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Decision letter and referee reports: first round

22nd Apr 20

Dear Prof Cho,

Thank you for submitting your manuscript, "Conformational flexibility of fatty acid-free BSA

proteins enables ultrathin film coatings with superior antifouling properties", to Communications

Materials. It has now been seen by 2 referees. You will see from their comments below that while

they find your work of interest, some important points are raised by Reviewer 1. We are interested

in the possibility of publishing your study in Communications Materials, but would like to consider

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Reviewers' comments:

Reviewer #1 (Remarks to the Author):

The manuscript describes the adsorption properties of various Bovin Serum Albumin (BSA) onto

silicium surfaces in order to determine the important parameters that affect the adsorption. Then

the antifouling property of the decorated surface in various nanotechnology applications is

determined. The BSA’s have been selected from a single manufacturer, and have different

purifications steps that affect their purity, their physical properties and conformational stability.

The adsorption properties are determined by three different techniques that allow good confidence

concerning the results. The conformational stability is determined by two technics that provide

similar results. All the experiments have been performed a significant number of times to provide

statistical analysis that strengthens the conclusions. The experimental details allows for a

researcher skilled in the art to reproduced and take benefit of the experiments, and their result. To

the best of my knowledge, such investigation has never been undertaken, and the finding are new

and of interest for the sensor community. The original result of the paper is that lipids inserted in

BSA stabilize the conformation of BSA and hamper their adsorption properties on Si surfaces, that

may reduce their antifouling properties. A careful removing of the lipids entrapped in the BSA

allows for an easiest destabilization of the proteins, that are more easily adsorbed on surfaces. The

nature of the lipid, being natural, does not seem to be of importance.

The paper offers a new methodology for the community involved in surface modification by BSA

adsorption. It includes sensors and all biotechnology analysis methods

Few questions then arise from the reading of the paper that may enlarge the potential interest of

readers involved in the field:

- The study focussed on one surface only. Would it be relevant to extend the adsorption properties

of unfatted BSA onto other surfaces that may allow for a more general coverage of the study?

- The selected BSA are all issued from one manufacturer, that is quite surprising. Furthermore, no

recombinant protein is used in the study. Recombinant protein are known to be less affected by

lipid entrapment after production. A comparison with this protein family, commercially available,

will also enlarge the coverage of the study.

- Adsorption is roughness dependant, and a comment on this point should be made somewhere in

the manuscript.

- Adsorption experiments are conducted at pH = 7,5 in buffer solution. Zapotocsny et col. vol.489 ,

20 January 2016, Pages 163-172 conducted similar experiment, with one BSA only, and found out

that the highest amount of adsorbed BSA on silica surface was obtained at pH= 4,5. The

Isoelectric point of BSA, and the surface charge of the surface do affect the deposition on the

surface. Would it be more relevant to run the adsorption experiments at lower pH, and would one

observe similar effect: indeed, the forces favouring adsorption would probably be higher, and the

reported effect of the present manuscript less important. Adsorption of BSA 1,2 or 3 should be run

at pH=4,5, and the adsorption efficiency compared to the one of BSA 4,5 and 6.

- The authors indicated that fatty acids were involved in the protein stabilization, however, no

quantitative determination of the fatty acid amount was provided. Instead, it was assumed that all

the impurities were fatty acids. The authors should reference a work supporting this assumption,

or making themselves the analysis. The IR analysis should reveal signal attributed to carboxylate,

however the provided spectra do not show the relevant region to determine their presence.

Furthermore, the IR spectra windows should be enlarged to see all the part of the spectra, and

inserts added. The titration of lipids can also be performed by colorimetric titration (Murphy and

Ripley)

- Related to the previous question, the authors conducted experiments using caprylic acid, that on

one sense, support the fact that impurities in BSA are mainly lipids, however, no quantification of

the inserted capr was undertaken: this quantification should then be related to the previous point.

- PI of BSA have been determined previously. what is the PI variation upon addition of carboxylic

lipids on a protein, knowing that at this pH, lipids are under the carboxylate form. A zeta potential

measurement is required to ensure that variation of the adsorption properties are attributed to

conformational stability, not to surface charge variation of the BSA.

