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A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice
Analytical Biochemistry 387 (2009) 82–86
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice Yun Tan, Xia Chu *, Guo-Li Shen, Ru-Qin Yu State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
a r t i c l e
i n f o
Article history: Received 18 October 2008 Available online 4 January 2009 Keywords: Aflatoxin B1 Electrochemical immunosensor Enzymatic silver deposition Linear sweep voltammetry
a b s t r a c t A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B1 (AFB1) in rice. The immunosensor was based on an indirect competitive format between free AFB1 and aflatoxin B1–bovine serum albumin (AFB1–BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB1 antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB1–BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB1 in real samples. Ó 2009 Elsevier Inc. All rights reserved.
Aflatoxins are a group of naturally occurring mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus, which can be found in a large variety of food and animal feed. Naturally occurring aflatoxins are composed of B1, B2, G1, and G2. Among them, aflatoxin B1 (AFB1)1, which is the most abundant and carcinogenic, was classified as a carcinogenic substance of group 1 by the International Agency for Research on Cancer (IARC) [1]. Many grains and foodstuffs, including corn, peanuts, cottonseed, cereals, beans, and rice, have been found to be contaminated with aflatoxins. Moreover, humans would be exposed to aflatoxins either directly by eating contaminated grains or indirectly via animal products. Because aflatoxins are relatively heat stable, it is hard to destroy them once they are formed. The significant threat to human health posed by contamination has motivated extensive research in this toxin. Several methods for the detection of aflatoxin B1 have been established, including thin* Corresponding author. Fax: +86 731 8821916. E-mail address: [email protected] (X. Chu). 1 Abbreviations used: AFB1, aflatoxin B1; IARC, International Agency for Research on Cancer; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; LC/ESI–MS/MS; OWLS, optical waveguide lightmode spectroscopy; AFB1–BSA, aflatoxin B1–bovine serum albumin; ALP, alkaline phosphatase; LSV, linear sweep voltammetry; IgG, immunoglobulin G; SCE, saturated calomel electrode; PBS, phosphate-buffered saline; SD, standard deviation. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.12.030
layer chromatography (TLC), high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA) [2–4]. TLC is a relatively economical method that requires little equipment. HPLC is often coupled with different cleanup procedures such as solid phase extraction, supercritical fluid extraction, immunoaffinity chromatography, and matrix solid phase dispersion [5–8]. Recently, a liquid chromatography–tandem mass spectrometry with electrospray ionization (LC/ESI–MS/MS) method [9] was reported. Although sensitive and accurate, most of the chromatographic methods require expensive equipment and extended cleanup steps. Immunoassays can offer advantages over chromatographic procedures because they are faster and cheaper. Thus, different immunoassay techniques for the detection of AFB1 have been developed. For instance, radioimmunoassay [10], a rapid colorimetric immunoassay [11], a surface plasmon resonance-based immunoassay [12], a strip liposome immunoassay [13], a filtration-based tyramide amplification immunoassay [14], and a fluorescence polarization immunoassay [15] have been reported. Immunosensors based on the optical waveguide lightmode spectroscopy (OWLS) technique, the impedimetric responses, and electrochemical arrays [16–18] have also appeared during recent years. There is an increasing demand for ultrasensitive methods of immunoassay. Electrochemical sensors may be a good choice due to their fast, simple, and low-cost detection capabilities for biological binding events [19]. During recent years, new schemes based
Immunosensor for aflatoxin B1 determination / Y. Tan et al. / Anal. Biochem. 387 (2009) 82–86
on coupling the biocatalytic amplification of enzyme labels with additional electrochemical detections have been developed [20]. The use of chronopotentiometry and Faradaic impedance spectroscopy coupled with biocatalyzed precipitation [21] or enzymatic deposition of silver for the amplified detection of a target DNA [22] has been demonstrated to be a sensitive method. In addition, immunoassays based on silver-enhanced gold nanoparticle label [23], biocatalytic deposition of silver nanoparticles [20], and biocatalytic metal deposition coupled with anodic stripping voltammetric detection [24] have been reported recently. The combination of enzyme catalysis and metal deposition seems to be a much more promising strategy for amplifying assay signal. In the current work, we attempted to use the signal-amplified method of enzymatic silver deposition for the detection of AFB1. The procedures are depicted in Scheme 1. Aflatoxin B1–bovine serum albumin (AFB1–BSA) conjugate was first immobilized on the gold electrode surface. An indirect competitive format between selected analyte in solution and AFB1–BSA on the electrode was performed. After the competitive step, monoclonal antibody against AFB1 was bound to the electrode and then conjugated to a secondary antibody–alkaline phosphatase (ALP) conjugate. The ALP could catalyze the substrate, ascorbic acid 2-phosphate, into ascorbic acid, and the latter can reduce silver ions in solution to metal silver deposited onto
83
the electrode surface. Finally, the metallic silver deposited onto the electrode was determined by linear sweep voltammetry (LSV).
