Excitation–Emission Fluorescence Mapping Analysis of Microplastics That Are Typically Pollutants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a study aiming at characterizing and identifying microplastics (MPs) based on fluorescence induced by multiple excitation wavelengths. The paper demonstrates scientific rigor, it is well written and well structured, with experimental methods and results presented clearly and coherently.

Given the extensive research on identifying various types of MPs in aquatic environments in recent years, the authors’ approach of using fluorescence spectroscopy with multiple excitation wavelengths to obtain specific spectroscopic fingerprints of polymer types is interesting and their results add value to the existing literature in this field. The FLE maps shown in figure 3 are especially informative, and I believe that these results could potentially be used in future studies to automate the identification of microplastics. The authors have validated their samples using various experimental techniques, including SEM, XPS and Raman spectroscopy, and the discussion of the origin of the spectroscopic peaks is thorough.

However, since the authors are studying MPs in DI water, which suggests that they believe their method could be applied directly to detect MPs in water samples, I have two issues that I would like the authors to comment, as doing so will further strengthen the manuscript:

1.      A discussion on the sensitivity of the proposed method, given that the authors used a concentration of 4.5% w/v. Is the sensitivity of the FL signal (without using a laser as an excitation source, as for example proposed in reference [37] and also in Appl. Phys. B 130, 168 (2024), https://doi.org/10.1007/s00340-024-08308-8) sufficient for direct application to real aquatic samples?

2.      A discussion on the method’s potential for distinguishing MPs from other organic materials in aqueous environments.

Finally, figure 3a,b,c will be more clear if emission intensity is shown with one decimal instead of three.

The topic of this work aligns well with the scope of Photochem, and I can recommend this manuscript for publication in the journal.

Author Response

Firstly, we would like to thank the editor for allowing us to improve the manuscript based on the invaluable comments of reviewer 1. We are very grateful to the reviewers for their feedback and constructive suggestions, which have further contributed to enhancing the quality of the manuscript. We have addressed all the comments raised by the reviewer 1 and have carefully revised the manuscript in accordance with their suggestions. For clarity, we have answered queries of reviewers point-by-point in the “Response to Reviewers’ Comments” and highlighted the changes in the revised manuscript.

Reviewer #1:

This manuscript presents a study aiming at characterizing and identifying microplastics (MPs) based on fluorescence induced by multiple excitation wavelengths. The paper demonstrates scientific rigor, it is well written and well structured, with experimental methods and results presented clearly and coherently.

Given the extensive research on identifying various types of MPs in aquatic environments in recent years, the authors’ approach of using fluorescence spectroscopy with multiple excitation wavelengths to obtain specific spectroscopic fingerprints of polymer types is interesting and their results add value to the existing literature in this field. The FLE maps shown in figure 3 are especially informative, and I believe that these results could potentially be used in future studies to automate the identification of microplastics. The authors have validated their samples using various experimental techniques, including SEM, XPS and Raman spectroscopy, and the discussion of the origin of the spectroscopic peaks is thorough.

However, since the authors are studying MPs in DI water, which suggests that they believe their method could be applied directly to detect MPs in water samples, I have two issues that I would like the authors to comment, as doing so will further strengthen the manuscript:

Comment:

A discussion on the sensitivity of the proposed method, given that the authors used a concentration of 4.5% w/v. Is the sensitivity of the FL signal (without using a laser as an excitation source, as for example proposed in reference [37] and also in Appl. Phys. B 130, 168 (2024), https://doi.org/10.1007/s00340-024-08308-8) sufficient for direct application to real aquatic samples?

Response:

Thank you for highlighting the critical aspect of sensitivity in our proposed method for microplastic detection in real aquatic samples. Fluorescence spectroscopy, as utilised in our study, typically achieves high sensitivity levels, often in the parts-per-billion (ppb) to parts-per-trillion (ppt) range. However, this sensitivity is influenced by several factors, including the detection capabilities of the instrument setup, the characteristics of the fluorescent molecules being analysed, the surrounding matrix or solvent, and the selected wavelength range.

In our study, we used a high-power but incoherent light source, successfully obtaining a strong emission signal from microplastics.  Thank you for bringing this research article (Laser Induced Fluorescence and Machine Learning: A Novel Approach to Microplastic Identification - Appl. Phys. B 130, 168 (2024), https://doi.org/10.1007/s00340-024-08308-8) to our attention; we have included it in our manuscript as reference number 68. We anticipate that using a laser source similar to the one used in these papers [37 and 68] but with the suggested excitation wavelengths determined in our current work could further enhance the fluorescence signal, thereby enhancing sensitivity and improving detection capability in real aquatic samples.

