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Ammonium-Induced Synthesis of Highly Fluorescent Hydroxyapatite Nanoparticles with Excellent Aqueous Colloidal Stability for Secure Information Storage

Coatings 2019, 9(5), 289; https://doi.org/10.3390/coatings9050289

by Cheng Cheng¹, Kun Tong², Yajun Fang¹, Jintao Wang¹, Yang Liu¹ and Junjun Tan^1,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Coatings 2019, 9(5), 289; https://doi.org/10.3390/coatings9050289

Submission received: 28 February 2019 / Revised: 21 April 2019 / Accepted: 23 April 2019 / Published: 27 April 2019

Round 1

Reviewer 1 Report

The paper titled “Ammonium-Induced Synthesis of Highly Fluorescent Hydroxyapatite Nanoparticles with Excellent Aqueous Colloidal Stability for Secure Information Storage” shows a very interesting application of hydroxyapatite nanoparticles. Nano-hydroxyapatite is a multi-purpose non-toxic material that has gained many new practical applications in recent years, which is shown in this paper. However, before I can accept the manuscript for publication, please refer to my comments.

1. In “Introduction” there is no information as to how the change in parameters of hydrothermal synthesis (duration, temperature) affects the size of the obtained HA NPs. Please add a few sentences of a comment about this topic. The impact of parameters of HA NPs synthesis was described in the following publications:

Synthesis and hydrothermal treatment of nanostructured hydroxyapatite of controllable sizes, J Mater Sci: Mater Med (2008) 19: 1389.

Influence of hydrothermal synthesis parameters on the properties of hydroxyapatite nanoparticles, Beilstein J. Nanotechnol. 2016, 7, 1586–1601.

2. In “Materials” there is no information about the following reagents: arginine, urea, ethylenediamine.

3. In “Preparation of hydroxyapatite nanoparticles” there is no information as to how exactly HA NPs are re-dispersed in water. What homogenisation method was applied? What was the homogenisation duration?

4. In “XRD patterns analysis” please determine the size of HA crystallites based on XRD data using e.g. the Scherrer’s formula and in “Discussion” please compare the achieved trend of change in HA NPs size with the results reported in the literature. Does the change in HA NPs size affect the fluorescence behaviour of the obtained samples?

5. “Colloidal stability analysis”:

- As a scholar, I personally do not use such words as “good” or “excellent” in scientific publications because they concern the qualitative analysis, not the quantitative analysis (confirmed in numerical results).

- In the abstract the authors did not include any information as to for how long the suspension stability was examined by them (confirmed stability).

- In “Figure” please supplement the results of zeta potential with the standard deviation value.

- I suggest placing photographs of the suspensions in a better resolution because this is the only result in this paper that confirms their “excellent” stability.

- Why do the authors not compare photographs of the suspensions just after they have been obtained (0 h) with photographs of the suspensions after 7 days in the paper?

- Why did the authors not provide the results of changes in particle/agglomerate size for the obtained suspensions for the duration “0h” and “7 days”? These results would unambiguously confirm the stability of the obtained suspensions.

6. “Elemental analysis”:

- No supporting information was appended to the paper.

7. Discussion (Table 5):

- Table no. 5 is unacceptable in the present form. The acronyms used there (DLS, EX, EM) must be explained in the table description.

- DLS is the acronym of a measurement method, not of the determined parameter.

- What was determined using DLS? (diameter or radius)

- I suggest rounding the results of the average particle/agglomerate size and of the zeta potential to first decimal place.

- The standard deviation value must be added to the results of the average size and zeta potential.

- The results for the polydispersity index were not provided.

- There is no information about the duration of synthesis of the samples provided in Table 5.

Author Response

Reviewers #1:

Synthesis and hydrothermal treatment of nanostructured hydroxyapatite of controllable sizes, J Mater Sci: Mater Med (2008) 19: 1389.

Influence of hydrothermal synthesis parameters on the properties of hydroxyapatite nanoparticles, Beilstein J. Nanotechnol. 2016, 7, 1586–1601.

Response 1:

Thank you for your valuable suggestion. We add the references mentioned above and provide a description of the synthesis of HA NPs along with experimental parameters in the revised manuscript (section of “Introduction”).