- The authors conducted a complete study of the adsorbed proteins by IR. The various BSA

domains have been identified, however, a more clear quantification and discussion of the domains

involved in the adsorption process could be provided. This point is of particular importance when

comparing both set of BSA.

- The authors conducted some DLS measurements, and concluded to oligomerization of the BSA.

Looking at the reference they provide, the authors prefer the term “aggregation”. I prefer to talk

about aggregation, except if the authors can provide some specific sites of interaction, or reaction.

The fig SI 1 should also provide a larger abscise scale to make sure that no aggregation occurs at

larger size.

- The authors claim that DLS measurements provided in Figure 2 SI provide an indication of

conformational stability of fatted-BSA. This measurement is providing only the information that

aggregation occurs at 60°C for the unfatted BSA. How could the authors discriminate the

conformational stability from a polyelectrolytic issue provided by a charge effect that is different

for unfatted and fatted-BSA.

- Fig.5 SI is providing the thickness of the layers as a function of the used BSA. It is measured

that the thickness of the BSA layer is higher for the unfatted-BSA. It is mentioned that monolayers

of BSA are adsorbed on the surface, and that unfatted BSA can spread more easily on the surface

thanks to its conformational lower stability. If these two last claims hold, the unfatted-BSA should

have a lower thickness than fatted one, that could not be easily spread on the surface. The authors

should comment this observation.

- The authors concluded p.14 that close-packed protein layers could be formed. AFM

characterization, for example, should support this conclusion. It will also may be provide some

inside to answer the previous question, i.e. the fatted-BSA could then be deposited on their flat

surface while unfitted-one could be adsorbed as random coil? This way of adsorption should be

clearly addressed in the manuscript.

The work provided in the manuscript may interest the community using BSA as an antifouling

material, and the manufacturer who may provide a new reference in its catalogue… I recommend

major modifications before considering this manuscript for publication.

Reviewer #2 (Remarks to the Author):

This communication reports interesting studies on the effect of fatty acids on BSA stability, with

important results showing that extensive defatting leads to less conformational stability upon

adsorption, a strategy with impact on its use for antifouling processes. The work is well conducted

to reveal the effect of fatty acids on BSA stability by using varied and complimentary techniques.

My comments are minor and, overall, I think this work deserves to the published after clarification

of the below:

- It is not clear to me whether commercially available fat-free BSA was studied as it was provided,

or after purification. If not, it would be important to test this widely used sample and to compare

its behavior with the purified BSA.

- In this respect, I found two earlier reports on BSA adsorption onto inorganic nanoparticles that

display CD curves agreeing with those shown here for fatted BSA. (Rial et al, Langmuir 2018 and

Galdino et al, Colloids Surf B, 2020 – the latter using Aldrich´s fat free BSA). For the benefit of the

community using these commercial samples, this clarification would be most helpful.

- The other curiosity is whether these findings could be transferred to HAS, due to its similarity

with BSA and, if possible, whether this would have impact of processes related to protein corona

formation onto surfaces ?

School of Materials Science and Engineering

MRS-Singapore Chair Professor School of Materials Science and Engineering, Nanyang Technological University 50 Nanyang Drive, CBSS, Research Techno Plaza, 06-05, Singapore 637553 Tel: +65 6592 7945 Fax: +65 6791 2274 E-mail: njcho@ntu.edu.sg

Reviewers’ Comments to Author:

Reviewer 1

Remarks to the Author:

The manuscript describes the adsorption properties of various Bovin Serum Albumin

(BSA) onto silicium surfaces in order to determine the important parameters that

affect the adsorption. Then the antifouling property of the decorated surface in

various nanotechnology applications is determined. The BSA’s have been selected

from a single manufacturer, and have different purifications steps that affect their

purity, their physical properties and conformational stability.

The adsorption properties are determined by three different techniques that allow

good confidence concerning the results. The conformational stability is determined

by two technics that provide similar results. All the experiments have been performed

a significant number of times to provide statistical analysis that strengthens the

conclusions. The experimental details allows for a researcher skilled in the art to

reproduced and take benefit of the experiments, and their result. To the best of my

knowledge, such investigation has never been undertaken, and the finding are new

and of interest for the sensor community. The original result of the paper is that lipids

inserted in BSA stabilize the conformation of BSA and hamper their adsorption

properties on Si surfaces, that may reduce their antifouling properties. A careful

removing of the lipids entrapped in the BSA allows for an easiest destabilization of

the proteins, that are more easily adsorbed on surfaces. The nature of the lipid, being

natural, does not seem to be of importance.