Materials and methods Materials and reagents Anti-AFB1 monoclonal antibody, AFB1–BSA conjugate, and AFB1 were purchased from Sigma–Aldrich (St. Louis, MO, USA). BSA and ALP-labeled anti-mouse immunoglobulin G (IgG) secondary antibody were purchased from the Beijing Dingguo Biotechnology Development Center (Beijing, China). Ascorbic acid 2-phosphate was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Glutaraldehyde was obtained from National Reagent (Shanghai, China). NaCl, KH2PO4, Na2HPO4, NaOH, glycine, AgNO3, and KNO3 were of analytical purity. Ultrapure water was obtained through a Nanopure Infinity Ultrapure water system (Barnstead/Thermolyne, Dubuque, IA, USA) with an electrical resistance greater than 18.3 MX. Apparatus All of the electrochemical experiments were carried out with a model CHI660C electrochemical workstation (Shanghai Chenhua Instruments, Shanghai, China). Electrochemical measurements were performed with a three-electrode system composed of a platinum foil as auxiliary electrode, a saturated calomel electrode (SCE) as reference electrode, and a modified gold electrode as working electrode. Electrode modification
Ascorbic acid 2-phosphate
Ascorbic acid
Gold electrode (99.99%, 2 mm diameter, Shanghai Chenhua Instruments) was immersed in freshly prepared Piranha solution (H2O2/H2SO4, 1:3, v/v) for 2 h, polished with 0.05 lm alumina slurry on microcloth, and rinsed with ultrapure water. The electrode was then sonicated in ultrapure water for 5 min, rinsed thoroughly with ultrapure water, and dried at room temperature. The cleaned gold electrode was immersed in 10 mM cysteamine solution overnight at 4 °C and rinsed thoroughly with ultrapure water to remove physically absorbed cysteamine. Subsequently, the electrode was immersed in 2.5% glutaraldehyde solution for 1 h at 37 °C and rinsed with ultrapure water. Then 5 ll of 100 lg/ml AFB1–BSA was dropped on the electrode surface and incubated for 1 h at 37 °C and washed with phosphate-buffered saline (PBS, pH 7.4) and ultrapure water. Finally, the electrode was incubated with 5 mg/ml BSA for 0.5 h to block the unreacted active sites.
Dehydroascorbic acid
Indirect competitive immunoassay Ag
Ag+
AFB1-BSA
AFB1
anti- AFB 1 antibody
Secondary antibody-ALP conjugate
Scheme 1. Schematic diagram of the developed electrochemical immunosensor for the detection of AFB1: (a) immobilization of AFB1–BSA conjugate on the gold electrode surface; (b) indirect competitive reaction between immobilized AFB1–BSA and free AFB1 in solution for binding to a fixed amount of anti-AFB1 antibody in solution; (c) association with secondary antibody–ALP conjugate and then enzymatic silver deposition.
To perform the assay, 2.5 ll of diluted anti-AFB1 antibody was first mixed with 2.5 ll of AFB1 standard solution, with the concentration ranging from 0 to 200 ng/ml in PBS (pH 7.4). The mixture was dropped at the surface of the modified electrode and incubated for 1 h at 37 °C in a humidity chamber. During incubation, a competition was carried out between free AFB1 in solution and the immobilized AFB1–BSA for binding to a fixed amount of antiAFB1 antibody in solution. After washing with PBS (pH 7.4), 5 ll of the secondary antibody–ALP conjugate (1:50, v/v) was added onto the electrode and incubated for 1 h at 37 °C. After rinsing with PBS (pH 7.4), the electrode was immersed in the freshly prepared 50 mM glycine–NaOH buffer (pH 9.08), containing 1 mM AgNO3 and 1 mM ascorbic acid 2-phosphate, at 37 °C for 30 min. Finally, the resulting electrode was rinsed with ultrapure water.
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Immunosensor for aflatoxin B1 determination / Y. Tan et al. / Anal. Biochem. 387 (2009) 82–86
tivity and the secondary antibody–ALP immobilized on the electrode.
Electrochemical detection LSV was performed at a potential range from 0 to 0.8 V (vs. SCE) with a 100-mV/s scanning rate in electrolyte of 0.6 M KNO3/0.1 M HNO3. All of the electrochemical detections were operated at room temperature. Sample preparation Noncontaminated rice (from a local market) was first ground in a household blender. Aliquots (1 g) of ground rice were spiked with AFB1 at different concentrations and mixed in a vortex mixer. After adding 5 ml of extraction solvent (80% methanol), the samples were mixed by shaking for 45 min and then centrifuged at 5000 rpm for 10 min. The supernatant was carefully removed and diluted with PBS (1:5, v/v). Safety AFB1 is a very potent carcinogen, so great care should be taken to avoid personal exposure. It is necessary to wear lab coat and gloves when doing experiments. The waste should be treated with hypochlorite before disposal. Results and discussion Characterization of electrochemical impedance spectrum Electrochemical impedance spectrum is an effective method to monitor the interface properties of modified electrodes. The impedance spectra were determined after each step. As can be seen in Fig. 1, the semicircle diameter of the Nyquist plots increased after immobilization of AFB1–BSA conjugate. This was due to a barrier for the electron transfer at the electrode interface resulting from insulated AFB1–BSA. After reaction with anti-AFB1 antibody, the resistance became further increased, indicating the immobilization of antibody on the electrode surface. Finally, after reaction with the secondary antibody–ALP conjugate, the enhanced resistance demonstrated that the anti-AFB1 antibody retained its bioac-
180 160 140
successfully