Previous edition(page 3, Introduction section)：

Revised edition:

Our previous studies have found that by balancing the molar ratio of sodium citrate to calcium, hydrothermal time, and temperature, rod-shaped hydroxyapatite nanoparticles with high crystallinity and excellent colloidal stability can be prepared ¹^,². On this basis, Eu³⁺-doped hydroxyapatite nanocrystals can be used in inkjet printing for secure information storage ³. Loo et al described that the size of the HA nanoparticles increases with increasing synthesis time and synthesis temperature⁴. Kuśnieruk et al also observed the diameter of HA nanoparticles influenced by the reaction time, pressure and temperature⁵.

2. In “Materials” there is no information about the following reagents: arginine, urea, ethylenediamine.

Response 2:

We add the related content in the revised manuscript.

Previous edition：

2.1 Materials

Calcium chloride anhydrous (CaCl₂, >96.0%), sodium phosphate tribasic dodecahydrate (Na₃PO₄·H₂O, >98.0%), ammonium phosphate tribasic ((NH₄)₃PO₄·3H₂O, >98.0%), sodium citrate tribasic dehydrate (C₆H₅Na₃O₇·2H₂O, >99.0%), and absolute ethanol (C₂H₆O, >99.7%) were purchased from Aladdin Industrial Corporation. All chemicals were used as received, without further purification. Deionized water was used throughout our experiments.

Revised edition:

2.1 Materials

Calcium chloride anhydrous (CaCl₂, >96.0%), sodium phosphate tribasic dodecahydrate (Na₃PO₄·H₂O, >98.0%), ammonium phosphate tribasic ((NH₄)₃PO₄·3H₂O, >98.0%), sodium citrate tribasic dehydrate (C₆H₅Na₃O₇·2H₂O, >99.0%), arginine (C₆H₅Na₃O₇·2H₂O, >98.0%), urea (CH₄N₂O, >99.0%), ethylenediamine (C₂H₈N₂, >99.0%), and absolute ethanol (C₂H₆O, >99.7%) were purchased from Aladdin Industrial Corporation. All chemicals were used as received, without further purification. Deionized water was used throughout our experiments.

Response 3:

We add the related content in the revised manuscript.

Previous edition：

2.2.1 Preparation of hydroxyapatite nanoparticles

Hydroxyapatite nanoparticles were prepared by using a hydrothermal method. In a typical experiment, a sodium citrate (C₆H₅Na₃O₇·2H₂O) solution (0.01 mol, 10 g water) was added, with continuous stirring, to an aqueous solution of CaCl₂ (0.01 mol, 10 g water) over 10 min. Then, an aqueous phosphate solution (Na₃PO₄×12H₂O, 0.006-x mol; (NH₄)₃PO₄×3H₂O, x mol; 15 g water) was added to the mixture with vigorous stirring over 15 min. Next, the mixed solution was transferred, as obtained, to a Teflon-lined stainless steel autoclave with a 50-mL capacity. The solution in the autoclave underwent hydrothermal treatment at 180°C for 0.5–4 h. After the hydrothermal treatment, the autoclave was allowed to cool down rapidly with water, and the resulting product was purified using a three-cycle process with deionized water and ethanol. Finally, the purified product was re-dispersed in deionized water to form an aqueous dispersion. The pH of the dispersion was adjusted to pH 9.5 by the addition of 0.1 M NaOH. A portion of the product was dried at 80°C for 12 h to obtain powder for future characterization. For the sake of simplicity, R_AMP represents the molar ratio of ammonium phosphate to total phosphate which one adds in a specific synthesis.

Revised edition:

2.2.1 Preparation of hydroxyapatite nanoparticles

Hydroxyapatite nanoparticles were prepared by using a hydrothermal method. In a typical experiment, a sodium citrate (C₆H₅Na₃O₇·2H₂O) solution (0.01 mol, 10 g water) was added, with continuous stirring, to an aqueous solution of CaCl₂ (0.01 mol, 10 g water) over 10 min. Then, an aqueous phosphate solution (Na₃PO₄×12H₂O, 0.006-x mol; (NH₄)₃PO₄×3H₂O, x mol; 15 g water) was added to the mixture with vigorous stirring over 15 min. Next, the mixed solution was transferred, as obtained, to a Teflon-lined stainless steel autoclave with a 50-mL capacity. The solution in the autoclave underwent hydrothermal treatment at 180°C for 0.5–4 h. After the hydrothermal treatment, the autoclave was allowed to cool down rapidly with water, and the resulting product was purified using a three-cycle process with deionized water and ethanol. Finally, the purified product was re-dispersed in deionized water to form an aqueous dispersion by magnetic stirring for 10 min. The pH of the dispersion was adjusted to pH 9.5 by the addition of 0.1 M NaOH. A portion of the product was dried at 80°C for 12 h to obtain powder for future characterization. For the sake of simplicity, R_AMP represents the molar ratio of ammonium phosphate to total phosphate which one adds in a specific synthesis.

