Incubation-free electrochemical immunoassay for diethylstilbestrol in ...

Microchim Acta (2014) 181:453–462 DOI 10.1007/s00604-013-1131-3

ORIGINAL PAPER

Incubation-free electrochemical immunoassay for diethylstilbestrol in milk using gold nanoparticle-antibody conjugates for signal amplification Ping Xiong & Ning Gan & Huan Cui & Jing Zhou & Yuting Cao & Futao Hu & Tianhua Li

Received: 16 July 2013 / Accepted: 15 November 2013 / Published online: 12 December 2013 # Springer-Verlag Wien 2013

Abstract We report on a novel enzyme-enhanced label for the electrochemical determination of diethylstilbestrol (DES). The label was obtained by orientation-controlled immobilization of a multiplex horseradisch peroxidase (HRP) conjugated polymer on gold nanoparticles (AuNPs) using the Envision reagent (EV) which is an enzyme-polymer complex that contains HRP and anti-IgG antibody in a polydextrin amine skeleton. The AuNPs were modified with Concanavalin A (Con A) and served as a carrier for immobilization of the EV−DES antibody composite. This resulted in a bioconjugate of the type AuNP−Con A−EV−DES Ab which was employed as the label. On exposure to samples containing DES, a sandwich immunocomplex is formed between antibody against DES (which was immobilized on a glassy carbon electrode and is acting as a capture probe), DES (the analyte), and the above label as the signal tracer. Hemin was used as an electronic mediator in the reaction of HRP. The HRP on the label catalyzes the oxidative formation of hydrogen peroxide at pH 7.0, and this induces an increased reductive current in the presence of hemin as an electron mediator. Under optimal conditions, the current increases linearly with increasing concentrations of DES in the range from 5 to 500 pg·mL−1, with a detection limit as low as 2 pg·mL−1 (at an S/N of 3). The method exhibits high selectivity and good stability. It works without incubation so that the time for an assay is shortened to

Electronic supplementary material The online version of this article (doi:10.1007/s00604-013-1131-3) contains supplementary material, which is available to authorized users. P. Xiong : N. Gan (*) : H. Cui : J. Zhou : Y. Cao : F. Hu : T. Li The State Key Laboratory Base of Novel Functional Materials and Preparation Science, School of Material Science and Chemical Engineering of Ningbo University, Ningbo 315211, People’s Republic of China e-mail: [email protected]

5 min. The assays was successfully applied to the determination of DES in milk samples. Keywords Envision complex . Diethylstilbestrol . Concanavalin A . Electrochemical immunoassay

Introduction Diethylstilbestrol (DES) is a synthetic nonsteroidal toluylene estrogen drug that is widely used in food animals, as a treatment for estrogen deficiency disorders in veterinary medicine, and for postcoital contraception [1, 2]. DES can affect reproductive capacity and the level of intracellular calcium ions [3, 4] and has been illegally used in animal production due to economic interests. Therefore, the use of DES in food and animal feed is prohibited by the Ministry of Agriculture of the People’s Republic of China, as evidenced by its inclusion in the list of banned veterinary drugs (Announcement No. 235). Additionally, the European Union (EU) requires that residual levels of DES in edible animal food should not exceed 2 ng· mL−1 [5]. However, there is no standard method for determination of residual DES [6]. Existing methods often include complex preconcentration processes prior to measurement. Thus, development of a rapid, sensitive, and inexpensive method for the determination of DES in milk samples is necessary. To date, various methods and strategies have been reported for determination of DES, such as immunohistochemistry [7], electrochemical detection [8], chemiluminescence [9], enzyme-linked immunosorbent assay (ELISA) [10], highperformance liquid chromatography (HPLC) [11, 12], and pressurized capillary electrochromatography [13]. Different chromatographic techniques, such as gas chromatography and HPLC, have extensive applications for simultaneous

