Biosensors and Bioelectronics 56 (2014) 97–103
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Photoelectrochemical sensor for pentachlorophenol on microfluidic paper-based analytical device based on the molecular imprinting technique Guoqiang Sun a, Panpan Wang a, Shenguang Ge b, Lei Ge a, Jinghua Yu a, Mei Yan a,n a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 10 October 2013 Received in revised form 29 December 2013 Accepted 1 January 2014 Available online 10 January 2014
Combining microfluidic paper-based analytical device (μ-PAD) and the molecular imprinting technique, a visible light photoelectrochemical (PEC) sensing platform for the detection of pentachlorophenol (PCP) was established on gold nanoparticles (AuNPs) decorated paper working electrode using polypyrrolefunctionalized ZnO nanoparticles. Ascorbic acid (AA) was exploited as an efficient and nontoxic electron donor for scavenging photogenerated holes under mild solution medium and facilitating the generation of stable photocurrent. The microfluidic molecular imprinted polymer-based PEC analytical origami device is developed for the detection of PCP in the linear range from 0.01 ng mL 1 to 100 ng mL 1 with a low detection limit of 4 pg mL 1. This disposable microfluidic PEC origami device would provide a new platform for sensitive, specific, and multiplex assay in public health, environmental monitoring, and the developing world. & 2014 Elsevier B.V. All rights reserved.
Keywords: Microfluidic paper-based analytical device Photoelectrochemical Molecular imprinting technique Pentachlorophenol
1. Introduction Pentachlorophenol (PCP) is a xenobiotic causing great environmental concern (Abramovitch and Capracotta, 2003). It enters into the environment as byproducts of industrial processes, such as production of antioxidants, dyes, and drugs, the chlorination of drinking water, and the chlorinated bleaching of paper. Furthermore, it can accumulate in living organisms and result in negative effects, including carcinogenicity and acute toxicity. Different analytical procedures based on thin layer chromatography (TLC) (Gremaud and Turesky, 1997), gas chromatography–mass spectrometry (GC–MS) (Mardones et al., 2003) and gas chromatography (GC) (Leblance et al., 1999) have been reported for the determination of PCP. However these methods, although highly sensitive and specific, are of high costs, time-consuming labor requirements, and the restriction of a limited analyte spectrum. Recently, photoelectrochemical (PEC) immunosensor has been established for the detection of PCP (Kang et al., 2010). The PEC sensor is considered to be a more sensitive technique with simple instrument and low cost to electronic detection. Meanwhile, while the PEC immunoassay showed promise, it relied on antibodies. Nevertheless, these biological recognition elements have some fundamental drawbacks, for instance, possible denaturation and instability during manufacture and transportation. To overcome
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Corresponding author. Tel.: þ 86 531 82767161. E-mail addresses: [email protected], [email protected] (M. Yan).
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the above limitations, a molecular imprinting technique, the design and construction of biomimetic receptor system with predetermined recognition for target molecule, has been proposed and developed rapidly (Tada et al., 2004; Kempe and Kempe, 2006). Owing to their high chemical and physical stability, ease of preparation, and low price, the synthesized molecularly imprinted polymers (MIPs) have widely been used as recognition elements for the development of chemical/biological sensors (Yang et al., 2009; Jin and Tang, 2009). In conventional immunoassays, commercialized electrodes were used, which was expensive, sometimes inconvenient. As screen-printed paper-electrodes can be simple, portable, lowcost, disposable and practical than commercialized ones (Nie et al., 2010; Delaney et al., 2011), it has become a preferred electrode material for biosensors. Microfluidic paper-based analytical devices (μ-PADs) based on screen-printed paper-electrodes, which combine the simplicity and low-cost of paper strip tests and the complexity of the conventional lab-on-chip devices, have been attracted more and more attention during the past five years (Zhao et al., 2008; Abe et al., 2008; Dungchai et al., 2009). Much effort has been directed toward the development of fabrication (Carrilho et al., 2009), functionalization (Hwang et al., 2011) and quantitative methods (Martinez et al., 2007) for μ-PADs. In the present work, MIPs-based sensor was established on a paper-based device which was based on low-cost screen-printed paper-electrodes. The gold nanoparticles (AuNPs) decorated paper working electrode (Au-PWE) was made through AuNPs layer growth on the surface of cellulose. Then, the synthesized ZnO
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spheres were attached to Au-PWE with the aid of 4-aminothiophenol (PATP) and a layer of polypyrrole (Ppy) was grafted on ZnO surface by electropolymerization. After the removal of PCP, the fabricated microfluidic MIPs-based origami device was used to detect PCP.
2. Experimental 2.1. Materials Zinc nitrate hexahydrate (Zn(NO3)2 6H2O), hexamethyleneteramine (HMT, (CH2)6N4), tripotassium citrate monohydrate (HOC (COOK)(CH2COOK)2 H2O), aniline, ascorbic acid (AA) and ammonia hydroxide (25 wt% NH3 in water) were purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. 0.1 mol L 1 AA was used as a blank solution for photocurrent measurements, which was degassed by highly pure nitrogen for 10 min before PEC experiments. Tetrachloroauric acid (HAuCl4 4H2O) was purchased from Sigma Chemical Co. (St. Louis, MO, USA) which used as a precursor for the formation of AuNPs seeds and growth solution. PATP was obtained from Acros Organics Chemical Co. Ultrapure water obtained from a Millipore water purification system (resistivity Z18.2 MΩ cm, Milli-Q, Millipore) was used in all assays and solutions. Whatman chromatography paper #1 (200.0 mm 200.0 mm) (pure cellulose paper) was obtained from GE Healthcare Worldwide (Pudong Shanghai, China) and used with further adjustment of size (A4 size). Pyrrole was distilled repeatedly under vacuum until a colorless liquid was obtained and kept under nitrogen in darkness at 4 1C in refrigerator. Other reagents were commercially available as analytical reagent grade and used without further purification.
2.2. Apparatus Scanning electron microscopy (SEM) images were recorded using a JEOL-JSM-6300 scanning electron microscope. Electrochemical impedance spectroscopy (EIS) was performed on a CHI 604D Electrochemical Workstation (Shanghai CH Instruments Inc., China). PEC measurements were performed with a home-built PEC system. A 500 W xenon arc lamp (CHF-XQ-500W, Beijing Changtuo Co. Ltd.) equipped with a monochromator was used as an irradiation source. Photocurrent was measured by the current– time curve experimental technique on a CHI660D electrochemistry workstation (Shanghai CH Instruments Co., China) with a three-electrode system: a modified paper-based electrode as the working electrode, a printing carbon and Ag/AgCl electrode were used as a counter electrode and reference electrode, respectively. All the photocurrent measurements were performed at a constant potential of 0 V (versus Ag/AgCl). All experiments were carried out at room temperature.