Response 4:

As suggested by the reviewer, we have added data (as shown in Table 2) on the hydroxyapatite grain size. At the same time, we also added relevant content in the description of the XRD method and in the results section and discussion section.

Table 2 Average size of crystal domains along the [002] and [310] directions of HA at different experimental condition, calculated applying the Scherrer equation.

Samples	D _[002] (nm)	D _[310] (nm)	D _[002]/D _[310]
100%-0.5h	19.5	8.4	2.3
50%-0.5h	17.7	5.6	3.2
50%-0.5h	23.4	6.9	3.4
0%-0.5h	14.8	4.1	3.6
100%-2h	28.4	17.0	1.7
50%-2h	27.3	12.6	2.2
5%-2h	24.9	19.9	1.3
0%-2h	25.8	8.7	3.0
100%-4h	31.1	14.0	2.2
50%-4h	30.7	13.3	2.3
5%-4h	32.0	8.1	4.0
0%-4h	33.3	8.2	4.1

The added content in the results section (page 5 ):

The average size of crystal domains along the c-axis (D[002]) and the a–b plane (orthogonal to the c-axis, D[310]), calculated by the Scherrer’s equation using the width at half height for the respective reflections 002 and non-overlapped 310, are shown in Table 2. D[002] strongly increased with hydrothermal time from 14.8 nm to 33.3 nm. D[310] increased with hydrothermal time and R_AMP from 8.1 nm to 19.9 nm. The D[002]/D[310] ratio of all samples was above 1, suggesting that the particles preferentially grow along the c-axis and rod-like shape of particles is formed.

The revised content in the discussions section (page 11):

A series of physicochemical properties of the product in accordance with changes in R_AMP and hydrothermal time have been investigated. Introducing ammonium ions has no appreciable effect on the crystallization, morphology, or colloidal stability of hydroxyapatite. Nevertheless, it impacts the fluorescence properties. In addition, increasing hydrothermal time (in the time range of our study) can greatly improve crystallization of the product, and has no appreciable effect on the colloidal stability. Nevertheless, there is a critical hydrothermal time limit for the fluorescence properties, which also decisively impacts the fluorescence properties. In other words, in addition to the citrate reported in previous work, ammonium ions and an appropriate hydrothermal time are critical for maximal fluorescent properties. At the same time, we noticed that the change in grain size did not significantly affect the fluorescence performance of the sample.

The added content in the results section (page 4 ):

The main peaks at (002) and (310) were generally used to calculate to investigate the average crystallite size of HA, according to the Debye-Scherrer formula (1):

D=κλ/(βcosθ) (1)

Where D is the average crystal size; κ is a constant related to crystal habit and it is set as 0.9; λ is the wavelength of the Cu Kα radiation (1.5406 Å) ; β is full width at half maximum (FWHM) of the XRD peak; θ is Bragg angle.

5. “Colloidal stability analysis”:

Response 5(1):

We accept the reviewer’s comment. We have corrected the corresponding expressions and the relevant changes have been marked in the manuscript.

- In the abstract the authors did not include any information as to for how long the suspension stability was examined by them (confirmed stability).

Response 5(2):

We modified the content related to colloidal stability in the part of abstract. The detailed is as following:

The corresponding dispersion is colloidally stable, and transparent for at least one week, and has an intense bright blue emission (centered at 440 nm, 11.6-ns lifetime, and 73.80% quantum efficiency) when excited by 340-nm UV light.

- In “Figure” please supplement the results of zeta potential with the standard deviation value.

Response 5(3):

We added the standard deviation on the Figure 3.

Previous edition

Revised edition

- I suggest placing photographs of the suspensions in a better resolution because this is the only result in this paper that confirms their “excellent” stability. Why do the authors not compare photographs of the suspensions just after they have been obtained (0 h) with photographs of the suspensions after 7 days in the paper? Why did the authors not provide the results of changes in particle/agglomerate size for the obtained suspensions for the duration “0h” and “7 days”? These results would unambiguously confirm the stability of the obtained suspensions.

