
7.94
99.3
1.1
2.0
8.0
10.08
100.8
3.4
2.0
4.0
6.32
105.3
1.9
2.0
6.0
8.10
101.3
2.8
2.0
8.0
98.8
4.0
9.88
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Average value of three determinations
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a
(10
-5
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Injection a
mol/L)
-5
Detect contenta
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(10
-5
Added
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The number of samples
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Fen Liu, Xianwen Kan PII: DOI: Reference:
S1572-6657(19)30065-7 https://doi.org/10.1016/j.jelechem.2019.01.050 JEAC 12880
To appear in:
Journal of Electroanalytical Chemistry
Received date: Revised date: Accepted date:
16 October 2018 14 January 2019 21 January 2019
Please cite this article as: F. Liu and X. Kan, Conductive imprinted electrochemical sensor for epinephrine sensitive detection and double recognition, Journal of Electroanalytical Chemistry, https://doi.org/10.1016/j.jelechem.2019.01.050
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ACCEPTED MANUSCRIPT Conductive imprinted electrochemical sensor for epinephrine sensitive detection and double recognition
Fen Liu, Xianwen Kan*
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College of Chemistry and Materials Science, Anhui Normal University, Wuhu
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241000, P.R. China; The Key Laboratory of Functional Molecular Solids, Ministry
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of Education; Anhui Laboratory of Molecule-Based Materials, Anhui Key
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Laboratory of Chemo-Biosensing.
*Corresponding author: Xianwen Kan
E-mail: [email protected]; Tel: +86-553-3937135; Fax: +86-553-3869303.
ACCEPTED MANUSCRIPT Abstract Epinephrine (EP), an important derivative of a neurotransmitter in the mammalian central nervous system and monitoring the concentration of EP is significant in biological and chemical researches. In this work, a molecularly imprinted polymer (MIP)/gold nanoparticles (AuNPs) composite (MIP/AuNPs) was
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modified on glassy carbon electrode (GCE) surface to fabricate a novel
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electrochemical sensor for EP detection. The modified AuNPs increased the surface
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area of the electrode and improved the sensitivity of the sensor. 3-thiophene boronic acid (3-TBA), as a monomer of conductive polymer, was employed to
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electropolymerize MIP in the presence of EP. Scanning electron microscope, cyclic
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voltammetry, and electrochemical impedance spectroscopy characterization results demonstrated that the prepared sensor possessed sensitive detection and selective
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recognition abilities toward EP. Unlike the other MIP based electrochemical sensors,
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conductive poly(3-TBA) matrix, as well as the conductive AuNPs can enhance the sensitivity of the sensor. Under the optimal conditions, the sensor can sensitively
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detect EP with a linear range and limit of detection of 9.0 × 10-8 ～ 1.0 × 10-4 mol/L
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and 7.6 × 10-8 mol/L, respectively. And the sensor showed double recognition ability to EP due to the following reasons: first, size and shape complementarity between the formed imprinted sites and the template molecules; second, the reversible covalent interaction between boronic acid of 3-TBA and cis-diol of EP. Thus, the sensor can recognize EP from its analogues. And the sensor has been applied to analyze EP in injection samples with satisfactory results.