2.3. Synthesis of ZnO spheres ZnO nanospheres were prepared according to the literature protocol with some modifications (Cho et al., 2011). In brief, an aqueous solution of 10 mmol L 1 zinc nitrate hexahydrate, 5 mmol L 1 HMT was prepared at room temperature. Then, 2.5 mmol L 1 tripotassium citrate monohydrate was added and dissolved by sonication. After the reaction at 90 1C for 20 min, the powders were acquired by centrifugation and dried in an oven at 60 1C. The as-prepared powders (0.07 g) were dissolved in 100 mL water with mild sonication. 91 μL ammonia was dropwise added and the solution was maintained at 90 1C for 1 h. After the reaction, the products were acquired by centrifugation at 10,000 rpm, subsequently, washed with water for three times and dried in an oven at 60 1C.
2.4. Fabrication of the molecularly imprinted PEC sensor on
μ-PAD
Scheme 1 shows the fabrication process of the microfluidic origami PEC device (the fabrication details could be found in the supporting information). To fabricate Au-PWE, an AuNPs layer was grown on the surface of cellulose fibers in the paper sample zone of PWE (Scheme 1A) to enhance the conductivity and enlarge the effective surface area of bare PWE. Firstly, the suspension of AuNPs seeds was prepared by using NaBH4 as a reductant and stabilized with sodium citrate according to the literature (Busbee et al., 2003). Then, 15.0 μL as-prepared AuNPs seeds solution was dropped into the paper sample zone of bare PWE. Then the origami device was equilibrated at room temperature for 1 h to optimize the surface immobilization of AuNPs seeds on cellulose fibers. After rinsing with water thoroughly according to the method in our previous work (Yan et al., 2012) to remove loosely bound AuNPs seeds, 15 μL freshly prepared growth aqueous solution of 10 mmol L 1 PBS (pH 7.0) containing 1.2 mmol L 1 HAuCl4, 2.0 mmol L 1 cetyltrimethylammonium chloride and 7.2 mmol L 1H2O2 for seeds growth were applied into the AuNPs seeded PWE, and incubated at room temperature for 15 min. Subsequently, the resulting porous Au-PWE was washed with water thoroughly. Thus a layer of interconnected AuNPs on cellulose fibers with good conductivity were obtained, which were dried at room temperature for 20 min (Scheme 1B). The ZnO/PATP composite was prepared by the following procedure: ZnO spheres were dissolved in 0.1 mmol L 1 PATP ethanol solution and the mixture was stirred for 1 h. Then the precipitate was centrifuged and rinsed with ethanol three times. For obtaining ZnO/Au-PWE assemblies, the prepared ZnO/PATP was dissolved in ethanol and dripped to the electrode surface (Scheme 1C). Finally, the electrode was rinsed with water and dried at room temperature.
Scheme 1. The fabrication process of the paper-based PEC sensor.
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The electropolymerization of pyrrole was performed on the
μ-PAD which was described in Scheme 1C. The aqueous electrolyte
solution comprised of 0.1 moL L 1 KCl, 0.5 mmoL L 1 pyrrole, 0.05 moL L 1 PBS (pH 6.86) and 10 mmoL L 1 PCP. After deoxygenating the solution by bubbling nitrogen gas for about 15 min, the electropolymerization (Scheme 1D) was performed by cyclic voltammetry in the potential range of 1.3 V to þ1.0 V, with a scan rate of 50 mV s 1 and 15 sweep cycles. After the electropolymerization process, the electrode was electrochemically treated at 1.3 V for 20 min in 0.2 mol L 1 K2HPO4 solution to remove template molecules (Xie et al., 2010). Then the molecularly imprinted PEC sensor (MIP/ZnO/Au-PWE) with empty sites for PCP recognition was obtained (Scheme 1E). As a control, the nonimprinted polymer (NIP) on ZnO modified Au-PWE (NIP/ZnO/AuPWE) was prepared in the same way except that the template molecules were omitted.
2.5. PEC activity determination The PEC detection procedures were described below. 20 μL spiked samples containing different concentrations of PCP were added into the electrode surface, and allowed to be absorbed for 200 s at room temperature to reach the equilibrium binding, followed by washing with water to prevent non-specific binding and to achieve the best possible signal-to-background ratio. The photocurrent was measured in 0.1 mol L 1 AA PBS solution (pH 7.0) under the 450 nm excited wavelength.
3. Results and discussions 3.1. Characterization of ZnO, Au-PWE, and MIP/ZnO/Au-PWE Fig. 1A showed the SEM image of ZnO sphere. The average diameter of these spherical structures was 300 nm. Visibly, the ZnO sphere we synthesized had a spherical shape with a large specific surface area which provides a good environment for the growth of Ppy. The Au-PWE was developed through the growth of an interconnected AuNPs layer on the surface of cellulose fibers in the paper sample zone to enhance the conductivity of paper sample zone. Fig. 1B showed that paper with porous structure and a high ratio of surface area to weight (9.5 m2 g 1) which could offer an excellent adsorption microenvironment for the AuNPs seeds. The AuNPs seeds were rapidly enlarged by incubating in the growth solution under the self-catalytic reduction mechanism of AuNPs growth. A continuous and dense conducting AuNPs layer with interconnected AuNPs was obtained completely on the cellulose fiber surfaces after 15 min of growth (Fig. 1C, D). In Fig. 1, the SEM images of ZnO/Au-PWE (Fig. 1E) and MIP/ ZnO/Au-PWE (Fig. 1F) were shown. Compared with the SEM image of Au-PWE, we can see apparent ZnO spheres on Au-PWE surface. There is no obvious change of the size of neat ZnO after the polymerization of Ppy.
