In Situ Assembly of Gold Nanoparticles in the Presence of Poly-DADMAC Resulting in Hierarchical and Highly Fractal Nanostructures
Round 1
Reviewer 1 Report
See attached file.
Comments for author File: 
Comments.pdf
Author Response
This communication describes experiments in which gold nanoparticle-containing structures are
produced during reduction of tetrachloroauric acid with ascorbic acid in the presence of varying
concentration of the poly-cationic macromolecule poly-DADMAC. Hierarchical and fractal
nanostructures were produced and characterized. The research topic is of interest but the
presentation could be improved to ensure that the message is delivered effectively. Below are a
number of recommended changes to the manuscript for the authors’ attention.
Recommended Changes
Line 19, should read:… acid with
Correction is done
Line 25, should read:…in the large aggregates..
Correction is done
Line 34, should be: motivated (remove all the dashes in the words in which they are used)
Correction is done
Line 35, provide examples of the bioanalytical applications.
Two examples are added:
They are used for bioanalytical applications as in pregnancy tests and immunoassays for virus proteins
Line 36, should read: …. influenced by their shape, size and morphology.
The sentence is completed, now:
The electromagnetic resonance of these so-called “plasmonic” particles is strongly influenced by their shape, size and morphology.
Line 37, Give examples of non-spherical gold nanoparticles.
The example is given now:
arise in case of non-spherical gold nanoparticles as nanorods, for example
Line 41, should read: The effect of these interactions is exploited in a variety of analytical
applications.
The sentence is modified following the recommendation:
The effect of these interactions is exploited in a variety of analytical applications.
Line 42, should read: The colour change due to aggregation of the negatively charged gold
nanoparticles can be used for the sensitive detection of low concentrations of polycationic
molecules.
The sentence is modified following the recommendation:
The colour change due to aggregation of the negatively charged gold nanoparticles can be used for the sensitive detection of low concentrations of polycationic molecules.
Line 49, should read: The interaction between the gold nanoparticles with oppositely charged
macromolecules offers a strategy for altering the morphology of the gold nanoparticles.
The sentence is modified following the recommendation:
The interaction between the gold nanoparticles with oppositely charged macromolecules offers a strategy for altering the morphology of the gold nanoparticles and to initiate nanoparticle assembling.
Line 51, give examples of the new types of nanomaterials; Line 51-53, re-write both sentences to make clear what you are trying to say; Line53-60, should read:
Beside factors which determine the crystallographic structures of the gold nanoparticles, electrostatic interactions between the particles play a crucial role in the formation of structures of different shapes and sizes. These interactions account for the colloidal stability and the
formation of non-spherical nanoparticles such as star-like nanoaggregates, networks formed by in situ interaction of the particles during the growth process, and formation of ellipsoidal, dumbbell
and astragal-like shapes of polymer-containing nanoparticles. The synthesis of nanoparticle
structures in the presence of macromolecules also depends on these electrostatic interactions.
These electrostatic interactions can occur between nascent gold nanoparticles and the
macromolecules during the formation and growth processes. (Outline the mechanisms of the
interactions between gold nanoparticles and poly-cations in general)
The paragraph is modified following the recommendations:
… In addition, the interaction between the gold nanoparticles with oppositely charged macromolecules offers a strategy for altering the morphology of the gold nanoparticles and to initiate nanoparticle assembling. Beside factors which determine the crystallographic structures and shape of the gold nanoparticles (polynuclear spheres, nanocubes, triangular or hexagonal nanoprisms and nanorods, for example), electrostatic interactions between the particles play a crucial role in the formation of structures and assemblies of different shapes and sizes [14-19]. These interactions account for the colloidal stability and the formation of non-spherical nanoparticles [20] such as star-like nanoaggregates [21], networks formed by in-situ interaction of the particles during the growth process [22], and formation of ellipsoidal, dumbbell and astragal-like shapes of polymer-containing nanoparticles [23]. The synthesis of nanoparticle structures in the presence of macromolecules also depends on these electrostatic interactions. These electrostatic interactions can occur between nascent gold nanoparticles and the macromolecules during the formation and growth processes [24-26]. ]. The binding between particles and macromolecules is supported by attractive Coulomb forces between negatively charged metal nanoparticles and positively charged macromolecules.
Line 90, should read: …. but the in-situ assemble behavior is dependent on the Poly-DADMAC
concentration.
The sentence is modified following the recommendation:
but the in-situ assembling behavior is dependent on the Poly-DADMAC concentration.
Line 99, should read: …. as confirmed by DLS measurements.
The sentence is modified following the recommendation:
a comparatively high yield as confirmed by DLS measurements
Line 100, Give an indication of the extent to which the concentration of Poly-DADMAC is lowered.
Line 106, should read: Evidence of the differences in the aggregate shapes of the particles can be
seen in the changes observed in the plasmon resonance spectra.
The sentence is modified following the recommendation:
Evidence of the differences in the aggregate shapes of the particles can be seen in the changes observed in the plasmon resonance spectra.