The paper offers a new methodology for the community involved in surface

modification by BSA adsorption. It includes sensors and all biotechnology analysis

methods

Few questions then arise from the reading of the paper that may enlarge the potential

interest of readers involved in the field:

Response: We sincerely thank the Reviewer for the positive evaluation of our manuscript

and for expert feedback to help us improve the manuscript. Below, we have provided point-

by-point responses describing how we have addressed each point made by the Reviewer

and how we have improved the manuscript accordingly.

Comments:

1) The study focussed on one surface only. Would it be relevant to extend the

adsorption properties of unfatted BSA onto other surfaces that may allow for a more

general coverage of the study?

Response: We thank the Reviewer for this excellent question. We selected silica as a

representative hydrophilic surface because it is a popular type of glass material that is a

widely used substrate in various biological and biotechnology assays, and silica surface

2

coatings are compatible with surface-sensitive measurement techniques such as QCM-D

and LSPR for the detailed adsorption measurements conducted in this work. In addition,

high-quality silica nanoparticles are also readily available and well-dispersed, so it was

possible to evaluate the effects of BSA antifouling coatings across surface- and

nanoparticle-based applications.

We agree with the Reviewer that there are also other types of surfaces that might be tested

in order to expand the general coverage of the study. For example, BSA is often used as a

surface passivation-related blocking reagent in blot membrane immunostaining

applications. One commonly used substrate for such applications is nitrocellulose

membranes, which typically have hydrophobic character and cause protein binding via

hydrophobic interactions. Therefore, to extend the general coverage of our study, we have

also tested the functional blocking performance of defatted and fatted BSA on

nitrocellulose substrates in an application-relevant context.

In particular, our Western blot experiments involved the use of a hydrophobic

nitrocellulose membrane as a substrate and demonstrated that defatted BSA also exhibited

superior blocking properties in this application context. These findings were presented as

Supplementary Figures 10-12 and Supplementary Note 1 in the original manuscript

although the description in the main text was concise and did not clearly mention the use

of the nitrocellulose substrate. In the revised manuscript, we have extended our description

of the Western blot results and use of the nitrocellulose substrate starting at line 259 of the

revised manuscript as follows:

“In addition, we tested the surface passivation (“blocking”) performance of fatted and

defatted BSA proteins in Western blot experiments involving a hydrophobic nitrocellulose

membrane surface (Supplementary Figs. 10-12 and Supplementary Note 3). We first

exposed the membrane surface to human serum by electrophoretic transfer, “blocked” the

remaining unexposed surface with BSA proteins, then incubated the membranes with a

primary C3 monoclonal antibody and a relevant secondary antibody before the intensities

of specific and nonspecific bands – produced by enhanced chemiluminescence – were

quantified. We found that the membranes “blocked” by defatted BSA proteins produced

lower intensity nonspecific bands, supporting that the higher packing density of adsorbed

defatted BSA proteins more effectively prevented nonspecific interactions between the

primary and/or secondary antibodies with the membrane surface. Together, these data

support that defatted BSA proteins exhibit superior surface passivation performance on

hydrophilic and hydrophobic surfaces.”

2) The selected BSA are all issued from one manufacturer, that is quite surprising.

Furthermore, no recombinant protein is used in the study. Recombinant protein are

known to be less affected by lipid entrapment after production. A comparison with

this protein family, commercially available, will also enlarge the coverage of the study.

Response: We thank the Reviewer for this suggestion. We selected BSA proteins from

Sigma-Aldrich because we have a long track record of using their BSA reagents in our

research (including in past protein adsorption-related works) and they have excellent

batch-to-batch consistency in our experience. They are also 1) well-established and widely

used within the research community, and 2) subject to stringent quality control. In

3

particular, we wanted to focus this study on naturally sourced BSA proteins purified via

different processing routes, without or with fatty acids, so we felt that selecting protein

options from one top manufacturer provides a focused set without additional concern about

manufacturer quality. At the same time, we do agree that the materials characterization

framework used in our study could be expanded to test even more proteins from other

commercial sources, although we believe that such efforts are outside the scope of the

present study. Indeed, our data across three different purification methods and without/with

fatty acid stabilizers (resulting in six different BSA types as prepared by the manufacturer)

along with our additional independent fatty acid doping control experiments provide strong

support that fatty acid-free BSA proteins exhibit distinct adsorption, conformational, and

antifouling-related application performance compared to fatty acid-containing BSA

proteins.

