Optimized NaYF₄: Er³⁺/Yb³⁺ Upconversion Nanocomplexes via Oleic Acid for Biomedical Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the authors synthesized NaYF4: Er3+/Yb3+ nanomaterials via a wet chemistry method and systematically examined the influence of oleic acid on their morphology and luminescent properties using SEM, XRD, FTIR, and other characterization techniques. They identified an optimal oleic acid concentration of 10-3 M for achieving enhanced green upconversion luminescence. Furthermore, they demonstrated the potential biomedical application of these nanomaterials in cancer cell detection using an inverted fluorescence microscope, highlighting their promising role in rapid pathogen detection. This study has been conducted in a highly systematic manner, with all characterizations of high quality. I suggest publication as soon as possible after addressing the following questions:

1. It would be beneficial to provide the absolute upconversion photoluminescence (PL) quantum yield efficiency under 980 nm excitation. Figure 6 currently presents only a relative comparison without quantitative values, making it difficult to assess the emissive performance of the optimal sample. Including absolute quantum yield measurements would enhance the study’s rigor and facilitate direct comparisons with other upconversion nanomaterials.

2. I am unable to identify the scale bar in Figure 5, and there are no visible scale bars in Figure 7. Please include clearly labelled scale bars in both figures which would improve the clarity and accuracy of the presented data.

Author Response

Dear Madam/Sir,

I and all my co-workers would like to thank you in deep for careful reading and very useful comments that help us to complete our manuscript with the article entitled: “Optimized NaYF₄: Er³⁺/Yb³⁺ Upconversion Nanocomplexes via Oleic Acid for Biomedical applications”. We have already revised and returned our manuscript to the Inorganics 's office. However, we would like to discuss in details all your comments as follows:

1. Comment 1: It would be beneficial to provide the absolute upconversion photoluminescence (PL) quantum yield efficiency under 980 nm excitation. Figure 6 currently presents only a relative comparison without quantitative values, making it difficult to assess the emissive performance of the optimal sample. Including absolute quantum yield measurements would enhance the study’s rigor and facilitate direct comparisons with other upconversion nanomaterials.

Response: We thank the reviewer for this insightful suggestion. We agree that providing the absolute upconversion photoluminescence quantum yield under 980 nm excitation would strengthen the rigor of our study and enable more direct comparisons with other upconversion nanomaterials. In this study, our primary goal is to demonstrate the kinetic effects influenced by chemical synthesis in order to highlight the potential applications of novel upconversion biomarker materials. More advanced dynamic characterizations, such as absolute quantum yield measurements, will be carried out in future work.

 

2. Comment 2: I am unable to identify the scale bar in Figure 5, and there are no visible scale bars in Figure 7. Please include clearly labelled scale bars in both figures which would improve the clarity and accuracy of the presented data.

Response:  We thank the reviewer for pointing out this important issue. We have revised both figures to include clearly labeled and properly sized scale bars, as shown in the updated manuscript.

Figure 5

Figure 7

 

All above items had been included in the revised version of our manuscript.

Thank you very much once again.

 

Yours sincerely,

 

Tran Thu Huong, Ph.D.

Corresponding author

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The author reported the synthesis of NaYF ₄: Er ³ ⁺/Yb ³ ⁺ upconversion luminescent nanomaterials by a wet chemistry method, and discussed the effect of oleic acid dosage on the synthesized materials. Afterwards, NaYF ₄ : Er ³ ⁺/Yb ³ ⁺ @ SNGA IgG synthesized was used to label HeLa cervical cancer cells. The article is rich in content, with detailed data and certain application prospects. But currently it cannot be directly published. The modification suggestion is:

1. The author reported that 10 ³ M is optimal at three concentrations of 10^-1, 10^-2, and 10^-3 M, but we did not see a turning point, such as whether 10^-4 would be better.
2. Please provide the supplementary information of the absorption spectrum of materials
3. Analyzing material energy levels and bands based on luminescence conditions
4. Add first principles
5. The imaging usually consists of 4 images, especially DAPI images. Please add.