We have provided an expanded discussion in the supplementary information section S0 of the revised manuscript on the sensitivity limitations of intrinsic fluorescence-based detection, along with the potential of laser sources to overcome these limitations.

See the following paragraph incorporated in the revised version of the manuscript supplementary information:

Fluorescence spectroscopy is known for high sensitivity, often reaching the detection level of parts per billion (ppb) [1] and, in some cases, even parts per trillion (ppt) [2]. This sensitivity depends on different factors such as instrument setup, the specific fluorescent molecules being analysed, the solvent or matrix, and the wavelength range used. For microplastics, the fluorescence technique has demonstrated the ability to detect concentrations as low as 0.2 µg/g (200 ppb) in natural sea salt samples [3]. This was achieved using pyrene as a fluorescent probe to detect polystyrene microparticles on the salt surface. Another study highlighted the use of Nile Red as a fluorescent dye,  using the Cyclops 7F fluorometer paired with the NR-H, which enabled the detection of MPs in environmental samples at levels from 100 parts per billion (ppb) down to 0.1 ppb [4]. While these studies demonstrated high sensitivity for microplastic detection using external dyes or probe-based surfaces, the sensitivity of fluorescence methods based on the intrinsic fluorescence of microplastics themselves remains unknown. Recent studies [37, 38, 69] employed fluorescence spectroscopy with a 405 nm laser source to acquire fluorescence fingerprints of microplastics, but precise sensitivity levels have not been reported.

Our current research used a high-power, incoherent light source to study intrinsic emissions from microplastics, yielding strong fluorescence signals. We believe that using laser sources of excitation wavelengths investigated in this study can further enhance intrinsic fluorescence emission, potentially enabling the detection of microplastics in real aquatic samples. However, further research is necessary to determine the precise sensitivity level of this technique for detecting microplastics solely based on intrinsic fluorescence.

References

1. Childress, E.S., et al., Ratiometric fluorescence detection of mercury ions in water by conjugated polymer nanoparticles. Analytical chemistry, 2012. 84(3): p. 1235-1239.

     2. Oki, Y., K. Furukawa, and M. Maeda, Extremely sensitive Na detection in pure water by laser ablation atomic fluorescence spectroscopy. Optics Communications, 1997. 133(1): p. 123-128.

3. Costa, C.Q., et al., Fluorescence sensing of microplastics on surfaces. Environmental Chemistry Letters, 2021. 19: p. 1797-1802.
4. Peinador, R.I., P.T. H.P, and J.I. Calvo, Innovative application of Nile Red (NR)-based dye for direct detection of micro and nanoplastics (MNPs) in diverse aquatic environments. Chemosphere, 2024. 362: p. 142609.

Comment:

A discussion on the method’s potential for distinguishing MPs from other organic materials in aqueous environments.

Response:

Thank you for raising the important question of this method's potential to distinguish microplastics (MPs) from other organic materials in aqueous samples. In our study, we emphasised fluorescence spectroscopy's ability to differentiate MPs based on unique excitation-emission characteristics. While many organic materials can exhibit fluorescence, the emission spectra of MPs generally display distinct profiles due to their polymeric structures and specific additives, which set them apart from natural organic matter (NOM) that are typically found in aquatic environments.

To further improve discrimination between MPs and organic materials, we carefully selected excitation wavelengths that are likely to enhance the intrinsic fluorescence of MPs while minimising overlap with other organic compounds. Additionally, we acknowledge that incorporating advanced techniques, such as machine learning algorithms or pattern recognition tools, could aid in more accurately distinguishing MPs from other materials based on spectral differences.

In the revised manuscript, we have expanded the discussion to include this distinction challenge and the methodological adaptations necessary to address it. A discussion on the method’s ability to differentiate MPs from NOM can be found in the revised version of the manuscript (Revision can be found in the Main Text, Section 3, Page 11-12, Lines 362-375).

Thank you for this valuable suggestion, which has allowed us to enhance our discussion of the method’s applicability in real-world conditions.