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determination of DES using a variety of detectors [14–17]. However, these methods have many drawbacks, including complex operating procedures, long analysis times, and expensive instruments. Therefore, they are not suitable for rapid screening tests. Among the currently available techniques, electrochemical immunoassays can offer a sensitive and fast means of detection for DES. Recently, electrochemical immunoassays have attracted enormous attention because of their intrinsic advantages: high sensitivity, low cost, and high compatibility with advanced micromachining technologies [18–22]. However, DES exhibits slow electron transfer at unmodified electrodes, leading to low sensitivity for its detection using electrochemical immunoassays. Therefore, some functional materials have been developed as matrices for immobilizing enzymes (such as horseradish peroxidase (HRP) and glucose oxidase enzyme) as labels to catalyze electronic mediators and amplify the current signal; this could then be used to develop sensitive electrochemical immunosensors. Thus, the preparation of novel signal amplification materials conjugated with highdensity enzymes as labels could increase electrochemical immunoassay sensitivity in the design of an ultrasensitive electrochemical immunosensor for DES. The Envision reagent (EV) is a type of enzyme-polymer complex that contains about 100 molecules of HRP and 15 molecules of anti-IgG antibody connected in a poly-dextrin amine skeleton [23, 24]. When EV is incubated with the DES antibody (DES Ab), the resulting copolymers (EV/DES Ab) can act as a highly sensitive label for the immunosensor because of its high enzyme-labeled density. With the development of nanotechnology, various types of nanomaterials, such as metal nanoparticles have been applied in the fabrication of immunosensors. Gold nanoparticles (AuNPs) are the most frequently used nanomaterials in electrochemical immunoassays due to their excellent biocompatibility and ease of functionalization with proteins. In this study, AuNPs were used as an efficient matrix for the immobilization of enzyme and antibody [25, 26]. Probes based on detection antibodies labeled on AuNPs have widely been used in clinical diagnosis. If AuNPs are employed as a matrix to immobilize more than one EV/DES Ab on them as a label, the prepared AuNPs-EV/DES Ab bioconjugate will have much higher density of HRP enzyme label than EV/ DES Ab, thereby further amplifying the detection current. However, EV/DES Abs bounded on AuNPs through physical adsorption rather than specific binding exhibit random orientation. Concanavalin A (Con A) is a sugar-binding protein of the lectin family and possesses multiple sites with high affinity for carbohydrates, which thus can specifically recognize various sugar, glycoprotein and glycolipids [26]. Con A can be immobilized on AuNPs, allowing oriented immobilization of EV/DES Ab due to the strong affinity between the Con A and

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the mannose residues of dextrin, therefore yielding the AuNPs-Con A-EV/DES Ab label. Therefore, the AuNPsCon A complex can be used as the carrier to immobilize the EV/DES Ab. However, no reports on the actual application of AuNPs-labeled EV copolymers as labels in the construction of electrochemical immunosensors have been published to date. Traditionally, competitive immunoassays are used to detect small molecule compounds, usually with only one antigenic determinant, such as clenbuterol or salbutamol. Electrochemical immunosensors based on competitive immunoassays have decreased signals after enhancing the concentration of antigens. However, many factors, such as non-electro-active proteins and nucleic acids, can lead to decreased signal intensities, which can result in false positive results. Moreover, nonspecific binding in competitive immunoassays would also give rise to false-positive errors. Sandwich-type immunoassays can overcome the shortcomings of traditional competitive immunoassays (such as sensitivity and the limited linear range); the method is also simple and reliable. Sandwich-type immunoassays require two monoclonal antibodies to identify different epitopes in the antigen, which can greatly improve the precision and specificity of the immunoreactions. Moreover, the signal of the electrochemical immunosensors in sandwich immunoassays would increase with increasing concentrations of antigen. The above features provide substantial minimization of matrix interferences. However, the assay is not very suitable for small molecules with only one epitope, which thus does not allow the combination of two kinds of antibodies due to steric exclusion. DES is symmetrical structure of the phenol, and we hypothesized that DES may conjugate with two monoclonal antibodies if the binding sites of DES antibody are the phenol functional groups. Recently, Liu et al. [27] developed a sandwich-type protocol to fabricate an electrochemical immunosensor for the detection of DES using two same DES antibodies with higher sensitivity and selectivity than those used for ELISA. Therefore, we use the sandwich protocol for fabricating an electrochemical immunosensor for DES. In this paper, we have successfully designed an ultrasensitive immunosensor for detecting DES without incubation at room temperature. The AuNPs-Con A-EV/DES Ab bioconjugate was employed as the label. AuNPs not only increased the surface area of the electrode and immobilized more antibodies on the electrode, but also allowed the maintenance of antibody activity due to their high biocompatibility. Upon achievement of the sandwich immunoreaction, the AuNPsCon A-EV/DES Ab label was attached onto the modified electrode surface through the antibody-antigen immunoreaction. The peak currents were dependent on the concentration of the corresponding antigen. The strategy allowed the direct detection of DES with incubation-free at room temperature. The whole detection process required less than 5 min, which greatly shortened the assay time.

