Journal of Chromatography B, 974 (2015) 147–154

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Ultrasensitive and quantitative gold nanoparticle-based immunochromatographic assay for detection of ochratoxin A in agro-products Marjan Majdinasab a,b , Mahmoud Sheikh-Zeinoddin b , Sabihe Soleimanian-Zad b , Peiwu Li a,c,d,e,∗ , Qi Zhang a,c,∗ , Xin Li a,c , Xiaoqian Tang a,c a Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, PR China b Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran c Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan 430062, PR China d Laboratory of Risk Assessment for Oilseeds Products (Wuhan), Ministry of Agriculture, Wuhan 430062, PR China e Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan 430062, PR China

a r t i c l e

i n f o

Article history: Received 5 July 2014 Accepted 25 October 2014 Available online 3 November 2014 Keywords: Immunochromatographic assay (ICA) Ochratoxin A Agro-products Gold nanoparticles

a b s t r a c t In most cases of mycotoxin detection, quantitation is critical while immunochromatographic strip tests are qualitative in nature. Moreover, the sensitivity of this technique is questioned. In order to overcome these limitations, an ultrasensitive and quantitative immunochromatographic assay (ICA) for rapid and sensitive quantitation of ochratoxin A (OTA) was developed. The assay was based on a competitive format and its sensitivity was improved by using a sensitive and selective OTA monoclonal antibody (OTA-mAb). The visible ICA results were obtained within 15 min, and in addition to visual examination, they were read by the rapid color intensity portable strip reader. The visual and computational detection limits (vLOD and cLOD, respectively) for ochratoxin A were 0.2 and 0.25 ng mL−1 , respectively. These values were lower than those reported by previous studies in a range 5–2500 folds. For validation, contaminated samples including wheat, maize, rice and soybean were assayed by ICA and a standard high performance liquid chromatography (HPLC). The results were in good agreement for both ICA and HPLC methods. The average recoveries of the HPLC were in the range 72–120% while the ICA values were from 76 to 104%, confirming the accuracy and sensitivity of this method. © 2014 Published by Elsevier B.V.

1. Introduction Humans and animals are continuously exposed to a variety of mycotoxins which naturally occur in various food products. Ochratoxins are secondary metabolites and a group of mycotoxins produced by several fungi of the Aspergillus and Penicillium families. Ochratoxin A (OTA), one of the most abundant and toxic members of this group [1], is produced by Penicillium verrucosum, Aspergillus carbonarius and Aspergillus niger [2]. This fungal toxin has nephrotoxic, hepatotoxic, teratogenic and immunotoxic properties and has been classified as possibly carcinogenic to humans (group 2B) by the International Agency for Research on Cancer (IARC) [3].

∗ Corresponding authors at: Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, PR China. Tel.: +86 27 86812943; fax: +86 27 86812862. E-mail addresses: [email protected] (P. Li), [email protected] (Q. Zhang). http://dx.doi.org/10.1016/j.jchromb.2014.10.034 1570-0232/© 2014 Published by Elsevier B.V.

OTA has been detected in raw materials such as cereals, coffee, grapes, and spices. In addition, it exists in their processed products such as bread and wine due to its chemical stability against heat treatments and hydrolysis [4]. The concern about food safety and harmful effects of consumption of the contaminated foods has caused most countries to set strict limits on the OTA level in various kinds of foodstuff. The maximum level of OTA for unprocessed cereals set by the European Commission [5] and World Health Organization is 5.0 ␮g kg−1 . This level has been specified 10 ␮g kg−1 in China [6]. Over the last few decades, many methods have been used for analysis and detection of OTA in food and feed. Due to high accuracy, instrumental methods such as high performance liquid chromatography (HPLC) [7–10] and liquid chromatography–tandem mass spectrometry (LC–MS) [11,12] have been widely used as reference methods. However, instrumental methods are time-consuming and require expensive equipment and skilled personnel. Alternatively, immunochemical methods have been developed for the