See the following paragraph incorporated in the revised version of the manuscript:

The challenge of distinguishing microplastics (MPs) from other organic materials in real aqueous environments can be catered using unique excitation-emission characteristics of MPs observed in this study. MPs exhibit distinct spectral profiles due to their polymeric structures and specific additives, differentiating them from natural organic matter that are commonly present in aquatic samples [65]. However, in some cases, the fluorescence spectra of microplastics overlap with the spectra obtained from organic matter, which can complicate the detection and identification process [35, 36]. By carefully selecting an optimised excitation wavelength, the intrinsic fluorescence of MPs can be enhanced while minimising interference from other organic compounds, a strategy also highlighted in a recent fluorescence-based detection study [66]. Furthermore, studies have also suggested that integrating machine learning or spectral pattern recognition could significantly improve this method’s ability to distinguish MPs from organic materials present in the aquatic sample [67, 68]. These methodological considerations will allow our approach to distinguish MPs better in real aqueous environmental matrices.

Comment:

Finally, figure 3a,b,c will be more clear if emission intensity is shown with one decimal instead of three.

Response:

Thank you for the suggestion. We have adjusted the emission intensity in Figure 3a, b, and c to display values with one decimal place instead of three. The updated figures can be found in the revised manuscript (Revision can be found in Main Text, see Fig- 3 (a,b, and c), Page 9).

Reviewer 2 Report

Comments and Suggestions for Authors

Label-free identification of microplastics without exogenous substances interference is vital. This manuscript proposed a fluorescence excitation-emission mapping method to reveal spectral fingerprints of various microplastics. The fingerprints of PP, PET and PS were exhibited, respectively. However, some issues still need to be considered.

1. Table 1 exhibited the existence of C─O in PS and PP samples. The analyses of XPS and impurity results need to be re-checked.

2. It seems that the fluorescence signals are derived from aqueous extraction. Please clarify the emission mechanism and highlight the novelty and superiority of this method.

3. Some text formatting errors exist in the manuscript.

Author Response

Responses to the comments from reviewer 2

Firstly, we would like to thank the editor for allowing us to improve the manuscript based on the invaluable comments of reviewer 2. We are very grateful to the reviewers for their feedback and constructive suggestions, which have further contributed to enhancing the quality of the manuscript. We have addressed all the comments raised by reviewer 2 and carefully revised the manuscript as per their suggestions. For clarity, we have answered reviewers' queries point-by-point in the “Response to Reviewers’ Comments” and highlighted the changes in the revised manuscript.

Reviewer #2:

Label-free identification of microplastics without exogenous substances interference is vital. This manuscript proposed a fluorescence excitation-emission mapping method to reveal spectral fingerprints of various microplastics. The fingerprints of PP, PET and PS were exhibited, respectively. However, some issues still need to be considered.

Comment:

Table 1 exhibited the existence of C─O in PS and PP samples. The analyses of XPS and impurity results need to be re-checked.

Response:

Thank you for raising this point regarding the existence of C─O bonds in the XPS analysis of polystyrene (PS) and polypropylene (PP) samples. We have carefully re-examined the XPS data and considered possible sources of the oxygenated group in PS and PP.

The presence of C─O bonds in these polymers can be attributed to surface oxidation, which is a known phenomenon when polymers are exposed to ambient air or during sample preparation. Studies have shown that polystyrene (PS) and polypropylene (PP), despite being primarily hydrocarbon-based, can develop surface oxidation through environmental exposure, leading to the formation of oxygen-containing groups such as C─O and C=O bonds. This oxidation can occur due to prolonged exposure to atmospheric oxygen, UV radiation, or handling processes that introduce minor surface modifications.

In our XPS analysis, the detected C─O bonds in PS and PP are thus consistent with this established behaviour and likely represent surface-level oxidation rather than bulk impurities. We have incorporated this in the revised manuscript (Revision can be found in the Main Text, Section 3, Page 4, Lines 176-183) to clarify this interpretation and include relevant references supporting this phenomenon in polymer surface chemistry.

See the following paragraph incorporated in the revised version of the manuscript:

In addition to these impurities, our results showed a considerable percentage of C-O bonds in polystyrene (7 %) and polypropylene (11.2 %) samples. The detected C─O bonds suggest surface-level oxidation, a well-established phenomenon in polymer surface chemistry. Previous studies indicate that PS and PP, though primarily hydrocarbon-based, can undergo oxidation when exposed to ambient air, resulting in oxygenated groups such as C─O and C=O on the polymer surface [38, 39]. This process is often initiated by prolonged atmospheric oxygen exposure, UV radiation, or handling during sample preparation.

Comment:

It seems that the fluorescence signals are derived from aqueous extraction. Please clarify the emission mechanism and highlight the novelty and superiority of this method.

