extfieldsofstudy: Medicine Physics

s2fieldsofstudy: Chemistry Physics

text:
High-resolution two-dimensional electronic spectroscopy reveals the homogeneous line profile of chromophores solvated in nanoclusters

Doped clusters in the gas phase provide nanoconfined model systems for the study of system-bath interactions. To gain insight into interaction mechanisms between chromophores and their environment, the ensemble inhomogeneity has to be lifted and the homogeneous line profile must be accessed. However, such measurements are very challenging at the low particle densities and low signal levels in cluster beam experiments. Here, we dope cryogenic rare-gas clusters with phthalocyanine molecules and apply action-detected two-dimensional electronic spectroscopy to gain insight into the local molecule-cluster environment for solid and superfluid cluster species. The high-resolution homogeneous linewidth analysis provides a benchmark for the theoretical modelling of binding configurations and shows a promising route for high-resolution molecular two-dimensional spectroscopy.

capabilities was exploited here. No cross-peak appeared in the maps and basically no dynamics was recorded (the time evolution of the studied systems didn't really show interesting features, which was expected considering the experimental conditions). Basically, a complex multidimensional technique has been downgraded to a static technique, and only two maps (figure 3) out of multidimensional matrixes (which I guess required a long time to be acquired) were effectively used to draw the conclusions.
In addition, the title and the introduction claimed an investigation on the role of the environment, but this is not what the paper does. There is no real comparison between different environments (except that recognizing that in Ne droplets there is a contribution by inhomogeneous broadening), nor a discussion about what the environment does on the system. Cooled gas nanodroplets are not a natural environment for molecules and therefore I found it inappropriate to claim that the role of environmental parameters affecting the dynamics could be studied in these conditions. Also because in the ultracold experimental conditions applied here there is basically no change in the system dynamics (as stated on page 5). I would suggest changing the tone of the introduction because it is bringing the reader somewhere else.
A final comment: most of the experimental details are lacking and I do not believe that enough detail is provided in the methods for the work to be reproduced.
Minor comments: 1. I suggest specifying better in the title that nanoparticles are intended as gas nanoclusters: in material science, 'nanoparticles' might indicate a completely different system. Figures 2c and 2d a bit misleading. While I understand that the scope is illustrating in general terms the meaning of the signals appearing in the 2DES maps, one would expect to see similar features also in the experimental data. Also, I found a bit confusing the labels 'molecule 1' and 'molecule 2', considering that only one molecular species was investigated here. I suggest removing these panels, not fully relevant here (since no cross-peaks were detected), and referring to previous literature (there are many clear and comprehensive reviews about the signal distribution in 2DES maps).

I found
3. there is a typo on page 6, second column, line 10: 'monochrmoator' Overall, this is a noteworthy and pioneering study demonstrating that local environments strongly affect the optical properties of an organic molecule chromophore. The authors use their results to report on the molecular-surface binding configurations, which is a key contribution. The authors have chosen to write their manuscript from the standpoint of spectroscopy method-development, which lowers the impact somewhat. The impact would have been higher had the authors focused their title and abstract on the scientific (new knowledge) advances with less focus on the tool used to generate that new knowledge. The method development aspect for cluster-isolation and highresolution 2D spectrometer is very strong.
Major comment: -The authors compare measurements of helium nano droplets and neon nanodroplets. There are two differences in these systems. One is the obvious change of temperature. The second is a change of solvation configuration, fully encompassed or on the surface. It is not clear how the authors disentangle these two effects without a temperature-dependent study.
-The generality of the results could have been enhanced had the authors chosen to conduct measurements of at least one more molecule.
Minor comments: -page 2 the authors write: "Peaks on the diagonal position of the 2D spectra reflect the linear absorption of the system, …". This is often repeated in many publications, but it is not strictly true for all systems. There are at least two complications: (1) The input laser pulse spectrum almost always provides different weights to the peaks because it is not fully de-convoluted. (2) ESA peaks can -in some systems -appear on or near the diagonal and thereby affect the relative peak heights. The authors could reword as, for example: "Peaks on the diagonal position of the 2D spectra mostly reflect the linear absorption…" - Fig. (1), panel (c). The caption could be enhanced by mentioning that the coupling refers to the blue dashed arrow.
- Fig. (2), panel (b). The unit of inverse centimeters is a wavenumber, not a frequency. This abuse of the words is endemic in spectroscopy, but that does not make it correct. Please adjust so that it is scientifically correct. There are at least two good options: (1) Replace the word "frequency" with "wavenumber", or (2) Adopt the approach of Tokmakoff and label as \omega/(2 \pi c).
-Page 3 the authors subtly introduce a control measurement of H2Pc in 1-chloronaphthalene. At what temperature were those measurements conducted? - Fig. (3). Why did the authors choose a waiting time of 1 ps? Time zero would in principle be better because the system would have not undergone any spectral diffusion at all.
- Fig (3) panel (a). How do the authors know that the blips along the diagonal are not simply due to scatter? -On page 4 the authors write "…except for an outlier at 15040 cm-1, which is due to a nonrecurring experimental artifact." What is the meaning of this? If it only occurred once, the authors should fit and present a different dataset that does not have the artifact.
Reviewer #3 (Remarks to the Author): Bangert et al. report photon echo study of free base phthalocyanine in helium and neon clusters. The authors found unexpectedly long electronic coherence of hundreds of picoseconds in helium and few picoseconds in neon. The photon echo technique enables attaining the homogeneous and heterogeneous linewidth. In both environments the homogeneous linewidths were found to be about 0.5 cm-1, which is very narrow. I feel these results may deserve publication in NC. However, the manuscript should be revised according to the following points.
1. The authors should remove uncorroborated claims and proposals.
In abstract and in the text the authors claim unprecedented spectral resolution without giving appropriate comparisons. I am rather sure that a factor of at least ten higher resolution was achieved in early hole burning experiments in solid matrices. Similar, the resolution in a conventional cw LIF spectrum of phthalocyanine in helium shown in Fig.1 of SI is higher than obtained in the reviewed manuscript.
Throughout the manuscript the authors implied that the photon echo technique could be applied to study: ..." local configurations in photo-chemical reactions and elucidating molecular dynamics in tailored nano-environments or at functional interfaces" (Introduction) or " the interaction of isolated molecules with metallic and semiconducting nanoclusters, of high relevance in catalysis and biomedical imaging" (Conclusions) or "to explore and control many-body mechanisms of high interest in photovoltaics, such as molecular exciton migration and annihilation processes, as well as in quantum information science which is, so far, predominately limited to atomic samples." (Conclusions) There is not any discussion of the applicability of the photon echo technique to any of the above systems. Therefore, the above propositions sound speculative to the present referee.
I suggest the authors focus on the studied system and provide more physical insights into the observed effects 2. The principle of the two-dimensional electronic spectroscopy should be introduced in the main text that should be accessible for non-specialists.
3. "A Fourier transform of the 1D coherence scans yield the linear absorption spectra of both systems (Fig. 2b). (p.3)" I feel this is not just FT, because the frequency of the oscillations in a) is much smaller than the peak frequency in b). The authors should better explain the application of the FT. 5. " We conclude that the molecules are mainly immobile on the (Ne) cluster surface on the time scale of 500 ps, which was previously only indirectly confirmed for ground state molecules [57]." (p.5) It would be nice to have some perspective on the limits imposed by the clusters and also to have measurements, such as in Fig. 5 with helium clusters. For example, although the molecules in neon are fixed in the cluster frame, the clusters nearly certainly are rotating in the laboratory frame. In case of helium clusters the molecules may rotate and translate inside owing to its superfluid nature, on the other hand the traditional phonon induced dephasing may be muted due to finite size effects.
Some more detailed discussion of the physical origin of the long coherence in clusters as compared to other previously studied condensed system will make this manuscript much stronger. 6. In methods, the modulation frequencies Omega(i) should be given. The explanation of the signal processing in the last two paragraphs is not understandable for a not specialist. It would be nice to have some equations for more clarity.

Response to reviewer comments
Manuscript NCOMMS-21-48836 Title: High-resolution two-dimensional electronic spectroscopy reveals homogeneous line profiles in isolated nanoparticles Authors: Ulrich Bangert, Frank Stienkemeier and Lukas Bruder.
We thank all reviewers for their time and efforts in critically evaluating our manuscript and their helpful advice for improvements.