ACCEPTED MANUSCRIPT Keywords: gold nanoparticles, conductive molecularly imprinted polymer, modified
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electrode, 3-thiophene boronic acid, epinephrine
ACCEPTED MANUSCRIPT 1. Introduction Molecular imprinting is a synthesis technique for artificial material preparation with specific recognition ability toward template molecule [1-3]. Prepared molecularly imprinted polymer (MIP) has lots of imprinted cavities with complementary sizes,
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shapes, and functional groups with template molecules, which make it can recognize
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template molecule from its analogues [4]. Thus, MIP has been applied in many fields [8, 9],
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including solid phase extraction [5-7], similar enzyme catalysis
chromatographic separation [10-15], and chemical sensing [16-21]. Due to the
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advantages of simplicity, sensitivity, and stability, electrochemical method has been
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combined with MIP for template molecule sensitive and selective detection [22-24]. In recent years, nanomaterials, such as nobel metal nanoparticles based nanocomposites
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(gold nano urchins/graphene oxide [25], platinum nanoparticles/carbon nitride
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nanotubes [26], silver nanoparticles/polyoxometalate functionalized reduced graphene oxide [27]) and quantum dots based nanocomposites (boron nitride quantum dots [28], nitride nanotubes/graphene quantum
dots
[29],
graphene quantum
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carbon
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dots/two-dimensional hexagonal boron nitride nanosheets [30]), have been modified on electrode for imprinted electrochemical sensors preparation to improve the sensitivity of the sensors. Besides the single template recognition and detection, dual templates have been involved into the imprinted polymer synthesis, in which dual templates can been simultaneously selectively detected [31]. Computational approach has also been used to select adequate functional monomers for imprinted electrochemical sensor preparation [32]. The results of these work demonstrated that
ACCEPTED MANUSCRIPT the MIP based electrochemical sensors have endowed the selectivity of the sensors besides the sensitivity for template molecules detection. Most MIP based electrochemical sensors were prepared by electropolymerizing functional monomer in the presence of template molecule. And the most interaction
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between functional monomer and template is non-covalent bond due to the flexible
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concerning the extraction and rebinding of template molecule. Compared with
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non-covalent interaction, covalent imprinting is superior in preventing leakage of template molecules during polymerization process because of the formation of stable
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covalent bonds between the template and monomer [33-35]. Thus, the precise
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imprinting sites facilitate the recognition ability of the prepared MIP [36-38]. Reversible covalent interaction has been employed for MIP preparation, which
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results in a rather homogenous population of binding sites with minimizing the
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existence of non-specific sites. However, the covalent interaction based MIP has been restricted since it’s difficult to design an appropriate template-monomer reversible
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covalent interaction under mild condition. It is worth mentioning that boronic acid
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derivatives exhibit high affinity to cis-diol containing compounds (CDCCs) to form cyclic esters in mild basic or neutral aqueous solution, while the cyclic esters dissociate once switching the medium to acidic solution [39, 40]. Thus, boronic acid based MIP has been investigated, which also has been applied for CDCCs electrochemical sensing [41-43]. And if a monomer possesses two functions of affinity and imprinted sites toward template molecules, it should simplify the procedure of MIP preparation process.
ACCEPTED MANUSCRIPT Thiophene, as a monomer of conductive polymer, has received a significant amount of attention in many fields, such as supercapacitor materials [44-46] and electrochemical sensing interface [47]. Also, thiophene has been used as functional monomer for MIP preparation [48]. To restrict the non-specific adsorption of MIP, the
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conductivity of MIP based electrochemical sensors is usually unsatisfying, which
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limits its sensitivity for template detection in turn. Thus, thiophene is seldom used as
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functional monomer for MIP based electrochemical sensor preparation due to the conductivity of poly(thiopnene) [49], which would provide a sensing interface for
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template electrochemical redox. As the consequences, the non-specific adsorption can
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not be well restrained and the selectivity of the sensor should be affected. It’s well-known that boronic acid formed cyclic esters with cis-diol containing
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compounds through covalent interaction in neutral or basic solution. Epinephrine (EP),
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a cis-diol compound, is an important derivative of a neurotransmitter in the mammalian central nervous system, which can lead to glycogenolysis in the liver and
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skeletal muscle, the mobilization of free fatty acid, the increase of plasma lactate and the
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improved force and rate of heart contraction [50, 51]. Numerous diseases are related to the concentration of EP, such as Parkinson’s disease, Alzheimer’s disease, hypertension and multiple sclerosis [52-54]. Thus, monitoring the concentration of EP is significant in biological and chemical researches. Poly(thiophene) matrix based MIP can form imprinted sites with complementary size and shape with EP [55, 56]. To achieve the sensitivity and selectivity, 3-thiophene boronic acid (3-TBA) was chosen as a novel functional monomer for MIP based electrochemical sensor
ACCEPTED MANUSCRIPT preparation in this work. Thus, the prepared electrochemical sensor can selectively detect EP due to the dual recognition ability. Besides, the modified gold nanoparticles (AuNPs) and the conductive poly(3-TBA) matrix would improve the sensitivity of the
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sensor.