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rapid detection of mycotoxins in food and feed samples due to their high sensitivity, specificity, short detection time and low cost. Some immunoassay methods such as enzyme-linked immunosorbent assay (ELISA) [8], enzyme-linked aptamer assays (ELAAs) [13], fluorescence polarization immunoassay (FPIA) [14], chemiluminenscent immunoassay (CL-IA) [15] and other biosensor based on immunoassay have been reported for OTA detection. Although some of these methods have high sensitivity, special and expensive instruments and specialized personnel are required in the detection process. Another immunoassay based on the reaction between an antibody and its antigen is immunochromatographic (ICG) technique which has been recently considered by researchers especially in the detection of mycotoxins. Immunochromatographic assay is a new immunochromatographic technology with several advantages such as procedural simplicity, rapid operation, immediate results, low cost, and no requirement for skilled technicians or expensive equipment [16]. Because of these advantages, immunochromatographic strip tests have been used in a wide range of applications. Due to concerns related to ochratoxin detection, up to now, few reports on ICA method have been published. Most of these evaluations were qualitative, not having good sensitivity for detection, and the antibodies used in these researches showed considerable cross-reactivities with other major mycotoxins. Recently, many efforts have been made to quantify and increase the sensitivity. The sensitivity of the ICA is mainly dependent on the affinity of the specific antibody; however, the material of the label can also contribute to improve it. In most cases, gold nanoparticles are used in these evaluations due to their high stability, easy synthesis and easy reading of result (detection with naked eyes) [17]. Despite of many advantages that ICAs offer, in most cases, especially in food samples, they cannot reach high sensitivity. Since food and feed samples have complex matrixes, extraction and dilution is required before application on the membrane so that the fluid can flow easily through the nitrocellulose membranes with micrometer pore size. On the other hand, organic solvents, especially methanol, are usually used in mycotoxin extraction solution. Published results [18] have indicated that due to negative effects of methanol on the intensity of the test strip, the extraction dilution factor should be increased, thereby reducing the final mycotoxin concentration in the test solution. Therefore, sometimes the final sample is diluted about 15–20 times so that test strip cannot detect contamination in samples with a low concentration of mycotoxin. It is quite evident the importance of a more sensitive test strip designation to detect analytes which are too risky, even in low concentrations. One way to increase the sensitivity of test strips is design and application of a sensitive and selective antibody. Lack of quantitation is considered as another disadvantage of the ICA method compared with HPLC or LC–MS/MS. Therefore, many researchers have applied ICA as a qualitative method for detection. In this work, a high sensitive and quantitative immunochromatographic assay is reported to improve the limitations of the ICA method for the rapid on-site quantitation of ochratoxin A in agro-products including wheat, maize, soybean and rice. The quantitation is obtained by a rapid color intensity portable strip reader, and by interpolating them onto a standard curve.

Ovalbumin (OVA), and rabbit anti-mouse immunoglobulin (IgG) were purchased from Sigma-Aldrich (St. Louis, USA). In addition, T2-toxin (Fermentek, Jerusalem, Israel), BSA (Roche Applied Science, Indianapolis, USA) and Anti-OTA monoclonal antibody (mAb) produced in our laboratory [19] were used in this study. Protein G SepharoseTM was purchased from GE Healthcare (Uppsala, Sweden). Water was obtained from a Milli Q purification system purchased from Millipore (Billerica, MA, USA). All other inorganic chemicals and organic solvents were of analytical reagent grade. Nitrocellulose (NC) membranes, glass fibers and absorbent pads were purchased from the Millipore Corporation (Bedford, USA). XYZ3050 Dispensing Platform, LM4000 Batch Laminator and CM4000 Guillotine Cutter from BioDot (Irvine, USA) were used to prepare test strips. The ultraviolet spectrum was obtained using a SpectraMax M2e microplate reader purchased from the Molecular Devices Corporation (Sunnyvale, USA). The vacuum freeze drier was obtained from the Thermo Electron Corporation (Rockford, USA). The high-speed freezing centrifuge (CF16RX) was from Hitachi (Tokyo, Japan). The color intensity of the lines in the strips was measured by using a rapid color intensity portable strip reader developed in our laboratory (Key Laboratory of Detection for Mycotoxins, Wuhan, China). The ICA results were validated with an Agilent 1100 high performance liquid chromatography system (Agilent Tech, Santa Clara, CA, USA). 2.2. Preparation of gold nanoparticles (GNPs) GNPs were prepared using the sodium citrate method with slight modifications [18]. In this method, the particle size is inversely proportional to the sodium citrate volume, so we used 4.8 mL of 1% sodium citrate to reduce 200 mL of 0.01% HAuCl4 . Briefly, 2 mL 1% (w/v) HAuCl4 solution was mixed with 200 mL ultrapure water, followed by heating for 4 min with high power in the microwave. Then, 1% sodium citrate (w/v) was added, and it was quickly shook and heated for another 4 min at medium-high power until the color of the solution changed into burgundy. The solution was left to cool down at room temperature. Then, ultrapure water was added so that the GNPs solution increased to the initial volume. The GNPs solution was stored at 4 ◦ C for further use. The solution was characterized with UV–vis spectra between 450 nm and 600 nm. 2.3. Preparation of the gold nanoparticles-labeled antibodies (GNPs-Ab) GNP-Ab conjugates were prepared according to the previous reports [18,20] with slight modifications. Briefly, the pH of the gold nanoparticles solution was adjusted with 0.1 M K2 CO3 . Then, 0.25 mg of purified anti-OTA monoclonal antibody (mAb), in 2.5 mL ultrapure water was added dropwise to 50 mL pH adjusted GNPs solution under gentle stirring. The mixture was stirred for another 1 h. Then, 5 mL of 10% (m/v) filtered BSA was added dropwise into the mixture and stirred for 1 h. The product solution was centrifuged (12,000 rpm at 4 ◦ C for 30 min) twice to remove the unconjugated protein from the solution. The final pellet (anti-OTAmAb-GNP conjugates) was concentrated and resuspended in 5 mL boric acid buffer solution (0.002 M boric acid, 0.2% PEG 20000 and 0.02% NaN3 ).

