Author’s Accepted Manuscript A novel “dual-potential” electrochemiluminescence aptasensor array using CdS quantum dots and luminol-gold nanoparticles as labels for simultaneous detection of malachite green and chloramphenicol Xiaobin Feng, Ning Gan, Huairong Zhang, Qing Yan, Tianhua Li, Yuting Cao, Futao Hu, Hongwei Yu, Qianli Jiang

PII: DOI: Reference:

www.elsevier.com/locate/bios

S0956-5663(15)30215-3 http://dx.doi.org/10.1016/j.bios.2015.06.048 BIOS7784

To appear in: Biosensors and Bioelectronic Received date: 19 May 2015 Revised date: 16 June 2015 Accepted date: 19 June 2015 Cite this article as: Xiaobin Feng, Ning Gan, Huairong Zhang, Qing Yan, Tianhua Li, Yuting Cao, Futao Hu, Hongwei Yu and Qianli Jiang, A novel “dual-potential” electrochemiluminescence aptasensor array using CdS quantum dots and luminol-gold nanoparticles as labels for simultaneous detection of malachite green and chloramphenicol, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.06.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel “dual-potential” electrochemiluminescence aptasensor array using CdS quantum dots and luminol-gold nanoparticles as labels for simultaneous detection of malachite green and chloramphenicol Xiaobin Fenga, Ning Gana,*, Huairong Zhanga, Qing Yana, Tianhua Lia, Yuting Caoa, Futao Hub, Hongwei Yub, Qianli Jiangc a

State Key Laboratory Base of Novel Functional Materials and Preparation Science,

Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211, PR China. b

c

Faculty of Marine, Ningbo University, Ningbo 315211, PR China. Department of Hematology, Nanfang Hospital, Southern Medical University,

Guangzhou, 510515, PR China * Corresponding author: Ning Gan Email: [email protected] Tel: + 86-574-87609987; Fax: + 86-574-87609987

Abstract A novel type of “dual-potential” electrochemiluminescence (ECL) aptasensor array was fabricated on a homemade screen-printed carbon electrode (SPCE) for simultaneous detection of malachite green (MG) and chloramphenicol (CAP) in one single assay. The SPCE substrate consisted of a common Ag/AgCl reference electrode, carbon counter electrode and two carbon working electrodes (WE1 and WE2). In the system, CdS quantum dots (QDs) were modified on WE1 as cathode ECL emitters 1

and luminol-gold nanoparticles (L-Au NPs) were modified on WE2 as anode ECL emitters. Then the MG aptamer complementary strand (MG cDNA) and CAP aptamer complementary strand (CAP cDNA) were attached on CdS QDs and L-Au NPs, respectively. The cDNA would hybridize with corresponding aptamer that was respectively tagged with cyanine dye (Cy5) (as quenchers of CdS QDs) and chlorogenic acid (CA) (as quenchers of L-Au NPs) using poly(ethylenimine) (PEI) as a bridging agent. PEI could lead to a large number of quenchers on the aptamer, which increased the quenching efficiency. Upon MG and CAP adding, the targets could induce strand release due to the highly affinity of analytes toward aptamers. Meanwhile, it could release the Cy5 and CA, which recovered cathode ECL of CdS QDs and anode ECL of L-Au NPs simultaneously. This “dual-potential” ECL strategy could be used to detect MG and CAP with the linear ranges of 0.1 - 100 nM and 0.2 150 nM, with detection limits of 0.03 nM and 0.07 nM (at 3sB), respectively. More importantly, this designed method was successfully applied to determine MG and CAP in real fish samples and held great potential in the food analysis.