Reviewer #1 (Remarks to the Author):
In this paper, the authors report high-resolution 2DES measurements performed on gas clusters doped with phthalocyanine molecules with the aim of determining with high precision the homogeneous line profile. The high-resolution 2DES setup is based on previous schemes (phase modulation by Marcus) but it represents indeed a new development, especially when applied to ultracold gas nanoclusters. In this sense, the presented data are novel, interesting, and worthy to be published. However, I do not believe that the quality of the analysis and the impact of the results are suitable for Nature Comm. Moreover, I have the feeling that the results and the conclusions are not yet fully mature.
Below are a few comments to support my opinion: On page 2 the authors explain the signals measured in 2DES defining the signal as a 'photon echo'. This is wrong. They do not measure a photon echo but a fluorescence. Moreover, they discuss the data (also in terms of homo and inhomogeneous broadening) referring to the wide body of theories developed for 2DES in the conventional photon echo BOXCARS configuration (where the response is stimulated by 3 laser pulses and the signal is a coherent electric filed, an 'echo'). Instead, what they do is an 'action-based' technique, where they employ 4 pulses. The 4th pulse brings the system into an excited state population from which fluorescence (the incoherent action signal) is recorded. The response function, in this case, is completely different from the one stimulated in the conventional photon echo experiments (see for example https://doi.org/10.1021/acs.jpclett.9b03851). Therefore, the analysis is based on wrong or at least inaccurate foundations. (an additional minor note in this context: also figure 1b is misleading because they define time intervals like in BOXCARS photon echo 2DES).
We disagree with the reviewer. The retrieval of homogeneous and inhomogeneous lineshapes is in coherent-detected and action-detected 2DES equivalent. The theoretical foundation for this is given e.g. in the seminal paper by Tan 1 , which provides a clear and solid foundation for our conclusions. In contrast, the reference pointed out by reviewer#1 refers to differences in excited state absorption (ESA) contributions. However, since these signals do not contribute in our experiment and thus do not affect the interpretation and conclusions of our study, we refrained from referring to this work in our initial manuscript.
To clarify this point we have added the following statement to the manuscript (p. 2, para. 4): "This concept is known from 2D photon echo spectroscopy as performed in coherence-detected 2DES [20,43]. The equivalence between coherence and action-detected 2DES in terms of lineshape information was shown in Ref. [44]. We note that coherence and action-detected 2DES may differ in the detection of excited state absorption and multiple quantum coherence signals [45,46] which are, however, not relevant in the current study." We agree with the reviewer, that on some occasions we used the term "photon echo" in an inaccurate context and have resolved this semantic inaccuracy in the revised manuscript.
About the impact of the results. I feel like the real exciting outcome of the paper is the possibility of pushing the resolution of multidimensional techniques. I do not find particularly impactful the determination of homogenous linewidth, which could have been measured with much easier techniques. Two of the advantages of using the complex 2DES technique rather than simpler monodimensional techniques are: (i) the possibility of achieving frequency and time resolution and (ii) getting info on the couplings through the appearance and the time dependence of cross-peaks. None of these capabilities was exploited here. No cross-peak appeared in the maps and basically no dynamics was recorded (the time evolution of the studied systems didn't really show interesting features, which was expected considering the experimental conditions). Basically, a complex multidimensional technique has been downgraded to a static technique, and only two maps (figure 3) out of multidimensional matrixes (which I guess required a long time to be acquired) were effectively used to draw the conclusions.
We thank the reviewer for the appreciation of the high resolution achieved with our experimental multidimensional spectroscopy approach. However, we strongly disagree on the other points. The low particle density at which the samples have to be prepared in order to yield cold and isolated quantum systems results in ultralow optical densities (OD~10 -11 ) 2 which prohibits absorption spectroscopy as well as dispersed fluorescence detection with sufficient high resolution. This prohibits the application of any of the established methods for homogeneous linewidth analysis (fluorescence line narrowing, photon echo techniques…) in our work. As such, action-detected 2DES is the only method which provides general access to the homogeneous linewidth in the studied isolated nanocluster samples. This by itself is an important achievement which exploits the unique capabilities of 2DES. Furthermore, we note that in several high impact 2DES experiments neither any off-diagonal peaks/couplings nor any dynamics were observed or even investigated 3 . The high impact of these studies is solely based on 2D lineshape analysis of diagonal peaks.
We have added the following statement to make this point clearer(p.1, para. 3): "Thereby, the key challenge is the extraction of the homogeneous line profile from the inhomogeneously broadened ensemble response. However, most methods for homogeneous linewidth retrieval [18] are not compatible with the low particle densities of gas-phase cluster samples. Hole burning, as an exception, has been applied to doped He nanodroplets, however did not resolve the homogeneous linewidth [19]. This technique faces the difficulty of adapting the laser parameters to the time and frequency scales of the target system, reducing the approach mainly to photochemical hole burning where photoreactive chromophores are probed on quasi infinite time scales [18]. These challenges may explain why in heterogeneous cluster samples the homogeneous linewidth has not been determined, so far." The reviewer also complained that "the time evolution of the studied systems didn't really show interesting features, which was expected considering the experimental conditions". Recent timeresolved experiments in He droplets have indeed shown a drastic change in the solute-solvent configuration upon electronic excitation leading to ultrafast dynamics. As such, the outcome of our time-resolved study was not clear and adds in our opinion valuable information to the manuscript. We made this clearer in the revised manuscript (p. 5, para. 3): "In this context the question arises, if an electronic excitation of the dopant can change the binding configuration with the environment. Such mechanisms are common for species dissolved in He droplets, leading e.g. to a migration towards the droplet surface followed by the ejection of the dopant on a pico to femtosecond time scale [64,65]." In addition, the title and the introduction claimed an investigation on the role of the environment, but this is not what the paper does. There is no real comparison between different environments (except that recognizing that in Ne droplets there is a contribution by inhomogeneous broadening), nor a discussion about what the environment does on the system. Cooled gas nanodroplets are not a natural environment for molecules and therefore I found it inappropriate to claim that the role of environmental parameters affecting the dynamics could be studied in these conditions. Also because in the ultracold experimental conditions applied here there is basically no change in the system dynamics (as stated on page 5). I would suggest changing the tone of the introduction because it is bringing the reader somewhere else.
As pointed out above, many important 2DES experiments focus on static properties of the sample otherwise inaccessible with other methods. According to the reviewer's suggestion we have modified the introduction of the revised manuscript to make the achievement of our work clearer. In particular we added the statement (p. 1, para. 4): "2DES and 2D infrared spectroscopy [21] have proven as very useful in condensed phase systems to extract line shape information where other methods are not applicable or do not provide the required time-frequency resolution [22][23][24][25][26]." A final comment: most of the experimental details are lacking and I do not believe that enough detail is provided in the methods for the work to be reproduced.
In order to increase the readability of the manuscript and to focus on the major results, we refrained from repeating experimental details mentioned in previous work. The individual ingredients (fluorescence-detected phase-modulated 2DES, cluster isolation spectroscopy) are described in detail in previous publications as cited in our manuscript (Refs. 6,38,75,76). Furthermore, we have recently published an extended review article about our experimental apparatus to which we refer in the manuscript (Ref. 29). We made this point clearer in the revised manuscript (p.6, para. 3): "The experimental apparatus including the cluster beam preparation are described in detail in a recent review article [29]." In addition, we have substantially extended our Methods Section to include more methodologic details (see colored text passages in revised manuscript).
Minor comments: 1. I suggest specifying better in the title that nanoparticles are intended as gas nanoclusters: in material science, 'nanoparticles' might indicate a completely different system.
We changed the wording accordingly. Figures 2c and 2d a bit misleading. While I understand that the scope is illustrating in general terms the meaning of the signals appearing in the 2DES maps, one would expect to see similar features also in the experimental data. Also, I found a bit confusing the labels 'molecule 1' and 'molecule 2', considering that only one molecular species was investigated here. I suggest removing these panels, not fully relevant here (since no cross-peaks were detected), and referring to previous literature (there are many clear and comprehensive reviews about the signal distribution in 2DES maps).

I found
From the reviewer comment we conclude that Fig. 1 was meant and we have modified Fig. 1 according to the reviewer's suggestion. However, we note that cross peak information is relevant in our study: the absence of cross peaks confirms the absence of vibrational excitations as well as the absence of inter-particle couplings among co-doped species (Fig. 3). Therefore, we have not fully removed the discussion of cross peaks in Fig. 1. 3. there is a typo on page 6, second column, line 10: 'monochrmoator' The typo is fixed in the revised manuscript.

