Development of a ratiometric fluorescence immunoassay based on manganese dioxide nanosheets for the detection of T-2 toxin

Background: T-2 toxin is a highly toxic mycotoxin that poses serious risks to food safety and human health. There is an urgent need for sensitive, selective, and reliable analytical methods for its detection in complex food matrices.

Methods: We developed a novel ratiometric fluorescence immunoassay based on gold nanoparticles encapsulated in manganese dioxide nanosheets (AuNP@MnO₂), which exhibit intrinsic oxidase-like activity. The assay utilizes dual-emission fluorescence (at 430 nm and 570 nm) and quantifies T-2 toxin by measuring the fluorescence intensity ratio (F₄₃₀/F₅₇₀), thereby minimizing environmental and instrumental variability.

Results: The method demonstrated excellent linearity over a wide concentration range (0.082–60.00 ng/mL) with a low detection limit of 0.068 ng/mL. High specificity was achieved, showing negligible cross-reactivity against common mycotoxins such as aflatoxin B₁, zearalenone, fumonisin B₁, ochratoxin A, and deoxynivalenol. In spiked corn samples, recoveries ranged from 76.94% to 99.48%, confirming good accuracy, stability, and reproducibility.

Conclusion: This ratiometric immunoassay significantly enhances detection precision by self-calibrating through an internal fluorescence reference. Its simplicity, sensitivity, and robustness in real food samples make it a promising tool for rapid and reliable monitoring of T-2 toxin in food safety applications.

1 Introduction

T-2 toxin, a potent trichothecene mycotoxin produced by various Fusarium species (Schrenk et al., 2022), poses a significant global threat to food safety due to its frequent contamination of cereals and cereal-based products such as wheat, barley, corn, and oats (Zhang et al., 2024). Regulatory agencies have therefore stringent limits for T-2 toxin levels, with the European Union recommending concentrations below 50–500 μg/kg in animal feed and not exceeding 200 μg/kg in cereals (Food and Drug Administration, 2015). Similarly, China’s “Feed Hygiene Standard” stipulates a maximum limit of 500 μg/kg in feed ingredients such as corn (Chinese Standard, 2017). The toxicological profile of T-2 toxin is particularly alarming due to its capacity to cross the blood–brain barrier and induce neuronal congestion, edema, and apoptosis (Wang et al., 2024). Chronic exposure has been associated with severe health risks including immunosuppression, gastrointestinal damage (Muñoz-Solano et al., 2024), and potential carcinogenicity (Zhang w. et al., 2021). In livestock, prolonged ingestion of T-2 toxin-contaminated feed can cause vomiting, feed refusal, weight loss, growth inhibition, and mortality in severe cases (Pang et al., 2021; Ding et al., 2025). Human exposure has similarly been linked to immune dysregulation, hepatotoxicity, and inflammatory diseases such as rheumatoid arthritis (Wu et al., 2020; Meneely et al., 2023a). Current analytical methods for T-2 toxin detection, including enzyme-linked immunosorbent assays (ELISA) and various chromatographic techniques, face significant limitations related to sensitivity, operational complexity, and susceptibility to matrix effects and environmental interferences (Feng et al., 2023). In particular, conventional fluorescence immunoassays relying on single-wavelength fluorescence intensities are vulnerable to signal fluctuations caused by variations in excitation light stability, probe concentrations, or instrument drift, leading to compromised accuracy and reproducibility (Meneely et al., 2023b). These challenges highlight the need for highly sensitive, accurate, and robust detection strategies suitable for practical applications in food safety monitoring.

Nanomaterials have emerged as versatile platforms for bioanalytical applications due to their unique physicochemical properties. Among them, gold nanoparticles (AuNPs) have attracted considerable interest because of their excellent biocompatibility, ease of surface functionalization, and strong localized surface plasmon resonance (LSPR), which enables signal amplification in various sensing modalities (Li et al., 2022; Kumar et al., 2024). Manganese dioxide nanosheets (MnO₂ NS), on the other hand, possess intrinsic oxidase-like activity and strong oxidizing capacity, which make them effective in catalytic signal generation and modulation in biosensing assays (Hu et al., 2024). Furthermore, MnO₂ can be selectively reduced by reducing agents such as ascorbic acid (AA), enabling controllable modulation of catalytic and optical properties. Hybrid nanostructures integrating AuNPs and MnO₂ nanosheets (AuNP@MnO₂) have been previously employed in biosensing platforms due to their synergistic properties, including enhanced catalytic activity, improved signal transduction, and increased assay sensitivity (Kong et al., 2023; Phoungsiri et al., 2023). However, many of these existing systems primarily rely on either colorimetric changes or single-emission fluorescence signals, which can still suffer from environmental interferences and lack sufficient ratiometric capabilities for precise quantitative analysis.

