Ratiometric Fluorescence Immunoassay Based on Carbon Quantum Dots for Sensitive Detection of Malachite Green in Fish

Malachite green (MG) is a synthetic poisonous organic compound that has been banned in many countries as a veterinary drug for aquaculture. An efficient, fast and sensitive method is urgently needed for monitoring the illegal use of malachite green (MG) in aquaculture. In this study, a novel ratiometric fluorescence immunoassay was established. Nitrogen-doped carbon quantum dots were used as ratiometric fluorescent probes with a fluorescence peak at 450 nm. Horseradish peroxidase was employed to convert o-phenylenediamine to 2,3-diaminophenazine, with a new fluorescence peak at 580 nm and a strong absorption at 420 nm. The inner filter effect between N-CQD fluorescence and DAP absorption was identified. It allows for the ratiometric detection of MG using a fluorescent immunoassay. The results demonstrated a linear ratiometric fluorescence response for MG between 0.1 and 12.8 ng·mL−1. The limit of detection of this method was verified to be 0.097 μg·kg−1 with recoveries ranging from 81.88 to 108%, and the relative standard deviations were below 3%. Furthermore, this method exhibited acceptable consistency with the LC-MS/MS results when applied for MG screening in real samples. These results demonstrated a promising application of this novel ratiometric fluorescence immunoassay for MG screening with the merits of rapid detection, simple sample preparation, and stable signal readout. It can be an alternative to other traditional methods if there are difficulties in the availability of expensive instruments, and achieve comparable results or even more sensitivity than other reported methods.


Introduction
MG is a synthetic triphenylmethane dye that has been widely used in aquaculture. It can protect aquatic animals from diseases caused by parasites, fungi, bacteria, etc. [1]. The compound can be easily absorbed by aquatic animals and rapidly metabolized into leucomalachite green (LMG), which accumulates in the organism, and remains in the body for several months. However, research has demonstrated that MG and LMG can lead to adverse effects such as carcinogenic, teratogenic, and mutagenic. This compound can remain in the body for a long time and have cumulative effects [2,3]. Although many countries have prohibited its use in aquaculture, the illegal use of MG still occurs due to its high therapeutic efficiency, low cost, and commercial availability [4]. Therefore, the residue

Preparation of N-CQDs from Wolfberry
N-CQDs were prepared through a hydrothermal carbonization technique following our previous work [27]. Briefly, 2 g of wolfberry was added to 5 mL of deionized water in a stainless steel autoclave hydrothermal synthesis reactor vessel, which was sealed and carbonized at 180 • C for 8 h. Then, the reactor was cooled down at room temperature, and the synthesized material was diluted with 5 mL of deionized water. The N-CQDs were obtained through centrifugation at 7104× g for 15 min to remove the precipitation. The clear supernatant was dialyzed for 24 h and lyophilized.

N-CQDs Characterization
The particle size of N-CQDs was analyzed on a high-resolution TEM (200 KV FEI-Tecnai G2 20 S-TWIN, Thermo Fisher Scientific, Waltham, MA, US). The Fourier transform infrared (FTIR) spectrum of N-CQDs was obtained on a spectrometer (Nicolet iS50, Thermo Fisher Scientific, Waltham, MA, USA). XPS (Axis Ultra DLD, Kratos, Manchester, UK) was used to determine the elemental composition. UV-vis absorption and fluorescence emission spectrum measurements were obtained with UV-vis spectrometry (Hitachi u4100, Tokyo, Japan) and fluorescence spectroscopy (F97Pro, Lengguang Tech, Shanghai, China). The X-ray diffraction (XRD) spectrum was acquired for structure analysis (Bruker D8 Advance, Karlsruhe, Germany).

Development of Immunoassay
First, 100 µL of MG-OVA in carbonate-buffered saline solution (pH 9.6, 0.05 mol·L −1 ) was added to a 96-well ELISA plate, and left standing at 37 • C for 1 h. Then, the plates were washed with 200 µL of phosphate-buffered saline containing 0.05% Tween-20 (PBST) three times. The plate was blocked with 200 µL blocking solution for 1 h. After another washing step with PBST, 50 µL diluted MG-Ab, and 50 µL of different concentrations of MG or the sample extract were added to the coated wells for competitive binding, and then the plate was incubated at 37 • C for 1 h. After washing the plates with PBST (200 µL) twice, 100 µL of diluted IgG-HRP was added to the plates, which were incubated at 37 • C for 1 h. These plates were washed with PBST (200 µL) twice, and the enzymatic reaction was carried out by adding 50 µL N-CQDs (0.2 mg·mL −1 ) and 200 µL OPD solution with H 2 O 2 (pH = 6.0) in each well. After incubation at 37 • C for 40 min, the fluorescence spectrum was recorded at 450 nm (I 450 ) and 580 nm (I 580 ) at the excitation wavelength of 380 nm, which was used for the calculation of I 580 /I 450 .

