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A label-free immunoassay protocol for aflatoxin B1 based on UV-induced fluorescence enhancement
Talanta 204 (2019) 261–265
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
A label-free immunoassay protocol for aflatoxin B1 based on UV-induced fluorescence enhancement
T
Qi Shu, Yue Wu, Lin Wang, Zhifeng Fu∗ Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing, 400716, China
ARTICLE INFO
ABSTRACT
Keywords: Aflatoxin B1 Fluorescent derivatization Addition reaction Fluorescent immunoassay Magnetic beads
As one of the most toxic chemical carcinogens, aflatoxin B1 (AFB1) has attracted extensive attention due to its severe impairment to human health. There exists urgent demand to develop facile and sensitive method for rapid screening of AFB1. Here magnetic beads modified with mouse monoclonal antibody (McAb) were adopted for capture and enrichment of the mycotoxin in sample matrix. Then UV radiation at 365 nm was utilized to induce the enhancement of fluorescent (FL) emission of the captured AFB1 with an addition reaction. The FL signal of the derivative at 435 nm was collected to quantify AFB1. The immunoassay method for AFB1 showed a wide detection range of 1.0–1000 ng mL−1, with a low detection limit of 0.21 ng mL−1 (3σ). It was applied to detect AFB1 in herbal medicines including Astragalus membranaceus and Salvia Miltiorrhiza, with acceptable recovery values of 95.4–107.7%. It shows many merits including facile manipulation, low cost, high sensitivity and ideal selectivity. Due to its simple detection mechanism, the UV-induced FL derivatization-based label-free immunoassay can be furtherly extended to detection of other mycotoxins with similar chemical structures.
1. Introduction Some fungal genera, including Penicillium, Aspergillus and Fusarium, produce highly toxic fungal secondary metabolites named as mycotoxins, which show severe health threats to human beings and animals [1–3]. Among these mycotoxins, aflatoxin is categorized as Group I chemical carcinogen by the International Agency of Research on Cancer [4]. It has attracted much attention because of its carcinogenic, mutagenic as well as teratogenic effects. To date, over 20 kinds of aflatoxins have already been identified, while aflatoxin B1 (AFB1) is the most predominant and harmful among them [5]. Exposure to the mycotoxin is believed to cause severe liver diseases including cirrhosis, necrosis as well as tumor [6]. Besides agricultural products and foods, herbal medicines have also been frequently reported to be polluted by AFB1 in the processes of collection, treatment and storage, which greatly affects the safety of herbal medicines [7,8]. Thus it is of great importance to develop sensitive and selective methods for detecting the mycotoxin. Over the past decades, various chromatographic methods such as HPLC [9–11] and LC-MS [12–14] have been frequently used in AFB1 detection. Though these chromatographic methods show excellent reliability and ideal accuracy, they usually require sophisticated instrumentations and well-trained operators. Moreover, they cannot be applied to screen massive samples with high throughput. Therefore, ∗
various immunoassay-based methods have also been established for AFB1 detection, such as ELISA [15], electrochemical immunoassay [16,17], fluorescent immunoassay (FIA) [18,19], chemiluminescent immunoassay [20], colorimetric immunoassay [21]. Nevertheless, all these previously established immunoassay methods for AFB1 detection require labelling of antibody/antigen with signal probe for achieving acceptable sensitivity. The labeling procedure is well known to be highcost, labor-intensive and time-consuming, and might lead to decreased binding activity between antibody and antigen. Furthermore, synthesis of structural analog and artificial antigen is required since the mycotoxin is a small-molecular substance that cannot be directly used in immunoassay. Thus it is essential to explore facile approach for immunoassay of AFB1 without using any signal probe. Due to the inherent advantages of high sensitivity, wide linear range and facile manipulation, fluorimetry has already been widely used for the detection of various compounds [22–24]. Many organic compounds containing aromatic rings or multiple conjugate structures show fluorescent (FL) emission characteristics. Usually, rigid planar structure leads to high efficiency of FL emission [25]. Additionally, the properties of substituents show huge effect on the FL emission characteristics. Generally, substitutions with electron-donating groups are believed to induce enhanced absorption and improved FL emission [26]. AFB1 is known to be a derivative of dihydrofuran coumarin composed of furan
Corresponding author. E-mail address: [email protected] (Z. Fu).