In the present study, we report a novel ratiometric fluorescence immunoassay for the detection of T-2 toxin that leverages the dual-functionality of AuNP@MnO₂ nanocomposites (Figure 1). In this system, MnO₂ nanosheets initially act as oxidase mimics that catalyze the oxidation of o-phenylenediamine (OPD) to generate 2,3-diaminophenazine (DAP), producing yellow fluorescence (λ_ex/λ_em = 423/570 nm) (Yu et al., 2023; Zhou et al., 2023) Upon the enzymatic generation of ascorbic acid, MnO₂ is sequentially reduced to Mn²⁺, leading to the release of embedded AuNPs and a decrease in oxidase activity. This reduction step occurs prior to the OPD redox transition, as previously reported for similar MnO₂-based fluorescence systems (Yang et al., 2020; Cao et al., 2021). The reduced environment then promotes the formation of quinoxaline (OPD_red), which emits blue fluorescence (λ_ex/λ_em = 350/430 nm). Therefore, the dual-emission response (F₄₃₀/F₅₇₀) arises from a sequential process involving MnO₂ reduction followed by OPD redox conversion, rather than a simultaneous reaction, which enhances the ratiometric sensitivity and signal resolution of the assay (Zhang s. et al., 2021). By calculating the fluorescence intensity ratio (F₄₃₀/F₅₇₀), our method effectively corrects for environmental and instrumental variability, offering significantly enhanced analytical reliability and sensitivity. Compared to previously reported AuNP@MnO₂-based assays, the innovation of our work lies in integrating ratiometric fluorescence readouts with the catalytic properties of the nanocomposite to achieve superior analytical performance for T-2 toxin detection. This dual-emission strategy not only improves quantification accuracy but also provides a visual colorimetric cue, facilitating rapid and preliminary assessment, especially in resource-limited settings. Thus, the proposed immunoassay represents a significant advancement toward practical, sensitive, and robust monitoring of T-2 toxin in food safety applications.

Figure 1

Figure 1. Schematic of fluorescence ratio immunological detection method for T-2 toxin. In the presence of ALP, p-AP reduces MnO₂ to Mn²⁺, exposing AuNPs and inducing aggregation, leading to a color change from brown to blue. The introduction of OPD enhances the assay’s sensitivity, producing distinct blue (quinoxaline) and yellow (2,3-diaminophenazine) fluorophores with unique excitation profiles. The target concentration is quantified by the fluorescence intensity ratio of these two substances.

2 Materials and methods

2.1 Materials and reagents

The T-2 toxin monoclonal antibody and T-2-BSA conjugate were synthesized in our laboratory. Alkaline phosphatase- labeled IgG (ALP-IgG) secondary antibody and T-2 toxin standards were obtained from Beijing Bioson Biotechnology Co., Ltd. (Beijing, China). All analytical grade reagents including Na₂CO₃, NaHCO₃, NaCl, Na₂HPO₄·12H₂O, NaH₂PO₄·2H₂O, KCl, KH₂PO₄, and H₂SO₄ were procured from Beijing Chemical Plant (Beijing, China). OPD was purchased from Aladdin (Shanghai, China). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl₄·3H₂O) was acquired from Sigma-Aldrich (St. Louis, MO, United States). Trisodium citrate was supplied by Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). 2-Aminoethyl phosphoric acid (AAP) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Equipment used included an Elx800 microplate reader (Bio-Tek Instruments, Winooski, VT, United States), a BSA124S analytical balance (Sartorius, Göttingen, Germany), and a Maxi MR1 magnetic stirrer (Thermo Scientific, Waltham, MA, United States).

