Copper-Enhanced Gold Nanoparticle Sensor for Colorimetric Histamine Detection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors propose a rapid, colorimetric sensor for copper-mediated histamine detection enhanced via gold nanoparticles. Numerous studies have been reported over the past five years on histamine detection using nanoparticles, both for electrochemical and colorimetric applications. I believe that this study currently lacks clear innovation and practical applicability. I suggest the possibility of resubmission and reconsideration if the authors address the following points. Please see the detailed comments below:

-The authors mentioned other reported AuNP-based detection assays, stating that they are pH-dependent. However, there are actually more studies than the single one they cited, including some recent reports. To support their claim, they wrote: “However, these systems generally rely on electrostatic interactions in unbuffered media, making them sensitive to pH variations and limiting their applicability in controlled analytical conditions.” Regarding applicability in real biological samples, which do exhibit pH variations, I do not believe that using buffered solutions is the main concern here, as these are typically employed only to characterize the developed sensors.

-A table summarizing all previously reported applications using gold nanoparticles should be added to the Discussion section to highlight any innovation of the present work. Does the novelty lie in the limit of detection, analysis time, or sample applicability?

-“The assay was performed in 1 mM MES buffer (pH 5.5) and 1mM HEPES buffer (pH 77 7.0) to evaluate pH effects on sensor performance. “ What about the buffer capacity in so small concentration? In this case this should be investigated as the usual concentration employed is at least 10 mM.

-In Figure 2 1) wavelength should be corrected and concentration units should be added for the calibrators, 2) Figure 2b should be rescaled as the initial calibrators are not visible, 3) figure quality should be improved, 4) what was the value selected in the cases that there is not absorbance peak at 620 nm?

-For figure 3 the authors state: “These results confirm that histamine alone cannot induce significant plasmonic changes in the AuNPs. Cu2+ is essential for rapid and complete sensor response.” This should be statistically assured and not just by seeing the increase of the response ratio as the statement is a bit vague.
-The Effect of Buffer Conditions section needs better explanation especially according to the innovation statement in comparison to the reported works from the authors.

-The authors state : “ The metal ion-enhanced colorimetric approach demonstrates sub-micromo lar sensitivity in the range relevant to biological histamine concentrations, with potential  applications in biosensing and analytical chemistry.” How is clinically relevant the limit of detection of the proposed study? The sensing system should be tested also in biological samples or at least spiked biofluids to prove the possibility for clinical applications.

Author Response

Thank you for your helpful comments. We have revised the manuscript accordingly, and our point-by-point responses are as follows.

 

The authors propose a rapid, colorimetric sensor for copper-mediated histamine detection enhanced via gold nanoparticles. Numerous studies have been reported over the past five years on histamine detection using nanoparticles, both for electrochemical and colorimetric applications. I believe that this study currently lacks clear innovation and practical applicability. I suggest the possibility of resubmission and reconsideration if the authors address the following points. Please see the detailed comments below:

 

1. The authors mentioned other reported AuNP-based detection assays, stating that they are pH-dependent. However, there are actually more studies than the single one they cited, including some recent reports. To support their claim, they wrote: “However, these systems generally rely on electrostatic interactions in unbuffered media, making them sensitive to pH variations and limiting their applicability in controlled analytical conditions.” Regarding applicability in real biological samples, which do exhibit pH variations, I do not believe that using buffered solutions is the main concern here, as these are typically employed only to characterize the developed sensors.

 

We thank the reviewer for this insightful comment. We agree that simply using buffered solutions is not an innovation in sensor development, and our previous wording overstated this point. In the revised manuscript, we have removed the sentence claiming that “these systems generally rely on electrostatic interactions in unbuffered media, making them sensitive to pH variations and limiting their applicability in controlled analytical conditions.” We have also added several AuNP-based histamine sensors [26, 27, 30, 31, 33–35], and expanded the Introduction (line 47–73), Discussion (260–265), and Conclusion (300–301).

 

 

2. A table summarizing all previously reported applications using gold nanoparticles should be added to the Discussion section to highlight any innovation of the present work. Does the novelty lie in the limit of detection, analysis time, or sample applicability?

 

We appreciate this suggestion. In the revised manuscript, we have added a new table in the Discussion (Table 1) summarizing representative AuNP-based colorimetric assays for histamine, including their surface modification, recognition mechanism, linear range and LOD. This comparison clarifies that most previous systems employ a single recognition mode, whereas our assay is explicitly designed as a Cu2+-assisted dual-recognition system at the nanoparticle interface. Thus, the main novelty of the present work lies in the mechanistic design and analysis of this dual-recognition scheme.