3.2. Electrochemical and PEC characteristics of MIP/ZnO/Au-PWE Electrochemical impedance spectroscopy (EIS) was used to monitor the interfacial properties of surface-modified electrode. This method was used to get information on the impedance changes of the sensor interface in the modification process and the EIS of variously modified electrodes were monitored in a solution of 5 mmol L 1 [Fe(CN)6]3 /4 and 0.1 mol L 1 KCl at a bias potential of 0.17 V. The frequency range was 100 MHz to 10 kHz. The typical impedance spectrum includes a semicircle
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portion at higher frequencies that corresponds to the electrontransfer resistance (Ret) and the linear part at lower frequencies that corresponds to the diffusion process. Fig. 2A shows the EIS (presented in the form of the Nyquist plot) of different surface conditions of the cellulose fibers in the PWE. The EIS of the bare PWE revealed a relatively small semicircle domain (curve a). After being coated with AuNPs layer, the interfacial resistance decreased (curve b), confirming the successful immobilization of AuNPs and it owns a high electronic conductivity. When the Au-PWE was modified with ZnO spheres with the aid of PATP, the resistance was greatly increased (curve c), which suggested that the ZnO spheres were successfully immobilization on the surface. Because the formed ZnO spheres block the electron transfer between the redox probe and electrode, so the charge transfer resistance increased. A remarkable increase in Ret value was observed after the electropolymerization of Ppy (curve d). This result indicated that Ppy membranes of low conductivity acted as a definite kinetic barrier for the charge transfer. In contrast, after the removal of PCP, the impedance decreased accordingly (curve e). The reason may be that the unoccupied binding cavities in the MIP layer are available for the hexacyanoferrate redox probe to diffuse through after the removal of the template from the MIP layer. As shown in Fig. 2B, curves a and b were the photocurrent responses of Au-PWE and ZnO/Au-PWE in 0.1 mol L 1 AA, respectively. Curves c and d were the photocurrent responses of MIP/ ZnO/Au-PWE in 0.1 mol L 1 AA after addition of different concentrations of PCP solution. Curve e was the photocurrent response of NIP/ZnO/Au-PWE in 0.1 mol L 1 AA after addition of PCP solution (10 ng mL 1). The ZnO/Au-PWE was excited by 365 nm wavelength light. The other electrodes were excited by 450 nm wavelength light. Almost no photocurrent was observed there upon irradiation of the Au-PWE (curve a). After being coated with ZnO spheres, the photocurrent was increased (curve b). As a photoelecchemically active material, ZnO owns good carrier mobility; the wide band gap of ZnO only allows it to absorb the ultraviolet light, after being illuminated with 365 nm wavelength light. The photogenerated electrons transfer to the Au-PWE, that showed an increased photocurrent compared to the Au-PWE before modified by ZnO. The photocurrent of MIP/ZnO modified Au-PWE (curves c and d) and NIP/ZnO modified Au-PWE (curve e) increased compared to that of ZnO modified electrode. This may result from the conjugation effect of Ppy, which can accelerate the electrontransfer and enhance the photocurrent. Furthermore, the photocurrent of MIP/ZnO/Au-PWE decreased with the increasing of the concentration of PCP (curve c). Therefore, it could be applied to sensitive determination of PCP in this disposable microfluidic PEC origami device. Meanwhile, the NIP/ZnO modified Au-PWE showed a higher photocurrent, the reason was that the recognition sites on MIP/ZnO/Au-PWE could specifically absorb PCP, which results in an increase in the steric hindrance toward the diffusion of quencher molecules and/or photogenerated holes on the electrode interface, and consequently a decrease in photocurrent. To compare the PEC performance built on different substrates, a control PEC sensor was fabricated on an indium tin oxide (ITO)coated glass electrode with the same area (6 mm in diameter) using an identical process. As shown in Fig. 2C, the sensor fabricated on the PWE exhibited superior PEC performance to the sensor based on ITO. The photocurrent of the MIP/ZnO/ITO (curve a) was lower (roughly 4-fold) than that in the PWE (curve b), which was mainly attributed to the enhanced immobilization capacity for Ppy/ZnO/Au in the modified macroporous paper sample zone with the 3D interwoven cellulose fiber network as well as the effective light transmission across the total thickness of paper sample zone which was filled with solution, resulting in the increased total number of excited Ppy/ZnO/Au in the whole PWE.
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Fig. 1. SEM images of (A) ZnO sphere; (B) bare paper sample zone of the PWE; (C) Au-PWE; (D) magnification SEM image of Au-PWE; SEM images of (E) ZnO/Au-PWE; and (F) MIP/ZnO/Au-PWE.
3.3. Optimization of experimental conditions To evaluate the influence of template molecules concentration, different concentrations of PCP was selected in the electropolymerizations of pyrrole. After removal of the template molecules, the fabricated sensors were used to detect PCP (10 ng mL 1). The results are given in Fig. 3A. When the concentration of template molecule (PCP) was below 10 mmol L 1, the photocurrent increased with increasing template molecule content due to an increase in the number of recognition cavities. However, a little decrease in the photocurrent could be obtained in the presence of
PCP concentration above 10 mmol L 1, which may be due to the interaction and impact between template molecules at a higher concentration of PCP in the process of PCP-MIPs fabrication and may cause a decrease of valid recognition cavities. So an optimized template molecule concentration of 10 mmol L 1 was selected. As is well-known, AA is a powerful antioxidant used widely in the food industry and for disease treatment because it is in general regarded to be safe and healthy. The formal potential of the redox couple of AA is 0.185 V (versus SCE), it can be easily oxidized by the holes in Ppy and facilitate the generation of stable photocurrent. Thus, AA was chosen as an electron donor in the experiment.
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Fig. 2. (A) EIS of the PWE under different condition in 5 mmol L 1 [Fe(CN)6]3 /4 solution containing 0.1 mol L 1 KCl: (a) bare PWE, (b) Au-PWE, (c) ZnO/Au-PWE, (d) molecularly imprinted Ppy/ZnO/Au-PWE, (e) the electrode ‘d’ after removal of PCP; (B) photocurrent responses of (a) Au-PWE, (b) ZnO/Au-PWE in 0.1 mol L 1 AA, (c) MIP/ZnO/Au-PWE in 0.1 mol L 1 AA after the addition of 10 ng mL 1 PCP, (d) MIP/ZnO/Au-PWE in 0.1 mol L 1 AA after addition of 7 ng mL 1 PCP, (e) NIP/ZnO/Au-PWE in 0.1 mol L 1 AA after the addition of 10 ng mL 1 PCP; (C) Photocurrent responses of (a) MIP/ZnO/ITO, (b) MIP/ZnO/Au-PWE in 0.1 mol L 1 AA after the addition of 10 ng mL 1 PCP for 200 s under 450 nm wavelength light excited.
Fig. 3. Effects of (A) template molecules concentration and (B) AA concentration on photocurrent response.
Fig. 4. (A) Photocurrent responses at MIP/ZnO/Au-PWE electrode in 0.1 mol L 1 AA PBS solution (pH 7.0) in the presence of 0.01, 0.1, 0.5, 1, 5, 10, 50, 100 ng mL 1 PCP (from left to right); and (B) linear calibration curve.
