Seedless gold nanostars with seed-like advantages for biosensing applications

Gold nanostars (AuNSs) are seen as promising building blocks for biosensors with potential for easy readouts based on naked-eye and ultraviolet–visible spectroscopy detection. We present a seedless synthesis strategy for AuNSs that has the advantages of the seeded methods. The method used ascorbic acid as a reducing agent and silver nitrate as an anisotropic growth control assisting agent. AuNSs with multiple branches and a diameter of 59 nm were produced. They showed good stability when capped with PVP and modified with an enzyme in relatively strong ionic conditions. We investigated their application in plasmonic sensing by modifying them with glucose oxidase and detection of glucose. The AuNSs were found to be a good scaffold for the enzyme, proved to be stable and sensitive as transducers. Thus, the AuNSs showed good promise for further applications in plasmonic biosensing for in vivo biomedical diagnosis.


2) In
, whilst the TEM is nice, the scalebars should be made clearer.
3) On p5 line 10, is there any sensitivity of this new method at all to the addition time of the Ag, or is the Ag the reaction initiator? (How immediate is "immediate addition of AuNS" to get good AuNS?) 4) Why was 0.3 M of NaCl used as the salt stability test concentration? Do the authors have a feel for that the upper limit of stability to salt and pH might be? 5) Typo on P7 line 45 "In the absence of glucose, the seedless AuNSs remained star shaped and progressively became more spherical as the glucose concentration was increased." This seems to conflate two different processes -absence = star shape, presence = AuNS become spherical? 6) Is Figure 6a  The editor assigned to your manuscript has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Authors report seedless synthesis of gold nanostars of 60 nm size and their application in biosensing. Typically they have used ascorbic acid reduction of gold salt in acidic medium in presence of Ag ion. Advantage and appropriateness of synthetic condition need to be varified/compared with various control conditions. In particular sequence of addition of various reagents and their consequence in optical property, particle size and quality of nanostars should be studied. Glucose detection approach via formation Ag form hydrogen peroxide, is interesting. The language is too difficult to follow.

Reviewer: 2
Comments to the Author(s) The authors (Phiri et al.) present an adapted technique for synthesis of gold nanostars (AuNS) to provide more control and stability than the recent HEPES method, through use of silver ions and citrate as the reductant. The nanoparticles produced are well characterised and applied in a silver deposition assay for glucose sensing.
The work seems novel, and sound and I have a only a few small comments for the authors: 1) It would be good to see the DLS intensity data curves plotted out in the paper or SI, rather than just the numbers, to get a better idea of the quality of fit and make it easier for the reader. The way the data is currently provided is hard to use. 2) In Figures 2 and 6, whilst the TEM is nice, the scalebars should be made clearer.
3) On p5 line 10, is there any sensitivity of this new method at all to the addition time of the Ag, or is the Ag the reaction initiator? (How immediate is "immediate addition of AuNS" to get good AuNS?) 4) Why was 0.3 M of NaCl used as the salt stability test concentration? Do the authors have a feel for that the upper limit of stability to salt and pH might be? 5) Typo on P7 line 45 "In the absence of glucose, the seedless AuNSs remained star shaped and progressively became more spherical as the glucose concentration was increased." This seems to conflate two different processes -absence = star shape, presence = AuNS become spherical? 6) Is Figure 6a

Referee 1
 Advantage and appropriateness of synthetic condition need to be verified/compared with various control conditions. In particular sequence of addition of various reagents and their consequence in optical property, particle size and quality of nanostars should be studied.
Response: It was experimentally observed that even when HCl was not added in the synthesis procedure, gold nanostars were still formed. However, the localised surfaced plasmon resonance of these nanostars were more blue-shifted compared to those synthesised with HCl added to it.  Glucose detection approach via formation Ag form hydrogen peroxide, is interesting. The language is too difficult to follow.

Response:
The language here was revised and simplified so as to make it easy to follow. It now reads as follows as quoted exactly from the manuscript: Using optimized conditions, this assay system was investigated for the determination of various concentrations of glucose as a proof of concept. As shown in Figure 6a

Referee 2
1) It would be good to see the DLS intensity data curves plotted out in the paper or SI, rather than just the numbers, to get a better idea of the quality of fit and make it easier for the reader. The way the data is currently provided is hard to use.
This was done and now added to Figure 3. Figures 2 and 6, whilst the TEM is nice, the scalebars should be made clearer.

2) In
The images were reworked and the scalebars are now clearer. Thus, the stability of the GOx-modified seedless-AuNSs was tested and proved in 0.3 M NaCl and pH 7 [4,5]. 0.3 M NaCl is deemed to be twice the ionic concentration of salt in plasma which is 0.135 M [6]. Therefore, this concentration has been in the cited publication to test the stability of the nanoparticles in ionic environment [4]. A critical flocculation concentration is usually determined as the threshold concentration of NaCl in the gold solution which causes rapid aggregation [5]. However, this was not done for the submitted manuscript. 5) Typo on P7 line 45 "In the absence of glucose, the seedless AuNSs remained star shaped and progressively became more spherical as the glucose concentration was increased." This seems to conflate two different processesabsence = star shape, presence = AuNS become spherical?
The line was revised and now reads as follows: "In the absence of glucose, the nanostructures remained star-shaped. Conversely, when the glucose was added to various tubes in increasing concentrations, the AuNS progressively became more spherical in shape." Figure 6a normalised? If so, please state in the caption.

6) Is
Yes. Figure 6a was normalized and has since been stated in the caption. The suggested references were reviewed and appropriately included in the manuscript. The suggestions are much appreciated as they threw some light on the subject matter.