A Chemiluminescent Magnetic Enzyme Immunoassay Method for 2 Triazole Pesticide Detection in Wheat

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

-The English needs extensive revision (grammar, typos, spacing issues).

-The extraction solvent is acetonitrile-PBS (20:80, v/v). The organic solvent content may affect antibody binding and enzyme activity. Indicate whether extracts are diluted before performing the immunoassay and report the final percentage of organic material in the reaction chamber.
-For validation of the HPLC-MS method, please provide the main parameters or clearly cite the standard method with sufficient details for comparison.-Please coordinate the units, for example: clearly reconcile the pollution levels in micrograms/milliliter and the reports in milligrams/kilogram, and show the conversion based on a sample of 5 grams and an extraction of 20 milliliters.
-The reported loading thickness of streptavidin of approximately 0.1 micrometers (100 nanometers) from TEM is unexpectedly large for a protein layer, and the units and explanation should be clarified. SEM/TEM images alone do not measure cohesion. 

Author Response

Comments 1: The English needs extensive revision (grammar, typos, spacing issues).

Response 1: Thank you sincerely for your valuable feedback on the language quality of this article. We fully acknowledge that there were grammar errors, spelling mistakes, and formatting issues in the initial draft. We have thoroughly revised it to comply with the academic standards of the journal. For example, maintaining subject verb agreement and tense consistency, paying attention to the format of spaces between professional terms, checking citation formats, abbreviation norms, and unit expressions, and eliminating inconsistencies between spaces and formats. The following are the modified parts in the manuscript:

1. In the summary section, “By optimizing the extraction solution and extraction time, acetonitrile-PBS was selected as the extraction solution with an extraction time of 5 minutes as the optimal pretreatment condition.” change to “Through optimization of the extraction solution and extraction time, acetonitrile-PBS was selected as the extraction solution with an extraction time of 5 minutes as the optimal pretreatment condition.” Avoid the problem of inconsistency between the logical subject of "optimization" and the subject of the main sentence (extraction solution).
2. In the introduction section, “Triazole fungicides, sush as triazolone and tebuconazole are widely used to combat these phytopathy[5-7].”change to “Triazole fungicides, such as triazolone and tebuconazole are widely used to combat these phytopathy[5-7].” We have corrected the spelling error.
3. In the introduction section, “traditional manual protocols involving centrifugation and repeated washing are time-intensive and suffer from reproducibility issues due to loss of analytes and residual contamination[32].”change to “traditional manual protocols involving centrifugation and repeated washing steps are time-consuming. Moreover, these manual operations suffer from poor reproducibility, primarily due to analyte loss and residual contamination during the washing process [32].”Split the lengthy concluding sentence of the original text into two clauses: "Traditional manual protocols... are time intensive and suffer from..." to avoid reading lag caused by multiple parallel predicates following the subject.
4. Spaces have been added between numbers and units in section 2.3 of the manuscript to standardize the spacing format.
5. In section 3.1, “Streptavidin-modified magnetic beads (MB-SA) servedassolid phase carriers to capture these complexes with signal amplification via MB-SA-biotin interaction.”change to “Streptavidin-modified magnetic beads (MB-SA) serve as solid-phase carriers to capture these complexes, enabling specific signal amplification through the MB-SA-biotin interaction.” We have corrected spelling and formatting errors, and noticed that compound adjectives in academic contexts require hyphens to better conform to terminology expression habits.
6. In section 3.3.1, “There are mycotoxins, heavy metals, endogenous macromolecules or other matrix interferents in the wheat extract, which could interfere with the antigen-antibody binding and thus affect chemiluminescence immunoassay performance. In order to eliminate these interferences, the extraction solvent was optimized. Three different extraction solvent system, including methanol-PBS (20:80, v/v), acetonitrile-PBS (20:80, v/v) and 0.01 M PBS were evaluated.” change to “There are mycotoxins, heavy metals, endogenous macromolecules or other matrix interferences in the wheat extract, which could interfere with the antigen-antibody binding and thus affect chemiluminescence immunoassay performance. To mitigate these interferences,  the extraction solvent was optimized. Three different extraction solvent system, including methanol-PBS (20:80, v/v), acetonitrile-PBS (20:80, v/v) and 0.01 M PBS were evaluated.” We have standardized the commonly used expressions for describing matrix interference in analytical chemistry. And change 'In order to eliminate these interferences' to' To mitigate these interferences', which is more realistic than 'eliminate' and makes the expression more rigorous.
7. In section 3.5.1, “As shown in Figure 7, there was a good dose-responserelationship between the concentration of pesticides and the corresponding RLU”. change to “As shown in Figure 7, there was a good dose-response relationship between the concentration of pesticides and the corresponding RLU”. We have eliminated the issue of inconsistent intervals and formats.

