Facile immobilization of glucose oxidase onto gold nanostars with enhanced binding affinity and optimal function

Gold nanoparticles provide a user-friendly and efficient surface for immobilization of enzymes and proteins. In this paper, we present a novel approach for enzyme bioconjugation to gold nanostars (AuNSs). AuNSs were modified with l-cysteine (Cys) and covalently bound to N-hydroxysulfosuccinimide (sulfo-NHS) activated intermediate glucose oxidase (GOx) to fabricate a stable and sensitive AuNSs–Cys–GOx bioconjugate complex. Such a strategy has the potential for increased attachment affinity without protein adsorption onto the AuNSs surface. Good dispersity in buffer suspension was observed, as well as stability in high ionic environments. Using the AuNSs–Cys–GOx bioconjugates showed greater sensitivity in the measuring of low concentrations of glucose based on plasmonic and colorimetric detection. Such a novel approach for enzyme immobilization can lead to AuNSs–Cys–GOx bioconjugate complexes that can be used as catalytic nanodevices in nanobiosensors based on oxidases in biomedical applications.


Page 3: -NMR section is very detailed and should be summerised
Page 3 lines 53-54 -"Comparison assays were done using AuNSs without any GOx, AuNSs with unattached GOx in solution" what is difference between these two states?
-"The thiol moiety has been found to be a very effective site for interaction" how this with one S atom of Cys?
-The text of "Stability of AuNSs-Cys-GOx bioconjugates" section needs the reference.
-Where are the activity results? Scheme 1 is very ambiguous: -Where is roles of sulfo-NHS and EDC in scheme 1.
-What is the role of EDC in the AuNSs-Cys-GOx synthesis? -Why in the GOx structure there are 2 NH3+ groups? -Why the arrow to belong from AuNS to sulfo-NHS? -Where is the pvp in this scheme? -Why the authors used the 2 linkers (NHS and EDC) in this work?
"The particle-size distribution was estimated by measuring the size of approximately 100 nanoparticles in different grid regions" -How? -Where is the figure including 100 NP and what was the software for this estimation? -All of components in the TEM images should be recognized and assigned?
In Figure 1: -the Roman numerals on the figure should be corrected.
-Zero point of the all spectra should be specified. I do not understand: -Spectrum (III) shows the peaks for the NHS-terminated GOx with sulfo-NHS in tandem with EDC.
Page 5 line 60: -I do not understand "No broadening of the GOx-modified AuNSs was observed which implies that the AuNSs maintained their structural integrity with good dispersity" I do not understand: -"How the Au nanostars formation was confirmed?" -All the UV-Vis spectra 400-0 nm were scanned -The figure 4 should be assigned and appeared consequently which explained in the text -Why the intensity for AuNSs-Cys-GOx is higher than the AuNSs intensity? -What is the goal of ionic strength investigation in this work? -What are the Gox, enzyme and protein roles in this work? -Generally the absorbance of AuNanoparticles have a max intensity before 600 nm wavelength but in this work this character has been appeared about 700 nm? Why? I cannot concluded this sentence: -"This showed the potential of the AuNSs-Cys-GOx bioconjugates fabricated in the proposed approach to be used to detect low concentrations of analytes with high sensitivity and stability" Minors: -What is Km? The in following mistakes in the text should be corrected: "Colorimentric" "by the use of intercalated" "linkers of different lengths" "is that is facile" "energy-dispersive X-ray spectroscopy (EDS)"

Is the language acceptable? Yes
Is it clear how to access all supporting data? Not Applicable 1. Why using gold star? As I understand in your work, the L-cysteine (Cys) plays a role on binding glucose oxidase. Then why did authors use gold star? Just because of "The method is simple and easy to replicate"? Did authors compare the enzyme response on normal gold nanoparticles (not star-shaped) also? Is the bare gold star bad for enzyme immobilization? If so, why? If the stars nanostructures play an important role, then I will have a question: How about the influence of the number of branches of stars on enzyme immobilization? 2. Please explain how to avoid nonspecific binding of the protein with the authors' methods. And which data show the sample reducing nonspecific binding of the protein?
3. I suggest the authors to show SEM images to check the aggregation of the samples, because TEM images just showed a small range. 4. Except for NMR, it is necessary to run XPS characterization also to check the modification and immobilization.