An incubation-free electrochemical immunosensor for Diethylstilbestrol

Experimental

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Preparation of AuNPs-Concanavalin A- EV/DES Ab (AuNPs-Con A-EV/DES Ab)

Instrumentation Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed by electrochemical analyzer (model CHI 660) (Shanghai Chen hua Instrumental Corp, China). Transmission electron microscope (TEM) image was obtained from H-7650 microscope (Hitachi Instruments, Japan). Scanning electron microscope (SEM) image was obtained from SU70 microscope (Hitachi Instruments, Japan) and S3400 microscope (Hitachi Instruments, Japan). A conventional three-compartment electrochemical cell consisting of a saturated calomel electrode as the reference electrode, a platinum wire electrode as the auxiliary electrode and a modified glassy carbon electrode (GCE, 3 mm in diameter) as a working electrode.

Reagents DES ELISA kit was supplied by Huaan Magnech Bio-techCo., Ltd (http://www.magnech.com/, Beijing, China). Envision™ Detection Kit was supplied by Gene Tech Co., Ltd (http:// www.genehk.com/, Shanghai, China). Bovine serum albumin (BSA, 96–99 %), and hydrogen tetrachloroaurate(III) tetrahydrate (HAuCl4), hydrogen peroxide (H2O2, 30 %), and trisodium citrate were obtained from Sinopharm Chemical Reagent Co., Ltd (http://www.sinoreagent.com/, Shanghai, China). Hemin was obtained from the Solarbio Bioscience & Technology Co., Ltd (http://www.solarbio.com/, Shanghai, China). Concanavalin A (Con A) was purchased from Sigma (http://www.sigmaaldrich.com/, USA), which is extracted from Canavalia ensiformis (Jack bean). Gamma alumina power 0. 05 μm was supplied by Chen hua Instrumental Corp (http:// w w w. c h i n s t r. c o m / , S h a n g h a i , C h i n a ) . P o l y diallyldimethylammonium chloride solution (PDDA) was purchased from Sigma (http://www.sigmaaldrich.com/, USA) Phosphate buffer saline (PBS, 0.1 mol·L−1 pH 7.0) containing 0.1 mol·L−1 KCl was used to prepare the protein solution. The blocking buffer solution was PBS (pH 7.0) containing 1 % (w/v) BSA. All other reagents were of analytical grade and were used without further purification. Distilled water was used throughout the study. All experiments were carried out at room temperature.

