Parathion methyl (PM),a type of organophosphate pesticides (OPPs),is widely used for the control of a broad range of pests in agricultural domain over the last several decades. Thus,OPPs played an important role in the increased agricultural productivity . However,OPPs are very toxic to organisms,and their residues have always been one of the most important problems in food security [2, 3]. Consequently,highly sensitive methods for the determination of OPPs in environmental and biological samples are highly desirable. Currently,several analytical methods for the OPPs detection including gas chromatography (GC) ,high performance liquid chromatography (HPLC) ,gas chromatography- mass spectroscopy (GC-MS)  and HPLC-mass spectroscopy ,have achieved low detection levels. However,these methods consume large amount of time and solvent,and require one or more cleanup steps involving liquid-liquid partition or solid-phase extraction [5, 6, 7].
Recently,various biosensors have been reported for the determination of OPPs [8, 9, 10]. Surface plasmon resonance (SPR) sensors have attracted attention in last two decades [11, 12]. SPR is an optical technique that measures changes in the refractive index occurring within approximately several hundred nanometers from the sensor surface . SPR was introduced in the early 1990s as the underlying technology in affinity biosensors for biomolecular interaction analysis . SPR sensors have important applications in the determination of affinity-binding constants [15, 16], monitoring  and diagnostic devices ,and genotype analysis .
Although many methods are used in sensitive SPR sensors,the most effective method is the molecular imprinting technique. The method relies on the principles of molecular recognition. It is a polymerization reaction that occurs around a template molecule. Molecularly imprinted polymers (MIPs),containing specific recognition sites with selectivity for analytes of interest,have various applications such as artificial enzymes ,solid-phase extraction ,bioseparation [21, 22],affinity detoxification  and sensor devices [24, 25, 26, 27].
In this study,we prepared a novel PM-imprinted film on gold surface of SPR chips. To date,although a number of approaches are utilized to prepare the MIF [28, 29, 30],few studies can achieve the low detectable concentration of PM (10-13 mol/L) that is performed using the thermally initiated polymerization reactions. Thus,this method has some important advantages compared to the traditional analytical methods. In addition,the developed SPR shows high sensitivity and high selectivity for the determination of PM. Furthermore,the MIF exhibits good reproducibility in the later experiments.2. Experimental 2.1. Material
Parathion methyl (PM),diuron,tetrachlorvinphose,fenitrothion, ethylene glycol dimethacrylate (EGDMA) were purchased from Aladdin Regent Company (Shanghai,China). Methacrylic acid (MAA) and 2,2-azobis-isobutyronitrile (AIBN) were purchased from J & K Scientific Ltd. (China). Acetonitrile,ethanol and acetic acid were of analytical grades and purchased from Beijing Tongguang Fine Chemicals Company. MAA and EGDMA were distilled under reduced pressure before use. All other reagents except MAA and EGDMA were used as received.2.2. Preparation of MIF
The MIF was prepared by thermo-polymerization on bare gold surface of the SPR substrate. Firstly,parathion methyl (0.025mol/L) and MAA (0.1 mol/L) monomer were mixed in acetonitrile (3mL) at roomtemperature for 2 h. The cross-linker ethylene glycol dimethacrylate (EGDMA,0.1 mol/L) and initiator 2,2-azobis-isobutyronitrile (AIBN,0.013 g/mL) were then added to the pre-polymerization solution,followed by ultra-sonication for 10 min. The SPR substrate was immediately placed on the glass slidewith the Au surface facing downwards and fastened tightly with chips. A piece of parafilm was sandwiched between the two glass slides as a spacer. Polymerization was carried out at 60℃for 6 hin awater bathundernitrogen. TheNIF was synthesized as a reference under identical conditions except for the absence of the template. After polymerization,the film coated on an SPR chip was washed with acetonitrile and ethanol (9:1,v/v) several times and dried under a stream of nitrogen gas.2.3. Detection of PM
The MIF with recognition cavities was immersed in acetonitrile to monitor the template PM. A peristaltic pump was used to pump the analyte or washing solution from a reservoir into the flow cell. Samples were prepared by spiking in acetonitrile with PM at concentrations ranging from 10-13 mol/L to 10-10 mol/L. Afterwards, each sample was pumped through the sensor surface for 20 min for the specific detection of PM,followed by rinsing of the sensor surface with acetonitrile for 10 min. For unspecific binding testing,diuron,tetrachlorvinphose and fenitrothion (at the concentrations of 10-9,10-8,10-7 mol/L) were employed using the same method.3. Results and discussion 3.1. Characterization of MIF
In order to create recognition cavities in the MIF,the template molecule removal experiments were carried out after polymerization. The imprinted parathionmethylmoleculeswere removed from the imprinted polymer films by injecting 5mL of acetonitrile/acetic acid solutions in a volume ratio of 9:1. The SPR angular reflectivity spectra were measured independently before and after the MIF rinse. Typical SPR curves obtained in acetonitrile are shownin Fig. 1. Because the release of parathion methyl templates in the MIF generated a decrease of refractive index,a shift of the coupling angle of theMIF decreasedby 0.5° (from63.1° to62.6°),suggesting that the parathion methyl molecules from MIF were removed.