Reviewer #2 (Remarks to the Author):
Overall, this is a noteworthy and pioneering study demonstrating that local environments strongly affect the optical properties of an organic molecule chromophore. The authors use their results to report on the molecular-surface binding configurations, which is a key contribution. The authors have chosen to write their manuscript from the standpoint of spectroscopy method-development, which lowers the impact somewhat. The impact would have been higher had the authors focused their title and abstract on the scientific (new knowledge) advances with less focus on the tool used to generate that new knowledge. The method development aspect for cluster-isolation and high-resolution 2D spectrometer is very strong.
We thank the reviewer for this suggestion and have modified title, abstract and introduction in our revised manuscript accordingly.
Major comment: -The authors compare measurements of helium nano droplets and neon nanodroplets. There are two differences in these systems. One is the obvious change of temperature. The second is a change of solvation configuration, fully encompassed or on the surface. It is not clear how the authors disentangle these two effects without a temperature-dependent study.
We agree that temperature-dependent studies are a common and frequently applied means to provide further inside into broadening mechanisms of condensed phase samples. However, for raregas matrices a temperature-dependent study is not possible due to the lacking access for heat injection (infrared inactivity and immediate cooling by evaporation). As such, our study resolving for the first time the phonon broadening at a given temperature represents the maximized information content achievable for isolated rare-gas nanoclusters.
We have added the following statement in our revised manuscript to make this point clearer (p. 4, para. 6): "A sample-temperature dependent study of the homogeneous linewidth would provide further insight into the phonon scattering as commonly done in the condensed phase [18]. However, the infrared inactivity and rapid evaporative cooling of the nanoclusters prevent any means for heat injection." -The generality of the results could have been enhanced had the authors chosen to conduct measurements of at least one more molecule.
We agree that it would be interesting to characterize the chromophore-cluster interaction for other molecular and cluster species. This is on our agenda for future experiments. However, at the current state further experiments would considerably delay the publication by at least 6 more months due to a laser brake down in our lab and difficulties to get a repair during the Corona pandemic. Given these circumstances and the timely theme of our work, we believe that it is important to share our results with the scientific community without further delay.
To account for the reviewer's comment, we have extended the comparison of our results with experiments in bulk matrices where different samples were studied (see comment #1 of reviewer #3).
Minor comments: -page 2 the authors write: "Peaks on the diagonal position of the 2D spectra reflect the linear absorption of the system, …". This is often repeated in many publications, but it is not strictly true for all systems. There are at least two complications: (1) The input laser pulse spectrum almost always provides different weights to the peaks because it is not fully de-convoluted. (2) ESA peaks can -in some systems -appear on or near the diagonal and thereby affect the relative peak heights. The authors could reword as, for example: "Peaks on the diagonal position of the 2D spectra mostly reflect the linear absorption…" We thank the reviewer for noting this inaccuracy and have changed this point in the revised manuscript, e.g. (p.2 para. 4): "Peaks on the diagonal position of the 2D spectra closely resemble the linear excitation spectrum of the system".
- Fig. (1), panel (c). The caption could be enhanced by mentioning that the coupling refers to the blue dashed arrow.
According to the suggestion of reviewer #1 we have removed this item in Fig. 1 (see also minor comment #2 of reviewer #1).
- Fig. (2), panel (b). The unit of inverse centimeters is a wavenumber, not a frequency. This abuse of the words is endemic in spectroscopy, but that does not make it correct. Please adjust so that it is scientifically correct. There are at least two good options: (1) Replace the word "frequency" with "wavenumber", or (2) Adopt the approach of Tokmakoff and label as \omega/(2 \pi c).
We have changed this in Fig. 2,4 and S1 of the revised manuscript.
-Page 3 the authors subtly introduce a control measurement of H2Pc in 1-chloronaphthalene. At what temperature were those measurements conducted?
We have added the information to the caption of Fig. 2. - Fig. (3). Why did the authors choose a waiting time of 1 ps? Time zero would in principle be better because the system would have not undergone any spectral diffusion at all. We investigated the system response also at smaller waiting times which did not reveal different behavior except for the common pulse-overlap effects observed at very short waiting times (<100fs). The latter is why we have refrained from choosing the waiting time at time zero. We clarify this in the revised manuscript (p.7, para. 3): "For the measurements in Fig. 5 the population time T was scanned from 1 to 501 ps. Values T < 100 fs were omitted to avoid the influence of parasitic pulse overlap effects." - Fig (3) panel (a). How do the authors know that the blips along the diagonal are not simply due to scatter?
To our knowledge and experimental experience scattering contributions do generally not contribute in action-detected 2DES especially if extremely dilute samples are investigated as in our case. All satellite peaks coincide with the peaks observed in light-induced fluorescence spectroscopy where they are identified as the molecular complexes. We clarify this point in the revised manuscript(p. 3, para. 4):" whose spectral positions coincide exactly with the weak features observed in the 2D spectra. Possible contributions from sample scattering can be excluded in action-detected 2DES [54]" -On page 4 the authors write "…except for an outlier at 15040 cm-1, which is due to a nonrecurring experimental artifact." What is the meaning of this? If it only occurred once, the authors should fit and present a different dataset that does not have the artifact.
The presented data set exhibits the highest resolution and was therefore chosen. The artifact does not occur in the 1D coherence scan (Fig. 2b). We agree that our statement is confusing and we have made this point clearer in the revised manuscript(p. 4, para. 5): "This outlier is not observed in our other data (Fig. 2, 5) and is attributed to an experimental artifact."

Reviewer #3 (Remarks to the Author):
Bangert et al. report photon echo study of free base phthalocyanine in helium and neon clusters. The authors found unexpectedly long electronic coherence of hundreds of picoseconds in helium and few picoseconds in neon. The photon echo technique enables attaining the homogeneous and heterogeneous linewidth. In both environments the homogeneous linewidths were found to be about 0.5 cm-1, which is very narrow. I feel these results may deserve publication in NC. However, the manuscript should be revised according to the following points.