Optimization of Experimental Conditions
To obtain the optimized parameters for ratiometric immunoassay, different concentrations of MG-OVA and MG-Ab, IgG-HRP, OPD, and H 2 O 2 were examined, respectively. In addition, the type of blocking buffer and reaction time were also evaluated separately.

Antibody Specificity Analysis
MG and six other common interferents (LMG, CV, LCV, MB, AZB, AZC) were used as competitors for the binding test with MG-Ab. The cross-reactivity ratios (CR) of MG-Ab were calculated as follows: Among the formula, IC 50 MG and IC 50 competitor are the concentration of MG and competitor, respectively, when the inhibition ratio reaches 50%.

Sample Preparation
Positive and negative fish samples were both obtained from the Aquatic Product Quality Supervision and Inspection and Testing Center (Shanghai), Ministry of Agriculture and Rural Affairs. First, two grams of fish samples were weighed in a 30 mL centrifuge tube. Then, 4 mL of acetonitrile was added. After a complete vortex of the tube for 3 min, the mixture was sonicated for 5 min and centrifuged at 7104× g for 5 min. The supernatant was collected in another tube. The solid remains were extracted again according to the above procedure and with the supernatant combined. To convert the LMG to MG, the supernatant was added with 300 µL of 2-chloro-1,4-benzoquinone (0.001 mol·L −1 , in acetonitrile) and vortexed for 1 min. Then, the extract was dried under a nitrogen stream and re-dissolved in 1 mL of PBS (pH = 6.0).

Characterizations of N-CQDs
The TEM image demonstrated that the N-CQDs were spherical nanoparticles ( Figure S1A in Supplementary Materials), with the size distribution ranging from 0.81 to 2.49 nm and the average diameter at 1.35 ± 0.15 nm ( Figure S1B). XRD results ( Figure S1C) exhibited a broad peak at 2θ = 20.9 • , corresponding to the (002) plane of graphite [28]. FTIR spectrum ( Figure S1D) showed the characteristic absorption bands of C-H, C=C/C=O, C-O, and C-N stretching peaks at around 2820, 1600, 1340, and 1080 cm −1 . The peak at 3460 cm −1 is attributed to O-H/N-H stretching. XPS was employed to further identify the elements of N-CQDs ( Figure S1E). Three prominent peaks at 285 eV, 400 eV, and 531 eV were fitted, corresponding to C1s, N1s, and O1s, respectively [29]. The high-resolution spectrum of O1s ( Figure S1F) showed three peaks at 530.9 eV, 531.6 eV, and 532.6 eV, which are attributed to C=O, C-O-H, C-O-C, respectively. The spectrum of N1s ( Figure S1G) showed three peaks at 399.2 eV, 399.7 eV, and 400.5 eV, corresponding to C=N-C, C-N-C, and N-C 3 , respectively. The spectrum of C1s ( Figure S1H) displayed three peaks at 284.6 eV, 285.7 eV, and 285.9 eV, coming from C-C, C=C, and C-N, respectively. The XPS data were in good agreement with FTIR results, which demonstrated abundant hydroxyl and other hydrophilic groups on the surface of N-CQDs. The excellent hydrophilicity of N-CQDs was also reasonably obtained.