In the experiments, changing the concentration of AA, different photocurrents were obtained. In order to obtain high and stable photocurrent under the same conditions, the sensor was used to detect PCP (10 ng mL 1) to optimize the concentration of AA. The intensity of the photocurrent increased with the increased concentrations of added electron donor AA and reached a maximum for 0.1 mol L 1 AA, then a weak decrease was observed for much higher concentrations of AA (see Fig. 3B). As a hole scavenger, AA in lower concentrations would result in a fall in electrical output because fewer reducing agent molecules were available for electron donation to photogenerated holes. While a much higher concentration of AA would also result in the increase of the absorbance of AA in solution, as a consequence, the intensity of
the irradiation arriving at the electrode surface decreased and the efficiency of excited Ppy would decrease. Other experimental conditions were also optimized. The potential and time of removing PCP, the electropolymerization time (sweep cycles) and the absorption time of PCP were usually important parameters to enhance the sensitivity of the sensor. 1.3 V and 20 min were chosen as the potential and time of removing PCP, respectively (Figs. S2A and S2B). In order to achieve the best selectivity and sensitivity, the thickness of Ppy was also optimized by adjusting the scanning cycles (Fig. S2C). From the Fig. S2C, we know that the most suitable sweep cycle was 15. The absorption time is an important parameter for the improvement of assay efficiency on the microfluidic PEC origami device. When the
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Table 1 Measurement results of PCP in real samples. Samples
Proposed methoda (ng mL 1)
GC–MSa (ng mL 1)
Added (ng mL 1)
Detected (ng mL 1)a
Recovery (%)
1 2 3 4
0.125 0.130 0.132 0.129
0.126 0.134 0.128 0.131
0.1 0.5 10 50
0.223 0.635 9.992 51.879
98.0 101.0 98.6 103.5
a
Average of 11 measurements.
absorption time was above 200 s, the photocurrent remained constant (Fig. S2D), which implied that the adsorption equilibrium was reached on this experimental condition. Thus, the optimum absorption time should be 200 s for the determination of PCP. 3.4. Analytical performance The analytical performance of MIP/ZnO/Au-PWE was verified by using samples of standard PCP solution at various concentrations at an applied potential of 0 V with visible light irradiation. Fig. 4A displays the typical photocurrent responses of the PEC biosensor in the presence of different concentrations of PCP. Under the optimal conditions, with the increasing concentration of PCP, the photocurrent decreases over a wide concentration range of 0.01–100 ng mL 1. Fig. 4B shows the derived calibration curve. The linear regression equation was I (μA)¼ 30.66 14.57 log cPCP (ng mL 1) with a correlation coefficient (R) of 0.9985. The detection limit was estimated to be 4 pg mL 1 at a signal-to-noise ratio of 3s (where s is the standard deviation of the blank, n ¼11). Obviously, the proposed sensor shows promise for application in the monitoring of PCP with its low detection limit and wide linear range. The PEC mechanism of the sensor we fabricated for PCP and the comparison with other pesticide sensors are summarized in Supporting information. 3.5. Stability, reproducibility, and selectivity of the PEC sensor The long-term stability of the PEC immunosensor was also examined. To investigate the stability of the modified electrode, continuous detection was carried out (10 min), and only 2.69% decrease of the initial response was observed. When the immunosensor was dried and stored at 4 1C over four weeks, no apparent change in photocurrent response in the same PCP concentration (10 ng mL 1) was found, illustrating its good longterm storage stability. There may be two reasons responsible for the excellent stability. (1) The porous structure of the paper based electrode, which can fix a mass of ZnO and Ppy. (2) The cross-link structure of Ppy is formed during the polymerization process, which makes Ppy particularly stable and further stabilizes the PEC sensor. The reproducibility of an assay was expressed in terms of values for a between-batch (interassay) relative standard deviation (RSD). The interassay RSD was estimated by measuring the same concentration of PCP with four immunosensors prepared independently at the identical experimental conditions. The interassay RSD obtained from 10 ng mL 1 of PCP was 4.9–6.2%. The results indicated a satisfactory precision and reproducibility of the proposed protocol. Because PCP is one of the most common environmental pollutants, the effects of other pollutants as interfering species on the PEC sensing response were examined. To evaluate the specificity of the MIP/ZnO/Au-PWE, 2,4-dichlorophenoxyacetic acid (2,4-D), aldrin, heptachlor, chlopyrifos (we use a, b, c and d representative these substances, respectively while adopt A representative PCP) were chosen as the interfering substances, and the i–t technique was employed to detect the photocurrent for
different systems on the PCP sensor. The signal was compared by assaying PCP (10 ng mL 1) with interfering agents (1 μg mL 1). The result was listed in Fig. S1 in Supporting information; significantly higher current response was observed with the target PCP than with other pesticides. When PCP coexisted with these sample interfering agents, no apparent signal change took place in comparison with that of only PCP even when the concentration of the interfering substance is 100 times that of PCP. These results indicate that the sensor was sufficiently selective for the detection of PCP. 3.6. Analysis of real samples The analytical reliability and applicable potentiality of the PEC immunoassay were evaluated by testing PCP in spiked samples containing different pollutants or prepared with pure drinking water and river water. As shown in Table 1, the results detected by the proposed method also accorded very well with those by GC– MS. The recoveries of 0.1, 0.5, 1.0, 10.0, and 50.0 ng mL 1 of PCP were determined by standard addition methods. The recoveries of added PCP can be quantitative and t-tests showed that there was no significant difference between recovery efficiency and was 100% at a confidence level of 95%. It can be concluded that the proposed method can offer accurate results and an analytical performance as good as the GC–MS. These data revealed that this method was comparable and acceptable for PCP detection, which means the developed immunoassay may provide a promising alternative tool for determining PCP in real samples.
4. Conclusion In summary, a disposable microfluidic PEC origami device, combining the simple, disposable, and low cost of μ-PAD and stability, and high selectivity of molecular imprinting technique, was developed here through PEC measurement. The Au-PWE was made through AuNPs layer growth on the surface of cellulose. Then the ZnO nanoparticles were modified to the electrode surface with the aid of PATP, the PATP could connect AuNPs and ZnO as it owned sulfydryl and amino. A layer of Ppy was grafted on the ZnO surface through the simple electropolymerization method. After the removal of the template molecule from the resulting crosslinked Ppy matrix the recognition sites to the PCP was generated, which can now specifically recognize and bind PCP. The paperbased PEC sensor with a short absorption time, exhibited good precision, acceptable stability and reproducibility, and could be used for the detection of PCP in real samples. We anticipate that this method can be extended for the determination of other proteins, pesticides and pollutants.
