Gold Nanoparticles/Carbon Nanotubes and Gold Nanoporous as Novel Electrochemical Platforms for L-Ascorbic Acid Detection: Comparative Performance and Application

Round 1

Reviewer 1 Report

Authors have added some additional results about NPG morphologies, which confirm the honeycomb feature of the deposited image. But, the main concerns I raised in my previous revisions are not addressed. There are still fundamental inaccuracies in the manuscript, which are highlighted below.

1) I don’t agree with the statement in line 178-181. “ In the case of the Au/SWCNTs electrode, it is possible to clearly note the presence of bundles of tubes, with an average diameter of about 1 nm, and clusters of AuNPs, with an average diameter of about 50 nm, onto the Au electrode surface (Figure 1, panel B).” I raised this point in my previous revision. However, the response provided by the authors is not convincing at all. I have the same question, how can one notice 1 nm dimension in a SEM image with a scale bar of 200 nm. Moreover, the image in 1B does not show any bundles of CNT.

2. Regarding the calibration curves extracted from Figure 7 and plotted in Figure 8, I am not convinced with the authors’ response. It is hard to see any oxidation wave of AA in Figure 7b and Figure 7c at lower concentration. The calibration curves in the following figure are not credible. Authors should show the CVs associated with low concentration of AA. Moreover, background current must be subtracted from the oxidation wave (which I don’t see at low AA concentration).
3. For the interference study, authors mentioned, “at h-nPG electrode the anodic peaks appear well resolved with peak potentials at 0, 0.20 and 0.32 V vs Ag/AgCl, respectively, as reported in Figure 9”. However, there is no point of recording a separate CV of the AA, dopamine and uric acid to justify a selective sensor. As I also stated in my previous revision, CV of the NPG coated electrode must be performed in solution containing AA, dopamine and uric acid together to confirm selectivity. This is because, oxidation product of AA are known to interact with dopamine and uric acid and interfere in the measurements.

Author Response

Authors have added some additional results about NPG morphologies, which confirm the honeycomb feature of the deposited image. But, the main concerns I raised in my previous revisions are not addressed. There are still fundamental inaccuracies in the manuscript, which are highlighted below.

1. I don’t agree with the statement in line 178-181. “ In the case of the Au/SWCNTs electrode, it is possible to clearly note the presence of bundles of tubes, with an average diameter of about 1 nm, and clusters of AuNPs, with an average diameter of about 50 nm, onto the Au electrode surface (Figure 1, panel B).” I raised this point in my previous revision. However, the response provided by the authors is not convincing at all. I have the same question, how can one notice 1 nm dimension in a SEM image with a scale bar of 200 nm. Moreover, the image in 1B does not show any bundles of CNT.

Thank you for your right observation. Figure 1B has been changed. It is now possible to notice the presence of nanoparticles in the diameter range 50-250 nm. The sentence in the main text (lines 170-171) has been accordingly modified with “…with a distribution of nanoparticles in the range 50-250 nm diameter.”  The presence of SWCNTs on the surface of the modified electrode has been confirmed by the Raman spectroscopy characterization (see paragraph 3.2), as the resolution of our SEM instrument does not allow the detection of SWCNTs with 1 nm diameter.

Unfortunately, as you correctly stated, the 1 nm dimension of our SWCNTs cannot be visualized with SEM image with a scale bar of 200 nm. In order to overcome this problem, Raman spectroscopy experiments were carried out to confirm the presence and to characterize the SWCNTS modified electrode surface.

1. Regarding the calibration curves extracted from Figure 7 and plotted in Figure 8, I am not convinced with the authors’ response. It is hard to see any oxidation wave of AA in Figure 7b and Figure 7c at lower concentration. The calibration curves in the following figure are not credible. Authors should show the CVs associated with low concentration of AA. Moreover, background current must be subtracted from the oxidation wave (which I don’t see at low AA concentration).

I agree with your observation but, unfortunately, with CV experiments is not possible to better visualize the oxidation peaks at low concentrations. However, the values reported in the calibration curves have been extrapolated by the Autolab Nova computer software directly from the voltammetric curves at the potential corresponding to the maximum current values for each electrode at all concentrations. Therefore, the calibration curves reported are reliable also at low concentration values. 