|Fig. 1.Angular reflectivity of before and after removal of the template PM.|
The adsorption properties of the sensor chip were characterized by SPR spectroscopy. Fig. 2 shows the SPR angular reflectivity spectra after rebinding the PM molecules with various concentrations of MIF (the inset graph is the enlarged range around coupling angles). Solutions of parathion methyl in acetonitrile with a range of concentrations from 10-13 mol/L to 10-10 mol/L were successively injected into the flow cell. The injection of each PM sample lasted for 20 min to reach equilibrium adsorption,and the MIF was rinsed with acetonitrile for 10 min. It can be clearly observed that the angles shifted towards higher values after the adsorption of higher concentrations of PM. It should be noticed that the cavities formed by departure of template PM in the MIF were gradually taken by the PM molecules.
|Fig. 2.SPR angular reflectivity spectra measured after rebinding with various concentrations of PM. The inset graph is the enlarged spectra around the coupling angles.|
As seen in Fig. 3,a plot of the change in resonance angle (△θ) versus the negative logarithmic concentration of PM exhibited a good linear relationship (R2 = 0.996) in the concentration range of 10-13-10-10 mol/L,which further suggested that the sensor chip based on grafted imprinted polymer possessed good adsorption to the template molecules. The error bar in the calibration curve indicated a good reproducibility of the MIF for the recognition of PM. In the experiment,the minimum detectable concentration was 10-13 mol/L.
|Fig. 3.Calibration curve for the resonant dips shift Du versus logarithmic concentration of PM (from 10-13 mol/L to 10-10 mol/L), fitted with linear function, coefficient R2 = 0.996, n = 5.|
In order to investigate the selectivity of the PM imprinted biosensor,we employed diuron,tetrachlorvinphose and fenitrothion as the analogous compounds. The molecular structures are shown in Fig. 4. The concentrations of the analogues used were 10-7,10-8,10-9 mol/L,respectively. The SPR reflectivity changes of MIF and NIF are shown in Fig. 5. The results in Fig. 5A showed that the refractive index had a gradual increase with higher concentrations of the template PM,which was possibly caused by the capture of more template molecules in the cavities in the MIF. Because of the selectivity of the cavities in polymer structure,the MIF has higher adsorption capacity forPM in comparison to diuron, tetrachlorvinphose and fenitrothion. As shown in Fig. 5B,the NIF produced a smaller response to the tested analytes compared to the MIF. These results demonstrated that the PM-imprinted films possessed good selectivity for the PM molecules.
|Fig. 5.Selectivity of SPR sensors: the SPR response of (A) adsorption parathion methyl (10-13, 10-12, 10-11, 10-10 mol/L) and diuron, tetrachlorvinphose and fenitrothion (each of 10-7, 10-8, 10-9 mol/L) on MIF and (B) adsorption parathion methyl, diuron, tetrachlorvinphose and fenitrothion (each of 10-9 mol/L) on NIF.|
In order to show the reproducibility of the MIF,five equilibration-adsorption-regeneration cycles were repeated by 10-10 mol/L and 10-12 mol/L PM respectively. As an example,the reproducibility of the MIF for adsorption 10-10 mol/L PM was shown in Fig. 6. It is can be seen from Fig. 6,PM-imprinted SPR nanosensor has demonstrated reproducible reflectivity responses during the cycles. The reproducibility of the MIF for adsorption 10-12 mol/L PM showed similar result as that of Fig. 6.