Author Response

Dear Madam/Sir,

I and all my co-workers would like to thank you in deep for careful reading and very useful comments that help us to complete our manuscript with the article entitled: “Optimized NaYF₄: Er³⁺/Yb³⁺ Upconversion Nanocomplexes via Oleic Acid for Biomedical applications”. We have already revised and returned our manuscript to the Inorganics 's office. However, we would like to discuss in details all your comments as follows:

1. Comment 1: The author reported that 10 ^-3M is optimal at three concentrations of 10^-1, 10^-2, and 10^-3 M, but we did not see a turning point, such as whether 10^-4 would be better.

Response: We thank the reviewer for this valuable suggestion. In our study, we investigated the effects of three representative oleic acid (OA) concentrations (10⁻¹, 10⁻², and 10⁻³ M) on the morphology and upconversion luminescence of NaYF₄:Er³⁺/Yb³⁺ nanomaterials. Among these, the 10⁻³ M concentration exhibited the very good performance in terms of emission intensity, particle uniformity, and size. Therefore, this sample was selected for cancer cell labeling experiments. We agree that exploring lower OA concentrations, such as 10⁻⁴ M, could provide additional insights into the optimal ligand concentration. Although this condition was not examined in the current study, we have addressed this limitation in the revised manuscript. Furthermore, we have added a discussion suggesting that future studies should investigate this lower concentration to determine whether further improvements in luminescence or particle size control can be achieved.

 

2. Comment 2: Please provide the supplementary information of the absorption spectrum of materials

Response: Thank you for your suggestion. Based on information from previous studies, such as [1, 2], the absorption region of the synthesized material is very close to 980 nm. Therefore, we did not perform absorption spectroscopic measurements in this work. In future studies, we will consider a more in-depth investigation of the relationship between the synthesis methodology and the material’s absorption properties.

• Qiang Wang, Mei Chee Tan, Rui Zhuo, G. A. Kumar, and Richard E. Riman “A Solvothermal Route to Size- and Phase-Controlled Highly Luminescent NaYF₄:Yb,Er Up-Conversion Nanocrystals” Journal of Nanoscience and Nanotechnology, 2010, Vol. 10, 1–8, DOI: 1166/jnn.2010.2120.
• Gunaseelan, S. Yamini, G. A. Kumar, D. K. Sardar, J. Senthilselvan “Reverse microemulsion synthesis of mixed α and β phase NaYF₄:Yb,Er nanoparticles: calcination induced phase formation, morphology, and upconversion emission” Journal of Sol-Gel Science and Technology, 2020, 96:550–563 https://doi.org/10.1007/s10971-020-05340-w.

 

3. Comment 3: Analyzing material energy levels and bands based on luminescence conditions.

Response: Thank you for your suggestion. The energy transfer mechanism between Yb³⁺ and Er³⁺ ions in NaYF₄:Yb³⁺/Er³⁺ under 980 nm excitation has been illustrated, accompanied by a brief discussion of the associated electronic transitions (²H₁₁_/₂ → ⁴I₁₅_⁄₂, ⁴S₃_⁄₂ → ⁴I₁₅_⁄₂, and ⁴F₉_⁄₂ → ⁴I₁₅_⁄₂). These transitions correspond to the observed green and red upconversion emission bands. This explanation helps clarify the relationship between the energy levels and the recorded photoluminescence spectra. The schematic illustration of the energy transfer mechanism and the related discussion are presented below:

 