Moreover, the more sensitive DPV technique has been also used and the values obtained with this technique has been reported. Fig. 1S in the supplementary shows the DP voltammograms at some concentrations of AA and the relative calibration curve is reported in the inset. The main text has been modified accordingly (331-335).

1. For the interference study, authors mentioned, “at h-nPG electrode the anodic peaks appear well resolved with peak potentials at 0, 0.20 and 0.32 V vs Ag/AgCl, respectively, as reported in Figure 9”. However, there is no point of recording a separate CV of the AA, dopamine and uric acid to justify a selective sensor. As I also stated in my previous revision, CV of the NPG coated electrode must be performed in solution containing AA, dopamine and uric acid together to confirm selectivity. This is because, oxidation product of AA are known to interact with dopamine and uric acid and interfere in the measurements.

As suggested by the referee, Fig. 9 has been changed. It shows the CVs of the three compounds present together in the same solution, as requested. Well separated peaks can be observed suggesting the high selectivity of h-nPG modified electrode towards DA and UA, which are the most common interferences present in physiological samples.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript reports a possibility of gold nano-structured electrochemical sensors for L-ascorbic acid detection. To prove of that, in the experimental sections, gold single-walled carbon 15 nanotubes (Au/SWCNTs) and nanoporous gold (h-nPG) film were designed and analyzed by FESEM and Raman spectroscopy. In addition, cyclic voltammetry characterize electrochemical signal from the electrodes with control.

This paper demonstrates with the developed h-nPG electrode to determine ascorbic acid spiked in human urine exhibiting relatively low limit of detection. The authors compared LOD of various current gold nano-structured sensors and claimed competitive sensing results detecting L ascorbic acid.  I recommend this paper to be publish as it is..

Author Response

Thank you for your revision.

Reviewer 3 Report

The paper is interesting and well written. Few comments are shown below.

Page 3; scheme 1.

Q1. Please re-draw Scheme 1; the words cannot be read.

Page 5, Figure 1

Q2.  If Fig. 1A represents bare Au surface, why is it full with Au nanoparticles?

Regarding Figure 1B, I cannot “clearly note the presence of bundles of tubes, with an average diameter of about 1 nm, and clusters of AuNPs, with an average diameter of about 50 nm…”.

Please provide an image with higher resolution (scale bar ~ 50 nm), where the mentioned details can be observed. According to the present image, the Au nanoparticles are around 200 nm, NOT 50 nm.

Page 8. Table 1 and main text

Q3. Please indicate the correct value for peak-to-peak separation (ΔEp) for h-nPG electrode, in the main text (87 mV, NOT 80 mV).

Page 10 and Figure 6.

Q4. There is no agreement between the main text and the legend of Figure 6. Please correct it!

“A classical Randles circuit was successfully applied to fit both Au bare and Au/SWCNTs modified electrodes (Fig. 6, circuit 1), while circuit 2 was proposed for better fitting h-nPG modified electrode.”

“Fig. 6. Equivalent circuits used for fitting the experimental data: circuit (1) for h-nPG modified electrode and Au/SWCNTs modified electrode (Randles circuit), and circuit (2) for Au bare electrode.”

Q5. In Figure 8, the current (I) should be replace with peak current (Ip).

Q6. I do NOT understand the legend of Fig. 9. In addition, the shape of AA in Figure 9 is different than that presented in Fig. 7C. Why?

Fig. 9. “Cyclic voltammograms of 4 mM AA, DA and AA 4 mM + DA 3 mM and UA 6 mM at h-nPG electrode in 0.1 M 471 PBS pH=7.4, KCl=0.1M, at υ = 10 mV s-1”- Is AA combined with DA in this figure?

Author Response

The paper is interesting and well written. Few comments are shown below.

Page 3; scheme 1.

Q1. Please re-draw Scheme 1; the words cannot be read.

As suggested by the referee, Scheme 1 has been redrawn (line 658).

Page 5, Figure 1

Q2.  If Fig. 1A represents bare Au surface, why is it full with Au nanoparticles?

