Single particles as resonators for thermomechanical analysis

Thermal methods are indispensable for the characterization of most materials. However, the existing methods require bulk amounts for analysis and give an averaged response of a material. This can be especially challenging in a biomedical setting, where only very limited amounts of material are initially available. Nano- and microelectromechanical systems (NEMS/MEMS) offer the possibility of conducting thermal analysis on small amounts of materials in the nano-microgram range, but cleanroom fabricated resonators are required. Here, we report the use of single drug and collagen particles as micro mechanical resonators, thereby eliminating the need for cleanroom fabrication. Furthermore, the proposed method reveals additional thermal transitions that are undetected by standard thermal methods and provide the possibility of understanding fundamental changes in the mechanical properties of the materials during thermal cycling. This method is applicable to a variety of different materials and opens the door to fundamental mechanistic insights.

are the most exciting and essential for the draft. The major advantage of the presented method is the possibility to achieve thermomechanical characterization at the single-particle level. The given difference between bulk TGA and DSC characterization and measurement at the single-particle level should also discuss in detail taking into account the difference between the nanoscopic and mesoscopic structure of polymers.
Answer: Understanding the fascinating and complex structure and dynamics of polymeric materials has been an ongoing challenge for many decades. From the point of view of molecular simulations, the spectrum of length and time scales associated with polymer melts of long chains poses a formidable challenge to studying their long-time dynamics. 1,2 The topological constraints arising from chain connectivity and uncrossability (entanglements) dominate intermediate and long-time relaxation 2 and transport phenomena when polymers become sufficiently long. Atomistic molecular simulations of dense phases of soft matter prove to be difficult for many systems across length and time scales of practical interest. Even coarse-grained particlebased simulation methods may not be applicable due to the lack of faithful descriptions of polymer-polymer and polymer-surface interactions. Since complex interactions between constituent phases at the atomic level ultimately manifest themselves in macroscopic properties, a broad range of length and time scales must be addressed and a combination of modelling techniques is therefore required to simulate meaningfully the bulk-level behaviour of nanocomposites. How do the ordering of polymers and the lack of significant entanglement of polymer chains at the singleparticle level affect thermomechanical properties?
Regarding the effect of "lack of significant entanglement of polymer chains at the single particle level on thermomechanical properties", we would like to attract the attention of the reviewer to below-mentioned points: Answer: We have applied compressed collagen construct as the polymeric model of a protein in this study.
With regard to the main aim of this system, i.e., using a much lower amount of the material for a comprehensive thermomechanical study, a tiny piece of the sheet was cut and used. To minimize the level of polymer chain entanglements, we have considered avoiding the use of cross-linkers to solidify the protein hydrogel. Instead, physical compression was used to remove the aqueous microenvironment surrounding the hydrophilic polymer chains to make the hydrogel 1 . Indeed the compressed collagen sheet is a nice example of a polymeric structure with a lower amount of entanglements compared with other conventional collagenbased hydrogels. Since entanglements impose topological constraints on polymer conformations 2 , no use of chemical crosslinking has reduced the level of intra-chain entanglements. However, we cannot consider the polymeric sample in the level of "single particle", from the molecular simulations point of view. In fact, polymers include long chains, where chain connectivity and uncrossability (entanglements) dominate intermediate and long-time relaxations in polymer melts 3 . It consequently poses challenges to study the longtime dynamics of the polymers, and any proposed method such as PMTA that is providing the possibility of using microgram amounts of material, whilst maintaining comprehensive data would be of great value.

Does the presented method allow us to investigate the property of the single polymer chain almost not accessible by other methods?
More deep physical discussion is needed for the journal like Nature Comm., and I encourage authors to provide deeper discussion in the revised version in order to make their case stronger.
Answer: Thank you for this important point. This has now been addressed on the entire text of the revised manuscript.
In Figures 2 and 4, the error of measurement should be presented, and the estimation of the error should be described and discussed in the main text.
Answer: The authors have now addressed this in Figure 2, 4, Table 1 and 2 of the revised manuscript.
Taking into account the importance of investigation at the single-particle level, which reveals the structure-