|Fig. 6.Reproducibility of the MIF: (A) adsorption and (B) desorption (for 10-10 mol/L PM).|
In summary,a novel SPR sensor containing a PM-imprinted polymer film as a recognition element was developed. We found that the sensor demonstrated high sensitivity and selectivity to detect PM. The MIF exhibited a linear response in the range of 10-13-10-10 mol/L (R2 = 0.996) for the detection of PM. The selectivity experiment of PM and other structurally related analogues indicated that the MIF has strong binding affinity only for the template. The method reported in this work can measure ultra-low concentrations of PM,and this approach may be used to develop field-based SPR sensors combined molecularly imprinted polymer for high-sensitivity detection of other OPPs.Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 20771015) and the National “111” Project of China’s Higher Education ([No. B07012).
|||C.P.Wei, H.Q. Zhou, J. Zhou, Ultrasensitively sensing acephate usingmolecular imprinting techniques on a surface plasmon resonance sensor, Talanta 83 (2011) 1422-1427.|
|||C.M. Torres, Y. Picó, J. Mañes, Determination of pesticide residues in fruit and vegetables, J. Chromatogr. A 754 (1996) 301-331.|
|||L. Pogačnik, M. Franko, Detection of organophosphate and carbamate pesticides in vegetable samples by a photothermal biosensor, Biosens. Bioelectron. 18 (2003) 1-9.|
|||A. Cappiello, G. Famiglini, P. Palma, F. Mangani, Trace level determination of organophosphorus pesticides in water with the new direct-electron ionization LC/MS interface, Anal. Chem. 74 (2002) 3547-3554.|
|||L. Chimuka, M. Van Pinxteren, J. Billing, E. Yilmaz, J.Å. Jönsson, Selective extraction of triazine herbicides based on a combination of membrane assisted solvent extraction and molecularly imprinted solid phase extraction, J. Chromatogr. A 1218 (2011) 647-653.|
|||D. Djozan, B. Ebrahimi, M. Mahkam, M.A. Farajzadeh, Evaluation of a new method for chemical coating of aluminum wire with molecularly imprinted polymer layer. Application for the fabrication of triazines selective solid-phase microextraction fiber, Anal. Chim. Acta 674 (2010) 40-48.|
|||L. Pallaroni, C. Von Holst, Determination of zearalenone from wheat and corn by pressurized liquid extraction and liquid chromatography-electrospray mass spectrometry, J. Chromatogr. A 993 (2003) 39-45.|
|||A.A. Ciucu, C. Negulescu, R.P. Baldwin, Determination of pesticides using an amperometric biosensor based on ferophthalocyanine chemically modified carbon paste electrode and immobilized bienzymatic system, Biosens. Bioelectron. 18 (2003) 303-310.|
|||Y.D. Zhang, S.B. Muench, H. Schulze, et al., Disposable biosensor test for organophosphate and carbamate insecticides in milk, J. Agric. Food Chem. 53 (2005) 5110-5115.|
|||Y.H. Qu, H. Min, Y.Y. Wei, et al., Au-TiO2/Chit modified sensor for electrochemical detection of trace organophosphates insecticides, Talanta 76 (2008) 758-762.|
|||Q.Q. Wei, T.X. Wei, A novel method to prepare SPR sensor chips based on photografting molecularly imprinted polymer, Chin. Chem. Lett. 22 (2011) 721-724.|
|||Y. Wang, T.X. Wei, Surface plasmon resonance sensor chips for the recognition of bovine serum albumin via electropolymerized molecularly imprinted polymers, Chin. Chem. Lett. 24 (2013) 813-816.|