Acknowledgments This work was financially supported by the Natural Science Research Foundation of China (21175058, 21277058, 51273084);
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Photoelectrochemical sensor for pentachlorophenol on microfluidic paper-based analytical device based on the molecular imprinting technique Guoqiang Sun a, Panpan Wang a, Shenguang Ge b, Lei Ge a, Jinghua Yu a, Mei Yan a,n a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, University of Jinan, Jinan 250022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 10 October 2013 Received in revised form 29 December 2013 Accepted 1 January 2014 Available online 10 January 2014
Combining microfluidic paper-based analytical device (μ-PAD) and the molecular imprinting technique, a visible light photoelectrochemical (PEC) sensing platform for the detection of pentachlorophenol (PCP) was established on gold nanoparticles (AuNPs) decorated paper working electrode using polypyrrolefunctionalized ZnO nanoparticles. Ascorbic acid (AA) was exploited as an efficient and nontoxic electron donor for scavenging photogenerated holes under mild solution medium and facilitating the generation of stable photocurrent. The microfluidic molecular imprinted polymer-based PEC analytical origami device is developed for the detection of PCP in the linear range from 0.01 ng mL 1 to 100 ng mL 1 with a low detection limit of 4 pg mL 1. This disposable microfluidic PEC origami device would provide a new platform for sensitive, specific, and multiplex assay in public health, environmental monitoring, and the developing world. & 2014 Elsevier B.V. All rights reserved.
Keywords: Microfluidic paper-based analytical device Photoelectrochemical Molecular imprinting technique Pentachlorophenol
1. Introduction Pentachlorophenol (PCP) is a xenobiotic causing great environmental concern (Abramovitch and Capracotta, 2003). It enters into the environment as byproducts of industrial processes, such as production of antioxidants, dyes, and drugs, the chlorination of drinking water, and the chlorinated bleaching of paper. Furthermore, it can accumulate in living organisms and result in negative effects, including carcinogenicity and acute toxicity. Different analytical procedures based on thin layer chromatography (TLC) (Gremaud and Turesky, 1997), gas chromatography–mass spectrometry (GC–MS) (Mardones et al., 2003) and gas chromatography (GC) (Leblance et al., 1999) have been reported for the determination of PCP. However these methods, although highly sensitive and specific, are of high costs, time-consuming labor requirements, and the restriction of a limited analyte spectrum. Recently, photoelectrochemical (PEC) immunosensor has been established for the detection of PCP (Kang et al., 2010). The PEC sensor is considered to be a more sensitive technique with simple instrument and low cost to electronic detection. Meanwhile, while the PEC immunoassay showed promise, it relied on antibodies. Nevertheless, these biological recognition elements have some fundamental drawbacks, for instance, possible denaturation and instability during manufacture and transportation. To overcome
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the above limitations, a molecular imprinting technique, the design and construction of biomimetic receptor system with predetermined recognition for target molecule, has been proposed and developed rapidly (Tada et al., 2004; Kempe and Kempe, 2006). Owing to their high chemical and physical stability, ease of preparation, and low price, the synthesized molecularly imprinted polymers (MIPs) have widely been used as recognition elements for the development of chemical/biological sensors (Yang et al., 2009; Jin and Tang, 2009). In conventional immunoassays, commercialized electrodes were used, which was expensive, sometimes inconvenient. As screen-printed paper-electrodes can be simple, portable, lowcost, disposable and practical than commercialized ones (Nie et al., 2010; Delaney et al., 2011), it has become a preferred electrode material for biosensors. Microfluidic paper-based analytical devices (μ-PADs) based on screen-printed paper-electrodes, which combine the simplicity and low-cost of paper strip tests and the complexity of the conventional lab-on-chip devices, have been attracted more and more attention during the past five years (Zhao et al., 2008; Abe et al., 2008; Dungchai et al., 2009). Much effort has been directed toward the development of fabrication (Carrilho et al., 2009), functionalization (Hwang et al., 2011) and quantitative methods (Martinez et al., 2007) for μ-PADs. In the present work, MIPs-based sensor was established on a paper-based device which was based on low-cost screen-printed paper-electrodes. The gold nanoparticles (AuNPs) decorated paper working electrode (Au-PWE) was made through AuNPs layer growth on the surface of cellulose. Then, the synthesized ZnO
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spheres were attached to Au-PWE with the aid of 4-aminothiophenol (PATP) and a layer of polypyrrole (Ppy) was grafted on ZnO surface by electropolymerization. After the removal of PCP, the fabricated microfluidic MIPs-based origami device was used to detect PCP.
2. Experimental 2.1. Materials Zinc nitrate hexahydrate (Zn(NO3)2 6H2O), hexamethyleneteramine (HMT, (CH2)6N4), tripotassium citrate monohydrate (HOC (COOK)(CH2COOK)2 H2O), aniline, ascorbic acid (AA) and ammonia hydroxide (25 wt% NH3 in water) were purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. 0.1 mol L 1 AA was used as a blank solution for photocurrent measurements, which was degassed by highly pure nitrogen for 10 min before PEC experiments. Tetrachloroauric acid (HAuCl4 4H2O) was purchased from Sigma Chemical Co. (St. Louis, MO, USA) which used as a precursor for the formation of AuNPs seeds and growth solution. PATP was obtained from Acros Organics Chemical Co. Ultrapure water obtained from a Millipore water purification system (resistivity Z18.2 MΩ cm, Milli-Q, Millipore) was used in all assays and solutions. Whatman chromatography paper #1 (200.0 mm 200.0 mm) (pure cellulose paper) was obtained from GE Healthcare Worldwide (Pudong Shanghai, China) and used with further adjustment of size (A4 size). Pyrrole was distilled repeatedly under vacuum until a colorless liquid was obtained and kept under nitrogen in darkness at 4 1C in refrigerator. Other reagents were commercially available as analytical reagent grade and used without further purification.
2.2. Apparatus Scanning electron microscopy (SEM) images were recorded using a JEOL-JSM-6300 scanning electron microscope. Electrochemical impedance spectroscopy (EIS) was performed on a CHI 604D Electrochemical Workstation (Shanghai CH Instruments Inc., China). PEC measurements were performed with a home-built PEC system. A 500 W xenon arc lamp (CHF-XQ-500W, Beijing Changtuo Co. Ltd.) equipped with a monochromator was used as an irradiation source. Photocurrent was measured by the current– time curve experimental technique on a CHI660D electrochemistry workstation (Shanghai CH Instruments Co., China) with a three-electrode system: a modified paper-based electrode as the working electrode, a printing carbon and Ag/AgCl electrode were used as a counter electrode and reference electrode, respectively. All the photocurrent measurements were performed at a constant potential of 0 V (versus Ag/AgCl). All experiments were carried out at room temperature.