Optimization of experimental conditions Protein molecules may adsorb nonspecifically on the sensor substrate. In this work, we used 5 mg/ml BSA to block the residually active site after the immobilization of AFB1–BSA conjugate, and the electrode was washed thoroughly after each step. These processes could effectively reduce the nonspecific adsorption. After the immobilization of 100 lg/ml AFB1–BSA on the electrode surface, one electrode was incubated with 5 mg/ml BSA for 0.5 h and then 5 ll of the secondary antibody–ALP conjugate (1:50, v/ v) was added onto the electrode and incubated for 1 h at 37 °C, whereas the other was directly incubated with 5 ll of the secondary antibody–ALP conjugate (1:50, v/v). The subsequent steps were the same as those described in Materials and Methods. The average peak current of the BSA-blocked electrode was 4.6 lA, which was significantly lower than that obtained with the electrode without BSA block (49.8 lA). So, 5 mg/ml BSA could effectively block the nonspecific adsorption in the proposed method. The concentration of AFB1–BSA conjugate is an important parameter because of the competitive reaction between AFB1– BSA on the electrode surface and free AFB1 in solution for binding to a fixed amount of anti-AFB1 antibody. As can be seen in Fig. 2, the peak current increased as the concentration of AFB1–BSA increased up to 100 lg/ml with fixed concentrations of anti-AFB1 antibody (1:10,000) and the secondary antibody–ALP conjugate (1:50, v/v). No significant increase in peak current was observed at a concentration higher than 100 lg/ml, so a 100-lg/ml AFB1– BSA solution was selected in the subsequent study. The amount of anti-AFB1 antibody also plays an important role in this approach. The more the AFB1 in solution reacts with antibody, the less the amount of antibody will immobilize on the electrode. As a result, the electrochemical signal would decrease significantly. So, the effect of the concentration of anti-AFB1 antibody on the peak current was also investigated. As shown in Fig. 3A, with fixed concentrations of AFB1–BSA (100 lg/ml) and the secondary antibody–ALP conjugate (1:50, v/v) and varying concentrations of anti-AFB1 antibody, the peak current increased rapidly with the increase of anti-AFB1 antibody concentration up to 1:10,000 and then tended to be stable. Dose–response curves obtained using various dilutions of anti-AFB1 antibody are shown in Fig. 3B; with fixed concentrations of AFB1–BSA (100 lg/ml) and the secondary antibody–ALP conjugate (1:50, v/v), the relatively
f
120
-z''/ohm
was
a
100
b
d
c
e 160
80
140
40 20 0 0
100
200
300
400
500
600
700
z'/ohm Fig. 1. Nyquist diagram of electrochemical impedance spectra: (a) bare gold electrode; (b) glutaraldehyde/cysteamine/gold electrode; (c) AFB1–BSA/glutaraldehyde/cysteamine/gold electrode; (d) BSA/AFB1–BSA/glutaraldehyde/cysteamine/ gold electrode; (e) anti-AFB1 antibody/BSA/AFB1–BSA/glutaraldehyde/cysteamine/ gold electrode; (f) secondary antibody–ALP conjugate/anti-AFB1 antibody/BSA/ AFB1–BSA/glutaraldehyde/cysteamine/gold electrode. Frequency range: 0.1 to 10 kHz. All of the measurements were performed in 1/15 M PBS containing 0.1 M KCl and 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (pH 7.4).
Peak current (µA)
60 120 100 80 60 40 20 0
50
100
150
200
250
Concentration of AFB 1-BSA (µg/ml) Fig. 2. Effect of concentration of AFB1–BSA conjugate on peak current.
300
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Immunosensor for aflatoxin B1 determination / Y. Tan et al. / Anal. Biochem. 387 (2009) 82–86 140
200 180
120
160 100
Current (µA)
Peak current (µA)
140 120 100 80 60 40
1 pg/ml 10 pg/ml 100 pg/ml 1 ng/ml 10 ng/ml 30 ng/ml 100 ng/ml
80 60 40 20
20 0
0 -20
1/30000
1/20000
1/10000
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0.0
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.2
c b
1.0
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100 80 60 40
.8
120
Peak current (µA)
Peak current (µA)
120
.6
140
160 140
.4
Potential (V vs. SCE)
Dilution ratio of anti-AFB1 antibody
a
80 60 40 20
20 0
0 .001
.01
.1
1
10
100
-20 .0001
1000
.001
Concentration of AFB 1 (ng/ml) Fig. 3. (A) Effect of dilution ratio of anti-AFB1 antibody on peak current. (B) Dose– response curves obtained using various dilutions of anti-AFB1 antibody: (a) 1:20,000; (b) 1:10,000; (c) 1:5000.
.01
.1
1
10
100
1000
Concentration of AFB 1 (ng/ml) Fig. 4. (A) LSV curves for determination of AFB1 in PBS (concentrations from 1 pg/ ml to 100 ng/ml). (B) Calibration curve for peak current versus concentration of AFB1 in PBS. Error bars represent SD for three experiments.
Sample analysis low concentration of antibody (1:20,000) significantly reduced electrochemical signal and the relatively high concentration (1:5000) could not obtain enough sensitivity. To get enough electrochemical signal and sensitivity, a 1:10,000 dilution ratio of anti-AFB1 antibody was chosen in the subsequent experiment.
The proposed method was applied to determine AFB1 in rice so as to test its performance in real matrix. The preparation of sample was described in detail in Materials and Methods. As can be seen in Figs. 4 and 5, the calibration curve of AFB1 prepared in extraction solution was nearly the same as that prepared in PBS (pH 7.4).
Performance of immunosensor
120
Peak current (µA)
A series of different concentrations of AFB1 prepared in PBS (pH 7.4) were detected to obtain the calibration curve. The LSV curves for different concentrations of AFB1 are depicted in Fig. 4A. As can be seen, the peak currents decreased with increases of the analyte concentration. The calibration curve of AFB1 prepared in PBS (pH 7.4) is shown in Fig. 4B. The peak currents were in inverse proportion to the concentrations of AFB1 in the range from 0.1 to 10 ng/ ml, and the detection limit was 0.054 ng/ml as calculated by the 3r rule (where r is the standard deviation [SD] of a blank solution and n = 6). The detection limit was superior to that of the AFB1 immunosensor based on impedimetric responses (100 ng/ml) [18] and was comparable to that of methods based on the electrochemical arrays (0.03 ng/ml) [16] and OWLS (0.5 ng/ml) [17]. Six repetitive assays of 1 ng/ml analyte were carried out to evaluate the reproducibility of the electrochemical immunosensor. The average of the peak current was 60 lA with a relative SD of 7.2%. So, the proposed method could be used with acceptable reproducibility.
