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Au(III)-promoted magnetic molecularly imprinted polymer nanospheres for electrochemical determination of streptomycin residues in food
Biosensors and Bioelectronics 41 (2013) 551–556
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Au(III)-promoted magnetic molecularly imprinted polymer nanospheres for electrochemical determination of streptomycin residues in food Bingqian Liu, Dianping Tang n, Bing Zhang, Xiaohua Que, Huanghao Yang, Guonan Chen n Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education and Fujian Province), Department of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350108, PR China
a r t i c l e i n f o
abstract
Article history: Received 8 August 2012 Received in revised form 13 September 2012 Accepted 14 September 2012 Available online 24 September 2012
Redox-active magnetic molecularly imprinted polymer (mMIP) nanospheres were first synthesized and functionalized with streptomycin templates for highly efficient electrochemical determination of streptomycin residues (STR) in food by coupling with bioelectrocatalytic reaction of enzymes for signal amplification. The mMIP nanospheres were synthesized by using Au(III)-promoted molecularly imprinted polymerization with STR templates on magnetic beads. Based on extraction of template molecules from the mMIP surface, the imprints toward STR templates were formed. The assay was implemented with a competitive-type assay format. Upon addition of streptomycin, the analyte competed with glucose oxidase-labeled streptomycin (GOX-STR) for molecular imprints on the mMIP nanospheres. With the increasing streptomycin in the sample, the conjugation amount of GOX-STR on the mMIP nanospheres decreased, leading to the change in the bioelectrocatalytic current relative to glucose system. Under optimal conditions, the catalytic current was proportional to STR level in the sample, and exhibited a dynamic range of 0.05–20 ng mL 1 with a detection limit of 10 pg mL 1 STR (at 3sB). Intra- and interassay coefficients of variation were below 12%. The assayed results for STR spiked samples including milk and honey with the mMIP-based sensor were received a good accordance with the results obtained from the referenced high-performance liquid chromatography (HPLC) method. & 2012 Elsevier B.V. All rights reserved.
Keywords: Magnetic molecularly imprinted polymer Electrochemical sensor Streptomycin residue Redox-active nanospheres Bioelectrocatalytic reaction
1. Introduction Antibiotic, as a veterinary drug and gardening medicine for treatment of bacterial diseases, has been widely used in agriculture, since it can usually kill or slow down the growth of bacteria (Barganska et al., 2011). However, there will inevitably be the antibiotic residues in the agricultural products. Although antibiotic residues in food have no direct toxic effect on human health, numerous adverse effects can range from fever and nausea to major allergic reactions including photodermatitis and anaphylaxis (Onal, 2011; Xu and Ying, 2011). Recently, various methods and strategies have been developed for detection of antibiotic residues in food, e.g., enzyme-based immunoassay (Xu et al., 2012), liquid chromatography–trandem mass spectrometry (Salvia et al., 2012), fluorimetric determination (Tang et al., 2012), amperometric immunosensor (Conzuelo et al., 2012), and surface plasmon resonance immunosensor (Fernandez et al., 2012). Despite many advances in this field, there is still the quest for new schemes and techniques to improve the accessibility and simplicity of the assays.
n
Corresponding authors. Tel.: þ 86 23 68138296; fax: þ 86 23 68254000. E-mail addresses: [email protected] (D. Tang), [email protected] (G. Chen). 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.09.021
Molecular imprinting technique, engaged in designing and synthesizing some artificial receptor molecules, has been demonstrated as a powerful technique for a focal research area to understand the molecular recognition phenomena in biological systems, and develop novel materials mimicking biological functions usable in analytical applications (Gauczinski et al., 2012; Schirhagl et al., 2012; Ansell et al., 1996; Wulff, 1995; Shea, 1994). The imprinting effect is achieved by polymerization of functional monomers and cross-linkers in the presence of template molecules, which is preferably the analyte itself, or an analogue molecule with suitable geometry and interaction sites (Pernites et al., 2012). After the templates are removed from the resultant polymer network to leave recognition cavity sites, the functional monomers used are expected to be laid out in the cavity as complementary to the chemical functionality of the template molecules, because the functional monomers are bound with the template molecules during the polymerization (Malaekeh-Nikouei et al., 2012; Wang et al., 2012). Consequently, resultant polymers exhibit the template-selective binding capacity. Hence, molecular imprinting technique has been used for detection of different molecules based on various signal generation principles, e.g., dopamine (Yu et al., 2010), lipopolysaccharides (Ogawa et al., 2010), and hazardous compounds and drugs of abuse (Sharma et al., 2012). Recently, molecularly imprinted electrochemical sensor holds great potential as the next-generation detection strategy because
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of its high sensitivity, simple instrumentation, and excellent compatibility with miniaturization techniques (Li et al., 2012). Typically, the analytical properties of molecularly imprinted electrochemical sensors heavily rely on the signal-transduction method. Various methods have been used for fabrication of molecularly imprinted polymer (MIP)-based electrochemical sensor, e.g., electropolymerization (Li et al., 2010, 2012; Viswanathan et al., 2012; Xing et al., 2012; Yuan et al., 2012), copolymerization (Mao et al., 2011), electrodeposition (Yang et al., 2011), codeposition (Casey et al., 2010), and photo-copolymerization (Wang et al., 2009). In these methods, the template molecules were basically imprinted on the electrode surface during the formation of polymers. In contrast, homogeneously MIP-based electrochemical sensors usually involve in the molecular imprints on the nano-/micro-beads, and take place in the solution, thus allowing the integration of multiple liquid handling processes (Guan et al., 2012). Especially combining with microfluidic device, the homogenous MIP-based sensors can be used for detection of complex samples without the large sample consumption and sample pretreatment, resulting in a relatively inexpensive and easy performance (Zhang et al., 2011). Herein, we synthesize a novel and redox-active magnetic molecularly imprinting polymer (mMIP) based on nanogoldencapsulated poly(o-phenylenediamine) shell on magnetic iron oxide cores by the one-pot method. The as-prepared mMIP nanospheres are used for determination of streptomycin (as a model analyte herein) in a man-made magneto-controlled detection cell. The assay is carried out with a competitive-type assay mode between target molecules and glucose oxidase-labeled streptomycin for molecular imprints on the magnetic beads. The signal is generated and amplified based on the catalytic oxidation of the labeled glucose oxidase toward glucose in the detection solution with the help of redox-active mMIP nanospheres. The aim of this work is to explore a novel, magneto-controlled, homogeneous MIP-based electrochemical sensor for simple and sensitive determination of small molecules.
