An on-site ultra-sensitive detection paper for multiclass chemical

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An on-site ultra-sensitive detection paper for multiclass chemical contaminants via universal bridge-antibody labelling: mycotoxin and illegal additives in milk as an example Guanghua Li, Lin Xu, Du Wang, Jun Jiang, Xiaomei Chen, Wen Zhang, Saranya Poapolathep, Amnart Poapolathep, Zhaowei Zhang, Qi Zhang, and Peiwu Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04290 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Analytical Chemistry

An on-site ultra-sensitive detection paper for multiclass chemical contaminants via universal bridge-antibody labelling: mycotoxin and illegal additives in milk as an example

Guanghua Lia†, Lin Xua†, Du Wanga,b,c,d, Jun Jianga,c, Xiaomei Chena, Wen Zhanga,b,c,d, Saranya Poapolathepe, Amnart Poapolathepe, Zhaowei Zhanga,*, Qi Zhanga,b,c,d*, Peiwu Lib,c,d,* a Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, P. R. China; b Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062, P. R. China; c Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan 430062, P. R. China; d Natonal Reference for Biotoxin Detection, Wuhan 430062, P. R. China; e Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, 10900, Thailand.

†

These authors contributed equally to this work.

* Corresponding authors Zhaowei Zhang, Qi Zhang, Peiwu Li, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China, E-mail: [email protected], [email protected], [email protected] Tel. +86 27 86812943; Fax +86 27 86812862.

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ABSTRACT Multiclass chemical contamination of food has aroused ever-increasing attention due to the increasingly common findings of the co-occurrence of multiclass contamination, such as mycotoxin (aflatoxin M1, AFM1) and illegal additives (melamine, MEL). In the present study, a rapid, ultra-sensitive detection paper was developed based on a unique bridge-antibody label to realize on-site simultaneous detection of AFM1 and MEL in milk. This detection paper used the bridge-antibody label on fluorescent particles (i.e., the fluorescent Eu nanoparticles were first conjugated with polyclonal antibodies and then with monoclonal antibodies). Dramatically enhanced sensitivity was recorded, probably due to the increase in immobilization of efficient monoclonal antibodies onto microspheres. Under optimal conditions, the lower limits of detection were 0.009 and 0.024 ng/mL for AFM1 and MEL in milk, respectively, in comparison with similar works. Moreover, the cut-off values were 0.4 and 150 ng/mL for AFM1 and MEL, respectively. The recoveries ranged from 88.7% to 105.0% for AFM1 and from 84.6% to 117.7% for MEL, with relative standard deviations (RSDs) of 0.5%–9.9% during the intraday and interday experiments. Comparison experiments conducted using the detection paper, HPLC and UPLC-MS/MS found excellent agreement in the simultaneous detection of AFM1 and MEL in milk. This proposed method can be extensively employed for simultaneous monitoring of multiclass chemical contaminants with to ensure food safety. Keywords: detection paper, multiclass chemical contaminants, bridge-antibody label, mycotoxin, illegal additives, milk


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Introduction Multiclass chemical contamination in food has attracted growing safety, economic and medical concerns due to changes in prevalence with variations in food processes 1. Two strategies are available to monitor multiple contaminants (i.e., lab-dependent and lab-independent methods). Lab-dependent methods, such as high performance liquid chromatography-tandem