Thank you for your observation. We changed Fig. 1A representing the Au bare electrode surface. Now the surface appears more flat with no nanoparticles present.

Regarding Figure 1B, I cannot “clearly note the presence of bundles of tubes, with an average diameter of about 1 nm, and clusters of AuNPs, with an average diameter of about 50 nm…”.Please provide an image with higher resolution (scale bar ~ 50 nm), where the mentioned details can be observed. According to the present image, the Au nanoparticles are around 200 nm, NOT 50 nm.

Thank you for your observation. We changed the figure 1B. It is now possible to notice the presence of nanoparticles of mainly 200 nm, as you stated, but also smaller nanoparticles are visible, in the diameter range 50-250 nm. The sentence in the main text (lines 170-171) has been accordingly modified with “…with a distribution of nanoparticles in the range 50-250 nm diameter.”  The presence of SWCNTs on the surface of the modified electrode has been confirmed by the Raman spectroscopy characterization (see paragraph 3.2), as unfortunately the resolution of our SEM instrument does not allow the detection of SWCNTs with 1 nm diameter.

Page 8. Table 1 and main text

Q3. Please indicate the correct value for peak-to-peak separation (ΔEp) for h-nPG electrode, in the main text (87 mV, NOT 80 mV).

We substituted the correct value in the main text (line 221).

Page 10 and Figure 6.

Q4. There is no agreement between the main text and the legend of Figure 6. Please correct it!

“A classical Randles circuit was successfully applied to fit both Au bare and Au/SWCNTs modified electrodes (Fig. 6, circuit 1), while circuit 2 was proposed for better fitting h-nPG modified electrode.”

“Fig. 6. Equivalent circuits used for fitting the experimental data: circuit (1) for h-nPG modified electrode and Au/SWCNTs modified electrode (Randles circuit), and circuit (2) for Au bare electrode.”

We changed the sentence in the main text (line 270-271) as “A classical Randles circuit was successfully applied to fit Au bare (Fig. 6, circuit 2), while circuit 1 was proposed for better fitting both h-nPG and Au/SWCNTs modified electrodes.”

Q5. In Figure 8, the current (I) should be replace with peak current (Ip).

Thanks for the referee observation, we replaced the current with I_p in Fig.8, pag. 29.

Q6. I do NOT understand the legend of Fig. 9. In addition, the shape of AA in Figure 9 is different than that presented in Fig. 7C. Why?

Fig. 9. “Cyclic voltammograms of 4 mM AA, DA and AA 4 mM + DA 3 mM and UA 6 mM at h-nPG electrode in 0.1 M 471 PBS pH=7.4, KCl=0.1M, at υ = 10 mV s-1”- Is AA combined with DA in this figure?

We changed Fig. 9 (pag. 30) that represents the three compounds present in the same solution (line 868) and the corresponding legend, as requested.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

NA

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The present manuscript describes development of ascorbic acid (AA) sensors based on conventional gold and screen-printed electrodes modified with carbon nanotube composite and nanoporous gold film. Authors report electrodeposition of the composite CNT/Au and NPG, their morphological characterizations by SEM and electrochemical studies by cyclic voltammetry and impedance spectroscopy. Finally, detection of AA by cyclic voltammetric method on these electrodes are reported. Although, the objective of the manuscript is timely and important as it promises to develop a sensing platforms for an important antioxidant which detection is sought in multitude of applications areas. However, the manuscript is way far off from the standard of scientifically precise and well-structured documents. Most of the reported claims in the interpretations are not validated by the results presented. Beside this, the main problem comes with the inaccurate interpretation of the results in every parts, including in electrodeposition, electrochemistry of AA and NPG and morphological characterizations. My comments are appended below.