removed using ultrafiltration for 12–15 times until the peak corresponding to GOX in the elution disappeared. Finally, the obtained GOX-STR was prepared into 1.0 mL pH 7.0 PBS. 2.3. Preparation of magnetic molecularly imprinted polymer nanospheres (mMIPs) Before preparation of mMIPs, nano-Fe3O4 particles (cores) (50 nm in diameter) were initially synthesized by co-precipitation of FeII and FeIII chlorides (FeII/FeIII ratio of 0.5) in alkaline solution, as described in the literature (Tang et al., 2006). Synthesis of the mMIPs was as follows: 50 mg of magnetic beads (MBs) were initially added into NaCl solution (15 mL, 0.5 M) containing 0.02 M sodium dodecylsulphate (SDS), and stirred for 4 h. After washing with distilled water, the SDS-MB composites were redispersed into poly(vinyl pyrrolidone) (PVP) aqueous solution (5.0 mL, 1.0 wt%), and the mixture was vigorously stirred for another 2 h at room temperature (RT) to obtain PVP-coated MBs. Excess SDS and PVP were removed in the supernatant fraction after centrifugation. Following that, the obtained precipitate was redispersed into HAuCl4 aqueous solution (1.0 mL, 1.0 wt%), and shaken slightly for 2 h at RT to make the [AuCl4] ions assemble on the PVP-MBs. Subsequently, 100 mL of STR standards (1.0 mg mL 1, as templates) were added into the resultant mixture, and gently stirred for 2 h at RT to enable positively charged STR adsorb onto the functionalized MBs. Afterwards, 5 mL of 10 mM o-phenylenediamine were injected into the mixture with gentle stirring. During this process, Au(III) ions could promote the polymerization of OPD monomers on the surface of magnetic beads, while Au(III) ions were reduced to zero-valent Au0 (Liu et al., 2012), following by the encapsulation of streptomycin monomers into the polymers. Finally, the suspension was collected by centrifugation. The streptomycin templates were removed by using ethanol (50%, v/v). The obtained mMIP nanospheres were used for detection of streptomycin. For comparison, magnetic poly(o-phenylenediamine) nanospheres (i.e., without streptomycin templates) were synthesized by using the above mentioned method. 2.4. Electrochemical measurement
2. Experimental 2.1. Reagents and chemicals Streptomycin sulfate (STR), chloramphenicol (Chl) and tetracycline (Tet) were purchased from Beijing Dingguo Biotechnol. Co. Ltd. (Beijing, China). Poly(vinylpyrrolidone) (PVP, K-30), o-phenylenediamine (OPD) and HAuCl4 4H2O were purchased from Sinopharm Chem. Re. Co. Ltd. (Shanghai, China). Glucose oxidase (GOX) was obtained from Sigma (USA). All reagents were of analytical grade and used as received without further purification. Ultrapure water obtained from a Millipore water purification system ( Z18 MO, Milli-Q, Millipore) was used in all runs. Phosphate-buffered saline (PBS, 0.01 M, pH 7.0) solution was prepared by adding 1.221 g K2HPO4, 0.1362 g KH2PO4, and 0.7455 g KCl into 1000 mL ultrapure water. 2.2. Preparation of glucose oxidase-streptomycin conjugation (GOX-STR) Glucose oxidase-streptomycin conjugates were prepared according to our previous reports with a little modification (Tang et al., 2009a, 2010). 200 mL of GOX (1.0 mg mL 1) and 200 mL of streptomycin (0.5 mg mL 1) were initially dissolved into 1 mL of 0.01 M PBS and the pH was adjusted to 9.5 by using 10 wt% K2CO3, and then 100 mL of gultaraldehyde solution (2.0 wt%) was added into the mixture. After stirred for 2 h, the mixture was adjusted to pH 7.0 by using 1.0 M NaH2PO4. The unbound GOX and streptomycin were
Electrochemical measurements were carried out with a CHI 630D Electrochemical Workstation (Shanghai, China) in combination with a flow-through detection cell. The detection system comprised of an Indium Tin Oxide (ITO) working electrode, a platinum wire as auxiliary electrode and an Ag/AgCl reference electrode (The schematic illustration is shown in our previous report (Tang et al., 2009b)). The flow injection system consists of a six-way connected valve equipped with a 1 mL syringe pump and connected through a Teflon tubing to the flow cell. The analytical flow stream entered from the other side into the center of the flow cell at 0.5 mL min 1. The ITO electrode was installed at the bottom of the cell, and an external permanent BaFe12O19 magnet with pot shape (10 mm in diameter and 5 mm in depth, 410–430 mT) was set under the working electrode. The carrier buffer containing glucose (PBS, pH 7.0), mMIPs, and GOX-STR were introduced at 500 mL min 1 via a control valve-based injection loop, respectively. The streptomycin analyte was directly injected into the detection cell by using a microsyringe. Scheme 1 represents the competitive-type assay protocol and measurement method. During the process, the collection, incubation and detection were performed in the flow cell. The electrochemical measurement was performed as follows: (i) 200 mL of mMIP nanospheres (C[mMIP] E50 mg mL 1) were flowed into the cell, and collected on the ITO surface with an external magnet; (ii) 100 mL of the above prepared GOX-STR and target analyte (streptomycin) with various concentrations were injected in the cell, and incubated for 10 min at RT without the magnet
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Scheme 1. Schematic illustration of nanogold-promoted magnetic molecularly imprinting polymer nanospheres for competitive-type electrochemical detection of streptomycin (STR) residues by coupling with bioelectrocatalytic reaction of glucose oxidase (GOX) for signal amplification .
(Note: During this process, the analyte competed with GOX-STR for molecular imprints on the mMIP nanospheres); and (iii) after washing with pH 7.0 PBS, square wave voltammetric (SWV) measurement was performed in pH 7.0 PBS containing 2 mM glucose with the aid of external magnet from 700 mV to 0 mV (vs. Ag/AgCl) (Amplitude: 25 mV; Frequency: 15 Hz; Increase E: 4 mV). All incubations and measurements were carried out using stopped-flow technique at RT (2571.0 1C). After each step, the detection cell was washed by using pH 7.0 PBS in the presence of the external magnet. Analyses were always made in triplicate.