mass

spectrometry

(HPLC-MS/MS)2

and

gas

chromatography-tandem mass spectrometry3, cannot be operated independently without special complex and expensive equipment, skilled operators, and extensive sample pre-treatment. These deficiencies hinder their uses for on-site detection. The lab-independent methods, which exhibit merits of robustness, rapidness, specificity, convenience, and low cost, include methods such as enzyme-linked immunosorbent assays4, microchips5 and detection papers6 and have been successfully applied in pointof-care testing7. Although the co-occurrence of multiclass chemical contaminants is growing, few on-site analytical methods for multiclass targets have been achieved using one-step detection. Milk is the essential nutritional and immunological resource for infants and adults and offers an excellent source of valuable proteins, essential amino acids, calcium, vitamins and antioxidants8. However, milk can be easily contaminated by multiclass chemical contaminants and thus requires an on-site rapid simultaneous assay. A growing number of milk safety incidents have been reported worldwide, typically for aflatoxin M1 (AFM1) and illegal ingredients such as melamine (MEL)9,10. AFM1 is found extensively in raw milk because dairy cattle are fed feedstuffs contaminated with aﬂatoxin B111. According to a survey report from China in 2016, the occurrence of AFM1 in milk was as high as 4.7% (267 positive cases in 5650 raw milk samples)12. AFM1 has been classified as a primary carcinogen by the International Agency for Research on Cancer13. Consumption of AFM1-contaminated milk can induce a wide spectrum of toxicological and carcinogenic effects that cause liver cirrhosis and tumors14. Fortunately, the maximum residual levels (MRLs) of AFM1 in milk and dairy products range from 0 to 1.0 ng/mL15. MEL (1,3,5-triazine-2,4,6-triamine) is an illegal



additive that increases the total nitrogen in milk to reduce its cost. MEL-contaminated milk and pet food can cause acute kidney failure, urolithiasis, and bladder cancer16 and has aroused broad attention due to recall issues of milk and pet food in the USA and China in 2007 and 2008, respectively17. China, the US Food and Drug Administration, and the European Union have set up MRLs for MEL residue. The MRLs of MEL have been set at 1.0 μg/mL for infant formula and 2.5 μg/mL for the remaining dairy products in many countries18. A paper-based immunoassay (PI) is one of the most attractive options for on-site rapid, simultaneous detection due to its properties, such as ease-of-use, simplicity, and low cost. Despite quite a few efforts to enhance the sensitivity of PIs using high quantum-yield chromophores and aggregation of multiple fluorescent materials, inevitable challenges result from unordered antibodies on the labeling materials. The major effective numbers of antibodies can heavily affect target recognition and reduce effective recognition between antibodies and antigens due to potential chaotic morphology (recumbent or vertical) via different potential interactions, such as hydrophobic interactions, ionic interactions and dative binding. Some pioneering work has been conducted using bi-antibody labeling to immobilize the monoclonal antibody (mAb) on a microsphere (MS)19, which dramatically improves the detection sensitivity. However, this qualitative method is not effective at detecting multiple targets in a quantitative manner. Similarly, tetrahedral programmable DNA nanostructures were introduced to engineer the surface and increase the detection sensitivity due to the increased number of effective probes20. Nevertheless, the precise fabrication of the tetrahedral DNA nanostructures was time/labor-consuming. These deficiencies hampered their wide application in monitoring multiclass targets in the field of food safety. Herein, the universal bridge-antibody (bAb) labelling method was introduced for the fabrication of on-site ultra-sensitive detection paper for multiclass chemical contaminants. The proof-of-concept was conducted in food safety using mycotoxin and illegal additives in milk as a real example. Under optimal conditions, the detection limit,


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linear range, recoveries, and specificity were evaluated. Comparisons between this proposed detection paper and typical HPLC for AFM1 and UPLC-MS/MS for MEL were performed using real milk samples. This detection paper opens an avenue to simultaneously monitor multiclass chemical contaminants and ensure food safety. Experimental Section Materials and regents N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), boric acid, bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), sucrose and Tween20 were purchased from Sigma-Aldrich Co., Ltd. (Urbana, IL, USA). MEL-BSA and a monoclonal antibody against melamine (mAbMEL) were acquired from Shenzhen Constant Medical Engineering Co., Ltd. (Shenzhen, China). AFM1-BSA and a monoclonal antibody against AFM1 (mAbAFM1) were produced as described previously21. Mouse immunoglobulin (IgG1) and goat anti-mouse immunoglobulin (IgG2, bridge-antibody) were purchased from Wuhan Boster Biological Technology, Co., Ltd. (Wuhan, China). The standard MEL solution was provided by Coast Hongmeng Standard Material Technology Co., Ltd. (Beijing, China). The carboxylmodified monodisperse polystyrene microspheres embedded with Eu were purchased from Shanghai Uni Biotechnology Co., Ltd. (Shanghai, China). The absorbent pad, nitrocellulose membrane (HFB 180), sample pad (fusion five) and plastic adhesive card were purchased from Millipore Corp. (Bedford, MA, USA). Ultrapure water was obtained from a Millipore Milli-Q system. Instruments An XYZ 3050 Dispensing Platform, CM 4000 Guillotine Cutter and LM 4000 Batch Laminator (Bio Dot, Irvine, CA, USA) were used to prepare the detection paper. The high-speed freezing centrifuge (CF16RX) was obtained from Hitachi (Tokyo, Japan). The vortex oscillator was made by VELP Corporation (Bohemia, NY, USA). The composition of the homemade portable reader was reported previously22. The excitation light wavelength was set at 365 nm, and the emission light was recorded at 613 nm.