Keywords:

Electrochemiluminescent

aptasensor

array;

quenching

simultaneous detection; malachite green and chloramphenicol

2

probes;

1. Introduction Residues of fish drugs may pose health risks to the public, as well as result in environmental contamination and the potential to encourage the proliferation of antibiotic resistant microbes in an aquatic environment. There are some commonly used fish drugs including industrial dyes and antibiotics such as malachite green (MG) and chloramphenicol (CAP). Although MG and CAP, as fungicide and disinfectant, are used all over the world in the fish farming industry, studies have indicated that MG is potential carcinogenic and mutagenic agents, while CAP has serious side effects such as gray baby syndrome, leukemia, and aplastic anemia on human beings (Zhang et al., 2012). For this reason, many countries have banned the use of MG and CAP in aquaculture. However, they are still being used illegally in a relatively large scale across the world because of the low cost and high effectiveness (Yu et al., 2014). Additionally, the two targets usually coexist in some substances such as fishery water, meat and feed, etc. Therefore, it is very necessary to develop a fast and simple method for simultaneous monitoring the amount of MG and CAP. The current analytical methods to detect these residues include gas chromatography-mass spectrometry (Basheer et al., 2005; Ma et al., 2013), liquid chromatography-mass spectrometry (Sniegocki et al., 2014; Mitani et al., 2005), enzyme-linked immunoassay (Xu et al., 2012; Kondo et al., 2012), raman

3

microfluidic sensor (Ji et al., 2015), chemiluminescent immunoassay (Xu et al., 2014), electrochemical method (Wang et al., 2014), and electrochemiluminescence (ECL) method (Fei et al., 2014; Muzyka et al., 2014). Among these methods, ECL has become one of the predominant analytical techniques because of its simple instrumentation, high sensitivity, and wide dynamic range (Feng et al., 2014). For example, Han et al. (2014) have successfully constructed a potential-resolved ECL immunosensor using Ru(bpy)32+ and luminol as probes for determination of two antigens at the cell surface on one electrode. Unfortunately, there is a strong ECL signals interference between Ru(bpy)32+ and luminol, which can affect the accuracy of the assay results. In order to avoid the occurrence of the above phenomenon, ECL combined with the spatially-resolved technique has aroused great interest because it not only could achieve detection of multiple analytes but exclude the crosstalk from the diffusion of the signal substance. Li et al. (2011) have proposed a SPCE array platform that used ECL to detect three targets by near-simultaneously based on spatially-resolved mode. Feng et al. (2014) have designed an ECL immunosensor array on a SPCE to perform multiplexed immunoassay of tumor markers using a homemade single-pore-two-throw switch. Although there are many advantages of this field, it can't detect two targets in one single assay and nor the ECL signal would be delayed because of using the switch to achieve signal switching. Therefore, it is still a challenge to explore new strategies for further reduction of detection process, and improvement the multiplexed capability. Recently, the “dual-potential” ratiometric ECL (R-ECL) has attracted increasing

4

concern due to it could well avoid false positive signal. For example, Zhang et al. (2013) have presented a “dual-potential” R-ECL sensing approach to detect DNA. Cheng et al. (2014) have fabricated an R-ECL biosensor to analyze Mg2+ based on the ratio of two ECL intensities. Hao et al. (2014) have designed an R-ECL biosensor for microRNA detection based on cyclic enzyme amplification and resonance energy transfer. These reported literatures all adopted one electrode modified by two probes then using R-ECL determination scheme to detect one target. Inspired by the idea that the probes can emit ECL signal at different potentials, we hope to employ this type of probes to simultaneously detect two samples at two potentials respectively. In this work, we fabricated an ECL aptasensor array on a homemade SPCE with for simultaneous detection of MG and CAP, which not only could expand the application fields of the “dual potential” analysis but solve the problems in simultaneous detection (description on second paragraph). The SPCE consisted of a common Ag/AgCl reference electrode, carbon counter electrode and two carbon working electrodes (WE1 and WE2). Compared with the previous reports (Li et al., 2011; Zhang et al., 2013; Wang, S., 2012; Ge, L., et al; 2012), we only used one conductive channel to connect with WE1 and WE2, which could achieve the simultaneous assay of analytes in a single run with improved diagnostic specificity. In addition, it could save costs, simplify experimental procedures and decrease the test time. ECL technique commonly adopts conventional “competitive immunoassay” to detect small molecules, and antibody is employed as capture probes. This type of assay has the following shortcomings (Ji et al., 2009): (1) high cost; (2) low stability;