Is the language acceptable? Yes
Is it clear how to access all supporting data? Not Applicable

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? I do not feel qualified to assess the statistics

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The article reports on Facile immobilisation of glucose oxidase onto gold nanostars with enhanced binding affinity and optimal function. It needs revision before accepting for the publications. The purification of AuNSs-Cys-GOx bioconjugates is not clear. It should be taken care in the revised paper.
Hydrogen bonding plays a vital role in between NH & CO groups present in GOx to Cysmodified AuNSs nanoparticle. Hence, I author should include FTIR data and to confirm structure the following relevant below references can be consider. Title: Facile immobilisation of glucose oxidase onto gold nanostars with enhanced binding affinity and optimal function Manuscript ID: RSOS-190205 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor: Comments to the Author: (There are no comments.) RSC Subject Editor: Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The work by Masauso Moses Phiri et al has been subjected on the immobilisation of glucose oxidase onto gold nanostars to produce of AuNSs-Cys-GOx bioconjugate complex for increased attachment affinity, Good dispersity and Greater sensitivity of it. I think this method is not a facile method to immobilisation of glucose oxidase onto gold nanostars and this manuscript need to major and minor revision with following details: Majors: page 3 lines 3-4: -"The PVP-stabilised seedless AuNSs were synthesized by reducing the HAuCl4 with ascorbic acid and AgNO3 added to control the growth of the branches based on the method reported by Phiri et al." The detailes of synthesis method should be clarified.

Page 3: -NMR section is very detailed and should be summerised
Page 3 lines 53-54 -"Comparison assays were done using AuNSs without any GOx, AuNSs with unattached GOx in solution" what is difference between these two states?
-"The thiol moiety has been found to be a very effective site for interaction" how this with one S atom of Cys?
-The text of "Stability of AuNSs-Cys-GOx bioconjugates" section needs the reference.
-Where are the activity results? Scheme 1 is very ambiguous: -Where is roles of sulfo-NHS and EDC in scheme 1.
-What is the role of EDC in the AuNSs-Cys-GOx synthesis? -Why in the GOx structure there are 2 NH3+ groups? -Why the arrow to belong from AuNS to sulfo-NHS? -Where is the pvp in this scheme?
-Why the authors used the 2 linkers (NHS and EDC) in this work?
"The particle-size distribution was estimated by measuring the size of approximately 100 nanoparticles in different grid regions" -How? -Where is the figure including 100 NP and what was the software for this estimation? -All of components in the TEM images should be recognized and assigned?
In I cannot concluded this sentence: -"This showed the potential of the AuNSs-Cys-GOx bioconjugates fabricated in the proposed approach to be used to detect low concentrations of analytes with high sensitivity and stability" Minors: -What is Km? The in following mistakes in the text should be corrected: "Colorimentric" "by the use of intercalated" "linkers of different lengths" "is that is facile" "energy-dispersive X-ray spectroscopy (EDS)" Reviewer: 2 Comments to the Author(s) This work synthesized gold nanostars based on the method reported in the literature. A stable AuNSs-Cys-GOx bioconjugate complex was obtained by modification with Cys and NHSterminated glucose oxidase. The authors need to clearly establish how the work presented here is novel compared to the previous work. And some conclusive evidence is needed by addressing the below concerns: 1. Why using gold star? As I understand in your work, the L-cysteine (Cys) plays a role on binding glucose oxidase. Then why did authors use gold star? Just because of "The method is simple and easy to replicate"? Did authors compare the enzyme response on normal gold nanoparticles (not star-shaped) also? Is the bare gold star bad for enzyme immobilization? If so, why? If the stars nanostructures play an important role, then I will have a question: How about the influence of the number of branches of stars on enzyme immobilization? 2. Please explain how to avoid nonspecific binding of the protein with the authors' methods. And which data show the sample reducing nonspecific binding of the protein?
3. I suggest the authors to show SEM images to check the aggregation of the samples, because TEM images just showed a small range. 4. Except for NMR, it is necessary to run XPS characterization also to check the modification and immobilization.
Reviewer: 3 Comments to the Author(s) The article reports on Facile immobilisation of glucose oxidase onto gold nanostars with enhanced binding affinity and optimal function. It needs revision before accepting for the publications. The purification of AuNSs-Cys-GOx bioconjugates is not clear. It should be taken care in the revised paper.
Hydrogen bonding plays a vital role in between NH & CO groups present in GOx to Cysmodified AuNSs nanoparticle. Hence, I author should include FTIR data and to confirm structure the following relevant below references can be consider. Polymer Engineering & Science 54 (1) AuNSs were cleaned-up by centrifugation for 90 minutes at 3000g. The pellet was then recovered and re-suspended in 2 mL of ddH 2 0.