Immobilization of mAbs on the MS The mAbs were immobilized on the MS via a two-step process. In the first step, the MS was conjugated with bAb, resulting in a MS-bAb conjugate. Briefly, in 400 μL of boric acid buffer solution (pH 8.18), 100 μL of carboxyl-modified monodisperse polystyrene MS (16.6 nM) and 20 μL of EDC solution (15 mg/mL) were mixed and incubated for 15 min at room temperature. The mixture was centrifuged at 13,300x g for 10 minutes. The precipitate was re-dissolved in 500 μL of boric acid buffer (pH 8.18). Subsequently, a serious volume (20, 40, 60, 80, 100, and 140 μL) of 1.0 mg/mL bAb was added for the optimization of the bAb loading. The mixture was shaken for 12 h prior to centrifugation at 13,300x g for 10 min. This precipitate was dissolved in 500 μL of boric acid buffer containing 0.5% (w/v) BSA and shaken for 3 h at room temperature. The second step was to conjugate the mAb with the bAb-MS. A total of 20 μL of 0.055 mg/mL mAbMEL (or 10 μL of 0.001 mg/mL mAbAFM1) was incubated with the bAb-MS in the running buffer (containing 2% sucrose, 0.5% BSA, 1% Tween20, and 1% PVP) for 12 h at 4°C. Conjugation between bAb and mAbMEL (or mAbAFM1) provided the mAb-functionalized bAb-MS (mAbAFM1-bAb-MS or mAbMEL-bAb-MS). Preparation of the detection paper Preparation of the working nitrocellulose membrane AFM1-BSA (0.25 mg/mL), MEL-BSA (0.48 mg/mL), and mouse IgG (0.1 mg/mL) were spotted onto the working nitrocellulose membrane to form the test lines (T1 and T2) and control line (C), respectively, at a rate of 0.7 μL/cm using the BioDot XYZ 3050 Dispensing Platform. The distance between each line was 5 mm. Assembly of the detection paper The detection paper consists of four components: a sample pad, a nitrocellulose membrane, an absorption pad, and a plastic adhesive card. HFB 180 and fusion five were used as the nitrocellulose membrane and sample pad, respectively. The nitrocellulose membrane, sample pad and absorbent pad were affixed onto the plastic adhesive card within a 2-mm overlap and cut into 4-mm width strips. Fluorescence detection procedure