The authors should remove uncorroborated claims and proposals.
In abstract and in the text the authors claim unprecedented spectral resolution without giving appropriate comparisons. I am rather sure that a factor of at least ten higher resolution was achieved in early hole burning experiments in solid matrices. Similar, the resolution in a conventional cw LIF spectrum of phthalocyanine in helium shown in Fig.1 of SI is higher than obtained in the reviewed manuscript.
We agree that higher resolution was achieved in other spectroscopy methods. However, these methods are either not applicable to the studied system (photochemical hole burning, see reviewer #1, comment #2) or do not yield the crucial information about the homogeneous linewidth (LIF measurements). To avoid confusion, we have omitted the term "unprecedented" in the revised manuscript wherever confusion may have been produced. The text passages are now (p.1, para. 5): "Our results provide insight into the molecule-surface binding configurations with a resolution far beyond the accuracy and resolution of current density functional theory approaches." And (p.6, para. 2) "The unique combination of 2DES with cluster-isolation techniques uncovered the homogeneous lineprofile beyond the ensemble average, providing insight into the molecule-surface binding configurations with very high resolution." Throughout the manuscript the authors implied that the photon echo technique could be applied to study: ..." local configurations in photo-chemical reactions and elucidating molecular dynamics in tailored nano-environments or at functional interfaces" (Introduction) or " the interaction of isolated molecules with metallic and semiconducting nanoclusters, of high relevance in catalysis and biomedical imaging" (Conclusions) or "to explore and control many-body mechanisms of high interest in photovoltaics, such as molecular exciton migration and annihilation processes, as well as in quantum information science which is, so far, predominately limited to atomic samples." (Conclusions) There is not any discussion of the applicability of the photon echo technique to any of the above systems. Therefore, the above propositions sound speculative to the present referee. I suggest the authors focus on the studied system and provide more physical insights into the observed effects To account for the reviewer's comment, we have modified the introduction and conclusions section of the manuscript and have extended our discussion about prospective applications of our approach. In particular, we added the following statements (p.6, para. 2): "Other methods for homogeneous linewidth retrieval cannot provide the combined time-frequency resolution of 2DES. The presented high-resolution 2DES method, thus, provides a promising perspective for the analysis of molecular dynamics as a function of the local environment with femtosecond time resolution." And "The potential of studying molecular networks on nanoconfined cluster surfaces was recently demonstrated [16,17] and the advantage of 2DES in uncovering intra-particle interactions and cooperative dynamics was proven in previous studies [72][73][74]." Furthermore, we extended the discussion of the observed effects (p. 5, para. 2): "In contrast, experiments in bulk Ne matrices showed an almost one order of magnitude broader inhomogeneous linewidth of ≈200 cm−1 [58]. The same behavior is observed for 3, 4, 9, 10-perylenetetracarboxylic dianhydride (PTCDA) [61]. The much broader inhomogeneous linewidth in the bulk environment may be explained by the formation of a solvation shell when the molecule is fully embedded in the bulk matrix compared to when bound to the surface of a cluster. At the same time, the homogeneous broadening of H2Pc-NeN seems in reasonable agreement with the values obtained in bulk matrices of heavier rare-gases where a factor of ≈ 5 smaller homogeneous broadening is observed [62]. The lower matrix temperature (4 K) in these experiments may explain the reduced phonon broadening than observed in our cluster samples with temperatures of ≈10 K." Further discussions can be found in comment #5 below.
2. The principle of the two-dimensional electronic spectroscopy should be introduced in the main text that should be accessible for non-specialists.
We agree that our work addresses an interdisciplinary readership which may not be familiar with the 2DES technique. However, in order to increase the readability of the manuscript we have spared experimental details described in previous work (see also response to reviewer #1, comment #4).
To account for the reviewer's comment, we have simplified the experimental description in section II A of the main text and have added references introducing the 2DES technique. In particular, we added the following paragraph (p. 2, para. 2): "So far, 2DES is mainly performed in the condensed phase and the desired nonlinear signals are separated from the background by non-collinear fourwave mixing geometries (coherent-detected 2DES) [37]. Conversely, for the dilute cluster beam samples a collinear beam geometry is needed combined with the detection of an action signal (action-detected 2DES) [29]. In the latter, the sample is excited with a sequence of four femtosecond laser pulses (Fig. 1b) and the fourth-order light-matter response is deduced from the detected fluorescence. The pulse delays τ,t are interferometrically scanned to track the free polarization decay of the sample induced by pulse 1 and 3, respectively. Accordingly, a Fourier transform with respect to τ,t yields 2D frequency-spectra, which directly correlate the excitation (x-axis) and detection (y-axis) frequencies, while the time delay T determines the time evolution of the correlation spectra (Fig.  1c,d) [20]." And (p. 2 para. 3): "The phase modulation leads to characteristic beat notes in the fluorescence yield based on which the linear and nonlinear signal contributions are efficiently separated and amplified using lock-in detection." 3. "A Fourier transform of the 1D coherence scans yield the linear absorption spectra of both systems (Fig. 2b). (p.3)" I feel this is not just FT, because the frequency of the oscillations in a) is much smaller than the peak frequency in b). The authors should better explain the application of the FT.
The responsible effect for this behavior is the rotating frame detection of the signal in the time domain. We have added the following explanation to the revised manuscript to make this point clearer (Fig. 2 caption): "In return, the frequency axis in the Fourier spectra in (b) are up-shifted by the same amount to recover the absolute transition frequencies of the system". Furthermore, we have added more information in the Methods section (see comment #6 below). 4. " Its far extending horizontal and vertical tails reflect the Lorentzian lineshape and are augmented by the saturated color scale." (p.3) I do not see any horizontal or vertical tails in Fig. 3 (a).
We meant the horizontal/vertical lines intersecting at the main peak of the 2D spectrum. A horizontal/vertical cut through the 2D spectrum reveals that these are the tails of the line shape of the main spectral peak. We modified our explanation in the manuscript to make this clearer (p. 3, para. 4): "The horizontal and vertical lines intersecting with the ZPL show the tails of the ZPL line profile, which are augmented by the saturated color scale." 5. " We conclude that the molecules are mainly immobile on the (Ne) cluster surface on the time scale of 500 ps, which was previously only indirectly confirmed for ground state molecules [57]." (p.5) It would be nice to have some perspective on the limits imposed by the clusters and also to have measurements, such as in Fig. 5 with helium clusters. For example, although the molecules in neon are fixed in the cluster frame, the clusters nearly certainly are rotating in the laboratory frame. In case of helium clusters the molecules may rotate and translate inside owing to its superfluid nature, on the other hand the traditional phonon induced dephasing may be muted due to finite size effects.
Some more detailed discussion of the physical origin of the long coherence in clusters as compared to other previously studied condensed system will make this manuscript much stronger.
We have added the suggested discussion in the revised manuscript (p.5, para. 5): "We note, that the rotation of the cluster in the laboratory frame adds in principle another contribution to the inhomogeneous broadening due to the cluster size distribution in the probed ensemble. This broadening is however expected to be negligible due to the large moment of inertia of the clusters.
On the contrary, dopant species are highly mobile on the surface of superfluid He droplets as reflected in the formation of alkali complexes on the droplet surface [66]. Likewise, species fully immersed in the quantum fluid clusters exhibit high mobility. Their rotational motion is well studied and served as a probe of the internal temperature of He clusters [35]. The solvation mechanisms of organic chromophores in superfluid He droplets have been previously studied with high-resolution frequency-domain spectroscopy [67]. It was rationalized that solutes are embedded in a nonsuperfluid He solvation layer. The different configuration of the solvation complexes lead to spectral finestructures [19,68] while the dispersive solute-cluster interaction causes asymmetric lineshapes due to the statistical cluster size distribution [52]. The homogeneous linewidth for the ZPL of solutes in superfluid He has not been determined to date. High-resolution 2DES may solve this problem and provide new insight into the solvation mechanisms in superfluid media. For Pc-HeN an inhomogeneous linewidth of ≥ 0.1 cm−1 was found and an estimate for the homogeneous broadening of 0.02 cm−1 was indirectly deduced [52]. Both are beyond our current experimental resolution. This could be solved by deploying advanced sampling strategies [69][70][71] or implementing frequency-comb technology [55] in order to increase the resolution to the required regime, thus, opening a promising perspective for the study of solvation mechanisms in superfluid environments." 6. In methods, the modulation frequencies Omega(i) should be given. The explanation of the signal processing in the last two paragraphs is not understandable for a not specialist. It would be nice to have some equations for more clarity.
We have added the frequency values in the Methods section. Moreover, we extended the explanation of the signal processing and included some equations to make it clearer to the reader. We changed the following paragraph to make this clearer (p. 7, para. 5): "As reference signal for the lock-in detection, a portion of the pulse train is spectrally filtered in a monochromator (0.025 nm spectral resolution) at a frequency of νR = c/662.33 nm and detected with a PMT. The reference signal records the optical interference of the pulses and, thus, tracks the phase changes and phase jitter in the optical setup at the frequency νR. The heterodyne demodulation of the fluorescence signal S(τ,T,t,Ω21 ±Ω43) with reference signal R(τ,T,t,Ω21 ±Ω43) inside the lock-in amplifier removes the modulation as well as the phase jitter from the signal S, which provides the required interferometric stability for the measurements. In addition, S is detected in the rotating frame of R, which down-shifts the signal frequencies νS by 2 orders of magnitude to ν = νS −νR. To recover the absolute frequencies in the Fourier spectra, the frequency axis are up-shifted again by νR."


s2fieldsofstudy:
Chemistry
Materials Science

text:
Synthesis of Xylan-Click-Quaternized Chitosan via Click Chemistry and Its Application in the Preparation of Nanometal Materials

For the high-valued utilization of hemicelluloses and for realizing the controllable synthesis of NPs, this paper’s aim is to combine xylan, chitosan and nanometal materials at the same time. In this research study, firstly, propargyl xylan was synthesized via nucleophilic substitution reaction between xylan and propargyl bromide in NaOH solution. On the other hand, a tosyl group was introduced onto the 6th position of synthesized quaternized chitosan (QCS), and the azide group replaced the tosyl group to obtain 6-amido-QCS (QCS-N3). The synthesis conditions of the above reactions were optimized. Subsequently, the novel xylan-click-QCS polymer was obtained via click reaction between terminal alkyne groups on the xylan chains and azide groups on QCS. Then, AgNPs and AuNPs were synthesized by adopting the xylan-click-QCS polymer as the reducing and stabilizing agent, and the reaction conditions were optimized to obtain well-dispersed and highly stable nanoparticles. There were two kinds of Ag nanomaterials, with diameters of 10~20 nm and 2~5 nm, respectively, indicating the formation of Ag nanoclusters, except for Ag nanoparticles, in this reaction. The diameter of the synthesized AuNPs was 20~30 nm, which possessed a more uniform size distribution. The Ag nanoclusters with a smaller size (2~5 nm) could inhibit MCF-7 cell proliferation effectively, indicating their application potential in cancer therapy. The study gives a new approach to the high-value utilization of biopolymers.