5

(3) lack of specific and sensitive. Correspondingly, aptamer has attracted considerable attention in the field of clinical diagnosis because they could recognize small molecules, proteins, viruses and even cells, with high affinity and specificity. Moreover, aptamers possess a lot of advantages, such as simple synthesis, high stability and ready modification. For example, Chen et al. (2010) have designed a aptasensor for adenosine based on the quenching of ECL of Ru(bpy)32+ by ferrocene. Wang et al. (2009) have fabricated an aptamer-modified ECL nanoprobe to detect thrombin. Liu et al. (2014) have designed an “off-on” ECL strategy for detection of adenosine 5′-triphosphate by using aptamer-involved sandwich structure as the signal switch. In this work, the double-strand probes were formed based on cDNA modified on WE1 and WE2 to hybridize with corresponding aptamers that were modified by quenchers. Due to the highly affinity of analytes toward aptamers, which could release the quenchers and result in the recovery of ECL signals. As shown in scheme 1, the two double-stranded structure were formed on WE1 and WE2 by hybridization reactions between the complementary DNA sequences (MG cDNA or CPA cDNA) immobilized on the luminophores (CdS QDs or L-Au) modified electrode and quencher (Cy5 or CA) modified aptamers. In the presence of target MG and CAP, the binding of MG and CAP with aptamers led to the disassembly of the corresponding double-stranded structure (Yan et al., 2012; Wang et al., 2014). As a result, the labeled Cy5 and CA were consequently kept away from the SPCE and the cathode and anode ECL intensity increased simultaneously. The increase of two ECL signals depended on the concentration of target MG and CAP,

6

respectively. This “dual-potential” strategy has been successfully used for simultaneous detection of MG and CAP in actual sample and possessed its potential application in food analysis. Preferred position for Scheme 1 2. Experimental 2.1. Chemicals and reagents The oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China), their sequences were as follow (Stead et al., 2010; Yadav et al., 2014): MG cDNA: 5’SH-(CH2)6-TTTTTGGATCCATTCGTT-3’ MG aptamer: 5’-GGAUCCCGACUGGCGAGAGCCAGGUAACGAAUGGAUC-C-COOH-3’ CAP cDNA: 5’SH-(CH2)6-TTTTTCTACCACCGACTC -3’; CAP aptamer:5’-ACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAG-COOH-3’

CAP and MG ELISA Kits were both purchased from Zhengzhou Biocell Biotechnology Co. Ltd. (China). Poly(ethylenimine) (PEI, Mw=4300-6500), chlorogenic acid (CA), cyanine dye (Cy5), polyethylene terephthalate (PET) substrate, luminol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroauric acid (HAuCl4·4H2O), Cadmium nitrate (Cd(NO3)2·4H2O), Sodium sulfide (Na2S.9H2O) and Bovine serum albumin (BSA, 96-99%) were bought from Sinophram Chemical Reagent Co. Ltd. (Shanghai, China). The Ag ink (737-H), Ag/AgCl ink (CNC-01) and carbon ink (ED-581-SS) were obtained from Henkel Acheson. The insulating ink was purchased