Comments 2: The extraction solvent is acetonitrile-PBS (20:80, v/v). The organic solvent content may affect antibody binding and enzyme activity. Indicate whether extracts are diluted before performing the immunoassay and report the final percentage of organic material in the reaction chamber.

Response 2: Thank you for pointing this out. We provide the following explanation for the issue you raised. We did not dilute the extract before conducting the immunoassay. However, during the immune reaction process, the initial reaction chamber contains 50 μL of AP-IgG, 50 μL of monoclonal antibody, 20 μL of sample, and 50 μL of biotinylated antigen solution for antigen antibody competition as described in Section 2.7 and Section 3.1. Due to acetonitrile accounting for 20% of the volume of the 20 μL sample, the percentage of organic solvent in the reaction chamber is approximately 2%.

Comments 3: For validation of the HPLC-MS method, please provide the main parameters or clearly cite the standard method with sufficient details for comparison.

Response 3: Thank you for pointing this out. Due to suggestions from other reviewers regarding the correlation analysis, we conducted repeated analyses on the samples in the accuracy evaluation and sample analysis sections. Due to the unavailability of the previous instrument, we have adopted another model of instrument for the experiment. We have revised to emphasize this point. To validate the UHPLC-MS/MS method, we provide the main parameters and clearly cite the standard method in section 2.8.4 of the manuscript, providing sufficient details for comparison. The content is as follows:

The supernatant of blank wheat samples that were spiked with at three levels of half time, one time, and two times the international maximum residue limits. The samples were analyzed by both UHPLC-MS/MS and chemiluminescence methods, and the accuracy of the methods was evaluated by calculating the average recovery rate and CVs. As shown in Table 1, UHPLC-MS/MS analysis was performed on a SCIEX 6500+ QTRAP™ MS/MS system operating in multiple reaction monitoring (MRM) mode and in positive mode with following source parameters: curtain gas 40, ion spray voltage 5500 V, source temperature 500 ℃, gas 1 and 2 both at 40 au, and CAD gas set to medium.                           

Triazolone and tebuconazole were separated on a Kinetex C18 column (100 × 2.1 mm, 1.8 μm, Phenomenex). The elutes were (A) water (0.1% FA, 1mM NH₄FA) and (B) methanol. The elution program was started with 10% B, after kept for 1min, the proportion of B was linearly increased to 90% in 9.0 min, which was held for 3 min. The proportion of B was then decreased back to 10 % in 0.5 min and kept for 3 min to equilibrate the column. The flow rate was set to 0.3 mL/min, and the column temperature was 40 ℃. The injection volume was 2 μL.

Table 1. Analytical parameters and retention time for determination of triazolone and tebuconazole with the UHPLC-MS/MS.

Analyte	Retention time/min	MRM Transition	Quantifier/Qualifier	CE/V	DP/V
triazolone	7.01	294.1-225.1	Quantifier	15	80
		294.1-197.1	Qualifier	22	80
tebuconazole	7.43	308.1-151.1	Quantifier	35	80
		310.1-127.1	Qualifier	55	80

 

Comments 4: Please coordinate the units, for example: clearly reconcile the pollution levels in micrograms/milliliter and the reports in milligrams/kilogram, and show the conversion based on a sample of 5 grams and an extraction of 20 milliliters.