Introduction
Noble-metal nanoparticles (NPs) have been widely used in various fields, such as catalysis [1,2], biosensors [3,4], antibacterial materials [5][6][7] and pharmaceuticals [8,9], due to their unique physical and chemical properties. Considering its wide application in biomedical and clinical practice, the green synthesis of NPs by adopting reducing sugars as reductants and stabilizers has attracted the attention of many [10][11][12][13]. Hemicelluloses are abundant and cheap natural polysaccharides with great potential application value [14]. The presence of hydroxyl groups (-OH) and carboxyl groups (-COOH) makes them candidates for the green synthesis of NPs. For example, green synthesized NPs were obtained by using plant extract containing hemicelluloses [15][16][17]. In our previous study, we prepared NPs in the presence of xylan [18,19], and the synthesized NPs with controllable shape were distributed uniformly after optimizing the reaction conditions. However, there were still some problems in those reactions; first, due to the relatively weak interaction of hydroxyl interaction of hydroxyl oxygen and NPs, excessive reducing sugars were added into the reaction system to stabilize the synthesized NPs, which may have affected the further application of the NPs. Second, the metal ions easily complexed with the reducing aldehyde groups, and the NPs synthesized in a short time (few minutes) with an uneven size distribution. The researchers, then, extended the reaction time to homogenize the size distribution, which made the reactions hard to control. Therefore, it is necessary to modify the reducing sugars to control the reaction rate and effectively stabilizing the synthesized NPs. Chitosan derivatives have been used to prepare noble-metal NPs. As a biocompatible polysaccharide with long molecular length, it forms a net structure packing the Ag NPs and effectively stabilizing the NPs [20,21]. Moreover, its biocompatibility extends its application in biomedicine, food chemistry, biosensors, etc. [22]. Hence, the xylan-chitosan complex may provide a more effective and controllable way for the green synthesis of NPs.
Nevertheless, hemicelluloses can be soluble in alkaline solution, while chitosan can only be soluble in acidic solution; most hemicelluloses-chitosan complexes have been prepared by aggregation or physical crosslinking rather than real chemical bonds [23][24][25][26][27], which may limit their homogeneous dispersion and stability. Proposed by Sharpless in the past decade, click chemistry is a class of chemical reactions well-known because of its full advantages of being effective, reliable and selective and its high yields with little or no byproducts [28][29][30]. One of the most popular reactions within the click chemistry philosophy is the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction between azide and terminal alkyne groups, which has received increasing attention because of its simple and powerful modular synthesis approach [30][31][32]. Moreover, there have been some reports about the combination of the click reaction and NPs for further applications [33][34][35]. For example, Dondi et al. [36] synthesized highly size-and shape-controlled AgNPs via sugar triazole templates, and found that sugar triazole ligands can control the nucleation and growth phases of the synthesis and passivate the surface of the resultant AgNPs. Therefore, we speculated that the hemicelluloses-chitosan click reaction product may overcome the disadvantages of reducing sugars for NP synthesizing.
In this article, xylan-the model compound of hemicelluloses-was used as raw material; a water-soluble chitosan derivative-quaternized chitosan (QCS) was prepared under microwave irradiation in aqueous solution. Then, terminal propargyl-functionalized xylan and the azido-functionalized QCS (QCS-N3) were synthesized. Subsequently, the novel xylan-click-QCS polymer was created via the one-step alkyne-azide click reaction in the presence of Cu(I) ( Figure S1). We also optimized the reaction conditions. The successful binding of xylan and QCS was confirmed and characterized by FT-IR and 13 C NMR. Then, AgNPs and AuNPs were synthesized by adopting the xylan-click-QCS polymer as the reducing and stabilizing agent, and the reaction conditions were optimized. Finally, the anticancer property of the Ag nanomaterials was evaluated ( Figure 1).  Xylan was propargylated with propargyl bromide in this paper. As shown in Table 1, the DS of Pg-xylan first increased and then decreased with the prolonging of the reaction time, and the maximum DS was 0.23 at 50 min. A longer reaction time benefitted the DS of Pg-xylan; however, the DS decreased slightly with the increase in reaction time after 50 min. A higher DS was obtained when the reaction temperature increased from 30 to 50 • C, but a further rise in temperature to 60 • C led to a fall in the DS from 0.65 to 0.44. This may have been induced by the degradation of xylan at a high temperature, which led to the decrease in the DS. This result also suggests that the preparation of Pg-xylan should be controlled at a mild temperature. Moreover, an increase in the ratio of PgBr/xylan from 0.5:1 to 2:1 resulted in an increase in the DS of Pg-xylan from 0.07 to 0.65; thereafter, the DS increased slightly to 0.70. It shows that a high amount of PgBr has a positive influence on the substitution of hydroxyl groups on the xylan chains. The reason could be that there was more PgBr reagent in contact with xylan molecules at a higher concentration. Moreover, the influence of the ratio of PgBr/xylan from 2:1 to 3:1 was negligible. As a result, the optimized Pg-xylan with a DS value of 0.65 could be obtained under the condition of a PgBr/xylan ratio of 2:1 at 50 • C for 50 min under microwave irradiation. As shown in Table 2, we also investigated the influence of the reaction conditions on the DS of QCS-N 3 under microwave irradiation. Increasing the reaction temperature from 50 to 80 • C resulted in an increase in the DS from 0.42 to 1.26, but a further rise in temperature to 90 and 100 • C had a slight influence on the DS. Moreover, the DS of QCS-N 3 exceeded 1 when the reaction temperature reached 80 • C, indicating that other than the 6th position, the 3rd position of QCS was substituted by the azide group in this reaction. Furthermore, the DS of QCS-N 3 increased from 1.26 to 1.57 with the increase in the reaction time from 60 to 80 min, after which the DS decreased to 1.22 and 0.88 with a reaction time of 100 and 120 min. The reason for this decrease is probably the partial degradation of QCS-6-OTs over a prolonged reaction time. Moreover, the DS of QCS-N 3 increased from 0.70 to 1.55 with the increase in the ratio of NaN 3 /QCS-6-OTs from 2:1 to 4:1. However, a higher NaN 3 concentration showed little effect on the DS of QCS-N 3 , which gave a slight increase in value from 1.55 to 1.61. Thus, the optimized QCS-N 3 with the DS value of 1.55 can be obtained under the conditions of a NaN 3 /QCS-6-OTs ratio of 4:1 at 80 • C for 80 min under microwave irradiation.  Figure 2a shows the FT-IR spectra of original xylan and Pg-xylan; clearly, the characteristic peaks at 3421, 2931, 1417, 1040 and 899 cm −1 originated from pure xylan. Compared with pure xylan, similar absorbances could be observed in the spectrum of Pg-xylan, indicating that the backbone structure of xylan remained after the reaction. Moreover, the characteristic peaks at 1417, 1040 and 899 cm −1 weakened after the reaction, which may have been induced by the effect of microwave irradiation [18]. On the other hand, a new adsorption band appeared at 2117 cm −1 , corresponding to C≡C stretching, indicating the successful incorporation of terminal alkyne groups onto the xylan chains [37,38].   Figure 2a shows the FT-IR spectra of original xylan and Pg-xylan; clearly, the characteristic peaks at 3421, 2931, 1417, 1040 and 899 cm −1 originated from pure xylan. Compared with pure xylan, similar absorbances could be observed in the spectrum of Pg-xylan, indicating that the backbone structure of xylan remained after the reaction. Moreover, the characteristic peaks at 1417, 1040 and 899 cm −1 weakened after the reaction, which may have been induced by the effect of microwave irradiation [18]. On the other hand, a new adsorption band appeared at 2117 cm −1 , corresponding to C≡C stretching, indicating the successful incorporation of terminal alkyne groups onto the xylan chains [37,38].  Figure 2b shows the FT-IR spectra of chitosan, QCS, QCS-6-OTs and QCS-N3. In the spectrum of chitosan, the absorption peak at 3480 cm −1 was ascribed to O-H stretching combined with N-H stretching vibration, while the characteristic absorption band at 1592 cm −1 was assigned to the vibration of −NH2 [39]. Compared with chitosan, the characteristic peak (1592 cm −1 ) of -NH2 was weakened in the spectrum of QCS, and a new peak at 1483 cm −1 appeared, attributable to the methyl groups in quaternary ammonium salt. The results prove that the chemical modification of chitosan with ETA resulted in the N-  Figure 2b shows the FT-IR spectra of chitosan, QCS, QCS-6-OTs and QCS-N 3 . In the spectrum of chitosan, the absorption peak at 3480 cm −1 was ascribed to O-H stretching combined with N-H stretching vibration, while the characteristic absorption band at 1592 cm −1 was assigned to the vibration of −NH 2 [39]. Compared with chitosan, the characteristic peak (1592 cm −1 ) of -NH 2 was weakened in the spectrum of QCS, and a new peak at 1483 cm −1 appeared, attributable to the methyl groups in quaternary ammonium salt. The results prove that the chemical modification of chitosan with ETA resulted in the N-monosubstitution of chitosan, which coincides with the previous study [40]. In the spectrum of QCS-6-OTs, there were characteristic peaks of the tosyl group at 1173 cm −1 and 900~600 cm −1 , indicating the success of the reaction between the tosyl group and QCS. In the spectrum of QCS-6-OTs, the characteristic peaks at 1173 cm −1 and 900~600 cm −1 were attributable to the disappearance of the tosyl group, indicating that the tosyl group was substituted by other groups. Noticeably, the presence of the new band between 2200 and 2000 cm −1 was assigned to -N 3 stretching. These new bands confirmed the successful introduction of azide groups onto the QCS backbone [41]. Figure 3 shows the FT-IR spectra of Pg-xylan, QCS-N 3 and xylan-click-QCS. After the click reaction, the band at 2117 cm −1 due to C≡C stretching and the bands at 2131 and 2038 cm −1 due to -N 3 stretching completely disappeared, and the characteristic band of C=C appeared at 775 cm −1 . Moreover, the characteristic absorbance bands of xylan and QCS still appeared in the spectrum of xylan-click-QCS, verifying that the reaction between alkyne groups on xylan and azide groups on QCS took place via click chemistry [42]. monosubstitution of chitosan, which coincides with the previous study [40]. In the spectrum of QCS-6-OTs, there were characteristic peaks of the tosyl group at 1173 cm −1 and 900~600 cm −1 , indicating the success of the reaction between the tosyl group and QCS. In the spectrum of QCS-6-OTs, the characteristic peaks at 1173 cm −1 and 900~600 cm −1 were attributable to the disappearance of the tosyl group, indicating that the tosyl group was substituted by other groups. Noticeably, the presence of the new band between 2200 and 2000 cm −1 was assigned to -N3 stretching. These new bands confirmed the successful introduction of azide groups onto the QCS backbone [41]. Figure 3 shows the FT-IR spectra of Pg-xylan, QCS-N3 and xylan-click-QCS. After the click reaction, the band at 2117 cm −1 due to C≡C stretching and the bands at 2131 and 2038 cm −1 due to -N3 stretching completely disappeared, and the characteristic band of C=C appeared at 775 cm −1 . Moreover, the characteristic absorbance bands of xylan and QCS still appeared in the spectrum of xylan-click-QCS, verifying that the reaction between alkyne groups on xylan and azide groups on QCS took place via click chemistry [42].      Figure 5a exhibits the 13 C-NMR spectrum of QCS. The peaks at δ = 102.3, 62.6, 73.3, 77.9, 74.9 and 60.8 ppm were attributed to C-1, C-2, C-3, C-4, C-5 and C-6 in the chitosan unit, respectively. The peaks at δ = 51.69, 54.19, 65.33 and 69.16 ppm were attributed to Ca, C-d, C-b and C-c of the quaternary ammonium group, suggesting that quaternary ammonium groups were successfully introduced onto the chitosan backbone [40]. Figure 5b shows the 13 C-NMR spectrum of QCS-OTs. There were strong signals between 120 and  unit, respectively. The peaks at δ = 51.69, 54.19, 65.33 and 69.16 ppm were attributed to C-a, C-d, C-b and C-c of the quaternary ammonium group, suggesting that quaternary ammonium groups were successfully introduced onto the chitosan backbone [40]. Figure 5b shows the 13 C-NMR spectrum of QCS-OTs. There were strong signals between 120 and 150 ppm, attributable to tosyl groups. Moreover, the signal at 20.55 ppm originated from the methyl group. All these results indicate that the reaction between QCS and TsCl was successfully carried out. Figure 5c shows the 13 C-NMR spectrum of QCS-N 3 ; the signals originated by tosyl groups decreased, indicating that azide groups replaced tosyl groups on the QCS chains. Figure 5d shows the 13 C NMR spectrum of xylan-click-QCS; after the Huisgen cycloaddition reaction, the signals of the triazole ring carbons were clearly identified between 120 and 140 ppm, supporting the success of the click reaction between Pg-xylan and QCS-N 3 .  Figure 6 shows the UV-Vis spectra of the xylan-click-QCS/AgNP composites obtained after irradiation under different reaction conditions. The typical surface plasmon resonance (SPR) band around 415 nm was observed, which indicated the formation of silver nanoparticles in the reaction. In Figure 6a, there was no SPR absorbance at 0 min, indicating that no AgNPs formed without heating. Then, the intensity of SPR peaks in-  Figure 6 shows the UV-Vis spectra of the xylan-click-QCS/AgNP composites obtained after irradiation under different reaction conditions. The typical surface plasmon resonance (SPR) band around 415 nm was observed, which indicated the formation of silver nanoparticles in the reaction. In Figure 6a, there was no SPR absorbance at 0 min, indicating that no AgNPs formed without heating. Then, the intensity of SPR peaks increased with the increase in reaction time and reached the maximum at 20 min, which confirmed the formation of more AgNPs. However, the absorption of the UV-Vis spectra increased slightly when further prolonging the heating time, indicating that an excessive reaction time has little influence on the formation of AgNPs. Thus, the optimal reaction time was determined at 20 min for this reaction. In Figure 6b, the absorption peak was weak at 60 °C, implying that this reaction temperature was too low to generate enough AgNPs. When the reaction temperature reached 70 °C, the absorption peak was strong and quite narrow, suggesting that more AgNPs were generated, and the AgNPs were monodispersed. At 80 °C, the absorption band became weak and broadened; we speculate that a high reaction temperature may have led to the aggregation of the formed AgNPs in this reaction. Therefore, 70 °C was the appropriate reaction temperature in this experiment. Figure 6c shows the influence of Ag + contents on the formation of AgNPs. Clearly, different Ag + contents had little influence on the intensity of UV-Vis spectra. However, the absorption band broadened with a slight decrease when the Ag + content exceeded 0.10 mmol, and there was silver glass in the reaction, implying that excessive Ag + contents may have led to the aggregation of the formed AgNPs, and the AgNPs became larger with a broader size distribution. Therefore, we chose 50 mg:0.08 mmol as the appropriate mass ratio in this experiment. Figure 6d shows the influence of ammonia concentration on the formation of AgNPs. The absorption peak was weak when the ammonia concentration was 0.5%; then, the intensity of the absorption peaks increased with the increase in ammonia concentration. However, the ammonia concentration had little influence on absorbance intensity from 1% to 4%. Thus, 1% was chosen as the optimized ammonia concentration in this reaction.