2.3. Synthesis of ZnO spheres ZnO nanospheres were prepared according to the literature protocol with some modifications (Cho et al., 2011). In brief, an aqueous solution of 10 mmol L 1 zinc nitrate hexahydrate, 5 mmol L 1 HMT was prepared at room temperature. Then, 2.5 mmol L 1 tripotassium citrate monohydrate was added and dissolved by sonication. After the reaction at 90 1C for 20 min, the powders were acquired by centrifugation and dried in an oven at 60 1C. The as-prepared powders (0.07 g) were dissolved in 100 mL water with mild sonication. 91 μL ammonia was dropwise added and the solution was maintained at 90 1C for 1 h. After the reaction, the products were acquired by centrifugation at 10,000 rpm, subsequently, washed with water for three times and dried in an oven at 60 1C.
2.4. Fabrication of the molecularly imprinted PEC sensor on
μ-PAD
Scheme 1 shows the fabrication process of the microfluidic origami PEC device (the fabrication details could be found in the supporting information). To fabricate Au-PWE, an AuNPs layer was grown on the surface of cellulose fibers in the paper sample zone of PWE (Scheme 1A) to enhance the conductivity and enlarge the effective surface area of bare PWE. Firstly, the suspension of AuNPs seeds was prepared by using NaBH4 as a reductant and stabilized with sodium citrate according to the literature (Busbee et al., 2003). Then, 15.0 μL as-prepared AuNPs seeds solution was dropped into the paper sample zone of bare PWE. Then the origami device was equilibrated at room temperature for 1 h to optimize the surface immobilization of AuNPs seeds on cellulose fibers. After rinsing with water thoroughly according to the method in our previous work (Yan et al., 2012) to remove loosely bound AuNPs seeds, 15 μL freshly prepared growth aqueous solution of 10 mmol L 1 PBS (pH 7.0) containing 1.2 mmol L 1 HAuCl4, 2.0 mmol L 1 cetyltrimethylammonium chloride and 7.2 mmol L 1H2O2 for seeds growth were applied into the AuNPs seeded PWE, and incubated at room temperature for 15 min. Subsequently, the resulting porous Au-PWE was washed with water thoroughly. Thus a layer of interconnected AuNPs on cellulose fibers with good conductivity were obtained, which were dried at room temperature for 20 min (Scheme 1B). The ZnO/PATP composite was prepared by the following procedure: ZnO spheres were dissolved in 0.1 mmol L 1 PATP ethanol solution and the mixture was stirred for 1 h. Then the precipitate was centrifuged and rinsed with ethanol three times. For obtaining ZnO/Au-PWE assemblies, the prepared ZnO/PATP was dissolved in ethanol and dripped to the electrode surface (Scheme 1C). Finally, the electrode was rinsed with water and dried at room temperature.
Scheme 1. The fabrication process of the paper-based PEC sensor.
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The electropolymerization of pyrrole was performed on the
μ-PAD which was described in Scheme 1C. The aqueous electrolyte
solution comprised of 0.1 moL L 1 KCl, 0.5 mmoL L 1 pyrrole, 0.05 moL L 1 PBS (pH 6.86) and 10 mmoL L 1 PCP. After deoxygenating the solution by bubbling nitrogen gas for about 15 min, the electropolymerization (Scheme 1D) was performed by cyclic voltammetry in the potential range of 1.3 V to þ1.0 V, with a scan rate of 50 mV s 1 and 15 sweep cycles. After the electropolymerization process, the electrode was electrochemically treated at 1.3 V for 20 min in 0.2 mol L 1 K2HPO4 solution to remove template molecules (Xie et al., 2010). Then the molecularly imprinted PEC sensor (MIP/ZnO/Au-PWE) with empty sites for PCP recognition was obtained (Scheme 1E). As a control, the nonimprinted polymer (NIP) on ZnO modified Au-PWE (NIP/ZnO/AuPWE) was prepared in the same way except that the template molecules were omitted.
2.5. PEC activity determination The PEC detection procedures were described below. 20 μL spiked samples containing different concentrations of PCP were added into the electrode surface, and allowed to be absorbed for 200 s at room temperature to reach the equilibrium binding, followed by washing with water to prevent non-specific binding and to achieve the best possible signal-to-background ratio. The photocurrent was measured in 0.1 mol L 1 AA PBS solution (pH 7.0) under the 450 nm excited wavelength.
3. Results and discussions 3.1. Characterization of ZnO, Au-PWE, and MIP/ZnO/Au-PWE Fig. 1A showed the SEM image of ZnO sphere. The average diameter of these spherical structures was 300 nm. Visibly, the ZnO sphere we synthesized had a spherical shape with a large specific surface area which provides a good environment for the growth of Ppy. The Au-PWE was developed through the growth of an interconnected AuNPs layer on the surface of cellulose fibers in the paper sample zone to enhance the conductivity of paper sample zone. Fig. 1B showed that paper with porous structure and a high ratio of surface area to weight (9.5 m2 g 1) which could offer an excellent adsorption microenvironment for the AuNPs seeds. The AuNPs seeds were rapidly enlarged by incubating in the growth solution under the self-catalytic reduction mechanism of AuNPs growth. A continuous and dense conducting AuNPs layer with interconnected AuNPs was obtained completely on the cellulose fiber surfaces after 15 min of growth (Fig. 1C, D). In Fig. 1, the SEM images of ZnO/Au-PWE (Fig. 1E) and MIP/ ZnO/Au-PWE (Fig. 1F) were shown. Compared with the SEM image of Au-PWE, we can see apparent ZnO spheres on Au-PWE surface. There is no obvious change of the size of neat ZnO after the polymerization of Ppy.