For antifouling-related surface passivation applications, naturally sourced BSA is far more

widely used than recombinant BSA due to its low cost and abundance. Indeed, recombinant

BSA can cost ~$2500 USD per mg, which is much higher than naturally sourced BSA

(typically <$0.16 USD per mg). On the other hand, recombinant human serum albumin

(HSA) is more commonly used than natural HSA for human therapeutic applications (not

surface passivation applications). This is due to the high demand for HSA-based

therapeutics, the limited supply of natural HSA from human plasma, and the potential risk

(even if low) of viral or prion contamination from naturally sourced HSA1,2.

For these reasons, selecting all naturally sourced BSA proteins from Sigma-Aldrich was

appropriate for this work.

Links to Costs

1. https://www.mybiosource.com/recombinant-protein/serum-albumin-alb/966488

2. https://www.sigmaaldrich.com/catalog/product/sigma/a0281?lang=en&region=US

3) Adsorption is roughness dependant, and a comment on this point should be made

somewhere in the manuscript.

Response: We thank the Reviewer for this suggestion. We agree that the scientific

literature supports that protein adsorption uptake, including that of BSA, generally

increases with surface roughness. To clarify this point and provide more context about

fundamental parameters that affect protein adsorption, we have added the following

sentence at line 68 of the revised manuscript:

“Coating performance is sensitive to various material parameters such as atomic

composition22, surface roughness23, and nano-curvature effects24, along with

environmental parameters such as solution pH25 and ionic strength20.”

Accordingly, we have also included the following references in the revised manuscript:

22 Givens, B. E., Xu, Z., Fiegel, J. & Grassian, V. H. Bovine serum albumin

adsorption on SiO2 and TiO2 nanoparticle surfaces at circumneutral and acidic pH:

4

A tale of two nano-bio surface interactions. J. Colloid Interface Sci. 493, 334-341

(2017).

23 Dolatshahi-Pirouz, A. et al. Bovine serum albumin adsorption on nano-rough

platinum surfaces studied by QCM-D. Colloids Surf., B 66, 53-59 (2008).

24 Satzer, P., Svec, F., Sekot, G. & Jungbauer, A. Protein adsorption onto

nanoparticles induces conformational changes: particle size dependency, kinetics,

and mechanisms. Eng. Life Sci. 16, 238-246 (2016).

25 Jachimska, B., Tokarczyk, K., Łapczyńska, M., Puciul-Malinowska, A. &

Zapotoczny, S. Structure of bovine serum albumin adsorbed on silica investigated

by quartz crystal microbalance. Colloids Surf., A 489, 163-172 (2016).

20 Park, J. H. et al. Controlling adsorption and passivation properties of bovine serum

albumin on silica surfaces by ionic strength modulation and cross-linking. Phys.

Chem. Chem. Phys. 19, 8854-8865 (2017).

4) Adsorption experiments are conducted at pH = 7,5 in buffer solution. Zapotocsny

et col. vol.489 , 20 January 2016, Pages 163-172 conducted similar experiment, with

one BSA only, and found out that the highest amount of adsorbed BSA on silica

surface was obtained at pH= 4,5. The Isoelectric point of BSA, and the surface charge

of the surface do affect the deposition on the surface. Would it be more relevant to

run the adsorption experiments at lower pH, and would one observe similar effect:

indeed, the forces favouring adsorption would probably be higher, and the reported

effect of the present manuscript less important. Adsorption of BSA 1,2 or 3 should be

run at pH=4,5, and the adsorption efficiency compared to the one of BSA 4,5 and 6.

