Novel Double-Potential Electrochemiluminescence Ratiometric

Feb 3, 2017 - Graphene oxide (GO) was obtained from Pioneer Nanotechnology Co. (Nanjing, China). Phosphate-buffered saline (PBS) solutions with ...
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Novel Double-Potential Electrochemiluminescence Ratiometric Strategy in Enzyme-Based Inhibition Biosensing for Sensitive Detection of Organophosphorus Pesticides Hongmei Chen, Han Zhang, Ruo Yuan,* and Shihong Chen* Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People’s Republic of China S Supporting Information *

ABSTRACT: Generally, electrochemiluminescence (ECL) ratiometric assays were based on the energy transfer (ET) between an emitter and a metal nanomaterial or between two different emitters. The choice of suitable energy donor−acceptor pair and the distance dependence of ET would greatly limit the practical application of ratiometric assays. This work explored a novel double-potential ECL ratiometry without the ET for organophosphorus pesticides (OPs) analysis, in which, reduced graphene oxide-CdTe quantum dots (RGO-CdTe QDs) and carboxylconjugated polymer dots (PFO dots) were chosen as cathodic and anodic ECL emitters, and the reactant (dissolved O2) and the product (H2O2) in enzymatic reactions served as their coreactants, respectively. With the occurrence of the enzymatic reactions induced by the acetylcholinesterase (AChE) and choline oxidase (ChOx), the cathodic ECL signal from RGO-CdTe QDs was at “signal off” state due to the consumption of dissolved O2. Meanwhile, the anodic ECL signal from PFO dots was at “signal on” state due to the in situ generation of H2O2. In the presence of OPs, the cathodic ECL signal would increase while the anodic ECL signal would decline correspondingly due to the inhibition of OPs on the activity of AChE. Using the reactant and the product in enzymatic reactions as the coreactants of two different ECL emitters, we conveniently achieved the opposite change trend in two ECL signals for the ratiometric detection of OPs, which exhibited a greatly improved accuracy, reliability and sensitivity, thus, showing a great attraction for developing ECL ratiometric systems for the bioanalysis.

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nology has been preliminarily introduced into enzyme-based inhibition biosensors for OPs detection.9−11 For example, Miao et al. achieved OPs detection based on H2O2 produced by enzymatic reactions as the coreactant to amplify the ECL signal of luminol.9 Gong’s groups constructed a highly sensitive ECL biosensor for OPs based on dissolved O2 as the coreactant of CdTe QDs.10 For these single signal-based construction strategies, false positive or negative errors are inevitable because of the instrumental or some environmental factors.12 The double signal-based construction strategies would be a superior choice to solve above problems. After the first reported by Xu,12 the ECL ratiometric sensing has been gotten growing attentions for detecting protein, cancer cell, microRNA, DNA, metal ion, antigen, and antibody to eliminate interferences and make the detection more convincing.12−15 Generally, ECL ratiometric assays were based on the energy transfer (ET) either between the emitter and the metal nanoparticles (NPs; such as Au NPs and Pt NPs) or between two different emitters. The ET not only requires a

rganophosphorus pesticides (OPs), as a kind of commonly used pesticide, have been widely used in agriculture for many years and caused prodigious pollution in environment, food, and water. OPs could inhibit the activity of acetylcholinesterase (AChE), thus, causing a serious human health problem. Therefore, reliable methods for quantification of trace level of OPs have become increasingly important. Over the past decades, the established methods for OPs monitoring include chromatography−mass spectrometry, liquid chromatography, enzyme-linked immunosorbent assay, electrochemical analysis, colorimetric and fluorometric detection.1−6 Among these methods, the enzyme-based inhibition biosensors based on AChE system were extensively used and have shown satisfactory results for the OPs analysis due to the fast response, high sensitivity, low cost, and easy on-site analysis.4 The enzymatic reactions involved in AChE system are as follows. The substrate acetylthiocholine (ATCl) is hydrolyzed to acetic acid (HAc) and thiocholine (RSH) by AChE. With the catalysis of choline oxidase (ChOx), RSH further reacts with the dissolved O2 to generate RSSR (dithio-bis-choline) and H2O2.7 With the remarkable features, including high sensitivity, good selectivity, low cost, low background signal, and spatial control,8 electrochemiluminescence (ECL) tech© 2017 American Chemical Society

Received: October 3, 2016 Accepted: February 3, 2017 Published: February 3, 2017 2823