Preparation and Characterization of Xylan-Click-QCS/AgNP Composites 2.2.1. Preparation of Xylan-Click-QCS/AgNP Composites
Based on the above analysis, the best reaction conditions were as follows: under microwave irradiation, the reaction time was 20 min; the reaction temperature was 70 °C; the ratio of xylan-click-QCS to AgNO3 was 50 mg:0.08 mmol; and the ammonia concentration was 1%. In Figure 6b, the absorption peak was weak at 60 • C, implying that this reaction temperature was too low to generate enough AgNPs. When the reaction temperature reached 70 • C, the absorption peak was strong and quite narrow, suggesting that more AgNPs were generated, and the AgNPs were monodispersed. At 80 • C, the absorption band became weak and broadened; we speculate that a high reaction temperature may have led to the aggregation of the formed AgNPs in this reaction. Therefore, 70 • C was the appropriate reaction temperature in this experiment. Figure 6c shows the influence of Ag + contents on the formation of AgNPs. Clearly, different Ag + contents had little influence on the intensity of UV-Vis spectra. However, the absorption band broadened with a slight decrease when the Ag + content exceeded 0.10 mmol, and there was silver glass in the reaction, implying that excessive Ag + contents may have led to the aggregation of the formed AgNPs, and the AgNPs became larger with a broader size distribution. Therefore, we chose 50 mg:0.08 mmol as the appropriate mass ratio in this experiment. Figure 6d shows the influence of ammonia concentration on the formation of AgNPs. The absorption peak was weak when the ammonia concentration was 0.5%; then, the intensity of the absorption peaks increased with the increase in ammonia concentration. However, the ammonia concentration had little influence on absorbance intensity from 1% to 4%. Thus, 1% was chosen as the optimized ammonia concentration in this reaction.
Based on the above analysis, the best reaction conditions were as follows: under microwave irradiation, the reaction time was 20 min; the reaction temperature was 70 • C; the ratio of xylan-click-QCS to AgNO 3 was 50 mg:0.08 mmol; and the ammonia concentration was 1%. Figure 7a shows the TEM image of the AgNPs prepared in this reaction. Most of the AgNPs were spherical, but the particle size was uneven. Bigger particles with a size range of 10-20 nm are generally called Ag nanoparticles, while other smaller particles with a size range of 2~5 nm are generally called Ag nanoclusters [43]. On the one hand, this phenomenon may have been induced by the complicated component of the xylan-click-QCS composites, which may have contained unreacted xylan and QCS. On the other hand, Ag + may have coordinated with the nitrogen atom of the triazole ring, which may rule out the coordination between Ag + and the reducing aldehyde groups at the end of the sugar chains [36]. A longer time was needed to generate big AgNPs for the xylan-click-QCS, while the generation time was short when xylan or QCS acted as the reducing agent; the longer reaction time may have led to the aggregation of xylan-or QCS-generated AgNPs. Thus, the xylan-or QCS-generated AgNPs possessed a size in the range of 10~20 nm, while the xylan-click-QCS-generated AgNPs possessed a size in the range of 2~5 nm under these reaction conditions.