Firstly, AuNPs colloide was prepared by using the Frens method [28] with modification. In brief, 0.01 wt% HAuCl4 (100 mL) was boiled with vigorous stirring in a flask. 1 wt% trisodium citrate solution (2.5 mL) was quickly added to the boiling solution, resulting in a color change from faint yellow to crimson, indicating that generated AuNPs. The solution was maintained for 5 minutes at boiling temperature. Figure 1b shows the procedure used to prepare the AuNPsCon A-EV/DES Ab bioconjugate. Initially, 5 mL of AuNPs colloid was adjusted to pH 8.2 using Na2CO3 solution. Thereafter, 200 μL of Con A at 1 mg·mL−1 was added to the dispersion, and the mixture was stirred 4 h at 4 °C. After centrifugation at 12,000 rpm for 10 min, the resulting mixture was redispersed in 1.0 mL of pH 7.0 PBS. Then 200 μL of EV/ DES Ab was added and the mixture was stirred overnight at 4 °C. After centrifugation at 12,000 rpm for 15 min, the resulting mixture was redispersed in 1.0 mL of pH 7.0 PBS solution containing 1 % BSA and stored at 4 °C for further use. Preparation of the modified electrode Before modification, the bare GCE was polished with γalumina down to 0.05 nm, then cleaned ultrasonically in deionized water for 2 min and finally cleaned with absolute alcohol and distilled water for 5 min, respectively. The polished GCE was immersed in the as-prepared 1 mmol· L−1 HAuCl4 solution, and the electrodeposition was performed by applying a constant voltage of −0.5 V for 60 s. After electrodeposition, the modified AuNPs/GCE was removed from the solution and rinsed with distilled water, then dried in the air at room temperature. Subsequently, 20 μL of PDDA solution was dropped onto the AuNPs/GCE surface and kept at room temperature for 30 min to produce a PDDA/AuNPs/GCE. Next, the modified electrode immersed in DES Ab solution at 4 °C overnight, the modified electrode (DES Ab/PDDA/AuNPs/GCE) was removed from the solution and rinsed with double distilled water. Finally, this modified electrode was incubated in 1 % BSA for 30 min in order to block possible remaining active sites and avoid non-specific adsorption. The finished immunosensor was stored at 4 °C when not in use. The fabricated procedure and detection principle of the immunosensor is summarized in Fig. 1c.

Preparation of envision/DES antibody (EV/DES Ab)

Electrochemical measurement

Figure 1a shows the procedure used to prepare the EV/ DES Ab bioconjugate. A certain volume of DES Ab and EV were mixed and stored overnight at 4 °C. After centrifuging, the EV/DES Ab was stored at 4 °C until further use.

The electrochemical properties of DES were measured by cyclic voltammetry (CV) in a standard three-electrode cell. About 5 mL PBS (pH 7.0) containing an appropriate amount of DES standard solution was added into the electrochemical cell. The CVs were recorded in the potential range from

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Fig. 1 Schematic representation of the preparation of a EV/DES Ab, b AuNPs-Con A-EV/DES Ab, and c the fabrication process of immunosensor

0.20 V to −0.60 V with a scan rate of 100 mV·s−1 after accumulating for 20 s.

Result and discussion Construction and characteristics of the electrochemical immunosensor The topography of the modified GCE and AuNPs-Con A-EV/ DES Ab bioconjugate was characterized by SEM, as depicted in Fig. 2. In Fig. 2a, we can see that the surface of PDDAmodified GCE was very smooth, indicating that PDDA formed a uniform film on the electrode. The image of the AuNPs/GCE (Fig. 2a) revealed that AuNPs with a homogeneous grain size were uniformly deposited on the surface of the electrode and were visible as many small, bright spots. When the DES Ab was modified on the electrode, a porous film was apparent on the surface of the immunosensor, suggesting that DES Ab had been immobilized (Fig. 2c). EV had a tree-like skeleton shape, with a large amount of HRP immobilized on it (Fig. 2d). When the EV was conjugated to

DES Ab, the EV/DES Ab copolymer exhibited skeleton-like polymerization, indicating that EV was connected to the skeleton of DES Ab (Fig. 2e). After AuNPs were employed as a matrix to immobilize Con A and the EV-DES Ab to produce the AuNPs-Con A-EV/DES Ab label, many small particles were seen (Fig. 2f), suggesting that many AuNPs were embedded in the protein layers. The signal label was characterized by TEM. The details were shown in the electronic supplementary material (ESM). Figure S1 (A) gives the TEM image of the AuNPs, it revealed that the AuNPs dispersed uniformly and that the average particle size was around 10 nm. Figure S1(B) showed that Con A, EV/DES Ab was bound onto the AuNPs surface, indicating that AuNPs-Con A-EV/DES Ab had been formed. Figure S2 shows the ultraviolet–visible absorption spectrometry (UV–Vis) for characterization of EV/DES Ab, AuNPs, AuNPs-Con A-EV/DES Ab, BSA, and BSA-blocked AuNPsCon A-EV/DES Ab. The EV/DES Ab had two absorption bands (curve a), one was at 260 nm and attributed to DES Ab, the other was at 404 nm and attributed to HRP. A strong absorption peak at 520 nm corresponding to the AuNPs was observed (curve b). After labeling with AuNPs, AuNPs-Con A-EV/DES Ab (curve