3. Results and discussion 3.1. Construction and characterizations of mMIP-based electrochemical sensors In our previous work (Liu et al., 2012), Au(III)-promoted core-shell iron oxide@poly(o-phenylendiamine) nanospheres (i.e., without streptomycin templates) were synthesized and characterized by using transmission electron microscope (TEM), fourier transfer infrared spectrometry (FTIR), Raman spectroscopy and X-ray diffraction (XRD). Experimental results revealed that o-phenylenediamine monomers could be successfully polymerized on the surface of magnetic beads with the help of Au(III), while Au(III) could be reduced to zero-valent Au0. However, another two questions herein are grown as follows: (i) whether streptomycin templates can be imprinted onto magnetic poly(o-phenylenediamine) nanospheres during the polymerization process, and (ii) streptomycin templates can be removed from the mMIP nanospheres by ethanol washing. To further demonstrate the issues, we also utilized XRD to investigate the mMIPs before and after the removal of streptomycin templates (Fig. 1A). Curve ‘a’ in Fig. 1A represents the XRD pattern of magnetic poly(o-phenylenediamine) nanospheres without streptomycin templates, which was in accordance with our previous report (Liu et al., 2012). In the presence of streptomycin templates, however, the as-prepared nanospheres exhibited another two characteristic peaks at 71 and 471 (curve ‘b’ in Fig. 1A), which might be derived from the imprinted streptomycin molecules in the polymers. Inspiringly, when the resultant magnetic nanospheres were washed by ethanol, two characteristic peaks at 71 and 471 were almost disappeared (curve ‘c’ in Fig. 1A), which was almost in correspondence with curve ‘a’. These results indicated that streptomycin molecules could be imprinted and eluted from the magnetic nanospheres by using the developed method. To further demonstrate this
issue, we also used electrochemical impedance spectroscopy (EIS) to investigate the change of resistance (Ret) before and after the removal of streptomycin templates from the magnetic nanospheres (Fig. 1B). Curve ‘a’ in Fig. 1B shows the EIS of the prepared mMIPs before the removal of streptomycin templates (Ret E800 O). After the removal of streptomycin templates, the resistance was decreased (Ret E510 O) (curve ‘b’ in Fig. 1B). This is most likely a consequence of the fact that the formed cavities on the mMIPs could facilitate the electron transfer. When the resultant mMIPs reacted with excess streptomycin targets, significantly, the resistance almost restored to the original value (Ret E761 O) (curve ‘c’ in Fig. 1B). The results further suggested that the prepared mMIPs could be preliminarily applied for screening of streptomycin. Next, the synthesized mMIPs were employed for detection of streptomycin (0 ng mL 1, 5 ng mL 1, and 10 ng mL 1 STR used as examples in this case) with a competitive-type assay format. As seen from curves ‘a to c’ in Fig. 1C, the SWV peak currents decreased with the increasing STR concentrations in the sample relative to pH 7.0 glucose-PBS system. The decrease in the SWV peak currents mainly derived from the competition between GOX-STR and STR analyte in the sample for the imprinting cavities on the mMIPs. The signal mainly originated from the conjugated GOX toward the bioelectrocatalytic reaction of glucose in the presence of poly(o-phenylenediamine). To further monitor the bioactivity of the conjugated GOX, SWV curves of mMIP-base sensor were measured in pH 7.0 PBS in the absence and presence of glucose relative to zero analyte. As indicated from curves ‘d’ to ‘a’ in Fig. 1C, upon addition of glucose in pH 7.0 PBS, SWV peak current was largely increased (Di E22 mA). Hence, use of bioactive enzyme could amplify the electrochemical signal of mMIP-based sensors. 3.2. Optimization of experimental conditions To ensure an optimal analytical performance of the electrochemical sensor, some experimental parameters including reaction time between the imprinting cavities and the analyte, and pH of the assay solution should be investigated. In this work, SWV peak currents derive from the conjugated GOX toward the oxidation of glucose. To maintain the bioactivity of the GOX and adequately fulfill its catalytic potential, a moderately acidic pH should be preferable. Fig. 2a displays the dependence of anodic currents on pH of PBS by using 5 ng mL 1 STR as an example. As indicated from Fig. 2a, an optimal current was obtained at pH 7.0 PBS. Higher or lower pHs resulted in the decrease of anodic
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Fig. 1. (A) XRD patterns of (a) magnetic poly(o-phenylenediamine) nanospheres without streptomycin templates, and (b) before and (c) after removal templates of the as-prepared magnetic molecularly imprinted polymer nanospheres. (B) Nyquist diagrams of the as-prepared magnetic molecularly imprinted polymer nanospheres-based ITO electrode: (a) before, (b) after removal templates, and (c) electrode ‘b’ after incubation with excess streptomycin, in 5 mM Fe(CN)46 /3 þ0.1 M KCl with the range from 10 2 Hz to 105 Hz at an alternate voltage of 5 mV. (C) SWV curves of the mMIP-based sensors after incubation with ((a), (d)) 0 ng mL 1 STR þ GOX-STR, (b) 5 ng mL 1 STR þGOX-STR, and (c) 10 ng mL 1 STR þ GOX-STR in pH 7.0 PBS in the (d) absence and ((a)–(c)) presence of 3 mM glucose.
Fig. 2. Effects of (a) pH of PBS, (b) incubation time between the as-prepared mMIP nanospheres and STR þGOX-STR, and (c) mass ratio of HAuCl4 and OPD on the electrochemical responses of the mMIP-based sensors (5.0 ng mL 1 STR used in this case).
currents. Thus, a pH 7.0 with PBS was chosen as the supporting electrolyte. Reaction time between the imprinting cavities and STR þSTRGOX usually affects the analytical properties of the electrochemical sensor. As indicated from Fig. 2b (5.0 ng mL 1 used in the case), SWV peak currents increased with the increment of incubation time, and tended to level off after 10 min. Hence, a reaction time of 10 min was selected for determination of STR at acceptable throughput. To achieve the enhancement of the electrochemical signal and stability of the mMIPs, the effect of HAuCl4/OPD ratio in the precursor mixture should be investigated. If OPD was too little, the formed MIP film on the magnetic beads was thin, which was not conducive to molecular imprints. If OPD was too much, however, the formed polymer film completely encapsulated the streptomycin templates, thus did not favor for the removal of template molecules. Meanwhile, the high-concentration polymer film on the magnetic beads might cause a relatively higher signalto-noise ratio. As shown in Fig. 2c, the highest signal-to-background current was obtained atM½HAuCl4 : M½OPD 1 : 1. Therefore, 1:1 of mass ratio of HAuCl4 and OPD was selected for the preparation of mMIPs. 3.3. Dose-response curves of the electrochemical sensor toward STR standards Under optimal conditions, the sensitivity and dynamic range of the mMIP-based electrochemical sensor was evaluated toward STR standards with a competitive-type assay mode. A square wave voltammetric (SWV) measurement was carried out in PBS, pH 7.0, containing 2.0 mM glucose after incubation with various
STR levels and excess GOX-STR for 10 min at RT. As indicated in Fig. 3a, the SWV peak currents of the mMIP-based sensor decreased with the increase of STR concentrations. The calibration plots displayed a good linear relationship between SWV peak currents and the logarithm of the analyte concentration in the range of 0.05–20 ng mL 1 for STR (Fig. 3b). The correlation coefficient was 0.986. The detection limit (LOD) value for STR was determined at 10 pg mL 1, estimated at the 3Sblank criterion, which were partially lower than those of colorimetric visualization using Au nanoparticles supramolecular assembly (LOD: 1160 pg mL 1) (Sun et al., 2011), surface plasmon resonance sensor using imprinted boronic acid-functionalized gold nanoparticle composites (LOD: 0.58 pg mL 1) (Fraseoni et al., 2010), gold-silica nanostructures-based electrochemical immunoassay (LOD: 5 pg mL 1) (Liu et al., 2011), and imaging surface plasmon resonance-based immunosensor (LOD: 370 pg mL 1) (Raz et al., 2009).