140
100 80 60 40 20 0 .0001
.001
.01
.1
1
10
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Concentration of AFB 1 (ng/ml) Fig. 5. Calibration curve for peak current versus concentration of AFB1 in extraction solution. Error bars represent SD for three experiments.
Immunosensor for aflatoxin B1 determination / Y. Tan et al. / Anal. Biochem. 387 (2009) 82–86
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Table 1 Applications of immunosensor for AFB1 determination in spiked sample. Sample number
Added (lg/kg)
Founda (lg/kg)
Recovery (%)
1 2 3 4
12.5 25 125 250
11.5 ± 1.2 27.9 ± 2.7 126.3 ± 7.3 221.3 ± 15.5
92 112 101 88.5
a
Mean ± SD of three measurements.
The peak current of the immunosensor exhibited a negative linear correlation to AFB1 concentration from 0.1 to 10 ng/ml, and the correlation coefficient was 0.993 with a detection limit of 0.06 ng/ml as calculated in terms of the 3r rule defined above. The calibration curve of AFB1 prepared in extraction solution was used for the detection of AFB1 in rice. The recovery of fortified rice samples ranged from 88.5 to 112%. The mean ± SD of each sample was calculated, and the values are reported in Table 1. According to the U.S. Food and Drug Administration, the AFB1 content of commodities used for human and animal consumption should not exceed 20 parts per billion (20 lg/kg). The proposed method could detect AFB1 in spiked rice samples as low as 2.5 lg/kg, indicating that the method is acceptable for analyses of AFB1 in spiked rice samples at a level of regulatory relevance. Conclusions A sensitive and simple electrochemical immunosensor coupled with a signal-amplified method of enzymatic silver deposition has been constructed for the detection of AFB1 in rice. A detection limit as low as 0.06 ng/ml can be obtained through combination with the biometallization and highly sensitive metal stripping voltammetry. The working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. The method also has advantages such as ease of use and cost effectiveness. Thus, it could serve as a screening approach for this type of mycotoxins. Future work will involve finding a new immobilization method with the aim of reaching a much lower detection limit. In addition, the application of this method to the detection of other mycotoxins was also attempted. Acknowledgment This work was supported by National Nature Science Foundation of China (20575020, U0632005). References [1] A.Y. Kolosova, W.B. Shim, Z.Y. Yang, S.A. Eremin, D.H. Chung, Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1 stabilization of ELISA kit components and application to grain samples, Anal. Bioanal. Chem. 384 (2006) 286–294.
[2] J. Jaimez, C.A. Fente, B.I. Vazquez, C.M. Franco, A. Cepeda, G. Mahuzier, P. Prognon, Application of the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis, J. Chromatogr. A 882 (2000) 1– 10. [3] J. Stroka, R.V. Otterdijk, E. Anklam, Immunoaffinity column clean-up prior to thin-layer chromatography for the determination of aflatoxins in various food matrices, J. Chromatogr. A 904 (2000) 251–256. [4] N.A. Lee, S. Wang, R.D. Allan, I.R. Kennedy, A rapid aflatoxin B1 ELISA: Development and validation with reduced matrix effects for peanuts, Corn, pistachio, and soybeans, J. Agric. Food Chem. 52 (2004) 2746–2755. [5] O.G. Roch, G. Blunden, R.D. Coker, S. Nawaz, The validation of a solid phase clean-up procedure for the analysis of aflatoxins in groundnut cake using HPLC, Food Chem. 52 (1995) 93–98. [6] M. Holcomb, H.C. Thompson, W.M. Cooper, M.L. Hopper, Extraction of aflatoxins (B1, B2, G1, and G2) from corn and analysis by HPLC, J. Supercrit. Fluids 9 (1996) 118–121. [7] M. Sharma, C. Marquez, Determination of aflatoxins in domestic pet foods (dog and cat) using immunoaffinity column and HPLC, Anim. Feed Sci. Technol. 93 (2001) 109–114. [8] J. Blesa, J.M. Soriano, J.C. Molto, R. Mann, J. Manes, Determination of aflatoxins in peanuts by matrix solid-phase dispersion and liquid chromatography, J. Chromatogr. A 1011 (2003) 49–54. [9] C. Cavaliere, P. Foglia, C. Guarino, M. Nazzari, R. Samperi, A. Lagana, Determination of aflatoxins in olive oil by liquid chromatography–tandem mass spectrometry, Anal. Chim. Acta 596 (2007) 141–148. [10] A. Korde, U. Pandey, S. Banerjee, H.D. Sarma, S. Hajare, M. Venkatesh, A.K. Sharma, M.R.A. Pillai, Development of a radioimmunoassay procedure for aflatoxin B1 measurement, J. Agric. Food Chem. 51 (2003) 843–846. [11] S.R. Garden, N.J.C. Strachan, Novel colorimetric immunoassay for the detection of aflatoxin B1, Anal. Chim. Acta 444 (2001) 187–191. [12] S.J. Daly, G.J. Keating, P.P. Dillon, B.M. Manning, R. Kennedy, H.A. Lee, M.R.A. Morgan, Development of surface plasmon resonance-based immunoassay for aflatoxin B1, J. Agric. Food Chem. 48 (2000) 5097–5104. [13] J.A. Ho, R.D. Wauchope, A strip liposome immunoassay for aflatoxin B1, Anal. Chem. 74 (2002) 1493–1496. [14] D. Saha, D. Acharya, D. Roy, T.K. Dhar, Filtration-based tyramide amplification technique: A new simple approach for rapid detection of aflatoxin B1, Anal. Bioanal. Chem. 387 (2007) 1121–1130. [15] M.S. Nasir, M.E. Jolley, Development of a fluorescence polarization assay for the determination of aflatoxins in grains, J. Agric. Food Chem. 50 (2002) 3116– 3121. [16] S. Piermarini, L. Micheli, N.H.S. Ammida, G. Palleschi, D. Moscone, Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection, Biosens. Bioelectron. 