Red-emitted AIEgens-based lateral flow immunoassay for the visual and quantitative detection of aflatoxin M1 in milk

Red-emitted AIEgens-based lateral flow immunoassay for the visual and quantitative detection of aflatoxin M1 in milk | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Red-emitted AIEgens-based lateral flow immunoassay for the visual and quantitative detection of aflatoxin M1 in milk Wenbo Guo, Qingyi Liu, Junhua Yang, Haijuan Zeng, Juan Liu, Lemei Zhu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7174022/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fluorescent material-based lateral flow immunochromatographic assay (LFIA) has been widespread applied for the point-of-care test of mycotoxins. However, aggregation-caused quenching (ACQ) phenomenon of fluorescent labels seriously affects the detection performance of the methods. Herein, a red-emitted aggregation-induced emission luminogens (AIEgens) based LFIA was developed and firstly applied for the visual and quantitative detection of aflatoxin M1 (AFM1) in milk. The fluorescent probe was constructed by directly coupling the carboxylated AIEgens with anti -AFM1-mAb, which demonstrated a strong AFM1-specific affinity and exceptional fluorescent characteristics. Under the optimal conditions, this AIEgens-LFIA could achieve a rapid detection of AFM1 within 15 min, with a visual limit of detection (vLOD) of 0.1 ng/mL and a quantitative limit of detection (qLOD) of 0.01 ng/mL. The accuracy and precision of the established AIEgens-LFIA method were assessed though the spiked milk samples, and the recoveries were in the range of 86.81%-106.37% with coefficients of variation below 10%. Furthermore, the test strip exhibited a wide detection range for AFM1 (0.01-1 ng/mL) and a remarkable stability for 180 days when stored at 4°C and 25°C. Importantly, the results for real milk sample were consistent with those of standard UPLC-MS/MS method. Overall, the newly developed AIEgens-LFIA demonstrates significant potential for the point-of-care test of AFM1 in milk. aflatoxin M1 aggregation-induced emission lateral flow immunoassay red emission milk Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Aflatoxin M1 (AFM1), formed through hepatic hydroxylation of aflatoxin B1 (AFB1), is typically secreted into milk by dairy animals after ingestion of AFB1-contaminated animal feeds. It is well-documented that AFM1 is a potential chemical contaminant in dairy products, posing adversely impact human health because of its carcinogenicity, teratogenicity, mutagenic, and immunotoxicity (Alvarez et al., 2020 ; Shabeer, Asad, Jamal, & Ali, 2022 ; Jaćević et al., 2023 ). Consequently, the International Agency for Research on Cancer has been classified AFM1 as Group Ⅰ carcinogen (Ostry, Malir, Toman, & Grosse, 2017 ). Considering its high toxicity and widespread occurrence, the United State, several other Asian countries, and China have established the regulatory limit standard for AFM1 as 0.5 µg/kg in milk, while a considerably lower level (0.05 µg/kg for raw milk and 0.025 µg/kg for infant formulae) was set by the European Commission (European Commission (EC), 2006 ). Despite these regulations, AFM1 contamination in milk is still a potential food safety risk factor, especially in many African (Kuboka, Imungi, Njue, Mutua, Grace, & Lindahl, 2019 ), South America (Peña-Rodas, Martinez-Lopez, & Hernandez-Rauda, 2018 ), and South Asian countries (Khaneghahi Abyaneh, Bahonar, Noori, Yazdanpanah, & Shojaee Aliabadi, 2019 ; Xiong et al., 2020 ). Therefore, the effective and timely AFM1 detection technologies are essential to safeguard food safety. At present, several analytical methods have been established for AFM1 detection, mainly including thin-layer chromatography (TLC) (Filazi, Sinan, & Temamogullari, 2010 ), high performance liquid chromatography coupled with fluorescence detector (HPLC-FLD) (Kolarič & Šimko, 2023 ), and liquid chromatography tandem mass spectrometry (LC-MS/MS) (Chen et al., 2022 ; Sun, Wu, Abdallah, Tan, Li, & Yang, 2023 ). Although sensitive, accurate, and reliable, these methods are only used in central laboratories due to the tedious sample pretreatment, large-scale instruments, and professional technicians. The enzyme-linked immunosorbent assay (ELISA) (Buzás et al., 2023 ; Kourti, Angelopoulou, Petrou, & Kakabakos, 2024 ) with the advantages of simple operation, high throughput, and low cost has also been developed for the detection of AFM1. However, the long incubation time and a high risk of false positives limit its applications. Compared with the abovementioned methods, the lateral flow immunochromatographic assay (LFIA) has been proved to be a mature on-site quick testing method with the outstanding properties of rapidity, simplicity, time-saving, and cost-effective, and has been applied for on-site detection of mycotoxins (Jiang et al., 2023 ; Sadeghi et al., 2024 ), pesticides (Kwon, Ruan, Yu, Lin, Du, & Van Wie, 2023 ), antibiotics (C. Liu et al., 2021 ), and pathogenic microorganism (Sohrabi et al., 2022 ). Up to now, numerous fluorescent signal tags, including colloidal gold nanoparticles (AuNPs) (Guliy & Dykman, 2024 ; Zeng et al., 2021 ), fluorescein isothiocyanate (FITC) (J. Wang et al., 2024 ), and quantum dots (QDs) (Fang, Xiong, Duan, Xiong, & Lai, 2022 ) have been used to label antibodies as the specific recognition elements and the tracer, which could convert the combined signal of antigen and antibody to visual signals to achieve qualitative and quantitative analysis. However, the chemical stability and repeatability of these luminescent materials are susceptible to environmental conditions, including pH and humidity, and the well-known aggregation-caused quenching (ACQ) could significantly affect the sensitivity and accuracy of the LFIA methods by limiting the signal intensity (Yuan et al., 2010 ; Zhang et al., 2020 ). As a new class of fluorescent nanomaterials, aggregation-induced emission luminogens (AIEgens) have been demonstrated success in larger Stokes shift, higher photostability, ultrahigh brightness and lower random blinking, which could not only effectively overcome the ACQ effect but also significantly improve the sensitivity and robustness of LFIA (Asad et al., 2022 ; Cai & Liu, 2020 ; Zheng et al., 2023 ). Herein, the red-emitted AIEgens were introduced as the fluorescence label of a sandwich LFIA for the visual and quantitative detection of AFM1 in milk for the first time. As shown in Scheme 1 , the red-emitted AIEgens were encapsulated in carboxylated polystyrene microspheres and then conjugated with anti -AFM1 monoclonal antibodies (mAbs) to prepare the AIEgens-mAb probes. Subsequently, the AIEgens-mAb probes, AFM1-BSA, and goat anti-mouse IgG were immobilized on the conjugate pad, control zone, and test zone, serving as the fluorescence probes, test line (T), and control line (C), respectively. The developed AIEgens-LFIA simultaneously achieved visual and quantitative detection of AFM1, offering a promising tool for on-site, rapid, accurate, and large-scale detection of AFM1 in milk. 2. Materials and methods 2.1 Chemical and Reagents The reference materials of AFM1 (0.502 µg/mL) and T-2 (100.0 µg/mL) were purchased from Romer labs (Tulln, Austria). The certified reference materials of DON (GBW(E)100464), ZEN (GBW(E)100465), FB1 (GBW(E)100551), FB2 (GBW(E)100909), and OTA (GBW(E)10310008) were got from Shanghai Academy of Agricultural Sciences (Shanghai, China). AIEgens were purchased from XFNANO (Nanjing, China). The goat anti-mouse IgG and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (MI, USA). The anti-AFM1 monoclonal antibody ( anti -AFM1-mAb) and AFM1-BSA were purchased from Landu (Binzhou, China). The 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) were sourced from Sangon (Shanghai, China). The nitrocellulose (NC) membrane, conjugate pad, sample pad, and absorbent pad were all from Shanghai Sailaikai (Shanghai, China). All other chemicals were analytical grade and obtained from Aladdin (Shanghai, China). 