key: cord-0028631-7ehq81t6 authors: Guo, Lingling; Xu, Xinxin; Zhao, Jing; Hu, Shudong; Xu, Liguang; Kuang, Hua; Xu, Chuanlai title: Multiple detection of 15 triazine herbicides by gold nanoparticle based-paper sensor date: 2022-03-15 journal: Nano Res DOI: 10.1007/s12274-022-4164-2 sha: 0254f0489e2a7a584406a5f0f33f15b2c0471eb0 doc_id: 28631 cord_uid: 7ehq81t6 Triazine herbicides have been widely used in agriculture, but their residues can harm the environment and human health. To help monitor these, we have developed an effective immunochromatographic strip test that can simultaneously detect 15 different triazines in grain samples (including ametryn, cyprazine, atraton, prometon, prometryn, atrazine, propazine, terbuthylazine, simetryn, trietazine, secbumeton, simazine, desmetryn, terbumeton and simetone). Based on our optimization procedure, the visual limit of detection (vLOD) for these triazines was found to be 2–10 ng/mL in assay buffer, and 0.02−0.1 mg/kg in grain samples. Four different grain matrices including corn, brown rice, wheat, and sorghum were studied and the test results showed no significant differences between the 15 triazines analyzed using this method. This test is simple, convenient, rapid, and low-cost, and could be an effective tool for primary screening of triazine residues in grain samples. [Image: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material (UV–vis spectra of 15 nm-GNP; K2CO3 usage; cross reactivity; strip images for spiked rice, wheat and sorghum samples, UPLC-MS/MS parameters; gray values for strip optimization) is available in the online version of this article at 10.1007/s12274-022-4164-2. Triazine-based herbicides are used world-wide for the control of annual grasses and broadleaf weeds in various crops, and account for an estimated 30% of agricultural herbicides [1] . Triazine herbicides have a common triazine ring ( Fig. 1(a) ), and depending on the substituent groups of X, they can be generally divided into three categories. The first group includes atrazine, cyprazine, propazine, terbutylazine, trietazine and simazine, whose X is Cl. The second group, in which X is O-CH 3 , includes atraton, prometon, secbumeton, terbumeton and simetone. The third group includes ametryn, prometryn, simetryn, and desmetryn, whose X is S-CH 3 . They have the characteristics of high mobility, persistence, and low degradation rate in the environment. Their widespread and excessive use have led to serious accumulation in soil and water, and they can be transferred directly or indirectly to human body through food chain. Several reports demonstrated that exposure to triazine herbicides could cause the disorder of reproductive system, nervous system, and immune system of mammals [2] [3] [4] .Consequently, many countries have set maximum residue limits (MRLs) for agricultural products according to the actual usage. Take atrazine for example, China enforces MRLs of 0.05 mg/kg for sorghum, corn and millet; but in Japan, the MRLs are 0.2 mg/kg in corn and 0.3 mg/kg in wheat. Based on the details mentioned above, it is important to develop effective detection methods to police this. Instrumental analytical methods, such as gas chromatographytandem mass spectrometry [5] and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) [6] [7] [8] , are the standard methods used for the detection of triazine residues. Although these methods are highly accurate, sensitive and reliable, some disadvantages such as the need for large amounts of organic solvents, complicated sample pre-processing steps, bulky instrumentation, long detection time, high cost and the need for professional operators, make them not suitable for onsite detection. In contrast, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA), are time saving, convenient and economic, and they have been widely applied to food quality control [9] [10] [11] [12] [13] [14] [15] , disease diagnosis [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] and environmental pollutant analysis [26] [27] [28] [29] [30] . Several immunoassays have also been developed for the determination of triazines (Table 1) [31] [32] [33] [34] [35] [36] [37] . These methods are all based on the use of a specific antibody (monoclonal antibody, polyclonal antibody, or antisera) against antigen. Enzymes (goat anti-mouse IgG horseradish peroxidase) or