Response 4: Thank you for pointing this out. Based on a sample of 5 grams and 20 milliliters of extraction solution, a contamination level of 1 milligrams/kilogram is equivalent to 1/4 microgram/milliliter. In this study, the LOD value was measured in micrograms/kilogram, which is converted to micrograms/milliliter. Therefore, a pollution level of 1 micrograms/kilogram is equivalent to 1/4000 micrograms/milliliter. We will unify the units in the manuscript to micrograms/milliliter. Therefore, after unifying the units, the determination results showed that the detection limit (LOD) of triazolone in wheat was 11.34 μg/kg, which is 0.002835 μg/mL. The LOD of tebuconazole is 2.56 μg/kg, which is 0.00064 μg/mL. In the national standard (GB 23200.121-2021), the detection limits for triazolone and tebuconazole in wheat are 200 μg/kg and 50 μg/kg, respectively. The detection limits for both are much lower than the national standard. And we have made specific data conversions in section 3.5.2 of the manuscript, as follows:

Under optimized conditions, LODs were determined to be 11.34 μg/kg (Namely 0.002835 μg/mL, converted from 5g sample/20mL extraction solution) for triazolone and 2.56 μg/kg (Namely 0.00064 μg/mL) for tebuconazole in wheat, while LOQ were far below the national standard.

Comments 5: The reported loading thickness of streptavidin of approximately 0.1 micrometers (100 nanometers) from TEM is unexpectedly large for a protein layer, and the units and explanation should be clarified. SEM/TEM images alone do not measure cohesion. 

Response 5: Thank you very much for pointing out these critical errors. You are absolutely correct, and we sincerely apologize for these inaccuracies. Due to the different scales in Figures 4c and 4d. The image in Figure 4c shows that the loading thickness limit of streptavidin is not clear enough. We magnified the transmission electron microscope (as shown in Figure 4d) and observed a clearer loading thickness of about 20-30nm for the modified layer. This protein layer can explain the successful loading of streptavidin onto magnetic beads. We completely agree that SEM and TEM imaging cannot measure cohesion. Our original intention was to describe the uniform morphology and surface coverage of the protein layer. And we have made revisions in section 3.5.2 of the manuscript.  

As shown in Figure 4c and 4d, streptavidin was loaded onto the surface of MB-SA with a thickness of approximately 20-30 nm. SEM and TEM images demonstrate the uniform morphology and complete surface coverage of the streptavidin layer, indicating stable attachment to the bead surface.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of this manuscript describe the development of an alkaline phosphatase immunoassay method, benchmarked against LC-MS to quantify triazole and tebuconazole in wheat. While the research is of potential interest to readers of Foods, there are several points that need to be addressed and clarified.  

1. There is an error in line 111 (Section 2.2), “Mettle Toledo” is incorrect, it is “Mettler Toledo”.
2. Section 2.2, line 117- Please provide the correct name of the MS instrument used in the study, is it HRMS? “U3000” is not recommended as shorthand. Please change this to Ultimate 3000, which is a common HPLC platform. I am assuming that the authors have shortened this to “U3000” erroneously. No information about the MS has been provided as such.
3. For table 3, precision test, it seems that there was only one reading per additive concentration, is this correct? Were replicates analyzed? It is recommended to have replicates for each measured concentration for greater confidence of RSD%. The same observation is made for Table 4. Please comment if replicates were analyzed for accuracy and precision of measurements.
4. It is recommended to include concluding remarks about the evaluation of the optimized reaction and extraction time in the conclusion section.

Author Response

Comments 1: There is an error in line 111 (Section 2.2), “Mettle Toledo” is incorrect, it is “Mettler Toledo”.

Response 1: Thank you very much for pointing this out, and we apologize for the error that occurred. We have made spelling changes in section 2.2 of the manuscript. The content is as follows:

The Mettler Toledo electronic analytical balance (Mettler Toledo Instruments (Shanghai) Co., Ltd.) was used to weigh samples. 

Comments 2: Section 2.2, line 117- Please provide the correct name of the MS instrument used in the study, is it HRMS? “U3000” is not recommended as shorthand. Please change this to Ultimate 3000, which is a common HPLC platform. I am assuming that the authors have shortened this to “U3000” erroneously. No information about the MS has been provided as such.