Evaluation of the Sensing Mechanism
In this study, the fluorescence signal was used to replace the absorbance signal in the traditional ELISA. As shown in Figure 1, MG and MG-OVA competitively bound with MG-Ab in the stage of antibody-antigen binding. With the increase of the concentration of MG, fewer MG-Ab reacted with the MG-OVA, resulting in less IgG-HRP binding. As a result, fewer DAP would be generated from OPD, which further affected the ratiometric fluorescence as mentioned above. The fluorescent signal at 580 nm emerged with DAP generated from the enzyme reaction, and the higher concentration of DAP led to higher fluorescent intensity. On the other hand, the fluorescent intensity of N-CQDs at 450 nm was quenched by DAP due to the IFE between these two substances. Interestingly, the ratio between these two fluorescent signals was related to the concentration of MG.
result, fewer DAP would be generated from OPD, which further affected the ratiometric fluorescence as mentioned above. The fluorescent signal at 580 nm emerged with DAP generated from the enzyme reaction, and the higher concentration of DAP led to higher fluorescent intensity. On the other hand, the fluorescent intensity of N-CQDs at 450 nm was quenched by DAP due to the IFE between these two substances. Interestingly, the ratio between these two fluorescent signals was related to the concentration of MG. During the enzymatic process in the immunoassay, the OPD was catalytically oxidized by HRP to generate yellow DAP in the presence of H2O2. The emission peak of N-CQDs was at 450 nm (λex = 380 nm), which broadly overlapped with the absorption spectrum of DAP at 420 nm ( Figure 2A). The IFE between N-CQDs and DAP occurred [30], leading to a decrease in fluorescence intensity of N-CQDs. Meanwhile, the fluorescence at 580 nm emerged and grew with the continual generation of DAP ( Figure 2B). For comparison, the separate addition of HRP, H2O2, or OPD into the N-CQDs solution did not affect its fluorescence. On the other hand, as the HRP, H2O2, and OPD were mixed with N-CQDs in parallel, the fluorescence at 450 nm decreased, followed by the generation of a new fluorescence peak at 580 nm. With the continuous generation of DAP, the fluorescence intensity of the N-CQDs gradually diminished. More addition of HRP led to During the enzymatic process in the immunoassay, the OPD was catalytically oxidized by HRP to generate yellow DAP in the presence of H 2 O 2 . The emission peak of N-CQDs was at 450 nm (λ ex = 380 nm), which broadly overlapped with the absorption spectrum of DAP at 420 nm ( Figure 2A). The IFE between N-CQDs and DAP occurred [30], leading to a decrease in fluorescence intensity of N-CQDs. Meanwhile, the fluorescence at 580 nm emerged and grew with the continual generation of DAP ( Figure 2B). For comparison, the separate addition of HRP, H 2 O 2, or OPD into the N-CQDs solution did not affect its fluorescence. On the other hand, as the HRP, H 2 O 2 , and OPD were mixed with N-CQDs in parallel, the fluorescence at 450 nm decreased, followed by the generation of a new fluorescence peak at 580 nm. With the continuous generation of DAP, the fluorescence intensity of the N-CQDs gradually diminished. More addition of HRP led to faster conversion of the OPD to DAP, and a stronger fluorescence at 580 nm and weaker fluorescence at 450 nm could be observed in a short time. Therefore, the ratiometric fluorescence response (I 580 /I 450 ) can be used to reflect the concentration of HRP, which is reciprocally related to the analytes in the traditional ELISA. Based on this principle, the ratiometric fluorescence method can be utilized for MG determination combined with ELISA.
faster conversion of the OPD to DAP, and a stronger fluorescence at 580 nm and wea fluorescence at 450 nm could be observed in a short time. Therefore, the ratiometric f orescence response (I580/I450) can be used to reflect the concentration of HRP, which is ciprocally related to the analytes in the traditional ELISA. Based on this principle, the tiometric fluorescence method can be utilized for MG determination combined w ELISA.