https://doi.org/10.1016/j.talanta.2019.05.109 Received 26 December 2018; Received in revised form 17 May 2019; Accepted 28 May 2019 Available online 30 May 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Talanta 204 (2019) 261–265
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ring and coumarin. Under the exposure of UV radiation, AFB1 undergoes an addition reaction, which converts a furan ring to an electrondonating hydroxyl group [27]. This process was found to greatly enhance the FL emission of AFB1 in the present work. Herein, we developed a UV-induced FL derivatization-based immunoassay method for the detection AFB1 utilizing the above mentioned FL enhancement effect. Mouse monoclonal antibody (McAb) for AFB1 was modified on MBs to selectively capture and enrich AFB1 from sample matrix. Then the captured AFB1 were derivatized by UV exposure to produce derivative with strong FL emission. Thus label-free FIA of AFB1 was easily achieved with facile manipulation.
2.4. Pretreatment of real samples One gram of Astragalus Membranaceus or Salvia Miltiorrhiza powder was dispersed in 5.0 mL of methanol, and vortex mixed with known amounts of AFB1 for 3 min. The supernatant was collected by 10-min centrifugal separation at 11000 r min−1, and then diluted to 50 mL using PBS. Lastly, the solution was filtered through a microfiltration membrane with a pore diameter of 0.22 μm. 3. Results and discussion 3.1. Principle of label-free FIA for AFB1 detection
2. Experimental
Besides the fluorophore in molecular structure, substituent also shows very strong influence on FL emission. Usually, electron-donating groups including amino group, hydroxyl group and alkoxyl group can enhance FL emission since their lone-pair electrons can combine with aromatic ring to form p-π conjugate structure that can enlarge the conjugate rigid planar structure of the aromatic ring [28]. According to published document [29], the C8-C9 of AFB1 can be converted to products with hydroxyl group (R1, R2 and R3) by an addition reaction under UV exposure (Fig. 1A), which greatly enhances the FL emission of AFB1 (Fig. 1B). The principle of UV-induced FL enhancement immunoassay for AFB1 detection is illustrated in Fig. 2. MBs-McAb were used to capture and enrich AFB1 from sample matrix. Then UV radiation at 365 nm was utilized to enhance the FL emission of AFB1 loaded on MBs-McAb according to the above described mechanism. Thus a label-free FIA can be easily established for AFB1 detection without using any signal probe.
2.1. Materials and instrumentations Carboxy groups-functionalized MBs with mean size of 5 μm were provided by Biospes Co., Ltd. (China). AFB1 and mouse McAb for AFB1 were both provided by Kuaijie Kang Biotechnology Co., Ltd (China). Nhydroxysuccinimide (NHS) and 2-(N-morpholino) ethanesulfonic acid (MES) were both provided by J&K Chemical Co., Ltd. (China). Bovine serum albumin (BSA) was a product of Gibco (USA). 1-(3Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was obtained from Aladdin Reagent Ltd. (China). Salvia Miltiorrhiza and Astragalus Membranaceus were both provided by a local pharmacy. Phosphate buffered saline (PBS) was provided by Dingguo Biotechnology Co., Ltd (China). Polystyrene high-affinity microplates were provided by Greiner Bio-One (Germany). The dilution buffer for all proteins and samples was 10 mmol L−1 PBS at pH 7.4. The activation buffer for carboxy groups-functionalized MBs was 10 mmol L−1 MES buffer at pH 5.5. The blocking buffer was 10 mmol L−1 PBS at pH 7.4 containing 1% BSA and 0.05% Tween-20. The washing buffer was 10 mmol L−1 PBS containing 0.05% Tween-20. An Infinite 200 PRO multifunctional microplate reader (Tecan, Austria) was used to detect FL signals. A LC-20A HPLC (Shimadzu Co., Ltd., Japan) was used to provide reference values for AFB1 detection. An UV camera obscura (ZF-20D) manufactured by Shanghai Guanghao Instrument Co., Ltd. (China) was used to provide 365-nm UV radiation with power of 40 W. An ELGA PURELAB classic system (France) was used for the preparation of ultra-pure water with a resistivity of 18.2 M Ω cm.