3.4. Specificity, precision, and stability of the electrochemical sensors To further investigate the selectivity of this new assay method, other antibiotic samples including tetracycline (Tet) and chloramphenicol (Chl) were analyzed via this strategy. In brief, these samples were mixed together by adding the desired dosage, and incubated with the mMIP nanospheres colloidal solution for 10 min at RT. Later, electrochemical response of the incubated mMIP nanospheres was checked and the concentration of target analyte (streptomycin) was calculated. The results are listed in Table 1. The date suggested some extent of non-specific blocking of STR-GOX by tetracycline and chloramphenicol.
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Fig. 3. (a) Typical SWV response curves of the mMIP-based electrochemical sensor toward various concentrations of STR standards, and (b) calibration plots of the mMIPbased sensor between SWV peak currents and the logarithm of target STR concentrations. Potential scanning was from 700 mV to 0 mV (vs. Ag/AgCl, Amplitude: 25 mV, Frequency: 15 Hz, Increase E: 4 mV). Measurements were performed in pH 7.0 PBS containing 2.0 mM glucose. Each data point represents the average value obtained from three different measurements. The error bars represent the 95% confidence interval of the mean for y-axis currents. The integrated currents in Fig. 3b were obtained from each peak current.
The precision of mMIP-based electrochemical sensor was assessed by estimating the variation coefficients (CVs) of intraand inter-assays. The intra-assay precision of the analytical method by using the same-batch mMIP nanospheres was evaluated by analyzing 3 STR standards. The CVs were 6.7%, 9.2%, and 8.3% at 0.1 ng mL 1, 1.0 ng mL 1, and 10.0 ng mL 1 STR, respectively. Similarly, the inter-assay CVs using different mMIP nanospheres with various batches were 9.5%, 11.7%, and 10.1% at the above-mentioned analytes, respectively. Hence, the precision of the mMIP-based electrochemical sensor was acceptable. In addition, the stability of the as-prepared mMIP nanospheres was investigated on a 30-day period. When mMIP nanospheres were stored at 4 1C and measured intermittently (every 3–5 days), they retained 90% of their initial responses after a storage period of 25 days. 3.5. Analysis of real samples and method validation To investigate the possible application of the mMIP-based electrochemical sensor for testing real food samples, STR spiked samples including milk and honey were assayed by using the developed sensor and HPLC as a reference method (finished by Dr J. Liao, SWU, Chongqing). The spiking process of real samples and detection method were described in our recently published paper (Liu et al., 2011). The experimental results are summarized in Table 2. The recovery in spiked samples is 81–129% for the mMIP-based electrochemical sensors. As analyzed from the recovery between two methods, however, we also found that the mMIP-based sensor is less accurate than that of HPLC.
4. Conclusions Herein, we have demonstrated the development of advanced mMIP-based sensors for electrochemical detection of STR, as a model analyte, on the functionalized magnetic beads by coupling with electrocatalytic reaction of bioactive enzymes for signal amplification. Compared with conventional MIP-modified electrodes, use of magnetic beads can facilitate the construction of the electrochemical sensors by using an external magnet. The asprepared mMIP nanospheres consist of a magnetic core and a redox-active organic shell doped with gold nanoparticles, thus can favor for electrochemical measurement and electron communication. Additionally, the present sensing system exhibits some advantages, such as easy preparation, simple operation, and low cost. By controlling the imprinted target molecules, this assay can
Table 1 Interference degree of the mMIP-based electrochemical sensors. STR concentration (ng mL 1)
Sample
Taken
Recovery (%) Found (mean 7SD, n ¼5)
STR 5.0 4.85 70.35 STR þTet 5.0 (STR)þ5.0 (Tet) 5.42 70.56 STR þTet þ Chl 5.0 (STR)þ 5.0 (Tet) þ5.0 (Chl) 5.76 70.42
97 108.4 115.2
Table 2 Comparison of determination results for STR in spiked food samples as obtained by the mMIPs-based sensors and HPLC as the reference method. Food Sample sample no.
Spiked STR (ng mL 1)
Methods (Mean 7 SD, ng mL 1)a and recovery (%) mMIP-based sensor
Milk
1b 2 3 4
0 1 5.0 10
0.037 0.02 0.817 0.19 4.397 0.15 12.21 7 0.13
Honey
5b 6 7 8
0 1 5.0 10
0.027 0.03 1.197 0.13 6.457 0.14 8.957 0.22
a b
HPLC
( ) 0.017 0.01 (81%) 0.94 7 0.07 (87.8%) 5.12 7 0.21 (122.1%) 11.31 7 0.37 ( ) (119%) (129%) (89.5%)
() (94%) (102.4%) (113.1%)
0.017 0.02 ( ) 0.98 7 0.09 (98%) 4.95 7 0.32(99%) 9.87 7 0.23 (98.7%)
Each sample was assayed in triplicate. Without spiking STR samples.
be easily extended for use with other small molecules and thus represents a versatile detection method.