2. Materials and methods

2.4. Preparation of the test strips

2.1. Reagent and equipment

Ochratoxin A-BSA conjugate and rabbit anti-mouse IgG were coated, respectively, on nitrocellulose (NC) membrane as the test line (T line) and control line (C line) by BioDot XYZ Platform at a proper jetting rate. The coated membranes were dried at 37 ◦ C for 2 h. The glass fiber, including the conjugate and sample pads, was treated with blocking buffer and was dried at 37 ◦ C overnight. The

Ochratoxin A (OTA) (Solid, HPLC grade, purity ≥98%), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), zearalenone (ZEN), OTA-BSA (bovine serum albumin) conjugate, hydrogen tetrachloroaurate (III) hydrate (HAuCl4 ·3H2 O),

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OTA-mAb-GNP conjugates were dispensed onto conjugate pads at a proper spray rate and dried with the vacuum freeze drier. The absorption pad was employed without treatment. Then, the NC membrane, conjugate pad, sample pad and absorbent pad were laminated and pasted onto a plastic backing plate. Finally, the whole assembled backings were cut lengthwise and divided into strips. ICA strips were inserted into rigid plastic cassettes, each one with a sample well and a reading window, and they were stored at plastic bags containing silica at room temperature.

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in our laboratory). Subsequently, the IAC was eluted with 1 mL of methanol at a flow rate of about one drop every two second. The eluate was used for HPLC-FLD detection. Sample preparation, extraction and HPLC/immunoaffinity column clean-up method for validation purposes were performed based on a reference method [22] with some modifications and optimization.

2.6. Immunochromatographic assay procedure, limits of detection and quantitation 2.5. Sample preparation for ICA and HPLC-FLD analysis Blank maize, wheat, soybean, and rice samples without OTA were confirmed by HPLC-FLD. In order to simulate samples to real contaminated samples and evaluate the test strips, the spiked samples were prepared as reported [21] with some modifications. Five grams of each ground blank sample was placed in a 50 mL centrifuge tube, and spiked with OTA at three levels, low (5 ␮g kg−1 ), medium (10 ␮g kg−1 ) and high (20 ␮g kg−1 ) and followed by vortex mixing for 1 min. The spiked samples were stored at room temperature overnight. Then, 5.0 g spiked sample was added to 25 mL of methanol/water (70:30, v/v). The samples were vortexed for 1 min, extracted by ultrasonic at 50 ◦ C for 10 min and filtered through double-filter paper. One milliliter of filtrate was diluted with 3 mL of water and after filtering with 0.22 ␮m filter membrane, it was used for test strips. For HPLC-FLD analysis, another 2 mL of filtrate was diluted with 12 mL of water and filtered through double-filter paper and 0.22 ␮m filter membrane. Then, it was cleaned up and concentrated through an immunoaffinity column (IAC) (developed