3.2. Optimization of experimental parameters To achieve high detection sensitivity, some conditions affecting the FL response were optimized with AFB1 standard sample at 250 ng mL−1. As seen in Fig. 3A, AFB1 showed a maximum absorption peak at around 360 nm. Therefore a UV lamp with a maximum emission wavelength of 365 nm was adopted to derivatize AFB1 and enhance the FL emission. Under a UV radiation at this wavelength, the I/I0 ratio of AFB1 FL emission was observed to rise with the derivatization time and achieve an almost constant value after 2-h exposure (Fig. 3B). Here I and I0 are the FL responses of 250 ng mL−1 AFB1 and a blank sample (PBS), respectively. Thus 2 h was adopted as the optimal FL derivatization time under UV exposure. During the FL derivatization procedure, the radiation power of the UV lamp was set as 40 W since stronger radiation can induce obvious decomposition of the polystyrene microplate. Another important factor affecting the signal response was the dosage of MBs-McAb. As shown in Fig. 3C, the I/I0 ratio rose with the dosage of MBs-McAb and achieved a maximum value when 10 μL of MBs-McAb was used. Then further increase of the dosage of MB-McAbs resulted in decreased I/I0 ratio. The result can be attributed to the fact that larger dosage of MBs-McAb blocked the FL detection. Thus, 10 μL was adopted as the optimal amount of MBs-McAb. Lastly, the effect of pH value on FL response was also investigated. As seen in Fig. 3D, the pH value did not show obvious effect on FL response. pH 7.4 was adopted since it was also adopted in the immunoreaction between AFB1 and MBs-McAb. Adoption of this pH value avoided replacement of reaction medium.
2.2. Modification of MBs with McAb for AFB1 MBs modified with McAb for AFB1 (MBs-McAb) were prepared by a conventional EDC/NHS amidization reaction. In brief, 1.0 mL of 10% carboxy groups-functionalized MBs was washed thoroughly using 1.0 mL of activation buffer, and dispersed in 1.0 mL of the same buffer added with 20 mg of NHS and the same amount of EDC. Following 20min reaction, the actived MBs were washed thoroughly using 1.0 mL of activation buffer and dispersed in 1.0 mL of the same buffer added with 200 μg of McAb. After overnight reaction with constant shake at 4 °C, the obtained MBs-McAb were washed thoroughly using 1.0 mL of washing buffer. Lastly the washed MBs-McAb were dispersed in 1.0 mL of blocking buffer till use. 2.3. Label-free FIA of AFB1 Ten microliters of MBs-McAb was mixed and incubated with 1.0 mL of AFB1 sample solution at 37 °C with constant shake for 1 h. Afterwards the MBs-McAb loading AFB1 were washed thoroughly using 1.0 mL of washing buffer, and dispersed in 300 μL of PBS. The MBs suspension was then pipetted into a microplate well, and exposed to UV radiation for 2 h to derivatize AFB1. Lastly, under the excitation at 360 nm, the FL signals at 435 nm were collected to quantify AFB1.
3.3. Performance of label-free FIA for AFB1 detection With the optimal detection conditions, the FL response rose with the concentration of AFB1 within a wide range of 1.0–1000 ng mL−1 (Fig. 4). The regressive equation for AFB1 detection was I-I0 = 4039 3968 × e−C/494.7 (R2 = 0.997), with a low detection limit of 0.21 ng mL−1 at 3σ. Afterwards relative standard deviation (RSD) tests 262
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Fig. 1. (A) Mechanism of FL enhancement of AFB1 under UV exposure. (B) FL emission of AFB1 before (black) and after (red) UV exposure. The concentration of AFB1 was 250 ng mL−1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
was performed with AFB1 standard samples at low (50 ng mL−1), medium (250 ng mL−1) and high (500 ng mL−1) concentrations, to estimate the repeatability for AFB1 detection. The RSD values obtained from AFB1 standard samples at the three concentrations were calculated to be 6.5%, 3.6% and 4.1% respectively.