Figure (*). The energy level diagram of Yb3+ and Er3+ ions and the corresponding upconversion mechanics in NaYF4:Yb3+/Er3+ nanoparticles under 980 nm near-infrared excitation.
Figure (*) presents the energy level diagram of Yb3+ and Er3+ ions and the corresponding upconversion mechanics in NaYF4:Yb3+/Er3+ nanoparticles under 980 nm near-infrared excitation. The Yb³⁺ ion acts as a sensitizer, efficiently absorbing photons and transferring energy to the Er³⁺ ion, which serves as the activator. Specifically, Yb³⁺ absorbs a 980 nm photon and transitions from ground state 2F7/2 -to 2F5/2​ level​, then transfers its energy to Er³⁺, exciting it from the ground state 4I15/2 to 4I11/2 level​. Upon absorbing a second photon (or receiving additional energy from another Yb³⁺ ion), Er³⁺ is further excited to higher energy states such as 4F7/2​, followed by non-radiative relaxation to lower excited states before emitting visible light. The main radiative transitions observed are 2H11/2→4I15/2​ (~520 nm, green), 4S3/2→4I15/2 (~540 nm, green), 4F9/2 →4I15/2 (~650 nm, red). This phenomenon results from a two-photon absorption process and cumulative energy transfer from Yb³⁺ ions, leading to visible upconversion luminescence, which holds great potential for biological labeling and biomedical imaging applications.

4. Comment 4: Add first principles

Response: We appreciate the reviewer’s suggestion. While first-principles calculations such as density functional theory (DFT) can provide valuable insights into the band structure and electronic properties of upconversion materials, our current study is focused on experimental synthesis, structural characterization, and photoluminescence behavior. The first-principles theoretical analysis would provide valuable insights into the fundamental understanding of the material system and is regarded as a promising direction for our future research.

 

5. Comment 5: The imaging usually consists of 4 images, especially DAPI images. Please add.

Response: We thank the reviewer for this suggestion. During the photoluminescence screening of all four samples, we selected the NaYF₄:Er³⁺/Yb³⁺-OA (10⁻³ M) nanoparticles as the most suitable candidate for cellular detection, owing to its optimal emission performance. Therefore, we focused the imaging analysis on this sample and presented only its result in the manuscript. We respectfully chose not to include the remaining three images to maintain clarity and conciseness.

However, we agree that multi-channel fluorescence imaging (e.g., including DAPI-stained nuclei) would provide additional insights, especially for co-localization analysis. We will consider incorporating such complementary imaging channels in future work.

 

All above items had been included in the revised version of our manuscript.

Thank you very much once again.

 

Yours sincerely,

Tran Thu Huong, Ph.D.

Corresponding author

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript prepared by H.T. Phuong et al. presents the study on the impact of the oleic acid (OA) concetration during the synthesis on the morphology of resulting structures. Moreover, optimized structures are then applied as luminescent labes in cancer cells.

Presented topic of lanthanide doped nanomaterials and their application in biomedical research is interesting, rapidly progressing field of studies. However, due to that fact, presented results are not characterized by high novelty - optimized lanthanide doped nanocrystals synthesis protocols are well-known, including also much more sophisticated core-shell architectures. Similarly, application of these nanomaterials as luminescent labels in biotissues is quite commonly applied. I would suggest the Authors to emphasize the aspects of their results, which present the new insight into these structures and their properties.

Detailed comments:

• One of the main aspects of this research is to investigate the role and impact of the OA concentration on the size and morphology of the synthesized materials. Thus, it should be discussed in a more detailed way. In particular, what is the reason that increased amount of OA promotes other crystla phase (cubic) to appear next to the hexagonal, which dominates in other structures, including also bare nanocrystals.
• Produced nanocrystals feature strong dependence of the size with the OA content, however even fot the lowest OA concentration, where the smallest nanocrystals were obtained, they are still of over 150 nm in size (150-250 nm). On the other hand, nanocrystals of nearly one order of magnitude smaller (20-50 nm) are frequently reported and studied. Authors should discuss, if further decrease of OA content, or other optimalization steps are required to obtain such smaller nanocrystals, and how their results confront with other reported nanomaterials of similar type.
• Only one of the structures, with the smallest OA content, is studied when it comes to uniformity of the structure and doping (Fig. 4, Fig. 5), is there any infomation, how it looks like in the case of the other studied materials?
• In the luminescence the materials were studied with the photoluminescence measuring system - it should be mentioned what was the form of the sample - was it powder, or solution in cuvette, or a layer? It can affect the emission of the lanthanide doped materials, epsecially when it comes to the emission intensity. Moreover, when preparation procedure of the nanocrystals is presented in Materials and methods section, is it mentioned that obtained products are centrifuged, and then washed with water, and dried. Howeve, OA-modified nanocrystals are hydrophobic, thus are typically dispersed in chloroform or other similar solvents. Could you somehow comment this procedure of sample preparation?
• Language and editing of the manuscript should be improved. In the lines 20 nad 22 wrong tabulator and double apperance of the same word appears. Moreover, in some lines different formating shpuld be applied, as them seem to be intended as the subchapters (line 145, 260, etc.)