The performance of the immunochromatographic test strips was evaluated by determining different concentrations of OTA in 10% methanol and subsequently using spiked samples. Limit of detection (LOD) was determined by two methods: qualitative (visual) and quantitative (instrumental using rapid color intensity portable strip reader). The test was carried out by adding 100 ␮L of various concentrations ranging from 0.0 to 10.0 ng mL−1 of OTA standard solution into the sample well. After 15 min of incubation at room temperature, the color of the test and control lines was visually evaluated. In addition, the color intensity values were measured and recorded by placing the cassettes into the rapid color intensity portable strip reader (Fig. 1a) equipped with color analysis software (Fig. 1b). The color analysis software was able to recognize lines color density and calculate the ratio between T line and C line. The values of T/C ratio were used for subsequent data analysis including calibration curves. Ochratoxin A concentration was obtained according to a calibration curve and T/C value.

Fig. 1. Schematic diagram of the (a) rapid color intensity portable strip reader, (b) color analysis software, (c) calibration curve using different concentrations of standard OTA and T/C values.

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The calibration curve was obtained by plotting the ratio between the intensity of T line and the C line (T/C) against the log of OTA concentration (Fig. 1c). The best data fit was obtained by linear regression of the standard points. The concentration of OTA in the samples was determined by interpolation on the linear calibration curve related to each sample. 2.7. HPLC-FLD analysis From each sample, one milliliter of eluate in methanol was injected into an HPLC equipped with a fluorescence detector (ex = 333 nm, em = 470 nm). The analytical column was an Alltima C18 (150 mm × 4.6 mm, particle size 5 ␮m, Alltech, Grace, IL, USA); the mobile phase consisted of a mixture of HPLC grade acetonitrile–water–acetic acid (49.5:49.5:1) eluted at a flow rate of 1.2 mL min−1 . The temperature of the column was 25 ◦ C and injection volume of suspended sample was 20 ␮L. Quantitation was done by interpolation on the linear calibration curve. 2.8. Matrix effects on ICA performance Matrix affects in the analysis of OTA in agro-products were investigated. Blank maize, wheat, soybean and rice samples without OTA were confirmed by HPLC-FLD. Five grams of each ground blank sample (maize, wheat, soybean and rice) was accurately weighed and extracted with 25 mL of methanol/water (70:30, v/v) by vortex mixing for 1 min. Then it was sonicated at 50 ◦ C for 10 min. After filtering with double-filter paper, 1 mL of the extract was diluted with 3 mL of water. Then, different concentrations of OTA were spiked into the diluted extract and 100 ␮L of each solution was used for OTA determination by ICA and for calibration curve plotting of the samples. 2.9. Statistical analysis Statistical analysis of data was performed using Microsoft Excel 2007 and regression analysis was used to plot graphs. 3. Result and discussion 3.1. Preparation of gold nanoparticles The basis of using noble metal particles as markers is their bright colors, which is due to the presence of a plasmon absorption band [23]. Among these particles, colloidal gold is used in most immunological applications due to its many advantages, including ease of production and conjugation to antibodies, stability, and intense color due to plasmonic effects [24]. In the present study, gold nanoparticles were used as label. The maximum absorbance of the GNP solution was at 519 nm, and mean diameter of gold nanoparticles was calculated about 12 nm according to the following equation:



d = exp B1

Aspr − B2 A450



(1)

where Aspr is absorption at the peak, A is absorption at 450 wavelength, B1 = 3.00 and B2 = 2.20 [25]. 3.2. Optimum pH and antibody concentration for GNP-antibody conjugate The definition of the optimum pH for conjugation of GNPs with antibody is the pH that colloidal GNPs solution is stable, and for the peak of the absorption curve, the absorbance is the highest. The optimum pH for the adsorption of antibodies on the surface

Fig. 2. Optimum pH of GNPs solution for conjugation with OTA-mAb.