Line 109, Give an indication of the extent of red shift at the lower concentration of Poly-DADMAC.
The sentence was modified:
moderately redshifted (to about 560 nm) in comparison with the well-known resonance
Line 111, should read: …. the increase of Poly-DADMAC.
Correction is done
Line 112, why is the scan limited to 900nm? Extending this to say 1100nm could have shown more of
the spectra.
The limitation cames from recent experimental limitations. But, the spectra show clearly the relative increase of absorption in the NIR range. For improvement, the discussion on Fig. 3 is modified:
The optical spectra show the appearance of a broad band above 800 nm with increasing Poly-DADMAC content. It is assumed that this increase is due to the electronic interaction of metallic nanoparticles coming in contact with each other by the aggregation process caused by the macromolecules
Line 116, should read: The DLS measurements indicate the formation of particles with apparent
diameter greater than two microns, although the sizes of the particles and clusters were in the midnanometer and submicron range, respectively.
The DLS spectra indicate both: particles with diameters in the submicron and nanometer range and particles larger than two microns. The sentence was modified:
The most DLS measurements indicate the formation of particles with apparent diameter greater than two microns and indicate the particles and clusters in the mid nanometer and submicron range, too.
Line 118, should read: The formation of the larger aggregates was confirmed by SEM, and by light
microscopy.
The sentence is modified following the recommendation:
was confirmed by SEM images (Fig. 4), and by light microscopy.
Line 120, should read: …. and consist of many single particles.
Correction is done
Line 124, replace by with between.
Here, I really mean, that the filaments bond the small metal nanoparticles and form a connection between them. The sentence is modified:
show that larger aggregates of metal nanoparticles are formed in which the connections between metal particles are due to small molecular filaments.
Line 127, should read: ….. indicates the presence of macromolecules.
The sentence is modified following the recommendation:
The structure of the filaments and the low contrast in SEM imaging indicates the presence of macromolecules.
Figure 5, use arrows to indicate the aspects of the image referred to in the figure legend.
Arrows are integrated in the figure and the figure caption is modified:
Figure 5. SEM images (overlay of contrast-enhanced images) of the aggregation of gold nanoparticles formed by reduction of HAuCl4 (20 µM) with ascorbic acid (60 µM) in the presence of Poly-DADMAC (100 ppm): a) larger cluster of metal nanoparticles, b) macromolecular filaments
Line 128, should read: In the absence of other reactants, the assumption is that the filaments are
formed from Poly-DADMAC.
In the absence of other reactants, the assumption is that the filaments are mainly formed by Poly-DADMAC.
The sentence is modified following the recommendation:
In Figure 6, show using arrows the different levels of structural complexity because it is not obvious
from the figure.
Fig. 6 was improved in order to illustrate better the hierarchical structure of aggregates.
Line 146, should read: …. consist of……
Correction is done.
Line 148, should read: …. support the assumption, that these…..
Correction is done.
Line 184, should read: These investigations have shown that the addition ……..
Correction is done.
Line 187, should read: As a result, different……
Correction is done.
Reviewer 2 Report
The manuscript "In-situ Assembling of Gold Nanoparticles in the Presence of Poly-DADMAC Resulting into Hierarchical and Highly Fractal Nanostructures " by Köhler and Kluitmann describes the formation of gold nanoparticles and nanoparticle assemblies in the presence of the polycationic macromolecule poly-DADMAC. The authors obtained different structures of gold nanoparticles at different ratios of the reagents.
However, I have questions and comments on the paper.
1) I did not notice the structuring of the received data in the manuscript. Everything looks very confusing and the choice of the ratio of reagents for the synthesis is not always clear.
2) Previously, there are a large number of publications where positively charged macromolecules are used in synthesis of gold nanoparticles. What is the fundamentally new strategy for the synthesis of gold nanoparticles using poly-DADMAC?
3) There are few references in the last 5 years (4 out of 35). Need to add more.
4) A large number of typos and auto-hyphenated words should be corrected. For example, "HAuCl4" change to "HAuCl4".
5) Need a detailed description of the methods for preparing samples for DLS and SEM with all the technical characteristics of the registration conditions.
6) It is necessary to add information about the average size and PDI of the systems measured by DLS.
7) It is not clear why the authors do not use TEM, XRD, XPS methods? Energy-dispersive X-ray spectroscopy should be used to determine the filament composition in Figure 8.
Author Response
The manuscript "In-situ Assembling of Gold Nanoparticles in the Presence of Poly-DADMAC Resulting into Hierarchical and Highly Fractal Nanostructures " by Köhler and Kluitmann describes the formation of gold nanoparticles and nanoparticle assemblies in the presence of the polycationic macromolecule poly-DADMAC. The authors obtained different structures of gold nanoparticles at different ratios of the reagents.
However, I have questions and comments on the paper.