Optimization of Experimental Conditions
A checkerboard method was used to optimize the concentration of MG-OVA a MG-Ab for immunoassay. The concentrations of MG-OVA and MG-Ab with the O value closest to 1.00 were selected for further experiments. In our study, the initial c centration of MG-OVA of 1 mg mL −1 and the initial concentration of MG-Ab of 1 mg•m were used. It can be seen in Table S1 that the optimal dilution factors for MG-OVA a MG-Ab were 2000 times (0.5 μg•mL −1 ) and 8000 times (0.125 μg•mL −1 ), respectively.
In order to optimize the experimental conditions, the type of blocking solution, concentration of IgG-HRP, OPD, and H2O2, and the enzymatic reaction time were inv tigated, respectively. The OD values of the negative control wells (OD0) corresponding different blocking solutions were compared, and the best blocking solution was selec with the lowest OD0. As shown in Figure 3A, OVA produced the lowest OD0 and th was chosen as the blocking solution. In order to obtain the maximum fluorescence ra (I580/I450), the fluorescence intensity at 580 nm under different concentrations of IgG-H was compared to select the suitable IgG-HRP concentration for the highest fluoresce as the optimal working concentration ( Figure 3B). The dilution factor of IgG-HRP was at 1000 (2 μg•mL −1 ), as the fluorescence intensity was the largest. Furthermore, OPD, H concentration, and reacting time were optimized based on I580/I450. With the increase OPD concentration, the value of I580/I450 became higher ( Figure S2). However, the h fluorescence intensity at 580 nm would cover the fluorescence at 450 nm, which co result in an unstable result of I580/I450. When the concentration of OPD reached 6.66 m the emission peak of 450 nm almost disappeared ( Figure 3C). In order to maintain c sistent and high results for I580/I450, the ideal concentration of OPD was determined to b mM. The increase of H2O2 concentration initially enhanced the growth of I580/I450 and th weakened the value at a higher concentration ( Figure 3D). The enzymatic reaction HRP would be inhibited if the concentration of H2O2 was increased too much [3 Therefore, the concentration of H2O2 (6.25 mM) corresponding to the maximum I580 was used for the following experiment. Besides, I580/I450 kept growing with the evolut of reaction time and reached a maximum after 40 min (Figures 3E and S3). Thus, the action time of 40 min was chosen for the immunoassay. As shown in Figure 3F, the co of fluorescence in the plates gradually evolved from blue (fluorescence of N-CQDs) yellow (fluorescence of DAP). Therefore, the concentration of MG can be determined measuring the fluorescence ratio between the produced DAP and the originally exist N-CQDs.

Optimization of Experimental Conditions
A checkerboard method was used to optimize the concentration of MG-OVA and MG-Ab for immunoassay. The concentrations of MG-OVA and MG-Ab with the OD value closest to 1.00 were selected for further experiments. In our study, the initial concentration of MG-OVA of 1 mg mL −1 and the initial concentration of MG-Ab of 1 mg·mL −1 were used. It can be seen in Table S1 that the optimal dilution factors for MG-OVA and MG-Ab were 2000 times (0.5 µg·mL −1 ) and 8000 times (0.125 µg·mL −1 ), respectively.
In order to optimize the experimental conditions, the type of blocking solution, the concentration of IgG-HRP, OPD, and H 2 O 2 , and the enzymatic reaction time were investigated, respectively. The OD values of the negative control wells (OD 0 ) corresponding to different blocking solutions were compared, and the best blocking solution was selected with the lowest OD 0 . As shown in Figure 3A, OVA produced the lowest OD 0 and thus was chosen as the blocking solution. In order to obtain the maximum fluorescence ratio (I 580 /I 450 ), the fluorescence intensity at 580 nm under different concentrations of IgG-HRP was compared to select the suitable IgG-HRP concentration for the highest fluorescence as the optimal working concentration ( Figure 3B). The dilution factor of IgG-HRP was set at 1000 (2 µg·mL −1 ), as the fluorescence intensity was the largest. Furthermore, OPD, H 2 O 2 concentration, and reacting time were optimized based on I 580 /I 450 . With the increase of OPD concentration, the value of I 580 /I 450 became higher ( Figure S2). However, the high fluorescence intensity at 580 nm would cover the fluorescence at 450 nm, which could result in an unstable result of I 580 /I 450 . When the concentration of OPD reached 6.66 mM, the emission peak of 450 nm almost disappeared ( Figure 3C). In order to maintain consistent and high results for I 580 /I 450 , the ideal concentration of OPD was determined to be 5 mM. The increase of H 2 O 2 concentration initially enhanced the growth of I 580 /I 450 and then weakened the value at a higher concentration ( Figure 3D). The enzymatic reaction of HRP would be inhibited if the concentration of H 2 O 2 was increased too much [31]. Therefore, the concentration of H 2 O 2 (6.25 mM) corresponding to the maximum I 580 /I 450 was used for the following experiment. Besides, I 580 /I 450 kept growing with the evolution of reaction time and reached a maximum after 40 min ( Figures 3E and S3). Thus, the reaction time of 40 min was chosen for the immunoassay. As shown in Figure 3F, the color of fluorescence in the plates gradually evolved from blue (fluorescence of N-CQDs) to yellow (fluorescence of DAP). Therefore, the concentration of MG can be determined by measuring the fluorescence ratio between the produced DAP and the originally existing N-CQDs.