 

 

3. “The assay was performed in 1 mM MES buffer (pH 5.5) and 1mM HEPES buffer (pH 77 7.0) to evaluate pH effects on sensor performance. “ What about the buffer capacity in so small concentration? In this case this should be investigated as the usual concentration employed is at least 10 mM.

 

We appreciate the reviewer’s concern regarding the use of 1 mM MES (pH 5.5) and 1 mM HEPES (pH 7.0). In our assay, the highest histamine concentration is 10 μM and the Cu²⁺ concentration is of the same order of magnitude. Thus, the total amount of acid/base equivalents introduced by the analyte is less than a few percent of the buffer capacity of a 1 mM solution. So, 1 mM buffer is sufficient to keep the pH essentially constant the micromolar concentration range used in this study.

We chose a low buffer concentration to minimize ionic strength, because high salt levels are known to strongly affect the electrostatic stabilization of citrate-stabilized AuNPs and to inhibit Cu²⁺-histamine coordination.

 

4. In Figure 2 1) wavelength should be corrected and concentration units should be added for the calibrators, 2) Figure 2b should be rescaled as the initial calibrators are not visible, 3) figure quality should be improved, 4) what was the value selected in the cases that there is not absorbance peak at 620 nm?

 

We thank the reviewer for these helpful comments on Figure 2.

(1) We have corrected the typo “Wavelangth” to “Wavelength” and added the concentration unit (μM) for the histamine calibrators in Figure 2a.

(2) To make the responses at the lowest histamine concentrations clearly visible, we have added an inset in Figure 2b that shows an expanded view of the low-concentration region.

(3) All graphs have been redrawn in GraphPad Prism, and the figures have been re-exported at a resolution of at least 600 dpi to improve image quality.

(4) We clarify that A620 and A520 were defined as the absorbance values at the fixed wavelengths of 620 nm and 520 nm, respectively, and the sensor response was calculated as the ratio A620/A520. The ratio was computed from the absorbance at these two fixed wavelengths, regardless of the detailed spectral shape.

 

 

5. For figure 3 the authors state: “These results confirm that histamine alone cannot induce significant plasmonic changes in the AuNPs. Cu²⁺ is essential for rapid and complete sensor response.” This should be statistically assured and not just by seeing the increase of the response ratio as the statement is a bit vague.

 

Thank you for this suggestion. We have added a bar graph including AuNPs alone (0 μM histamine), histamine without Cu²⁺, and histamine with Cu²⁺ as Figure 3a, and we analyzed these data using one-way ANOVA with Tukey’s post hoc test (line 157–161). This analysis showed statistical difference between AnNPs alone and histamine without Cu²⁺, but the increase induced by histamine alone was still much smaller than that observed for histamine with Cu²⁺.  These results support our conclusion that histamine alone produces only a modest plasmonic change, whereas Cu²⁺ markedly amplifiers the sensor response.

 

 

6. . The Effect of Buffer Conditions section needs better explanation especially according to the innovation statement in comparison to the reported works from the authors.

 

Thank you for this comment. We have expanded the “Effect of Buffer Conditions” section to explain in more detail how pH and ionic strength influence the sensor response in the context of the Cu²⁺-mediated dual-recognition mechanism (line 165–169, 174–179).

 

 

7. The authors state : “ The metal ion-enhanced colorimetric approach demonstrates sub-micromolar sensitivity in the range relevant to biological histamine concentrations, with potential applications in biosensing and analytical chemistry.” How is clinically relevant the limit of detection of the proposed study? The sensing system should be tested also in biological samples or at least spiked biofluids to prove the possibility for clinical applications.
   
   We appreciate this important comment. Our intention was not to claim that the present system is ready for direct clinical diagnostics, but rather that its sensitivity is compatible with micromolar histamine levels typically sued in model biological studies. We agree that truly clinical applications would require validation in complex biofluids and, in many cases, lower detection limits. To avoid any overstatement, we have revised our wording in both the Abstract (line 17–22) and the Discussion (line 256–259).

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors report copper-enhanced gold nanoparticle sensor for colorimetric histamine detection. The organization of the paper is good, and the results are clearly presented. Some concerns need to be addressed to improve the manuscript.