Acknowledgements Support by the Research Fund for the National Science Foundation of Fujian Province (2011J06003), the Doctoral Program of Higher Education of China (20103514120003), the National Natural Science Foundation of China (21075019 and 41176079), the ‘‘973’’ National Basic Research Program of China (2010CB732403), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1116) is gratefully acknowledged. Dr. J. Liao (SWU, Chongqing) is thanked for HPLC analysis of real samples for the method-comparison study.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Au(III)-promoted magnetic molecularly imprinted polymer nanospheres for electrochemical determination of streptomycin residues in food Bingqian Liu, Dianping Tang n, Bing Zhang, Xiaohua Que, Huanghao Yang, Guonan Chen n Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education and Fujian Province), Department of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350108, PR China
a r t i c l e i n f o
abstract
Article history: Received 8 August 2012 Received in revised form 13 September 2012 Accepted 14 September 2012 Available online 24 September 2012
Redox-active magnetic molecularly imprinted polymer (mMIP) nanospheres were first synthesized and functionalized with streptomycin templates for highly efficient electrochemical determination of streptomycin residues (STR) in food by coupling with bioelectrocatalytic reaction of enzymes for signal amplification. The mMIP nanospheres were synthesized by using Au(III)-promoted molecularly imprinted polymerization with STR templates on magnetic beads. Based on extraction of template molecules from the mMIP surface, the imprints toward STR templates were formed. The assay was implemented with a competitive-type assay format. Upon addition of streptomycin, the analyte competed with glucose oxidase-labeled streptomycin (GOX-STR) for molecular imprints on the mMIP nanospheres. With the increasing streptomycin in the sample, the conjugation amount of GOX-STR on the mMIP nanospheres decreased, leading to the change in the bioelectrocatalytic current relative to glucose system. Under optimal conditions, the catalytic current was proportional to STR level in the sample, and exhibited a dynamic range of 0.05–20 ng mL 1 with a detection limit of 10 pg mL 1 STR (at 3sB). Intra- and interassay coefficients of variation were below 12%. The assayed results for STR spiked samples including milk and honey with the mMIP-based sensor were received a good accordance with the results obtained from the referenced high-performance liquid chromatography (HPLC) method. & 2012 Elsevier B.V. All rights reserved.
Keywords: Magnetic molecularly imprinted polymer Electrochemical sensor Streptomycin residue Redox-active nanospheres Bioelectrocatalytic reaction
1. Introduction Antibiotic, as a veterinary drug and gardening medicine for treatment of bacterial diseases, has been widely used in agriculture, since it can usually kill or slow down the growth of bacteria (Barganska et al., 2011). However, there will inevitably be the antibiotic residues in the agricultural products. Although antibiotic residues in food have no direct toxic effect on human health, numerous adverse effects can range from fever and nausea to major allergic reactions including photodermatitis and anaphylaxis (Onal, 2011; Xu and Ying, 2011). Recently, various methods and strategies have been developed for detection of antibiotic residues in food, e.g., enzyme-based immunoassay (Xu et al., 2012), liquid chromatography–trandem mass spectrometry (Salvia et al., 2012), fluorimetric determination (Tang et al., 2012), amperometric immunosensor (Conzuelo et al., 2012), and surface plasmon resonance immunosensor (Fernandez et al., 2012). Despite many advances in this field, there is still the quest for new schemes and techniques to improve the accessibility and simplicity of the assays.
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Corresponding authors. Tel.: þ 86 23 68138296; fax: þ 86 23 68254000. E-mail addresses: [email protected] (D. Tang), [email protected] (G. Chen). 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.09.021
Molecular imprinting technique, engaged in designing and synthesizing some artificial receptor molecules, has been demonstrated as a powerful technique for a focal research area to understand the molecular recognition phenomena in biological systems, and develop novel materials mimicking biological functions usable in analytical applications (Gauczinski et al., 2012; Schirhagl et al., 2012; Ansell et al., 1996; Wulff, 1995; Shea, 1994). The imprinting effect is achieved by polymerization of functional monomers and cross-linkers in the presence of template molecules, which is preferably the analyte itself, or an analogue molecule with suitable geometry and interaction sites (Pernites et al., 2012). After the templates are removed from the resultant polymer network to leave recognition cavity sites, the functional monomers used are expected to be laid out in the cavity as complementary to the chemical functionality of the template molecules, because the functional monomers are bound with the template molecules during the polymerization (Malaekeh-Nikouei et al., 2012; Wang et al., 2012). Consequently, resultant polymers exhibit the template-selective binding capacity. Hence, molecular imprinting technique has been used for detection of different molecules based on various signal generation principles, e.g., dopamine (Yu et al., 2010), lipopolysaccharides (Ogawa et al., 2010), and hazardous compounds and drugs of abuse (Sharma et al., 2012). Recently, molecularly imprinted electrochemical sensor holds great potential as the next-generation detection strategy because
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of its high sensitivity, simple instrumentation, and excellent compatibility with miniaturization techniques (Li et al., 2012). Typically, the analytical properties of molecularly imprinted electrochemical sensors heavily rely on the signal-transduction method. Various methods have been used for fabrication of molecularly imprinted polymer (MIP)-based electrochemical sensor, e.g., electropolymerization (Li et al., 2010, 2012; Viswanathan et al., 2012; Xing et al., 2012; Yuan et al., 2012), copolymerization (Mao et al., 2011), electrodeposition (Yang et al., 2011), codeposition (Casey et al., 2010), and photo-copolymerization (Wang et al., 2009). In these methods, the template molecules were basically imprinted on the electrode surface during the formation of polymers. In contrast, homogeneously MIP-based electrochemical sensors usually involve in the molecular imprints on the nano-/micro-beads, and take place in the solution, thus allowing the integration of multiple liquid handling processes (Guan et al., 2012). Especially combining with microfluidic device, the homogenous MIP-based sensors can be used for detection of complex samples without the large sample consumption and sample pretreatment, resulting in a relatively inexpensive and easy performance (Zhang et al., 2011). Herein, we synthesize a novel and redox-active magnetic molecularly imprinting polymer (mMIP) based on nanogoldencapsulated poly(o-phenylenediamine) shell on magnetic iron oxide cores by the one-pot method. The as-prepared mMIP nanospheres are used for determination of streptomycin (as a model analyte herein) in a man-made magneto-controlled detection cell. The assay is carried out with a competitive-type assay mode between target molecules and glucose oxidase-labeled streptomycin for molecular imprints on the magnetic beads. The signal is generated and amplified based on the catalytic oxidation of the labeled glucose oxidase toward glucose in the detection solution with the help of redox-active mMIP nanospheres. The aim of this work is to explore a novel, magneto-controlled, homogeneous MIP-based electrochemical sensor for simple and sensitive determination of small molecules.