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polyfluorene derivative poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO, MW 147000, polydispersity 3.0), the functional copolymer poly(styrene-co-maleicanhydride) (PSMA, average molecular weight ∼ 1700, styrene content 68%), and ethyl paraoxon (EP) were obtained from Aladdin Ltd. (Shanghai, China). N-Hydroxysuccinimide (NHS), ̀ trisodium citrate hydrate (C6H5Na3O7·2H2O), glutaraldehyde (GA), L-cysteine, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), sodium borohydride (NaBH4), acetylcholinesterase (AChE), choline oxidase (ChOx), tetrahydrofuran (THF, anhydrous, ≥99.9%), and acetylthiocholine chloride (ATCl) were purchased from Sigma-Aldrich Co. (Shanghai, China). Graphene oxide (GO) was obtained from Pioneer Nanotechnology Co. (Nanjing, China). Phosphate-buffered saline (PBS) solutions with different pH were prepared with 0.10 M Na2HPO4 and 0.10 M KH2PO4 (containing 0.10 M supporting electrolyte KCl). All the solutions were prepared in ultrapure water, which was purified by a Milli-Q water purification system (Millipore Corp., Bedford, MA). Stock solutions of 250 U/mL ChOx and 125 U/mL AChE were prepared in the Tris-HCl and stored at −20 °C before use. A 1.0 M ATCl was prepared with PBS and stored at 4 °C before use. The vegetables (cabbage, pakchoi, and lettuce) were bought from a local Yonghui supermarket. Apparatus. The MPI-E electrochemical analyzer (Xi’an Remax Analyze Instrument Co. Ltd., Xi’an, China) was used to perform the ECL measurements in PBS (0.10 M, pH 7.4) with the voltage of the photomultiplier tube (PMT) set at 800 V in the detection. Cyclic voltammetry (CV) measurements were performed with a CHI 600D electrochemical workstation (Shanghai Chenhua Instruments Co., China) in 3.0 mL 0.10 M PBS (pH 7.0) containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6]. Transmission electron microscopy (TEM) images were obtained from a Hitachi H-800 microscope (Japan). Fluorescence spectrometry was performed on a FR-5301-PC spectrophotometer (Shimadzu, Tokyo, Japan) at room temperature with the range of 400−700 nm. The UV−visible (UV− vis) spectrometry was performed on a Lambda 17 UV−vis spectrometer 8500 (PECo., U.S.A.) with the range of 200−800 nm. Fourier transform infrared spectroscopy (FT-IR) was performed on an IFS 66 V/S (Bruker) IR spectrometer in the range of 400−4000 cm−1. VG Scientific ESCALAB 250 spectrometer (Thermoelectricity Instruments, U.S.A.) was used to perform X-ray photoelectron spectroscopy (XPS). Raman spectra were obtained for 800−2000 cm−1 using Bruker optics model Senterra employing 532 nm wavelength incident laser light and power 20 mW. Preparation of RGO-CdTe QDs. Reduced graphene oxideCdTe quantum dots (RGO-CdTe QDs) were obtained by onestep method (Scheme 1). First, CdCl2 solution was prepared by dissolving 73.78 mg CdCl2·2.5H2O in 100 mL of ultrapure water. Subsequently, CdCl2 solution and GO dispersion (440 mL, 1 mg/mL) were mixed under stirring to make plenty of Cd2+ be adsorbed onto the GO. Afterward, under stirring, C6H5Na3O7·2H2O (100 mg), Na2TeO3 (0.010 M, 2 mL), Lcysteine (0.10 mM, 50 mL), and NaBH4 (200 mg) were added gradually. The mixture solution was refluxed in the roundbottom flask at 150 °C for 10 h. The resultant solution was separated by centrifugation (8000 rpm, 5 min) to obtain RGOCdTe QDs, which were washed three times with absolute ethanol and then dispersed in the ultrapure water. Finally, the dispersion of RGO-CdTe QDs was stored at 4 °C refrigerator for further use. During the preparation process of RGO-CdTe