2.2 Apparatus The XYZ 3050 dispensing platform equipped with guillotine cutter, dispenser, and motion controller were provided by BioDot (Irvine, USA). The morphology characterizations of AIEgens were performed on a Zeiss Sigma 300 scanning electron microscope (SEM) (Oberkochen, Germany). The 8WBLB UV light table lamp (365 nm) was provided by Kelishi medical Equipment Co., Ltd. (Shenzhen, China). The high-speed freezing centrifuge (5430 R) was from Eppendorf (Hamburg, Germany). UPLC-MS/MS analysis was achieved using a Waters ACQUITY UPLC system coupled with a Waters T-QS mass spectrometer (Milford, USA). 2.3 Preparation of the AIEgens-mAb probes The AIEgens-mAb probes were prepared via the formation of the amide bond between the carboxyl group of AIEgens and the amino group of anti -AFM1-mAb as shown in Scheme. 1A. In brief, 100 µL carboxylated AIEgens (5 µM) and 100 µL MES (100 mM, pH 6) were stirred evenly in a brown bottle. Then, 10 µL NHS (0.1 M) and 4 µL EDC (0.1 M) were introduced into the mixed solution. After activation for 15 min, the mixture was supplemented with 100 µL of anti-AFM1-mAb (2 mg/mL), followed by 2 h of incubation at room temperature to form the AIEgens-mAb conjugates. Subsequently, the non-specific binding sites were blocked by adding 27 µL of 25% BSA (w/v). Following incubation for 45 min, the AIEgens-mAb probes were obtained by dialyzing the mixture in PBS solution (0.01 M, pH 7.4), and stored in darkness at 4°C. 2.4 Fabrication of the AIEgens-LFIA strip AIEgens-LFIA strip is mainly composed by nitrocellulose (NC) membrane, sample pad, conjugate pad, backing card, and absorbent pad (Scheme. 1B). The goat anti-mouse IgG (1 mg/mL) and AFM1-BSA (1.5 mg/mL) antibody were spotted onto the NC membrane (2.5×30 cm) at 1 µL/cm to form T line and C line, respectively. The AIEgens-mAb (0.5 mg/mL) probes were deposited at 4 µL/cm on the conjugate pad. Finally, the NC membrane, absorbent pad, sample pad, and conjugate pad were aligned on the PVC backing card (8×30 cm) with a 1.5 ± 0.3 mm overlap, and then cut into 3 mm-wide sections. The prepared AIEgens-LFIA was stored at 4°C with desiccant in a zip-lock bag. 2.5 Detection procedure of AIEgens-LFIA for AFM1 The principle of the AIEgens-LFIA is depicted in Scheme 1 . An 80 µL sample solution was applied dropwise onto the sample pad of the AIEgens-LFIA strip. Along with the migration of positive sample, AFM1 was captured by the AIEgens-mAb in the conjugate pad. The immunocomplex was then captured by the goat anti-mouse IgG coated in the C line, presenting a positive result with a clear purple strip under the excitation of 365 nm. It is worth noting that the T line now does not display fluorescent strip due to the principle of competitive reaction (Scheme. 1C). Correspondingly, the AIEgens-mAb would be directly captured by the AFM1-BSA in T line when AFM1 was absent in the sample solution. And then, two red strips would be visualized by the naked eye and the sample was judged as negative as shown in Scheme. 1D. For quantitative detection, the grayscale value of the T and C lines were scanned and calculated by ImageJ software, representing the fluorescence intensity (FI) at T line (FI T ) and C line (FI C ). The concentration of AFM1 could be accurately quantified based on the competitive inhibition curve, which was built by plotting FI T /FI C against the logarithm of AFM1 concentration. 2.6 Sample preparation for AIEgens-LFIA and UPLC-MS/MS All pasteurized milk samples were randomly collected from supermarkets in Shanghai. Sample pretreatment was conducted as follows: 5.0 mL of milk sample was accurately weighed and centrifuged at 12000 rpm for 5 min to remove the upper fat layer. Subsequently, 20 µL of trifluoroacetic acid was added and centrifuged for protein precipitation. The supernatant was neutralized to pH 7.0 with the solution of NaOH (1 mol/L) and finally applied for AIEgens-LFIA detection. For validation and comparison, the pretreatment of milk samples was conducted according to the National Standard GB/T 5009.24–2016), and the UPLC-MS/MS conditions were presented in Supporting Information. 2.7 Validation of the AIEgens-LFIA To evaluate the performance of the developed AIEgens-LFIA, the key parameters including sensitivity, specificity, accuracy, precision, and stability were seriatim validated. For the sensitivity, blank milk sample was processed according to the aforementioned pre-treatment method and the solutions with different concentrations of AFM1 were prepared by diluting the standard solution using the blank matrix. The visual limit of detection (vLOD) could be observed by naked eyes within 15 min. Meanwhile, the ratio values of FI T /FI C were obtained by Image J software. The quantitative calibration curve was established by plotting the FI T /FI C ratios against the AFM1 concentrations. The quantitative limit of detection (qLOD) was defined as the concentration of AFM1 that induce a 10% decrease of the FI T /FI C value compared with that induced by PBS buffer (Jia, Liao, Sun, Fang, Zhou, & Kong, 2021 ). For the specificity, AFM1 (0.5 ng/mL), and other six frequent-occurring mycotoxins, including DON, OTA, ZEN, T-2, FB1, and FB2, were employed as the competitors with a concentration of 5 ng/mL. For the accuracy and precision (intra-assay and inter-assay), blank milk samples were fortified with AFM1 at various concentrations, and the mean value, standard deviation (SD) and coefficient of variation (CV) were then calculated. The stability of AIEgens-LFIA stored at 4°C and 25°C was evaluated by testing the spiked samples (0.5 ng/mL) at different points in time. 3. Results and discussions 3.1 Characterization of the AIEgens As illustrated in SEM images, the AIEgens nanospheres were well-dispersed spherical particles with smooth surface and an average diameter of approximately 170 nm (Fig. 1 A). The fluorescent property of the AIEgens was confirmed by fluorescence spectroscopy. As shown in Fig. 1 B, the AIEgens exhibited typical excitation and emission peaks at 510 nm and 625 nm. The relatively large Stokes shift effectively minimized interference between excitation and emission signals (Hu et al., 2021 ). In addition, polystyrene-coated AIE molecules were isolated from the external environment, which greatly improved the fluorescence stability and durability. 3.2. Optimization of Key Parameters of AIEgens-LFIA 3.2.1 Optimization of the pH of reaction buffer The pH value of reaction buffer (0.1 mol/L MES) significantly influenced the coupling efficiency between amino groups on anti -AFM1-mAb and carboxyl groups on AIEgens, which could future affect the detection sensitivity of the AIEgens-LFIA. To explore the effect of pH on performance of the fluorescent probes, various reaction buffers with pH values ranging from 5 to 7 were successively evaluated. As depicted in Fig. 2 A, the FI C , FI T and FI T /FI C values of the negative sample progressively increased as the pH values ranging from 5 to 6, and finally achieved its peak at pH 6, indicating the optimal signal intensity. As the pH continued to increase, the FI sharply declined, possibly due to protein denaturation under strong alkaline conditions (Yang et al., 2023 ). Considering the requirements for fluorescence intensity and stability, a MES solution (0.1 M) at pH 6 was chosen as the optimal pH for the reaction buffer for coupling. 