2.2 Synthesis of AuNPs

Gold nanoparticles (AuNPs) with a diameter of 13 nm were synthesized following a previously reported protocol (Zha et al., 2021). A solution of HAuCl₄·3H₂O (100 μL, 250 mmol/L) was added to ultrapure water (100 mL) in a siliconized conical flask. The solution was brought to boil under continuous stirring, followed by the rapid addition of filtered trisodium citrate solution (3.5 mL, 34 mmol/L). Upon addition, the reaction mixture underwent a color transition to red, indicating the formation of gold nanoparticles. The solution was maintained at boiling temperature for an additional 5 min to ensure complete reduction. Subsequently, the colloidal suspension was cooled to room temperature and stored at 4 °C until further use.

2.3 Synthesis of AuNP@MnO₂

AuNP@MnO₂ nanocomposites were synthesized following a previously established protocol with minor modifications (Zhang et al., 2014; Yang et al., 2020). Briefly, 20 mL of potassium permanganate solution (10 mmol/L KMnO4) was added dropwise to 64 mL of as-prepared AuNPs colloidal solution under vigorous magnetic stirring (800 rpm) at room temperature. The resulting mixture was then heated in an open glass vessel (non-reflux condition) in a water bath at 80 °C for 30 min to ensure complete reduction of KMnO₄ and formation of MnO₂ nanosheets on the AuNP surface. During heating, the vessel was loosely covered to prevent contamination while allowing gas exchange. After cooling to room temperature, the resulting AuNP@MnO₂ nanocomposites were stored at 4 °C for further use. Based on prior reports, the synthesized MnO₂ nanosheets typically exhibit lateral dimensions of 100–300 nm with a thickness of 2–5 nm (Zhang et al., 2014; Yang et al., 2020), while the AuNPs used in this study have a diameter of approximately 13 nm.

2.4 Proof of principle

To verify the mechanism of the AuNP@MnO₂-based sensing platform, a series of control experiments were conducted systematically. Six experimental groups were designed: (1) ALP + AAP, (2) OPD, (3) AuNP@MnO₂, (4) AuNP@MnO₂ + ALP + AAP, (5) AuNP@MnO₂ + OPD, and (6) AuNP@MnO₂ + ALP + AAP + OPD. Fluorescence measurements (λ ex = 340 nm, λ em = 425 nm) were performed to evaluate: (i) the intrinsic oxidative capacity of AuNP@MnO₂ toward OPD substrate, (ii) the effect of AuNP@MnO₂ in combination with OPD on fluorescence signal generation, and (iii) the impact of enzymatic reaction products from the ALP-AAP system on the AuNP@MnO₂-mediated oxidation of OPD. The fluorogenic reaction was monitored using a spectrofluorometer under optimized conditions, and the resulting fluorescence intensities were compared to elucidate the underlying sensing mechanism of the developed nanozyme-based detection system.

2.5 Optimization of the amount of OPD in the substrate and the optimal reaction time

The OPD concentration was determined by evaluating a concentration gradient (100, 50, 25, 12.5, 6.25, 3.13, and 1.56 mmol/L) in a 96-well microplate format. OPD solution (30 μL/well) was combined with AuNP@MnO₂ nanocomposites (1 μL/well) and incubated at 37 °C for 1 h. Fluorescence intensity measurements were recorded using a microplate reader at excitation and emission wavelengths of 423 and 570 nm, respectively. All measurements were performed in triplicate. The OPD concentration yielding maximum fluorescence intensity was selected for subsequent experiments. Each concentration was tested in three independent replicates (n = 3), and fluorescence data were expressed as mean ± standard deviation (SD).

2.6 Optimization of AAP dosage and reaction time in substrate

Under previously established conditions, ascending concentrations of AAP solutions (8, 4, 2, 1, 0.5, 0.25, and 0.125 mmol/L; 80 μL/well) were incubated with alkaline phosphatase-conjugated immunoglobulin G (ALP-IgG, 2.5 μg/mL; 20 μL/well) in a 96-well microplate at 37 °C for 1 h. Following incubation, optimal OPD concentration and AuNP@MnO₂ nanocomposites were added sequentially. Fluorescence measurements were recorded at dual wavelengths: excitation 350 nm/emission 430 nm (F₄₃₀) and excitation 423 nm/emission 570 nm (F₅₇₀). A negative control (AAP = 0 mmol/L) was included for background correction. The ratiometric fluorescence response (F₄₃₀/F₅₇₀) was calculated to determine the optimal AAP concentration. For reaction kinetics optimization, AAP and ALP-IgG were incubated for varying time intervals (100, 80, 70, 60, 50, 40, 30, and 20 min), followed by identical fluorescence measurements. The optimal reaction duration was established based on the maximum F₄₃₀/F₅₇₀ ratio. All assays were performed in triplicate, and results are presented as mean ± SD.

