Exploring the Feasibility of a Microchip Laser Ablation Method for the Preparation of Biopolymer-Stabilized Gold Nanoparticles: Case Studies with Gelatin and Collagen

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The presented research is interesting for the scientific community working in the field of laser ablation. It is well prepared and it was interesting to read. There are only few points that could enrich the current version of the article.

1) The circular dichroism measurements are not sufficiently described in the article. 

2) Eventhough the gelatin concentration was changed, the nanoparticle size did not changed. Could authors elaborate more on this point why the concentration is not crucial here. 

3) What is the yield of the process? Whether particles are stable for long time?

Author Response

The presented research is interesting for the scientific community working in the field of laser ablation. It is well prepared and it was interesting to read. There are only few points that could enrich the current version of the article.

• Thank you for your positive evaluation and for recognizing the relevance of our work. A detailed point-by-point response is provided below.

1) The circular dichroism measurements are not sufficiently described in the article. 

• The missing/conclusive discussion of circular dichroism (CD) was added as yellow highlights in the manuscript. In addition, we added a new reference 18 for help.

2) Eventhough the gelatin concentration was changed, the nanoparticle size did not changed. Could authors elaborate more on this point why the concentration is not crucial here. 

• We appreciate the reviewer’s insightful question. As noted in the main text (lines 141–145), the nanoparticle size remained consistent across different gelatin concentrations due to the distinctive features of the microchip laser PLAL. Its short pulse duration (0.9 ns) and low pulse energy generate small, short-lived cavitation bubbles that govern particle formation. These bubbles facilitate nucleation and growth independently of the stabilizer (gelatin) concentration, resulting in uniform particle sizes. In contrast to conventional chemical or high-power laser methods—where matrix concentration significantly affects NP growth—the MCL-PLAL approach suppresses this dependency, consistent with previous findings in the PVP system (ref. 14: B. S. Hettiarachchi et al., Ind. Chem. Mater., 2024, 2, 340–347).

3) What is the yield of the process? Whether particles are stable for long time?

• The nanoparticle yield under each condition is summarized in Table 1. For instance, AuNPs synthesized in 0.2 wt% gelatin solution yielded approximately 4 × 10⁻⁵ mmol, as determined by ICP-AES (Table 1). In terms of stability, AuNPs prepared in pure water remained stable for at least one month, showing no signs of aggregation. In contrast, those prepared in PBS aggregated within the same period, underscoring the critical role of the dispersion medium in ensuring long-term stability (see Section 3, Table 2, and discussion lines 206–209).

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors present a PLAL study in which they employ a microchip laser (1064 nm, 180 µs pulse width, 140 mW, 80 Hz) to generate AuNP colloids in gelatin and type I collagen. The manuscript is well written, and the work clearly presented. As the Authors note, microchip lasers offer a small footprint, robustness, and a low cost of entry, so demonstrating their effective use in PLAL nanoparticle synthesis is noteworthy. The Authors conduct the ablations in aqueous solutions of two biopolymers, gelatin and type I collagen. They provide convincing evidence that ablation in gelatin produces a stable, monodisperse colloid of very small AuNPs and identify an optimal gelatin concentration for the liquid medium. I appreciate that the Authors extend the study to include phosphate-buffered saline, since PBS is often required in mammalian cell studies. Unfortunately, as demonstrated, adding PBS to the ablation medium adversely affects colloid stability, leading to large aggregates and the solution became more basic. Ablation in Col1 appears to fare even worse, with larger aggregates and lower productivity, although the Authors show that the supernatant still contains very small, stable AuNPs. I have only a few comments and concerns:

1. In the Introduction (line 56), the Authors highlight the benefits of microchip lasers in minimizing decomposition during PLAL. However, in the present experiments, the liquid environment exhibits photodegradation. They also cite short pulse durations of 0.9 ns, yet the laser used in their study has a duration of 180 µs (as noted in the supplemental document), which is long compared with the nanosecond or shorter pulses typical of most PLAL work.
2. The purpose of the zirconia beads is not stated.
3. In line 116, the current is listed as 70 A. Is this value correct?
4. The Authors claim that the short pulse duration leads to a small cavitation bubble and therefore consistent nanoparticle formation. This reasoning is questionable. A 180 µs pulse is not short for PLAL, so the cavitation bubble may not be small. No data on bubble size are provided, and the connection between bubble size and consistent nanoparticle size is not demonstrated and should not be asserted.

Author Response

The Authors present a PLAL study in which they employ a microchip laser (1064 nm, 180 µs pulse width, 140 mW, 80 Hz) to generate AuNP colloids in gelatin and type I collagen. The manuscript is well written, and the work clearly presented. As the Authors note, microchip lasers offer a small footprint, robustness, and a low cost of entry, so demonstrating their effective use in PLAL nanoparticle synthesis is noteworthy. The Authors conduct the ablations in aqueous solutions of two biopolymers, gelatin and type I collagen. They provide convincing evidence that ablation in gelatin produces a stable, monodisperse colloid of very small AuNPs and identify an optimal gelatin concentration for the liquid medium. I appreciate that the Authors extend the study to include phosphate-buffered saline, since PBS is often required in mammalian cell studies. Unfortunately, as demonstrated, adding PBS to the ablation medium adversely affects colloid stability, leading to large aggregates and the solution became more basic. Ablation in Col1 appears to fare even worse, with larger aggregates and lower productivity, although the Authors show that the supernatant still contains very small, stable AuNPs. I have only a few comments and concerns:

• We appreciate Reviewer 2 for the positive and thoughtful feedback. We are glad the potential of microchip lasers and our findings in gelatin, PBS, and collagen matrices were well recognized. Below we address the specific comments point by point.

1. In the Introduction (line 56), the Authors highlight the benefits of microchip lasers in minimizing decomposition during PLAL. However, in the present experiments, the liquid environment exhibits photodegradation. They also cite short pulse durations of 0.9 ns, yet the laser used in their study has a duration of 180 µs (as noted in the supplemental document), which is long compared with the nanosecond or shorter pulses typical of most PLAL work.

We thank the reviewer for the valuable comment and apologize for the confusion. We assume the reviewer may have inferred the laser parameters from Figure S1. To clarify, as noted in both the Introduction and Results sections, the laser used in this study was a microchip laser with a pulse duration of 0.9 ns. The value “180” mentioned in the supplemental document refers to the laser repetition rate in Hz, not the pulse duration. We have revised the relevant text to eliminate this ambiguity.

Based on our previous reports (refs. 14, 15, and 16), the short pulse duration (0.9 ns) and low pulse energy of the current MCL system generate small, short-lived cavitation bubbles that dominate the particle formation process. These bubbles facilitate nucleation and growth independently of the stabilizer (gelatin) concentration, resulting in uniform particle sizes.

 

2. The purpose of the zirconia beads is not stated.

• The purpose of the Zirconia beads is to stabilize the position of the gold target during stirring and to maximize the efficiency of laser irradiation. We added the comment in Lines 111-112.

3. In line 116, the current is listed as 70 A. Is this value correct?

• We confirm that the current value of 70 A stated in line 116 is correct.

4. The Authors claim that the short pulse duration leads to a small cavitation bubble and therefore consistent nanoparticle formation. This reasoning is questionable. A 180 µs pulse is not short for PLAL, so the cavitation bubble may not be small. No data on bubble size are provided, and the connection between bubble size and consistent nanoparticle size is not demonstrated and should not be asserted.

• As noted in the previous response, we used very short pulse duration conditions. Therefore, we believe the resulting cavitation bubbles were extremely small—similar to those observed in the PVP system—such that they could not be detected even with a high-speed camera.