1) I did not notice the structuring of the received data in the manuscript. Everything looks very confusing and the choice of the ratio of reagents for the synthesis is not always clear.
A more detailed description of the reactand mixing for nanoparticle synthesis is added in order to avoid any confusion about the application of chemicals:
The synthesis experiments have been carried out at room temperature (20oC). For the formation of assembling particles, at first the polymer solution was diluted with water. The mixing ratio was chosen to achieve a final volume of 1 mL in each experiment. At second, the reducing agent (ascorbic acid) was added and mixed by vigorous vortexing for about 20 s. In the last step, the metal precursor (HAuCl4) was added in a concentration of 1 mM in aqueous solution.
2) Previously, there are a large number of publications where positively charged macromolecules are used in synthesis of gold nanoparticles. What is the fundamentally new strategy for the synthesis of gold nanoparticles using poly-DADMAC?
Most of these investigations had been focused on the effect of ionic surfactants and polyionic macromolecules on the formation of non-spherical metal particles, but had hardly been focused on the formation of assemblies.
The related paragraphs of introduction had been modified:
Beside this analytical aspect, the interaction of forming metal nanoparticles with ionic surfactants and macromolecules was of interest for modulating the formation nanoparticles and to achieve different nanoparticle shapes. In addition, the interaction between the gold nanoparticles with oppositely charged macromolecules offers a strategy for altering the morphology of the gold nanoparticles and to initiate nanoparticle assembling. Beside factors which determine the crystallographic structures and shape of the gold nanoparticles (polynuclear spheres, nanocubes, triangular or hexagonal nanoprisms and nanorods, for example), electrostatic interactions between the particles play a crucial role in the formation of structures and assemblies of different shapes and sizes [14-19]. These interactions account for the colloidal stability and the formation of non-spherical nanoparticles [20] such as star-like nanoaggregates [21], networks formed by in-situ interaction of the particles during the growth process [22], and formation of ellipsoidal, dumbbell and astragal-like shapes of polymer-containing nanoparticles [23]. The synthesis of nanoparticle structures in the presence of macromolecules also depends on these electrostatic interactions. These electrostatic interactions can occur between nascent gold nanoparticles and the macromolecules during the formation and growth processes [24-26]. ]. The binding between particles and macromolecules is supported by attractive Coulomb forces between negatively charged metal nanoparticles and positively charged macromolecules.
3) There are few references in the last 5 years (4 out of 35). Need to add more.
Added.
4) A large number of typos and auto-hyphenated words should be corrected. For example, "HAuCl4" change to "HAuCl4".
The typos have been corrected.
5) Need a detailed description of the methods for preparing samples for DLS and SEM with all the technical characteristics of the registration conditions.
The missed information is added to the experimental part and the related paragraph is re-organized, now:
DLS-Measurements were conducted with a Zetasizer Nano-ZS (Malvern Panalytical). For the DLS measurements (25 oC, aqueous solution, absorbance: 0.3, viscosity, 0.8872 cP, equilibration time: 120 s, measurement angle: 173o, backscattering mode, three single measurements per sample, 70 size classes between 1 nm and 2500 nm), 60 µl of the as obtained samples were transferred to a single use cuvette and the cuvette capped off. .In 33 DLS measurements PDIs between 1.93 and 4.93 have been found and a mean PDI of these 33 samples of 2.53 (inverse: 0.494) with a standard deviation of 0.68 (27%) was observed. SEM-images were obtained in a Hitachi S-4800 FESEM (secondary electron signal, acceleration voltage: 25 kV), optical spectra were measured in a SPECORD 200 (Analytik Jena). For the SEM measurements, the as obtained samples were drop casted onto silicon chips, dried in air and washed with de-ionized water.
6) It is necessary to add information about the average size and PDI of the systems measured by DLS.
The information on PDI measurement results by DLS is added to the experimental part:
In 33 DLS measurements PDIs in the range between 1.93 and 4.93 have been found and a mean PDI of these 33 samples of 2.53 (inverse: 0.494) with a standard deviation of 0.68 (27%) was observed.
7) It is not clear why the authors do not use TEM, XRD, XPS methods? Energy-dispersive X-ray spectroscopy should be used to determine the filament composition in Figure 8.
XRD measurements are well suited for characterization of metal lattices, XPS for bond characterization. For characterization of assemblies, we found the combination of high contrast SEM imaging, optical spectroscopy and DLS measurements particular suited. An additional sentence in the experimental part is added in order to clear the selection of investigation methods:
From the point of view of characterization of nanoparticle assemblies, it was decided to choosing a combination of spectrophotometry, DLS measurements and contrast-enhanced SEM imaging (measurement by secondary electron detector). These three methods address very different aspects of the formation of particle aggregates: Spectrophotometry supplies information about the electronic states, the so-called particle plasmonic resonances and their change due to electronic coupling in case of particle contact. DLS measurements supply information about particle mobility (Brownian motion), which is related to the mass and shape of particles and the properties of embedding medium (solution, swelling). Finally, the contrast-enhanced SEM measurements allow to combining signals from optimal imaging of materials with high yield of secondary electrons and low transparency (metals) with signals from optimal imaging of materials with low yield of secondary electrons and high electron beam transparency.