1.8%, 6.7%, 2.8% and 9.9%, respectively (Fig. 5). We also prepared a mixture of the five mycotoxins and tested its interference to the detection of AFB1. The DI value of the mycotoxins mixture was 3.2%. All these results demonstrated the acceptable selectivity of the established label-free FIA method for AFB1 detection.
3.4. Estimation of specificity for AFB1 detection
3.5. Application in real samples detection
To estimate the selectivity of the FIA method for label-free detection of AFB1, some other mycotoxins frequently found in herbal products were studied as the potential interfering chemicals, including mycotoxins zearalenone, ochratoxin A, patulin, citrinin, T-2 toxin. In the selectivity study, 250 ng mL−1 was adopted as the concentration of all mycotoxins. The degree of interference (DI) values of these mycotoxins were obtained by the following formula (1):
DI = (C
A)/(C
B ) × 100%
To further estimate its application potential for real samples, Astragalus Membranaceus and Salvia Miltiorrhiza were used as sample matrixes to conduct recovery test of AFB1. The two herbal medicine samples were treated with a method provided by Chinese Pharmacopoeia and confirmed to be free of AFB1 by HPLC. Afterwards, recovery tests were performed with the herbal medicines spiked with AFB1 at low (50 ng mL−1), medium (250 ng mL−1) and high (500 ng mL−1) concentrations. The results shown as Table 1 displayed acceptable recovery values from 95.4% to 107.7%, with RSD values not higher than 3.3%. From these results, the method was believed to be reliable enough for real sample detection.
(1)
Here, A, B and C are the FL signals produced by the interfering agents, AFB1 and a blank sample (PBS), respectively. The DI values of zearalenone, ochratoxin A, patulin, citrinin and T-2 toxin were 3.0%, 263
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Fig. 4. Linearity curve for AFB1 detection. All conditions were the chosen optimal conditions, n = 3. Fig. 2. Schematic illustration of the principle of UV-induced FL enhancement immunoassay for label-free detection of AFB1 on MBs platform.
Fig. 5. FL responses of five interfering mycotoxins and their mixture by the label-free FIA. The concentration of all mycotoxins was 250 ng mL−1. All tests were conducted under the chosen optimal conditions, n = 3. Fig. 3. (A) Absorbance spectrum of AFB1 at 250 ng mL−1. (B) Effect of the UV exposure time on the I/I0 ratio for AFB1 detection. (C) Effect of the amount of MBs-McAb on the I/I0 ratio for AFB1 detection. (D) Effect of the pH value on the I/I0 ratio for AFB1 detection. I and I0 represent the FL responses of AFB1 at 250 ng mL−1 and 0, respectively. All other conditions were the chosen optimal conditions, n = 3.
Table 1 Results of recovery tests for AFB1 spiked in herbal medicines (n = 3). Sample Initial (ng/mL) Added (ng/mL) Found (ng/mL) RSD (%) Recovery (%)
4. Conclusions In summary, a label-free method was established for FIA of mycotoxins by using AFB1 as a model analyte. For this method, only one McAb was required to capture the target analyte, and the signal readout can be easily achieved by a very facile UV-induced FL derivatization of AFB1. With the combination of both the selectivity of McAb and the detection wavelength of the derivative of AFB1, the label-free method showed acceptable selectivity. Compared with the traditional immunoassays for small-molecular substances, it avoided time-consuming and labor-intensive procedures of signal probe labelling and artificial antigen/structural analog synthesis. We believe that it can be potentially extended to immunoassay of many other mycotoxins containing similar conjugated structures as well in the future works. However, this platform is not suitable for the detection of some molecules without
Astragalus Membranaceus a
50.0 48.6 2.3 97.2
– 250.0 243.9 2.5 97.6
a
500.0 477.0 1.6 95.4
Salvia Miltiorrhiza – 50.0 53.8 0.9 107.6
250.0 262.8 3.3 105.1
500.0 516.6 2.4 103.2
Undetected. a The concentration values detected by reference HPLC method.