Response 5(4):

To evaluate the colloidal stability of the prepared samples, we characterized the zeta potential values of the samples and observed the sedimentation after seven days of sample placement. The results show that the absolute values of the zeta potential of all the samples are greater than 30mV. Besides, there is no observable precipitate at the bottom of the container, and the dispersions are nearly transparent for more than a week observation, which fully demonstrates the colloidal stability of the samples.

6. “Elemental analysis”:

- No supporting information was appended to the paper.

Response 6:

You can see the information in the uploaded support material.

7. Discussion (Table 5):

- Table no. 5 is unacceptable in the present form. The acronyms used there (DLS, EX, EM) must be explained in the table description.

- DLS is the acronym of a measurement method, not of the determined parameter.

- What was determined using DLS? (diameter or radius)

- I suggest rounding the results of the average particle/agglomerate size and of the zeta potential to first decimal place.

- The standard deviation value must be added to the results of the average size and zeta potential.

- The results for the polydispersity index were not provided.

- There is no information about the duration of synthesis of the samples provided in Table 5.

Response 7:

Thanks to the reviewer's suggestion, we have revised the Table 5, as follows:

Previous edition

Table 5. Synthesizing fluorescent hydroxyapatite using other compounds containing an NH₂ group

Source of nitrogen	DLS (nm)	Zeta potential (mV)	EX (nm)	EM (nm)	XRD
Arginine	84.96	−36.03	340	420	Pure HA
Urea	101.1	-36.17	340	427	Pure HA
Ethylenediamine	107.5	−33.75	340	443	Pure HA

Revised edition

Table 5. Synthesizing fluorescent hydroxyapatite using other compounds containing an NH₂ group

Source of nitrogen	D (nm)	PDI	Zeta potential (mV)	EX (nm)	EM (nm)	XRD
Arginine	85.0	0.124	−36.0 (±1.2)	340	420	Pure HA
Urea	101.1	0.117	−34.2(±0.6)	340	427	Pure HA
Ethylenediamine	107.5	0.112	−33.8(±0.9)	340	443	Pure HA

Notes: “D” is hydrodynamic diameter; “PDI” is polydispersity index; “EX” is excitation wavelength; “EM” is emission wavelength

Revised content (Page 13)

To confirm the aforementioned mechanism, three different nitrogen-containing compounds were chosen, in which the molecular structure contains rich –NH₂ groups, to evaluate the effectiveness of fluorescent hydroxyapatite particle synthesis (Table 5). The synthetic steps are essentially the same, except that phosphate is used in sodium phosphate, and the nitrogen-containing compound is added in a 20% molar ratio with respect to sodium citrate. The hydrothermal time is 2 hours. All of the samples prepared with different –NH₂ sources are pure HA, with excellent colloidal stability and high fluorescence (Fig. S3; see supporting information).

Author Response File: Author Response.docx

Reviewer 2 Report

There are two quite distinctively separate parts of paper: 1) synthesis of HA NPs in a form of stable colloids and 2) luminescent properties of synthesized materal.

Synthetic part is quite interesting and sound in my opinion. Though, approximately the same technique was published earlier by the same authors, the new insights on preparation and study of the colloidal stability of HA NPs in the current paper are definitely worth publishing as they provide the important information on a subject, which is quite interesting for many researchers nowadays.

On the other hand, study of luminescent properties is not that convincing. In my opinion obtained results most likely could be explained by formation of N-doped carbon dots and there's no need in complicated explanation of the HA "influence" or "participation" in this process. Dependence on ammonium phosphate concentration is due to the facts that 1) carbon dots synthesis is very sensitive to pH, 2) N-doped dots synthesis is very sensitive to NH₄⁺ concentration.

The degree to which these N-doped carbon dots are linked to the HA NPs is unclear. My guess is that most likely they are just physically sorbed on the surface (to some extent may be trapped in the volume). Provided mechanism of formation of chain-like molecules on the surface of HA is rather unlikely. Due to a small size and polydenticity of citrate ions, they very much tend to form globular cross-linked macromolecules, carbon dots. And that is what most likely was happening in the experiments under discussion. One can only guess, how strong is the conjunction between c-dots and HA NPs. It could be possible, actually, that they are just a mechanical mixture. Anyway, there's no need for HA NPs for whatever luminescent applications authors are keeping in mind - c-dots alone will do the same and even better.