removed using ultrafiltration for 12–15 times until the peak corresponding to GOX in the elution disappeared. Finally, the obtained GOX-STR was prepared into 1.0 mL pH 7.0 PBS. 2.3. Preparation of magnetic molecularly imprinted polymer nanospheres (mMIPs) Before preparation of mMIPs, nano-Fe3O4 particles (cores) (50 nm in diameter) were initially synthesized by co-precipitation of FeII and FeIII chlorides (FeII/FeIII ratio of 0.5) in alkaline solution, as described in the literature (Tang et al., 2006). Synthesis of the mMIPs was as follows: 50 mg of magnetic beads (MBs) were initially added into NaCl solution (15 mL, 0.5 M) containing 0.02 M sodium dodecylsulphate (SDS), and stirred for 4 h. After washing with distilled water, the SDS-MB composites were redispersed into poly(vinyl pyrrolidone) (PVP) aqueous solution (5.0 mL, 1.0 wt%), and the mixture was vigorously stirred for another 2 h at room temperature (RT) to obtain PVP-coated MBs. Excess SDS and PVP were removed in the supernatant fraction after centrifugation. Following that, the obtained precipitate was redispersed into HAuCl4 aqueous solution (1.0 mL, 1.0 wt%), and shaken slightly for 2 h at RT to make the [AuCl4] ions assemble on the PVP-MBs. Subsequently, 100 mL of STR standards (1.0 mg mL 1, as templates) were added into the resultant mixture, and gently stirred for 2 h at RT to enable positively charged STR adsorb onto the functionalized MBs. Afterwards, 5 mL of 10 mM o-phenylenediamine were injected into the mixture with gentle stirring. During this process, Au(III) ions could promote the polymerization of OPD monomers on the surface of magnetic beads, while Au(III) ions were reduced to zero-valent Au0 (Liu et al., 2012), following by the encapsulation of streptomycin monomers into the polymers. Finally, the suspension was collected by centrifugation. The streptomycin templates were removed by using ethanol (50%, v/v). The obtained mMIP nanospheres were used for detection of streptomycin. For comparison, magnetic poly(o-phenylenediamine) nanospheres (i.e., without streptomycin templates) were synthesized by using the above mentioned method. 2.4. Electrochemical measurement
2. Experimental 2.1. Reagents and chemicals Streptomycin sulfate (STR), chloramphenicol (Chl) and tetracycline (Tet) were purchased from Beijing Dingguo Biotechnol. Co. Ltd. (Beijing, China). Poly(vinylpyrrolidone) (PVP, K-30), o-phenylenediamine (OPD) and HAuCl4 4H2O were purchased from Sinopharm Chem. Re. Co. Ltd. (Shanghai, China). Glucose oxidase (GOX) was obtained from Sigma (USA). All reagents were of analytical grade and used as received without further purification. Ultrapure water obtained from a Millipore water purification system ( Z18 MO, Milli-Q, Millipore) was used in all runs. Phosphate-buffered saline (PBS, 0.01 M, pH 7.0) solution was prepared by adding 1.221 g K2HPO4, 0.1362 g KH2PO4, and 0.7455 g KCl into 1000 mL ultrapure water. 2.2. Preparation of glucose oxidase-streptomycin conjugation (GOX-STR) Glucose oxidase-streptomycin conjugates were prepared according to our previous reports with a little modification (Tang et al., 2009a, 2010). 200 mL of GOX (1.0 mg mL 1) and 200 mL of streptomycin (0.5 mg mL 1) were initially dissolved into 1 mL of 0.01 M PBS and the pH was adjusted to 9.5 by using 10 wt% K2CO3, and then 100 mL of gultaraldehyde solution (2.0 wt%) was added into the mixture. After stirred for 2 h, the mixture was adjusted to pH 7.0 by using 1.0 M NaH2PO4. The unbound GOX and streptomycin were
Electrochemical measurements were carried out with a CHI 630D Electrochemical Workstation (Shanghai, China) in combination with a flow-through detection cell. The detection system comprised of an Indium Tin Oxide (ITO) working electrode, a platinum wire as auxiliary electrode and an Ag/AgCl reference electrode (The schematic illustration is shown in our previous report (Tang et al., 2009b)). The flow injection system consists of a six-way connected valve equipped with a 1 mL syringe pump and connected through a Teflon tubing to the flow cell. The analytical flow stream entered from the other side into the center of the flow cell at 0.5 mL min 1. The ITO electrode was installed at the bottom of the cell, and an external permanent BaFe12O19 magnet with pot shape (10 mm in diameter and 5 mm in depth, 410–430 mT) was set under the working electrode. The carrier buffer containing glucose (PBS, pH 7.0), mMIPs, and GOX-STR were introduced at 500 mL min 1 via a control valve-based injection loop, respectively. The streptomycin analyte was directly injected into the detection cell by using a microsyringe. Scheme 1 represents the competitive-type assay protocol and measurement method. During the process, the collection, incubation and detection were performed in the flow cell. The electrochemical measurement was performed as follows: (i) 200 mL of mMIP nanospheres (C[mMIP] E50 mg mL 1) were flowed into the cell, and collected on the ITO surface with an external magnet; (ii) 100 mL of the above prepared GOX-STR and target analyte (streptomycin) with various concentrations were injected in the cell, and incubated for 10 min at RT without the magnet
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Scheme 1. Schematic illustration of nanogold-promoted magnetic molecularly imprinting polymer nanospheres for competitive-type electrochemical detection of streptomycin (STR) residues by coupling with bioelectrocatalytic reaction of glucose oxidase (GOX) for signal amplification .
(Note: During this process, the analyte competed with GOX-STR for molecular imprints on the mMIP nanospheres); and (iii) after washing with pH 7.0 PBS, square wave voltammetric (SWV) measurement was performed in pH 7.0 PBS containing 2 mM glucose with the aid of external magnet from 700 mV to 0 mV (vs. Ag/AgCl) (Amplitude: 25 mV; Frequency: 15 Hz; Increase E: 4 mV). All incubations and measurements were carried out using stopped-flow technique at RT (2571.0 1C). After each step, the detection cell was washed by using pH 7.0 PBS in the presence of the external magnet. Analyses were always made in triplicate.