1. There is no data for Au NPs. FESEM/TEM and XRD should be included.
2. How was the pH of 5.5 selected for the MES buffer? What happens at higher or lower pH?
3. The sensor showed a linear response up to 10 μM concentration. Why was this concentration selected for the selectivity test?
4. Why is the limit of detection higher compared with the previous report listed in Table 1?
5. Illustrate the number of experimental data points chosen for plots containing error bars.
6. Post-analysis of the sample could also be interesting to see.
7. The conclusions should be improved and explained in detail. This section contains sentences similar to those in the abstract and the last paragraph of the introduction.

Author Response

Thank you for your helpful comments. We have revised the manuscript accordingly, and our point-by-point responses are as follows.

 

In this study, the authors report copper-enhanced gold nanoparticle sensor for colorimetric histamine detection. The organization of the paper is good, and the results are clearly presented. Some concerns need to be addressed to improve the manuscript.

1. There is no data for Au NPs. FESEM/TEM and XRD should be included.

 

RESPONSE: We appreciate the reviewer’s suggestion. In this study, we did not synthesize AuNPs inhouse; instead, we use commercially available citrate-stabilized AuNPs (nominal diameter 20 nm, Sigma-Aldrich) as received. The AuNPs themselves are not a variable in our experimental design, and no chemical or thermal treatments that could affect their size or crystallinity were applied. We routinely confirmed the quality of each batch by UV-vis spectroscopy, which consistently showed a LSPR peak at = 520 nm without noticeable shift or broadening, indicating that the particle size and aggregation state remained unchanged during the experiments. For this reason, we did not include additional FESEM/TEM or XRD data in the manuscript.

 

1. How was the pH of 5.5 selected for the MES buffer? What happens at higher or lower pH?
   
   

RESPONSE: We thank the reviewer for this question. Our choice of pH 5.5 was guided by the speciation of histamine and the dual-recognition mechanism. According to reported stability constants [30,31], histamine is predominantly in a doubly protonated form (H₂His²⁺) around pH 5–6, which enhances electrostatic adsorption onto the negatively charged citrate-coated AuNPs, while the imidazole group can still coordinate to Cu²⁺. We therefore selected pH 5.5 in MES as a representative mildly acidic condition.

As shown in the Section 3.4, the A₆₂₀/A₅₂₀ response at pH 7.0 is much lower than at pH 5.5, which we attribute to the reduced positive charge of histamine and weaker electrostatic adsorption. At more lower pH values than 5.5, the imidazole ring becomes fully protonated, which is expected to diminish Cu²⁺–imidazole coordination and weaken the dual-recognition effect.

1. The sensor showed a linear response up to 10 μM concentration. Why was this concentration selected for the selectivity test?
   
   

RESPONSE: Thank you for this comment. We selected 10 μM because it is the highest concentration within the linear response range of our sensor. Using this upper limit ensures that the histamine signal is sufficiently large for a clear comparison with potential interferents, while still remaining within the calibrated working range.

1. Why is the limit of detection higher compared with the previous report listed in Table 1?

 

RESPONSE: We appreciate this comment. To report lower LODs often employ optimized condition such as optimization of size and concentration of AuNPs. In contrast, our system uses label-free commercial AuNPs without further optimization and a straightforward absorbance measurement, which is sufficient to monitor histamine in the micromolar range in simple buffered system. We agree that there is substantial room for further improvement of sensitivity by adjusting AuNP size and concentration.

1. Illustrate the number of experimental data points chosen for plots containing error bars.
   
   

RESPONSE: Thank you for your pointing this out. We have clarified the number of data points by adding the statement “All data with error bars are presented as mean ± SD from three independent measurements (n = 3)” in the Materials and Methods section (line 109–110 in the revised manuscript).

1. Post-analysis of the sample could also be interesting to see.

 

RESPONSE: We agree that post-analysis of the samples after histamine/Cu²⁺-induced aggregation would be interesting and could provide additional structural insight (e.g., by TEM or DLS). However, the present study was designed as a simple mechanistic model focusing on the optical response, so we did not perform further structural characterization of the aggregates. In the revised Discussion, we now explicitly mention that such post-analysis will be an important direction for future work to better correlate aggregate structure with the observed plasmonic changes.

1. The conclusions should be improved and explained in detail. This section contains sentences similar to those in the abstract and the last paragraph of the introduction.

 

RESPONSE: Thank you for your comment. We have rewritten the Conclusions section to provide a more detailed summary of the dual-recognition mechanism, assay performance, and positioning of the present proof-of-concept within the context of previously reported AuNP-based histamine sensors. The revised Conclusions now emphasize the mechanistic framework and the potential adaptability of the metal ion–assisted dual-recognition concept, rather than simply repeating the Abstract.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed most of the reviewers' suggestions and concerns and the article is now suggested towards publication.