3.2. Electrochemical and PEC characteristics of MIP/ZnO/Au-PWE Electrochemical impedance spectroscopy (EIS) was used to monitor the interfacial properties of surface-modified electrode. This method was used to get information on the impedance changes of the sensor interface in the modification process and the EIS of variously modified electrodes were monitored in a solution of 5 mmol L 1 [Fe(CN)6]3 /4 and 0.1 mol L 1 KCl at a bias potential of 0.17 V. The frequency range was 100 MHz to 10 kHz. The typical impedance spectrum includes a semicircle
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portion at higher frequencies that corresponds to the electrontransfer resistance (Ret) and the linear part at lower frequencies that corresponds to the diffusion process. Fig. 2A shows the EIS (presented in the form of the Nyquist plot) of different surface conditions of the cellulose fibers in the PWE. The EIS of the bare PWE revealed a relatively small semicircle domain (curve a). After being coated with AuNPs layer, the interfacial resistance decreased (curve b), confirming the successful immobilization of AuNPs and it owns a high electronic conductivity. When the Au-PWE was modified with ZnO spheres with the aid of PATP, the resistance was greatly increased (curve c), which suggested that the ZnO spheres were successfully immobilization on the surface. Because the formed ZnO spheres block the electron transfer between the redox probe and electrode, so the charge transfer resistance increased. A remarkable increase in Ret value was observed after the electropolymerization of Ppy (curve d). This result indicated that Ppy membranes of low conductivity acted as a definite kinetic barrier for the charge transfer. In contrast, after the removal of PCP, the impedance decreased accordingly (curve e). The reason may be that the unoccupied binding cavities in the MIP layer are available for the hexacyanoferrate redox probe to diffuse through after the removal of the template from the MIP layer. As shown in Fig. 2B, curves a and b were the photocurrent responses of Au-PWE and ZnO/Au-PWE in 0.1 mol L 1 AA, respectively. Curves c and d were the photocurrent responses of MIP/ ZnO/Au-PWE in 0.1 mol L 1 AA after addition of different concentrations of PCP solution. Curve e was the photocurrent response of NIP/ZnO/Au-PWE in 0.1 mol L 1 AA after addition of PCP solution (10 ng mL 1). The ZnO/Au-PWE was excited by 365 nm wavelength light. The other electrodes were excited by 450 nm wavelength light. Almost no photocurrent was observed there upon irradiation of the Au-PWE (curve a). After being coated with ZnO spheres, the photocurrent was increased (curve b). As a photoelecchemically active material, ZnO owns good carrier mobility; the wide band gap of ZnO only allows it to absorb the ultraviolet light, after being illuminated with 365 nm wavelength light. The photogenerated electrons transfer to the Au-PWE, that showed an increased photocurrent compared to the Au-PWE before modified by ZnO. The photocurrent of MIP/ZnO modified Au-PWE (curves c and d) and NIP/ZnO modified Au-PWE (curve e) increased compared to that of ZnO modified electrode. This may result from the conjugation effect of Ppy, which can accelerate the electrontransfer and enhance the photocurrent. Furthermore, the photocurrent of MIP/ZnO/Au-PWE decreased with the increasing of the concentration of PCP (curve c). Therefore, it could be applied to sensitive determination of PCP in this disposable microfluidic PEC origami device. Meanwhile, the NIP/ZnO modified Au-PWE showed a higher photocurrent, the reason was that the recognition sites on MIP/ZnO/Au-PWE could specifically absorb PCP, which results in an increase in the steric hindrance toward the diffusion of quencher molecules and/or photogenerated holes on the electrode interface, and consequently a decrease in photocurrent. To compare the PEC performance built on different substrates, a control PEC sensor was fabricated on an indium tin oxide (ITO)coated glass electrode with the same area (6 mm in diameter) using an identical process. As shown in Fig. 2C, the sensor fabricated on the PWE exhibited superior PEC performance to the sensor based on ITO. The photocurrent of the MIP/ZnO/ITO (curve a) was lower (roughly 4-fold) than that in the PWE (curve b), which was mainly attributed to the enhanced immobilization capacity for Ppy/ZnO/Au in the modified macroporous paper sample zone with the 3D interwoven cellulose fiber network as well as the effective light transmission across the total thickness of paper sample zone which was filled with solution, resulting in the increased total number of excited Ppy/ZnO/Au in the whole PWE.
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Fig. 1. SEM images of (A) ZnO sphere; (B) bare paper sample zone of the PWE; (C) Au-PWE; (D) magnification SEM image of Au-PWE; SEM images of (E) ZnO/Au-PWE; and (F) MIP/ZnO/Au-PWE.
3.3. Optimization of experimental conditions To evaluate the influence of template molecules concentration, different concentrations of PCP was selected in the electropolymerizations of pyrrole. After removal of the template molecules, the fabricated sensors were used to detect PCP (10 ng mL 1). The results are given in Fig. 3A. When the concentration of template molecule (PCP) was below 10 mmol L 1, the photocurrent increased with increasing template molecule content due to an increase in the number of recognition cavities. However, a little decrease in the photocurrent could be obtained in the presence of
PCP concentration above 10 mmol L 1, which may be due to the interaction and impact between template molecules at a higher concentration of PCP in the process of PCP-MIPs fabrication and may cause a decrease of valid recognition cavities. So an optimized template molecule concentration of 10 mmol L 1 was selected. As is well-known, AA is a powerful antioxidant used widely in the food industry and for disease treatment because it is in general regarded to be safe and healthy. The formal potential of the redox couple of AA is 0.185 V (versus SCE), it can be easily oxidized by the holes in Ppy and facilitate the generation of stable photocurrent. Thus, AA was chosen as an electron donor in the experiment.
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Fig. 2. (A) EIS of the PWE under different condition in 5 mmol L 1 [Fe(CN)6]3 /4 solution containing 0.1 mol L 1 KCl: (a) bare PWE, (b) Au-PWE, (c) ZnO/Au-PWE, (d) molecularly imprinted Ppy/ZnO/Au-PWE, (e) the electrode ‘d’ after removal of PCP; (B) photocurrent responses of (a) Au-PWE, (b) ZnO/Au-PWE in 0.1 mol L 1 AA, (c) MIP/ZnO/Au-PWE in 0.1 mol L 1 AA after the addition of 10 ng mL 1 PCP, (d) MIP/ZnO/Au-PWE in 0.1 mol L 1 AA after addition of 7 ng mL 1 PCP, (e) NIP/ZnO/Au-PWE in 0.1 mol L 1 AA after the addition of 10 ng mL 1 PCP; (C) Photocurrent responses of (a) MIP/ZnO/ITO, (b) MIP/ZnO/Au-PWE in 0.1 mol L 1 AA after the addition of 10 ng mL 1 PCP for 200 s under 450 nm wavelength light excited.
Fig. 3. Effects of (A) template molecules concentration and (B) AA concentration on photocurrent response.
Fig. 4. (A) Photocurrent responses at MIP/ZnO/Au-PWE electrode in 0.1 mol L 1 AA PBS solution (pH 7.0) in the presence of 0.01, 0.1, 0.5, 1, 5, 10, 50, 100 ng mL 1 PCP (from left to right); and (B) linear calibration curve.