Response: We thank the Reviewer for this excellent question. While it is known from

fundamental studies that BSA adsorption near its isoelectric point (pI) of BSA (pH 4-5) is

typically greater due to reduced protein-protein and protein-surface charge repulsion, most

biological and biotechnology assays involving BSA surface passivation protocols,

including antifouling coatings, are performed under near-physiological pH conditions (pH

7-8). Thus, we decided to focus on investigating BSA adsorption in application relevant

conditions around pH 7.5. To clarify this point, alongside our choice of BSA concentration

and ionic strength conditions, we have added the following sentence at line 156 of the

revised manuscript:

“The experiments were conducted using a BSA protein concentration of 100 µM (~6.6 mg

mL-1) in aqueous buffer (10 mM Tris [pH 7.5] with 150 mM NaCl) which are representative

of the typical solution conditions used in BSA coating protocols8,19,34.”

We have also included the following references:

8 Mahmood, T. & Yang, P.-C. Western blot: technique, theory, and trouble shooting.

N. Am. J. Med. Sci. 4, 429 (2012).

19 Jeyachandran, Y., Mielczarski, J., Mielczarski, E. & Rai, B. Efficiency of blocking

of non-specific interaction of different proteins by BSA adsorbed on hydrophobic

and hydrophilic surfaces. J. Colloid Interface Sci. 341, 136-142 (2010).

34 Jeyachandran, Y., Mielczarski, E., Rai, B. & Mielczarski, J. Quantitative and

qualitative evaluation of adsorption/desorption of bovine serum albumin on

hydrophilic and hydrophobic surfaces. Langmuir 25, 11614-11620 (2009).

5

5) The authors indicated that fatty acids were involved in the protein stabilization,

however, no quantitative determination of the fatty acid amount was provided.

Instead, it was assumed that all the impurities were fatty acids. The authors should

reference a work supporting this assumption, or making themselves the analysis. The

IR analysis should reveal signal attributed to carboxylate, however the provided

spectra do not show the relevant region to determine their presence. Furthermore,

the IR spectra windows should be enlarged to see all the part of the spectra, and

inserts added. The titration of lipids can also be performed by colorimetric titration

(Murphy and Ripley)

Response: We thank the Reviewer for this excellent suggestion. There are several

references in the scientific publication and patent literature that indicate fatty acids are

added as the main stabilizer in the purification process. We have included these references

in line 80 of the revised manuscript:

16 Mannuzza, F. J. & Montalto, J. G. Is bovine albumin too complex to be just a

commodity? BioProcess Int. (2010).

27 Reid, A. F. Method of purifying albumin. US Patent 2,705,230 (1955).

28 Porsche, J. D., Lesh, J. B. & Grossnickle, M. D. Recovery of serum albumin. US

Patent 2,765,299 (1956).

29 Schneider, W., Lefevre, H., Fiedler, H. & McCarty, L. J. An alternative method of

large scale plasma fractionation for the isolation of serum albumin. Blut 30, 121-

134 (1975).

In addition, we have completed more detailed evaluation of the FTIR data in line with the

Reviewer’s suggestions. In particular, we have added Supplementary Fig. 1, which shows

a much larger wavenumber range of the FTIR spectra for BSA proteins 1-6. The insets

show the absorbance peaks from the asymmetric stretch of CH2 (νas(CH2)) and CH3

(νas(CH3)) functional groups, which originate from the fatty acid tail’s methylene chain and

terminal methyl group, respectively. A higher νas(CH2)/νas(CH3) ratio indicates more

bound fatty acids present in the BSA sample. Our data clearly show that fatted BSA

proteins have greater νas(CH2)/νas(CH3) values than their defatted counterparts. Another

indication is the presence of the peak arising from asymmetric stretching of the fatty acids’

COO- functional group, which coincides with the amide II peak. It can be observed that the

height of the amide II peak relative to the amide I peak is higher for fatted BSA proteins

than for defatted BSA proteins, indicating a greater amount of bound fatty acids in fatted

BSA proteins. We have added related discussion remarks in Supplementary Note 1.

Accordingly, we have extended our discussion of this aspect in the revised manuscript by

adding the following sentence to the main text at line 102 of the revised manuscript:

“Infrared spectroscopic characterization – through the analysis of relevant spectral

features – confirmed the removal of fatty acids from defatted BSA proteins, which is in line

with gas chromatography results (Supplementary Fig. 1 and Supplementary Note 1).”

We have also included the following references in the revised Supplementary Information:

6

1 Yu, P. & Damiran, D. Heat-induced changes to lipid molecular structure in Vimy

flaxseed: Spectral intensity and molecular clustering. Spectrochim. Acta, Part A 79,

51-59 (2011).