perfect spectral overlap of donor−acceptor pair, but also is a distance-dependent process.15,16 The choice of suitable energy donor−acceptor pair and the distance dependence of ET both would greatly limit the practical application of ratiometric assays. Therefore, it is of great significance to design a convenient and simple ECL ratiometry protocol excluding the ET. In earlier ECL ratiometric systems, the cathodic and anodic ECL emitter groups were confined to the following cases: CdS nanocrystal and luminol,12,15,17,18 graphene quantum dots and luminol,19 K2S2O8 and luminol,20 or CdTe quantum dots and Ru(bpy)32+.21 Furthermore, the cathodic and anodic ECL emitters often shared the common coreactant such as H2O2 or K2S2O8. The limitations on the selection of both the two ECL emitters with a potential-dependent property and corresponding coreactant strongly restricted the development and application of ECL ratiometric detection, especially in the enzyme sensing field. The enzyme-based ECL ratiometric biosensors for small molecules analysis were not associated with the formation of sandwich immune model or DNA hybridization, so it was difficult to introduce the second ECL signal probe. Furthermore, the oxidereductase induced enzymatic reactions usually involved the consumption of dissolved O2 and the generation of H2O2. As is well-known, O2 and H2O2 could simultaneously serve as the coreactants of many emitters, thus make the signal becoming disorganized because the consumption of dissolved O2 would cause a decrease in ECL signal, while the in situ generation of H2O2 would cause an increase in ECL signal. However, if dissolved O2 and produced H2O2 can be used as the coreactants of two different emitters, respectively, such a disadvantage may be transformed into a favorable factor to conveniently achieve an opposite change trend in the two ECL signals. Inspired by above idea, we screened out two desirable emitters. One is conjugated polymer poly(9,9-dioctylfluorene) dots (PFO dots), which have potential advantages such as nontoxic features, high luminescence efficiency, and easy functionalization.22−24 Their anodic ECL mechanisms with H2O2 as the coreactant have been explored in our previous work.25 The other one is CdTe QDs, which have been widely used as an ECL emitter.10,26 The luminous potentials of 1.96 V for PFO dots and −1.67 V for CdTe QDs can be separated well. Moreover, H2O2 and dissolved O2 could serve as their coreactants, respectively. More importantly, there was no ET between them (see Results and Discussion). Thus, in this work, the nanocomposites of PFO dots and RGO-CdTe QDs were prepared, and further used as matrix for immobilizing the bienzymes (AChE and ChOx) to obtain enzyme-based inhibition ECL ratiometric biosensing for OPs detection. Upon the inhibition of OPs on AChE enzyme activity, the consumption of dissolved O2 and generation of H2O2 would be prevented, thus, resulting in an increase in cathodic ECL signal and a decrease in anodic ECL signal. By calculating the ratio of two ECL signal intensities, the quantitative determination of OPs would be achieved with a greatly improved accuracy, reliability, and sensitivity. This is a pioneering work of applying the opposite change trend between the reactant and the product to easily achieve the ratio detection model.

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EXPERIMENTAL SECTION Material and Reagents. Cadmium chloride hemipentahydrate (CdCl2·2.5H2O) was obtained from Alfa Aesar Chemical Co. (Tianjin, China). Sodium tellurite(IV) (Na2TeO3), 2824

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GCE, which was stored at 4 °C when not in use. The preparation process was illustrated in Scheme 1. Measurement Procedure. The biosensor AChE-ChOx/ PFO-RGO-CdTe QDs/GCE was employed for the detection of the ethyl paraoxon (EP) using ECL technique. The proposed biosensor was incubated with the EP solution with different concentrations for 15 min. Then, the obtained electrode was detected in the detecting cell with 3.0 mL PBS (0.10 M, pH 7.4) containing 0.090 mM ATCl. Preparation of Real-Life Samples. The real-life samples were prepared as fellows. The cabbage, pakchoi and lettuce were washed with ultrapure water. Then, 10 g samples were ground into vegetable juice and added into the mixed solution which was prepared with 9 mL of PBS (0.1 M, pH 7.4) and 1 mL of acetone. Then, the obtained suspension was sonicated for 10 min and centrifuged (8000 rpm, 15 min). The supernatant was used for further experiments.

Scheme 1. Schematic Description of the Biosensor Fabrication and Response Mechanism

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RESULTS AND DISCUSSION Characterization of Nanomaterials. TEM was used to characterize the nanomaterials, and the results were presented in Figure 1. As seen in Figure 1A, the PFO dots were spheroidal

QDs, GO was reduced to reduced graphene oxide (RGO), which was proved by Raman spectroscopy (seen in the Supporting Information, Figure S1). Preparation of PFO Dots and PFO-RGO-CdTe QDs. First, PFO polymer and functional polymer PSMA were dissolved in THF to make solutions with 1 mg/mL, respectively. Two solutions were mixed and diluted with THF to make a solution mixture with a PFO concentration of 100 μg/mL and a PSMA concentration of 20 μg/mL. The mixture was sonicated to form a homogeneous solution. Subsequently, 1 mL of mixed polymer dispersions was injected quickly into 1 mL of water. This two-phase solution was then emulsified using a tip sonicator (30 W, 10 min). THF was removed by partial vacuum evaporation, the carboxylconjugated PFO dots were obtained after filtered with a 2.7 μm syringe filter. The preparation process was illustrated in Scheme 1. As shown in Scheme 1, the carboxyl-conjugated PFO dots and RGO-CdTe QDs were cross-linked with EDC/NHS (molar ratio 4:1) under magnetic stirring to obtain PFORGO-CdTe QDs nanocomposites, which were separated by centrifugation (12000 rpm, 10 min) and washed with the mixture of ultrapure water and ethanol (V/V = 1:1) three times. Finally, the PFO-RGO-CdTe QDs nanocomposites were dispersed in 1 mL of ultrapure water to obtain the dispersion, which was stored in dark at 4 °C refrigerator. Preparation of AChE-ChOx Biocomposite. The AChEChOx biocomposite was prepared through the cross-linking between the amino-groups of bienzymes using cross-linking agent GA. Briefly, 10 μL of ChOx (250 U/mL), 2 μL of AChE (125 U/mL), and 2 μL of GA (0.75% V/V) were mixed and then incubated for 8 h at 4 °C. Finally, the AChE-ChOx conjugates were obtained by filtering with a dialysis membrane (MWCO: 3500 Da) to remove GA. Fabrication of the ECL Biosensor. First, the GCE (Φ = 4.0 mm) was carefully polished with 0.3 and 0.05 μm alumina slurry repeatedly, ultrasonically cleaned in ethanol and ultrapure water thoroughly and dried in air. Subsequently, 15 μL of PFO-RGO-CdTe QDs dispersion was dropped onto the surface of GCE and air-dried at room temperature to achieve the modified electrode (denoted as PFO-RGO-CdTe QDs/ GCE). Finally, 10 μL of AChE-ChOx biocomposite was incubated on the PFO-RGO-CdTe QDs/GCE surface for 8 h to prepare the biosensor AChE-ChOx/PFO-RGO-CdTe QDs/