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The 150-μL milk samples were spiked with varying concentrations of AFM1 (0, 0.02, 0.04, 0.1, 0.2, 0.3, and 0.4 ng/mL) or MEL (0, 5, 10, 25, 50, 100, and 150 ng/mL). The standard curve was a fitted function expressed as the fluorescence intensity of the test line versus the natural logarithm of the AFM1/MEL concentration (X) with the equation: YAFM1= b1 XAFM1 (ln cAFM1) + a1, YMEL = b2 XMEL (ln cMEL) + a2. The average value for each spot was based on five experiments. The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the mean value from 20 negative samples23. The linear ranges were recorded from the LOQ to the cut-off value. The cut-off value was the AFM1/MEL concentration at which the color faded on the test line or exceeded the linear range. Optimization of the mAb dosage The mAb dosage was optimized with 100 μL of 1 mg/mL bAb immobilized on the MS. Series volumes (1, 5, 10, and 20 μL) of 0.055 mg/mL of mAbMEL and 0.001 mg/mL of mAbAFM1 were conjugated to the bAb-MS for 12 h at 4°C. After purification, 37.5 μL of mAbAFM1-bAb-MS and 37.5 μL of mAbMEL-bAb-MS were mixed with 75 μL of milk, and the fluorescence intensity was recorded after a 10-minute incubation. Specificity, accuracy, and precision Blank milk samples were confirmed by UPLC-MS/MS analysis. Six chemicals with similar structures (cyanuric acid, proline, β-lactam, chloramphenicol, MEL and AFM1, 200 ng/mL each) were spiked in the same milk samples. The fluorescence intensity on the test line was recorded. To estimate the accuracy and precision, blank milk samples were spiked with MEL (5, 50, and 150 ng/mL) and AFM1 (0.01, 0.2, and 0.4 ng/mL). The spiked samples were subsequently detected with the detection paper. For the intraday precision study, 5 replicate experiments were conducted with each single sample, whereas the replicate analyses were performed on five successive days. Comparison of results obtained via detection paper, HPLC and UPLC-MS/MS To validate its application in milk samples, nine milk samples from markets were analyzed with the detection paper, HPLC and UPLC-MS/MS methods24-26. All results



for AFM1 and MEL were compared between the detection paper and the HPLC and UPLC-MS/MS methods based on the Chinese National Standards (GB 5413.37-2010 and GB/T 22388-2008). Results and Discussion Working principle of the detection paper The detection paper consisted of three capture reagents, including a MEL-BSA antigen, an AFM1-BSA antigen on two independent test lines and mouse immunoglobulin (IgG1) on the control line (Figure 1). The functionalized MS was prepared through a two-step process (Figure 2). First, the bAb was directly immobilized on the MS. Then, specific monoclonal antibodies (mAbAFM1 or mAbMEL) were conjugated to the bAb-MS via specific recognition, resulting in mAbMEL-bAb-MS or mAbAFM1-bAb-MS. The merits of the bAb could probably be attributed to two factors. First, multiple bAbs (around 213 bAbs according to Coomassie blue staining) are immobilized on a single MS as the first layer, which increases the effective number of mAbs and thus enhances the mAb validity. Second, the bAbs allow a universal conjugation strategy by alternating mAbs for the detection of other targets of interest. Milk samples (75 μL) with or without analytes (MEL or AFM1) were mixed with two functionalized MSs (37.5 μL of mAbMEL-bAb-MS and 37.5 μL of mAbAFM1-bAbMS) and then detected at room temperature. In the absence of analytes, mAbMEL-bAbMS or mAbAFM1-bAb-MS was trapped by the immobilized MEL-BSA or AFM1–BSA on the test line, and two intense red strips are observed under UV light. In the presence of analytes, MEL (or AFM1) is in competition with MEL (or AFM1)-BSA for mAbMEL-bAb-MS (mAbAFM1-bAb-MS) during the lateral flow. With the increase in MEL or AFM1, a small amount of functionalized MS conjugates to MEL (or AFM1)BSA on the test line, resulting in reduced coloring of the test lines. Absence of the control line suggests the invalidation of the detection paper. Optimization of the mAb dosage The loading amount of mAbMEL or mAbAFM1 on the MS plays a vital role in improving the sensitivity and linear range. Increasing the dosage of mAb(MEL or AFM1)