Response 2: Thank you for pointing out this point. I am sorry that we did miss the relevant information of the mass spectrometer. Due to suggestions from other reviewers regarding the correlation analysis, we conducted repeated analyses on the samples in the accuracy evaluation and sample analysis sections. Due to the unavailability of the previous instrument, we have adopted another model of instrument for the experiment. We have made detailed modifications and added information on the instrument model in manuscript 2.2.. The content is as follows:

The SCIEX 6500+ QTRAP™ UHPLC-MS/MS system (MA, USA) equipped with an electrospray ionization (ESI) source was used for UHPLC-MS/MS analysis.

And, in section 2.8.4, we supplemented the main parameters of specific liquid phase and mass spectrometry conditions.

As shown in Table 1, UHPLC-MS/MS analysis was performed on a SCIEX 6500+ QTRAP™ MS/MS system operating in multiple reaction monitoring (MRM) mode and in positive mode with following source parameters: curtain gas 40, ion spray voltage 5500 V, source temperature 500 ℃, gas 1 and 2 both at 40 au, and CAD gas set to medium.                           

Triazolone and tebuconazole were separated on a Kinetex C18 column (100 × 2.1 mm, 1.8 μm, Phenomenex). The elutes were (A) water (0.1% FA, 1mM NH₄FA) and (B) methanol. The elution program was started with 10% B, after kept for 1min, the proportion of B was linearly increased to 90% in 9.0 min, which was held for 3 min. The proportion of B was then decreased back to 10 % in 0.5 min and kept for 3 min to equilibrate the column. The flow rate was set to 0.3 mL/min, and the column temperature was 40 ℃. The injection volume was 2 μL.

Table 1. Analytical parameters and retention time for determination of triazolone and tebuconazole with the UHPLC-MS/MS.

Analyte	Retention time/min	MRM Transition	Quantifier/Qualifier	CE/V	DP/V
triazolone	7.01	294.1-225.1	Quantifier	15	80
		294.1-197.1	Qualifier	22	80
tebuconazole	7.43	308.1-151.1	Quantifier	35	80
		310.1-127.1	Qualifier	55	80

 

Comments 3: For table 3, precision test, it seems that there was only one reading per additive concentration, is this correct? Were replicates analyzed? It is recommended to have replicates for each measured concentration for greater confidence of RSD%. The same observation is made for Table 4. Please comment if replicates were analyzed for accuracy and precision of measurements.

Response 3: Thank you for this important observation. We are sorry for the confusion caused by the unclear presentation. For precision test (Table 3)，we analyzed seven independent replicates at each concentration level and three replicates for accuracy tests (Table 4). The values presented in the tables are the mean concentrations, and the RSD% values were calculated from these replicate measurements, providing statistically reliable precision data. To correct this, we have revised Tables 3 and 4 as follows: Table 3 lists the readings of 7 independent experiments. Table 4 only includes three parallel groups. Due to suggestions from other reviewers regarding correlation analysis, we increased the sample size in Table 4 and conducted a repeat analysis of the experiment, calculating the recovery rate and coefficient of variation (CV) values. But due to the addition of content to the front part of the manuscript, we have changed Table 3 to Table 4 and Table 4 to Table 5. The content is as follows:

Table 4. Test precision of the method.(n=7)

Pesticides	Spiked/ μg·mL-1	RLU value							RSD/%
		1	2	3	4	5	6	7
Triazolone	0.0000	301135	289002	291783	301825	273961	289467	292766	3.2
	0.0500	173294	163296	183961	172697	174061	169201	183661	4.3
	0.1000	106320	115290	101265	99027	110756	102656	108346	5.4
	0.2000	66892	68247	67248	66024	65288	65024	66024	1.7
Tebuconazole	0.0000	431067	412942	405817	424917	405901	433886	419688	2.7
	0.0125	275247	266912	246028	251708	271378	254869	269078	4.3
	0.0250	175924	166923	184609	174249	173402	158217	167042	4.9
	0.0500	91248	96589	92247	89347	88248	90347	89235	3.1

Table 5. Recovery test for MB-CLIA and UHPLC-MS/MS. (n=7)