7

from Acheson Co. Ltd. Other chemicals were analytically pure and used as received. The electrolyte was 0.1 M phosphate buffer solutions (PBS, pH 8.0) containing 0.1 M KCl. PBS (pH 7.4) with 0.05% (v/v) Tween 20 (PBST) was used as the washing buffer solution. Deionized water was used throughout the experiments. 2.2. Instrumentation ECL signals were obtained by using a MPI-B model ECL analyzer (Remax) equipped with a photomultiplier tube (PMT) and a potentiostat. A homemade SPCE consisted of a common Ag/AgCl reference electrode, a common carbon counter electrode and two carbon working electrodes (as shown in scheme 1). A transmission electron microscope (TEM, Tecnai G20, Philip) and UV-vis spectrophotometer (TU 1910, Beijing Purkinje General Instrument Co., Ltd.) were employed to characterize the surface morphology and stepwise synthesis of the nanoparticles. 2.3. Preparation of CdS QDs and L-Au NPs CdS QDs and L-Au NPs were prepared according the previous literature (Zhang et al., 2014; Cheng et al., 2014) (shown in supporting information). 2.4. Synthesis of quenching probes The quenching probes were synthesized by a one-step method according to references with some modification (Chen et al., 2010; Zhou et al., 2013). Briefly, an mixture solution of 20 mM EDC and 10 mM NHS were added to the mixture solution of Cy5 (15 μL, 10 mM) and MG aptamer (75 μL, 20 μM) for 45 min to activate carboxylic group and then mixed with PEI solution (15μL, 10 μM) to incubate for 16 h with stirring at room temperature, which could produce Cy5-PEI-MG/aptamer

8

compounds. In a parallel manner, the CA-PEI-CAP/aptamer compounds were fabricated. The quenching probes were stored in refrigerator at 4 oC for the following experiments. 2.5. Pretreatment of the real fish sample Chloramphenicol (CAP) was extracted based upon the method of Takino et al. (2003). Firstly, 5 g fish meat, 5 g anhydrous sodium sulphate and 10 ml ethyl acetate and were mixed into a centrifuge tube. Then, the mixture was homogenized for 1 min with a meat grinder. After centrifugation for 5 min at 6000 rpm, the supernatant was removed and transferred to a round flask. This extraction step was repeated twice, each time with 10 ml ethyl acetate. The combined ethyl acetate extract was then rotated in a rotary evaporator at 40 °C under vacuum to evaporate to dryness. After that, 1 ml acetonitrile and 1 ml n-hexane were added the residue. The dissolved residue was transferred into a graduated glass stopped reagent bottle and shaken. After that, the n-hexane phase was discarded. This step was repeated with a further 1 ml portion of n-hexane. The acetonitrile phase was evaporated to dryness under a stream of dry nitrogen using a heating block at 50 °C and the dry residue was dissolved in 0.5 ml of PBS (pH 7.4) and the solution was filtered through a 0.22 μm nylon centrifuge filter. Finally, 30 μL of CAP in PBS were used to ECL measurement. Malachite green (MG) was extracted according to the previously reported (Xie et al., 2013). Firstly, 10.0 g fish meat, 5 mL ammonium acetate buffer (0.1 mol/L, pH 4.5), 1 mL hydroxylamine hydrochloride solution (0.25 g/mL), 100 μL p-toluene sulfonic acid solution (1 mol/L) and 15 mL acetonitrile were mixed into a centrifuge

9

tube. Then, the mixture was homogenized for 1 min. After that, 10 mL acetonitrile and 10 g alumina were added and the mixture was shaken periodically for 30 min followed by centrifuging at 5000 rpm for 5 min. The supernatant was decanted into a 200-mL separatory funnel. This extraction step was repeated twice, each time with 25 ml acetonitrile. The supernatant was decanted into the separatory funnel containing the first extract. The resulted supernatant was liquid-liquid extracted twice, each time with 25 mL dichloromethane. The lower dichloromethane layer was collected into a 200-mL pear-shaped boiling flask and then the dichloromethane phase was evaporated to dryness under a stream of dry nitrogen using a heating block at 50 °C. After that, the dry residue was dissolved in 0.5 ml of PBS (pH 7.4) and the solution was filtered through a 0.22 μm nylon centrifuge filter. Finally, 30 μL of MG in PBS were used to ECL measurement. 2.6. Fabrication of SPCE and the ECL aptasensor array The SPCE was prepared by reference of the slightly modified literature (Li et al., 2011) (shown in supporting information). It consisted of two parallelly arranged carbon working electrodes (WE1 and WE2), a carbon counter electrode (CE) and an Ag/AgCl reference electrode (RE) (as shown in scheme 1). To prepare ECL aptasensor array, 10 μL CdS QDs and 10 μL L-Au NPs were respectively drop-cast on the WE1 and WE2, and then air-dried at room temperature to get the modified WE1 and WE2. After that, 20 μL of 1 × 10−5 M MG cDNA and 20 μL of 1 × 10−5 M CPA cDNA were pretreated by 2 μL of TCEP, and then dropped on the CdS QDs modified electrode and L-Au NPs modified electrode for 24 h at 4 °C,

10

respectively. The obtained WE1 and WE2 were rinsed with 0.1 M PBS buffer to remove

the

unspecified

MG

cDNA

and

CPA

cDNA.