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results in a higher fluorescence intensity on the test line in the negative samples, which allows a wider linear dynamic range. However, when the dosage of mAbMEL or mAbAFM1 is over the dosage of MEL or AFM1, it may negatively affect the fluorescence reduction on the test lines. To ensure clear test lines and reduce background interference, the optimized mAbs were 10 μL of 0.001 mg/mL mAbAFM1 and 20 μL of 0.055 mg/mL mAbMEL, before they were conjugated with 100 μL of 1 mg/mL bAb on the MS surface, respectively. Performance evaluation of the detection paper The performance was evaluated without any sample pretreatment, additional solution and manipulations. A linear range from 0.02 to 0.4 ng/mL with an acceptable correlation coefficient (R2 = 0.994) was obtained for AFM1 (Figure 3a), and a linear range from 5 to 150 ng/mL (R2= 0.986) was obtained for MEL (Figure 3b). The LODs of the detection paper for AFM1 and MEL were 0.009 ng/mL and 0.024 ng/mL, respectively, which fully satisfied the MRLs established for AFM1 and MEL residues in liquid milk in China. Compared to the detection system without bAb, the LOD was improved one order of magnitude.21,27 The major detection parameters are shown in Table S1. Compared with other literatures9,28-30, this work provided a pioneering onsite detection paper for simultaneous detection of multiclass chemical contaminants via universal bridge-antibody labelling with the higher sensitivity (Table 1). Simultaneous detection of AFM1 and MEL in milk samples was performed by the detection paper (Figure 4a). As expected, with the increase in AFM1/MEL, the fluorescence intensity of the test line decreased. The cut-off value was defined as the lowest concentration at which the test line was completely invisible. The cut-off values of AFM1 and MEL were 0.4 ng/mL and 1.5 ng/mL, respectively. The results can also be seen by the naked eye under UV light, suggesting the applicability of this test for on-site detection. Concerning the rigorous residue regulation for AFM1 and MEL in liquid infant formula in major countries, the portable reader was used to detect AFM1 and MEL at trace levels. To ensure no other interference occurred during the simultaneous detection of



AFM1 and MEL, milk samples containing cyanuric acid, proline, β-lactam, and chloramphenicol were detected using the detection paper. The results demonstrated that the presence of these potential chemical residues in the milk samples did not affect the AFM1 and MEL signals (Figure 4b). The detection paper exhibited high specificity as a result of the high specificity of mAbAFM1 and mAbMEL. In terms of its accuracy, an intraday study was performed and found recoveries of 88.7%-105% and 91.4%-101.9% for AFM1 and MEL, respectively. When precision was evaluated, the recoveries were 96%-102.8% and 84.6%-117.7% for AFM1 and MEL, respectively (Table S2). The acceptable accuracy and precision indicate that this assay can be extensively applied for on-site detection of AMF1 and MEL. To validate its use in practice, 9 milk samples were used to compare the results between this detection paper and HPLC for AFM1 and UPLC-MS/MS for MEL. The detection paper and the HPLC and UPLC-MS/MS method results were in good agreement, with recoveries ranging from 91.4% to 118.2% for AFM1 and from 84.5% to 104.3% for MEL (Table 2). The results indicated that the proposed detection paper was applicable for the simultaneous detection of AFM1 and MEL in real milk samples. Conclusions A bridge Ab was introduced for the first time to conjugate the MS and mAb for the simultaneous determination of AFM1 and MEL in milk without any sample pretreatment, additional solution and manipulations. Compared to other paper-based immunoassays, this detection paper has several appealing features. First, immobilization of the bAb on the MS as the first layer increased the number of effective mAbs, which was probably beneficial for the binding sites of the specific mAbs. Secondly, the bAb-MS label, which serves as a universal capture probe, can be readily adapted for the detection of other targets by conjugating to an appropriate mAb via a recognition reaction. Taken together, our results suggest that the proposed detection paper can be used as a universal, simple, and sensitive screening tool for on-site simultaneous detection of multiclass chemical contaminants via universal bridgeantibody labelling.


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Note Guanghua Li is the exchanging graduate student from School of Materials Science and Engineering, Wuhan Institute of Technology, Wuhan 430074, P. R. China. Conflict There is no conflict to declare. Acknowledgements This work was supported by the National Program for Support of Top-notch Young Professionals.