Response:

Thank you for raising this point regarding the fluorescence signals and their derivation from aqueous extraction. We have carefully re-evaluated the fluorescence data of the previously conducted experiment of only DI water to confirm that the fluorescence signals are indeed emitted solely from the microplastics.

In the control experiment, we used pure deionized (DI) water without any microplastics, as shown in Figure S2 in the supplementary information. This confirmed that DI water alone exhibited no fluorescence emission, validating that all observed fluorescence signals originated exclusively from the microplastics rather than the aqueous medium (Please refer to the Main Text, Section 3, Page 8, Lines 252-256). Studies have shown that microplastics when exposed to certain excitation wavelengths, produce intrinsic fluorescence due to specific chemical structures within the polymer that can absorb and re-emit light. This intrinsic fluorescence is valuable for detecting microplastics without additional labelling or modification, making it a powerful label-free method.

The fluorescence emission mechanism involves interactions between light and the polymeric structure, often enhanced by environmental factors like surface oxidation or ageing, which introduce oxygen-containing groups that can modify fluorescence intensity and emission wavelengths. These modifications provide a unique fluorescence profile that aids in distinguishing different types of microplastics.

This study utilises excitation-emission mapping, an advanced fluorescence-based technique, which enables comprehensive spectral analysis across multiple excitation and emission wavelengths. This mapping technique provides detailed fluorescence "fingerprints" for each microplastic type, allowing high specificity in polymer identification without the need for labelling. The novelty of our approach lies in optimising excitation wavelengths, as conventional methods typically use a single excitation wavelength, yielding a weaker emission signal. By using optimised wavelengths, our study achieves a significantly enhanced emission signal, thereby improving both the sensitivity and applicability of this method for detecting microplastics in real aquatic samples. Furthermore, this excitation-emission mapping approach is superior because it offers rapid and label-free identification of microplastics with minimal sample preparation, offering a non-destructive approach for accurate detection and characterisation of microplastics in aqueous environments.

We have incorporated these clarifications and relevant references in the revised manuscript (Revision can be found at three different places in the Main Text, references to those pages and line numbers are provided below) to emphasise the unique advantages of this intrinsic fluorescence detection method.

See the following paragraph incorporated in the revised version of the manuscript:

Main Text, Section 3, Page 8, Lines 253-256:

The FLE mapping of deionized (DI) water in a cuvette without microplastic particles shows no intrinsic FL emission (Supplementary information, Figure S1). This confirms that the recorded emissions from the microplastic samples exclusively originate from the fragmented microplastics themselves.

Main Text, Section 3, Page 8, Lines 261-273:

In general, the fluorescence emission mechanism from microplastics, including polystyrene (PS), polyethylene terephthalate (PET), and polypropylene (PP), arises from intrinsic photophysical processes associated with the chemical structure of these polymers. Each polymer contains chromophoric groups that, upon excitation, emit characteristic fluorescence signals due to π-conjugated bonds or other reactive functional groups introduced through polymerisation and environmental ageing. For example, PS contains aromatic rings that facilitate π-π* transitions, resulting in distinctive fluorescence under UV excitation, whereas PET's carbonyl and ester linkages contribute to strong n-π* and π-π* transitions that result in detectable fluorescence emissions when excited within certain wavelength ranges [51, 52]. In PP, fluorescence is typically weak due to its non-aromatic structure. However, environmental factors such as surface oxidation and UV exposure can induce oxygenated groups (C–O, C=O) on the PP surface, enhancing the fluorescence signal and enabling detection [53].

Main Text, Section 3, Page 11, Lines 351-360:

The novelty of our approach lies in optimising excitation wavelengths, as conventional methods typically use a single excitation wavelength, yielding a weaker emission signal. Our study achieves a significantly enhanced emission signal by using optimised wavelengths, thereby improving the sensitivity and applicability of this method for detecting microplastics in real aquatic samples. Furthermore, This technique enables the rapid and label-free identification of microplastics with minimal sample preparation, offering a non-destructive approach for accurately detecting and characterising microplastics in aqueous environments. Moreover, environmental factors such as surface oxidation can further enhance fluorescence signals, offering spectral fingerprints unique to each polymer type [64].

Comment:

Some text formatting errors exist in the manuscript.

Response:

Thank you for pointing out the text formatting errors in the manuscript. We have carefully reviewed and corrected the formatting errors. All text alignment, font styles, and spacing issues have been addressed, and we have verified consistency throughout the document. The revised manuscript reflects these improvements for a more polished final version.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I think the authors have improved the quality of the manuscript after considering reviewer's comments.