conjugated structure that can be derived to fluorophore by UV radiation. Acknowledgements The authors are grateful to the financial support from the National Natural Science Foundation of China (21775125), the Natural Science Foundation of Chongqing (cstc2018jcyjAX0175) and the Fundamental Research Funds for the Central Universities (XDJK2017A008). 264
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Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
A label-free immunoassay protocol for aflatoxin B1 based on UV-induced fluorescence enhancement
T
Qi Shu, Yue Wu, Lin Wang, Zhifeng Fu∗ Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing, 400716, China
ARTICLE INFO
ABSTRACT
Keywords: Aflatoxin B1 Fluorescent derivatization Addition reaction Fluorescent immunoassay Magnetic beads
As one of the most toxic chemical carcinogens, aflatoxin B1 (AFB1) has attracted extensive attention due to its severe impairment to human health. There exists urgent demand to develop facile and sensitive method for rapid screening of AFB1. Here magnetic beads modified with mouse monoclonal antibody (McAb) were adopted for capture and enrichment of the mycotoxin in sample matrix. Then UV radiation at 365 nm was utilized to induce the enhancement of fluorescent (FL) emission of the captured AFB1 with an addition reaction. The FL signal of the derivative at 435 nm was collected to quantify AFB1. The immunoassay method for AFB1 showed a wide detection range of 1.0–1000 ng mL−1, with a low detection limit of 0.21 ng mL−1 (3σ). It was applied to detect AFB1 in herbal medicines including Astragalus membranaceus and Salvia Miltiorrhiza, with acceptable recovery values of 95.4–107.7%. It shows many merits including facile manipulation, low cost, high sensitivity and ideal selectivity. Due to its simple detection mechanism, the UV-induced FL derivatization-based label-free immunoassay can be furtherly extended to detection of other mycotoxins with similar chemical structures.
1. Introduction Some fungal genera, including Penicillium, Aspergillus and Fusarium, produce highly toxic fungal secondary metabolites named as mycotoxins, which show severe health threats to human beings and animals [1–3]. Among these mycotoxins, aflatoxin is categorized as Group I chemical carcinogen by the International Agency of Research on Cancer [4]. It has attracted much attention because of its carcinogenic, mutagenic as well as teratogenic effects. To date, over 20 kinds of aflatoxins have already been identified, while aflatoxin B1 (AFB1) is the most predominant and harmful among them [5]. Exposure to the mycotoxin is believed to cause severe liver diseases including cirrhosis, necrosis as well as tumor [6]. Besides agricultural products and foods, herbal medicines have also been frequently reported to be polluted by AFB1 in the processes of collection, treatment and storage, which greatly affects the safety of herbal medicines [7,8]. Thus it is of great importance to develop sensitive and selective methods for detecting the mycotoxin. Over the past decades, various chromatographic methods such as HPLC [9–11] and LC-MS [12–14] have been frequently used in AFB1 detection. Though these chromatographic methods show excellent reliability and ideal accuracy, they usually require sophisticated instrumentations and well-trained operators. Moreover, they cannot be applied to screen massive samples with high throughput. Therefore, ∗
various immunoassay-based methods have also been established for AFB1 detection, such as ELISA [15], electrochemical immunoassay [16,17], fluorescent immunoassay (FIA) [18,19], chemiluminescent immunoassay [20], colorimetric immunoassay [21]. Nevertheless, all these previously established immunoassay methods for AFB1 detection require labelling of antibody/antigen with signal probe for achieving acceptable sensitivity. The labeling procedure is well known to be highcost, labor-intensive and time-consuming, and might lead to decreased binding activity between antibody and antigen. Furthermore, synthesis of structural analog and artificial antigen is required since the mycotoxin is a small-molecular substance that cannot be directly used in immunoassay. Thus it is essential to explore facile approach for immunoassay of AFB1 without using any signal probe. Due to the inherent advantages of high sensitivity, wide linear range and facile manipulation, fluorimetry has already been widely used for the detection of various compounds [22–24]. Many organic compounds containing aromatic rings or multiple conjugate structures show fluorescent (FL) emission characteristics. Usually, rigid planar structure leads to high efficiency of FL emission [25]. Additionally, the properties of substituents show huge effect on the FL emission characteristics. Generally, substitutions with electron-donating groups are believed to induce enhanced absorption and improved FL emission [26]. AFB1 is known to be a derivative of dihydrofuran coumarin composed of furan
Corresponding author. E-mail address: [email protected] (Z. Fu).