Reviewer 3 Report
The authors present a study on the influence of PDADMAC on the nucleation and growth of gold colloids. They report on the observation of different levels of aggregation as a function of the PDADMAC concentration. Apart from small quasi-spherical colloids, larger clusters, and fractal/hierarchical assemblies are reported on. This is not surprising as the competition of positive and negative surface charges present will ultimately result in the aggregation of the formed particles into larger structures. In fact, the addition of polyelectrolytes as linkers and the use of oppositely charged building blocks are commonly applied strategies. However, electrostatic assembly needs to be distinguished in regard to the amount of available structural control, and thus the uniformity of the obtained morphologies. For example, mixing negatively charged nanospheres with positively charged core building blocks (spheres, rods, triangles) leads to very regular hierarchical superstructures - and this in the absence of uncontrolled aggregation, as recently shown by Höller R. et al. However, the method presented by the authors is challenging, since the generation of the building blocks and their surface functionalization (and also their assembly) is performed in a single step in a one-pot fashion. For this reason, it is not surprising that the superstructures show less uniformity. For the sake of clarity, the authors might want to discuss these special challenges of their method in detail in the light of the above mentioned methods, which perform particle formation, functionalization and assembly in seperate steps. Another point not taken into account is the colloidal stability of the structures obtained. This should be quantified and discussed especially in the view of application for SERS sensing and/or colorimetric sensing.
Overall I encourage the publication in Applied Sciences. Nevertheless, I advocate to consider the following comments and open questions before final acceptance can be recommended.
Comments/questions:
1) The molecular weight of PDADMAC is not clear. The term "very low molecular weight" is highly undefined. This parameter is an essential parameter and needs to be disclosed. If it is not provided by the supplier the authors need to perform standard techniques of macromolecular analysis to quantify the Mw, Mn, and PDI for the sake of reproducibility.
2) As a polyelectrolyte the molecular conformation of PDADMAC depends on the ionic strength of the medium. The authors need to discuss the observed processes in the view of the ionic strength in their synthesis media and the to be expected conformations of their polycations.
3) The surface chemistry of the obtained structures is not clear. The authors might want to perform zeta potential measurements to allow a judgment of the colloidal stability of the formed particle aggregates.
4) The abbreviation PSSS is discouraged, as the commonly accepted abbreviation is PSS. This is because the couterion is commonly not considered.
5) The experimental procedures are not entirely clear. Please state clearly which volumes are added at which times.
6) DLS is inherently assuming a spherical shape of the scatterer by definition. If the formed aggregates are of non-spherical shape, how does this affect the hydrodynamic radii measured. Rigorous averaging of sizes in all directions needs to be taken into account here (rotational averaging).
7) The authors jump back and forth between decimal points and comma.
8) Could it be expected that the addition of PDADMAC does not completely invert the surface charges of the formed particles? This could result in patchy particles as described by Lunn et al. (10.1039/C5SC01141H) or Brasili et al. (10.1016/j.jcis.2020.07.006) using biomacomolecular linkers.
9) The authors might want to consider performing these experiments in microfluidic cells, which is known to be their field of expertise. Here, similar approaches by Popp et al. might be a source of inspiration as the aggregation of Ag colloids can be directed to proceed in a very controlled manner by means of a lab-on-a-chip approach.
10) Fig. 3 is not predominately showing a redshifting but a broadening of the LSPR into a band. This clearly stems from the (uncontrolled nature of the) aggregation process. Because the structure exhibit a wide variety, the optical properties average out to a large extent. This is evidence by the poor intensity of the LSPR in the presence of PDADMAC). Please revise the discussion.
11) Fig. 4: How can these fractal structures be distinguished from random aggregates formed by or during drying?
12) Fig. 5 is not entirely clear. If an image overlay has been applied, this must be described and shown in detail so that the reader can correctly assess the effects of the image manipulation on the original data.
13) Fig. 6: The authors might want to consider performing time-dependent UV/vis/NIR spectroscopy (showing full spectra, not only the extinction at 550 and 800 nm) to follow the proposed process of multi-level aggregation directly and provide more quantitative evidence for their claims.
14) Fig. 7: It would be very helpful to normalised the optical data by the extinction at 400 nm (which reflects the Au0 content) to allow for a more accurate comparison of the data.
15) The conclusions need revision. The terms "molecule modulation of nucleation“, "in-situ assembling“ and "levels of nanoparticles“ are in a way misleading and need further explanations. Also, the authors might want to acknowledge that the conclusion that additives such as polycations affect the nucleation and growth behaviour of gold nanoparticles has been known for more than a decade, and thus is not a novel finding at all.