3.2.2 Optimization of the amount of anti -AFM1-mAb The sensitivity of AIEgens-LFIA could be affected by the amount of anti -AFM1-mAb. Insufficient or excessive antibodies could gravely affect the detection sensitivity of AIEgens-LFIA. In this experiment, 1, 1.5, 2, 2.5, and 3 mg/mL of anti -AFM1-mAb were investigated. As shown in Fig. 2 B, when the amounts of anti -AFM1-mAb were below 2 mg/mL, numerous AIEgens were unlabeled, resulting in a lower FI in the negative sample. As the amount of anti -AFM1-mAb increased, the amount of labeled AIEgens also increased. The values of FI C , FI T and FI T /FI C reached a platform at the dosage of 2.5 mg/mL and then decreased when the anti -AFM1-mAb dosage was increased to 3 mg/mL. This result indicated that excessive anti -AFM1-mAb could decrease fluorescence efficiency of antibodies due to the compression of antibody spatial structure or spatial resistance. Thus, 2 mg/mL of anti -AFM1-mAb was selected. 3.2.3 Optimization of the amount of the AIEgens-mAb probe The amount of AIEgens-mAb could directly affect the performance of AIEgens-LFIA due to the critical role of recognition and indicator. To optimize the probe dosage, various volumes of 2, 3, 4, 5 µL of AIEgens-mAb (0.5 mg/mL) were investigated. As shown in Fig. 2 C, the values of FI C and FI T for negative sample gradually increased with the increasing volume of AIEgens-mAb until it reached its highest point at 5 µL. When the volume of AIEgens-mAb was 2 µL, the probes demonstrated optimal binding affinity to the antigen immobilized on the T line, resulting in the highest FI T /FI C ratio. However, as the volume of AIEgens-mAb continued to increase from 3 µL to 5 µL, the FI T /FI C ratio gradually decreased and entered a stable state. It was noteworthy that the background fluorescence on the NC membrane significantly increased when the volume of AIEgens-mAb was increased to 5 µL, which could interfere with the FI of T line and C line. Fig S1 also demonstrated that as the volume of AIEgens-LFIA decreased, the FI of the T and C lines gradually strengthened, with two clear red bands appearing. Consequently, 4 µL of AIEgens-mAb was selected as the optimal probe dosage for establishing AIEgens-LFIA. 3.3 Detection performance of the established AIEgens-LFIA 3.3.1 Sensitivity and specificity The sensitivity is a crucial performance parameter of the developed AIEgens-LFIA method. In this study, spiked milk samples were prepared with a series concentration (0.01-1 ng/mL) of AFM1 and assayed under optimal conditions. As shown in inset of Fig. 3 A, the FI of the T line gradually strengthened as the AFM1 concentration decreased. The detection concentration of 0.1 ng/mL was determined as the vLOD, defined as the minimum concentration at which the AIEgens-LFIA test line exhibits a significantly shallower color intensity compared to the negative control. For quantitative detection, the grayscale values of the T line and C line were scanned by Image J software, and the FI T /FI C value was calculated. The calibration curve was fitted by plotting FI T /FI C values against the logarithm of AFM1 concentration from 0.01 to 1 ng/mL. As presented in Fig. 3 A, the regression equation with a good correlation coefficient ( R 2 = 0.9928) was expressed as y = 0.4315 + 2.3123/[1+( x /0.0085) 0.7426 ], where y represents the FI T /FI C value and x is the AFM1 concentration. Based on this calibration curve, the qLOD was determined to be 0.01 ng/mL, which is 10-fold lower than the vLOD. To evaluate the specificity of the established AIEgens-LFIA, both AFM1 and six commonly-found mycotoxins (ZEN, DON, OTA, FB1, FB2, and T-2) were simultaneously tested. As displayed in Fig. S2, the presence of AFM1 could induce significant fluorescent signal decreases to almost invisible, whereas ZEN, DON, OTA, FB1, FB2, and T-2 exhibited negligible changes in fluorescence signal response compared to PBS. In addition, Fig. 3 B showed that the FI T /FI C values of AFM1 was significantly lower than those of other mycotoxins, indicating excellent specificity and selectivity. These findings confirmed that the AIEgens-LFIA platform achieved both exceptional specificity and anti-interference ability. 3.3.2 Accuracy and precision The accuracy and precision of the AIEgens-LFIA were assessed using the blank milk samples spiked with AFM1 at low (0.1 µg/kg), medium (0.5 µg/kg), and high (1 µg/kg) concentration. Intra-assay and inter-assay were carried out on the same day and over three successive days with triplicate of each spiked concentration (n = 5). As presented in Table 1 , the average recoveries for both intra- and inter-assay ranged from 86.81–106.37% with the CV from 7.06–14.71%. These results confirmed that the proposed AIEgens-LFIA provides acceptable accuracy and reproducibility for AFM1 detection in milk. Table 1 Accuracy and precision of the established AIEgens-LFIA for detection of AFM1 in milk. Spiked levels (ng/mL) Intra-Assay Inter-Assay Mean (ng/mL) Recovery ± SD (%) CV (%) Mean (ng/mL) Recovery ± SD (%) CV (%) 0.1 0.087 86.81 ± 8.53 9.92 0.101 100.73 ± 14.82 14.71 0.5 0.475 94.93 ± 10.58 11.15 0.496 99.13 ± 10.30 10.39 1 1.064 106.37 ± 7.51 7.06 1. 012 101.17 ± 9.06 8.96 3.3.3 Methods comparison To facilitate a thorough understanding of the advantages and disadvantages of the established detection platform, a comprehensive table was presented to compare it with previously reported rapid detection methods for AFM1 analysis (Table 2 ). The sensitivity of the proposed method matches or surpasses that of previously reported technologies, thus showing superior application prospects for rapid detection of AFM1 in milk. Table 2 Comparison of the developed AIEgens-LFIA with previous reported methods for AFM1 detection. Label LOD (ng/mL) Linear range (ng/mL) Reference AuNPs 0.5 - (Pimpitak, Rengpipat, Phutong, Buakeaw, & Komolpis, 2020 ) AuNPs 0.1 - (B.-H. Liu, Chu, & Yu, 2016 ) AuNPs 1.0 - (J.-J. Wang, Liu, Hsu, & Yu, 2011 ) TRFM 0.019 0.05-2 (M. Li et al., 2021 ) 0.0423 0.05–0.6 (Wu et al., 2017 ) QDs 0.0932 0.1-1 Silver nanoparticles (AgNPs) 0.21 0.2–1.4 (J. Li et al., 2021 ; Z. Li, Li, Jiang, & Xu, 2017 ) Eu-modified fluorescent microspheres 0.009 0.02–0.4 (G. Li et al., 2018 ) (PEI/PSS) 4 red SiNPs 0.01 - (Su, Zhao, & Dou, 2020 ) AIEgens 0.01 0.01-1 This study 3.3.4 Stability The stability of AIEgens-LFIA stored at 4°C and 25°C was evaluated. A stable fluorescence signal was observed and the values of FI T /FI C basically remained unchanged with the CV of 8.12% for 4°C and 10.67% for 25°C (Table S1 ). These results demonstrated that the developed AIEgens-LFIA could remain stable for at least 180 days at 4°C and 25°C, and fully meet the requirements of storage and transport in the practical use. 3.3.5 Analysis of actual samples by AIEgens-FICS and UPLC-MS/MS For further verify the reliability, milk samples spiked with different concentrations of AFM1 (0.1, 0.5, and 1.0 ng/mL) were simultaneously determined by AIEgens-LFIA and UPLC-MS/MS. The recoveries of AIEgens-LFIA ranged from 97.53–104.13%, while those obtained from UPLC-MS/MS were in the range of 96.47%-107.37% (Table S2). Moreover, high correlation coefficients ( R 2 > 0.99) were obtained for both methods (Fig. 4 ), proving the accuracy and reliability of the established AIEgens-LFIA for AFM1 detection in real milk samples 4. Conclusions In this study, a novel fluorescent test strip with red-emitted AIEgens as signal reporters was constructed for the rapid detection of AFM1 in milk. Benefiting from the excellent fluorescence properties, the designed AIEgens-LFIA not only enabled a rapid visual recognition but also achieved an accurately quantitative detection of AFM1. Under the optimal conditions, the established AIEgens-LFIA exhibited high selectivity and sensitivity, satisfactory linearity and accuracy, good reproducibility, and stability during AFM1 detection. With the various merits, the proposed AIEgens-LFIA platform could be a suitable alternative for the point-of-care testing and high-throughput screening of AFM1 in milk in food safety supervision, risk assessments, and the early-stage diagnosis of some diseases in future applications. Declarations Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding Declaration The research was supported by the Shanghai Rising-Star Program (Grant No. 23QB1403500) and the National Key R&D Program of China (Grant No. 2024YFF1105802). Author Contribution Wenbo Guo: Conceptualization Ideas, Methodology, Writing-Original draft. Qiyi Liu: Formal analysis. Junhua Yang: Investigation, Data curation. Haijuan Zeng: Methodology, Writing-review & editing, Supervision, Funding. Juan Liu: Visualization. Lemei Zhu: Writing-review & editing. Dongxia Nie: Validation. Zhihui Zhao: Writing-review & editing. Zheng Han: Writing-review & editing, Funding, All authors reviewed the manuscript. References Alvarez CS, Hernández E, Escobar K, Villagrán CI, Kroker-Lobos MF, Rivera-Andrade A, Smith JW, Egner PA, Lazo M, Freedman ND (2020) Aflatoxin B1 exposure and liver cirrhosis in Guatemala: A case–control study. 