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Fig. 2 SEM images of a PDDA/ GCE; b AuNPs/PDDA/GCE; c Ab/AuNPs/PDDA/GCE, d EV; e EV/DES Ab; f AuNPs-Con AEV/DES Ab, respectively

c) showed a characteristic peak of AuNPs at 520 nm, which indicated the capture of AuNPs. A peak at 280 nm was attributed to BSA (curve d). When BSA blocked the nonspecific binding sites, the BSA-blocked AuNPs-Con A-EV/DES Ab showed peaks at 520, 404, and 260 nm (curve e). From the above results, it can be concluded that the AuNPs-Con A-EV/DES Ab label had been constructed successfully. Electrochemical characterization of the immunosensor Electrochemical impedance spectroscopy (EIS) has been used to study the interfacial properties of modified electrodes [29]. Impedance measurements were performed at a DC potential of 0.223 V in 5 mmol·L−1 [Fe(CN)6]4−/3−containing 0.1 mol· L−1 KCl (pH 6.5) in 0.1 mol·L−1 PBS as a supporting electrolyte. An alternating voltage (peak-to-peak) of 5 mV was superimposed on the applied DC potential. Nyquist plots were recorded over the frequency range 10 kHz to 0.1 Hz.

Simulations were performed using the SIM software program (MEP Instruments Pty Ltd, NSW, Australia) [30]. In EIS, the semicircle diameter equals the electron-transfer resistance (Ret). The results are shown in Fig. 3 at different stages of the modified electrode in 5 mmol · L −1 [Fe(CN) 6 ] 4−/3 − containing 0.1 mol · L−1 KCl (pH 6.5). The bare GCE displayed a small semicircle at high frequencies, suggesting a low Ret to [Fe(CN)6]4−/3−(curve a). After the electrode was modified by PDDA, the Ret was increased, which indicated that PDDA constituted a barrier for electron transfer from [Fe(CN)6]4 −/3− at the electrode surface (curve b). When the AuNPs were bound to the surface of electrode, the Ret was decreased, which indicated that the AuNPs dramatically accelerated electron transfer (curve c). The Ret increased in the same way after the DES Ab was immobilized on the electrode surface (curve d); this was ascribed to the nonconducting properties of the DES Ab, hindering the electron transfer reaction. After BSA was used to block nonspecific sites, Ret increased in the same way

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reduced (curve b). Following the deposition of AuNPs on the electrode, the CV (curve c) revealed an increase in the peak current as a result of AuNPs facilitating the electron transfer reaction. As shown in curve d, the peak current was decreased after immobilization of the DES Ab on the electrode, indicating hindered electron transfer by the DES Ab. Similarly, the reduction peak current was further decreased (curve e) after BSA was used to block nonspecific binding. Subsequently, the peak current decreased after the immunosensor was incubated in a DES antigen solution (curve f), as the immunocomplex hindered tunneling mass and electron transfer at the electrode. These results showed that the DES Ab and DES were successfully immobilized on the electrode surface and are also in agreement with the results of EIS. Fig. 3 EIS of the different electrodes measured in 5.0 mmol·L−1 [Fe(CN)6]4−/3− (pH 6.5): a bare GCE; b PDDA/GCE; c AuNPs/ PDDA/GCE; d DES Ab/AuNPs/PDDA/GCE; e BSA/DES Ab/AuNPs/ PDDA/GCE; f DES/BSA/DES Ab/AuNPs/PDDA/GCE, respectively