2.7 Detection of ALP-IgG

To assess the analytical performance of the method, ALP-IgG at defined concentrations (2, 1, 0.5, 0.25, 0.125, and 0.0625 μg/mL; 20 μL/well) was combined with optimized AAP solution (4 mmol/L; 80 μL/well) in a 96-well microplate. After incubation at 37 °C for 40 min, AuNP@MnO₂ nanocomposites (1 μL/well) and OPD solution (50 mmol/L; 80 μL/well) were sequentially introduced, followed by further incubation at 37 °C for 50 min. Dual-wavelength fluorescence measurements were acquired at excitation 423 nm/emission 570 nm (F₅₇₀) and excitation 350 nm/emission 430 nm (F₄₃₀). The ratiometric fluorescence response (F₄₃₀/F₅₇₀) was plotted against ALP-IgG concentration to construct a calibration curve for quantitative analysis.

2.8 Fluorescence immunoassay procedure

Microtiter plates were functionalized by immobilizing T-2-BSA conjugate (100 μL/well) diluted in coating buffer and incubated at 37 °C. After incubation, plates were washed three times with PBST buffer (200 μL/well). T-2 toxin standards and T-2 toxin-specific monoclonal antibodies (50 μL/well each) were added to respective wells, followed by incubation and subsequent washing steps. ALP-conjugated secondary antibody (ALP-IgG, 100 μL/well) diluted in PBS buffer was added and incubated at 37 °C, followed by washing. AAP substrate solution (80 μL/well) was introduced and incubated at 37 °C. After the enzymatic reaction, AuNP@MnO₂ nanocomposites and OPD were sequentially added, followed by incubation at 37 °C. Dual-wavelength fluorescence measurements (F₄₃₀ and F₅₇₀) were recorded using a microplate reader, and the ratiometric fluorescence response (F₄₃₀/F₅₇₀) was calculated for quantitative analysis of T-2 toxin.

2.9 Checkerboard optimization of antigen–antibody working concentrations

T2-BSA conjugate was serially diluted in coating buffer to final concentrations of 4, 2, 0.5, and 0.25 μg/mL, and dispensed vertically (100 μL/well) into a 96-well ELISA microplate. Following incubation for 2 h at 37 °C, the plates were decanted and washed with phosphate-buffered saline containing 0.05% tween-20 (PBST) (200 μL/well). Concurrently, anti-T-2 toxin monoclonal antibody was diluted in PBS to corresponding concentrations and added horizontally (50 μL/well) to the antigen-coated microplate. The final row received 100 μL PBS as a negative control. After incubation for 1 h at 37 °C, fluorescence ratios (F₄₃₀/F₅₇₀) were measured, and optimal concentration pairs were determined through signal-to-noise ratio analysis.

2.10 Optimization of the optimal ALP-IgG dosage

ALP-IgG conjugate was serially diluted in PBS to concentrations of 4, 2, 1, 0.5, 0.25, and 0.125 μg/mL, with a negative control group (without monoclonal antibody) included. Fluorescence intensities at 430 nm (F₄₃₀) and 570 nm (F₅₇₀) were quantified using a microplate reader. The fluorescence ratio (F₄₃₀/F₅₇₀) was calculated for both experimental and negative control groups, denoted as (F₄₃₀/F₅₇₀) and (F₄₃₀/F₅₇₀)₀, respectively. The optimal ALP-IgG concentration was determined as the concentration yielding the maximum ratio of (F₄₃₀/F₅₇₀)/(F₄₃₀/F₅₇₀)₀.

2.11 Establishment of standard curve

A ratiometric fluorescence immunoassay for T-2 toxin was developed under optimized conditions. Competitive immunoassays were performed with T-2 toxin concentrations of 60, 20, 6.67, 2.22, 0.741, 0.247, 0.0823, and 0 ng/mL in triplicate. A standard calibration curve was constructed by plotting the fluorescence ratio (F₄₃₀/F₅₇₀) against the logarithm of T-2 toxin concentration. The SD of the blank measurements was determined at 0 ng/mL, and the slope (S) of the standard curve was calculated.

The limit of detection (LOD) was calculated according to the following equation:

$\mathrm{LOD}=3\times \frac{\mathrm{SD}}{\mathrm{S}}$

SD is the standard deviation of the blank value measured without adding T-2 for competition, and S is the slope of the established standard curve.