2 Oleszko, A., Hartwich, J., Gąsior-Głogowska, M. & Olsztyńska-Janus, S. Changes

of albumin secondary structure after palmitic acid binding: FT-IR spectroscopic

study. Acta Bioeng. Biomech. 20 (2018).

3 Grdadolnik, J. & Maréchal, Y. Bovine serum albumin observed by infrared

spectrometry. I. methodology, structural investigation, and water uptake.

Biopolymers 62, 40-53 (2001).

6) Related to the previous question, the authors conducted experiments using caprylic

acid, that on one sense, support the fact that impurities in BSA are mainly lipids,

however, no quantification of the inserted capr was undertaken: this quantification

should then be related to the previous point.

Response: We thank the Reviewer for this helpful comment. We added caprylic acid to

defatted BSA 5 (CA-BSA 5) in a 10:1 CA:BSA molar ratio for all experiments. In the

revised manuscript, we have added Supplementary Fig. 14, which quantifies the

νas(CH2)/νas(CH3) ratio of CA-BSA 5 and BSA 5 proteins based on their ATR-FTIR spectra.

This analysis confirmed the presence of additional bound fatty acids (i.e., caprylic acid)

for CA-BSA 5 protein as compared to BSA 5 protein.

In addition, the amount of caprylic acid bound to BSA can be quantified by taking into

account past isothermal titration calorimetry measurements that measured the binding of

caprylic acid to BSA, as reported in Ref. 3. Specifically, the binding constant and

stoichiometry were determined to be 3.65×104 M-1 and 2, respectively. Based on the one-

site binding model4 and by taking into account the BSA and caprylic acid concentrations

of 100 µM and 1000 µM, respectively, the fraction of sites in BSA occupied by caprylic

acid was calculated to be ~0.97 in our system.

In line with the Reviewer’s suggestions, we have added more detailed remarks about

quantifying the amount of bound caprylic acid in Supplementary Note 4 of the revised

Supplementary Information:

“In order to quantify the amount of caprylic acid bound to BSA, we note that the binding

constant and stoichiometry for caprylic acid binding to BSA is 3.65× 104 M-1 and 2

respectively as previously determined by isothermal titration calorimetry11. By applying

these parameters to the one-site binding model12, and taking BSA and caprylic acid

concentrations of 100 µM and 1000 µM respectively, the fraction of sites in BSA occupied

by caprylic acid was calculated to be ~0.97.

We have also included the following references in the revised Supplementary Information:

11 Zhu, T.-T. et al. Difference in binding of long-and medium-chain fatty acids with

serum albumin: The role of macromolecular crowding effect. J. Agric. Food Chem.

66, 1242-1250 (2018).

7

12 Freyer, M. W. & Lewis, E. A. Isothermal titration calorimetry: experimental design,

data analysis, and probing macromolecule/ligand binding and kinetic interactions.

Methods Cell Biol. 84, 79-113 (2008).

7) PI of BSA have been determined previously. what is the PI variation upon addition

of carboxylic lipids on a protein, knowing that at this pH, lipids are under the

carboxylate form. A zeta potential measurement is required to ensure that variation

of the adsorption properties are attributed to conformational stability, not to surface

charge variation of the BSA.

Response: We thank the Reviewer for this excellent question. As the Reviewer points out,

it has been reported that the pI of fatty acid-free serum albumins are usually higher (pI >

5) than fatted ones (pI < 5), as reported in Ref. 5 and Ref. 6 and also noted in the

manufacturer’s product information sheet (Ref. 7). In the discussion of our manuscript, we

discuss how both conformational stability and surface charge variation play important roles

in mediating the different adsorption properties of fatty acid-free and fatty acid-containing

BSA proteins. In particular, our ATR-FTIR data support that defatted BSA denatures more

than fatted BSA in the adsorbed state. At the same time, it is important to point out that the

QCM-D and LSPR data indicate greater total adsorption of defatted BSA as opposed to

fatted BSA. These findings support that defatted BSA undergoes greater denaturation in

the adsorbed state and forms more tightly packed adlayers while fatted BSA undergoes

less denaturation in the adsorbed state and forms less tightly packed adlayers.