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2. Experimental
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2.1 Chemicals
3-Thiophene boronic acid (3-TBA), thiophene (Th), and epinephrine (EP) were
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purchased from Aladdin Reagent Co. Ltd. Dopamine (DA), ascorbic acid (AA), and
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uric acid (UA) were ordered from Sigma-Aldrich Reagent Co. Ltd. All other reagents were of analytical grade and used without further purification. All aqueous solutions
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2.2 Apparatus
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purification system.
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were prepared with ultrapure water (18.25 MΩ cm) from a Millipore water
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Electrochemical experiments, such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS), were performed on CHI 660C workstation (ChenHua Instruments Co., Shanghai, China) with a conventional three-electrode system. A bare or modified glassy carbon electrode (GCE) was served as a working electrode. A saturated calomel electrode and a platinum wire electrode were used as a reference electrode and a counter-electrode, respectively. Field emission scanning electron microscope
ACCEPTED MANUSCRIPT (FE-SEM) images were obtained on an S-4800 field emission scanning electron microanalyzer (Hitachi, Japan).
2.3 Fabrication of gold nanoparticles modified GCE (AuNPs/GCE)
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Prior to fabricating gold nanoparticles (AuNPs) modified electrode, a bare GCE
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was successively polished with 1.0, 0.3, and 0.05 μm α-Al2O3 powder. Then the clean
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electrode was immersed into 4 mmol/L HAuCl4 and treated by the use of a constant
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potential of − 0.2 V for 500 s, obtaining the AuNPs modified GCE (AuNPs/GCE).
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2.4 Fabrication of MIP modified AuNPs/GCE (MIP/AuNPs/GCE) The clean AuNPs/GCE was immersed into 5.0 mL phosphate buffer solution (PBS,
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0.1 mol/L, pH 6.0) containing 400 μL 0.05 mol/L 3-TBA and 100 μL 0.05 mol/L EP
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[57]. Then cyclic voltammetry (CV) was performed from - 0.2 V to + 1.2 V for 20 cycles at a scan rate of 50 mV/s, obtaining polymer film modified electrode.
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Subsequently, the embedded EP was extracted by immersing the modified electrode
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into 0.05 mol/L HCl aqueous solution and was electrochemically scanned between 0.0 V – 1.5 V for several cycles, getting MIP modified AuNPs/GCE (MIP/AuNPs/GCE). As a control, non-molecular imprinting polymer (NIP) modified AuNPs/GCE (NIP/AuNPs/GCE) was prepared in exactly the same way except for the omitting of EP in the electropolymerization process. And the MIP and NIP film prepared using thiophene (Th) as functional monomer was also modified on AuNPs/GCE in the same way, which were labeled as MIP(Th)/AuNPs/GCE and NIP(Th)/AuNPs/GCE,
ACCEPTED MANUSCRIPT respectively. The procedures for the construction of the MIP/AuNPs/GCE and MIP(Th)/AuNPs/GCE were illustrated in Scheme 1. Also, MIP/GCE and NIP/GCE were prepared by direct electropolymerization of MIP and NIP on GCE, respectively.
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2.5 Electrochemical properties measurements
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Electrochemical characterization of the prepared modified electrodes were
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carried out in 5.0 mmol/L [Fe(CN)6]3-/[Fe(CN)6]4- using CV and EIS methods. Electrochemical detection of EP was performed in 0.1 mol/L PBS (pH 7.0) by using
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CV and DPV approaches. AA, UA, and DA were selected as coexisted or structural
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similar compounds to evaluate the recognition capacity of the prepared sensor.
D
2.6 Regeneration of MIP/AuNPs/GCE
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After the detection of 5.0 × 10-5 mol/L EP, MIP/AuNPs/GCE was immersed in 0.05 M HCl and electrochemically scanned between 0.0 V – 1.5 V for several cycles
CE
to remove adsorbed EP molecules. The eluted sensor was then immersed in the same
AC
concentration of EP for detection.