Author Response
The authors present a study on the influence of PDADMAC on the nucleation and growth of gold colloids. They report on the observation of different levels of aggregation as a function of the PDADMAC concentration. Apart from small quasi-spherical colloids, larger clusters, and fractal/hierarchical assemblies are reported on. This is not surprising as the competition of positive and negative surface charges present will ultimately result in the aggregation of the formed particles into larger structures. In fact, the addition of polyelectrolytes as linkers and the use of oppositely charged building blocks are commonly applied strategies. However, electrostatic assembly needs to be distinguished in regard to the amount of available structural control, and thus the uniformity of the obtained morphologies. For example, mixing negatively charged nanospheres with positively charged core building blocks (spheres, rods, triangles) leads to very regular hierarchical superstructures - and this in the absence of uncontrolled aggregation, as recently shown by Höller R. et al.
However, the method presented by the authors is challenging, since the generation of the building blocks and their surface functionalization (and also their assembly) is performed in a single step in a one-pot fashion. For this reason, it is not surprising that the superstructures show less uniformity. For the sake of clarity, the authors might want to discuss these special challenges of their method in detail in the light of the above mentioned methods, which perform particle formation, functionalization and assembly in seperate steps.
A section in the introduction was modified and completed in order to clear the motivation of investigations and the strategy of forming hierarchical organized particle aggregates by polyionic molecules:
Beside this analytical aspect, the interaction of forming metal nanoparticles with ionic surfactants and macromolecules was of interest for modulating the formation nanoparticles and to achieve different nanoparticle shapes. In addition, the interaction between the gold nanoparticles with oppositely charged macromolecules offers a strategy for altering the morphology of the gold nanoparticles and to initiate nanoparticle assembling. Beside factors which determine the crystallographic structures and shape of the gold nanoparticles (polynuclear spheres, nanocubes, triangular or hexagonal nanoprisms and nanorods, for example), electrostatic interactions between the particles play a crucial role in the formation of structures and assemblies of different shapes and sizes [14-19]. These interactions account for the colloidal stability and the formation of non-spherical nanoparticles [20] such as star-like nanoaggregates [21], networks formed by in-situ interaction of the particles during the growth process [22], and formation of ellipsoidal, dumbbell and astragal-like shapes of polymer-containing nanoparticles [23]. The synthesis of nanoparticle structures in the presence of macromolecules also depends on these electrostatic interactions. These electrostatic interactions can occur between nascent gold nanoparticles and the macromolecules during the formation and growth processes [24-26]. ]. The binding between particles and macromolecules is supported by attractive Coulomb forces between negatively charged metal nanoparticles and positively charged macromolecules.
Another point not taken into account is the colloidal stability of the structures obtained. This should be quantified and discussed especially in the view of application for SERS sensing and/or colorimetric sensing.
An additional sentence was added at the end of the conclusionsection in order to clarify this point:
The colloidal solutions are rather stable under the applied experimental conditions and the cyclic photospectrometric measurements have shown that the sedimentation speed of aggregates is not very high.
Overall I encourage the publication in Applied Sciences. Nevertheless, I advocate to consider the following comments and open questions before final acceptance can be recommended.
Comments/questions:
1) The molecular weight of PDADMAC is not clear. The term "very low molecular weight" is highly undefined. This parameter is an essential parameter and needs to be disclosed. If it is not provided by the supplier the authors need to perform standard techniques of macromolecular analysis to quantify the Mw, Mn, and PDI for the sake of reproducibility.
The missed information was added to the experimental part:
(ALDRICH, molecular weight <= 100 kDA, 35wt % in water),
2) As a polyelectrolyte the molecular conformation of PDADMAC depends on the ionic strength of the medium. The authors need to discuss the observed processes in the view of the ionic strength in their synthesis media and the to be expected conformations of their polycations.
A short discussion about the solution conditions and ionic strength is added to the experimental part:
The ionic strength is comparable low, because all applied reactants have concentrations in the sub-millimolar or micromolar range in the final reaction mixture. Additional chlorid ions are generated by the reaction of tetrachloroaurate. Therefore, it has to be expected that the Poly-DADMAC is highly ionized under the applied experimental conditions.
3) The surface chemistry of the obtained structures is not clear. The authors might want to perform zeta potential measurements to allow a judgment of the colloidal stability of the formed particle aggregates.
The optical measurements show that the aggregates are forming a rather stable colloidal solution over several minutes, at least (see, for example, the additional Fig. 6b). The advice concerning the Zeta potentials is very valuable - related measurements will made in future investigations.
4) The abbreviation PSSS is discouraged, as the commonly accepted abbreviation is PSS. This is because the couterion is commonly not considered.
“PSSS” is always substituted by “PSS”, now.
5) The experimental procedures are not entirely clear. Please state clearly which volumes are added at which times.
The experimental part was completed by describing the order of mixing of the reactants:
The synthesis experiments have been carried out at room temperature (20oC). For the formation of assembling particles, at first the polymer solution was diluted with water. The mixing ratio was chosen to achieve a final volume of 1 mL in each experiment. At second, the reducing agent (ascorbic acid) was added and mixed by vigorous vortexing for about 20 s. In the last step, the metal precursor (HAuCl4) was added in a concentration of 1 mM.