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Toxins 14(5):307. 10.3390/toxins14050307 Sohrabi H, Majidi MR, Fakhraei M, Jahanban-Esfahlan A, Hejazi M, Oroojalian F, Baradaran B, Tohidast M, de la Guardia M, Mokhtarzadeh A (2022) Lateral flow assays (LFA) for detection of pathogenic bacteria: A small point-of-care platform for diagnosis of human infectious diseases. Talanta 243:123330. 10.1016/j.talanta.2022.123330 Su Z, Zhao G, Dou W (2020) Determination of trace aflatoxin M1 (AFM1) residue in milk by an immunochromatographic assay based on (PEI/PSS) 4 red silica nanoparticles. Microchim Acta 187(12):658. 10.1007/s00604-020-04636-6 Sun F, Wu P, Abdallah MF, Tan H, Li Y, Yang S (2023) One sample multi-point calibration curve as a novel approach for quantitative LC-MS analysis: The quantitation of six aflatoxins in milk and oat-based milk as an example. Food Chem 420:135593. 10.1016/j.foodchem.2023.135593 Wang J-J, Liu B-H, Hsu Y-T, Yu F-Y (2011) Sensitive competitive direct enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin M1 in milk. Food Control 22(6):964–969. 10.1016/j.foodcont.2010.12.003 Wang J, Wang Y, Hu X, Chen Y, Jiang W, Liu X, Liu J, Zhu L, Zeng H, Liu H (2024) A dual-RPA based lateral flow strip for sensitive, on-site detection of CP4-EPSPS and Cry1Ab/Ac genes in genetically modified crops. Food Sci Hum Wellness 13(1):183–190. 10.26599/FSHW.2022.9250015 Wu C, Hu L, Xia J, Xu G, Luo K, Liu D, Duan H, Cheng S, Xiong Y, Lai W (2017) Comparison of immunochromatographic assays based on fluorescent microsphere and quantum-dot submicrobead for quantitative detection of aflatoxin M1 in milk. J Dairy Sci 100(4):2501–2511. 10.3168/jds.2016-12065 Xiong J, Peng L, Zhou H, Lin B, Yan P, Wu W, Liu Y, Wu L, Qiu Y (2020) Prevalence of aflatoxin M1 in raw milk and three types of liquid milk products in central-south China. Food Control 108:106840. 10.1016/j.foodcont.2019.106840 Yang X, Cheng X, Wei H, Tu Z, Rong Z, Wang C, Wang S (2023) Fluorescence-enhanced dual signal lateral flow immunoassay for flexible and ultrasensitive detection of monkeypox virus. J Nanobiotechnol 21(1):450. 10.1186/s12951-023-02215-4 Yuan WZ, Lu P, Chen S, Lam JW, Wang Z, Liu Y, Kwok HS, Ma Y, Tang BZ (2010) Changing the behavior of chromophores from aggregation-caused quenching to aggregation‐induced emission: development of highly efficient light emitters in the solid state. Adv Mater 22(19):2159–2163. 10.1002/adma.200904056 Zeng H, Wang J, Jia J, Wu G, Yang Q, Liu X, Tang X (2021) Development of a lateral flow test strip for simultaneous detection of BT-Cry1Ab, BT-Cry1Ac and CP4 EPSPS proteins in genetically modified crops. Food Chem 335:127627. 10.1016/j.foodchem.2020.127627 Zhang Y, Zhao Y, Han Z, Zhang R, Du P, Wu Y, Lu X (2020) Switching the photoluminescence and electrochemiluminescence of liposoluble porphyrin in aqueous phase by molecular regulation. Angew Chem Int Ed 59(51):23261–23267. 10.1002/anie.202010216 Zheng Z, Yang T, Li D, Cao H, Gong J, Liu H, Zhou C, Liu L, Wei P, Gu X (2023) Molecular and aggregate synergistic engineering of aggregation-induced emission luminogens to manipulate optical/electronic properties for efficient and diversified functions. ACS Nano 17(9):8782–8795. 10.1021/acsnano.3c02134 Additional Declarations No competing interests reported. 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(A) pH value of the reaction buffers. (B) Various amounts of \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb. (C) Various volumes of AIEgens-mAb.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7174022/v1/6bd2f91fca931e5ca7d7317b.jpg"},{"id":88125402,"identity":"e4e6e6e3-d83f-46bd-8c34-9bce36acdd9c","added_by":"auto","created_at":"2025-08-01 16:53:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94888,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The logistic calibration curve for quantitative detection of AFM1 and (B) the specificity of the AIEgens-LFIA to other mycotoxins.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7174022/v1/0935d6919bb3221ce92ac2f9.jpg"},{"id":88125401,"identity":"96051282-07ab-449f-9455-be89b682e1ff","added_by":"auto","created_at":"2025-08-01 16:53:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74998,"visible":true,"origin":"","legend":"\u003cp\u003eDetection results of AIEgens-LFIA and UPLC-MS/MS for AFM1 in milk samples spiked with low (0.1 ng/mL), medium (0.5 ng/mL), and high (1 ng/mL) levels.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7174022/v1/35c0137b360821db629872e5.jpg"},{"id":88263177,"identity":"b61042ea-fd34-41ab-bab3-3864a60f9c3e","added_by":"auto","created_at":"2025-08-04 15:47:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1279036,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7174022/v1/20f24ee6-d35f-44ca-8dff-cb1ccfad4987.pdf"},{"id":88124868,"identity":"7fede6f9-0a80-4003-b51c-297ee003a3a7","added_by":"auto","created_at":"2025-08-01 16:45:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1586708,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7174022/v1/3387cbd7f29696cef6b17305.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Red-emitted AIEgens-based lateral flow immunoassay for the visual and quantitative detection of aflatoxin M1 in milk","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAflatoxin M1 (AFM1), formed through hepatic hydroxylation of aflatoxin B1 (AFB1), is typically secreted into milk by dairy animals after ingestion of AFB1-contaminated animal feeds. It is well-documented that AFM1 is a potential chemical contaminant in dairy products, posing adversely impact human health because of its carcinogenicity, teratogenicity, mutagenic, and immunotoxicity (Alvarez et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shabeer, Asad, Jamal, \u0026amp; Ali, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jaćević et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consequently, the International Agency for Research on Cancer has been classified AFM1 as Group Ⅰ carcinogen (Ostry, Malir, Toman, \u0026amp; Grosse, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Considering its high toxicity and widespread occurrence, the United State, several other Asian countries, and China have established the regulatory limit standard for AFM1 as 0.5 \u0026micro;g/kg in milk, while a considerably lower level (0.05 \u0026micro;g/kg for raw milk and 0.025 \u0026micro;g/kg for infant formulae) was set by the European Commission (European Commission (EC), \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Despite these regulations, AFM1 contamination in milk is still a potential food safety risk factor, especially in many African (Kuboka, Imungi, Njue, Mutua, Grace, \u0026amp; Lindahl, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), South America (Pe\u0026ntilde;a-Rodas, Martinez-Lopez, \u0026amp; Hernandez-Rauda, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and South Asian countries (Khaneghahi Abyaneh, Bahonar, Noori, Yazdanpanah, \u0026amp; Shojaee Aliabadi, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, the effective and timely AFM1 detection technologies are essential to safeguard food safety.