3. Results and discussion 3.1 Characterization of MIP/AuNPs/GCE The morphologies of AuNPs/GCE and MIP/AuNPs/GCE were characterized by SEM, as shown in Fig. 1A and B. It can be found that a layer of nanoparticles coated onto the electrode surface, which can improve the surface area of the electrode and
ACCEPTED MANUSCRIPT facilitate the electronic transfer. With the electropolymerization of MIP, an obvious layer of film can be observed on the surface of AuNPs, indicating the preparation of MIP/AuNPs/GCE. [Fe(CN)6]3-/[Fe(CN)6]4- is a commonly used probe for the characterization of
PT
MIP based electrochemical sensor preparation procee, which was also chosen to
RI
investigate the prepared process of MIP/AuNPs/GCE. The reaction between boronic
SC
acid and cis-diol containing compound can produce boronate ester [58], [59], which causes the conversion from sp2 to sp3 hybridization of boron atom [60]. Thus,
NU
the electrostatic repulsion between boronate moieties and [Fe(CN)6]3-/[Fe(CN)6]4-
MA
couple should block the arrival of [Fe(CN)6]3-/[Fe(CN)6]4- to sensor surface [61]. As shown in Fig. 2A, before the extraction of EP, a pair of weak redox peak of be
found
on
MIP/AuNPs/GCE
(Fig.2Aa)
or
D
[Fe(CN)6]3-/[Fe(CN)6]4- can
PT E
NIP/AuNPs/GCE (Fig. 2Ac), although the polymer matrix (poly(3-TBA) or poly(thiphene)) was conductive. Compared with NIP/AuNPs/GCE (Fig. 2Ad), a
CE
remarkably increased peak current of probe can be found on MIP/AuNPs/GCE (Fig.
AC
2Ab) after the removal of template molecules. It may implied that the cavities were left by the extraction of template molecules, forming channels for the probe to pass through the cavities. EIS is a valuable way for the characterization of the sensor preparation process. Fig. 2B shows the Nyquist diagrams of the modified electrode fabricated at each step in the presence of [Fe(CN)6]3-/[Fe(CN)6]4-. Compared with bare GCE (Fig. 2Ba), the slope of the line part of AuNPs/GCE (Fig. 2Bb) dramatically increased, indicating
ACCEPTED MANUSCRIPT that AuNPs had good electric conducting property and could enhance the electron transfer rate [62, 63]. Before the extraction, MIP/AuNPs/GCE (Fig. 2Bc) exhibited an obvious interfacial resistance because the polymer film decreased the electron transfer rate. A remarkable decrease of the interfacial resistance can be
PT
observed on MIP/AuNPs/GCE after the removal of template molecules (Fig. 2Bd),
RI
which implied that the formed imprinted sites enhanced the diffusion rate of
SC
[Fe(CN)6]3-/[Fe(CN)6]4- through the MIP film and made it easier for electronic transfer. With the addition of EP, the impedance increased (Fig. 2Be), which can be
NU
ascribed to the block of the channels for probe to reach the electrode surface by the
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adsorbed EP molecules. Thus, the impedance and CV changes of the modified process, as well as the SEM characterization demonstrated the successful preparation of the
D
sensor.
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Fig. 3 exhibits the UV-vis spectra of Th (light blue), TBA (red), EP (green), Th-EP (blue), and TBA-EP (black) aqueous solution. A characteristic absorption peak
CE
at 290 nm for EP can be observed. When EP was mixed with Th, no obvious shift of
AC
adsorption peak can be found, indicating no interaction between EP and Th. Although TBA has no adsorption like Th, the adsorption peak of TBA-EP was red-shifted from 290 nm to 296 nm, compared with individual EP. The results implied the obvious interaction between TBA and EP [64].