Characterization of Xylan-Click-QCS/AgNP Composites
Molecules 2022, 27, x FOR PEER REVIEW 9 of 17 may have coordinated with the nitrogen atom of the triazole ring, which may rule out the coordination between Ag + and the reducing aldehyde groups at the end of the sugar chains [36]. A longer time was needed to generate big AgNPs for the xylan-click-QCS, while the generation time was short when xylan or QCS acted as the reducing agent; the longer reaction time may have led to the aggregation of xylan-or QCS-generated AgNPs. Thus, the xylan-or QCS-generated AgNPs possessed a size in the range of 10~20 nm, while the xylan-click-QCS-generated AgNPs possessed a size in the range of 2~5 nm under these reaction conditions. For further application of the AgNPs prepared in this reaction, the xylan-click-QCS/AgNP composites were centrifuged to obtain supernatant and sediment, respectively. The sediment dissolved in water again, and the TEM images of the sediment and supernatant AgNPs are shown in Figure 7b,c. In Figure 7b, most of the AgNPs were bigger with a size range of 5~20 nm. In Figure 7c, the Ag nanoclusters with a size range of 2~5 nm were well-dispersed in the supernatant. In addition, the high-resolution transmission electron microscopy (HRTEM) analysis of the Ag nanoclusters in the supernatant is shown in Figure 7d; the Ag nanoclusters were spherical with a clear lattice fringe, indicating the formation of Ag nanoclusters. Due to their ultra-small sizes, Ag nanoclusters possess unique physical and chemical properties compared with those of larger-sized nanoparticles, and there have been reports about Ag nanoclusters being used as electrochemical catalysts [44,45], fluorescence-based sensors [46,47], preparation materials for optical fibers [48], etc. Therefore, the prepared Ag nanoclusters could have wide applications in many areas. For further application of the AgNPs prepared in this reaction, the xylan-click-QCS/AgNP composites were centrifuged to obtain supernatant and sediment, respectively. The sediment dissolved in water again, and the TEM images of the sediment and supernatant AgNPs are shown in Figure 7b,c. In Figure 7b, most of the AgNPs were bigger with a size range of 5~20 nm. In Figure 7c, the Ag nanoclusters with a size range of 2~5 nm were well-dispersed in the supernatant. In addition, the high-resolution transmission electron microscopy (HRTEM) analysis of the Ag nanoclusters in the supernatant is shown in Figure 7d; the Ag nanoclusters were spherical with a clear lattice fringe, indicating the formation of Ag nanoclusters. Due to their ultra-small sizes, Ag nanoclusters possess unique physical and chemical properties compared with those of larger-sized nanoparticles, and there have been reports about Ag nanoclusters being used as electrochemical catalysts [44,45], fluorescence-based sensors [46,47], preparation materials for optical fibers [48], etc. Therefore, the prepared Ag nanoclusters could have wide applications in many areas. Figure 8a-c show the UV-Vis spectra of xylan-click-QCS/AuNP composites prepared under microwave irradiation; the strong absorption peaks centered around 520 nm, relating to the characteristic surface plasma resonance (SPR) peak of AuNPs, indicating the formation of AuNPs. In Figure 8a, the absorption peak was weak at 70 • C, indicating that few AuNPs generated at this temperature. The absorbance of the UV-Vis spectra reached the maximum when the reaction temperature was 80 and 90 • C. Moreover, the UV-Vis spectrum at 90 • C was narrower with a blue shift compared with that at 80 • C, suggesting that the AuNPs became smaller with a narrow size distribution at 90 • C. However, the absorption peak became weak and broadened at 100 • C, and the solution turned to purple during the reaction, implying that part of the AuNPs aggregated under these reaction conditions. When the reaction temperature reached 100 • C, the water in the reaction system evaporated quickly, resulting in an unstable reaction system, and the generated AuNPs were uneven under this condition. Therefore, 90 • C was chosen as the optimal reaction temperature in this experiment. few AuNPs generated at this temperature. The absorbance of the UV-Vis spectra reached the maximum when the reaction temperature was 80 and 90 °C. Moreover, the UV-Vis spectrum at 90 °C was narrower with a blue shift compared with that at 80 °C, suggesting that the AuNPs became smaller with a narrow size distribution at 90 °C. However, the absorption peak became weak and broadened at 100 °C, and the solution turned to purple during the reaction, implying that part of the AuNPs aggregated under these reaction conditions. When the reaction temperature reached 100 °C, the water in the reaction system evaporated quickly, resulting in an unstable reaction system, and the generated AuNPs were uneven under this condition. Therefore, 90 °C was chosen as the optimal reaction temperature in this experiment. In Figure 8b, the intensity of SPR peaks increased with the increase in HAuCl4·4H2O concentration and reached the maximum when the mass ratio of xylan-click-QCS to HAuCl4·4H2O was 50 mg: 10 mg, which confirmed the formation of more AuNPs. Then, the absorption band became weak and broadened with a red shift when the HAuCl4·4H2O concentration further increased to 15 mg; this may have been induced by the aggregation of the AuNPs. Therefore, we chose 50 mg: 10 mg as the appropriate mass ratio in this experiment. In Figure 8c, the absorbance was weak before 30 min; we speculate that this reaction may need a longer reaction time. It is worth noting that the absorbance decreased slightly when the reaction time reached 70 min. Thus, the optimal reaction time was determined to be 70 min for this reaction.

Preparation of Xylan-Click-QCS/AuNP Composites
In Figure 8d, we compared the UV-Vis spectrum curves of xylan-click-QCS/AuNP composites and xylan /AuNP composites. Clearly, the AuNPs prepared by xylan-click-QCS possessed a narrow absorption band; this may be attributed to the triazole ring. Au 3+ In Figure 8b, the intensity of SPR peaks increased with the increase in HAuCl 4 ·4H 2 O concentration and reached the maximum when the mass ratio of xylan-click-QCS to HAuCl 4 ·4H 2 O was 50 mg:10 mg, which confirmed the formation of more AuNPs. Then, the absorption band became weak and broadened with a red shift when the HAuCl 4 ·4H 2 O concentration further increased to 15 mg; this may have been induced by the aggregation of the AuNPs. Therefore, we chose 50 mg:10 mg as the appropriate mass ratio in this experiment. In Figure 8c, the absorbance was weak before 30 min; we speculate that this reaction may need a longer reaction time. It is worth noting that the absorbance decreased slightly when the reaction time reached 70 min. Thus, the optimal reaction time was determined to be 70 min for this reaction.
In Figure 8d, we compared the UV-Vis spectrum curves of xylan-click-QCS/AuNP composites and xylan /AuNP composites. Clearly, the AuNPs prepared by xylan-click-QCS possessed a narrow absorption band; this may be attributed to the triazole ring. Au 3+ easily coordinated with the nitrogen atom of the triazole ring, which in turn provided a template for the nucleation and growth of the AuNPs. Moreover, the triazole ring can passivate the surface of the resultant AuNPs [49] and can finally present functional groups on the surface of the AuNPs [36]. Figure 9a shows the TEM image of fresh AuNPs; we can clearly see that most of the AuNPs were spherical and well-dispersed, with the diameters of 20~30 nm. Figure 9b is the high-resolution transmission electron microscopy (HRTEM) image of a single AuNP; the AuNP is spherical with a lattice fringe. In Figure 9c, the Au element in the EDS profile originated from the AuNPs reduced by xylan-click-QCS, demonstrating the formation of AuNPs. Moreover, the Cr element at 5.4 keV and Fe element at 6.4 and 6.9 keV may have originated from the specimen holder or contaminant.  Figure 9a shows the TEM image of fresh AuNPs; we can clearly see that most of the AuNPs were spherical and well-dispersed, with the diameters of 20~30 nm. Figure 9b is the high-resolution transmission electron microscopy (HRTEM) image of a single AuNP; the AuNP is spherical with a lattice fringe. In Figure 9c, the Au element in the EDS profile originated from the AuNPs reduced by xylan-click-QCS, demonstrating the formation of AuNPs. Moreover, the Cr element at 5.4 keV and Fe element at 6.4 and 6.9 keV may have originated from the specimen holder or contaminant.  Figure 10 shows the transportation of sediment and supernatant Ag NPs during cell incubation. Obviously, sediment and supernatant Ag NPs transported into cells over a certain incubation time. It was noted that supernatant Ag NPs could be observed in the cell even after 1 h, which may be attributed to their smaller size [40]. As a result, the cells exposed to supernatant Ag NPs lost their typical shape and shrunk to a smaller size [50]. Figure 11 shows that the sediment and supernatant Ag NPs significantly inhibited the proliferation of MCF-7. Moreover, the supernatant showed stronger cell-proliferation inhibition ability than the sediment, which coincides well with the Ag NPs dispersion results during cell incubation. The result indicates that xylan-click-QCS/AgNPs could inhibit cancer.   Figure 10 shows the transportation of sediment and supernatant Ag NPs during cell incubation. Obviously, sediment and supernatant Ag NPs transported into cells over a certain incubation time. It was noted that supernatant Ag NPs could be observed in the cell even after 1 h, which may be attributed to their smaller size [40]. As a result, the cells exposed to supernatant Ag NPs lost their typical shape and shrunk to a smaller size [50]. Figure 11 shows that the sediment and supernatant Ag NPs significantly inhibited the proliferation of MCF-7. Moreover, the supernatant showed stronger cell-proliferation inhibition ability than the sediment, which coincides well with the Ag NPs dispersion results during cell incubation. The result indicates that xylan-click-QCS/AgNPs could inhibit cancer. cell even after 1 h, which may be attributed to their smaller size [40]. As a result, the cells exposed to supernatant Ag NPs lost their typical shape and shrunk to a smaller size [50]. Figure 11 shows that the sediment and supernatant Ag NPs significantly inhibited the proliferation of MCF-7. Moreover, the supernatant showed stronger cell-proliferation inhibition ability than the sediment, which coincides well with the Ag NPs dispersion results during cell incubation. The result indicates that xylan-click-QCS/AgNPs could inhibit cancer.