Pesticides	Spiked/ μg·mL-1	MB-CLIA			UHPLC-MS/MS
		Measured/ μg·mL-1(Mean±SD)	Recovery/%	CV/%	Measured/ μg·mL-1(Mean±SD)	Recovery/%	CV/%
Triazolone	0.0500	0.0515±0.0034	103.1	6.5	0.0511±0.0020	102.3	3.9
	0.1000	0.1006±0.0065	100.1	6.5	0.1033±0.0041	103.3	4.0
	0.2000	0.1801±0.0043	90.1	2.4	0.1813±0.0056	90.7	3.1
Tebuconazole	0.0125	0.0129±0.0011	103.6	8.5	0.0127±0.0010	101.6	8.1
	0.0250	0.0250±0.0015	100.1	6.1	0.0250±0.0014	100.0	5.4
	0.0500	0.0502±0.0015	100.4	3.0	0.0499±0.0018	99.8	3.5

 

Comments 4: It is recommended to include concluding remarks about the evaluation of the optimized reaction and extraction time in the conclusion section.

Response 4: Thank you for making this suggestion. Due to the precipitation effect of acetonitrile on interfering macromolecules in wheat matrix, it can effectively reduce the extraction of matrix impurities and improve the enrichment of target pesticide residues. At the same time, a reasonable ratio with PBS can maintain a mild environment required for subsequent immune reactions, avoiding damage to antibody activity due to excessive solvent polarity. And 5 min vortex extraction is sufficient to completely extract the target triazole pesticide. Triazole pesticides bind to wheat substrate with weak interaction forces, resulting in low desorption activation energy. The extraction solvent can quickly penetrate the substrate and achieve solid-liquid mass transfer equilibrium of the target substance within 5 min. Extending the extraction time does not significantly improve the recovery rate, but instead increases the co extraction rate of impurities such as lipids and proteins in the matrix, exacerbating the matrix effect in subsequent testing. Extending the extraction time may also increase the experimental time and operating costs. Therefore, we made revisions and added a concluding statement in section 3.3 of the manuscript to demonstrate the reliability of the conclusion. The content is as follows:

As shown in Figure 5, the acetonitrile-PBS (20:80, v/v) system has a significantly better extraction efficiency for the target triazole pesticides than the other two systems. It is speculated that its advantage stems from the precipitation effect of acetonitrile on interfering macromolecules in the wheat substrate, which can effectively reduce the co-extraction of substrate impurities. Meanwhile, the reasonable ratio with PBS can maintain the mild environment required for subsequent immune reactions, avoiding the damage to antibody activity caused by overly strong solvent polarity. Thus, while improving the extraction efficiency, it can also reduce the impact of substrate interference on chemiluminescence signals. Based on this, acetonitrile-PBS (20:80, v/v) was determined as the optimal extraction solvent for the subsequent analysis.

As shown in Figure 6, there was no significant difference in the chemiluminescence signal values measured at different extraction times, indicating that 5 min vortex extraction is sufficient to completely extract the target triazole pesticide. The reason is that the binding force between triazole pesticides and wheat substrate is weak, resulting in low desorption activation energy. The extraction solvent can quickly penetrate the substrate and achieve solid-liquid mass transfer equilibrium of the target substance within 5 min. Extending the extraction time does not significantly improve the recovery rate, but instead increases the co-extraction rate of impurities such as lipids and proteins in the matrix, exacerbating the matrix effect in subsequent testing.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents the development and validation of a magnetic bead-based chemiluminescence immunoassay (MB-CLIA) for the detection of triazolone and tebuconazole in wheat. The work addresses a relevant need for rapid, high-throughput, and sensitive methods for pesticide residue monitoring.
However, there is no statistical analysis section in this manuscript. The manuscript includes basic descriptive statistics, such as RSD, recovery percentages, and 4-parameter logistic calibration fitting. No hypothesis testing, such as ANOVA or equivalent is applied.
The accuracy is assessed by recovery rates (Table 4), which is good, but to fully validate the new CLIA method against the reference HPLC-MS method, a proper correlation analysis is required.

Therefore, the authors are encouraged to strengthen the statistical rigor by applying appropriate inferential statistics, reporting confidence intervals, and providing a quantitative comparison with the reference method in accordance with established analytical validation guidelines.