2.12 Specificity analysis

The specificity of the immunoassay was evaluated by testing T-2 toxin alongside other mycotoxins including AFB1, ZEN, FB1, ochratoxin A (OTA), and deoxynivalenol (DON) using the optimized ratiometric fluorescence immunoassay protocol described above. All experiments were performed in triplicate, and statistical analysis was conducted using one-way analysis of variance (ANOVA) with SPSS software.

2.13 Determination of spiked recoveries

Prior to analysis, negative cornmeal samples (verified using a commercially validated T-2 toxin ELISA kit, consistent with previously reported recovery studies) were dried overnight in an oven (Renaud et al., 2019; Raza et al., 2025). Spike concentrations were established at three levels: 100, 50, and 2 μg/kg. The highest spiking level (100 μg/kg) was selected to represent a realistic high-end contamination scenario in animal feed, consistent with regulatory guidance levels (Food and Drug Administration, 2015). Importantly, after 20-fold dilution of the corn extract prior to assay, the final T-2 toxin concentration entering the immunoassay was 5 μg/kg, which falls well within the linear detection range of 0.082–60 ng/mL. For extraction, 0.5 g of each spiked sample was suspended in 1 mL of PBS buffer containing 20% methanol and subjected to mechanical shaking for 10 min, followed by ultrasonication for 10 min. The suspensions were then centrifuged at 12,000 g for 15 min at room temperature. The resulting supernatants were diluted 20-fold with PBS buffer before analysis using the established ratio fluorescence immunoassay method.

The recovery formula is:

$\text{Recovery rate}=\frac{\begin{array}{l}\text{amount detected after spiked}-\\ \text{amount not detected after spiked}\end{array}}{\text{actual amount added}}\times 100\%$

3 Results

3.1 Principal verification

To characterize the optical properties of the nano biocatalytic system, fluorescence spectroscopy was employed to conduct a comprehensive wavelength analysis of AAP + ALP, OPD, AuNP@MnO₂, AuNP@MnO₂ + AAP + ALP, AuNP@MnO₂ + OPD, and AuNP@MnO₂ + OPD + AAP + ALP in the presence of enzymatic markers. The spectroscopic results are presented in Figure 2 The reaction products of AuNP@MnO₂ and OPD exhibited a distinct emission peak at 570 nm (Figure 2, curve d), confirming the oxidative capability of AuNP@MnO₂ to convert OPD to DAP. Curve a represents the fluorescence spectrum of OPD alone, which serves as the substrate for the oxidation reaction. Figure 2, curve b shows the fluorescence spectrum of AAP in the presence of ALP, demonstrating the enzymatic hydrolysis process. Figure 2, curve c corresponds to the fluorescence spectrum of AuNP@MnO₂ nanocomposites, highlighting their intrinsic optical properties. Notably, the reaction mixture containing AuNP@MnO₂ + OPD + AAP + ALP demonstrated a characteristic emission peak at 430 nm (Figure 2, curve f), indicating a different reaction pathway in the presence of all components. Further analysis revealed that the reaction products generated from AuNP@MnO₂, AAP, and ALP effectively reduced OPD to OPD red, while control experiments with other components showed negligible fluorescence signals. These spectroscopic findings collectively demonstrate that ALP can effectively mediate both the catalytic oxidation and reduction of OPD within the substrate, resulting in distinguishable ratiometric fluorescence signals suitable for analytical applications.

Figure 2

Figure 2. Verification of the dual-emission mechanism in the ratiometric fluorescence system based on AuNP@MnO₂. (a). OPD; (b). AAP + ALP; (c). AuNP@MnO2; (d). AuNP@MnO2 + OPD; (e). AuNP@MnO2 + AAP + ALP; (f). AuNP@MnO2 + OPD + AAP + ALP; Fluorescence intensity scans of various reaction systems show that AuNP@MnO₂ oxidizes OPD to DAP (emission peak at 570 nm), while the presence of AAP + ALP reduces OPD to OPDred (emission peak at 430 nm).