Synthesis of Propargyl Xylan (Pg-Xylan)
Propargyl hemicellulose was synthesized using the method by Nakagawa et al. with some modifications [51]. Briefly, 0.33 g of xylan was dissolved in 8 mL of 2% NaOH; then, we added 3 mL of H2O and stirred for 1 h to guarantee uniform solubilization. Subsequently, the required quantities of propargyl bromide (the molar ratios of PgBr to xylan were 0.5:1, 1:1, 2:1 and 3:1) were slowly added to the xylan solution. The reaction temperatures were subsequently kept at 30, 45, 50 and 60 °C under microwave irradiation, and the reaction was run for 30, 40, 50 and 60 min, respectively. After the required time, the product was precipitated with ethanol. Then, the precipitate was washed with ether and freeze-dried to obtain Pg-xylan. The degrees of substitution (DSs) were calculated using the following equation:

Materials
Chitosan was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd. (Jinan, China). Its degree of deacetylation was 85%, and its weight average molecular weight (Mw) was 2.0 × 10 5 g/mol. Xylan isolated from bagasse was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Its Mw was 4.9 × 10 4 g/mol.  Propargyl hemicellulose was synthesized using the method by Nakagawa et al. with some modifications [51]. Briefly, 0.33 g of xylan was dissolved in 8 mL of 2% NaOH; then, we added 3 mL of H 2 O and stirred for 1 h to guarantee uniform solubilization. Subsequently, the required quantities of propargyl bromide (the molar ratios of PgBr to xylan were 0.5:1, 1:1, 2:1 and 3:1) were slowly added to the xylan solution. The reaction temperatures were subsequently kept at 30, 45, 50 and 60 • C under microwave irradiation, and the reaction was run for 30, 40, 50 and 60 min, respectively. After the required time, the product was precipitated with ethanol. Then, the precipitate was washed with ether and freeze-dried to obtain Pg-xylan. The degrees of substitution (DSs) were calculated using the following equation: where C is the ratio of C content in the Pg-xylan products determined by elemental analysis using an elemental analyzer (Elementar, Langenselbold, Germany), and C 0 is the ratio of C content in xylan.

Synthesis of Azide-6-Quaternized Chitosan (QCS-N 3 )
Synthesis of quaternized chitosan (QCS): QCS was synthesized according to the previous work [40]. Chitosan powder (1.0 g) was dissolved in acetic acid (2 wt%; 100 mL) and filtered using a 200-mesh cloth; sodium hydroxide (15 wt%) was added dropwise till complete precipitation; then, the precipitation was washed to neutral. The obtained purified chitosan was added into the mixture of isopropanol and water and then placed in a microwave reaction system. After reacting for a certain time, the reaction product was washed three times with acetone and dissolved in water. At last, the production was obtained after lyophilization at −40 • C.
The DS of quaternization groups was estimated using the deposit-titration method, which is used to determine the concentration of the chloride ion on QCS in AgNO 3 standard solution with K 2 CrO 4 as the indicator. The DS was calculated as follows [40]: where M 1 stands for the glucosamine molar mass of the structural unit, g/mol; M 2 stands for the quaternized chitosan molar mass of the structural unit, g/mol; and C AgNO3 (mol/L) and V AgNO3 (mL) represent the molarity and volume of AgNO 3 standard solution, respectively. The DS of QCS in this study was 96%. Synthesis of quaternized chitosan-6-p-sulfonyl ester (QCS-6-OTs): an amount of 0.5 g of chitosan was dissolved in 50 mL of H 2 O, and 1.830 g of toluene sulfonyl chloride (TsCl) was dissolved in 3 mL of pyridin. Then, we mixed the above solution and allowed it to react at room temperature for 8 h. The reaction mixture was filtered, washed with acetone and then freeze-dried to obtain QCS-6-OTs.
Synthesis of azide-6-quaternized chitosan (QCS-N 3 ): QCS-6-OTs (0.5 g) was dissolved in 30 mL of H 2 O; then, the required quantities of sodium azide (the molar ratios of NaN 3 to QCS-6-OTs were 2:1, 4:1, 6:1 and 8:1) were added to the solution. The reaction temperatures were subsequently kept at 60, 70, 80 and 90 • C under microwave irradiation, and the reaction was run for 60, 80, 100 and 1200 min, respectively. After the required time, the product was precipitated with acetone. Then, the precipitate was washed with ether and freeze-dried to obtain QCS-N 3 . The DS was calculated using the following equation: where N%/C% is the ratio of N/C contents in the QCS-N 3 products, determined by elemental analysis using an elemental analyzer (Elementar, Langenselbold, Germany), and terms 70 and 28 are the nitrogen elements in QCS-N 3 and QCS, respectively. Term 144 is the carbon element in QCS-N 3 and QCS. The mixture was stirred at 40 • C for 48 h and dialyzed for two days. Then, it was filtered, washed sequentially with acetone and ether and freeze-dried to obtain xylan-click-QCS.

Characterization of the Production
Elemental analyses were carried out using an elemental analyzer (Elementar, Langenselbold, Germany). The C, H, and N contents were determined, and the substitution degree was calculated.
Fourier transform infrared (FT-IR) spectra were obtained using a Vector 33 spectrophotometer (Bruker, Bremen, Germany) in dry air at room temperature using the KBr pellets method. The spectra were collected over the range from 4000 to 400 cm −1 with a resolution of 4 cm −1 .
13 C-NMR spectra were recorded on a DRX-400 spectrometer (Bruker, Bremen, Germany) at 100 MHz, and the solvent was NaOD.

Preparation of Xylan-Click-QCS/AgNP Composites
First, a few drops of diluted sodium hydroxide were added into some aqueous silver nitrate (1 mmol/mL). Then, different concentrations of aqueous ammonia (0.5, 1, 2, 3 and 4%) were added until all the brown silver oxide was dissolved. At this point, the mixture was clear, and aqueous silver ions existed as [Ag(NH 3 ) 2 ]OH in the mixture.
JEM-2010HR transmission electron microscopy (TEM) (JEOL, Tokyo, Japan) was used to investigate the microstructure of the AgNPs at an accelerating voltage of 200 kV. TEM samples were prepared by diluting colloidal xylan-click-QCS/AgNP composites with water with sonication. A few drops of the suspended AgNPs were placed on a copper grid coated with an ultrathin carbon film. The particle size was measured with ImageJ software v.1.50d (NIH, Bethesda, MD, USA).
JEM-2010HR transmission electron microscopy (TEM) (JEOL, Tokyo, Japan) was used to investigate the microstructure of the AuNPs at an accelerating voltage of 200 kV. TEM samples were prepared by diluting colloidal xylan-click-QCS/AuNP composites with water with sonication. A few drops of the suspended AuNPs were placed on a copper grid coated with an ultrathin carbon film. The particle size was measured with ImageJ software.

Cell Assay
FITC (fluorescein isothiocyanate)-marked AgNP sediment and AgNP supernatant were obtained by a reaction (12 h), centrifugal and redispersion (water) process. MCF-7 cells were treated with 50 µL of FITC-marked Ag NP sediment and Ag NP supernatant for 1, 4, 12 and 24 h. The morphology of cells and Ag NP dispersion in cells were determined with a microscope (Carl Zeiss, Jena, Germany) at a 200× magnification.
Human breast adenocarcinoma cells (MCF-7) were cultured with Ag NP sediment (50 µL) and Ag NP supernatant (50 µL) for 24 h, respectively. The cell growth was evaluated with a PrestoBlue kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Each test was measured in three separate wells.

Conclusions
Functional biopolymers were successfully prepared by the introduction of terminal alkyne and azide groups onto xylan and QCS chains, respectively. Subsequently, the xylanclick-QCS polymer was successfully synthesized via the Cu(I)-catalyzed click reaction. AgNPs and AuNPs were prepared by adopting xylan-click-QCS as the reducing and stabilizing agent. The prepared AuNPs with a diameter of 20~30 nm were mostly spherical and distributed uniformly. More interestingly, Ag nanoclusters generated along with AgNPs in this reaction. Ag nanoclusters could transport into cells and inhibited cell proliferation effectively. These results propose that Ag nanoclusters may be utilized as anticancer agents for the treatment of MCF-7-type cancer. The study provides a new way to combine two biopolymers for high-value applications.