nanoparticles (gold nanoparticles (GNPs), up-conversion materials, or mesoporous core-shell palladium@platinum nanoparticles) have all been used for signal visualization and amplification before detection [38] [39] [40] . Due to their advantage as visual detection systems, these methods need no specialist equipment and are of low cost. Furthermore, ICAs are more suitable for rapid detection than other types of immunoassays [41, 42] . However, current ICAs can only detect one type of triazine rather than multiple forms. In this work, we prepared a GNP basedimmunochromatographic strip for the multiple determination of 15 triazine herbicides using a series of optimization steps. Furthermore, its usefulness with different grains (corn, brown rice, wheat, and sorghum) and the effects of different grain matrices were also determined. Triazine standards (ametryn, cyprazine, atraton, prometon, prometryn, atrazine, propazine, terbuthylazine, simetryn, trietazine, secbumeton, simazine, desmetryn, terbumeton, simetone, cyanazine, dipropetryn and metribuzin) were purchased from TanMo Quality Testing Technology Co., Ltd (Beijing, China). Goat anti-mouse IgG antibody, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and chloroauric acid (HClO 4 ) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-((4-chloro-6-(isopropylamino)-1,3,5-triazin-2yl)amino)butanoic acid was prepared by Sandia Pharmaceutical Technology (Shanghai) Co., LTD. Nitrocellulose (NC) membrane was purchased from Sartorius Stedim Biotech GmbH (Gὅttingen, Germany). Sample pad, absorption pad, glass fiber anline-height-add:-0.5ptd polyvinychloride (PVC) backing plates were purchased from JieYi Biotechnology Co., Ltd. (Shanghai, China) and the XYZ dispensing platform and guillotine cutting module for immunochromatographic strip preparation were from Shanghai Kinbio Tech Co., Ltd. According to a previous report [31] , 4-((4-chloro-6-(isopropylamino)-1,3,5-triazin-2-yl)amino)butanoic acid were used as hapten ( Fig. 1(b) ). Immunogen (Hapten-KLH) and coating antigen (Hapten-BSA) were prepared by the active ester method [43] . Through immunization, cell fusion, and ascites purification, anti-triazine monoclonal antibody (mAb) was obtained. GNPs were prepared according to our previous study [43, 44] . Briefly, 12.5 mL, 10 mmol/L HClO 4 solution was added to 487.5 mL ultrapure water. After boiling and constant stirring for 15 min, 1 mL of sodium citrate tribasic dihydrate (10%, w/v) was added quickly. Then the solution was boiled until it turned to a red-wine color (approximately 30 min) and allowed to cool to room temperature and kept at 4 °C for further use. The prepared GNP solution was characterized using an ultraviolet-visible (UV-vis) spectrophotometer and transmission electron microscope (TEM). The GNP-labelled mAb was prepared as follows [45] [46] [47] . First, K 2 CO 3 (0.2 mol/L) was used to adjust the pH of the GNP solution (10 mL) to 8.2. Then, the anti-triazine mAb diluted with 0.2 mol/L borate saline buffer (BSB) was added at room temperature. After 1 h at room temperature, 0.5 mL of BSA solution (10%, w/v) was added to the solution to block any unbound site. 2 h later, the solution was centrifuged at 4 °C, 8,000 g for 55 min to remove unconjugated mAb. The supernatant was then removed and the pellet was resuspended in 10 mL of resuspension solution. This was added into a microwell plate (50 µL/well). The solution was finally lyophilized using a vacuum freeze-dryer, and stored in a drying cabinet until use. This ICA strip was composed of a sample pad, an absorbent pad, and a NC membrane with a PVC backing plate, and was assembled in layers (Fig. 2) . The NC membrane was placed on the center of the PVC backing plate and then the sample pad and absorbent pad were stuck to either end and overlapped with the NC membrane by 2 mm. Then Hapten-BSA and goat anti-mouse IgG antibody were sprayed onto the NC membrane forming the test T and control C lines. After drying for 2 h at 37 °C, it was cut into strips of 2.95 mm in width and then stored in a drying cabinet at room temperature until use. For the ICA test, 200 µL of standard or sample solution was added into a microwell plate containing freeze-dried GNP-labelled mAb. 2 min later, the ICA strip was placed into the well. Due to capillarity action, the solution flowed from the sample pad to the absorbent pad, and after 4 min, the strip was removed and the color of the T line was observed. 