Figure 1. TEM images of (A) PFO dots, (B) RGO-CdTe QDs, and (D) PFO-RGO-CdTe QDs; (C) HRTEM image of RGO-CdTe QDs (inset: the interplanar spacing of CdTe QDs).

particle with the diameter of 60−160 nm. Figure 1B presented the TEM image of RGO-CdTe QDs. Many CdTe QDs with the average size of 3.5 nm were uniformly grown on the surface of RGO. Figure 1C was the HRTEM images of CdTe QDs. As seen, a good crystal structure were observed with the interplanar spacing of about 0.325 nm, which was consistent with the previous literature.27,28 As shown in Figure 1D, both the PFO dots and RGO-CdTe QDs were clearly observed. PFO dots exhibited a uniform distribution in the TEM image of RGO-CdTe QDs. Furthermore, flaky RGO-CdTe QDs tended to cover onto the surface of PFO dots, indicating the preparation of PFO-RGO-CdTe QDs nanocomposite. X-ray photoelectron spectroscopy (XPS) was performed to gain the information concerning the chemical composition of the PFO-RGO-CdTe QDs nanocomposite. As shown in Figure 2A, the C 1s peak and O 1s peak were obviously observed from the survey of PFO dots. The O 1s was owed to the carboxyl of PFO dots, which was resulted from the functional copolymer PSMA. In order to further confirm the functionalization of PFO 2825

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investigations. First, the ECL spectrum of PFO dots and UV−vis absorption spectrum of RGO-CdTe QDs were measured. As seen in Figure 3A, the ECL spectrum of PFO

Figure 2. XPS survey curves of (A) PFO dots and (C) PFO-RGOCdTe QDs (insets: the enlarged curves of Cd and Te). (B) Highresolution XPS curves of the carbon of PFO dots. (D) FL spectra of (a) PFO dots, (b) RGO-CdTe QDs, and (c) PFO-RGO-CdTe QDs. Figure 3. (A) ECL spectrum of (a) PFO dots and (b) UV−vis absorption spectrum of RGO-CdTe QDs. (B) Normalized ECL spectra of (a) PFO dots (scan range: 0−2 V) and (b) RGO-CdTe QDs (scan range: −1.7−0 V). (C) ECL responses of bare GCE (black curve), bare GCE with 15 μL of PFO dots (red curve), RGO-CdTe QDs/GCE (green curve), and RGO-CdTe QDs/GCE with 15 μL of PFO dots (blue curve) in air-saturated PBS (0.10 M, pH 7.4). (D) ECL intensity curves of CdTe QDs/GCE (red curve), CdTe QDs/ GO/GCE (blue curve), and RGO-CdTe QDs/GCE (green curve) in air-saturated PBS (0.10 M, pH 7.4).

by carboxyl-containing PSMA, the high-resolution C 1s XPS was performed and the results were shown in Figure 2B. As seen, raw data showed two peaks of carbon atoms with the binding energy at 288.1 and 284.8 eV. These two peaks can be fitted by four components with the binding energy at 284.7, 285.6, 286.6, and 288.2 eV, which were associated with the sp2 C atoms, C−OH, C−O epoxy/ether groups, and the carbonyl groups (O−CO), respectively, indicating the functionalization of PFO by carboxyl-containing PSMA. In addition, the FTIR spectroscopy was also performed to characterize the formation of the carboxyl-conjugated PFO dots (Figure S2 in the Supporting Information). The XPS survey curve of PFORGO-CdTe QDs nanocomposite was presented in Figure 2C, and the characteristic peaks of S 2p, Cd 3d, and Te 3d were obtained. The S 2p located at 161.5 eV was originated from Lcysteine in the RGO-CdTe QDs. The characteristic peaks of Cd 3d and Te 3d were displayed in the insets of Figure 2C, which were 405.2 eV (Cd 3d5/2), 412.3 eV (Cd 3d3/2), 572.8 eV (Te 3d5/2), and 583.4 eV (Te 3d3/2). The peaks at 576.3 and 586.9 eV were due to the Te 3d levels in Te4+. To further confirm the synthesis of PFO-RGO-CdTe QDs nanocomposite, fluorescence emission spectra were performed and the results were shown in Figure 2D. For PFO dots (curve a), a narrow and sharp fluorescence peak was observed at 441.4 nm due to the β-phase of PFO, and another two characteristic vibronic peaks were observed at 468 and 499.2 nm, respectively. The FL emission peak of RGO-CdTe QDs was obviously observed at 598.4 (curve b). For the PFO-RGOCdTe QDs (curve c), the characteristics emission of both PFO dots and RGO-CdTe QDs were clearly observed, indicating the successful synthesis of PFO-RGO-CdTe. Additionally, the FTIR spectroscopy was also performed to confirm the covalent conjugation between PFO dots and RGO-CdTe QDs, and the results are shown in Figure S3 in the Supporting Information. UV−vis absorption spectra were used to characterize the synthesis of PFO-RGO-CdTe QDs and the results are shown in Figure S4 in the Supporting Information. Characterization of the Stepwise Fabrication and Feasibility of Ratiometric ECL Biosensor. In order to confirm whether there was energy transfer between the PFO dots and RGO-CdTe QDs, we performed the following