1. The line 88-92, is very long. I don’t not understand, how gold is less expensive materials. Moreover, i don't agree with the ranges of pore sizes of NPG given here. Most of the AA sensors, utilizing NPG reported pore sizes much larger. The references 49 and 50 cited to support the statement are not specific to this.
2. In line 99, authors mentioned that NPG films by self-templating method were realized mainly at -2 V. Why only -2 V? There are many reports of NPG synthesis at higher negative potential. For instance, groups of M. Bertotti and others synthesized many NPG at more negative potential, which are in fact more sensitive for electroanalytical applications (Bioelectrochemistry 112 (2016) 125–131, (Talanta 226 (2021) 122130), (International Journal of Hydrogen Energy 44 (2019) 15001 – 15008), (Journal of Electroanalytical Chemistry 805 (2017) 18–23).
3. The statement in line 102-104 is not correct. There are previous researches reporting fabrication of NPG electrodes by hydrogen bubble template for AA detection (Analytica Chimica Acta 1095 (2020) 61-70).
4. There is a problem with bottom right part of the scheme-1. Since NPG are electrodeposited like a films on the electrode surface, it is not an appropriate representation.
5. The method of bare electrode treatment, NPG synthesis and its further activation are wrongly interpreted. For instance, application of a few CV cycles from 0.8 V to 0 V will deposit some gold clusters on the bare electrode surface. For subsequent potentiostatic deposition at -4 V potential, these initially formed aggregates act as nucleation centers and will result in much larger aggregates and thickening of the electrode modification. What is the meaning of the activation after potentiostatic deposition?
6. The SEM images reported in Fig. 1 does not support the claimed pore sizes of 1 nm in NPG and sizes of SWNT and Au NP in the text. These are completely filmsy imagination. Moreover, the observation made from SEM images are wrongly interpreted (line 190-194). In fact, the roughness of SPE is generated by the deposition/adsorption of Au nanoclusters at potential less than its equilibrium potential of formation (because of underpotential deposition UPD). Later, when -4 V is applied, these roughness centers act as a nucleation centers for the deposition of Au atoms and simaltaneous evolution of H2 bubbles. The reference 58 given to support the statement is not appropriate.
7. Impedance circuits are wrongly selected. CPE are used to explain the dispersion of time constants of eauivalent circuit parameters. This is applied to the system where there is distribution in the microscopic properties of materials defined by a time constant (Electrochimica Acta 363 (2020) 137199). Given the experimental nature, the impedance curves can be simply fitted with combination of Rs (solution resistance), Rct (charge transfer resistance), W (Warburb) and C.
8. How one can say by looking at the CV that irreversible redox reaction of AA is accompanied by 2H and 2e. A reference or interpretation should be given.
9. How can authors justify the statement “It appears evident that both peak current and peak position of AA oxidation is strictly related to the electrode morphology.”
10. How can one say that AA exhibits slow kinetics without a quantitative reference value. In fact, Nanoconfinement is not the right reason for the electrocatalysis. Structural defects and exposure to low index cystalline planes play a dominant role (Journal of The Electrochemical Society, 166 (14) H704-H711 (2019)), (Nanoscale, 2018, 10, 11091).
11. In lines 361-364, This comment is not so straightforward. There is no linear correlation between pH and AA electrode process. Moreover, the reference 43 is not relevant in this context because there is no reported work on pH studies. For reference 40, there is pH studies for AA electrode process but it does not give the mechanistic answers. An appropriate mechanistic answer can be found in the work of Compton et al. J. Phys. Chem. B 1998, 102, 38, 7442–7447.
12. For Fig. 7, It is impossible to extract such calibration curve from the voltammograms shown for AA. For instance, i can't see any peak for AA oxidation at low concentrations like 10, 20 or 50 uM for any electrodes. Moreover, in all these CVs, capacitive contribution is not subtracted. So the claimed Faraidic peak of AA is not true. In Fig. 6b, the capacitive contribution is so high that it is impossible to justify that if there is any AA oxidation.
13. The values of sensitivity for different electrodes given in the abstract are not supported by the presented data in the manuscript. A change in the Faradaic current is not noticed even at much higher AA concentration in Fig. 5.
14. In line 400-402, this is a wrong statement. At first, electrode process of ferrycyanide is purely diffusional mass transport process, while for AA, DA and UA, there are followed chemical reactions after electron transfer. Moreover, how authors can compare the electron transfer kinetics of AA, DA and UA with ferrycyanide without having any quantitative or qualitative data.
15. For the interference study, CV of the solution containing AA, DA and UA should be recorded. Recording separate CV is not correct because presence of DA and UA influences the electrode process of AA.
16. The manuscript contains too many citations, many of which are not precise to the referred context. Some of them are completely not linked at all to the said statements.