Author Response

Comment: This manuscript presents the development and validation of a magnetic bead-based chemiluminescence immunoassay (MB-CLIA) for the detection of triazolone and tebuconazole in wheat. The work addresses a relevant need for rapid, high-throughput, and sensitive methods for pesticide residue monitoring.

However, there is no statistical analysis section in this manuscript. The manuscript includes basic descriptive statistics, such as RSD, recovery percentages, and 4-parameter logistic calibration fitting. No hypothesis testing, such as ANOVA or equivalent is applied.

The accuracy is assessed by recovery rates (Table 4), which is good, but to fully validate the new CLIA method against the reference HPLC-MS method, a proper correlation analysis is required.

Therefore, the authors are encouraged to strengthen the statistical rigor by applying appropriate inferential statistics, reporting confidence intervals, and providing a quantitative comparison with the reference method in accordance with established analytical validation guideline.

Response: Thank you sincerely for pointing out this issue. Correlation analysis is essential for comparing new methods with traditional methods. We increased the sample size and conducted repeated analyses. Confirm the accuracy of the results by measuring the recovery rate and coefficient of variation (CV) values of this method. Due to the unavailability of the previous instrument, we have adopted another model of instrument for the experiment. Comparison was validated using traditional UHPLC-MS/MS. We conducted a correlation analysis between the results of our developed method and traditional UHPLC-MS/MS method using reported confidence intervals (as shown in Figure 8) to enhance statistical rigor and validate the consistency of the results. In addition, we also cited a reference on correlation analysis in this section. We have added experimental content to this in section 2.8.4. and section 3.5.4.. But due to the addition of content to the front part of the manuscript, we have changed Table 4 to Table 5. The content is as follows:

To verify method accuracy, the supernatant of blank wheat samples was spiked with triazolone and tebuconazole at 0.5×, 1×, and 2× the international maximum residue limits and then analyzed by MB-CLIA, the results were obtained by UHPLC-MS/MS[40]. As shown in Table 5, the recovery rates of MB-CLIA and UHPLC-MS/MS ranged from 90.1% to 103.6% and 90.7% to 103.3%, respectively, with coefficients of variance (CV) between 2.4% to 8.5% and 3.1% to 8.1%. The above recovery rate and CV are within an acceptable range, indicating that the established method meets the requirements for quantitative analysis of triazolone and tebuconazole. As shown in Figure 8, by establishing a linear equation (Y represents the concentration determined by UHPLC-MS/MS method, X represents the concentration determined by MB-CLIA method), the correlation coefficient was calculated to be greater than 0.99. Figure 8a shows that the 95% confidence intervals (CI) for slope (b=1.00576, 95% CI: 0.9728-1.03872) and intercept (a=0.000692057, 95% CI: -0.00335-0.00474) include ideal values of 1 and 0, respectively. Figure 8b shows that the 95% confidence intervals (CI) for slope (b=0.99515, 95% CI: 0.96722-1.02308) and intercept (a=0.0000450932, 95% CI: -0.000974054-0.000883868) also include ideal values of 1 and 0, respectively, indicating that there is no proportional or systematic bias between the two methods. The detection results of the two methods showed a good correlation, indicating that the developed MB-CLIA is highly consistent with the standard instrument method, demonstrating the accuracy and practicality of the detection.

Table 5. Recovery test for MB-CLIA and UHPLC-MS/MS. (n=7)

Pesticides	Spiked/ μg·mL-1	MB-CLIA			UHPLC-MS/MS
		Measured/ μg·mL-1(Mean±SD)	Recovery/%	CV/%		Measured/ μg·mL-1(Mean±SD)	Recovery/%	CV/%
Triazolone	0.0500	0.0515±0.0034	103.1	6.5		0.0511±0.0020	102.3	3.9
	0.1000	0.1006±0.0065	100.1	6.5		0.1033±0.0041	103.3	4.0
	0.2000	0.1801±0.0043	90.1	2.4		0.1813±0.0056	90.7	3.1
Tebuconazole	0.0125	0.0129±0.0011	103.6	8.5		0.0127±0.0010	101.6	8.1
	0.0250	0.0250±0.0015	100.1	6.1		0.0250±0.0014	100.0	5.4
	0.0500	0.0502±0.0015	100.4	3.0		0.0499±0.0018	99.8	3.5

Figure 8. Comparison between MB-CLIA and UHPLC-MS/MS. (a) triazolone (b) tebuconazole

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have satisfactorily addressed this reviewer's comments.