1 g of crushed grain sample was extracted with 4 mL of extraction solution with vibration for 3 min. After centrifugation at 3,000 g for 3 min, the supernatant was removed and diluted using a sample diluent for ICA analysis. Different triazines-negative grain samples (purchased from a local supermarket: corn, wheat, brown rice, and sorghum) were confirmed by UPLC-MS/MS. Details of the UPLC-MS/MS method are given in the Electronic Supplementary Material (ESM), including liquid chromatography (LC) and mass spectrometry (MS) conditions (Table S1 ) and MS/MS parameters ( Table S2 in the ESM). Triazine standard solutions at different concentrations (0, 0.02, 0.1, 0.5, and 2.5 mg/kg) were spiked into the negative grain samples for ICA analysis after the pretreatment described above. Hapten-KLH and Hapten-BSA were conjugated successfully and characterized by ultraviolet (UV) spectrum (Figs. 1(c) and 1(d)), which had both the characteristic UV absorption peaks of Hapten and carrier protein (KLH or BSA). The half-maximal inhibition concentration values (IC 50 ) of 15 triazines were tested by ELISA [31] . As shown in Table 2 , this prepared mAb can recognize 15 triazines. The mainly reason is that the hapten retained the common structure in maximum, and meanwhile the space arm (-NH-CH 2 -CH 2 -CH 2 -CO-) is good for the exposure of the common structure to produce group-selective monoclonal antibody. Due to their high specific surface area, chemical inertness and biocompatibility, GNPs are one of the most attractive materials for use as a label for detecting analytes with an ICA, and can provide a visual signal on the T line when binding between antibody and antigen occurs. Herein, we have adopted the classical citrate reduction method for the synthesis of GNPs [48] , and citrate ions not only act as a reducing agent for the GNP formation, but also play a role in preventing the GNPs from aggregating [49] . With this method, the amount of citrate is related to the GNP size and GNPs of 10-40 nm in diameter are reported to be optimal for ICA analysis, as smaller particles have unacceptable color development and larger particles are unstable for long-term storage [50] . Based on our previous research [51] , GNPs with a diameter of 15 nm were found to be stable and the wine color produced was suitable for ICA analysis. As shown in Fig. 3(a) under the TEM, our GNPs appeared uniform and well dispersed and the UV-vis spectrophotometer in Fig. S1 in the ESM showed a strong absorption peak at 520 nm, which is also consistent with previous reports. The preparation of GNP labeled mAb mainly utilizes electrostatic adsorption between the negatively charged surface of the GNPs and the positively charged groups on the mAb. This bioconjugation is highly susceptible to pH when the pH of the system is greater than or equal to the isoelectric point of mAb (pH 8.0), and the GNP labeled mAb has good stability. Thus, the amount of K 2 CO 3 used for the adjustment of pH needs be optimized. According to a previous report [52], the optimum amount of K 2 CO 3 used for the labelling process is also defined as the minimum amount preventing GNP accumulation under the addition of 10% NaCl (w/w). As can be seen from Fig. S2 in the ESM, when the K 2 CO 3 content in GNP solution was lower than 5 µL/mL, the particles began to clump, and the color of the solution turned very dark. When the K 2 CO 3 content was greater than or equaled 5 µL/mL, the particles were well dispersed. Furthermore, excess K 2 CO 3 may also influence the conjugation of mAb and GNPs, which is illustrated in Fig. 3(b) . We can see that the color of the T line was darker when K 2 CO 3 content was 5 µL/mL. The amount of mAb used was also optimized and herein an increase in mAb concentration also gave a darker T line, and when the mAb content in GNP solution was 10 µg/mL the T line color became maximally dark. The inhibition when ametryn (20 ng/mL) was added was also distinct. Besides, the gray values (T line and C line) obtained by processing strip images using Photoshop software could further confirm the result. The gray value was not very different, but the inhibition ratio was better under the condition of 5 µL of K 2 CO 3 and 10 µg of mAb (Table S3 in the ESM). Thus, 5 µL/mL