Author Response

Dear Madam/Sir,

All my co-workers and I would like to thank you in deep for careful reading and very useful comments that help us to complete our manuscript with the article entitled: “Optimized NaYF₄: Er³⁺/Yb³⁺ Upconversion Nanocomplexes via Oleic Acid for Biomedical applications”. We have already revised and returned our manuscript to the Inorganics 's office. However, we would like to discuss in details all your comments as follows:

1. Comment 1: One of the main aspects of this research is to investigate the role and impact of the OA concentration on the size and morphology of the synthesized materials. Thus, it should be discussed in a more detailed way. In particular, what is the reason that increased amount of OA promotes other crystal phase (cubic) to appear next to the hexagonal, which dominates in other structures, including also bare nanocrystals.

Response: We thank the reviewer for this insightful comment highlighting the importance of discussing in more detail the influence of oleic acid (OA) concentration on the size, morphology, and particularly on the phase evolution of the synthesized nanomaterials. In the revised manuscript, we have expanded the discussion on this point. Specifically, the formation of different crystal phases cubic (α) versus hexagonal (β) is strongly influenced by the concentration of OA due to its role as a surface-capping ligand and growth modulator. At low OA concentrations, partial surface coverage allows more anisotropic crystal growth, which favors the formation of the thermodynamically stable hexagonal β-phase, known for its superior upconversion luminescence. However, as the OA concentration increases, the nanocrystal surfaces become more densely capped with OA molecules, which alters the surface energy landscape and inhibits anisotropic growth, thus kinetically favoring the formation or persistence of the cubic α-phase. This cubic phase is known to dominate at early nucleation stages or under conditions where crystal growth is restricted. Moreover, a high amount of OA can reduce the rate of crystal growth and phase transition from cubic to hexagonal, resulting in the coexistence of both phases. This explanation has now been added to the “Results and Discussion” section of the revised manuscript.

2. Comment 2: Produced nanocrystals feature strong dependence of the size with the OA content, however even fot the lowest OA concentration, where the smallest nanocrystals were obtained, they are still of over 150 nm in size (150 - 250 nm). On the other hand, nanocrystals of nearly one order of magnitude smaller (20 - 50 nm) are frequently reported and studied. Authors should discuss, if further decrease of OA content or other optimization steps are required to obtain such smaller nanocrystals, and how their results confront with other reported nanomaterials of similar type.

Response: We thank the reviewer for pointing out the issue regarding the relatively large size (150 - 250 nm) of the synthesized nanocrystals, even at the lowest OA concentration. This is an important observation, and we have now addressed it in the revised manuscript. Indeed, many reports have demonstrated that NaYF₄-based upconversion nanocrystals can be synthesized with significantly smaller sizes (e.g., 20 - 50 nm) by employing different synthetic strategies [1, 2]. To further reduce particle size, several optimization approaches can be considered, such as: decreasing the OA concentration; adjusting the reaction temperature and time; employing a co-surfactant or alternative ligands using microwave-assisted synthesis. In our work, the primary focus was to ensure strong upconversion luminescence and phase purity (especially stabilizing the β-phase), we have investigated the effects of three representative oleic acid (OA) concentrations (10⁻¹, 10⁻², and 10⁻³ M) on the morphology and upconversion luminescence of NaYF₄:Er³⁺/Yb³⁺ nanomaterials. Among these, the 10⁻³ M concentration exhibited the very good performance in terms of emission intensity, particle uniformity, and size. Therefore, this sample was selected for cancer cell labeling experiments. We agree that exploring lower OA concentrations, such as 10⁻⁴ M, could provide additional insights into the optimal ligand concentration. Although this condition was not examined in the current study, we have addressed this limitation in the revised manuscript. Furthermore, we have added a discussion suggesting that future studies should investigate this lower concentration to determine whether further improvements in luminescence or particle size control can be achieved.