Author Response

Comments 1: The title should be changed to “A Chemiluminescent Magnetic Enzyme Immunoassay Method for 2 Triazole Pesticide Detection in Wheat.”

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have changed the title to “A Chemiluminescent Magnetic Enzyme Immunoassay Method for 2 Triazole Pesticide Detection in Wheat.”

Comments 2: Abstract: A sentence should be included stating that the two analytes under study have not been detected in the wheat samples analyzed.

Response 2: Thank you for pointing this out. We have added the following content to the abstract section：

In actual sample detection, this method did not detect triazolone and tebuconazole, and the results were consistent with UHPLC-MS/MS.

Comments 3: The units should be written in the same way. For example, Section 3.5.2 includes µg/kg, whereas in Table 3 or Table 5 the units are written as µg·kg^-1 or µg·ml^-1, respectively. Therefore, the entire text should be revised.

Response 3: Thank you for pointing this out. We apologize for the issue of inconsistent units in the manuscript. We have standardized the units in Table 3 to µg·ml^-1_. The detailed revisions to the abstract, 3.5.2., Table 3, and conclusion sections of the manuscript are as follows:

Abstract:

Under the optimal conditions, the method demonstrated that the limits of detection (LOD) of triazolone and tebuconazole were 0.002835 μg/mL and 0.00064 μg/mL, respectively.

3.5.2.

After condition optimization, the method was further developed. As shown in Table 3, under optimized conditions, LODs were determined to be 0.002835 μg/mL for triazolone and 0.00064 μg/mL for tebuconazole in wheat, while LOQ were far below the national standard. It indicated that the method had high sensitivity and could detect trace triazole residue in wheat samples.

Table 3. The detected limit of triazolone and tebuconazole in wheat blank.

	Triazolone/ μg·mL-1	Tebuconazole/ μg·mL-1
Average	0.0008200	0.0002025
SD	0.0006725	0.0001450
LOD	0.0028350	0.0006400
LOQ	0.0075450	0.0016525

Conclusion:

The detection limits of triazolone and tebuconazole in wheat were 0.002835 μg/mL and 0.00064 μg/mL, respectively, with recoveries ranging from 90.1% to 103.6%.

Comments 4: In Section 3.5.6, the authors should describe in greater detail the type of wheat samples that have been analyzed.

Response 4: Thank you for pointing this out. We apologize for not knowing the variety of the collected wheat samples, but all wheat samples were collected from fields in Shandong and Henan provinces. We have made the following modifications in section 2.1 of the manuscript:

Five wheat samples were collected from Shandong Province and Henan Province respectively. All samples were collected in the field.

Comments 5: Figures 2, 3, 5, 7, 8: The size of the words should be increased.

Response 5: Thank you for pointing this out. We have increased the font size in Figures 2, 3, 5, 7, and 8. And, in order to unify the fonts in the figure, we also made modifications to the font size in Figure 1 and 6. We have inserted new images into the manuscript. And we have packaged the modified original image in a word.

Comments 6: References: In some cases, the full title of the journal is included, and in others, abbreviations are used. It should be written in the same way and following the publisher's guidelines. The number on the last page of the references should be complete.

Response 6: Thank you for pointing this out. We have comprehensively revised the entire reference list in accordance with the publisher's guidelines. Specifically, we have made all journal names complete. In addition, we carefully checked and supplemented the incomplete page number on the last page. We also carefully examined the entire reference list to confirm consistency between the format, such as author name, journal name, publication year, volume code, page number, and DOI. The following is the modified references.

References

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Comments 7: The DOI of the articles should be included in the References Section.

Response 7: Thank you for pointing this out. We have added DOIs to each article in the reference section as required, verifying the accuracy of each DOI to ensure its effectiveness and proper formatting. We also carefully examined the entire reference list to confirm consistency in all formatting aspects (such as author name, journal name, publication year, volume, page number, DOI) with journal requirements. We have made detailed revisions in the manuscript.