K 2 CO 3 and 10 µg/mL mAb were chosen for the labeling of the mAb with the GNPs. The principle of the ICA is based on the competition between the fixed antigen on the T line and the free analytes in the sample solution for binding to the GNP labeled mAb, as shown in Fig. 2 . For weakly positive or positive samples, the analyte in the sample solution firstly conjugates with the GNP labeled mAb during incubation. Due to its capillary effect, the sample solution flows from the sample pad to absorption pad and when it reaches the T line, the unconjugated or non-additional GNP labeled mAb can conjugate with the Hapten-BSA on the T line, making the color of the T line light or even disappear. The C line should always be red because of the binding between the goat anti-mouse IgG and the GNP labelled mAb. For negative samples, no analyte in sample solution conjugates with the GNP labelled mAb, so the T line color is the same as or darker than that of the C line. The resuspension solution for the GNP labelled mAb and assay buffer was optimized for ICA analysis. Surfactants play an important role in ICA analysis, and can improve their performance by facilitating sample flow along the strip and increasing the analyte solubility. In this study, the GNP labeled mAb was resuspended in suspension buffer (0.01 M phosphate buffer saline (PBS) solution containing 5% trehalose, 0.2% BSA and 0.04% sodium azide), which was mixed with four different surfactants: 5% Tween-20, 5% Triton X-100, 5% ON-870 and 5% Brij-35. Based on the color intensity at 0 and 20 ng/mL for ametryn in 0.01 M, pH 7.4 PBS, the most stable deep red was obtained using Brij-35 (Fig. 3(c) and Table S3 in the ESM) . Assay buffers at different pH and ionic strengths can also influence the binding of the antibody and antigen, and further affect the sensitivity of the ICA. Thus, four kinds of assay buffer, including tris(hydroxymethyl)aminomethane hydrochloride solution (Tris-HCl, pH 6.0), PBS (pH 7.4), BSB (pH 8.2) and carbonate buffer solution (CBS, pH 9.6), with the same concentration (0.01 M) and Brij-35 content (3%, w/w), were optimized. The darkest red color on the T line was produced using CBS, but the inhibition with the addition of ametryn (20 ng/mL) was less obvious than that seen with PBS ( Fig. 3(d) and Table S3 in the ESM). While the color intensities in Tris-HCl and BSB were slightly lower than that for PBS. Therefore, PBS (0.01 M, pH 7.4) was chosen as the assay buffer for triazine analysis. Based on the optimization of 15 types of triazines, including ametryn, cyprazine, atraton, prometon, prometryn, atrazine, propazine, terbuthylazine, simetryn, trietazine, secbumeton, simazine, desmetryn, terbumeton and simetone, we used ICA for their determination. The standard concentrations of each triazine used were 0, 2, 10, 50 and 250 ng/mL and from the original strip images (Fig. 4) , we can determine the visual limit of detection (vLOD) for each triazine, which is defined as the concentration, at which the T line color is obviously less than that of the blank solution. The vLOD values in buffer for cyprazine, atraton, prometon, atrazine, propazine, terbuthylazine, simazine, Figure 4 The strip images of 15 triazines in assay buffer. The 15 triazines are in order (1-15) as follows: ametryn, cyprazine, atraton, prometon, prometryn, atrazine, propazine, terbuthylazine, simetryn, trietazine, secbumeton, simazine, desmetryn, terbumeton and simetone. The standard concentrations of each triazine are 0, 2, 10, 50 and 250 ng/mL from left to right. desmetryn, and terbumeton were all 2 ng/mL, and the other triazines including ametryn, prometryn, simetryn, trietazine, secbumeton, and simetone were all 10 ng/mL (Table 2) . Furthermore, three other triazines (cyanazine, dipropetryn and metribuzin) were also tested, which showed no obvious color change at a concentration of 5,000 ng/mL (Fig. S3 in the ESM), suggesting that the strips could not be used to detect cyanazine, dipropetryn and metribuzin. Different organic solvents can influence not only the extraction efficiency but also the binding of antigen to antibody. Five organic solvents, including acetonitrile (ACN), dimethyl formamide (DMF), methanol (MT), acetone (CP) and dimethylsulfoxide (DMSO), were optimized, as shown in Fig. 3(e) ; the order of the color intensity in the blank sample was shown to be