Therefore, my recommendation is to revise and resubmit the manuscript with more emphasis on the synthetic part and colloidal stability, e.g. more information could be provided on the evolution of z-potential over longer time intervals, as well as structure study could be more thorough and detailed. And at the same time cut the luminescent part severely, leaving only a brief discussion of this luminescent effect as an interesting feature of the synthesis.

Author Response

Reviewers #2:

There are two quite distinctively separate parts of paper: 1) synthesis of HA NPs in a form of stable colloids and 2) luminescent properties of synthesized materal.

Response 1:

Thanks to the reviewer's suggestion. In this article we mainly want to express two points: one is based on sodium citrate assisted hydrothermal method in the presence of ammonium ions that can achieve hydroxyapatite nanoparticles with both colloidal stability and fluorescent properties. The other is the fluorescent properties of hydroxyapatite that we believe to be primarily due to the formation of carbon dots. The carbon dots are not separately formed and adsorbed on the surface of the hydroxyapatite particles, but are derived from the citrate ions complexed with the amorphous calcium phosphate. Otherwise, once the particles and carbon dots are formed independently, both sides are negatively charged, and it is difficult to produce adsorption behavior.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

1. "Finally, the purified product was re-dispersed in deionized water to form an
aqueous dispersion by magnetic stirring for 10 min."

- There is no information about the magnetic stirrer (model, manufacturer) and rotational speed (RPM).

2. Discussion (Table 5):

- The standard deviation value must be added to the results of the hydrodynamic diameter.

Author Response

Dear editor Lilith Wu and reviewers:We are pleased to submit the revised manuscript (Manuscript Number, coatings-464166), “Ammonium-Induced Synthesis of Highly Fluorescent Hydroxyapatite Nanoparticles with Excellent Aqueous Colloidal Stability for Secure Information Storage” to Coatings.

We appreciate the effort done by editor and reviewers with our manuscript again. Herein, the detailed responses to the reviewers’ comments are as following:

Reviewers #1:

1. "Finally, the purified product was re-dispersed in deionized water to form an aqueous dispersion by magnetic stirring for 10 min."- There is no information about the magnetic stirrer (model, manufacturer) and rotational speed (RPM).

Response 1:

We have added the related information in the revised manuscript, as follows:

Previous edition(page 3, Introduction section)：

Finally, the purified product was re-dispersed in deionized water to form an aqueous dispersion by magnetic stirring for 10 min.

Revised edition:

Finally, the purified product was re-dispersed in deionized water to form an aqueous dispersion by magnetic stirring (MS-H280-Pro, DLAB Scientific Co., Ltd) with 600 RPM for 10 min.

2. Discussion (Table 5):

- The standard deviation value must be added to the results of the hydrodynamic diameter.

Response 2:

We have revised the Table 5 with adding the standard deviation value of hydrodynamic diameter, as follows:

Previous edition

Table 5. Synthesizing fluorescent hydroxyapatite using other compounds containing an NH₂ group

Source of nitrogen	D (nm)	PDI	Zeta potential (mV)	EX (nm)	EM (nm)	XRD
Arginine	85.0	0.124	−36.0 (±1.2)	340	420	Pure HA
Urea	101.1	0.117	−34.2(±0.6)	340	427	Pure HA
Ethylenediamine	107.5	0.112	−33.8(±0.9)	340	443	Pure HA

Notes: “D” is hydrodynamic diameter; “PDI” is polydispersity index; “EX” is excitation wavelength; “EM” is emission wavelength

Revised edition

Table 5. Synthesizing fluorescent hydroxyapatite using other compounds containing an NH₂ group

Source of nitrogen	D (nm)	PDI	Zeta potential (mV)	EX (nm)	EM (nm)	XRD
Arginine	85.0 (±38.3)	0.124	−36.0 (±1.2)	340	420	Pure HA
Urea	101.1 (±40.1)	0.117	−34.2 (±0.6)	340	427	Pure HA
Ethylenediamine	107.5(±46.5)	0.112	−33.8 (±0.9)	340	443	Pure HA

Notes: “D” is hydrodynamic diameter; “PDI” is polydispersity index; “EX” is excitation wavelength; “EM” is emission wavelength

Reviewer 2 Report

Authors did not revise manuscript according to my recommendations. The conclusion about formation of composite between HA and carbon-based material is yet not proved experimentally and does not seem likely. I recommend to reject this manuscript.