dots presented a strong ECL emission peak at about 438 nm (curve a), and other two peaks appeared at 468 and 502 nm. The typical absorption spectrum of RGO-CdTe QDs presented an obvious and wide absorption band with an absorption inflection point at 583 nm (Figure 3A, curve b). We can see that the ECL emission of PFO dots nearly had no spectral overlap with the absorption spectrum of RGO-CdTe QDs. Second, the ECL emission spectra of PFO dots and RGOCdTe QDs were compared. Figure 3B, curve a, depicted the ECL emission of PFO dots. As for RGO-CdTe QDs (Figure 3B, curve b), the maximum emission wavelength of ECL peak was at 595 nm in the scan potential from −1.7 to 0 V. Clearly, there was also no spectral overlap between the ECL spectra of PFO dots and RGO-CdTe QDs. Third, the effect of PFO dots on the ECL emission intensity of RGO-CdTe QDs/GCE was investigated in air-saturated pH 7.4 PBS. As seen from Figure 3C, compared with the bare electrode (black curve), an anodic ECL signal was observed at 1.96 V after adding 15 μL of PFO dots in the detector cell (red curve), which was ascribed to the ECL emission of PFO dots. The RGO-CdTe QDs modified electrode (green curve) presented a strong cathodic ECL signal at −1.67 V in the presence of the coreactant dissolved O2 (green curve). After adding 15 μL of PFO dots in the detector cell, no obvious change in the cathodic ECL signal was observed at RGO-CdTe QDs/GCE, meanwhile an anodic ECL signal from PFO dots appeared and its intensity was proximity to that of the bare electrode in the presence of 15 μL of PFO dots (red curve). Based on above results, we can concluded that there was no energy transfer between the PFO dots and RGOCdTe QDs. 2826

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compared to the electrodes modified only with PFO dots (curve a) or RGO-CdTe QDs (curve b). When AChE-ChOx was modified onto the electrode (curve d), both the anodic signal and cathodic signal decreased because of the nonconductive of AChE-ChOx. In the presence of ATCl in PBS solution (curve e), the anodic signal markedly increased, while cathodic signal markedly decreased as result of the fact that ATCl was hydrolyzed to thiocholine, and thiocholine further reacted with O2 under the catalysis of (ChOx) to produce H2O2. The consumption of dissolved O2 and in situ generation of H2O2 in enzymatic reactions caused a decrease in cathodic ECL signal from RGO-CdTe QDs and an increase in anodic ECL signal from PFO dots since O2 and H2O2 served as the coreactant of CdTe QDs and PFO dots, respectively. When the biosensor was incubated with EP (curve f), the consumption of dissolved O2 and the generation of H2O2 were inhibited due to the inhibition of EP on the activity of AChE system, thus, resulting in an increase in cathodic ECL signal and a decrease in anodic ECL signal, respectively. Above results proved that the biosensor for EP detection was reasonable and feasible. The detailed mechanisms of RGO-CdTe QDs with dissolved O2 as the coreactant and PFO dots with H2O2 as the coreactant were elaborated in the Supporting Information. Optimization of Experimental Conditions. The concentration of ATCl, a vital factor for OPs detection, was optimized. The ECL signals were tested at the biosensor in airsaturated pH 7.4 PBS containing ATCl with different concentration (0−120 μM; Figure S6A in the Supporting Information). As seen from Figure S6A, the cathodic ECL intensity from RGO-CdTe QDs decreased, while the anodic ECL intensity from PFO dots increased, respectively, with increasing the ATCl concentration from 0 to 90 μM. Then both the two ECL signals reached a relatively stable value when the concentration of ATCl exceeded 90 μM. The reason was as follows. AChE hydrolyzed the substrate ATCl to acetic acid (HAc) and thiocholine (RSH), which further reacts with dissolved O2 to in situ generate H2O2 under the catalysis of ChOx. With the increase in ATCl concentrations, the more dissolved O2 was consumed and the more H2O2 was produced, resulting in an increase from the ECL signal of PFO dots and a decrease in the ECL signal from RGO-CdTe QDs since in situ produced H2O2 and dissolved O2 served as the coreactants of the anodic and cathodic ECL emitters, respectively. When the concentration of ATCl exceeded 90 μM, almost all active centers of AChE would be occupied by the substrate and insensible to a higher concentration of substrate ATCl; thus, the two ECL signals leveled off after 90 μM. Therefore, 90 μM was chosen as the optimum concentration of ATCl. Since the catalytic activity of AChE and ChOx was deeply affected by pH, the pH dependence of the ECL response was investigated over the pH range from 6.0 to 9.0 in air-saturated PBS (0.10 M, pH 7.4) containing 90 μM ATCl (Figure S6B in the Supporting Information). As shown in Figure S6B, with increasing pH from 6.0 to 7.4, the ECL intensity of RGO-CdTe QDs showed a rapid decrease and the ECL intensity of PFO dots showed a rapid increase. Subsequently, PFO dots signal decreased and RGO-CdTe QDs signal increased when pH exceeded 7.4. The reason may be as follows. It was reported that the optimum pH for AChE and ChOx activity was 8.0−9.0 and 7.0−8.0, respectively. On the one hand, a lower pH (pH < 7.4) was unfavorable to the activity of AChE and ChOx, resulting in a less yield of thiocholine, accompanied by a less consumption of O2 and less product of H2O2. On the other