3.2 The specific adsorption of the sensor
ACCEPTED MANUSCRIPT The specific adsorption is a natural property of MIP, which was investigated by immersing different modified electrodes in 5.0 × 10-5 mol/L EP solution, as shown in Fig. 4A. It’s different from other reported MIP based electrochemical sensors, a pair of redox peaks of EP can be observed when NIP/AuNPs/GCE was used as the
PT
working electrode (blue and red) due to the conductivity of poly(thiophene) matrix of
RI
NIP film. Thus, the non-specific adsorbed EP molecules can be redoxed on NIP/GCE
SC
and NIP/AuNPs/GCE. An increased current of EP was found on MIP/GCE (green), which can be attributed to the specific adsorption of EP into imprinted sites. Owing to
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the good conductivity of AuNPs, MIP/AuNPs/GCE (black) showed much higher
MA
current of EP than that of MIP/GCE. By contrast, the same experiments were performed on MIP(Th)/AuNPs/GCE and NIP(Th)/AuNPs/GCE. Although the
D
NIP(Th)/AuNPs/GCE (rose red) and NIP/AuNPs/GCE (red) showed low peak
PT E
currents of EP, MIP(Th)/AuNPs/GCE (light blue) exhibited a much lower current than that of MIP/AuNPs/GCE. The oxidation current of EP was chosen to calculate
CE
imprinted factor (IF, IF=ΔiMIP/ΔiNIP). As shown in Fig. 4B, MIP/AuNPs/GCE and
AC
MIP/GCE exhibit almost the same IF of 2.18 and 2.25, respectively. Compared with MIP/AuNPs/GCE, MIP(Th)/AuNPs/GCE possessed a lower IF of 1.66. These phenomena can be explained that the MIP prepared using 3-TBA as the functional monomer possessed double recognition capacity toward template molecule. Besides the removal of template molecules left imprinted sites with complementary sizes, shapes, and functional groups with template molecules, boronic acid formed cyclic
ACCEPTED MANUSCRIPT esters with EP through covalent interaction, which made the MIP/AuNPs/GCE show a higher specific adsorption capacity toward EP than that of MIP(Th)/AuNPs/GCE. 3.3 Optimization of the conditions for sensor preparation To
obtain
an
ideal
imprinted
sensor,
experimental
conditions
for
PT
MIP/AuNPs/GCE preparation, such as the pH value of the prepolymerization solution,
RI
the molar ratio of functional monomer to template molecule, the number of
SC
electropolymerization cycles and the electropolymerization rate were optimized. The change of current response of EP on each electrode was calculated by subtracting the
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current response recorded in the absence of EP from current response caused in the
MA
presence of 5.0 × 10-5 mol/L EP.
Prepolymerization solution pH is an important factor for sensor preparation,
D
which influences both of the formation of polymer and the interaction between EP
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and 3-TBA. Fig. 5A shows the oxidation current of EP on the sensor prepared under different pH value. It’s unlike the result described in common literatures, in which the
CE
optimal pH value for cyclic esters formed by boronic acid with cis-diol compound is
AC
in neutral or basic solution. In the present work, the sensor exhibited the highest current when the sensor was prepared in prepolmerizatblyion solution with the pH of 6.0. This was probably because proton is significantly important in the electropolymerization of thiophene [65]. If the electroploymerization in neutral or base solution, there are no sufficient amounts of protons for the growth of poly(thiophene) chain [66]. Thus, it was speculated that the poly(3-TBA) matrix of
ACCEPTED MANUSCRIPT the present MIP film can not be well formed in neutral or base solution. Based on these, the optimized pH value of electropolymerization solution was chosen as 6.0. The molar ratio of functional monomer to template molecule directly affects the number of imprinted sites in the MIP/AuNPs/GCE. The sensors were prepared by
PT
electropolymerizing MIP in the electrolyte solution containing varied concentration of
RI
3-TBA and a consistent concentration of EP (1.0 × 10-3 mol/L ) with the mole ratios
SC
of 1:1, 2:1, 3:1, 4:1, and 5:1. It can be found in Fig. 5B, with the increase of mole ratio, the current of EP increased, indicating the increase of imprinted sites. However,
NU
the current of EP decreased when the mole ratio was over 4:1, which probably
MA
because too many functional monomer decreased the imprinted sites in turn. Thus, the optimal mole ratio between functional monomer and template molecule was chosen as
D
4:1.
PT E
As important factors for MIP preparation, the scan cycles and rate of electropolymerization are related to the thickness and density of the film, respectively.
CE
When the sensor is prepared under less electropolymerization scan cycle, low current
AC
of EP can be found due to the formation of less imprinted sites (Fig. 5C). Too many scan cycles would cause the formation of thick film, in which embedded EP molecules is hard to be extracted. The highest current of EP can be observed when the number of electropolymerzation cycle was 20, which was selected as optimized cycle. A dense MIP film would be formed if the sensor was prepared under a low scan rate, which was also difficult for template molecules removal. However, too fast scan rate would cause a loose film, which was probably not stable. Fig. 5D shows that the
ACCEPTED MANUSCRIPT highest current of EP can be obtained when scan rate was 50 mV/s, which was chosen as optimal scan rate.