Then,

30

μL

Cy5-PEI-MG/aptamer and 30 μL AC-PEI-CPA/aptamer were respectively dropped onto WE1 and WE2 to react for 1h, by this means, the Cy5-PEI-MG/aptamer and AC-PEI-CPA/aptamer could hybridize with the corresponding cDNA. The resulting WE1 and WE2 were washed with PBS and then blocked with 10.0 μL of 1% BSA for 50 min at room temperature. The prepared ECL aptasensor array was kept at 4 °C. 2.7. Measurement procedure The detailed assay of MG and CPA was illustrated in Scheme 1. Firstly, 30 μL of different concentrations of MG and CPA in PBS (0.1M, pH 7.4) were individually dropped onto WE1 and WE2 of the array and kept for 120 min at room temperature. Then, the array was washing thrice with PBS to remove the forming target-aptamer complexes. After that, the obtained biosensor array was immersed into 20 μL of 0.1 M PBS (pH 8.0) containing 12 mM H2O2 for ECL detection, in which the PBS was previously bubbled with highly pure N2 for 60 min at room temperature to remove dissolved oxygen. The voltage of the PMT was set at 800 V, and the potential range was set from 0.6 to -1.2 V. 3. Results and discussion 3.1. Characterization of L-Au NPs and CdS QDs TEM images were used to characterize the size and the morphology of the obtained L-Au NPs and CdS QDs. Figure S1A and Figure S1B showed a homogeneous distribution of around 20 nm for L-Au NPs and 6 nm for CdS QDs in

11

diameter, respectively. Figure S1C was the results of energy-dispersive X-ray (EDX) analysis. Compared with Au NPs (inset of Figure S1C), an obvious N peak (400 eV) could be observed in L-Au NPs, which verified L-Au NPs were successful preparation. Besides, a significant increase in the EDX peak of C element in L-Au NPs was clearly detected. Both of them were ascribed to luminol in the L-Au NPs. These results were further confirmed by UV-vis spectra of L-Au NPs and Au NPs. As shown in Figure S1D, compared with the spectrum of Au NPs (curve a), L-Au NPs (curve c) exhibited a new absorption band at 250-400 nm which was attributed to luminol (curve b). It was consistent with the data described previously (Tian et al., 2010; Chai et al., 2010; Li et al., 2013), indicating that L-Au NPs has been prepared. 3.2. Feasibility of dual-potential ECL aptasensor array In order to confirm the feasibility of the method, ECL-potential responses were investigated by stepwise, and the results were shown in Figure 1. Firstly, WE1 and WE2 were respectively modified by CdS QDs and L-Au NPs, which showed two ECL emission peaks at -1.15 V and +0.6 V in air-saturated pH 8.0 PBS buffer with H2O2 as co-reactant (curve a). The corresponding ECL mechanism of CdS QDs (A) and luminol (B) were as follow (Jie et al., 2010; Chu et al., 2009): A CdS QDs + ne− → nCdS•−

(1)

H2O2 + 2e− → •OH + OH−

(2)

2CdS•− + 2•OH → 2CdS* + H2O2

(3)

CdS* → CdS QDs + hv

(4)

Or

12

2CdS•− + H2O2 → 2CdS* + 2OH−

(5)

CdS* → CdS QDs + hv

(6)