of gold nanoparticles was adjusted by 0.1 M K2 CO3 . In this experiment, 4–12 ␮L of 0.1 M K2 CO3 was added to 1 mL of GNPs solution. Then, 20 ␮L of OTA-mAb at a concentration of 1 mg mL−1 was added to the tubes. After reaction, the effects of pH values on the conjugation were investigated by measuring the absorbance between 450 nm and 650 nm. As shown in Fig. 2, GNPs solution with 5 ␮L of K2 CO3 had the maximum absorbance. Accordingly, the suitable pH for conjugation of GNPs with OTA-mAb was obtained 5.0. The definition of the optimum concentration of the mAb was the one that gave the required visibility and the best sensitivity. During antibody concentration optimization, firstly, the minimal stable mAb concentration for mAb-GNPs conjugation was evaluated by 10% NaCl solution, and then the optimal concentration was studied on the prepared strips. Appropriate antibody concentration was determined when a clear, red visible line occurred in the test zone for negative samples within 15 min, and the weakest color intensity was formed in the test line for positive samples containing 2.0 ng mL−1 OTA. The lowest mAb concentration that complied with the standards was considered the optimum amount. In fact, because immunochromatographic tests for small molecules such as OTA are based on a competitive format, if the less amount of antibody is used, the higher sensitivity will result. Based on this, the optimum concentration for OTA-mAb was 5 ␮g mL−1 to produce the conjugate. When the OTA antibody was conjugated on the GNPs surface, its size increased and the absorbance peak shifted from 519 to 527 nm. 3.3. Optimization of the ultrasensitive ICA strips and its principle The strips were composed as follows: from the top; the absorbent pad, the nitrocellulose membrane, the conjugate pad and the sample pad. The sample pad, conjugate pad, NC membrane and absorbent pad had a length of about 11, 11, 25 and 20 mm, respectively. The pads were pasted onto an adhesive polyester layer with appropriate overlap. The strip composition and its sizes are shown in Fig. 3a. The membrane is probably the most important material used in an immunochromatographic test strip. Nitrocellulose membranes are the most commonly used for immunochromatographic assays. Capillary flow rate is an important property of nitrocellulose membranes affected by membrane pore size. There is a range of commercial NC membranes with different pore sizes and subsequently, different flow rates. In the present study, in order to obtain high sensitivity of the assay along with an acceptable flow rate, HiFlow135 NC membrane was chosen. Sample pad and conjugate pad both of which were made of glass fiber before applying the reagents, were treated with blocking buffer. Blocking buffer composition was evaluated to study its effect

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Fig. 3. Schematic illustration of the ICA, (a) Dimensions of ICA strip, (b) Configuration of the ICA: reagents deposited in each part and putting of sample (c) Immunoassay procedure for negative samples, (d) Immunoassay procedure for positive samples.

on OTA-mAb and analyte (OTA). One of the compounds found in the blocking buffer is blocking protein. Usually, serum proteins and in most cases, BSA are effectively used for this purpose; however unlike other mycotoxins, OTA can bind to serum proteins, especially serum albumin, with different affinities in various species [26]. So, three serum albumin proteins including BSA, OVA and alpaca were evaluated by indirect competitive ELISA (icELISA) to study the degree of interaction with OTA. Results indicated that OVA and alpaca behaved very similarly in binding to OTA, and their behavior was different from BSA. In other words, unlike both OVA and alpaca, BSA would strongly react with OTA. Therefore, OVA was chosen as the blocking protein in the blocking buffer because it is more common and available than alpaca. Finally, 0.01 M phosphate buffer solution (PBS) (pH 7.4) containing 1% OVA, 2% sucrose, 0.4% tween 20 and 0.02% NaN3 was chosen as the optimal blocking buffer for treatment of the sample and conjugate pads. The concentrations of immunoreagents were optimized according to the following points. A kind of background color appeared maybe due to incomplete upward moving of GNP-mAb conjugates across the membrane within 15 min. Therefore, it is necessary to complete release of GNP-mAb conjugates from conjugate pad. On the other hand, clearness of the lines, along with appearing of red color on the test line for negative samples, high sensitivity and minimum immunoreagents consumption should be taken into consideration. Based on these parameters, the immunoreagents amount was evaluated as the “checkerboard titration” in ELISA. Finally, GNP-mAb conjugates containing 5.0 ␮g mL−1 of the OTA monoclonal antibody and spray rate of 8.0 ␮L cm−1 was selected as the optimum concentration. The optimum combination of 0.60 ␮L cm−1 of the OTA-BSA (0.4 mg mL−1 ) and 0.60 ␮L cm−1 of rabbit anti-mouse IgG (0.25 mg mL−1 ) were dispensed on the T line and C line, respectively.