(curve e), which may also be attributed to the nonconducting properties of the DES Ab. The Ret further increased (curve f) after the resulting immunosensor was incubated in the DES solution, suggesting that formation of the immunocomplex layer enhanced electron transfer. Figure 4 illustrates the cyclic voltammograms obtained at different stages of the modified electrode, recorded using CV in PBS (pH 7.0) containing 80 μmol·L−1 hemin from 0.2 to −0.6 V (versus. the saturated calomel electrode, SCE) at 100 mV·s−1. As expected, a clean GCE in PBS yielded a current peak at −0.32 V (curve a). When the PDDA solution was used to modify the electrode, reduction peaks was

Comparison of electrochemical responses using various labels To further investigate the effect of the synthesized AuNPsCon A-EV/DES Ab on the sensitivity of the electrochemical immunosensors, four types of detection antibodies were used, such as HRP-DES Ab, AuNPs-HRP-DES Ab, AuNPs-EV/ DES Ab and AuNPs-Con A-EV/DES Ab (Fig. 5). As shown in Fig. 5a–d, upon addition of H2O2 to the substrate solution, an obvious catalytic characteristic appeared, with a distinct increase in the reduction current and a decrease in the oxidation current. The catalytic current is mainly due to the labeled HRP toward the reduction of H2O2 with the help of hemin as an electron mediator. As shown in Fig. 5, we found that the electrochemical immunosensor exhibited a larger current shift using EV/DES Ab (Fig. 5b) as the detection antibody than when using HRP-DES Ab (Fig. 5a). The reason for this might be that EV contains large number of HRP molecules. The electrochemical immunosensor exhibited a larger current shift using AuNPs-EV/DES Ab (Fig. 5c) as the detection antibody than when using EV/DES Ab (Fig. 5b). The reason is that the AuNPs with a high surface-to-volume ratio and good biocompatibility can load more EV/DE Ab. Moreover, AuNPs have good conductivity, which can enhance enzyme activity [31]. More significantly, the electrochemical responses could be improved by using AuNPs-Con A-EV/DES Ab (Fig. 5d) rather than AuNPs-EV/DES Ab (Fig. 5c). A possible reason is that Con A can oriented immobilization of EV/DES Ab, which indirectly increases the amount of bound DES Ab and HRP. Optimization of the immunoassay conditions

Fig. 4 CVs obtained at 5 mL 0.1 mol·L−1 PBS (pH 6.5) containing 80 μmol·L−1 hemin: a bare GCE; b PDDA/GCE; c AuNPs/PDDA/ GCE; d DES Ab/AuNPs/PDDA/GCE; e BSA/DES Ab/AuNPs/PDDA/ GCE; f DES/BSA/Ab/AuNPs/PDDA/GCE, respectively

In order to obtain the best analytical performance for DES, experimental conditions were optimized. The pH of the detection solution is an important parameter because the acidity of the solution is greatly affected by the activity of immobilized proteins. CVs were recorded in PBS


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Fig. 5 CV of the electrochemical sandwich immunosensors toward 100 pg·mL−1 DES in the absence (a) and presence (b) of 80 μmol·L−1 H2O2 in an electrolytic cell in PBS containing 80 μmol·L−1 hemin by

using various detection antibodies a HRP-DES Ab, b EV/DES Ab, c AuNPs-EV/DES Ab, and d AuNPs-Con A-EV/DES, respectively. Scan rate: 100 mV·s−1

prepared with different pH values. The effects of the pH of the solution on the amperometric response for DES are shown in Figure S3 (A). As shown, the current response increased rapidly with increasing pH value from 5.0 to 7.0 and then decreased at higher pH condition. This resulted from the influence of the pH on protein denaturation, and a highly acidic or alkaline environment would be expected to damage the immobilized protein [32]. Therefore, pH 7.0 was optimal and corresponding PBS was used for the following measurements. As illustrated in Figure S3 (B), the concentration of hemin was optimized by varying its concentration from 16 to 96μ mol·L−1. The current value increased to a maximum when hemin reached a concentration of 80μ mol·L−1. Therefore, 80μ mol·L−1 was chosen as the optimum concentration of hemin for use in further experiments. The concentration of H2O2 is also an important parameter affecting response of the immunosensor to hemin. Different concentrations of H2O2 from 16 to 240μmol·L−1 were evaluated. Figure S3 (C) shows the effects of different