In order to confirm the effect of GO on the ECL response of CdTe QDs, we performed following comparative study. The CdTe QDs were directly dropped onto the bare GCE to obtain the CdTe QDs/GCE. The bare GCE was modified with GO and further modified with CdTe QDs to prepare CdTe QDs/ GO/GCE. The bare GCE was modified with RGO-CdTe QDs to obtain RGO-CdTe QDs/GCE. As seen in Figure 3D, compared with the CdTe QDs/GCE (red curve), CdTe QDs/ GO/GCE displayed a decreased ECL emission of CdTe QDs (blue curve), which indicated that GO without the reduction treatment indeed quenched the cathodic ECL intensity of CdTe QDs. Compared with CdTe QDs/GCE, the RGO-CdTe QDs/GCE exhibited a greatly increased cathodic ECL emission (green curve), which enough confirmed that RGO can greatly improve the ECL emission of CdTe QDs. In fact, Ju group has expounded the facts that GO could quench the ECL emission of CdTe QDs due to the structural defects of GO and its blocking off the electron transfer between CdTe QDs and the electrode, and RGO could significantly increase the ECL emission of CdTe QDs due to the restoration of structural conjugation of RGO, the adsorption of dissolved O2 on RGO and the facilitated electron transfer.29 The stepwise assembly processes of the biosensor was characterized by cyclic voltammetry (CV) and the results indicated that the biosensor was successfully constructed (Figure S5 in the Supporting Information). In order to verify the feasibility of the ratiometric strategy, ECL behaviors of different modified electrodes were investigated in air-saturated pH 7.4 PBS, including PFO dots/GCE, RGO-CdTe QDs/GCE, PFO-RGO-CdTe QDs/GCE, and AChE-ChOx/PFO-RGO-CdTe QDs/GCE. As shown in Figure 4, PFO dots/GCE presented an obvious anodic ECL

Figure 4. ECL responses of (a) PFO dots/GCE, (b) RGO-CdTe QDs/GCE, (c) PFO-RGO-CdTe QDs/GCE, and (d) AChE-ChOx/ PFO-RGO-CdTe QDs/GCE in air-saturated PBS (0.10 M, pH 7.4). ECL responses of AChE-ChOx/PFO-RGO-CdTe QDs/GCE without (e) and with (f) incubation of 5.0 × 10−11 M EP in air-saturated PBS (0.10 M, pH 7.4) containing 90 μM ATCl.

signal at 1.96 V in air-saturated pH 7.4 PBS; meanwhile, no ECL signal was observed in the cathodic potential range of −1.7−0 V (curve a). RGO-CdTe QDs modified electrode presented an intensive cathodic ECL emission peak at −1.67 V in air-saturated pH 7.4 PBS; meanwhile, no anodic signal appeared in the range of 0−2 V (curve b). Such an intensive cathodic ECL emission was ascribed to the high luminescence efficiency of emitter (RGO-CdTe QDs) and the influence of the coreactant dissolved O2 on the ECL emission of RGOCdTe QDs. However, for PFO-RGO-CdTe QDs nanocomposite modified GCE (curve c), both the anodic signal from PFO at 1.96 V and the cathodic signal from CdTe QDs at −1.67 V were obtained. Furthermore, there were no obvious changes in ECL peaks potential and peak intensity when 2827