The ICA is based on the competitive theory (Fig. 3). The liquid sample is applied to the sample pad and quickly diffuses into the conjugate pad (Fig. 3b). The GNP-mAb conjugate is solubilized and moves forward with the sample flow chromatographically across the membrane. For negative sample solution, when the mixture passes over the T line on which OTA-BSA conjugate is immobilized, the GNP-mAb conjugate is captured by OTA-BSA. Then, due to capillary action, excess GNP-mAb conjugate move continuously to the C line and is captured by rabbit anti-mouse IgG. Therefore, for a negative sample, two red bands appear due to the accumulation of red colored GNP-mAb conjugates (Fig. 3c). In contrast, for positive sample solutions containing OTA, firstly, the antibody reacts with the OTA. Then, as the mixture of free GNP-mAb conjugates, GNP-mAb-ochratoxin A (GNP-mAb-OTA) and free OTA pass over the test line, the OTA competes with OTA-BSA for a limited number of antibody binding sites. Consequently, less GNP-mAb conjugate will remain on the test line (Fig. 3d). So, the intensity of the color in test zone is inversely proportional to the OTA concentration in the sample. 3.4. Evaluation of immunochromatographic assay 3.4.1. Sensitivity and specificity The sensitivity of ICA strips was measured by two methods: visual and computational. In visual method, the visual limit of detection (vLOD) of competitive ICA could be defined as the lowest ochratoxin A concentration producing the color on the test line significantly weaker than that of the negative control strip. According to this definition, as deduced from Fig. 4, vLOD of the strip test for OTA was 0.2 ng mL−1 and the cut-off level which gave complete disappearance of pink band of test line was 2 ng mL−1 . For quantitative measurement and evaluation of computational LOD (cLOD),

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Fig. 4. Visual detection limit (vLOD) of the ochratoxin A standard solutions. A series of OTA concentrations (0–10 ng mL−1 ).

various concentrations of standard OTA/10% methanol solutions and 20 negative control solutions (OTA-free 10% methanol solution) were prepared. One hundred microliter of each solution was loaded onto the strips. They were put aside for 15 min at room temperature (nearly 25 ◦ C). Then, the strips were inserted into the rapid color intensity portable strip reader (Fig. 1a). The strip reader recognized lines color density and calculated the ratio between T line and C line. The calibration curve was plotted using the T/C ratio versus natural logarithm of different concentrations of the OTA (Fig. 1c). The cLOD was calculated as three times the standard deviation of T/C values of the 20 negative control strips. A more accurate determination of the LOD was carried out with the calibration curve and using the following equation: cLOD =

3 × SD b

(2)

where SD is the standard deviation of the 20 negative controls, and b is the slope of the calibration curve. Based on these calculations, cLOD of the ICA strip test for OTA was 0.25 ng mL−1 . To evaluate the specificity of the test strip, some toxins such as aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, ochratoxin B and T-2 toxin in 10% methanol were tested for crossreactivity at final concentrations of 10, 20, 50 and 100 ng mL−1 for each toxin. The results showed there is no cross reactivity with these toxins, and color intensity of the test lines was similar to that of the negative control test strip. Obtained results using strip reader also confirmed the visual results. Therefore, the assay showed high selectivity which mainly was attributed to the OTA-mAb. 3.4.2. Matrix effects on ICA performance Foodstuffs have a complex matrix that could have a significant effect on the application of rapid diagnostic techniques including immunochromatographic assays. For the evaluation of matrix effects, blank samples of rice, wheat, maize and soybean, were pretreated as explained earlier. As deduced from Fig. 5, rice displayed minimum matrix effect with almost the same limit of detection. In contrast to rice, wheat, maize and soybean matrices could be effective in OTA detection. The difference of various samples (Fig. 5) is related to the type of samples and its components. For example, the presence of a large content of starch as well as colored components in maize and oily nature of soybean can affect the detection process by ICA and the assay sensitivity. Despite the matrix effects, the developed ICA was sensitive enough for OTA detection, and the OTA concentrations could be accurately calculated by calibration curves constructed in the agro-products matrices.