https://doi.org/10.1016/j.talanta.2019.05.109 Received 26 December 2018; Received in revised form 17 May 2019; Accepted 28 May 2019 Available online 30 May 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Talanta 204 (2019) 261–265
Q. Shu, et al.
ring and coumarin. Under the exposure of UV radiation, AFB1 undergoes an addition reaction, which converts a furan ring to an electrondonating hydroxyl group [27]. This process was found to greatly enhance the FL emission of AFB1 in the present work. Herein, we developed a UV-induced FL derivatization-based immunoassay method for the detection AFB1 utilizing the above mentioned FL enhancement effect. Mouse monoclonal antibody (McAb) for AFB1 was modified on MBs to selectively capture and enrich AFB1 from sample matrix. Then the captured AFB1 were derivatized by UV exposure to produce derivative with strong FL emission. Thus label-free FIA of AFB1 was easily achieved with facile manipulation.
2.4. Pretreatment of real samples One gram of Astragalus Membranaceus or Salvia Miltiorrhiza powder was dispersed in 5.0 mL of methanol, and vortex mixed with known amounts of AFB1 for 3 min. The supernatant was collected by 10-min centrifugal separation at 11000 r min−1, and then diluted to 50 mL using PBS. Lastly, the solution was filtered through a microfiltration membrane with a pore diameter of 0.22 μm. 3. Results and discussion 3.1. Principle of label-free FIA for AFB1 detection
2. Experimental
Besides the fluorophore in molecular structure, substituent also shows very strong influence on FL emission. Usually, electron-donating groups including amino group, hydroxyl group and alkoxyl group can enhance FL emission since their lone-pair electrons can combine with aromatic ring to form p-π conjugate structure that can enlarge the conjugate rigid planar structure of the aromatic ring [28]. According to published document [29], the C8-C9 of AFB1 can be converted to products with hydroxyl group (R1, R2 and R3) by an addition reaction under UV exposure (Fig. 1A), which greatly enhances the FL emission of AFB1 (Fig. 1B). The principle of UV-induced FL enhancement immunoassay for AFB1 detection is illustrated in Fig. 2. MBs-McAb were used to capture and enrich AFB1 from sample matrix. Then UV radiation at 365 nm was utilized to enhance the FL emission of AFB1 loaded on MBs-McAb according to the above described mechanism. Thus a label-free FIA can be easily established for AFB1 detection without using any signal probe.
2.1. Materials and instrumentations Carboxy groups-functionalized MBs with mean size of 5 μm were provided by Biospes Co., Ltd. (China). AFB1 and mouse McAb for AFB1 were both provided by Kuaijie Kang Biotechnology Co., Ltd (China). Nhydroxysuccinimide (NHS) and 2-(N-morpholino) ethanesulfonic acid (MES) were both provided by J&K Chemical Co., Ltd. (China). Bovine serum albumin (BSA) was a product of Gibco (USA). 1-(3Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was obtained from Aladdin Reagent Ltd. (China). Salvia Miltiorrhiza and Astragalus Membranaceus were both provided by a local pharmacy. Phosphate buffered saline (PBS) was provided by Dingguo Biotechnology Co., Ltd (China). Polystyrene high-affinity microplates were provided by Greiner Bio-One (Germany). The dilution buffer for all proteins and samples was 10 mmol L−1 PBS at pH 7.4. The activation buffer for carboxy groups-functionalized MBs was 10 mmol L−1 MES buffer at pH 5.5. The blocking buffer was 10 mmol L−1 PBS at pH 7.4 containing 1% BSA and 0.05% Tween-20. The washing buffer was 10 mmol L−1 PBS containing 0.05% Tween-20. An Infinite 200 PRO multifunctional microplate reader (Tecan, Austria) was used to detect FL signals. A LC-20A HPLC (Shimadzu Co., Ltd., Japan) was used to provide reference values for AFB1 detection. An UV camera obscura (ZF-20D) manufactured by Shanghai Guanghao Instrument Co., Ltd. (China) was used to provide 365-nm UV radiation with power of 40 W. An ELGA PURELAB classic system (France) was used for the preparation of ultra-pure water with a resistivity of 18.2 M Ω cm.