|||J. Homola, Surface plasmon resonance sensors for detection of chemical and biological species, Chem. Rev. 108 (2008) 462-493.|
|||M. Malmqvist, BIACORE: an affinity biosensor system for characterization of biomolecular interactions, Biochem. Soc. Trans. 27 (1999) 335-340.|
|||L.X. Zhang, J.Y. Liu, E.K. Wang, A new method for studying the interaction between chlorpromazine and phospholipid bilayer, Biochem. Biophys. Res. Commun. 373 (2008) 202-205.|
|||L. Quan, D.G. Wei, X.L. Jiang, et al., Resurveying the Tris buffer solution: the specific interaction between tris(hydroxymethyl)aminomethane and lysozyme, Anal. Biochem. 378 (2008) 144-150.|
|||C.F. Mandenius, R.H. Wang, A. Aldén, et al., Monitoring of influenza virus hemagglutinin in process samples using weak affinity ligands and surface plasmon resonance, Anal. Chim. Acta 623 (2008) 66-75.|
|||G. Hayashi, M. Hagihara, K. Nakatani, Genotyping by allele-specific L-DNA-tagged PCR, J. Biotechnol. 135 (2008) 157-160.|
|||F. Bonini, S. Piletsky, A.P.F. Turner, A. Speghini, A. Bossi, Surface imprinted beads for the recognition of human serum albumin, Biosens. Bioelectron. 22 (2007) 2322-2328.|
|||C. Esen,M. Andac, N. Bereli, et al., Highly selective ion-imprinted particles for solid-phase extraction of Pb2+ ions, Mater. Sci. Eng. C: Mater. 29 (2009) 2464-2470.|
|||Y. Saylan, M.M. Sari, S. Özkara, L. Uzun, A. Denizli, Hydrophobic microbeads as an alternative pseudo-affinity adsorbent for recombinant human interferon-alpha via hydrophobic interactions, Mater. Sci. Eng. C: Mater. 32 (2012) 937-944.|
|||S. Asliyuce, L. Uzun, R. Say, A. Denizli, Immunoglobulin G recognition with Fab fragments imprintedmonolithic cryogels: evaluation of the effects ofmetal-ion assisted-coordination of template molecule, React. Funct. Polym. 73 (2013) 813-820.|
|||G. Ertürk, L. Uzun, M.A. Tümer, R. Say, A. Denizli, Fab fragments imprinted SPRbiosensor for real-time human immunoglobulin G detection, Biosens. Bioelectron. 22 (2007) 2322-2328.|
|||H.X. Hao, H. Zhou, J. Chang, J. Zhu, T.X. Wei, Molecularly imprinted polymers for highly sensitive detection of morphine using surface plasmon resonance spectroscopy, Chin. Chem. Lett. 22 (2011) 477-480.|
|||V.K. Gupta, M.L. Yola, N.Özaltın, et al., Molecularimprinted polypyrrole modified glassy carbon electrode for the determination of tobramycin, Electrochim. Acta 112 (2013) 37-43.|
|||M.L. Yola, L. Uzun, N. Özaltın, A. Denizli, Development of molecular imprinted nanosensor for determination of tobramycin in pharmaceuticals and foods, Talanta 120 (2014) 318-324.|
|||M.L. Yola, T. Eren, N. Atar, Molecular imprinted nanosensor based on surface plasmon resonance: application to the sensitive determination of amoxicillin, Sens. Actuators: B 195 (2014) 28-35.|
|||M. Frasconi, R. Tel-Vered, M. Riskin, I. Willner, Surface plasmon resonance analysis of antibiotics using imprinted boronic acid-functionalized Au nanoparticle composites, Anal. Chem. 82 (2010) 2512-2519.|
|||B.M. Riskin, R. Tel-Vered, I. Willner, Imprinted Au-nanoparticle composites for the ultra-sensitive surface plasmon resonance detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), Adv. Mater. 22 (2010) 1387-1391.|
|||R.B. Pernites, R.R. Ponnapati, R.C. Advincula, Surface plasmon resonance (SPR) detection of theophylline via electropolymerized molecularly imprinted polythiophenes, Macromolecules 43 (2010) 9724-9735.|