Author Response

We appreciate the effort done by editor and reviewers with our manuscript again. Herein, the detailed responses to the reviewers’ comments are as following:

Reviewers #2:

(first round)

There are two quite distinctively separate parts of paper: 1) synthesis of HA NPs in a form of stable colloids and 2) luminescent properties of synthesized materal.

(second round)

Response:

I am very grateful to the reviewers for their suggestions, and we also respect the reviewers' suggestions. As the reviewer said, the fluorescence mechanism of the hydroxyapatite nanoparticles synthesized by this method is still at a development stage. Recently, Fang et al has also explored its fluorescence mechanism through a series of hydrothermal synthesis comparison experiments based on sodium citrate (J. Mater. Chem. B, 2017, 5, 3749-3757). They verify which combination of N-rich carbon dots and hydroxyapatite nanocrystals (1) physical adsorption (2) Forming chemical bond between the carbon dots and the surface of HA particles (3) The carbon dots as impurities in HA crystal structure. The authors believe that the current results are more biased towards carbon dots (mainly is polymer clusters and/or molecular fluorophores constituents) trapped in the hydroxyapatite crystal structure. By comparing the fluorescence efficiency, the authors believe that the luminescent center may be one of the fluorescent molecular state, the amorphous carbon dot and the crystalline carbon dot depending on the hydrothermal conditions. In any case, there are still some shortcomings in the current understanding of the mechanism.

In view of this, we accept the comments of reviewers. In the revised draft, we will strengthen the discussion on the colloidal stability and simplify the discussion of its fluorescence mechanism. Details as follows:

Previous edition (Discussion section):

A series of physicochemical properties of the product in accordance with changes in R_AMP and hydrothermal time have been investigated. Introducing ammonium ions has no appreciable effect on the crystallization, morphology, or colloidal stability of hydroxyapatite. Nevertheless, it impacts the fluorescnce properties. In addition, increasing hydrothermal time (in the time range of our study) can greatly improve crystallization of the product, and has no appreciable effect on the colloidal stability. Nevertheless, there is a critical hydrothermal time limit for the fluorescence properties, which also decisively impacts the fluorescence properties. In other words, in addition to the citrate reported in previous work, ammonium ions and an appropriate hydrothermal time are critical for maximal fluorescent properties. At the same time, we noticed that the change in grain size did not significantly affect the fluorescence performance of the sample.

Currently, there are primarily two perspectives to explain this type of fluorescence phenomenon for hydroxyapatite. One perspective is that, due to the possible decomposition of citrate ions in the hydrothermal treatment to produce different levels of carbon dioxide radicals, these carbon dioxide radicals enter the hydroxyapatite lattice and cause defects, which are the fluorescence emission centers ^1-5. Another perspective is that the dehydration of sodium citrate to form carbon dots is the cause of luminescence, which is supported by the observation with high-resolution TEM that carbon dots are distributed on the surface of hydroxyapatite nanoparticles ⁶.

It is informative to first compare literature results of HA fluorescence (prepared via the hydrothermal method) with our results (Table S2; see supporting information). One can see that there are some common features:

· All involve ammonium and citrate ions.

· The maximum excitation and emission wavelengths are 340 and 440 nm, respectively.

· All require hydrothermal treatment.

Table S2 also shows that our research affords the highest fluorescence efficiency and quantum yield. This may be attributable to the shorter hydrothermal time of our method ⁷.

Citrate or ammonium ions often are used as raw materials for synthesizing fluorescent carbon nanodots. Consequently, Table S3 (see supporting information) compares our findings with examples of fluorescent carbon dot synthesis in the literature.

At present, one of the widely accepted luminescence mechanisms (namely, molecule state) considers that the photoluminescence (PL) center of carbon nanodots is an organic fluorophore, on the surface or interior of the carbon backbone, and can exhibit direct PL emission. At low hydrothermal temperatures, small fluorophore molecules are formed by dehydration of the initial molecules, which exhibit strong PL emission with high QYs. At high hydrothermal temperatures, the carbon core forms by dehydration of the initial molecules or consumption of the fluorophores, which exhibit high photostability and low QYs.