3.2 Selection of OPD dosage in substrate and optimal reaction time

To determine the optimal concentration, various OPD concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.5625 mmol/L) were investigated in reaction with 1 μL AuNP@MnO₂. As illustrated in Figure 3A, fluorescence intensity exhibited a concentration-dependent increase that plateaued when OPD concentration exceeded 50 mmol/L. Beyond this threshold concentration, no significant enhancement in fluorescence intensity was observed, indicating saturation of the reaction system. Consequently, 50 mmol/L was established as the optimal OPD concentration for subsequent experiments.

Figure 3

Figure 3. Selection of optimal dosage and action time of OPD in substrate. (A) Set different concentrations of AAP (8, 4, 2, 1, 0.5, 0.25, 0.125 mmol/L) for substrate reaction and the change in fluorescence intensity; (B). The fluorescence intensity of the reaction product of OPD and AuNP@MnO2 was recorded, reaching the maximum value at 50 min.

Additionally, reaction kinetics were evaluated by varying the incubation time between OPD and AuNP@MnO₂ (30, 40, 50, 60, and 70 min). Figure 3B demonstrates that fluorescence intensity progressively increased with extended reaction duration, reaching maximum signal intensity at 50 min, after which no substantial enhancement was detected. Based on these results, a reaction time of 50 min was selected as optimal for the OPD-AuNP@MnO₂ interaction to ensure maximum analytical sensitivity while maintaining experimental efficiency.

3.3 Selection of AAP dosage in substrate and optimal reaction time

To determine the optimal parameters for the assay, we systematically investigated the effect of varying AAP concentrations (0.125–8 mmol/L) on the substrate reaction. As illustrated in Figure 4A, the ratiometric fluorescence intensity (F₄₃₀/F₅₇₀) exhibited a concentration-dependent response, with signal intensity increasing proportionally with AAP concentration until reaching saturation at 4 mmol/L. Beyond this threshold, no significant enhancement in signal intensity was observed, indicating complete substrate utilization. Consequently, 4 mmol/L was established as the optimal AAP concentration for subsequent experiments.

Figure 4

Figure 4. Selection of the best dosage and action time of AAP in substrate. (A) Set different concentrations of AAP (8, 4, 2, 1, 0.5, 0.25, 0.125 mmol/L) for substrate reaction and the changes in the ratio fluorescence value F430/F570. (B) Setting different reaction times for ALP-IgG with AAP, and the changes in the ratio fluorescence F₄₃₀/F₅₇₀.

The reaction kinetics were evaluated by monitoring the ratiometric fluorescence response at various time intervals (20, 30, 40, 50, and 60 min) during the incubation of ALP-IgG with AAP. As demonstrated in Figure 4B, the F₄₃₀/F₅₇₀ signal intensity progressively increased with extended reaction times until plateauing at 40 min, suggesting the reaction had reached completion. No significant enhancement in signal intensity was observed with prolonged incubation beyond this timepoint. Therefore, a reaction time of 40 min was selected as optimal, balancing assay sensitivity with practical throughput considerations.

3.4 Detection of ALP-IgG

The optimized substrate was validated by evaluating varying concentrations of ALP-IgG (2, 1, 0.5, 0.25, 0.125, and 0.0625 μg/mL). Fluorescence measurements were performed using a spectrofluorometer with excitation at 423 nm. As illustrated in Figure 5A, the characteristic emission peak at 570 nm exhibited a concentration-dependent increase, correlating positively with ALP-IgG concentration, which confirms DAP as an effective substrate for ALP detection. Additionally, fluorescence spectroscopy with excitation at 350 nm revealed an inverse relationship between ALP-IgG concentration and the emission intensity at 430 nm (Figure 5B). This decrease in the 430 nm signal indicates reduced OPDred production at higher ALP-IgG concentrations. To establish a quantitative relationship, we calculated the ratiometric fluorescence (F₄₃₀/F₅₇₀) for each concentration and plotted these values against ALP-IgG concentration. The resulting calibration curve (Figure 5C) demonstrated excellent linearity (Y = 13.722x − 0.036, R² = 0.99) across the tested concentration range. These findings validate that our designed ratiometric fluorescence sensor provides a reliable analytical platform for ALP-IgG quantification.

Figure 5

Figure 5. The fluorescent spectra of test results for ALP-IgG. (A) The microplate reader scans the fluorescence intensity of the optimized ALP-IgG at an ex of 423 nm over the entire wavelength. (B) The microplate reader scans the fluorescence intensity of the optimized ALP-IgG at full wavelength at ex of 350 nm. (C) The microplate reader reads and calculates the ratio fluorescence of F430 and F570. The linear regression equation is: Y = 13.722x − 0.036, R² = 0.99.