3.4 Analytical performances of MIP/AuNPs/GCE
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To explore the ability of MIP/AuNPs/GCE for EP quantitative detection, DPV
RI
method was chosen to examine the relationship between the current response of EP
SC
and the concentration of EP on MIP/AuNPs/GCE. As shown in Fig. 6A, with the addition of EP, the oxidation peak current of EP continually increased. A linear range
NU
and a limit of detection (LOD) were 9.0 × 10-8 ～ 1.0 × 10-4 mol/L and 7.6 × 10-8
MA
mol/L, respectively (Inset of Fig. 6A). And the sensor prepared using thiophene as functional monomer was also used for EP detection with the linear range and LOD of
D
4.0 × 10-6 ～ 1.0 × 10-4 mol/L and 3.2 × 10-7 mol/L, respectively (Fig. 6B). It’s
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obvious that MIP/AuNPs/GCE possessed higher sensitivity for EP detection. The results of the reported electrochemical sensors for EP detection were summarized in
CE
Table S1. Overall, the present sensor showed a wider linear range or a lower LOD
AC
than that of the other electrochemical sensors, which can be attributed to the conductivity of polymer film and AuNPs, as well as the double specific adsorption of the prepared MIP. Thus, MIP/AuNPs/GCE is a promising candidate of fabricating sensitive biosensors for cis-diol compounds selective detection.
3.5 Reproducibility, stability, and selectivity of MIP/AuNPs/GCE
ACCEPTED MANUSCRIPT The reproducibility of the sensor was investigated by detecting 5.0 × 10 -5 mol/L EP on eight sensors, which were fabricated under the same conditions. A relative standard deviation (RSD) of 4.1% was obtained, implying the good reproducibility of the sensor. When a sensor was used to detect 5.0 × 10-5 mol/L EP for six times with
PT
subsequent cycles of extraction and measurement operations. The obtained RSD was
RI
5.9%. And EP kept about 87.2% of the initial current response after being stored for
SC
fifteen days at room temperature. These results demonstrated that the present sensor possessed good reproducibility, regeneration, and stability, which should facilitate
NU
itself to be used for real sample determination.
MA
Selectivity is another essential property of MIP, which was investigated using AA, UA, and DA as the structural analogues or coexisted compounds. Fig. 7A and B the
current
of
EP
or
each
analogue
on
MIP/AuNPs/GCE
and
D
show
PT E
MIP(Th)/AuNPs/GCE, respectively. It can be seen that each compound showed its current on both MIP based sensors and NIP based sensors. This was consistent with
CE
the aforementioned description that poly(thiophene) possesses good conductivity.
AC
However, the highest IF of 2.18 can be found on MIP/AuNPs/GCE, indicating the good selectivity of the prepared sensor.
3.6 Application of MIP/AuNPs/GCE In order to evaluate the practical application of the sensor, two kinds of EP hydrochloride injections were taken as real samples, which were obtained from the first affiliated hospital of Wannan Medical College. The DPV was used to detect EP
ACCEPTED MANUSCRIPT in the real sample. The results were summarized in Table 1. The test value is close to the labeled standard value of the injection. In order to evaluate the recovery of the sensor, 4.0 × 10−5 mol/L, 6.0 × 10−5 mol/L, and 8.0 × 10−5 mol/L EP were added to the samples, respectively. The obtained recoveries ranged from 90.6% to 103.5%,
RI
PT
indicating that the prepared sensor was reliable for EP detection in real samples.
SC
4. Conclusion
In summary, 3-TBA was used as a novel functional monomer for the MIP based
NU
electrochemical sensor preparation. Thiophene and boronic acid groups endowed the
MA
sensor with imprinted effect and boronic acid affinity to EP, respectively. Thus, the sensor possessed double recognition capacity, which made it selectively detect EP
D
from its analogues. Due to the conductivity of AuNPs and poly(3-TBA), the sensor
PT E
can sensitively detect EP in the concentration range of 9.0 × 10-8 ～ 1.0 × 10-4 mol/L. The results implied that 3-TBA can be expected to become a promising
CE
functional monomer for cis-diol containing compounds sensitive and selective
AC
detection with double recognition capacity.