\u003c/p\u003e\u003cp\u003eAt present, several analytical methods have been established for AFM1 detection, mainly including thin-layer chromatography (TLC) (Filazi, Sinan, \u0026amp; Temamogullari, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), high performance liquid chromatography coupled with fluorescence detector (HPLC-FLD) (Kolarič \u0026amp; Šimko, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and liquid chromatography tandem mass spectrometry (LC-MS/MS) (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sun, Wu, Abdallah, Tan, Li, \u0026amp; Yang, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although sensitive, accurate, and reliable, these methods are only used in central laboratories due to the tedious sample pretreatment, large-scale instruments, and professional technicians. The enzyme-linked immunosorbent assay (ELISA) (Buz\u0026aacute;s et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kourti, Angelopoulou, Petrou, \u0026amp; Kakabakos, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) with the advantages of simple operation, high throughput, and low cost has also been developed for the detection of AFM1. However, the long incubation time and a high risk of false positives limit its applications. Compared with the abovementioned methods, the lateral flow immunochromatographic assay (LFIA) has been proved to be a mature on-site quick testing method with the outstanding properties of rapidity, simplicity, time-saving, and cost-effective, and has been applied for on-site detection of mycotoxins (Jiang et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sadeghi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), pesticides (Kwon, Ruan, Yu, Lin, Du, \u0026amp; Van Wie, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), antibiotics (C. Liu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and pathogenic microorganism (Sohrabi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUp to now, numerous fluorescent signal tags, including colloidal gold nanoparticles (AuNPs) (Guliy \u0026amp; Dykman, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zeng et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), fluorescein isothiocyanate (FITC) (J. Wang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and quantum dots (QDs) (Fang, Xiong, Duan, Xiong, \u0026amp; Lai, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have been used to label antibodies as the specific recognition elements and the tracer, which could convert the combined signal of antigen and antibody to visual signals to achieve qualitative and quantitative analysis. However, the chemical stability and repeatability of these luminescent materials are susceptible to environmental conditions, including pH and humidity, and the well-known aggregation-caused quenching (ACQ) could significantly affect the sensitivity and accuracy of the LFIA methods by limiting the signal intensity (Yuan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a new class of fluorescent nanomaterials, aggregation-induced emission luminogens (AIEgens) have been demonstrated success in larger Stokes shift, higher photostability, ultrahigh brightness and lower random blinking, which could not only effectively overcome the ACQ effect but also significantly improve the sensitivity and robustness of LFIA (Asad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cai \u0026amp; Liu, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHerein, the red-emitted AIEgens were introduced as the fluorescence label of a sandwich LFIA for the visual and quantitative detection of AFM1 in milk for the first time. As shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the red-emitted AIEgens were encapsulated in carboxylated polystyrene microspheres and then conjugated with \u003cem\u003eanti\u003c/em\u003e-AFM1 monoclonal antibodies (mAbs) to prepare the AIEgens-mAb probes. Subsequently, the AIEgens-mAb probes, AFM1-BSA, and goat anti-mouse IgG were immobilized on the conjugate pad, control zone, and test zone, serving as the fluorescence probes, test line (T), and control line (C), respectively. The developed AIEgens-LFIA simultaneously achieved visual and quantitative detection of AFM1, offering a promising tool for on-site, rapid, accurate, and large-scale detection of AFM1 in milk.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemical and Reagents\u003c/h2\u003e\u003cp\u003eThe reference materials of AFM1 (0.502 \u0026micro;g/mL) and T-2 (100.0 \u0026micro;g/mL) were purchased from Romer labs (Tulln, Austria). The certified reference materials of DON (GBW(E)100464), ZEN (GBW(E)100465), FB1 (GBW(E)100551), FB2 (GBW(E)100909), and OTA (GBW(E)10310008) were got from Shanghai Academy of Agricultural Sciences (Shanghai, China). AIEgens were purchased from XFNANO (Nanjing, China). The goat anti-mouse IgG and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (MI, USA). The anti-AFM1 monoclonal antibody (\u003cem\u003eanti\u003c/em\u003e-AFM1-mAb) and AFM1-BSA were purchased from Landu (Binzhou, China). The 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) were sourced from Sangon (Shanghai, China). The nitrocellulose (NC) membrane, conjugate pad, sample pad, and absorbent pad were all from Shanghai Sailaikai (Shanghai, China). All other chemicals were analytical grade and obtained from Aladdin (Shanghai, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Apparatus\u003c/h2\u003e\u003cp\u003eThe XYZ 3050 dispensing platform equipped with guillotine cutter, dispenser, and motion controller were provided by BioDot (Irvine, USA). The morphology characterizations of AIEgens were performed on a Zeiss Sigma 300 scanning electron microscope (SEM) (Oberkochen, Germany). The 8WBLB UV light table lamp (365 nm) was provided by Kelishi medical Equipment Co., Ltd. (Shenzhen, China). The high-speed freezing centrifuge (5430 R) was from Eppendorf (Hamburg, Germany). UPLC-MS/MS analysis was achieved using a Waters ACQUITY UPLC system coupled with a Waters T-QS mass spectrometer (Milford, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Preparation of the AIEgens-mAb probes\u003c/h2\u003e\u003cp\u003eThe AIEgens-mAb probes were prepared via the formation of the amide bond between the carboxyl group of AIEgens and the amino group of \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb as shown in Scheme. 1A. In brief, 100 \u0026micro;L carboxylated AIEgens (5 \u0026micro;M) and 100 \u0026micro;L MES (100 mM, pH 6) were stirred evenly in a brown bottle. Then, 10 \u0026micro;L NHS (0.1 M) and 4 \u0026micro;L EDC (0.1 M) were introduced into the mixed solution. After activation for 15 min, the mixture was supplemented with 100 \u0026micro;L of anti-AFM1-mAb (2 mg/mL), followed by 2 h of incubation at room temperature to form the AIEgens-mAb conjugates. Subsequently, the non-specific binding sites were blocked by adding 27 \u0026micro;L of 25% BSA (w/v). Following incubation for 45 min, the AIEgens-mAb probes were obtained by dialyzing the mixture in PBS solution (0.01 M, pH 7.4), and stored in darkness at 4\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Fabrication of the AIEgens-LFIA strip\u003c/h2\u003e\u003cp\u003eAIEgens-LFIA strip is mainly composed by nitrocellulose (NC) membrane, sample pad, conjugate pad, backing card, and absorbent pad (Scheme. 1B). The goat anti-mouse IgG (1 mg/mL) and AFM1-BSA (1.5 mg/mL) antibody were spotted onto the NC membrane (2.5\u0026times;30 cm) at 1 \u0026micro;L/cm to form T line and C line, respectively. The AIEgens-mAb (0.5 mg/mL) probes were deposited at 4 \u0026micro;L/cm on the conjugate pad. Finally, the NC membrane, absorbent pad, sample pad, and conjugate pad were aligned on the PVC backing card (8\u0026times;30 cm) with a 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mm overlap, and then cut into 3 mm-wide sections. The prepared AIEgens-LFIA was stored at 4\u0026deg;C with desiccant in a zip-lock bag.