3.4.3. Precision and accuracy To evaluate the precision and accuracy of the developed ICA, spiked blank rice samples at three levels of spiking (low: 5 ␮g kg−1 ; medium: 10 ␮g kg−1 and high: 20 ␮g kg−1 ) were evaluated. Quantitation of OTA in spiked blank samples was obtained by means of appropriate calibration curves prepared by fortifying the solution belonging to the extraction of blank maize, wheat, soybean and rice samples at various levels (as explained earlier). Linear calibration curves were obtained by plotting the ratio T/C against natural logarithm of OTA concentration (Fig. 5). After obtaining the T/C values by the rapid color intensity portable strip reader and according to the calibration curve, the concentration of OTA in spiked rice samples was determined by interpolation on the linear calibration curve. As can be inferred from the results in Table 1, the results of ICA were in conformity with the spiked ochratoxin A concentrations. Although the OTA in the samples were diluted at 1:20 during extraction, consistent results were obtained for the spiking levels with twenty replications. This indicates good precision (reproducibility), strip-to-strip performance of the assay. 3.5. Comparison of the LOD with published results Immunochromatographic method has been widely used for mycotoxin determination. Therefore, optimization of the analytical

Fig. 5. Standard calibration curves for different concentrations of OTA standard solutions and blanck samples extracts containing different concentrations of OTA along with matrix effects of the agro-product samples. All points are means ± SD of six independent measurements for each concentration.

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Table 1 Results of a precision and accuracy evaluation of the developed ICA with the ochratoxin A spiked rice samples. Spiking level (␮g kg−1 )

Dilution factor

Mean concentration (␮g kg−1 )

SD

%Accuracy

5.0 10 20

20 20 20

4.6a 8.6 17.8

0.12 0.09 0.18

92 86 89

SD, standard deviation. a Mean value (n = 20).

Table 2 Comparison of the published gold nanoparticles-based ICA for OTA detection. References

Target analyte

Label

Antibody

vLOD (ng mL−1 )

cLOD (ng mL−1 )

[28] [29] [30] [6] [31] [27] [2] [32] –a

OTA OTA OTA OTA OTA and ZEA OTA OTA OTA OTA

GNPs GNPs GNPs GNPs GNPs GNPs GNPs GNPs GNPs

Monoclonal Polyclonal Monoclonal Monoclonal Monoclonal Monoclonal Polyclonal Monoclonal Monoclonal

500 1.0 5.0 10 OTA: 5 – – 10 0.2

– – – – – 5 1.5

a

0.25

The data obtained in this study.

performance of such a system is necessary. The sensitivity and the specificity of the ICA are controlled by the choice of immunoreagents. In this context, antibodies can play an important role. The general recommendation is to choose antibodies with the highest affinity and appropriate sensitivity and specificity. During recent years, some ICAs have been developed for OTA detection. But some of the assays did not have a good sensitivity and some others had no specificity. In some literatures, the vLOD was considered to be the lowest toxin concentration that resulted in no color intensity in the test line. In order to compare them under the same conditions, the minimum concentrations which caused a slight but distinguishable difference compared to the negative control were identified and used here. By comparison, the ICA developed in our research showed the highest sensitivity (Table 2). In most cases, the developed ICA was only qualitative, and the antibody used in the assay did not provide a suitable sensitivity. Thus, the sensitivity was higher than/equal to maximum limits for OTA in cereals proposed by the World Health Organization (5 ␮g kg−1 ) because in the ICA assays for food applications, sample extraction and several times dilution are inevitable. Anfossi et al. [2] achieved a cLOD of 1.5 ng mL−1 for OTA. Although the sensitivity was almost suitable, they used polyclonal antibody for the detection. The NC membrane

that they used was also HiFlow180 that increased the sensitivity and detection time. In addition, Urusov et al. [27] and Anfossi et al. [2] developed a quantitative ICA for OTA detection. However, they used a scanner and a computer for the ICA strips analysis which is not portable and usable everywhere for on-site detection. In our research, we developed a quantitative ICA using a portable and easy to use color intensity strip reader which can detect OTA amount in agro-products in a short time. In addition, the method is very sensitive and selective. The ultrasensitivity and specificity is mainly due to the top-quality OTA-mAb used in this assay. In the optimized indirect competitive enzyme-linked immunosorbent assay (icELISA), the OTA-mAb showed a 50% inhibition concentration (IC50 ) value of 0.058 ng mL−1 and a detection limit (IC10 ) of 0.001 ng mL−1 . 3.6. Comparative analysis of ICA and HPLC in agro-products In order to evaluate the test strips, spiked samples including wheat, rice, maize and soybean were detected by the developed ICA and compared with HPLC as reference method. OTA contents in samples were diluted 20 times in test solutions for ICA and were calculated by the calibration curves after putting on strips