22 (2007) 1434– 1440. [17] N. Adanyi, I.A. Levkovets, S. Rodriguez, A. Ronald, M. Varadi, I. Szendro, Development of immunosensor based on OWLS technique for determining aflatoxin B1 and ochratoxin A, Biosens. Bioelectron. 22 (2007) 797–802. [18] J.H.O. Owino, A. Ignaszak, A. Al-Ahmed, P.G.L. Baker, H. Alemu, J.C. Ngila, E.I. Iwuoha, Modelling of the impedimetric responses of an aflatoxin B1 immunosensor prepared on an electrosynthetic polyaniline platform, Anal. Bioanal. Chem. 388 (2007) 1069–1074. [19] E. Bakker, Y. Qin, Electrochemical sensors, Anal. Chem. 78 (2006) 3965–3983. [20] Z.P. Chen, Z.F. Peng, Y. Luo, B. Qu, J.H. Jiang, X.B. Zhang, G.L. Shen, R.Q. Yu, Successively amplified electrochemical immunoassay based on biocatalytic deposition of silver nanoparticles and silver enhancement, Biosens. Bioelectron. 23 (2007) 485–491. [21] E. Katz, L. Alfonta, I. Willner, Chronopotentiometry and Faradaic impedance spectroscopy as methods for signal transduction in immunosensors, Sens. Actuat. B 76 (2001) 134–141. [22] S. Hwang, E. Kim, J. Kwak, Electrochemical detection of DNA hybridization using biometallization, Anal. Chem. 77 (2005) 579–584. [23] X. Chu, X. Fu, K. Chen, G.L. Shen, R.Q. Yu, An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label, Biosens. Bioelectron. 20 (2005) 1805–1812. [24] Z.P. Chen, Z.F. Peng, J.H. Jiang, X.B. Zhang, G.L. Shen, R.Q. Yu, An electrochemical amplification immunoassay using biocatalytic metal deposition coupled with anodic stripping voltammetric detection, Sens. Actuat. B 129 (2008) 146–151.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice Yun Tan, Xia Chu *, Guo-Li Shen, Ru-Qin Yu State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
a r t i c l e
i n f o
Article history: Received 18 October 2008 Available online 4 January 2009 Keywords: Aflatoxin B1 Electrochemical immunosensor Enzymatic silver deposition Linear sweep voltammetry
a b s t r a c t A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B1 (AFB1) in rice. The immunosensor was based on an indirect competitive format between free AFB1 and aflatoxin B1–bovine serum albumin (AFB1–BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB1 antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB1–BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB1 in real samples. Ó 2009 Elsevier Inc. All rights reserved.
Aflatoxins are a group of naturally occurring mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus, which can be found in a large variety of food and animal feed. Naturally occurring aflatoxins are composed of B1, B2, G1, and G2. Among them, aflatoxin B1 (AFB1)1, which is the most abundant and carcinogenic, was classified as a carcinogenic substance of group 1 by the International Agency for Research on Cancer (IARC) [1]. Many grains and foodstuffs, including corn, peanuts, cottonseed, cereals, beans, and rice, have been found to be contaminated with aflatoxins. Moreover, humans would be exposed to aflatoxins either directly by eating contaminated grains or indirectly via animal products. Because aflatoxins are relatively heat stable, it is hard to destroy them once they are formed. The significant threat to human health posed by contamination has motivated extensive research in this toxin. Several methods for the detection of aflatoxin B1 have been established, including thin* Corresponding author. Fax: +86 731 8821916. E-mail address: [email protected] (X. Chu). 1 Abbreviations used: AFB1, aflatoxin B1; IARC, International Agency for Research on Cancer; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; LC/ESI–MS/MS; OWLS, optical waveguide lightmode spectroscopy; AFB1–BSA, aflatoxin B1–bovine serum albumin; ALP, alkaline phosphatase; LSV, linear sweep voltammetry; IgG, immunoglobulin G; SCE, saturated calomel electrode; PBS, phosphate-buffered saline; SD, standard deviation. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.12.030
layer chromatography (TLC), high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA) [2–4]. TLC is a relatively economical method that requires little equipment. HPLC is often coupled with different cleanup procedures such as solid phase extraction, supercritical fluid extraction, immunoaffinity chromatography, and matrix solid phase dispersion [5–8]. Recently, a liquid chromatography–tandem mass spectrometry with electrospray ionization (LC/ESI–MS/MS) method [9] was reported. Although sensitive and accurate, most of the chromatographic methods require expensive equipment and extended cleanup steps. Immunoassays can offer advantages over chromatographic procedures because they are faster and cheaper. Thus, different immunoassay techniques for the detection of AFB1 have been developed. For instance, radioimmunoassay [10], a rapid colorimetric immunoassay [11], a surface plasmon resonance-based immunoassay [12], a strip liposome immunoassay [13], a filtration-based tyramide amplification immunoassay [14], and a fluorescence polarization immunoassay [15] have been reported. Immunosensors based on the optical waveguide lightmode spectroscopy (OWLS) technique, the impedimetric responses, and electrochemical arrays [16–18] have also appeared during recent years. There is an increasing demand for ultrasensitive methods of immunoassay. Electrochemical sensors may be a good choice due to their fast, simple, and low-cost detection capabilities for biological binding events [19]. During recent years, new schemes based