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Detection procedure of AIEgens-LFIA for AFM1\u003c/h2\u003e\u003cp\u003eThe principle of the AIEgens-LFIA is depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. An 80 \u0026micro;L sample solution was applied dropwise onto the sample pad of the AIEgens-LFIA strip. Along with the migration of positive sample, AFM1 was captured by the AIEgens-mAb in the conjugate pad. The immunocomplex was then captured by the goat anti-mouse IgG coated in the C line, presenting a positive result with a clear purple strip under the excitation of 365 nm. It is worth noting that the T line now does not display fluorescent strip due to the principle of competitive reaction (Scheme. 1C). Correspondingly, the AIEgens-mAb would be directly captured by the AFM1-BSA in T line when AFM1 was absent in the sample solution. And then, two red strips would be visualized by the naked eye and the sample was judged as negative as shown in Scheme. 1D. For quantitative detection, the grayscale value of the T and C lines were scanned and calculated by ImageJ software, representing the fluorescence intensity (FI) at T line (FI\u003csub\u003eT\u003c/sub\u003e) and C line (FI\u003csub\u003eC\u003c/sub\u003e). The concentration of AFM1 could be accurately quantified based on the competitive inhibition curve, which was built by plotting FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e against the logarithm of AFM1 concentration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Sample preparation for AIEgens-LFIA and UPLC-MS/MS\u003c/h2\u003e\u003cp\u003eAll pasteurized milk samples were randomly collected from supermarkets in Shanghai. Sample pretreatment was conducted as follows: 5.0 mL of milk sample was accurately weighed and centrifuged at 12000 rpm for 5 min to remove the upper fat layer. Subsequently, 20 \u0026micro;L of trifluoroacetic acid was added and centrifuged for protein precipitation. The supernatant was neutralized to pH 7.0 with the solution of NaOH (1 mol/L) and finally applied for AIEgens-LFIA detection. For validation and comparison, the pretreatment of milk samples was conducted according to the National Standard GB/T 5009.24\u0026ndash;2016), and the UPLC-MS/MS conditions were presented in Supporting Information.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Validation of the AIEgens-LFIA\u003c/h2\u003e\u003cp\u003eTo evaluate the performance of the developed AIEgens-LFIA, the key parameters including sensitivity, specificity, accuracy, precision, and stability were seriatim validated. For the sensitivity, blank milk sample was processed according to the aforementioned pre-treatment method and the solutions with different concentrations of AFM1 were prepared by diluting the standard solution using the blank matrix. The visual limit of detection (vLOD) could be observed by naked eyes within 15 min. Meanwhile, the ratio values of FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e were obtained by Image J software. The quantitative calibration curve was established by plotting the FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e ratios against the AFM1 concentrations. The quantitative limit of detection (qLOD) was defined as the concentration of AFM1 that induce a 10% decrease of the FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e value compared with that induced by PBS buffer (Jia, Liao, Sun, Fang, Zhou, \u0026amp; Kong, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor the specificity, AFM1 (0.5 ng/mL), and other six frequent-occurring mycotoxins, including DON, OTA, ZEN, T-2, FB1, and FB2, were employed as the competitors with a concentration of 5 ng/mL. For the accuracy and precision (intra-assay and inter-assay), blank milk samples were fortified with AFM1 at various concentrations, and the mean value, standard deviation (SD) and coefficient of variation (CV) were then calculated. The stability of AIEgens-LFIA stored at 4\u0026deg;C and 25\u0026deg;C was evaluated by testing the spiked samples (0.5 ng/mL) at different points in time.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussions","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Characterization of the AIEgens\u003c/h2\u003e\u003cp\u003eAs illustrated in SEM images, the AIEgens nanospheres were well-dispersed spherical particles with smooth surface and an average diameter of approximately 170 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The fluorescent property of the AIEgens was confirmed by fluorescence spectroscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the AIEgens exhibited typical excitation and emission peaks at 510 nm and 625 nm. The relatively large Stokes shift effectively minimized interference between excitation and emission signals (Hu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, polystyrene-coated AIE molecules were isolated from the external environment, which greatly improved the fluorescence stability and durability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Optimization of Key Parameters of AIEgens-LFIA\u003c/h2\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Optimization of the pH of reaction buffer\u003c/h2\u003e\u003cp\u003eThe pH value of reaction buffer (0.1 mol/L MES) significantly influenced the coupling efficiency between amino groups on \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb and carboxyl groups on AIEgens, which could future affect the detection sensitivity of the AIEgens-LFIA. To explore the effect of pH on performance of the fluorescent probes, various reaction buffers with pH values ranging from 5 to 7 were successively evaluated. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the FI\u003csub\u003eC\u003c/sub\u003e, FI\u003csub\u003eT\u003c/sub\u003e and FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e values of the negative sample progressively increased as the pH values ranging from 5 to 6, and finally achieved its peak at pH 6, indicating the optimal signal intensity. As the pH continued to increase, the FI sharply declined, possibly due to protein denaturation under strong alkaline conditions (Yang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Considering the requirements for fluorescence intensity and stability, a MES solution (0.1 M) at pH 6 was chosen as the optimal pH for the reaction buffer for coupling.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 Optimization of the amount of \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb\u003c/h2\u003e\u003cp\u003eThe sensitivity of AIEgens-LFIA could be affected by the amount of \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb. Insufficient or excessive antibodies could gravely affect the detection sensitivity of AIEgens-LFIA. In this experiment, 1, 1.5, 2, 2.5, and 3 mg/mL of \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb were investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, when the amounts of \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb were below 2 mg/mL, numerous AIEgens were unlabeled, resulting in a lower FI in the negative sample. As the amount of \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb increased, the amount of labeled AIEgens also increased. The values of FI\u003csub\u003eC\u003c/sub\u003e, FI\u003csub\u003eT\u003c/sub\u003e and FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e reached a platform at the dosage of 2.5 mg/mL and then decreased when the \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb dosage was increased to 3 mg/mL. This result indicated that excessive \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb could decrease fluorescence efficiency of antibodies due to the compression of antibody spatial structure or spatial resistance. Thus, 2 mg/mL of \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb was selected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 Optimization of the amount of the AIEgens-mAb probe\u003c/h2\u003e\u003cp\u003eThe amount of AIEgens-mAb could directly affect the performance of AIEgens-LFIA due to the critical role of recognition and indicator. To optimize the probe dosage, various volumes of 2, 3, 4, 5 \u0026micro;L of AIEgens-mAb (0.5 mg/mL) were investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, the values of FI\u003csub\u003eC\u003c/sub\u003e and FI\u003csub\u003eT\u003c/sub\u003e for negative sample gradually increased with the increasing volume of AIEgens-mAb until it reached its highest point at 5 \u0026micro;L.