3.5 Selection of the best working concentration of antigen and antibody

The results of the checkerboard method are shown in Table 1, where the ratiometric fluorescence F₄₃₀/F₅₇₀ increases with increasing antigen–antibody concentration. At the same antigen–antibody dosage, the measured ratiometric fluorescence F₄₃₀/F₅₇₀ is greater than any combination of antigen–antibody concentrations that are arbitrarily lower than the experimental results. Therefore, antigen–antibody concentrations of 2, 1, 0.5, and 0.25 μg/mL were chosen, and a standard curve was established using the logarithm of the T-2 toxin concentration as the horizontal coordinate and the ratiometric fluorescence F₄₃₀/F₅₇₀ as the vertical coordinate. As shown in Figure 6, when the antigen–antibody concentration is lower than 1 μg/mL, the fluorescence signal generated is weak, and the slope is basically 0, which cannot be used for the detection of T-2 toxin. When comparing the standard curves established at 1 μg/mL and 2 μg/mL, the antigen–antibody concentration of 1 μg/mL has a wider detection range and lower detection limit. Therefore, the optimal working concentration for both antigen and antibody is 1 μg/mL.

Table 1

Table 1. Selection of the best working concentration of antigen and antibody.

Figure 6

Figure 6. Standard curves established for antigen antibody concentrations of 2, 1, 0.5, and 0.25 μg/mL. A standard curve was established with the logarithm of T2 concentration as the horizontal axis and the ratio fluorescence F430/F570 as the vertical axis.

3.6 Selection of optimal ALP-IgG dosage

The concentration of ALP-IgG was set at 4, 2, 1, and 0.5 μg/mL for ratiometric fluorescence immunological detection. The results are shown in Figure 7 When the concentration of ALP-IgG is 2 μg/mL, (F₄₃₀/F₅₇₀)/(F₄₃₀/F₅₇₀)₀ is the largest. Therefore, 2 μg/mL was chosen as the optimal ALP-IgG concentration.

Figure 7

Figure 7. Selection of optimum concentration of ALP-IgG. The concentrations of ALP-IgG were set at 4, 2, 1, and 0.5 μg/mL for ratiometric fluorescence immunoassay.

3.7 Establishment of standard curves

The competitive concentrations of T-2 toxin were set to 180, 60, 20, 6.66, 2.22, 0.74, 0.25, 0.08, and 0 ng/mL for ratio fluorescence immunoassay. The experimental results are shown in Figure 8. The T-2 concentration has good linearity in the range of 0.25–60.00 ng/mL. The linear regression equation is: Y = − 0.0874 X + 0.1974, and R2 is 0.99. The detection range is 0.25–60.00 ng/mL. The minimum detection limit (LOD) was calculated to be 0.068 ng/mL.

Figure 8

Figure 8. Establishment of standard curve. The competitive concentrations of T2 were set to: 180, 60, 20, 6.66, 2.22, 0.74, 0.25, 0.08, and 0 ng/mL perform ratiometric fluorescence immunoassay.

3.8 Specificity analysis

T-2 toxin and AFB1, ZEN, FB1, OTA, DON were detected according to the optimized ratiometric fluorescence immunological assay as described above, and SPSS was used for one-way ANOVA, and the results of cross-reactivity are shown in Figure 9, which showed that T-2 toxin was significantly different from AFB1, ZEN, FB1, OTA, and DON, with good specificity.

Figure 9

Figure 9. Determination of cross reaction. T-2 toxin and AFB1, ZEN, FB1, OTA, DON were detected according to the above-mentioned optimized ratio fluorescence immunoassay method.

3.9 Determination of spiked recoveries

The results of the spiked recovery experiments were shown in Table 2, the recoveries of this method were from 76.94 to 99.48%, which were in accordance with the AOAC regulations. The average coefficient of variation (CV) of the spiked recoveries was 7.38%, and the accuracy of the method was good. Notably, the CV at the highest spiking level (100 μg/kg) was slightly higher (9.85%) compared to that at 2 μg/kg (6.42%), which may be attributed to minor heterogeneity in sample spiking or matrix effects at elevated concentrations. Nevertheless, both recovery and CV values remain well within acceptable limits, confirming the robustness of the assay across the tested concentration range.

Table 2

Table 2. Results of spiked recovery test.