concentrations of H2O2 on the amperometric response of 80μ mol·L−1 hemin for the detection of 100 pg·mL−1 DES. With increasing concentrations of H2O2 from 16 to 80μmol· L−1, the electrocatalytic activity increased sharply and reached a plateau after 80μ mol·L−1. At H2O2 concentrations greater than 80μ mol·L−1, the response increased only slightly. Therefore, 80μ mol·L−1 was used as the optimal H2O2 concentration in further experiments. When using the AuNPs-Con A-EV/DES Ab as a label, the density of AuNPs-Con A-EV/DES Ab has an effect on the interaction efficiency between the detection antibody and the DES antigen. To determine the optimal density of AuNPs-Con A-EV/DES Ab, various volumes of AuNPs-Con A-EV/DES Ab were used in conjunction with 20 μL of the 100 pg·mL−1 DES solution. As shown in Figure S3 (D), the peak current increased to a maximum when the volume of AuNPs-Con AEV/DES Ab reached 10 μL and then decreased as AuNPsCon A-EV/DES Ab was further elevated to 16 μL. Thus, the optimal volume of AuNPs-Con A-EV/DES Ab was chosen as 10 μL.

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Fig. 6 Calibration plots for different concentration of DES under optimal conditions in PBS, pH 7.0. Scan rate: 100 mV·s−1

Differences between the performances of immunosensors incubation and incubation-free

Electrochemical responses of the electrochemical immunosensor

The temperature and incubation time have a great influence on the sensitivity of the sandwich-type immunoassay. The immunosensor current was investigated at 100 pg·mL−1 of DES. As shown in Figure S4, curve b was the reduction current following a 30-min incubation at 37 °C±1.0 °C, and there was no obvious increase compared to the current of the sample incubation-free at room temperature (curve a), indicating that the formation of the sandwich-type immunocomplex was rapid and convenient when using AuNPs-Con A-EV/ DES Ab as a label. Longer incubation times did not improve the response. Therefore, detection of DES was carried out incubation-free at room temperature.

Under the optimal conditions, the immunosensor was used for the detection of various concentrations of DES. CVs were used for quantification. As shown in Fig. 6a, the catalytic peak revealed by CV in the presence of DES (curve b) was higher than that in the absence of DES (curve a) and increased gradually with increasing concentrations of DES (curves b→p). A standard calibration curve for DES detection is shown in Fig. 6b. The detection limit of the method was 2 pg·mL−1 DES (S/N=3). The correlation coefficients were 0.999 and 0.980 for the linear ranges, respectively. The calibration curve presented with two slopes, likely because the amount of antibody immobilized on the electrode surface was

Table 1 Comparison of analytical properties of various DES immunosensors or immunoassays References

Label

Detection limit (ng·mL−1)

Linear range (ng·mL−1)

Present study

AuNPs-Con A-EV/DES Ab

0.002

0.005–0.5

[8] [26] [34] [35] Specification of DES ELISA kit

Cyclodextrin-reduced grapheme oxide hybrid nanosheets HRP-functionalized nanoparticles bioconjugate Multi-wall carbon nanotubes ZnSe Quantum-dots ELISA

3

1.07×10 0.12 6.71×102 5.37×102 0.1

Incubation time (min) 0

2.68×10 –3.49×10 0.33–4.5×103 2.68×103–5.37×105 1.61×103–1.07×107 0–8.1 3

6

3.5 5 4 Not mentioned 30


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Table 2 Recoveries of DES comparison of two methods obtained in the spiked milk samples (n =3)

EV/DES Ab was prepared and stored in refrigerator at 4 °C. When the immunosensor was prepared and not in use, it was stored in refrigerator at 4 °C. After 3 weeks, the catalytic current of the immunosensor using AuNPs-Con A-EV/DES Ab as label decreased to about 90 % of its initial value. The decrease in the current response maybe due to the gradual denature of HRP and Ab.