Page 3:
-NMR section is very detailed and should be summarized

Response:
The above-mentioned section has since been summarised and reads as follows:

Page 3 lines 53-54
-"Comparison assays were done using AuNSs without any GOx, AuNSs with unattached GOx in solution" what is difference between these two states?

Response:
This section has been rewritten in order to clarify the difference between the two states. The following is an extract from the section: Three comparison assays were done to assess if the AuNSs-Cys-GOx bioconjugation method offered any advantage in biosensing; 1) AuNSs only without the addition of any GOx to it as assay controls. 2) AuNSs with 5 µL GOx added to the reaction solutions. 3) Lastly, AuNSs-Cys-GOx bioconjugates in solution for both oxidation and detection of glucose.
4. -"The thiol moiety has been found to be a very effective site for interaction" how this with one S atom of Cys?

Response:
Reference is made to the two following publications, listed below, that explain this in greater detail. In brief: Zhao et al., performed a comprehensive analysis of cysteine-gold cluster complexes. In contrast to the conventional understanding of the covalent nature of the S-Au bond, using computational methods, they present that the S-Au bond exhibits both covalent and donoracceptor characters. One stable isomer of Au 3 -Cys S was specially designed to demonstrate these two bonding components explicitly. They concluded that generally, the bonding strength between gold clusters and cysteine is positively correlated with the S-Au overlap-weighted bond order, but negatively correlated with the S-Au bond length.

Response:
The section has been rewritten and citation included in the manuscript.
6. -Where are the activity results?

Response:
The heading on page 3 line 49 has been modified to "Glucose sensing using AuNSs-Cys-GOx bioconjugates" so that it is not misleading to the reader what exactly was done.

Scheme 1 is very ambiguous:
i.
Where is roles of sulfo-NHS and EDC in scheme 1. ii.
What is the role of EDC in the AuNSs-Cys-GOx synthesis?
iii. Why in the GOx structure there are 2 NH3+ groups? iv.
Why the arrow to belong from AuNS to sulfo-NHS? v.
Where is the pvp in this scheme? -All of components in the TEM images should be recognized and assigned?

Response:
In the revised manuscript, the authors have removed this part from the methodology so as to narrow down to the main focus of the studythe immobilisation of enzymes onto the nanoparticles. 10. In Figure 1: -the Roman numerals on the figure should be corrected.

Response:
This has since been corrected.
-Zero point of the all spectra should be specified.

Response:
Only spectral regions of differentiation between L-Cysteine, Cysteine-modified AuNSs, NHS-terminated glucose oxidase, and AuNSs-Cys-GOx bioconjugates is shown. This is in accordance with the accepted norm of reported NMR data.

I do not understand:
-Spectrum (III) shows the peaks for the NHS-terminated GOx with sulfo-NHS in tandem with EDC.

Response:
The sentence has been modified to read as: "Spectrum (III) shows the peaks for the NHS-terminated GOx." 12. In figure2: -No difference between UV of AuNS-pvp and AuNS-Cys.

Response:
There was a slight red-shift from 712 nm to 716 nm after the addition of 10 -6 M L-cysteine. Using both the NMR and UV-vis spectroscopy characterisation, a good confirmation can be stated this shift is likely from the absorption of Lcysteine molecules on gold nanostars surface.
13. Page 5 line 60: -I do not understand "No broadening of the GOx-modified AuNSs was observed which implies that the AuNSs maintained their structural integrity with good dispersity"

Response:
The sentence has been corrected and now reads as: "No broadening of the LPSR spectra for GOx-modified AuNSs was observed which implies that the AuNSs maintained their structural integrity with good dispersity." 14. I do not understand: -"How the Au nanostars formation was confirmed?"

Response:
The UV-vis spectra of the synthesized AuNSs were observed to have typical -All the UV-Vis spectra 400-0 nm were scanned

Response:
This was corrected and now reads as follows: "All the UV-vis spectra scans were from 400 -990 nm." -The figure 4 should be assigned and appeared consequently which explained in the text

Response:
The exact request here is however, not fully understood by us. The position of the figure relative to the text has nonetheless been adjusted.
-Why the intensity for AuNSs-Cys-GOx is higher than the AuNSs intensity?

Response:
The UV-vis spectra in Figure 4 are not normalised. With the functionalisation of the AuNSs with GOx there is an increase in the absorbance.
-What is the goal of ionic strength investigation in this work?

Response:
Literature requires that the functionalized nanoparticles are tested for stability -What are the Gox, enzyme and protein roles in this work?