References (1) Rather, I. A.; Koh, W. Y.; Paek, W. K.; Lim, The Sources of Chemical Contaminants in Food and Their Health Implications. Frontiers in Pharmacology. J. Front. Pharmacol. 2017, 8, 830. (2) Dasenaki, M. E.; Thomaidis, N. S. Multi-residue determination of 115 veterinary drugs and pharmaceutical residues in milk powder, butter, fish tissue and eggs using liquid chromatography-tandem mass spectrometry. Anal. Chim. Acta 2015, 880, 103121. (3) Petrarca, M. H.; Fernandes, J. O.; Godoy, H. T.; Cunha, S. C. Multiclass pesticide analysis in fruit-based baby food: A comparative study of sample preparation techniques previous to gas chromatography–mass spectrometry. Food Chem. 2016, 212, 528-536. (4) Hu, R.; Liu, T.; Zhang, X. B.; Yang, Y.; Chen, T.; Wu, C.; Liu, Y.; Zhu, G.; Huan, S.; Fu, T. DLISA: A DNAzyme-Based ELISA for Protein Enzyme-Free Immunoassay of Multiple Analytes. Anal. Chem. 2015, 87, 7746-7753. (5) Soares, R. R. G.; Santos, D. R.; Chu, V.; Azevedo, A. M.; Aires-Barros, M. R.; Conde, J. P. A point-of-use microfluidic device with integrated photodetector array for immunoassay multiplexing: Detection of a panel of mycotoxins in multiple samples. Biosens. Bioelectron. 2016, 87, 823-831. (6) Liu, L.; Lin, H. Paper-Based Colorimetric Array Test Strip for Selective and Semiquantitative Multi-Ion Analysis: Simultaneous Detection of Hg2+, Ag+, and Cu2+. Anal. Chem. 2014, 86, 8829-8834. (7) Banerjee, R.; Jaiswal, A. Recent advances in nanoparticle-based lateral flow immunoassay as a point-of-care diagnostic tool for infectious agents and diseases. Analyst 2018, 143, 1970-1996. (8) Giorgio, D.; Di Trana, A.; Claps, S. Oligosaccharides, polyamines and sphingolipids in ruminant milk. Small Ruminant Res. 2018, 160, 23-30. (9) Guo, H.; Zhou, X.; Zhang, Y.; Song, B.; Zhang, J.; Shi, H. Highly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by multiplexed


Page 12 of 27

Page 13 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


planar waveguide fluorescence immunosensor (MPWFI). Food Chem. 2016, 197, 359366. (10) Spiegel, M. V.; Sterrenburg, P.; Haasnoot, W.; Fels-Klerx, H. J. V. Towards a decision support system for control of multiple food safety hazards in raw milk production. Trends Food Sci. Technol. 2013, 34, 137-145. (11) Qian, G. S.; Yasei, P.; Yang, G. C. Rapid extraction and detection of aflatoxin M1 in cow's milk by high-performance liquid chromatography and radioimmunoassay. Anal. Chem. 1984, 56, 2079-2080. (12) Li, S.; Min, L.; Wang, P.; Zhang, Y.; Zheng, N.; Wang, J. Aflatoxin M1 contamination in raw milk from major milk-producing areas of China during four seasons of 2016. J. Food Control 2017, 82, 121-125. (13) Neal, G.; Eaton, D.; Judah, D.; Verma, A. Metabolism and toxicity of aflatoxins M1 and B1 in human-derived in vitro systems. Toxicol. Appl. Pharmacol. 1998, 151, 152-158. (14) Parker, C. O.; Lanyon, Y. H.; Manning, M.; Arrigan, D. W. M.; Tothill, I. E.; Chem, A. Electrochemical immunochip sensor for aflatoxin M1 detection. Anal. Chem. 2009, 81, 5291-5298. (15) Iqbal, S. Z.; Jinap, S.; Pirouz, A. A.; Faizal, A. R. A. Aflatoxin M-1 in milk and dairy products, occurrence and recent challenges: A review. Trends Food Sci. Technol. 2015, 46, 110-119. (16) Shuang, H.; Zhu, S.; Liu, Z.; Hu, L.; Parveen, S.; Xu, G. Oligonucleotide-stabilized fluorescent silver nanoclusters for turn-on detection of melamine. Biosens. Bioelectron. 2012, 36, 267-270. (17) Choi, J. W.; Min, K. M.; Hengoju, S.; Kim, G. J.; Chang, S. I.; Demello, A. J.; Choo, J.; Kim, H. Y. A droplet-based microfluidic immunosensor for high efficiency melamine analysis. Biosens. Bioelectron. 2015, 80, 182-186. (18) Rahman, M. M.; Ahmed, J. Cd-doped Sb2O4 nanostructures modified glassy carbon electrode for efficient detection of melamine by electrochemical approach. Biosens. Bioelectron. 2017, 102, 631-636.