In the case of fluorescent HA, the PL mechanism may be more complex. Based on literature reports on the crystallization of bio-inspired hydroxyapatite nanoparticles ^9-12, once can infer that in the entire hydrothermal synthesis process, there are two parallel aspects of the reaction. The first aspect is based on the formation of hydroxyapatite and consists of two parts: (1) calcium ion complexation with citrate ions, followed by phosphate ion addition to form amorphous calcium phosphate; and (2) amorphous calcium phosphate transforms into hydroxyapatite along with hydrothermal treatment. The second aspects is that due to the higher density of citrate and ammonium ions in amorphous calcium phosphate, the citrate ions complexed with calcium ions undergo an intermolecular dehydration reaction in the presence of ammonium ions during the hydrothermal treatment. Since both citrate and ammonium contain more than three reactive groups (such as O–H and N–H), the reaction proceeds gradually as the hydrothermal time increases, to form polymer-like fluorescent molecules ⁷. With an extended hydrothermal time, the transformation from an amorphous calcium phosphate phase to HA phase, and the dehydration reaction of ammonium ions and citrate ions, were carried out simultaneously. The degree of dehydration depends on the concentration of ammonium ions as well as the hydrothermal time and temperature. Fig. 6 describes the detailed mechanism.

Figure 6. Schematics of the mechanism for forming fluorescent HA particles during hydrothermal treatment.

A prolonged hydrothermal time is advantageous for forming hydroxyapatite with superior fluorescence properties. However, if the hydrothermal time is too long, the colloidal stability of hydroxyapatite decreases, and settlement or delamination occurs. Thus, the fluorescence properties can be controlled by controlling the dehydration condensation reaction rate, via the concentration of ammonium ions. In our case, given that that hydrothermal time is only 4 h, and no carbon dots are observed on the surface of HA nanoparticles based on an analysis of HR–TEM images, one can speculate that the dehydration reaction of citrate ions occurs in the polymer cluster stage. This also explains why the fluorescent hydroxyapatite nanorods in our study have a high fluorescence efficiency.

Revised edition (Discussion section)::

For the colloidal stability of the particles, we believe that the contribution is mainly due to the following two aspects^9-12. The first aspect is based on the formation of hydroxyapatite and consists of two parts: (1) calcium ion complexation with citrate ions, followed by phosphate ion addition to form amorphous calcium phosphate; and (2) amorphous calcium phosphate transforms into hydroxyapatite along with hydrothermal treatment. Thus, the particle shape and size of the hydroxyapatite are relatively uniform, and the size of the particles is also relatively small. The second aspect is a large amount of citrate ions are adsorbed on the surface of the hydroxyapatite particles after completion of crystallization. In a weak alkaline environment, a good electrostatic repulsion force can be maintained between the particles. These two aspects make the particles with excellent colloidal stability in aqueous phase.

For the fluorescence of the particles, the PL mechanism is not completely clear. It is informative to first compare literature results of HA fluorescence (prepared via the hydrothermal method) with our results (Table S2; see supporting information). One can see that there are some common features:

· All involve ammonium and citrate ions.

· The maximum excitation and emission wavelengths are 340 and 440 nm, respectively.

· All require hydrothermal treatment.

Table S2 also shows that our research affords the highest fluorescence efficiency and quantum yield. This may be attributable to the shorter hydrothermal time of our method ⁷.

Both the hydrothermal treatment and the resulting fluorescence characteristics are similar to those of our hydroxyapatite research (Table S3). Ammonium ion is also necessary. The fluorescence properties herein should be more biased toward the luminescence mechanism of fluorescent carbon dots ⁶. The current results are more biased towards carbon dots (mainly is polymer clusters and/or molecular fluorophores constituents) trapped in the hydroxyapatite crystal structure. The luminescent center may be one of the fluorescent molecular state, the amorphous carbon dot and the crystalline carbon dot depending on the hydrothermal conditions.

Previous edition (conclusion section):

In summary, uniform HA nanoparticles, with excellent aqueous colloidal stability and high fluorescence, have been synthesized via a citrate-assisted hydrothermal method. Introduction of ammonium ions has no appreciable effect on the crystallization, morphology, or colloidal stability of hydroxyapatite. Nevertheless, it is critical to the fluorescence properties. An increased hydrothermal time (in the time range of our study) considerably improved crystallization of the product, and had no appreciable effect on the colloidal stability. Nevertheless, there was a critical hydrothermal time limit for the fluorescence properties. In other words, in addition to the citrate reported in previous work, ammonium ions and an appropriate hydrothermal time are critical for optimum fluorescence properties. The cause of HA fluorescence is the dehydration of citrate molecules, caused by ammonium ions, along with transformation from amorphous calcium phosphate to HA during hydrothermal treatment. The polymer-like fluorescent molecules generated in situ on the surface of hydroxyapatite crystals are likely to be their fluorescent centers. Owing to these properties, a highly fluorescent HA colloidal dispersion was applied toward secure information storage.