4 Discussion

In this study, we successfully developed a novel ratiometric fluorescence immunoassay for the highly sensitive and accurate detection of T-2 toxin. This method utilizes manganese dioxide nanosheets (MnO₂ NS) as an oxidase mimic, ingeniously combining the high catalytic activity of alkaline phosphatase (ALP) with the unique properties of the newly synthesized AuNP@MnO₂ nanomaterials. Compared to traditional fluorescence immunoassays, the ratiometric fluorescence immunoassay developed in this study offers significant advantages (Chen et al., 2023; Wang et al., 2025). Traditional fluorescence immunoassays typically rely on single-wavelength fluorescence intensity measurements, which are susceptible to interference from factors such as excitation light stability fluctuations or probe concentration variations, leading to inaccurate detection results (Lippolis et al., 2019; Liu et al., 2020). In contrast, the ratiometric fluorescence immunoassay employed in this study effectively eliminates these interference factors by simultaneously monitoring fluorescence signals at two different wavelengths (F₄₃₀ and F₅₇₀) and calculating their ratio, thereby enhancing the accuracy and reliability of the detection results. Furthermore, we optimized various experimental parameters to ensure the optimal performance of the detection method. By optimizing the usage of OPD and AAP, as well as the reaction time, we achieved the highest detection sensitivity and efficiency. The experimental results demonstrate that under the optimized conditions, the method exhibits a linear detection range for T-2 toxin of 0.08–60.00 ng/mL, a detection limit as low as 0.069 ng/mL, and recovery rates ranging from 76.94 to 99.48%. These results indicate that the ratio metric fluorescence immunoassay developed in this study possesses high sensitivity, accuracy, and reproducibility, meeting the demands for T-2 toxin detection in the field of food safety.

Notably, the detection method developed in this study also exhibits excellent specificity. The cross-reactivity experiments demonstrate that the method has high selectivity for T-2 toxins, with negligible cross-reactivity to other common mycotoxins, such as AFB1, ZEN, and FB1. This characteristic enables the method to accurately detect T-2 toxin present in food samples without interference from other similar substances (Khan et al., 2023). Although this study has achieved encouraging results, there are still some areas that require further research and improvement (Lai et al., 2023). For example, this study only validated the performance of the method under laboratory conditions, and future studies should further investigate its application in the detection of actual food samples. Additionally, exploring the combination of this method with other detection techniques could further enhance its detection sensitivity and efficiency.

Moreover, while liquid chromatography–tandem mass spectrometry (LC–MS/MS) is widely regarded as the gold standard for mycotoxin quantification, its high cost, complex sample preparation, and requirement for skilled operators limit its use in routine on-site screening. In contrast, immunoassay-based methods are commonly validated against regulatory limits and commercial ELISA kits when LC–MS/MS data are unavailable, as demonstrated in recent studies (Ding et al., 2025; Khan et al., 2023). Our method meets AOAC-recommended recovery (70–120%) and precision (CV < 15%) criteria for mycotoxin analysis at μg/kg levels, supporting its suitability as a rapid screening tool. Nevertheless, direct comparison with LC–MS/MS using real contaminated samples will be pursued in future work to further confirm its accuracy and reliability in complex matrices.

In summary, this study developed a novel ratio metric fluorescence immunoassay based on manganese dioxide nanosheets for the highly sensitive and accurate detection of T-2 toxin. This method offers advantages such as simple operation, high sensitivity, and good specificity, demonstrating promising applications in the field of food safety.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

CM: Data curation, Formal analysis, Methodology, Software, Visualization, Writing – original draft. HO: Investigation, Validation, Writing – review & editing. HT: Data curation, Visualization, Writing – review & editing. YW: Conceptualization, Project administration, Writing – original draft. ZZ: Methodology, Writing – original draft. YL: Funding acquisition, Supervision, Writing – review & editing. JD: Conceptualization, Project administration, Resources, Writing – review & editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This study was supported by Innovation and Entrepreneurship Training Program for University Students (S202413706034) Jilin Province, China; Key Project of Jilin Provincial Science and Technology Department (20230203088SF) Jilin, China; Natural Science Foundation of China (NSFC82302563), China. Jilin Province Health Science and Technology Capacity Improvement Plan (2025WS-KB003) Jilin Province, China.

Conflict of 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.

Generative AI statement

The authors declare that no Gen AI was used in the creation of this manuscript.

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