\u003c/p\u003e\u003cp\u003eWhen the volume of AIEgens-mAb was 2 \u0026micro;L, the probes demonstrated optimal binding affinity to the antigen immobilized on the T line, resulting in the highest FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e ratio. However, as the volume of AIEgens-mAb continued to increase from 3 \u0026micro;L to 5 \u0026micro;L, the FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e ratio gradually decreased and entered a stable state. It was noteworthy that the background fluorescence on the NC membrane significantly increased when the volume of AIEgens-mAb was increased to 5 \u0026micro;L, which could interfere with the FI of T line and C line. Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e also demonstrated that as the volume of AIEgens-LFIA decreased, the FI of the T and C lines gradually strengthened, with two clear red bands appearing. Consequently, 4 \u0026micro;L of AIEgens-mAb was selected as the optimal probe dosage for establishing AIEgens-LFIA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Detection performance of the established AIEgens-LFIA\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1 Sensitivity and specificity\u003c/h2\u003e\u003cp\u003eThe sensitivity is a crucial performance parameter of the developed AIEgens-LFIA method. In this study, spiked milk samples were prepared with a series concentration (0.01-1 ng/mL) of AFM1 and assayed under optimal conditions. As shown in inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the FI of the T line gradually strengthened as the AFM1 concentration decreased. The detection concentration of 0.1 ng/mL was determined as the vLOD, defined as the minimum concentration at which the AIEgens-LFIA test line exhibits a significantly shallower color intensity compared to the negative control.\u003c/p\u003e\u003cp\u003eFor quantitative detection, the grayscale values of the T line and C line were scanned by Image J software, and the FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e value was calculated. The calibration curve was fitted by plotting FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e values against the logarithm of AFM1 concentration from 0.01 to 1 ng/mL. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the regression equation with a good correlation coefficient (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9928) was expressed as \u003cem\u003ey\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.4315\u0026thinsp;+\u0026thinsp;2.3123/[1+(\u003cem\u003ex\u003c/em\u003e/0.0085)\u003csup\u003e0.7426\u003c/sup\u003e], where y represents the FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e value and \u003cem\u003ex\u003c/em\u003e is the AFM1 concentration. Based on this calibration curve, the qLOD was determined to be 0.01 ng/mL, which is 10-fold lower than the vLOD.\u003c/p\u003e\u003cp\u003eTo evaluate the specificity of the established AIEgens-LFIA, both AFM1 and six commonly-found mycotoxins (ZEN, DON, OTA, FB1, FB2, and T-2) were simultaneously tested. As displayed in Fig. S2, the presence of AFM1 could induce significant fluorescent signal decreases to almost invisible, whereas ZEN, DON, OTA, FB1, FB2, and T-2 exhibited negligible changes in fluorescence signal response compared to PBS. In addition, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB showed that the FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e values of AFM1 was significantly lower than those of other mycotoxins, indicating excellent specificity and selectivity. These findings confirmed that the AIEgens-LFIA platform achieved both exceptional specificity and anti-interference ability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2 Accuracy and precision\u003c/h2\u003e\u003cp\u003eThe accuracy and precision of the AIEgens-LFIA were assessed using the blank milk samples spiked with AFM1 at low (0.1 \u0026micro;g/kg), medium (0.5 \u0026micro;g/kg), and high (1 \u0026micro;g/kg) concentration. Intra-assay and inter-assay were carried out on the same day and over three successive days with triplicate of each spiked concentration (n\u0026thinsp;=\u0026thinsp;5). As presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the average recoveries for both intra- and inter-assay ranged from 86.81\u0026ndash;106.37% with the CV from 7.06\u0026ndash;14.71%. These results confirmed that the proposed AIEgens-LFIA provides acceptable accuracy and reproducibility for AFM1 detection in milk.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAccuracy and precision of the established AIEgens-LFIA for detection of AFM1 in milk.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpiked levels (ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eIntra-Assay\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eInter-Assay\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003cp\u003e(ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRecovery\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCV\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003cp\u003e(ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRecovery\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCV\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.087\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e86.81\u0026thinsp;\u0026plusmn;\u0026thinsp;8.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e100.73\u0026thinsp;\u0026plusmn;\u0026thinsp;14.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e14.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.475\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e94.93\u0026thinsp;\u0026plusmn;\u0026thinsp;10.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.496\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e99.13\u0026thinsp;\u0026plusmn;\u0026thinsp;10.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e10.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.064\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e106.37\u0026thinsp;\u0026plusmn;\u0026thinsp;7.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1. 012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e101.17\u0026thinsp;\u0026plusmn;\u0026thinsp;9.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e8.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.3.3 Methods comparison\u003c/h2\u003e\u003cp\u003eTo facilitate a thorough understanding of the advantages and disadvantages of the established detection platform, a comprehensive table was presented to compare it with previously reported rapid detection methods for AFM1 analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The sensitivity of the proposed method matches or surpasses that of previously reported technologies, thus showing superior application prospects for rapid detection of AFM1 in milk.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of the developed AIEgens-LFIA with previous reported methods for AFM1 detection.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLabel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLOD\u003c/p\u003e\u003cp\u003e(ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLinear range\u003c/p\u003e\u003cp\u003e(ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAuNPs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(Pimpitak, Rengpipat, Phutong, Buakeaw, \u0026amp; Komolpis, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAuNPs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(B.