B

After bound by MG cDNA (CdS QDs/MG cDNA) and CAP cDNA (L-Au NPs/CAP cDNA), respectively, the cathode and anode ECL intensity were slightly decreased by 8.9% and 9.3% (curve b) for the reason that the increased impedance and inhibition of H2O2 diffusion to the electrode surface. However, after hybridizing with corresponding Cy5-PEI-MG/aptamer and AC-PEI-CPA/aptamer compounds, the modified electrodes (WE1 and WE2) respectively exhibited a significant 83.9% and 86.3% decrease in the cathode and anode ECL intensity (curve c). Sequentially, after adding 10 nM MG to WE1 and 10 nM CAP to WE2, the cathode and anode ECL signals enhanced obviously (curve d), which were attributed to the inhibition of electrochemiluminescence resonance energy transfer (ERET) between CdS QDs and Cy5, and the quenching effect between L-Au NPs and CA (Cheng et al., 2014; Sun et

13

al., 2000). Therefore, the “dual-potential” of ECL intensities could be used for simultaneously determine of MG and CAP in a single run. Preferred position for Figure 1 3.3. Comparison of the quenching effect To investigate the quenching efficiency of Cy5-PEI-MG/aptamer and AC-PEI-CPA/aptamer compounds, we conducted the contrast experiment to compare the different aptamer compounds for the ability of quenching the ECL signal under the same conditions. Six kinds of quencher-functionalized aptamers were prepared, and the results were shown in Figure S2. The quencher-functionalized aptamers were PEI-MG/aptamer,

PEI-CPA/aptamer,

Cy5-MG/aptamer,

AC-CPA/aptamer,

Cy5-PEI-MG/aptamer and AC-PEI-CPA/aptamer. The ECL responses of the aptasensor array with PEI-MG/aptamer and PEI-CPA/aptamer (curve b) were reduced to about 8576 au (-1.15 V) and 10243 au (+0.6 V) compared with background values (curve a). Then about 6157 au (-1.15 V) and 7156 au (+0.6 V) ECL signals were produced by the aptasensor array with Cy5-MG/aptamer and AC-CPA/aptamer (curve c), which could be attributed to the fact that the quenchers were loaded on aptamers via the amide bond. When the aptasensor array was incubated with the as-prepared Cy5-PEI-MG/aptamer and AC-PEI-CPA/aptamer (curve d), the ECL emissions were noticeably reduced to about 1633 au (-1.15 V) and 1836 au (+0.6 V), which was attributed to the increase of the loading amount of quencher on aptamers via PEI as connection agent. Thus, these comparison results indicated that the as-prepared Cy5-PEI-MG/aptamer and AC-PEI-CPA/aptamer could be utilized for sensitive

14

detection of MG and CAP. 3.4. Optimization of detection conditions In order to obtain a better response of ECL, we used a single variable method to optimize the detection conditions (including incubation time of the quenching probes, the incubation time of MG and CAP, the concentrations of H2O2 and the pH value of the supporting electrolyte). It is known that the detection sensitivity depended on the formation of DNA and targets reaction on the electrode surface (Cheng et al., 2014). Thus, the incubation times of the aptasensor array with both quenching probes and then targets MG and CAP were optimized. As shown in Figure 2A, the quenching efficiency trended to the maximum value at 40 min, which was attributed to the limitation of the saturated hybridization site on the surface of CdS QDs and L-Au NPs. Therefore, 40 min was used for hybridization time between aptamer and complementary strand. In addition, the ECL peak intensity had no obvious change after the time of targets reaction reached 50 min (Figure 2B), indicating that 50 min was sufficient for specific reaction during the ECL detection, which was chosen as the optimal condition. Figure 2C illustrated the signal intensity versus different concentrations of H2O2 from 6 mM to 16 mM. It could be seen that ECL intensity gradually increases with the increasing concentration of H2O2, and then reached an approximate plateau above 12 mM. Therefore, 12 mM H2O2 solution was used as the performed concentration in further experiments. As shown in Figure 2D, the ECL intensity of the aptasensor array increased with