Response:
The GOxwhich is both a protein and enzyme in this case, provides surface functionalisation that offers stability to the gold nanoparticlesin our case, gold nanostars (Wangoo et al., 2008).
-Generally, the absorbance of Au Nanoparticles have a max intensity before 600 nm wavelength but in this work this character has been appeared about 700 nm? Why?

Response:
AuNSs colloids display a wide and distinct LSPR feature, including an intense

Response:
Again, we did not fully understand the comment here. However, the figures have since been presented as separate figures.
I cannot concluded this sentence: -"This showed the potential of the AuNSs-Cys-GOx bioconjugates fabricated in the proposed approach to be used to detect low concentrations of analytes with high sensitivity and stability"

Response:
The section has been rewritten in order to increase the clarity.

Minors:
 What is Km? The Michaelis constant (K m ) The in following mistakes in the text should be corrected: "Colorimentric" Corrected "by the use of intercalated" Corrected "linkers of different lengths" Corrected "is that is facile" Corrected "energy-dispersive X-ray spectroscopy (EDS)" Corrected All the minor corrections have been dealt with accordingly in the manuscript.

Reviewer: 2
Comments to the Author(s) 1. Why using gold star? As I understand in your work, the L-cysteine (Cys) plays a role on binding glucose oxidase. Then why did authors use gold star?
Just because of "The method is simple and easy to replicate"? Did authors compare the enzyme response on normal gold nanoparticles (not star-shaped) also?
Is the bare gold star bad for enzyme immobilization? If so, why?
If the stars nanostructures play an important role, then I will have a question: How about the influence of the number of branches of stars on enzyme immobilization?

Response:
Gold nanostars were used mostly for two reasons; (i) signal transduction, and enhance the attachment affinity of the enzyme to the nanostars. In short, the aim was an attempt at finding optimal enzyme immobilisations strategies to our synthesised gold nanostars.
The attachment approach that we used in this study was a covalent attachment to the surface of L-cysteine-modified nanostars. In this case, we postulate that the branches have no effect on the immobilisation process.
2. Please explain how to avoid nonspecific binding of the protein with the authors' methods. And which data show the sample reducing nonspecific binding of the protein?

Response:
Experimentally, it has been reported that functionalisation of gold nanoparticles with thiolated ligands leads to the formation of self-assembled monolayers of thiols on the gold surfaces that has the potential to reduce nonspecific protein absorption drastically (Lahiri et al., 1999). Thus, this study was building on such literature studies that have leveraged this knowledge to functionalise proteins in such a way as to reduce the potential for nonspecific absorption (Li et al., 2007;Pandey et al., 2007;Bezbradica et al., 2014). For example, cysteine was used together with glutaraldehyde to functionalise enzymes on an epoxyactivated support. Mercaptoundecanoic acid (MUA) has is reported to have been used to optimise enzyme immobilisation with thermal stability. It is therefore expected that there was no unspecific protein absorption based on the addition of cysteine that typically has the tendency to prevent it.
3. I suggest the authors to show SEM images to check the aggregation of the samples, because TEM images just showed a small range.

Response:
SEM images were not taken at the time of imaging but TEM images with lower magnifications covering a number of AuNSs were taken during microscopic characterisation of the glucose oxidasemodified AuNSs. Such an image has since been added as supplementary information.
4. Except for NMR, it is necessary to run XPS characterization also to check the modification and immobilization.

Response:
Noted with thanks. Although this technique is currently unavailable to us, it could in future be done in collaboration with others.

Reviewer: 3
Comments to the Author(s) The article reports on Facile immobilisation of glucose oxidase onto gold nanostars with enhanced binding affinity and optimal function. It needs revision before accepting for the publications.
The purification of AuNSs-Cys-GOx bioconjugates is not clear. It should be taken care in the revised paper.

Response:
The section has been adjusted accordingly to include those details.

Hydrogen bonding plays a vital role in between NH & CO groups present in
GOx to Cys-modified AuNSs nanoparticle. Hence, I author should include FTIR data and to confirm structure the following relevant below references can be consider. Polymer

Response:
FTIR technique was our method-of-choice but was unfortunately not available to us at the time of characterisation. Hence, we resorted to using various techniques such as the NMR, UV-vis spectroscopy, microscopy, and dynamic light scattering (although the data was not included in the manuscript).
However, we shall try to include FTIR in our on-going work of functionalisation of enzymes to nanoparticles.
The suggested references are well appreciated and appropriately referred to in the manuscript.
English and grammatical errors should be rectified during the revision of the paper.