-H. Liu, Chu, \u0026amp; Yu, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAuNPs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(J.-J. Wang, Liu, Hsu, \u0026amp; Yu, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTRFM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.05-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(M. Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0423\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.05\u0026ndash;0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e(Wu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQDs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0932\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1-1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSilver nanoparticles (AgNPs)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.2\u0026ndash;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(J. Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Z. Li, Li, Jiang, \u0026amp; Xu, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEu-modified fluorescent microspheres\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.02\u0026ndash;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(G. Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(PEI/PSS)\u003csub\u003e4\u003c/sub\u003e red SiNPs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(Su, Zhao, \u0026amp; Dou, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAIEgens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.01-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.3.4 Stability\u003c/h2\u003e\u003cp\u003eThe stability of AIEgens-LFIA stored at 4\u0026deg;C and 25\u0026deg;C was evaluated. A stable fluorescence signal was observed and the values of FI\u003csub\u003eT\u003c/sub\u003e/FI\u003csub\u003eC\u003c/sub\u003e basically remained unchanged with the CV of 8.12% for 4\u0026deg;C and 10.67% for 25\u0026deg;C (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results demonstrated that the developed AIEgens-LFIA could remain stable for at least 180 days at 4\u0026deg;C and 25\u0026deg;C, and fully meet the requirements of storage and transport in the practical use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.3.5 Analysis of actual samples by AIEgens-FICS and UPLC-MS/MS\u003c/h2\u003e\u003cp\u003eFor further verify the reliability, milk samples spiked with different concentrations of AFM1 (0.1, 0.5, and 1.0 ng/mL) were simultaneously determined by AIEgens-LFIA and UPLC-MS/MS. The recoveries of AIEgens-LFIA ranged from 97.53\u0026ndash;104.13%, while those obtained from UPLC-MS/MS were in the range of 96.47%-107.37% (Table S2). Moreover, high correlation coefficients (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.99) were obtained for both methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), proving the accuracy and reliability of the established AIEgens-LFIA for AFM1 detection in real milk samples\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn this study, a novel fluorescent test strip with red-emitted AIEgens as signal reporters was constructed for the rapid detection of AFM1 in milk. Benefiting from the excellent fluorescence properties, the designed AIEgens-LFIA not only enabled a rapid visual recognition but also achieved an accurately quantitative detection of AFM1. Under the optimal conditions, the established AIEgens-LFIA exhibited high selectivity and sensitivity, satisfactory linearity and accuracy, good reproducibility, and stability during AFM1 detection. With the various merits, the proposed AIEgens-LFIA platform could be a suitable alternative for the point-of-care testing and high-throughput screening of AFM1 in milk in food safety supervision, risk assessments, and the early-stage diagnosis of some diseases in future applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThe research was supported by the Shanghai Rising-Star Program (Grant No. 23QB1403500) and the National Key R\u0026amp;D Program of China (Grant No. 2024YFF1105802).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eWenbo Guo: Conceptualization Ideas, Methodology, Writing-Original draft. Qiyi Liu: Formal analysis. Junhua Yang: Investigation, Data curation. Haijuan Zeng: Methodology, Writing-review \u0026amp; editing, Supervision, Funding. Juan Liu: Visualization. Lemei Zhu: Writing-review \u0026amp; editing. Dongxia Nie: Validation. Zhihui Zhao: Writing-review \u0026amp; editing. Zheng Han: Writing-review \u0026amp; editing, Funding, All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlvarez CS, Hern\u0026aacute;ndez E, Escobar K, Villagr\u0026aacute;n CI, Kroker-Lobos MF, Rivera-Andrade A, Smith JW, Egner PA, Lazo M, Freedman ND (2020) Aflatoxin B1 exposure and liver cirrhosis in Guatemala: A case\u0026ndash;control study. 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Angew Chem Int Ed 59(51):23261\u0026ndash;23267. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/anie.202010216\u003c/span\u003e\u003cspan address=\"10.1002/anie.202010216\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng Z, Yang T, Li D, Cao H, Gong J, Liu H, Zhou C, Liu L, Wei P, Gu X (2023) Molecular and aggregate synergistic engineering of aggregation-induced emission luminogens to manipulate optical/electronic properties for efficient and diversified functions. ACS Nano 17(9):8782\u0026ndash;8795. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acsnano.3c02134\u003c/span\u003e\u003cspan address=\"10.1021/acsnano.3c02134\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"aflatoxin M1, aggregation-induced emission, lateral flow immunoassay, red emission, milk","lastPublishedDoi":"10.21203/rs.3.rs-7174022/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7174022/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFluorescent material-based lateral flow immunochromatographic assay (LFIA) has been widespread applied for the point-of-care test of mycotoxins. However, aggregation-caused quenching (ACQ) phenomenon of fluorescent labels seriously affects the detection performance of the methods. Herein, a red-emitted aggregation-induced emission luminogens (AIEgens) based LFIA was developed and firstly applied for the visual and quantitative detection of aflatoxin M1 (AFM1) in milk. The fluorescent probe was constructed by directly coupling the carboxylated AIEgens with \u003cem\u003eanti\u003c/em\u003e-AFM1-mAb, which demonstrated a strong AFM1-specific affinity and exceptional fluorescent characteristics. Under the optimal conditions, this AIEgens-LFIA could achieve a rapid detection of AFM1 within 15 min, with a visual limit of detection (vLOD) of 0.1 ng/mL and a quantitative limit of detection (qLOD) of 0.01 ng/mL. The accuracy and precision of the established AIEgens-LFIA method were assessed though the spiked milk samples, and the recoveries were in the range of 86.81%-106.37% with coefficients of variation below 10%. Furthermore, the test strip exhibited a wide detection range for AFM1 (0.01-1 ng/mL) and a remarkable stability for 180 days when stored at 4\u0026deg;C and 25\u0026deg;C. Importantly, the results for real milk sample were consistent with those of standard UPLC-MS/MS method. Overall, the newly developed AIEgens-LFIA demonstrates significant potential for the point-of-care test of AFM1 in milk.\u003c/p\u003e","manuscriptTitle":"Red-emitted AIEgens-based lateral flow immunoassay for the visual and quantitative detection of aflatoxin M1 in milk","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-01 16:45:22","doi":"10.21203/rs.3.rs-7174022/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d0a8c33-1c50-40fe-8fde-3d852e10c4fa","owner":[],"postedDate":"August 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T15:39:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-01 16:45:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7174022","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7174022","identity":"rs-7174022","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}