15

the pH value from 6.4 to 8.0, and then decreased when the pH value exceed 8.0. Therefore, pH 8.0 was selected for the following ECL measurements. Preferred position for Figure 2 3.5. Evaluation of cross-reactivity and crosstalk Crosstalk and cross-reactivity are two important factors that affect the specificity, and the reliability of ECL aptasensor array (Nyfeler et al., 1997; Xie et al., 2006; Wu et al., 2007). In this study, the ECL emitters were from the CdS QDs and L-Au NPs, and they were respectively modified on the surface of WE1 and WE2. Besides, the ECL signals were respectively produced at cathode and anode potentials. Thus, the potential crosstalk and cross-reactivity should be avoided as much as possible. To validate the above claims, the multiplex capability of aptasensor array for the detection of MG and CAP was studied by comparing the ECL intensities of the aptasensor array incubated with blank control (10 nM PBS), 10 nM MG, 10 nM CAP or a mixture of the two analytes. As shown in Figure S3, the ECL intensities exhibited minimal difference when the incubation solution contained one or two types of analytes. Moreover, since each working electrode only responded to its corresponding analyte, the cross-reactivity between each analyte and its non-cognate aptamers was also negligible. These results indicated that the aptasensor array could be used for multiplexed detection of MG and CAP. 3.6. Analytical performance Under the optimal conditions, the ECL intensity became larger with the increasing targets (MG and CAP) concentrations because more aptamers modified by quenching

16

agent released from the aptasensor array surface (Figure 3). The standard calibration curves for MG and CAP detection (inset) showed a good linear relationship between the ΔECL intensity and the logarithmic values of the analyte concentrations. The dynamic range of MG and CAP were 0.1 - 100 nM and 0.2 - 150 nM, and the limits of detection for MG and CAP were 0.03 nM and 0.07 nM (estimated at the 3sB criterion), respectively. The detection limits of the developed method for multiplexed detection of MG and CAP are comparable with or even more sensitive than some previously reported methods (Table 1) Preferred position for Figure 3 and Table 1 The specificity of the aptasensor array was investigated by analyzing a standard solution of 10 nM MG, 10 nM CPA and other compounds such as ascorbic acid (AA), tetracycline (TC), methylene blue (MB), dopamine (DA), streptomycin (ST), ciprofloxacin (CF). The foreign interference were added into the incubation solution with same concentration (100 nM), respectively. Figure S4 showed that the foreign substances did not interfere with the determination of MG and CAP, respectively. The reproducibility of the aptasensor array was studied with inter-assay precision. Inter-assay precision of the array was examined at 10 nM MG and 10 nM CAP. The relative standard deviations (RSD) were calculated to be 4.28% for MG and 5.63% for CPA, respectively, which indicated acceptable precision and high reproducibility. From the repeatability experiment, thirteen ECL measurements of the biosensor array upon continuous cyclic scans respectively showed signal with RSD of 1.26% for CdS QDs and 1.19% for L-Au NPs, indicating quite satisfying stability. Moreover, the

17

storage stability of the aptasensor array was also investigated. After 8 weeks of storage in PBS (pH 7.4) at 4 ℃ , over 90% of the initial response remained, demonstrating acceptable stability. 3.7. Application in fish samples analyses To verify the feasibility and application potential of the developed aptasensor array, three kinds of fish samples were analyzed. The results analyzed by using the proposed method were compared with the reference values obtained by using the commercially ELISA method. As shown in Table S1, the recovery of the proposed method ranged from 92.6-100.5% for MG and 94.7-101.5% for CAP, and the corresponding RSD ranged from 4.1-6.6% for MG and 3.9-5.7% for CAP, respectively. These results indicated that the proposed ECL aptasensor array could be used for sensitive and reliable detection of MG and CAP in real samples. 4. Conclusions In this work, we have demonstrated a novel “dual-potential” ECL aptasensor array consisting of two screen-printed carbon working electrodes (WE1 and WE2) for simultaneous detection of MG and CAP in one single assay. The employment of the CdS QDs and L-Au NPs labels with distinct ECL signal potentials were respectively modified on WE1 and WE2, which not only reflected the identity of the targets MG and CAP, enabled the multiplexed capability of the aptasensor array, but also could avoid the potential crosstalk and cross-reactivity as much as possible. Besides, the involvement of quenching probes enriched by PEI polymer in the assay which could effective cut down the original ECL signal of aptasensor array. Therefore, it