Immunosensor for aflatoxin B1 determination / Y. Tan et al. / Anal. Biochem. 387 (2009) 82–86
on coupling the biocatalytic amplification of enzyme labels with additional electrochemical detections have been developed [20]. The use of chronopotentiometry and Faradaic impedance spectroscopy coupled with biocatalyzed precipitation [21] or enzymatic deposition of silver for the amplified detection of a target DNA [22] has been demonstrated to be a sensitive method. In addition, immunoassays based on silver-enhanced gold nanoparticle label [23], biocatalytic deposition of silver nanoparticles [20], and biocatalytic metal deposition coupled with anodic stripping voltammetric detection [24] have been reported recently. The combination of enzyme catalysis and metal deposition seems to be a much more promising strategy for amplifying assay signal. In the current work, we attempted to use the signal-amplified method of enzymatic silver deposition for the detection of AFB1. The procedures are depicted in Scheme 1. Aflatoxin B1–bovine serum albumin (AFB1–BSA) conjugate was first immobilized on the gold electrode surface. An indirect competitive format between selected analyte in solution and AFB1–BSA on the electrode was performed. After the competitive step, monoclonal antibody against AFB1 was bound to the electrode and then conjugated to a secondary antibody–alkaline phosphatase (ALP) conjugate. The ALP could catalyze the substrate, ascorbic acid 2-phosphate, into ascorbic acid, and the latter can reduce silver ions in solution to metal silver deposited onto
83
the electrode surface. Finally, the metallic silver deposited onto the electrode was determined by linear sweep voltammetry (LSV).
Materials and methods Materials and reagents Anti-AFB1 monoclonal antibody, AFB1–BSA conjugate, and AFB1 were purchased from Sigma–Aldrich (St. Louis, MO, USA). BSA and ALP-labeled anti-mouse immunoglobulin G (IgG) secondary antibody were purchased from the Beijing Dingguo Biotechnology Development Center (Beijing, China). Ascorbic acid 2-phosphate was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Glutaraldehyde was obtained from National Reagent (Shanghai, China). NaCl, KH2PO4, Na2HPO4, NaOH, glycine, AgNO3, and KNO3 were of analytical purity. Ultrapure water was obtained through a Nanopure Infinity Ultrapure water system (Barnstead/Thermolyne, Dubuque, IA, USA) with an electrical resistance greater than 18.3 MX. Apparatus All of the electrochemical experiments were carried out with a model CHI660C electrochemical workstation (Shanghai Chenhua Instruments, Shanghai, China). Electrochemical measurements were performed with a three-electrode system composed of a platinum foil as auxiliary electrode, a saturated calomel electrode (SCE) as reference electrode, and a modified gold electrode as working electrode. Electrode modification
Ascorbic acid 2-phosphate
Ascorbic acid
Gold electrode (99.99%, 2 mm diameter, Shanghai Chenhua Instruments) was immersed in freshly prepared Piranha solution (H2O2/H2SO4, 1:3, v/v) for 2 h, polished with 0.05 lm alumina slurry on microcloth, and rinsed with ultrapure water. The electrode was then sonicated in ultrapure water for 5 min, rinsed thoroughly with ultrapure water, and dried at room temperature. The cleaned gold electrode was immersed in 10 mM cysteamine solution overnight at 4 °C and rinsed thoroughly with ultrapure water to remove physically absorbed cysteamine. Subsequently, the electrode was immersed in 2.5% glutaraldehyde solution for 1 h at 37 °C and rinsed with ultrapure water. Then 5 ll of 100 lg/ml AFB1–BSA was dropped on the electrode surface and incubated for 1 h at 37 °C and washed with phosphate-buffered saline (PBS, pH 7.4) and ultrapure water. Finally, the electrode was incubated with 5 mg/ml BSA for 0.5 h to block the unreacted active sites.
Dehydroascorbic acid
Indirect competitive immunoassay Ag
Ag+
AFB1-BSA
AFB1
anti- AFB 1 antibody
Secondary antibody-ALP conjugate
Scheme 1. Schematic diagram of the developed electrochemical immunosensor for the detection of AFB1: (a) immobilization of AFB1–BSA conjugate on the gold electrode surface; (b) indirect competitive reaction between immobilized AFB1–BSA and free AFB1 in solution for binding to a fixed amount of anti-AFB1 antibody in solution; (c) association with secondary antibody–ALP conjugate and then enzymatic silver deposition.
To perform the assay, 2.5 ll of diluted anti-AFB1 antibody was first mixed with 2.5 ll of AFB1 standard solution, with the concentration ranging from 0 to 200 ng/ml in PBS (pH 7.4). The mixture was dropped at the surface of the modified electrode and incubated for 1 h at 37 °C in a humidity chamber. During incubation, a competition was carried out between free AFB1 in solution and the immobilized AFB1–BSA for binding to a fixed amount of antiAFB1 antibody in solution. After washing with PBS (pH 7.4), 5 ll of the secondary antibody–ALP conjugate (1:50, v/v) was added onto the electrode and incubated for 1 h at 37 °C. After rinsing with PBS (pH 7.4), the electrode was immersed in the freshly prepared 50 mM glycine–NaOH buffer (pH 9.08), containing 1 mM AgNO3 and 1 mM ascorbic acid 2-phosphate, at 37 °C for 30 min. Finally, the resulting electrode was rinsed with ultrapure water.