Table 3 Comparison of the analysis results for the OTA in spiked samples by the developed ICA and reference HPLC. Sample

Spiking level (␮g kg−1 )

ICA strips

HPLC

OTA ± SD

RC (%)

OTA ± SD

RC (%)

Wheat

5 10 20

4.5 ± 0.02 8.2 ± 0.11 16.8 ± 0.03

90 82 84

5.07 ± 0.14 12.02 ± 0.07 21.84 ± 0.36

101 120 109

Maize

5 10 20

4.4 ± 0.04 9.0 ± 0.05 17.4 ± 0.01

88 90 87

4.36 ± 0.22 10.04 ± 0.15 24.53 ± 0.06

87 100 122

Rice

5 10 20

4.7 ± 0.04 10.4 ± 0.05 20.3 ± 0.08

94 104 102

3.77 ± 0.11 10.4 ± 0.08 25.08 ± 0.20

75 104 125

Soybean

5 10 20

3.8 ± 0.06 9.4 ± 0.08 17.6 ± 0.12

76 94 88

3.6 ± 0.07 10.07 ± 0.27 23.62 ± 0.22

72 100 118

SD, standard deviation; RC, recovery. a Mean value (␮g kg−1 ) ±SD (n = 6).

a

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M. Majdinasab et al. / J. Chromatogr. B 974 (2015) 147–154

as described previously. The results of two methods are listed in Table 3. The comparison between the results obtained via both methods showed the results of ICA were in good agreement with those of HPLC. It confirms that the developed ICA method can be proposed as a screening method for the quantitative determination of OTA in agro-product samples. The results showed that the average recoveries of the HPLC were ranging from 72 to 120% while the ICA values were from 76 to 104%, confirming the accuracy and sensitivity of this method. So, it can be used for both qualitative and quantitative analyses. Moreover, some data showed the ICA recoveries were lower than the HPLC, maybe caused by effects of some things extracted from samples on the immunoreactions. Therefore, a proper pretreatment can decrease the effects and increase the assay recoveries in the future. Anyway, according to the European Commission [33], the ICA recovery data are in the range of “accuracy” (acceptable). 4. Conclusions Previously developed ICAs used for qualitative and visual detection of positive or negative samples did not have a good sensitivity and specificity. In this study, an ultrasensitive and quantitative immunochromatographic assay for ochratoxin A was developed. The ICA was optimized and evaluated. The vLOD and cLOD level of optimized ICA for OTA was 0.2 and 0.25 ng mL−1 , respectively, showing the highest sensitivities so far. By comparison with the results in the literature, it can be observed that there are about 5–2500 folds difference on vLOD. This represents a considerable improvement in the sensitivity of the ICA due to the application of a high sensitive monoclonal antibody against ochratoxin A. Quantitation of the assay was obtained by using a rapid color intensity portable strip reader developed in our laboratory. For validation, both the ICA test strip and the standard HPLC method were used to detect the same spiked samples. It showed good agreement of the results. Although the matrix of agro-products can affect the sensitivity of assay, the high sensitivity of the assay can reduce or eliminate these effects. Liu et al. [34] studied the matrix effects in the analysis of zearalenone in baby food, maize, wheat, and feeds by ICA method. They concluded that wheat and cereal-based baby foods display negligible matrix effects. In contrast to literature, the detection of zearalenone could be affected by feed and maize extracts. In summary, the developed ICA in this study can provide an alternative tool for sensitive, quantitative, specific, rapid, low cost and convenient on-site detection of ochratoxin A in different agroproduct matrices. Acknowledgments This work was supported by the Project of National Science & Technology Pillar Plan (2012BAB19B09), the Special Fund for Agroscientific Research in the Public Interest (201203094), and the Key Project of the Ministry of Agriculture (2011-G5).

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Ultrasensitive and quantitative gold nanoparticle-based immunochromatographic assay for detection of ochratoxin A in agro-products.

In most cases of mycotoxin detection, quantitation is critical while immunochromatographic strip tests are qualitative in nature. Moreover, the sensit...
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