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Analytical Chemistry

ratiometric strategy and the excellent ECL performance of PFO dots and RGO-CdTe QDs. Stability and Reproducibility. As presented in Figure 5D, the operational stability of the ECL biosensor incubated with 5.0 × 10−11 M EP was evaluated under consecutive cyclic potential scans for 10 cycles in air-saturated pH 7.4 PBS containing 90 μM ATCl. The ECL signals tended to stable with a relative standard deviation (RSD) of 2.26% for anodic signal and 2.85% for cathodic signal, respectively, indicating an acceptable stability of such a ratiometric ECL biosensor. The storage stability of the biosensor was also monitored. The biosensor was stored in 4 °C when not in use, and detected every 3 days. One month later, the ECL response of the anodic and cathode ECL signal decreased 9.8% and 9.4%, respectively, indicating an acceptable storage stability of the biosensor. Additionally, the reproducibility of the sensing system was valuated. We prepared the biosensors with five batches of different PFO dots and detected their ECL responses toward 5.0 × 10−11 M EP. As seen in Figure S7, the relative standard deviation (RSD) of the lg(ECLPFO/ECLCdTe) for the five batches of different PFO dot modified biosensors was 3.41%, indicating a good repeatability for different batches of PFO dots although the distribution of particle size of PFO dots was relatively large. Real Samples Analysis. To further evaluate the practicality of the proposed biosensor in the detection of real-life samples, the recovery experiments were performed using different vegetable samples through standard addition method in airsaturated pH 7.4 PBS buffer. First, EP solutions with different concentrations were prepared using real vegetable samples. The biosensor was incubated 15 min by the above different known concentrations EP solutions, respectively. Washed with ultrapure water, the resultant biosensors were detected in airsaturated pH 7.4 PBS with 90 μM ATCl. As presented in Table S1, no OPs were detected in the vegetable samples. The recoveries of EP in the cabbage, pakchoi, and lettuce samples were varied in the range of 94.0∼104%, 95.7∼102%, and 98∼105%, respectively, demonstrating that the ECL biosensor has a potential application to analyze practical samples.

hand, a higher pH (pH > 8.0) was also unfavorable to the activity of AChE and ChOx, thus, the yield of thiocholine, the consumption of O2, and the product of H2O2 decreased, causing the anodic signal decreased and cathodic signal increased. Therefore, pH 7.4 was selected as the optimal pH. ECL Response of the Biosensor to EP. In this work, EP was chosen as the model to investigate the ECL response of the biosensor AChE-ChOx/PFO-RGO-CdTe QDs/GCE toward OPs in air-saturated PBS. As shown in Figure 5, under the

Figure 5. (A) ECL response of the biosensor to EP with different concentration: (a) 0 M, (b) 5.0 × 10−13 M, (c) 1.0 × 10−12 M (d) 5.0 × 10−12 M, (e) 1.0 × 10−11 M, (f) 5.0 × 10−11 M, (g) 1.0 × 10−10 M, (h) 5.0 × 10−9 M, (i) 1.0 × 10−9 M, and (j) 5.0 × 10−8 M in airsaturated PBS (0.10 M, pH 7.4) containing 90 μM ATCl. (B) The change of anodic ECL intensity and cathodic ECL intensity with the logarithm of EP concentration. (C) The calibration curve for EP. (D) The stability of the biosensor incubated with 5.0 × 10−11 M EP in airsaturated PBS (0.10 M, pH 7.4) containing 90 μM ATCl.

optimal conditions, the cathodic ECL intensity from RGOCdTe QDs increased with increasing the concentrations of EP while the anodic ECL intensity from PFO dots decreased accordingly (Figure 5A). Figure 5B showed the change of the anodic ECL intensityand cathodic ECL intensity with the logarithm of EP concentration. As a result, a linear relationship between the logarithm of EP concentration and the logarithm of the ratio of anodic to cathodic ECL intensity (IPFO/ICdTe) was obtained in the concentration range from 5.0 × 10−13 M to 1.0 × 10−8 M (Figure 4C). The regression equation was lg(IPFO/ICdTe) = −6.49115−0.50511 lg c, with a correlation coefficient of 0.995. Here, c was the concentration of EP. The detection limit was 1.25 × 10−13 M at signal-to-noise ratio S/N = 3. This work was compared with the previous research.11,30−32 As seen in Table 1, the proposed biosensor exhibited a lower LOD, which may be attributed to the

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CONCLUSION This work demonstrated a novel double-potential ECL ratiometry in enzyme-based inhibition biosensing based on PFO dots and RGO-CdTe QDs as the anodic and cathodic emitters and, in situ, produced H2O2 and dissolved O2 as the coreactants of two emitters, respectively. Without the involvement of ET, this construction strategy conveniently achieved the opposite change trend in two ECL signals using the reactant and the product in enzymatic reactions as the coreactants of cathodic and anodic ECL emitters, respectively. With the EP as the model of OPs, upon the inhibition of EP toward the activity of AChE system, a double-potential ECL ratiometric detection for OPs was achieved with greatly