DMF < DMSO < CP < MT < ACN and the order of inhibition for the spiked sample (0.1 mg/kg ametryn) was MT > DMSO > CP > ACN. However, the inhibition when using DMF as the extraction solvent was not clear because it was invisible to the naked eye. Because of these tests, MT with the highest color intensity and inhibition was chosen as the solution to be used for the grain extraction when using the ICA. An appropriate sample dilution can reduce the interference between MT and the grain matrix in the ICA. The color strips with blank and spiked samples (0.1 mg/kg of ametryn in corn extracting solution) had almost identical color intensities when diluted two, three, and four times, respectively (Fig. 3(f) ). This result suggested that our developed ICA based on a triazines-mAb had the characteristics of grain matrix resistance. And high dilution ratio signified low sensitivity. Thus, grains samples were diluted two times for triazine residue detection. Four grain samples, including corn, wheat, brown rice, and sorghum, purchased from thr local supermarket were triazinesnegative, which can be seen from the chromatograms of the quantitative ions for the 15 triazines from 1 ng/mL standard solution and four grain samples (Fig. 5) . Matrix effects were evaluated based on the vLOD values of the four types of grains spiked with the 15 different triazines after a pretreatment and analysis as described above. From the original strip images for the spiked corn samples (Fig. 6) , the vLOD values for cyprazine, atraton, prometon, atrazine, propazine, and terbuthylazine, were all 0.02 mg/kg, and the other triazines including ametryn, prometryn, simetryn, trietazine, secbumeton, simazine, desmetryn, terbumeton and simetone, were all 0.1 mg/kg. Moreover, when comparing the strip results (Figs. S4-S6 in the ESM), we found that each triazine had the same vLOD values in corn maize, brown rice, wheat and sorghum, indicating that the result with the developed ICA strips is not affected by grain matrices. Thus, our assembled ICA strips can be used for the detection of 15 triazines simultaneously from grain samples, and the test results are consistent between different grain types. The vLOD values for the 15 triazines in grain are listed in Table 2 . Except for prometryn with an MRL of 0.02 mg/kg in corn and simetryn with an MRL of 0.05 mg/kg in brown rice, all the other triazine vLOD values satisfy Chinese MRL requirements. Compared with single-residue ICA previously reported, this developed multi-residue ICA cannot quantitate triazines, but it has advantages of high detection efficiency, low cost and short detection time. Furthermore, the whole testing process including pretreatment was no more than 15 min. Conclusively, this technique represents a simple-to-operate and lower-cost assay and is suitable for the fast screening of triazine residues in various grain samples. Herein, we have successfully prepared rapid detection strips and developed an ICA for the rapid and simultaneous detection of 15 different triazines in grain samples, and our results showed no significant differences between different grain matrices. Moreover, the whole operation is convenient and simple, and can be completed within 15 min. Thus, the developed ICA can provide a rapid, low-cost, and efficient tool for the on-site determination of triazine herbicides in grain samples. +MRM: Trietazine2 +MRM: Terbumeton1− (226.0/170.0) Wheat, +MRM: Terbumeton1− (226.0/170.0) Sorghum, +MRM: Terbumeton1− Herbicide micropollutants in surface, ground and drinking waters within and near the area of Zagreb A comparison of symmetrical and asymmetrical triazine herbicides for enhanced degradation in three midwestern soils Chronic toxicity of 1, 3, 5-triazine herbicides in the postembryonic development of the western clawed The 15 triazines are in order (1-15) as follows: ametryn, cyprazine, atraton, prometon, prometryn, atrazine, propazine, terbuthylazine, simetryn, trietazine, secbumeton, simazine, desmetryn, terbumeton and simetone. 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FITC as the labelling in lateral flow immunoassays for rapid detection of Ralstonia solanacearum This work was financially supported by the National Key R&D Program (No. 2019YFC1604703) and the Natural Science Foundation of Jiangsu Province (Nos. BK20200598, CMB21S1614, and CSE11N1310).