In the experiments, changing the concentration of AA, different photocurrents were obtained. In order to obtain high and stable photocurrent under the same conditions, the sensor was used to detect PCP (10 ng mL 1) to optimize the concentration of AA. The intensity of the photocurrent increased with the increased concentrations of added electron donor AA and reached a maximum for 0.1 mol L 1 AA, then a weak decrease was observed for much higher concentrations of AA (see Fig. 3B). As a hole scavenger, AA in lower concentrations would result in a fall in electrical output because fewer reducing agent molecules were available for electron donation to photogenerated holes. While a much higher concentration of AA would also result in the increase of the absorbance of AA in solution, as a consequence, the intensity of
the irradiation arriving at the electrode surface decreased and the efficiency of excited Ppy would decrease. Other experimental conditions were also optimized. The potential and time of removing PCP, the electropolymerization time (sweep cycles) and the absorption time of PCP were usually important parameters to enhance the sensitivity of the sensor. 1.3 V and 20 min were chosen as the potential and time of removing PCP, respectively (Figs. S2A and S2B). In order to achieve the best selectivity and sensitivity, the thickness of Ppy was also optimized by adjusting the scanning cycles (Fig. S2C). From the Fig. S2C, we know that the most suitable sweep cycle was 15. The absorption time is an important parameter for the improvement of assay efficiency on the microfluidic PEC origami device. When the
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Table 1 Measurement results of PCP in real samples. Samples
Proposed methoda (ng mL 1)
GC–MSa (ng mL 1)
Added (ng mL 1)
Detected (ng mL 1)a
Recovery (%)
1 2 3 4
0.125 0.130 0.132 0.129
0.126 0.134 0.128 0.131
0.1 0.5 10 50
0.223 0.635 9.992 51.879
98.0 101.0 98.6 103.5
a
Average of 11 measurements.
absorption time was above 200 s, the photocurrent remained constant (Fig. S2D), which implied that the adsorption equilibrium was reached on this experimental condition. Thus, the optimum absorption time should be 200 s for the determination of PCP. 3.4. Analytical performance The analytical performance of MIP/ZnO/Au-PWE was verified by using samples of standard PCP solution at various concentrations at an applied potential of 0 V with visible light irradiation. Fig. 4A displays the typical photocurrent responses of the PEC biosensor in the presence of different concentrations of PCP. Under the optimal conditions, with the increasing concentration of PCP, the photocurrent decreases over a wide concentration range of 0.01–100 ng mL 1. Fig. 4B shows the derived calibration curve. The linear regression equation was I (μA)¼ 30.66 14.57 log cPCP (ng mL 1) with a correlation coefficient (R) of 0.9985. The detection limit was estimated to be 4 pg mL 1 at a signal-to-noise ratio of 3s (where s is the standard deviation of the blank, n ¼11). Obviously, the proposed sensor shows promise for application in the monitoring of PCP with its low detection limit and wide linear range. The PEC mechanism of the sensor we fabricated for PCP and the comparison with other pesticide sensors are summarized in Supporting information. 3.5. Stability, reproducibility, and selectivity of the PEC sensor The long-term stability of the PEC immunosensor was also examined. To investigate the stability of the modified electrode, continuous detection was carried out (10 min), and only 2.69% decrease of the initial response was observed. When the immunosensor was dried and stored at 4 1C over four weeks, no apparent change in photocurrent response in the same PCP concentration (10 ng mL 1) was found, illustrating its good longterm storage stability. There may be two reasons responsible for the excellent stability. (1) The porous structure of the paper based electrode, which can fix a mass of ZnO and Ppy. (2) The cross-link structure of Ppy is formed during the polymerization process, which makes Ppy particularly stable and further stabilizes the PEC sensor. The reproducibility of an assay was expressed in terms of values for a between-batch (interassay) relative standard deviation (RSD). The interassay RSD was estimated by measuring the same concentration of PCP with four immunosensors prepared independently at the identical experimental conditions. The interassay RSD obtained from 10 ng mL 1 of PCP was 4.9–6.2%. The results indicated a satisfactory precision and reproducibility of the proposed protocol. Because PCP is one of the most common environmental pollutants, the effects of other pollutants as interfering species on the PEC sensing response were examined. To evaluate the specificity of the MIP/ZnO/Au-PWE, 2,4-dichlorophenoxyacetic acid (2,4-D), aldrin, heptachlor, chlopyrifos (we use a, b, c and d representative these substances, respectively while adopt A representative PCP) were chosen as the interfering substances, and the i–t technique was employed to detect the photocurrent for
different systems on the PCP sensor. The signal was compared by assaying PCP (10 ng mL 1) with interfering agents (1 μg mL 1). The result was listed in Fig. S1 in Supporting information; significantly higher current response was observed with the target PCP than with other pesticides. When PCP coexisted with these sample interfering agents, no apparent signal change took place in comparison with that of only PCP even when the concentration of the interfering substance is 100 times that of PCP. These results indicate that the sensor was sufficiently selective for the detection of PCP. 3.6. Analysis of real samples The analytical reliability and applicable potentiality of the PEC immunoassay were evaluated by testing PCP in spiked samples containing different pollutants or prepared with pure drinking water and river water. As shown in Table 1, the results detected by the proposed method also accorded very well with those by GC– MS. The recoveries of 0.1, 0.5, 1.0, 10.0, and 50.0 ng mL 1 of PCP were determined by standard addition methods. The recoveries of added PCP can be quantitative and t-tests showed that there was no significant difference between recovery efficiency and was 100% at a confidence level of 95%. It can be concluded that the proposed method can offer accurate results and an analytical performance as good as the GC–MS. These data revealed that this method was comparable and acceptable for PCP detection, which means the developed immunoassay may provide a promising alternative tool for determining PCP in real samples.
4. Conclusion In summary, a disposable microfluidic PEC origami device, combining the simple, disposable, and low cost of μ-PAD and stability, and high selectivity of molecular imprinting technique, was developed here through PEC measurement. The Au-PWE was made through AuNPs layer growth on the surface of cellulose. Then the ZnO nanoparticles were modified to the electrode surface with the aid of PATP, the PATP could connect AuNPs and ZnO as it owned sulfydryl and amino. A layer of Ppy was grafted on the ZnO surface through the simple electropolymerization method. After the removal of the template molecule from the resulting crosslinked Ppy matrix the recognition sites to the PCP was generated, which can now specifically recognize and bind PCP. The paperbased PEC sensor with a short absorption time, exhibited good precision, acceptable stability and reproducibility, and could be used for the detection of PCP in real samples. We anticipate that this method can be extended for the determination of other proteins, pesticides and pollutants.
Acknowledgments This work was financially supported by the Natural Science Research Foundation of China (21175058, 21277058, 51273084);
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