3. Results and discussion 3.1. Construction and characterizations of mMIP-based electrochemical sensors In our previous work (Liu et al., 2012), Au(III)-promoted core-shell iron oxide@poly(o-phenylendiamine) nanospheres (i.e., without streptomycin templates) were synthesized and characterized by using transmission electron microscope (TEM), fourier transfer infrared spectrometry (FTIR), Raman spectroscopy and X-ray diffraction (XRD). Experimental results revealed that o-phenylenediamine monomers could be successfully polymerized on the surface of magnetic beads with the help of Au(III), while Au(III) could be reduced to zero-valent Au0. However, another two questions herein are grown as follows: (i) whether streptomycin templates can be imprinted onto magnetic poly(o-phenylenediamine) nanospheres during the polymerization process, and (ii) streptomycin templates can be removed from the mMIP nanospheres by ethanol washing. To further demonstrate the issues, we also utilized XRD to investigate the mMIPs before and after the removal of streptomycin templates (Fig. 1A). Curve ‘a’ in Fig. 1A represents the XRD pattern of magnetic poly(o-phenylenediamine) nanospheres without streptomycin templates, which was in accordance with our previous report (Liu et al., 2012). In the presence of streptomycin templates, however, the as-prepared nanospheres exhibited another two characteristic peaks at 71 and 471 (curve ‘b’ in Fig. 1A), which might be derived from the imprinted streptomycin molecules in the polymers. Inspiringly, when the resultant magnetic nanospheres were washed by ethanol, two characteristic peaks at 71 and 471 were almost disappeared (curve ‘c’ in Fig. 1A), which was almost in correspondence with curve ‘a’. These results indicated that streptomycin molecules could be imprinted and eluted from the magnetic nanospheres by using the developed method. To further demonstrate this
issue, we also used electrochemical impedance spectroscopy (EIS) to investigate the change of resistance (Ret) before and after the removal of streptomycin templates from the magnetic nanospheres (Fig. 1B). Curve ‘a’ in Fig. 1B shows the EIS of the prepared mMIPs before the removal of streptomycin templates (Ret E800 O). After the removal of streptomycin templates, the resistance was decreased (Ret E510 O) (curve ‘b’ in Fig. 1B). This is most likely a consequence of the fact that the formed cavities on the mMIPs could facilitate the electron transfer. When the resultant mMIPs reacted with excess streptomycin targets, significantly, the resistance almost restored to the original value (Ret E761 O) (curve ‘c’ in Fig. 1B). The results further suggested that the prepared mMIPs could be preliminarily applied for screening of streptomycin. Next, the synthesized mMIPs were employed for detection of streptomycin (0 ng mL 1, 5 ng mL 1, and 10 ng mL 1 STR used as examples in this case) with a competitive-type assay format. As seen from curves ‘a to c’ in Fig. 1C, the SWV peak currents decreased with the increasing STR concentrations in the sample relative to pH 7.0 glucose-PBS system. The decrease in the SWV peak currents mainly derived from the competition between GOX-STR and STR analyte in the sample for the imprinting cavities on the mMIPs. The signal mainly originated from the conjugated GOX toward the bioelectrocatalytic reaction of glucose in the presence of poly(o-phenylenediamine). To further monitor the bioactivity of the conjugated GOX, SWV curves of mMIP-base sensor were measured in pH 7.0 PBS in the absence and presence of glucose relative to zero analyte. As indicated from curves ‘d’ to ‘a’ in Fig. 1C, upon addition of glucose in pH 7.0 PBS, SWV peak current was largely increased (Di E22 mA). Hence, use of bioactive enzyme could amplify the electrochemical signal of mMIP-based sensors. 3.2. Optimization of experimental conditions To ensure an optimal analytical performance of the electrochemical sensor, some experimental parameters including reaction time between the imprinting cavities and the analyte, and pH of the assay solution should be investigated. In this work, SWV peak currents derive from the conjugated GOX toward the oxidation of glucose. To maintain the bioactivity of the GOX and adequately fulfill its catalytic potential, a moderately acidic pH should be preferable. Fig. 2a displays the dependence of anodic currents on pH of PBS by using 5 ng mL 1 STR as an example. As indicated from Fig. 2a, an optimal current was obtained at pH 7.0 PBS. Higher or lower pHs resulted in the decrease of anodic
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Fig. 1. (A) XRD patterns of (a) magnetic poly(o-phenylenediamine) nanospheres without streptomycin templates, and (b) before and (c) after removal templates of the as-prepared magnetic molecularly imprinted polymer nanospheres. (B) Nyquist diagrams of the as-prepared magnetic molecularly imprinted polymer nanospheres-based ITO electrode: (a) before, (b) after removal templates, and (c) electrode ‘b’ after incubation with excess streptomycin, in 5 mM Fe(CN)46 /3 þ0.1 M KCl with the range from 10 2 Hz to 105 Hz at an alternate voltage of 5 mV. (C) SWV curves of the mMIP-based sensors after incubation with ((a), (d)) 0 ng mL 1 STR þ GOX-STR, (b) 5 ng mL 1 STR þGOX-STR, and (c) 10 ng mL 1 STR þ GOX-STR in pH 7.0 PBS in the (d) absence and ((a)–(c)) presence of 3 mM glucose.
Fig. 2. Effects of (a) pH of PBS, (b) incubation time between the as-prepared mMIP nanospheres and STR þGOX-STR, and (c) mass ratio of HAuCl4 and OPD on the electrochemical responses of the mMIP-based sensors (5.0 ng mL 1 STR used in this case).
currents. Thus, a pH 7.0 with PBS was chosen as the supporting electrolyte. Reaction time between the imprinting cavities and STR þSTRGOX usually affects the analytical properties of the electrochemical sensor. As indicated from Fig. 2b (5.0 ng mL 1 used in the case), SWV peak currents increased with the increment of incubation time, and tended to level off after 10 min. Hence, a reaction time of 10 min was selected for determination of STR at acceptable throughput. To achieve the enhancement of the electrochemical signal and stability of the mMIPs, the effect of HAuCl4/OPD ratio in the precursor mixture should be investigated. If OPD was too little, the formed MIP film on the magnetic beads was thin, which was not conducive to molecular imprints. If OPD was too much, however, the formed polymer film completely encapsulated the streptomycin templates, thus did not favor for the removal of template molecules. Meanwhile, the high-concentration polymer film on the magnetic beads might cause a relatively higher signalto-noise ratio. As shown in Fig. 2c, the highest signal-to-background current was obtained atM½HAuCl4 : M½OPD 1 : 1. Therefore, 1:1 of mass ratio of HAuCl4 and OPD was selected for the preparation of mMIPs. 3.3. Dose-response curves of the electrochemical sensor toward STR standards Under optimal conditions, the sensitivity and dynamic range of the mMIP-based electrochemical sensor was evaluated toward STR standards with a competitive-type assay mode. A square wave voltammetric (SWV) measurement was carried out in PBS, pH 7.0, containing 2.0 mM glucose after incubation with various
STR levels and excess GOX-STR for 10 min at RT. As indicated in Fig. 3a, the SWV peak currents of the mMIP-based sensor decreased with the increase of STR concentrations. The calibration plots displayed a good linear relationship between SWV peak currents and the logarithm of the analyte concentration in the range of 0.05–20 ng mL 1 for STR (Fig. 3b). The correlation coefficient was 0.986. The detection limit (LOD) value for STR was determined at 10 pg mL 1, estimated at the 3Sblank criterion, which were partially lower than those of colorimetric visualization using Au nanoparticles supramolecular assembly (LOD: 1160 pg mL 1) (Sun et al., 2011), surface plasmon resonance sensor using imprinted boronic acid-functionalized gold nanoparticle composites (LOD: 0.58 pg mL 1) (Fraseoni et al., 2010), gold-silica nanostructures-based electrochemical immunoassay (LOD: 5 pg mL 1) (Liu et al., 2011), and imaging surface plasmon resonance-based immunosensor (LOD: 370 pg mL 1) (Raz et al., 2009).