Samples

1

2

3

Containing Added (pg·mL−1) Found (pg·mL−1) Recovery (%)

ND 20 20.6±0.5 103.0

ND 50 49.8±0.7 98.0

ND 100 86.6±1.0 86.6

ELISA (pg·mL−1)

ND

ND

88.0

Application in milk samples analysis

ND not detected

constant. As the antigen concentration increased, the active sites of the antibodies immobilized on the electrode surface became fewer, leading to a decrease in sensitivity [33]. Table 1 provides a comparison of the analytical properties of various DES immunosensors and immunoassays. Reproducibility, selectivity, interference and stability of the electrochemical immunosensor Intra- and inter-assay coefficients of variation (C.V) were employed to evaluate the reproducibility of the immunosensor. By analyzing five concentration levels, three times for each concentration, the precision of the used method was given. The C.V of intraassay by the immunosensor were 3.1, 3.4, 3.5, 3.6, and 3.2 % at 10, 20, 40, 60, 80 pg·mL−1 of DES, respectively. And the interassay C.V in three immunosensors were 3.5, 3.8, and 3.9 % at 10, 20, 40, 60, 80 pg·mL−1 of DES, respectively. The results indicated that the precision and reproducibility of the immunosensor were quite good. To investigate the selective of the fabricated immunosensor, the immunoassay was performed in a 100 pg·mL−1 DES sample solution containing some interfering substances with DES 100 pg·mL−1 of DES solution containing 1 ng·mL−1 of interfering substance (Estradiol, Estriol, Oestrone, respectively) was measured by the immunosensor. The current variation due to the interfering substances was not more than 9.4 % that without interferences (Figure S5, ESM). Such high selectivity was due to the highly specific binding affinity of the antigenantibody immunoreactions and the minimization of nonspecific adsorption by the efficient blocking. It determined that the developed immunosensor had excellent selectivity. 100 mL pure milk without DES was chosen as blank sample for spiking experiment which contains 3.1 grams (g) of protein, 3.7 g fat and 4.8 g carbohydrate and others metal ions, such as sodium and calcium etc. When 50 pg·mL−1 DES was added, the current variation due to the interfering substances was not more than 5 % of that without interferences. The result suggested that the immunosensor had excellent reproducibility and selectivity. The stability of the immunosensor was also checked by periodically checking the current responses. AuNPs-Con A-

In order to further investigate the applicability and reliability of the newly developed immunosensor, the electrochemical immunosensor was used to detect of DES in milk products from one manufactures purchased from a supermarket in Ningbo (China). The detection was performed by the standard addition method. These analyses were performed for three times under the same conditions, and the results listed in Table 2 were also compared with the traditional ELISA method. The mean recoveries of DES are in the range from 86.6 % to 103.0 %, the results are in good agreement with the ELISA method, thus suggesting that the new method is reliable and exact. The result indicated the developed immunosensor had high accuracy and could be applied for the detection of real sample.

Conclusions In this work, we designed a novel sandwich electrochemical immunoassay for the detection of DES based on the use of a novel AuNPs-Con A-EV/Ab label synthesized using Con A as an anchoring reagent for orientation-controlled immobilization of EV. Through a sandwich immunoreaction, the AuNPs-Con A-EV/Ab label was captured on the modified electrode surface. The CV of hemin was used to quantify the concentration of DES after the addition of H2O2. This method showed excellent performance for the detection of DES and permitted low detection limits. The current approach has provided a new avenue for DES detection and has great potential for biosensor applications. Acknowledgments This work was supported by the Natural Science Foundation of Zhejiang (No. LY13C200017, LY12C20004, Y3110479), the National Science Foundation of China (No. 81273130), the Science and Technology Project of Zhejiang province (2011C50038, 2012C23101, 2013A610241), and the K.C. Wong Megna Fund in Ningbo University (China).

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