6) DLS is inherently assuming a spherical shape of the scatterer by definition. If the formed aggregates are of non-spherical shape, how does this affect the hydrodynamic radii measured. Rigorous averaging of sizes in all directions needs to be taken into account here (rotational averaging).
This is absolute true. The DLS gives rough information only about the real particle size. But, the appearance of DLS signals for larger objects gives a good confirmation of formation of micrometer aggregates reflected by the SEM images. For discussing the problem of accuracy of DLS data following addition is made in the third section (Results and discussions):
It has to reconsider that the DLS measurements supply comparatively good data for compact particles in the mid nanometer and submicron range, whereas it supplies only a rough estimation for the size of larger non-spherical aggregates consisting of particle clusters and macromolecular filaments.
7) The authors jump back and forth between decimal points and comma.
The mistakes are corrected, now.
8) Could it be expected that the addition of PDADMAC does not completely invert the surface charges of the formed particles? This could result in patchy particles as described by Lunn et al. (10.1039/C5SC01141H) or Brasili et al. (10.1016/j.jcis.2020.07.006) using biomacomolecular linkers.
For our opinion. it is expected that the macromolecules are not covering completely the surface of metal particles. For this point an additional short discussion is added in connection with the discussion on Fig. 5:
Probably, there should take place a competition between solvent and nanoparticle interactions with the ammonium groups. This effect, the mobility of polymer chains and repulsive forces between free ammonium groups lead to the formation of the observed non-compact aggregates in the lower micrometer range as they had been observed by the SEM images.
9) The authors might want to consider performing these experiments in microfluidic cells, which is known to be their field of expertise. Here, similar approaches by Popp et al. might be a source of inspiration as the aggregation of Ag colloids can be directed to proceed in a very controlled manner by means of a lab-on-a-chip approach.
Indeed, such experiments are planned in our further research activities.
10) Fig. 3 is not predominately showing a redshifting but a broadening of the LSPR into a band. This clearly stems from the (uncontrolled nature of the) aggregation process. Because the structure exhibit a wide variety, the optical properties average out to a large extent. This is evidence by the poor intensity of the LSPR in the presence of PDADMAC). Please revise the discussion.:
The discussion was modified following the recommendations:
The optical spectra show the appearance of a broad band above 800 nm with increasing Poly-DADMAC content. It is assumed that this increase is due to the electronic interaction of metallic nanoparticles coming in contact with each other by the aggregation process caused by the macromolecules
11) Fig. 4: How can these fractal structures be distinguished from random aggregates formed by or during drying?
It is true that we cannot be absolutely sure about the formation of such aggregates during the drying process. But two evidences support our interpretation: At first these clusters in this order of magnitude of size are observed in the presence of Poly-DADMAC preferably whereas in other preparations of SEM chips non aggregated particles or larger size distributions of clusters were observed. At second, the order of magnitude of these clusters matches approximately with the peak of larger particles from DLS measurements. For clearing this point, a sentence was added into the paragraph with discussion on Fig. 4:
The preferential appearance of clusters in this size range and the rough estimation of size ranges in solution by DLS measurements speak for the fact that the clusters are present in the solution, already and not formed during the drying process in the preparation of chips for SEM imaging.
12) Fig. 5 is not entirely clear. If an image overlay has been applied, this must be described and shown in detail so that the reader can correctly assess the effects of the image manipulation on the original data.
I apologize for the mistake. Fig. 5 was completed by arrows and the figure caption was modified.
13) Fig. 6: The authors might want to consider performing time-dependent UV/vis/NIR spectroscopy (showing full spectra, not only the extinction at 550 and 800 nm) to follow the proposed process of multi-level aggregation directly and provide more quantitative evidence for their claims.
Fig. 6 is completed by an example of an experiment showing the evolution of absorption over time.
14) Fig. 7: It would be very helpful to normalised the optical data by the extinction at 400 nm (which reflects the Au0 content) to allow for a more accurate comparison of the data.
The figure was improved according to the reviewers suggestion.
15) The conclusions need revision. The terms "molecule modulation of nucleation“, "in-situ assembling“ and "levels of nanoparticles“ are in a way misleading and need further explanations. Also, the authors might want to acknowledge that the conclusion that additives such as polycations affect the nucleation and growth behaviour of gold nanoparticles has been known for more than a decade, and thus is not a novel finding at all.
The conclusion section was improved in order to respect the known effect of polyionic macromolecules on nanoparticles aggregation and avoiding the above mentioned misleading terms "molecule modulation of nucleation“, "in-situ assembling“ and "levels of nanoparticles“ :
The investigations confirm the fact that the addition of the poly-cationic water-soluble polymer Poly-DADMAC causes significant changes in the growth and aggregation behavior of gold nanoparticles. The charged polymer is responsible for the modulation of nucleation and growth rate of gold nanoparticles as well as for their interaction and incorporation in larger clusters and assemblies. As a result, different types of nanoparticle aggregates with sizes between about 20 nm and several microns are formed. SEM images suggest at least three different aggregation levels of nanoparticles can be distinguished which can be described as hierarchically organized aggregate structures.