3.4. Specificity, precision, and stability of the electrochemical sensors To further investigate the selectivity of this new assay method, other antibiotic samples including tetracycline (Tet) and chloramphenicol (Chl) were analyzed via this strategy. In brief, these samples were mixed together by adding the desired dosage, and incubated with the mMIP nanospheres colloidal solution for 10 min at RT. Later, electrochemical response of the incubated mMIP nanospheres was checked and the concentration of target analyte (streptomycin) was calculated. The results are listed in Table 1. The date suggested some extent of non-specific blocking of STR-GOX by tetracycline and chloramphenicol.
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Fig. 3. (a) Typical SWV response curves of the mMIP-based electrochemical sensor toward various concentrations of STR standards, and (b) calibration plots of the mMIPbased sensor between SWV peak currents and the logarithm of target STR concentrations. Potential scanning was from 700 mV to 0 mV (vs. Ag/AgCl, Amplitude: 25 mV, Frequency: 15 Hz, Increase E: 4 mV). Measurements were performed in pH 7.0 PBS containing 2.0 mM glucose. Each data point represents the average value obtained from three different measurements. The error bars represent the 95% confidence interval of the mean for y-axis currents. The integrated currents in Fig. 3b were obtained from each peak current.
The precision of mMIP-based electrochemical sensor was assessed by estimating the variation coefficients (CVs) of intraand inter-assays. The intra-assay precision of the analytical method by using the same-batch mMIP nanospheres was evaluated by analyzing 3 STR standards. The CVs were 6.7%, 9.2%, and 8.3% at 0.1 ng mL 1, 1.0 ng mL 1, and 10.0 ng mL 1 STR, respectively. Similarly, the inter-assay CVs using different mMIP nanospheres with various batches were 9.5%, 11.7%, and 10.1% at the above-mentioned analytes, respectively. Hence, the precision of the mMIP-based electrochemical sensor was acceptable. In addition, the stability of the as-prepared mMIP nanospheres was investigated on a 30-day period. When mMIP nanospheres were stored at 4 1C and measured intermittently (every 3–5 days), they retained 90% of their initial responses after a storage period of 25 days. 3.5. Analysis of real samples and method validation To investigate the possible application of the mMIP-based electrochemical sensor for testing real food samples, STR spiked samples including milk and honey were assayed by using the developed sensor and HPLC as a reference method (finished by Dr J. Liao, SWU, Chongqing). The spiking process of real samples and detection method were described in our recently published paper (Liu et al., 2011). The experimental results are summarized in Table 2. The recovery in spiked samples is 81–129% for the mMIP-based electrochemical sensors. As analyzed from the recovery between two methods, however, we also found that the mMIP-based sensor is less accurate than that of HPLC.
4. Conclusions Herein, we have demonstrated the development of advanced mMIP-based sensors for electrochemical detection of STR, as a model analyte, on the functionalized magnetic beads by coupling with electrocatalytic reaction of bioactive enzymes for signal amplification. Compared with conventional MIP-modified electrodes, use of magnetic beads can facilitate the construction of the electrochemical sensors by using an external magnet. The asprepared mMIP nanospheres consist of a magnetic core and a redox-active organic shell doped with gold nanoparticles, thus can favor for electrochemical measurement and electron communication. Additionally, the present sensing system exhibits some advantages, such as easy preparation, simple operation, and low cost. By controlling the imprinted target molecules, this assay can
Table 1 Interference degree of the mMIP-based electrochemical sensors. STR concentration (ng mL 1)
Sample
Taken
Recovery (%) Found (mean 7SD, n ¼5)
STR 5.0 4.85 70.35 STR þTet 5.0 (STR)þ5.0 (Tet) 5.42 70.56 STR þTet þ Chl 5.0 (STR)þ 5.0 (Tet) þ5.0 (Chl) 5.76 70.42
97 108.4 115.2
Table 2 Comparison of determination results for STR in spiked food samples as obtained by the mMIPs-based sensors and HPLC as the reference method. Food Sample sample no.
Spiked STR (ng mL 1)
Methods (Mean 7 SD, ng mL 1)a and recovery (%) mMIP-based sensor
Milk
1b 2 3 4
0 1 5.0 10
0.037 0.02 0.817 0.19 4.397 0.15 12.21 7 0.13
Honey
5b 6 7 8
0 1 5.0 10
0.027 0.03 1.197 0.13 6.457 0.14 8.957 0.22
a b
HPLC
( ) 0.017 0.01 (81%) 0.94 7 0.07 (87.8%) 5.12 7 0.21 (122.1%) 11.31 7 0.37 ( ) (119%) (129%) (89.5%)
() (94%) (102.4%) (113.1%)
0.017 0.02 ( ) 0.98 7 0.09 (98%) 4.95 7 0.32(99%) 9.87 7 0.23 (98.7%)
Each sample was assayed in triplicate. Without spiking STR samples.
be easily extended for use with other small molecules and thus represents a versatile detection method.
Acknowledgements Support by the Research Fund for the National Science Foundation of Fujian Province (2011J06003), the Doctoral Program of Higher Education of China (20103514120003), the National Natural Science Foundation of China (21075019 and 41176079), the ‘‘973’’ National Basic Research Program of China (2010CB732403), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1116) is gratefully acknowledged. Dr. J. Liao (SWU, Chongqing) is thanked for HPLC analysis of real samples for the method-comparison study.
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