3.2. Optimization of experimental parameters To achieve high detection sensitivity, some conditions affecting the FL response were optimized with AFB1 standard sample at 250 ng mL−1. As seen in Fig. 3A, AFB1 showed a maximum absorption peak at around 360 nm. Therefore a UV lamp with a maximum emission wavelength of 365 nm was adopted to derivatize AFB1 and enhance the FL emission. Under a UV radiation at this wavelength, the I/I0 ratio of AFB1 FL emission was observed to rise with the derivatization time and achieve an almost constant value after 2-h exposure (Fig. 3B). Here I and I0 are the FL responses of 250 ng mL−1 AFB1 and a blank sample (PBS), respectively. Thus 2 h was adopted as the optimal FL derivatization time under UV exposure. During the FL derivatization procedure, the radiation power of the UV lamp was set as 40 W since stronger radiation can induce obvious decomposition of the polystyrene microplate. Another important factor affecting the signal response was the dosage of MBs-McAb. As shown in Fig. 3C, the I/I0 ratio rose with the dosage of MBs-McAb and achieved a maximum value when 10 μL of MBs-McAb was used. Then further increase of the dosage of MB-McAbs resulted in decreased I/I0 ratio. The result can be attributed to the fact that larger dosage of MBs-McAb blocked the FL detection. Thus, 10 μL was adopted as the optimal amount of MBs-McAb. Lastly, the effect of pH value on FL response was also investigated. As seen in Fig. 3D, the pH value did not show obvious effect on FL response. pH 7.4 was adopted since it was also adopted in the immunoreaction between AFB1 and MBs-McAb. Adoption of this pH value avoided replacement of reaction medium.
2.2. Modification of MBs with McAb for AFB1 MBs modified with McAb for AFB1 (MBs-McAb) were prepared by a conventional EDC/NHS amidization reaction. In brief, 1.0 mL of 10% carboxy groups-functionalized MBs was washed thoroughly using 1.0 mL of activation buffer, and dispersed in 1.0 mL of the same buffer added with 20 mg of NHS and the same amount of EDC. Following 20min reaction, the actived MBs were washed thoroughly using 1.0 mL of activation buffer and dispersed in 1.0 mL of the same buffer added with 200 μg of McAb. After overnight reaction with constant shake at 4 °C, the obtained MBs-McAb were washed thoroughly using 1.0 mL of washing buffer. Lastly the washed MBs-McAb were dispersed in 1.0 mL of blocking buffer till use. 2.3. Label-free FIA of AFB1 Ten microliters of MBs-McAb was mixed and incubated with 1.0 mL of AFB1 sample solution at 37 °C with constant shake for 1 h. Afterwards the MBs-McAb loading AFB1 were washed thoroughly using 1.0 mL of washing buffer, and dispersed in 300 μL of PBS. The MBs suspension was then pipetted into a microplate well, and exposed to UV radiation for 2 h to derivatize AFB1. Lastly, under the excitation at 360 nm, the FL signals at 435 nm were collected to quantify AFB1.
3.3. Performance of label-free FIA for AFB1 detection With the optimal detection conditions, the FL response rose with the concentration of AFB1 within a wide range of 1.0–1000 ng mL−1 (Fig. 4). The regressive equation for AFB1 detection was I-I0 = 4039 3968 × e−C/494.7 (R2 = 0.997), with a low detection limit of 0.21 ng mL−1 at 3σ. Afterwards relative standard deviation (RSD) tests 262
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Fig. 1. (A) Mechanism of FL enhancement of AFB1 under UV exposure. (B) FL emission of AFB1 before (black) and after (red) UV exposure. The concentration of AFB1 was 250 ng mL−1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
was performed with AFB1 standard samples at low (50 ng mL−1), medium (250 ng mL−1) and high (500 ng mL−1) concentrations, to estimate the repeatability for AFB1 detection. The RSD values obtained from AFB1 standard samples at the three concentrations were calculated to be 6.5%, 3.6% and 4.1% respectively.