Round 2
Reviewer 2 Report
The authors have significantly improved the manuscript.
Regarding the DLS data (Q5,6), the authors should add a table of all sizes and PDI of nanoparticles. The PDI values obtained with the Zetasizer devices are usually given in the range of 0.00-1.00. It is also worth adding information and discussion about the zeta potential (electrokinetic potential) of AuNPs-polyDADMAC systems in different concentrations of polyDADMAC. Since the DLS method is one of the main in this study, these data are very important.
Regarding the stability of the obtained systems, the authors did not provide in the experimental part information about how long after the preparation of the solutions the DLS data were obtained?
The conclusion "the sedimentation speed of aggregates is not very high" is not correct due to the abscence of quantitative data in the text of the manuscript. Stability must also be confirmed by other methods, for example DLS.
Author Response
Response to reviewer comments
Reviewer 2, Runde 2
The authors have significantly improved the manuscript.
Regarding the DLS data (Q5,6), the authors should add a table of all sizes and PDI of nanoparticles. The PDI values obtained with the Zetasizer devices are usually given in the range of 0.00-1.00.
A supplementary table of two new series of DLS measurements including the PDI data is added, now. All measurements have been made in triplicate. The text in the experimental section is adapted:
… In the DLS measurements PDIs in the range between about 0.1 and 0.5 have been found. Details are given in the supplementary table.
It is also worth adding information and discussion about the zeta potential (electrokinetic potential) of AuNPs-polyDADMAC systems in different concentrations of polyDADMAC. Since the DLS method is one of the main in this study, these data are very important.
In the new measurements, Zeta potentials have been registered, too. Always the expected significant positive Zeta potentials could be confirmed. The new data including Zeta potentials are given in the revised Fig. 2. The text was slightly extended:
Zeta potentials in the positive range (about +30 to +50 mV) have been observed in all cases of particle preparation in the presence of PDADMAC, as expected (Fig. 2).
Regarding the stability of the obtained systems, the authors did not provide in the experimental part information about how long after the preparation of the solutions the DLS data were obtained?
We observed a sedimentation process over several hours (see below). The particles can be re-dispersed. The DLS measurements have been made four days after particle preparation.The information is now given in the experimental part:
The DLS measurements have been performed four days after particle preparation and a re-dispersion (3 minutes shaking with ultrasound).
The conclusion "the sedimentation speed of aggregates is not very high" is not correct due to the abscence of quantitative data in the text of the manuscript. Stability must also be confirmed by other methods, for example DLS.
An additional experiment was made in order to evaluate the sedimentation speed. Therefore fresh prepared a colloidal solution was measured by cyclic photometry over several hours. The results are shown in the supplementary Fig.1. They reflect a slow sedimentation which can be proved photometrically after about five hours.The results are mentioned in the experimental part:
The sedimentation of particles is comparatively slow. In a standard UV/VIS spectrometric cuvette (1 cm) beginning sedimentation was proved after about five hours (supplementary Fig. 1). The particles are stable and can be re-dispersed by support of ultrasound.
Reviewer 3 Report
The authors have revised the manuscript on the basis of the concerns and comments expressed and have improved it considerably. However, some aspects have only been considered rather superficially and I recommend that they be revised again:
(1) This concerns the clear presentation of the impact and challenges of their method and the differentiation from previously established alternative approaches. I repeat that I am convinced that the authors should discuss in detail the particular challenges of their one-pot method in light of other methods of electrostatic assembly -- especially those with high structural control (by performing particle formation, functionalization and assembly in separate steps as for example by Höller and coauthors; ACS Photonics 10.1021/acsnano.5b07533; Nanoscale 10.1039/C9NR06102A).
(2) Several revised passages contain vague expressions that lack scientific precision; see the following examples: The colloidal solutions are “rather stable“ under the applied experimental conditions (rather stable is undefined, how stable?); the sedimentation speed of aggregates is “not very high” (not very high is undefined, how high?); ionic strength is “comparable low“ (compared to what? how low?); the DLS measurements supply „comparatively good data“ (compared to what?; what means good?). Please be more specific.
(3) I have to reiterate my concern expressed in initial comment 2 that the authors need to discuss the observed processes in the view of the ionic strength in their synthesis media and the to be expected conformations of their polycations. What is the ionic strength in the different growth media and which conformations could thus be expected for the PDADMAC chains?
(4) In their reply the authors indicate that they envision Zeta potential for future measurements. Again, I must emphasize the high importance of this parameter for the results presented in this manuscript and their scientific soundness. I recommend that this information not be withheld and advocate its addition in this manuscript rather than a future one.