Detection of MG
After optimization of the ratiometric fluorescent immunoassay, its capacity for the detection of MG was examined. A series of concentrations of PBS solution were prepared through dilution of the concentrated MG solution. A good linear response of the I580/I450 to the logarithm of the MG concentration (lgMG) ranging from 0.1 to 12.8 ng•mL −1 was observed ( Figure 4A), with a high correlation coefficient (R 2 = 0.9902). It was obvious that the linearity with only I450 or I580 showed data points of high error for calibration, as well as relatively poorer linearity with R 2 at 0.9290 and 0.8027, respectively ( Figure 4B,C). This demonstrated that the good linear response and low errors using ratiometric fluorescent calibration could result from the stable signal ratio between DAP and N-CQDs. Despite some unstable factors such as the light source variation, and the slight change of the fluorescent molecules due to solution evaporation, the intensity at 450 nm changed, in parallel with the emission intensity change at 580 nm, which led to a relatively stable ratio of I580/I450. Therefore, the use of ratiometric fluorescence for immunoassay might greatly improve the linearity and stability of the calibration curve and further lead to more accurate and precise test results. The limit of detection (LOD) was calculated based on LOD = 3δ/K, where δ represents the standard deviation at the low spiked concentration in the blank samples, and K is the slope of the fitted curve. The LOD was calculated to be 0.097 μg•kg −1 , which was below the required sensitivity on the official regulation of MG [32,33]. Therefore, this method can be an alternative to other traditional methods if there are difficulties in the availability of expensive instruments. This method can achieve comparable results or even more sensitivity than other reported methods (Table S2).

Detection of MG
After optimization of the ratiometric fluorescent immunoassay, its capacity for the detection of MG was examined. A series of concentrations of PBS solution were prepared through dilution of the concentrated MG solution. A good linear response of the I 580 /I 450 to the logarithm of the MG concentration (lgMG) ranging from 0.1 to 12.8 ng·mL −1 was observed ( Figure 4A), with a high correlation coefficient (R 2 = 0.9902). It was obvious that the linearity with only I 450 or I 580 showed data points of high error for calibration, as well as relatively poorer linearity with R 2 at 0.9290 and 0.8027, respectively ( Figure 4B,C). This demonstrated that the good linear response and low errors using ratiometric fluorescent calibration could result from the stable signal ratio between DAP and N-CQDs. Despite some unstable factors such as the light source variation, and the slight change of the fluorescent molecules due to solution evaporation, the intensity at 450 nm changed, in parallel with the emission intensity change at 580 nm, which led to a relatively stable ratio of I 580 /I 450. Therefore, the use of ratiometric fluorescence for immunoassay might greatly improve the linearity and stability of the calibration curve and further lead to more accurate and precise test results. The limit of detection (LOD) was calculated based on LOD = 3δ/K, where δ represents the standard deviation at the low spiked concentration in the blank samples, and K is the slope of the fitted curve. The LOD was calculated to be 0.097 µg·kg −1 , which was below the required sensitivity on the official regulation of MG [32,33]. Therefore, this method can be an alternative to other traditional methods if there are difficulties in the availability of expensive instruments. This method can achieve comparable results or even more sensitivity than other reported methods (Table S2).

Specificity Test
The specificity of this ratiometric fluorescence immunoassay was evaluated b adding six interferents with a similar structure to MG; that is, LMG, CV, LCV, MB, AZB and AZC. During the competitive immunoreaction between antibody and antigen rea tion, 50 μL of each interferent (1.6 ng•mL −1 ) was individually added to the MG-Ab, fo lowed by the addition of IgG-HRP, N-CQDs, OPA, and H2O2 for the record of I580/I450. Th cross-reaction ratios (CR) were calculated according to the formula shown at 2.6. A shown in Table 1, apart from the high cross-reactivity of this antibody to CV (121% other interferents induced less than 10% cross-reactivity, indicating excellent specificit of this method to MG. CV has a similar structure to MG, which led to the high CR of CV

Specificity Test
The specificity of this ratiometric fluorescence immunoassay was evaluated by adding six interferents with a similar structure to MG; that is, LMG, CV, LCV, MB, AZB, and AZC. During the competitive immunoreaction between antibody and antigen reaction, 50 µL of each interferent (1.6 ng·mL −1 ) was individually added to the MG-Ab, followed by the addition of IgG-HRP, N-CQDs, OPA, and H 2 O 2 for the record of I 580 /I 450 . The crossreaction ratios (CR) were calculated according to the formula shown at 2.6. As shown in Table 1, apart from the high cross-reactivity of this antibody to CV (121%), other interferents induced less than 10% cross-reactivity, indicating excellent specificity of this method to MG. CV has a similar structure to MG, which led to the high CR of CV.