1.8%, 6.7%, 2.8% and 9.9%, respectively (Fig. 5). We also prepared a mixture of the five mycotoxins and tested its interference to the detection of AFB1. The DI value of the mycotoxins mixture was 3.2%. All these results demonstrated the acceptable selectivity of the established label-free FIA method for AFB1 detection.
3.4. Estimation of specificity for AFB1 detection
3.5. Application in real samples detection
To estimate the selectivity of the FIA method for label-free detection of AFB1, some other mycotoxins frequently found in herbal products were studied as the potential interfering chemicals, including mycotoxins zearalenone, ochratoxin A, patulin, citrinin, T-2 toxin. In the selectivity study, 250 ng mL−1 was adopted as the concentration of all mycotoxins. The degree of interference (DI) values of these mycotoxins were obtained by the following formula (1):
DI = (C
A)/(C
B ) × 100%
To further estimate its application potential for real samples, Astragalus Membranaceus and Salvia Miltiorrhiza were used as sample matrixes to conduct recovery test of AFB1. The two herbal medicine samples were treated with a method provided by Chinese Pharmacopoeia and confirmed to be free of AFB1 by HPLC. Afterwards, recovery tests were performed with the herbal medicines spiked with AFB1 at low (50 ng mL−1), medium (250 ng mL−1) and high (500 ng mL−1) concentrations. The results shown as Table 1 displayed acceptable recovery values from 95.4% to 107.7%, with RSD values not higher than 3.3%. From these results, the method was believed to be reliable enough for real sample detection.
(1)
Here, A, B and C are the FL signals produced by the interfering agents, AFB1 and a blank sample (PBS), respectively. The DI values of zearalenone, ochratoxin A, patulin, citrinin and T-2 toxin were 3.0%, 263
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Fig. 4. Linearity curve for AFB1 detection. All conditions were the chosen optimal conditions, n = 3. Fig. 2. Schematic illustration of the principle of UV-induced FL enhancement immunoassay for label-free detection of AFB1 on MBs platform.
Fig. 5. FL responses of five interfering mycotoxins and their mixture by the label-free FIA. The concentration of all mycotoxins was 250 ng mL−1. All tests were conducted under the chosen optimal conditions, n = 3. Fig. 3. (A) Absorbance spectrum of AFB1 at 250 ng mL−1. (B) Effect of the UV exposure time on the I/I0 ratio for AFB1 detection. (C) Effect of the amount of MBs-McAb on the I/I0 ratio for AFB1 detection. (D) Effect of the pH value on the I/I0 ratio for AFB1 detection. I and I0 represent the FL responses of AFB1 at 250 ng mL−1 and 0, respectively. All other conditions were the chosen optimal conditions, n = 3.
Table 1 Results of recovery tests for AFB1 spiked in herbal medicines (n = 3). Sample Initial (ng/mL) Added (ng/mL) Found (ng/mL) RSD (%) Recovery (%)
4. Conclusions In summary, a label-free method was established for FIA of mycotoxins by using AFB1 as a model analyte. For this method, only one McAb was required to capture the target analyte, and the signal readout can be easily achieved by a very facile UV-induced FL derivatization of AFB1. With the combination of both the selectivity of McAb and the detection wavelength of the derivative of AFB1, the label-free method showed acceptable selectivity. Compared with the traditional immunoassays for small-molecular substances, it avoided time-consuming and labor-intensive procedures of signal probe labelling and artificial antigen/structural analog synthesis. We believe that it can be potentially extended to immunoassay of many other mycotoxins containing similar conjugated structures as well in the future works. However, this platform is not suitable for the detection of some molecules without
Astragalus Membranaceus a
50.0 48.6 2.3 97.2
– 250.0 243.9 2.5 97.6
a
500.0 477.0 1.6 95.4
Salvia Miltiorrhiza – 50.0 53.8 0.9 107.6
250.0 262.8 3.3 105.1
500.0 516.6 2.4 103.2
Undetected. a The concentration values detected by reference HPLC method.
conjugated structure that can be derived to fluorophore by UV radiation. Acknowledgements The authors are grateful to the financial support from the National Natural Science Foundation of China (21775125), the Natural Science Foundation of Chongqing (cstc2018jcyjAX0175) and the Fundamental Research Funds for the Central Universities (XDJK2017A008). 264
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