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Immunosensor for aflatoxin B1 determination / Y. Tan et al. / Anal. Biochem. 387 (2009) 82–86
tivity and the secondary antibody–ALP immobilized on the electrode.
Electrochemical detection LSV was performed at a potential range from 0 to 0.8 V (vs. SCE) with a 100-mV/s scanning rate in electrolyte of 0.6 M KNO3/0.1 M HNO3. All of the electrochemical detections were operated at room temperature. Sample preparation Noncontaminated rice (from a local market) was first ground in a household blender. Aliquots (1 g) of ground rice were spiked with AFB1 at different concentrations and mixed in a vortex mixer. After adding 5 ml of extraction solvent (80% methanol), the samples were mixed by shaking for 45 min and then centrifuged at 5000 rpm for 10 min. The supernatant was carefully removed and diluted with PBS (1:5, v/v). Safety AFB1 is a very potent carcinogen, so great care should be taken to avoid personal exposure. It is necessary to wear lab coat and gloves when doing experiments. The waste should be treated with hypochlorite before disposal. Results and discussion Characterization of electrochemical impedance spectrum Electrochemical impedance spectrum is an effective method to monitor the interface properties of modified electrodes. The impedance spectra were determined after each step. As can be seen in Fig. 1, the semicircle diameter of the Nyquist plots increased after immobilization of AFB1–BSA conjugate. This was due to a barrier for the electron transfer at the electrode interface resulting from insulated AFB1–BSA. After reaction with anti-AFB1 antibody, the resistance became further increased, indicating the immobilization of antibody on the electrode surface. Finally, after reaction with the secondary antibody–ALP conjugate, the enhanced resistance demonstrated that the anti-AFB1 antibody retained its bioac-
180 160 140
successfully
Optimization of experimental conditions Protein molecules may adsorb nonspecifically on the sensor substrate. In this work, we used 5 mg/ml BSA to block the residually active site after the immobilization of AFB1–BSA conjugate, and the electrode was washed thoroughly after each step. These processes could effectively reduce the nonspecific adsorption. After the immobilization of 100 lg/ml AFB1–BSA on the electrode surface, one electrode was incubated with 5 mg/ml BSA for 0.5 h and then 5 ll of the secondary antibody–ALP conjugate (1:50, v/ v) was added onto the electrode and incubated for 1 h at 37 °C, whereas the other was directly incubated with 5 ll of the secondary antibody–ALP conjugate (1:50, v/v). The subsequent steps were the same as those described in Materials and Methods. The average peak current of the BSA-blocked electrode was 4.6 lA, which was significantly lower than that obtained with the electrode without BSA block (49.8 lA). So, 5 mg/ml BSA could effectively block the nonspecific adsorption in the proposed method. The concentration of AFB1–BSA conjugate is an important parameter because of the competitive reaction between AFB1– BSA on the electrode surface and free AFB1 in solution for binding to a fixed amount of anti-AFB1 antibody. As can be seen in Fig. 2, the peak current increased as the concentration of AFB1–BSA increased up to 100 lg/ml with fixed concentrations of anti-AFB1 antibody (1:10,000) and the secondary antibody–ALP conjugate (1:50, v/v). No significant increase in peak current was observed at a concentration higher than 100 lg/ml, so a 100-lg/ml AFB1– BSA solution was selected in the subsequent study. The amount of anti-AFB1 antibody also plays an important role in this approach. The more the AFB1 in solution reacts with antibody, the less the amount of antibody will immobilize on the electrode. As a result, the electrochemical signal would decrease significantly. So, the effect of the concentration of anti-AFB1 antibody on the peak current was also investigated. As shown in Fig. 3A, with fixed concentrations of AFB1–BSA (100 lg/ml) and the secondary antibody–ALP conjugate (1:50, v/v) and varying concentrations of anti-AFB1 antibody, the peak current increased rapidly with the increase of anti-AFB1 antibody concentration up to 1:10,000 and then tended to be stable. Dose–response curves obtained using various dilutions of anti-AFB1 antibody are shown in Fig. 3B; with fixed concentrations of AFB1–BSA (100 lg/ml) and the secondary antibody–ALP conjugate (1:50, v/v), the relatively
f
120
-z''/ohm
was
a
100
b
d
c
e 160
80
140
40 20 0 0
100
200
300
400
500
600
700
z'/ohm Fig. 1. Nyquist diagram of electrochemical impedance spectra: (a) bare gold electrode; (b) glutaraldehyde/cysteamine/gold electrode; (c) AFB1–BSA/glutaraldehyde/cysteamine/gold electrode; (d) BSA/AFB1–BSA/glutaraldehyde/cysteamine/ gold electrode; (e) anti-AFB1 antibody/BSA/AFB1–BSA/glutaraldehyde/cysteamine/ gold electrode; (f) secondary antibody–ALP conjugate/anti-AFB1 antibody/BSA/ AFB1–BSA/glutaraldehyde/cysteamine/gold electrode. Frequency range: 0.1 to 10 kHz. All of the measurements were performed in 1/15 M PBS containing 0.1 M KCl and 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (pH 7.4).
Peak current (µA)
60 120 100 80 60 40 20 0
50
100
150
200
250
Concentration of AFB 1-BSA (µg/ml) Fig. 2. Effect of concentration of AFB1–BSA conjugate on peak current.