Revised edition (conclusion section):

In summary, uniform HA nanoparticles, with excellent aqueous colloidal stability and high fluorescence, have been synthesized via a citrate-assisted hydrothermal method. Introduction of ammonium ions has no appreciable effect on the crystallization, morphology, or colloidal stability of hydroxyapatite. Nevertheless, it is critical to the fluorescence properties. An increased hydrothermal time (in the time range of our study) considerably improved crystallization of the product, and had no appreciable effect on the colloidal stability. Nevertheless, there was a critical hydrothermal time limit for the fluorescence properties. In other words, in addition to the citrate reported in previous work, ammonium ions and an appropriate hydrothermal time are critical for optimum fluorescence properties. For the colloidal stability of the particles, The transformation of amorphous calcium phosphate to hydroxyapatite and surface adsorption of citrate are two important factors for the good colloidal stability of the particles. The cause of HA fluorescence are more biased towards carbon dots (mainly is polymer clusters and/or molecular fluorophores constituents) trapped in the hydroxyapatite crystal structure. The luminescent center may be one of the fluorescent molecular state, the amorphous carbon dot and the crystalline carbon dot depending on the hydrothermal conditions. Owing to these properties, a highly fluorescent HA colloidal dispersion was applied toward secure information storage.

Previous edition (Abstract section):

In this paper, uniform hydroxyapatite (HA) nanoparticles, with excellent aqueous colloidal stability and high fluorescence, have been successfully synthesized via a citrate-assisted hydrothermal method. The effect of the molar ratio of ammonium phosphate in phosphate (R_AMP), and hydrothermal time, on the resultant products were characterized in terms of crystalline structure, morphology, colloidal stability, and fluorescence behavior. When the R_AMP is 50% and the hydrothermal time is 4 h, the product consists of a pure hexagonal HA phase and a uniform rod-like morphology, with 120- to 150-nm length and approximately 20-nm diameter. The corresponding dispersion is colloidally stable, and transparent for at least one week, and has an intense bright blue emission (centered at 440 nm, 11.6-ns lifetime, and 73.80% quantum efficiency) when excited by 340-nm UV light. Although prolonging the hydrothermal time and increasing the R_AMP had no appreciable effect on the aqueous colloidal stability of HA nanoparticles, the fluorescence intensity was enhanced. Fluorescence from HA is attributable to the dehydration of citrate molecules by ammonium ions, and transformation from an amorphous calcium phosphate (ACP) to HA during hydrothermal treatment. Owing to these properties, a highly fluorescent HA colloidal dispersion could find applications in secure information storage.

Revised edition (Abstract section):

In this paper, uniform hydroxyapatite (HA) nanoparticles, with excellent aqueous colloidal stability and high fluorescence, have been successfully synthesized via a citrate-assisted hydrothermal method. The effect of the molar ratio of ammonium phosphate in phosphate (R_AMP), and hydrothermal time, on the resultant products were characterized in terms of crystalline structure, morphology, colloidal stability, and fluorescence behavior. When the R_AMP is 50% and the hydrothermal time is 4 h, the product consists of a pure hexagonal HA phase and a uniform rod-like morphology, with 120- to 150-nm length and approximately 20-nm diameter. The corresponding dispersion is colloidally stable, and transparent for at least one week, and has an intense bright blue emission (centered at 440 nm, 11.6-ns lifetime, and 73.80% quantum efficiency) when excited by 340-nm UV light. Although prolonging the hydrothermal time and increasing the R_AMP had no appreciable effect on the aqueous colloidal stability of HA nanoparticles, the fluorescence intensity was enhanced. The cause of HA fluorescence are more biased towards carbon dots (mainly is polymer clusters and/or molecular fluorophores constituents) trapped in the hydroxyapatite crystal structure. Owing to these properties, a highly fluorescent HA colloidal dispersion could find applications in secure information storage.

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Junjun Tan

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Round 3

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In its current form I have no objections against publication of the manuscript.

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Ammonium-Induced Synthesis of Highly Fluorescent Hydroxyapatite Nanoparticles with Excellent Aqueous Colloidal Stability for Secure Information Storage

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