(19) Urusov, A. E.; Petrakova, A. V.; Zherdev, A. V.; Dzantiev, B. B. "Multistage in one touch" design with a universal labelling conjugate for high-sensitive lateral flow immunoassays. Biosens. Bioelectron. 2016, 86, 575-579. (20) Lin, M.; Wang, J.; Zhou, G.; Wang, J.; Wu, N.; Lu, J.; Gao, J.; Chen, X.; Shi, J.; Zuo, X.; Fan, C. Programmable engineering of a biosensing interface with tetrahedral DNA nanostructures for ultrasensitive DNA detection. Angew Chem. Int. Ed. 2015, 54, 2151-2155. (21) Tang, X.; Zhang, Z.; Li, P.; Zhang, Q.; Jiang, J.; Wang, D.; Lei, J. amplepretreatment-free based high sensitive determination of aflatoxin M1 in raw milk using a time-resolved fluorescent competitive immunochromatographic assay. Rsc Adv. 2014, 5, 558-564. (22) Zhang, Z.; Tang, X.; Wang, D.; Zhang, Q.; Li, P.; Ding, X. Rapid on-site sensing aflatoxin B1 in food and feed via a chromatographic time-resolved fluoroimmunoassay. PLoS One 2015, 10, e0123266. (23) Wang D.; Zhang Z.; Li P.; Zhang Q.; Ding X.; Zhang W. Europium Nanospheresbased Time-resolved Fluorescence for Rapid and Ultrasensitive Determination of Total Aflatoxin in Feed. J. Agric. Food Chem. 2015, 63, 10313-10318. (24) Lee, D.; Lee, K.-G. Analysis of aflatoxin M1 and M2 in commercial dairy products using high-performance liquid chromatography with a fluorescence detector. Food Control 2015, 50, 467-471. (25) Mao, J.; Lei, S.; Liu, Y.; Xiao, D.; Fu, C.; Zhong, L.; Ouyang, H. Quantification of aflatoxin M1 in raw milk by a core-shell column on a conventional HPLC with large volume injection and step gradient elution. Food Control 2015, 51, 156-162. (26) Ge, X.; Wu, X.; Liang, S.; Su, M.; Sun, H. Trace residue analysis of dicyandiamide, cyromazine, and melamine in animal tissue foods by ultra-performance liquid chromatography. J. Food Drug Anal. 2016, 24, 579-585. (27) Li G.; Wang D.; Zhou A.; Sun Y.; Zhang Q.; Poapolathep A.; Zhang L.; Fan Z.; Zhang Z.; Li P. A rapid, onsite, ultrasensitive melamine quantitation method for protein beverages using time-resolved fluorescence detection paper. J. Agric. Food Chem. 2018,


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5671-5676. (28) Zhou, Q.; Liu, N.; Qie, Z.; Wang, Y.; Ning, B.; Gao, Z. Development of Gold Nanoparticle-Based Rapid Detection Kit for Melamine in Milk Products. J. Agric. Food Chem. 2011, 59, 12006-12011. (29) Wu, C.; Hu, L.; Xia, J.; Xu, G.; Luo, K.; Liu, D.; Duan, H.; Cheng, S.; Xiong, Y.; Lai, W. Comparison of immunochromatographic assays based on fluorescent microsphere and quantum-dot submicrobead for quantitative detection of aflatoxin M1 in milk. J. Dairy Sci. 2017, 100, 2501-2511. (30) Li, Z.; Li, Z.; Jiang, J.; Xu, D. Simultaneous detection of various contaminants in milk based on visualized microarray. Food Control 2017, 73, 994-1001.



Figures and Tables

Figure 1 Principle of detection paper.


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Figure 2 Preparation of mAb-bAb-MS.



Figure 3 Standard curve of (a) T1: AFM1 and (b) T2: MEL.