300
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200 180
120
160 100
Current (µA)
Peak current (µA)
140 120 100 80 60 40
1 pg/ml 10 pg/ml 100 pg/ml 1 ng/ml 10 ng/ml 30 ng/ml 100 ng/ml
80 60 40 20
20 0
0 -20
1/30000
1/20000
1/10000
1/5000
0.0
1/3000
.2
c b
1.0
100
100 80 60 40
.8
120
Peak current (µA)
Peak current (µA)
120
.6
140
160 140
.4
Potential (V vs. SCE)
Dilution ratio of anti-AFB1 antibody
a
80 60 40 20
20 0
0 .001
.01
.1
1
10
100
-20 .0001
1000
.001
Concentration of AFB 1 (ng/ml) Fig. 3. (A) Effect of dilution ratio of anti-AFB1 antibody on peak current. (B) Dose– response curves obtained using various dilutions of anti-AFB1 antibody: (a) 1:20,000; (b) 1:10,000; (c) 1:5000.
.01
.1
1
10
100
1000
Concentration of AFB 1 (ng/ml) Fig. 4. (A) LSV curves for determination of AFB1 in PBS (concentrations from 1 pg/ ml to 100 ng/ml). (B) Calibration curve for peak current versus concentration of AFB1 in PBS. Error bars represent SD for three experiments.
Sample analysis low concentration of antibody (1:20,000) significantly reduced electrochemical signal and the relatively high concentration (1:5000) could not obtain enough sensitivity. To get enough electrochemical signal and sensitivity, a 1:10,000 dilution ratio of anti-AFB1 antibody was chosen in the subsequent experiment.
The proposed method was applied to determine AFB1 in rice so as to test its performance in real matrix. The preparation of sample was described in detail in Materials and Methods. As can be seen in Figs. 4 and 5, the calibration curve of AFB1 prepared in extraction solution was nearly the same as that prepared in PBS (pH 7.4).
Performance of immunosensor
120
Peak current (µA)
A series of different concentrations of AFB1 prepared in PBS (pH 7.4) were detected to obtain the calibration curve. The LSV curves for different concentrations of AFB1 are depicted in Fig. 4A. As can be seen, the peak currents decreased with increases of the analyte concentration. The calibration curve of AFB1 prepared in PBS (pH 7.4) is shown in Fig. 4B. The peak currents were in inverse proportion to the concentrations of AFB1 in the range from 0.1 to 10 ng/ ml, and the detection limit was 0.054 ng/ml as calculated by the 3r rule (where r is the standard deviation [SD] of a blank solution and n = 6). The detection limit was superior to that of the AFB1 immunosensor based on impedimetric responses (100 ng/ml) [18] and was comparable to that of methods based on the electrochemical arrays (0.03 ng/ml) [16] and OWLS (0.5 ng/ml) [17]. Six repetitive assays of 1 ng/ml analyte were carried out to evaluate the reproducibility of the electrochemical immunosensor. The average of the peak current was 60 lA with a relative SD of 7.2%. So, the proposed method could be used with acceptable reproducibility.
140
100 80 60 40 20 0 .0001
.001
.01
.1
1
10
100
1000
Concentration of AFB 1 (ng/ml) Fig. 5. Calibration curve for peak current versus concentration of AFB1 in extraction solution. Error bars represent SD for three experiments.
Immunosensor for aflatoxin B1 determination / Y. Tan et al. / Anal. Biochem. 387 (2009) 82–86
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Table 1 Applications of immunosensor for AFB1 determination in spiked sample. Sample number
Added (lg/kg)
Founda (lg/kg)
Recovery (%)
1 2 3 4
12.5 25 125 250
11.5 ± 1.2 27.9 ± 2.7 126.3 ± 7.3 221.3 ± 15.5
92 112 101 88.5
a
Mean ± SD of three measurements.
The peak current of the immunosensor exhibited a negative linear correlation to AFB1 concentration from 0.1 to 10 ng/ml, and the correlation coefficient was 0.993 with a detection limit of 0.06 ng/ml as calculated in terms of the 3r rule defined above. The calibration curve of AFB1 prepared in extraction solution was used for the detection of AFB1 in rice. The recovery of fortified rice samples ranged from 88.5 to 112%. The mean ± SD of each sample was calculated, and the values are reported in Table 1. According to the U.S. Food and Drug Administration, the AFB1 content of commodities used for human and animal consumption should not exceed 20 parts per billion (20 lg/kg). The proposed method could detect AFB1 in spiked rice samples as low as 2.5 lg/kg, indicating that the method is acceptable for analyses of AFB1 in spiked rice samples at a level of regulatory relevance. Conclusions A sensitive and simple electrochemical immunosensor coupled with a signal-amplified method of enzymatic silver deposition has been constructed for the detection of AFB1 in rice. A detection limit as low as 0.06 ng/ml can be obtained through combination with the biometallization and highly sensitive metal stripping voltammetry. The working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. The method also has advantages such as ease of use and cost effectiveness. Thus, it could serve as a screening approach for this type of mycotoxins. Future work will involve finding a new immobilization method with the aim of reaching a much lower detection limit. In addition, the application of this method to the detection of other mycotoxins was also attempted. Acknowledgment This work was supported by National Nature Science Foundation of China (20575020, U0632005). References [1] A.Y. Kolosova, W.B. Shim, Z.Y. Yang, S.A. Eremin, D.H. Chung, Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1 stabilization of ELISA kit components and application to grain samples, Anal. Bioanal. Chem. 384 (2006) 286–294.
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