Minor points

1. In the abstract line 13-19, sentence is very long. It can be split into three.
2. There are typos in line 133, 137, 263, 349.
3. In equation 1, arrow is not properly indicated.

Author Response

The manuscript has been reviewed according to the referee's suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this paper two nanostructured gold-based electrodes have been developed and used to detect L-ascorbic acid. The voltammetric and impedimetric responses have been evaluated in AA solutions and in human urine.

The paper is interesting, the methodology is adequate, and the conclusions are sound and justified. For this reasons I recommend to publish the paper. However, some aspects must be clarified or justified.

1. Lines 84-87: authors indicate that gold-based materials such as gold nanoparticles and gold nanoporous have the advantage of minimizing electrode biofouling due to protein adsorption. This statement requires an explanation because gold nanomaterials have a high affinity towards biomaterials (proteins, Ab, DNA…). Besides this, after reading the introduction, one could expect that authors include a study on the fouling effects occurring in their electrodes, but this is not the case. So, I suggest or a) to remove this paragraph of b) include the study of the fouling at least in urine samples.
2. Scheme 1. The image of the sponge is not very professional. I suggest to change it.
3. SEM characterization: The scale of the SEM images is quite large. However, authors describe really small structures that cannot be seen at that scale. For this reason, the surface characterization is not adequate
   •  in the case of the Figure 1.b nanotubes of 200 nm diameter can be seen.  This is weird since in the experimental section it is stated that the diameter of the SWCNTs  is 0.83 nm. Moreover,  the structures observed in that image have diameters of 1 micron or more and their shape does not coincide with that of rigid nanotubes
   • Similarly in Figure 1.C. it is impossible to observe well-defined pores of 1 nm diameter.

In order to characterize such small structures authors must provide TEM or AFM images registered at the appropriate scale and with enough resolution.

4. My main concern about this paper is that all the differences observed in the performance of the AuNP-SWCNT and the hnPG electrodes are attributed to the porous structure of the hnPG. It is evident that porosity is playing a main role, but both electrodes have a different chemical structure and the presence of SWCNT must also be important (many papers have been published highlighting the electrocatalytic properties of SWCNTs) . Authors should include data of a AuNP electrode free of SWCNT. This will help to evaluate the real role of the porosity and also the possible role of the SWCNTs

Author Response

The manuscript has been reviewed according to the referee's suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I am not convinced with answers provided by the authors and modifications made in the article. For the revised submission, authors have to be rigorous and precise in their answers. I am mentioning few inaccuracies in the author’s rebuttal letter addressed to my previous review report. Although there are much more but it is time consuming and frustrating highlighting the same errors.

1. In the rebuttal letter, authors say that they are following the synthesis procedure given by Bollella et al. (Anal. Chem. 2018, 90, 20, 12131). I read this referenced article, which shows a typical pattern (honeycomb feature) of h-nPG film obtained by dynamic hydrogen bubble template deposition. However, the SEM image provided in the present article does have a honeycomb like morphology. By looking at the image, it appears that there is no templating effect by H2 bubbles.

 Moreover, the claimed statement in the manuscript “ a homogeneous highly curved morphology of well-defined pores with diameters of  approximately 1 µm is clearly observed” is not corroborated by the provided SEM image in Fig. 1c.

Further, in SEM image 1B, I can not see any CNT of tube diameter 1 nm and length 50 nm as claimed by the author. How one can visualize 1 nm dimension in a SEM image provided on a scale of 200 nm.

Again, for SEM image in Fig. 1a, authors mentioned in the title that it is for bare SPE. I suspect, because it is full of spherical clusters. Usually a clean bare SPE surface is very flat.

2. I don’t agree with authors response about Fig. 6 and 7. By looking at the voltammograms in Fig. 6, it is hard to confirm the data points given in the calibration curve.

Author Response

The manuscript has been modified according to referee' suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Author have made the changes requested

Author Response

Thank you for accepting the mofidied version of the manuscript.