Table 1. Comparison of Different Modified Electrode for the Determination of EP electrode materials

determine method

linear range/nM

AChE-ChOx/Au−Pt NPs/3-APTES/GCE AChE/Au-PPy-rGO/GCE AChE/sol gel-TCNQ-MWCNT/SPE AChE/g-C3N4-CD-Fc-COOH/GCE AChE-ChOx/PFO-RGO-CdTe/GCE

amperometry amperometry amperometry ECL ECL

150−200 1−5000 0.1−500 0.001−500 5 × 10−4∼50 2828

detection limit/pM

ref

500 30 0.3 0.125

30 31 32 11 this work DOI: 10.1021/acs.analchem.6b03883 Anal. Chem. 2017, 89, 2823−2829

Article

Analytical Chemistry

(15) Hao, N.; Li, X. L.; Zhang, H. R.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2014, 50, 14828−14830. (16) Shan, Y.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2009, 905−907. (17) Zhang, H. R.; Wu, M. S.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2014, 86, 3834−3840. (18) Cheng, Y.; Huang, Y.; Lei, J. P.; Zhang, L.; Ju, H. X. Anal. Chem. 2014, 86, 5158−5163. (19) Zhao, H. F.; Liang, R. P.; Wang, J. W.; Qiu, J. D. Chem. Commun. 2015, 51, 12669−12672. (20) Zhang, Q. R.; Dai, H.; Wang, T.; Li, Y. L.; Zhang, S. P.; Xu, G. F.; Chen, S. H.; Lin, Y. Y. Electrochim. Acta 2016, 196, 565−571. (21) Dai, H.; Xu, G. F.; Zhang, S. P.; Hong, Z. S.; Lin, Y. Y. Chem. Commun. 2015, 51, 7697−7700. (22) Feng, Y. Q.; Dai, C. H.; Lei, J. P.; Ju, H. X.; Cheng, Y. X. Anal. Chem. 2016, 88, 845−850. (23) Li, Q.; Sun, K.; Chang, K. W.; Yu, J. B.; Chiu, D. T.; Wu, C. F.; Qin, W. P. Anal. Chem. 2013, 85, 9087−9091. (24) Wu, C. F.; Chiu, D. T. Angew. Chem., Int. Ed. 2013, 52, 3086− 3109. (25) Chen, H. M.; Lu, Q. Y.; Liao, J. Y.; Yuan, Y.; Chen, S. H. Chem. Commun. 2016, 52, 7276−7279. (26) Yu, Y. Q.; Zhang, H. Y.; Chai, Y. Q.; Yuan, R.; Zhuo, Y. Biosens. Bioelectron. 2016, 85, 8−15. (27) Li, J.; Hong, X.; Li, D.; Zhao, K.; Wang, L.; Wang, H. Z.; Du, Z. L.; Li, J. H.; Bai, Y. B.; Li, T. J. Chem. Commun. 2004, 1740−1741. (28) Dagtepe, P.; Chikan, V.; Jasinski, J.; Leppert, V. J. J. Phys. Chem. C 2007, 111, 14977−14983. (29) Deng, S. Y.; Lei, J. P.; Cheng, L. X.; Zhang, Y. Y.; Ju, H. X. Biosens. Bioelectron. 2011, 26, 4552−4558. (30) Upadhyay, S.; Rao, G. R.; Sharma, M. K.; Bhattacharya, B. K.; Rao, V. K.; Vijayaraghavan, R. Biosens. Bioelectron. 2009, 25, 832−838. (31) Yang, Y. Q.; Asiri, A. M.; Du, D.; Lin, Y. H. Analyst 2014, 139, 3055−3060. (32) Rotariu, L.; Zamfir, L. G.; Bala, C. Anal. Chim. Acta 2012, 748, 81−88.

improved accuracy, reliability, and sensitivity. The integration of anodic PFO/H2O2 and cathodic RGO-CdTe QDs/O2 ECL systems would open up a new avenue to design a facile dualpotential ECL ratiometric biosensor without ET for bioanalysis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03883. The Raman spectroscopy characterization, the FT-IR spectroscopy characterization, UV−vis absorption spectrum characterization, the detailed ECL mechanisms, cyclic voltammetry (CV) characterization, and supplementary figures and tables (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86 23 68252277. Fax: +86 23 68253172. E-mail: [email protected]. *Tel.: +86 23 68252277. Fax: +86 23 68253172. E-mail: [email protected]. ORCID

Ruo Yuan: 0000-0003-3664-6236 Shihong Chen: 0000-0003-4375-5836 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (51473136, 21575116) and Natural Science Foundation Project of Chongqing City (CSTC2014JCYJA20005), China.

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DOI: 10.1021/acs.analchem.6b03883 Anal. Chem. 2017, 89, 2823−2829