(5) This may be a matter of taste, but from my linguistic understanding the term “assembling” should be replaced by “assembly” in almost all cases; as for example in the title.
I am still convinced of the scientific value of this manuscript and I support its publication in Applied Sciences, but after the revision of the points mentioned.
Author Response
The authors have revised the manuscript on the basis of the concerns and comments expressed and have improved it considerably. However, some aspects have only been considered rather superficially and I recommend that they be revised again:
(1) This concerns the clear presentation of the impact and challenges of their method and the differentiation from previously established alternative approaches. I repeat that I am convinced that the authors should discuss in detail the particular challenges of their one-pot method in light of other methods of electrostatic assembly -- especially those with high structural control (by performing particle formation, functionalization and assembly in separate steps as for example by Höller and coauthors; ACS Photonics 10.1021/acsnano.5b07533; Nanoscale 10.1039/C9NR06102A).
The introduction was modified and the approach of R.P.M. Höller et al. (2016) is discussed now:
A regular connection between smaller and larger metal nanoparticles succeeded by a protein-assisted assembling using bovine serum albumin (BSE) [36]. In this case, a homogeneous population of raspberry-like nanoclusters with a large core and a shell of satellite particles had been prepared, whereby the electronic coupling between particles could be concluded from the detection of a bathochromic shift in the optical absorption spectrum. The bathochromic shift of plasmonic resonance is typical for clustered gold nanoparticles as found, for example, in formerly studies in the presence of polyvinylalcohol [37] and BSE [38]. Beside the adhesive effect or protein molecules, an electrostatic interaction of gold nanoparticles and molecules can take place during the formation and growth process in a wet chemical nanoparticle synthesis with synthetic macromolecules. The silver triangle formation by addition of PSS (poly(sodium 4-styrenesulfonate)) [39], might be facilitated by an electrostatic support of particle self-polarization by the polyanions [40]. A strong effect on the formation and assembling behavior of metal nanoparticles should also be expected in the presence of positively charged polyionic molecules. Beside the formation of regular assembly structures, it is of interest, how larger aggregates between macromolecules and nanoparticles are formed and which structures can arise. Therefore, here the formation of gold nanoparticles and nanoparticle assemblies in the presence of the polycationic macromolecule poly-DADMAC is investigated.
(2) Several revised passages contain vague expressions that lack scientific precision; see the following examples: The colloidal solutions are “rather stable“ under the applied experimental conditions (rather stable is undefined, how stable?); the sedimentation speed of aggregates is “not very high” (not very high is undefined, how high?); ionic strength is “comparable low“ (compared to what? how low?); the DLS measurements supply „comparatively good data“ (compared to what?; what means good?). Please be more specific.
The concerned sentences have been improved:
… The total ion concentration is in the sub-millimolar or micromolar range in the final reaction mixture. …
… It has to be reconsidered that the DLS measurements supplies only a rough estimation for the size of larger non-spherical aggregates consisting of particle clusters and macromolecular filaments. …
… The cyclic photospectrometric measurements do not reflect sedimentation of aggregates after 15 minutes.
(3) I have to reiterate my concern expressed in initial comment 2 that the authors need to discuss the observed processes in the view of the ionic strength in their synthesis media and the to be expected conformations of their polycations. What is the ionic strength in the different growth media and which conformations could thus be expected for the PDADMAC chains?
In the particle preparation experiments, no additional salt was added. The information on ionic strength is given in the experimental part, now:
The total ion concentration is in the sub-millimolar or micromolar range in the final reaction mixture, typically. No additional salt is added. The ionic strength is given by the ionic reactants PDADMAC and [HAuCl4] , which was in the range up to 400 ppm (PDADMAC) and 0.2 mM ([HAuCl4]).
(4) In their reply the authors indicate that they envision Zeta potential for future measurements. Again, I must emphasize the high importance of this parameter for the results presented in this manuscript and their scientific soundness. I recommend that this information not be withheld and advocate its addition in this manuscript rather than a future one.
New experimental series have been performed in order to prove that the prepared particles have the expected positive Zeta potential. The detailed data are given in the revised Fig. 2 and mentioned in the text, now:
…. Zeta potentials in the positive range (about +30 to +50 mV) have been observed in all cases of particle preparation in the presence of PDADMAC, as expected (Fig. 2).
(5) This may be a matter of taste, but from my linguistic understanding the term “assembling” should be replaced by “assembly” in almost all cases; as for example in the title.
The title is modified as suggested. “Assembling” is substituted in three other cases by “assembly”, but in other cases we would like to keep “assembling”:
Titel: In-situ Assembly of Gold Nanoparticles in the Presence of Poly-DADMAC Resulting into Hierarchical and Highly Fractal Nanostructures
I am still convinced of the scientific value of this manuscript and I support its publication in Applied Sciences, but after the revision of the points mentioned.
Round 3
Reviewer 2 Report
The authors added the required data and answered all questions.