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Figure 4 Under UV light, (a) Simultaneous detection of AFM1 and MEL with serious concentration (b) Specificity evaluation results. 1: cyanuric acid, 2: proline, 3: βlactam, 4: chloramphenicol, 5: MEL, and 6: AFM1.



Page 20 of 27

Table 1 Comparison of the detection paper with other methods. Method Visualized microarray Multiplexed planar waveguide fluorescence immunosensor Fluorescent microsphere immunochromatographic assay and quantum-dot submicrobead immunochromatographic assay Gold nanoparticle-based rapid detection kit This work

LOD (ng/mL) 0.210 16.31 0.055 15.60

Linear range (ng/mL) 0.200-1.400 16.30-152.6 0.098-0.709 33.20-439.6

AFM1

0.042 0.093

0.050-0.600 0.100-1.000

MEL AFM1 MEL

1000 0.009 0.024

/ 0.020-0.400 5.000-150.0

Targets AFM1 MEL AFM1 MEL


Reference [30] [9] [29] [28] /

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Table 2 Comparison of detection paper, HPLC, and UPLC-MS/MS.

AFM1 (ng/mL) Analyte

MEL (ng/mL)

HPLC

Detection paper

Recovery (%)

UPLCMS/MS

Detection paper

Recovery (%)

Milk 1

0.11

0.13

118.2

4.57

3.86

84.5

Milk 2

0.14

0.16

114.3

19.63

17.25

87.9

Milk 3

ND

ND

/

ND

ND

/

Milk 4

0.54

/a

/

15.24

13.38

88.0

Milk 5

0.40

0.38

95.0

ND

ND

/

Milk 6

ND

ND

/

ND

ND

/

Milk 7

ND

ND

/

ND

ND

/

Milk 8

0.35

0.32

91.4

45.09

47.13

104.3

Milk 9

ND

ND

/

ND

ND

/

ND: Not detected a: Exceed the linear range of detection paper



Figures and Tables

Figure 1 Principle of detection paper.


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Figure 2 Preparation of mAb-bAb-MS.



Figure 3 Standard curve of (a) T1: AFM1 and (b) T2: MEL.


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Figure 4 Under UV light, (a) Simultaneous detection of AFM1 and MEL with serious concentration (b) Specificity evaluation results. 1: cyanuric acid, 2: proline, 3: βlactam, 4: chloramphenicol, 5: MEL, and 6: AFM1.



Page 26 of 27

Table 1 Comparison of the detection paper with other methods. Method Visualized microarray Multiplexed planar waveguide fluorescence immunosensor Fluorescent microsphere immunochromatographic assay and quantum-dot submicrobead immunochromatographic assay Gold nanoparticle-based rapid detection kit This work

LOD (ng/mL) 0.210 16.31 0.055 15.60

Linear range (ng/mL) 0.200-1.400 16.30-152.6 0.098-0.709 33.20-439.6

AFM1

0.042 0.093

0.050-0.600 0.100-1.000

MEL AFM1 MEL

1000 0.009 0.024

/ 0.020-0.400 5.000-150.0

Targets AFM1 MEL AFM1 MEL


Reference [30] [9] [29] [28] /

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Table 2 Comparison of detection paper, HPLC, and UPLC-MS/MS.

AFM1 (ng/mL) Analyte

MEL (ng/mL)

HPLC

Detection paper

Recovery (%)

UPLCMS/MS

Detection paper

Recovery (%)

Milk 1

0.11

0.13

118.2

4.57

3.86

84.5

Milk 2

0.14

0.16

114.3

19.63

17.25

87.9

Milk 3

ND

ND

/

ND

ND

/

Milk 4

0.54

/a

/

15.24

13.38

88.0

Milk 5

0.40

0.38

95.0

ND

ND

/

Milk 6

ND

ND

/

ND

ND

/

Milk 7

ND

ND

/

ND

ND

/

Milk 8

0.35

0.32

91.4

45.09

47.13

104.3

Milk 9

ND

ND

/

ND

ND

/

ND: Not detected a: Exceed the linear range of detection paper


An on-site ultra-sensitive detection paper for multiclass chemical

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