18

significantly reduced the background signal to achieve sensitivity for MG and CAP detection. Such an ECL aptasensor array provides new opportunities for multiplexed detection of MG and CAP, and it seems to be of great potential application in the food analysis. Acknowledgements This work was supported by the Natural Science Foundation of Zhejiang and Ningbo (Y15B050008, LY13C200017, 2013A610241, 2014A610184, and 2013A610163), and K.C. Wong Magna Fund in Ningbo University.

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Figure captions Scheme 1. Schematic illustration of the “dual-potential” ECL aptasensor array for

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simultaneous detection of MG and CAP.

Figure 1. ECL-potential curves of (a) CdS QDs and L-Au NPs, (b) CdS QDs/MG cDNA and L-Au NPs/CAP cDNA, (c) CdS QDs/MG cDNA/Cy5-PEI-MG/aptamer and L-Au NPs/CAP cDNA/AC-PEI-CPA/aptamer in the absence and presence (d) of 10 nM MG and 10 nM CAP in pH 8.0 PBS buffer containing 12 mM H2O2.

Figure 2. Effects of (A) incubation time of quenching probe, (B) incubation time of MG and CAP, (C) concentration of H2O2 solution, (D) pH of detection solution on ECL response of aptasensor array in air-saturated PBS in absence (A) and presence (B, C, D) of 10 nM MG and 10 nM CAP.

Figure 3. ECL responses of the proposed aptasensor array for multiplexed detection of MG and CAP (nM) at: (a) 0 and (a‫ )׳‬0, (b) 0.1 and (b‫ )׳‬0.2, (c) 0.3 and (c‫ )׳‬0.6, (d) 1.0 and (d‫ )׳‬2.0, (e) 3.0 and (e‫ )׳‬6.0, (f) 10 and (f‫ )׳‬20, (g) 30 and (g‫ )׳‬60, (h) 100 and (h‫)׳‬ 200. Inset: calibration curve.

Figures

24

Scheme 1

25

Figure 1

26

Figure 2

27

Figure 3

Table Table 1 Comparison of the this method to MG and CAP with other immunoassay 28

Detection methods

Linear range

Detection limit

Reference

MG: 0.03-3.27 ng mL-1

0.01 ng mL-1

Zhang et al., 2015

CAP: 0.6-10 μM

0.16 μM

Yang et al., 2011

MG: 10-510 nM

6 nM

Qu et al., 2012

CAP: 1-1000 nM

0.29 nM

Yan et al., 2012

MG: 0.8-800 nM

0.1 nM

Huang et al., 2013

CAP: 0.05-1 mM

0.01 mM

Lindino et al., 2004

MG: 0.01-5 nM

0.003 nM

Guo et al., 2011

CAP: 0.01-5 ng mL-1

0.4 ng mL-1

Xu et al., 2012

MG: 1.83-200 ng mL−1

1.61 ng mL-1

Shen et al., 2012

CAP: 0.2-150 nM

0.07 nM

Chemiluminescent immunoassay

Electrochemical immunoassay

Electrochemiluminescence immunoassay

Enzyme immunoassay

This method

MG: 0.1-100 nM

0.03 nM

Highlights (1) A novel “dual-potential” ECL aptasensor array based on a homemade screen-printed carbon electrode was designed. (2) The MG aptamer connect with Cy5 was employed as quenching probes of CdS QDs. 29

(3) The CAP aptamer connect with CA was used as quenching probes of L-Au NPs. (4) The MG and CAP combination with corresponding quenching probes could restore cathode ECL of CdS QDs and anode ECL of L-Au NPs simultaneously. (5) The CdS QDs and L-Au NPs can display high signal intensity at different potentials.

30