Spiking and Real Sample Test
The established method was further examined with a spiking experiment by adding different amounts of MG in the blank fish samples (yellow catfish) and testing the recovered MG after sample preparation. As shown in Table 2, the recoveries of MG ranged from 81.88 to 108% with the relative standard deviation (RSD) lower than 3%. The recoveries and RSDs meet the requirements of the guideline SANTE/12682/2019; therefore, indicating good accuracy and precision for use as a quantitative method [34]. Furthermore, the concentration at 1.6 ng·g −1 was selected to examine the inter-batch and intra-batch variation. The spiking experiment at this concentration was repeated three times in three batches of measurement, and the result showed recoveries between 80.21 and 87.92%, with the RSD at 2.97 and 2.84%, respectively. At the spiking concentration of 1.6 ng· g −1 , 3 sets of intra-batch data and 9 sets of inter-batch data were analyzed with t-testing. The t-test result was 0.82 with a confidence level of 95% (alpha = 0.05), indicating that the inter-batch and intra-batch results had no significant difference. These results suggest that the developed method has good stability. These results also demonstrate the good feasibility of this method for MG screening in real samples.
To verify the practicability of the method, this new method was applied to real fish samples to test the total MG (LMG and MG) residue after pre-treatment of the samples. The detected results by this method were compared with the results from the gold standard of LC-MS/MS [35]. As shown in Table 3, three positive samples were found with MG concentrations at 4.79, 2.99, and 3 ng·g −1 using the established method, with RSD lower than 5%. The three detected positive results had a 7.5, 13.3, and 7.0% relative discrepancy to the LC-MS/MS results, and other negative results agreed well with the LC-MS/MS method. As the results of these samples were measured with both our method and the LC-MS/MS five times, the t-test for these results of the same samples between the two methods did not show significant difference. These results demonstrated good consistency between the ratiometric fluorescent immunoassay and the LC-MS/MS assay. We performed the stability experiment by storing the blocked antigen-coated microplates at 4 • C. They could provide a stable signal output in a week with the RSD value lower than 15%.

Conclusions
In summary, a ratiometric fluorescence immunoassay based on N-CQDs was developed for high-sensitivity detection of MG. In the fluorescence immunoassay, the IFE between N-CQDs and DAP occurred, leading to the evolution of the ratiometric fluorescence as the signal output. This ratiometric fluorescence detection mode enabled the detection of MG with higher resistance to environmental interference and resulted in more stable measurement. Furthermore, the specific recognition between antibody and antigen confirms the fluorescence method had good selectivity for MG. This method provided a good linear range between 0.1 and 12.8 ng·mL −1 , high sensitivity with the LOD of 0.097 µg· kg −1 , and recoveries between 81 and 108%, with RSD <3% for the spiking experiment in pretreated fish. In addition, the method was successfully verified for MG detection in real fish samples, where it displayed results consistent with those of LC-MS/MS assay. These results demonstrated a promising application of this novel ratiometric fluorescence immunoassay for MG screening with the merits of rapid detection, simple sample preparation, and stable signal readout.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios13010038/s1, Figure S1: (A) TEM image, (B) Particle size distribution, (C) XRD spectrum, (D) FTIR spectrum, (E) Full scan XPS, (F) O1s of XPS spectrum, (G) N1s of XPS spectrum, and (H) C1s of XPS spectrum of N-CQDs, Figure S2: The I 580 /I 450 of the ratiometric fluorescent immunoassay for the addition of different amounts of OPD for optimization, Figure S3: The I 580 /I 450 of the ratiometric fluorescent immunoassay with the evolution of reaction time, Table S1: The optimization of the concentrations of MG-OVA and MG-Ab, Table S2: Comparison of the method with those in the previous literature for the detection of MG. References [36][37][38][39] are cited in Supplementary information.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data will be made available on request.

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.