Acknowledgements We greatly appreciate the support of the National Natural Science Foundation of China (21005002, 21575003) and Foundation for Innovation Team of Bioanalytical Chemistry of Anhui Province.
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Acta 121 (2014) 369-375.
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polymers, Polymer 52 (2011) 4631-4643.
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coupling with sol-gel molecularly imprinted polymer as a recognition element of an electrochemical sensor, Sens. Actuators B 238 (2017) 420-426.
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ACCEPTED MANUSCRIPT [65] M. Can, K. Pekmez, N. Pekmez, A. Yildiz, Electropreparation and electrochemical stability of polythiophenes in acetonitrile containing anhydrous HBF4, J. Appl. Polym. Sci. 77 (2000) 312-322. [66] M. Can, F. Sevin, A. Yildiz, The effect of the proton on electropolymerization of
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ACCEPTED MANUSCRIPT Figure Captions Scheme 1 Preparation process of MIP/AuNPs/GCE using 3-TBA and thiophene as functional monomers, respectively. Fig. 1 (A) SEM images of AuNPs/GCE and (B) MIP/AuNPs/GCE.
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Fig. 2 (A) CV curves of MIP/AuNPs/GCE before (a) and after (b) the extraction of
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EP, CV curves of NIP/AuNPs/GCE before (c) and after (d) the extraction of EP in
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[Fe(CN)6]3-/[Fe(CN)6]4-. (B) EIS curves of different modified electrodes in [Fe(CN)6]3-/[Fe(CN)6]4-. GCE (a), AuNPs/GCE (b), MIP/AuNPs/GCE before (c) and
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after (d) the extraction of EP, and MIP/AuNPs/GCE after incubating in 5.0 × 10-5
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mol/L EP solution (e).
Fig. 3 UV-vis spectra of Th (light blue), TBA (red), EP (green), Th-EP (blue), and
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TBA-EP (black) aqueous solution.
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Fig. 4 (A) CV curves of MIP/AuNPs/GCE (black), NIP/AuNPs/GCE (red), MIP/GCE (green), NIP/GCE (blue), MIP(Th)/AuNPs/GCE (light blue), NIP(Th)/AuNPs/GCE
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(rose red) in [Fe(CN)6]3-/[Fe(CN)6]4-. (B) Imprinted factor of MIP/GCE,
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MIP/AuNPs/GCE, MIP(Th)/AuNPs/GCE. Fig. 5 Effect factors for the preparation of MIP/AuNPs/GCE. (A) the mole ratio between monomer and template molecule of the electropolymerization solution, (B) electropolymerization scan cycles (C) and scan rate, (D) pH value. Fig. 6 (A) DPV curves of MIP/AuNPs/GCE with the increase of EP concentration (a～k: 9.0×10-8 ～1×10-4 mol/L) (Inset: the calibration plot of the concentration of EP vs. peak current); (B) DPV curves recorded on MIP(Th)/AuNPs/GCE with the
ACCEPTED MANUSCRIPT increase of EP concentration (a～h: 7.0×10-7 ～ 1×10-4 mol/L) (Inset: the calibration plot of the concentration of EP vs. peak current).
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Fig.7 Selectivity of MIP/AuNPs/GCE (A) and MIP(Th)/AuNPs/GCE (B).
ACCEPTED MANUSCRIPT Table 1 The application of the sensor for EP detection in real samples. Theoretical value
Injection b
mol/L)
(10
mol/L)
Recovery (%)
RSD (%)
2.0
4.0
6.53
108.9
1.5
2.0
6.0
7.94
99.3
1.1
2.0
8.0
10.08
100.8
3.4
2.0
4.0
6.32
105.3
1.9
2.0
6.0
8.10
101.3
2.8
2.0
8.0
98.8
4.0
9.88
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Average value of three determinations
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a
(10
-5
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Injection a
mol/L)
-5
Detect contenta
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(10
-5
Added
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The number of samples
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7