• Zhang, Y.; Sun, X. & Zhang, H. Size, phase-controlled synthesis, the nucleation and growth of NaYF₄Materials Chemistry and Physics, 2018, 210, 1–7. https://doi.org/10.1016/j.matchemphys.2017.05.012​
• Wang, F.; Han, Y.; Lim, C. S.; Lu, Y; Wang, J.; Xu, J.; Chen, H.; Zhang, C.; Hong, M., & Liu, X. “Simultaneous phase and size control of upconversion nanocrystals through lanthanide doping”. Nature, 2010, 463, 1061–1065. https://doi.org/10.1038/nature08777

3. Comment 3: Only one of the structures, with the smallest OA content, is studied when it comes to uniformity of the structure and doping (Fig. 4, Fig. 5), is there any information, how it looks like in the case of the other studied materials?

Response: We appreciate the reviewer’s valuable suggestion. Figures 4 and 5 specifically focus on the selected sample, as it demonstrated the most promising characteristics in terms of particle uniformity, phase purity, and upconversion luminescence performance. This sample was selected for further biomedical experiments, which necessitated more detailed characterization. In contrast, the other samples (prepared with 10⁻¹ and 10⁻² M OA) exhibited significantly larger particle sizes, as shown in the FESEM images (Figure 1). Additionally, their upconversion luminescence spectra (Figure 6) revealed weaker emission intensities. Therefore, we did not perform elemental mapping for those samples.

4. Comment 4: In the luminescence the materials were studied with the photoluminescence measuring system - it should be mentioned what was the form of the sample - was it powder, or solution in cuvette, or a layer? It can affect the emission of the lanthanide-doped materials, epsecially when it comes to the emission intensity. Moreover, when preparation procedure of the nanocrystals is presented in Materials and methods section, is it mentioned that obtained products are centrifuged, and then washed with water, and dried. Howeve, OA-modified nanocrystals are hydrophobic, thus are typically dispersed in chloroform or other similar solvents. Could you somehow comment this procedure of sample preparation?

Response: We thank the reviewer for this detailed and thoughtful comment regarding the form of the sample used during photoluminescence measurements and the clarification needed for the sample preparation procedure. We agree that the form of the sample (powder, solution in cuvette, or a layer) can significantly affect emission intensity, particularly for lanthanide-doped materials. Regarding the measurement form, we confirm that all photoluminescence spectra were measured using solid powder samples. The dried nanocrystal powders were placed on a flat Cooper holder and measured under 980 nm excitation using the photoluminescence system described. We have now clarified this information in the revised "Materials and Methods" section.

5. Comment 5: Language and editing of the manuscript should be improved. In the lines 20 nad 22 wrong tabulator and double apperance of the same word appears. Moreover, in some lines different formating shpuld be applied, as them seem to be intended as the subchapters (line 145, 260, etc.)

Response: We have revised the manuscript for grammar, style, and consistency. The formatting of subheadings (e.g., lines 145 and 260) has been corrected, and typographical errors such as those on lines 20 and 22 have been fixed.

 

All above items had been included in the revised version of our manuscript.

Thank you very much once again.

 

Yours sincerely,

Tran Thu Huong, Ph.D.

Corresponding author

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I am satisfied with the response from the Authors, the essential concerns regarding the manuscript have been clarified. Revised version of the manuscript is of higher quality and scientific soundness than orginal one. However, quality of the figures in general should be improved to be more uniform and presented with higher graphical resolution to easly read all the labels and scale bars