To clarify that both conformational stability and surface charge variation play important

roles in affecting BSA adsorption behavior, we have added the following sentence starting

from line 326 of the revised manuscript:

“These findings support that bound fatty acids enhance not only BSA conformational

stability but also charge repulsion for protein molecules in the adsorbed state.”

We have also added “and charge repulsion” to lines 336 and 338 of the revised manuscript.

We have also edited the following sentence starting from line 346 of the revised manuscript

as follows:

“Mechanistically, we show that defatted BSA proteins are superior because they have

lower solution-phase conformational stability and reduced charge repulsion in the

adsorbed state, which translates into greater surface-induced denaturation and adsorption

uptake, resulting in tighter adlayer packing that yields superior passivation coatings.”

8) The authors conducted a complete study of the adsorbed proteins by IR. The

various BSA domains have been identified, however, a more clear quantification and

discussion of the domains involved in the adsorption process could be provided. This

point is of particular importance when comparing both set of BSA.

Response: We thank the Reviewer for the excellent suggestion, and we understand that

the various domains involved in the adsorption process that the Reviewer mentioned refers

to the various secondary structure changes of BSA protein due to the adsorption process.

8

In this respect, ATR-FTIR spectroscopy can be used to quantify the various secondary

structure elements present in a protein molecule. We have completed a more detailed

evaluation and discussion of the relevant secondary structure elements based on the ATR-

FTIR spectroscopy data. We have included the percentage values of other secondary

structure elements in Supplementary Table 3 for the 6 BSA proteins and in

Supplementary Table 5 for BSA 5 vs. CA-BSA 5. We have also added Supplementary

Note 2 to discuss adsorption-related changes related to other secondary structure elements.

Briefly, a loss in α-helical structure due to surface-induced denaturation is balanced by an

increase in random coil fraction, and the addition of fatty acid stabilizers reduces the extent

of this transition from α-helix to random coil. On the other hand, the fractions of other

secondary structure elements were not appreciably different between fatted and defatted

BSA proteins, indicating that fatty acid stabilization mainly conferred protection to α-

helical regions. We have added two sentences beginning at line 235 of the revised

manuscript on this discussion with a reference to Supplementary Note 2:

“The adsorption-related decrease in α-helicity was related to protein unfolding, as

indicated by a corresponding increase in the random coil fraction21,41,42. Specifically,

fatted and defatted BSA proteins experienced an increase in random coil fraction by ~9%

and ~12%, respectively, supporting that fatty acids partially stabilize BSA proteins against

surface-induced denaturation by reducing the extent of the helix-to-random coil secondary

structure transition (Supplementary Note 2).”

The following references within this text were included in the revised manuscript:

22 Givens, B. E., Xu, Z., Fiegel, J. & Grassian, V. H. Bovine serum albumin

adsorption on SiO2 and TiO2 nanoparticle surfaces at circumneutral and acidic pH:

A tale of two nano-bio surface interactions. J. Colloid Interface Sci. 493, 334-341

(2017).

41 Zeng, H., Chittur, K. K. & Lacefield, W. R. Analysis of bovine serum albumin

adsorption on calcium phosphate and titanium surfaces. Biomaterials 20, 377-384

(1999).

42 Roach, P., Farrar, D. & Perry, C. C. Surface tailoring for controlled protein

adsorption: effect of topography at the nanometer scale and chemistry. J. Am. Chem.

Soc. 128, 3939-3945 (2006).

These references were also included in the revised Supplementary Information:

4 Zeng, H., Chittur, K. K. & Lacefield, W. R. Analysis of bovine serum albumin

adsorption on calcium phosphate and titanium surfaces. Biomaterials 20, 377-384

(1999).

5 Roach, P., Farrar, D. & Perry, C. C. Surface tailoring for controlled protein

adsorption: effect of topography at the nanometer scale and chemistry. J. Am. Chem.

Soc. 128, 3939-3945 (2006).

6 Givens, B. E., Xu, Z., Fiegel, J. & Grassian, V. H. Bovine serum albumin

adsorption on SiO2 and TiO2 nanoparticle surfaces at circumneutral and acidic pH:

A tale of two nano-bio surface interactions. J. Colloid Interface Sci. 493, 334-341

(2017).

9

9) The authors conducted some DLS measurements, and concluded to

oligomerization of the BSA. Looking at the reference they provide, the authors prefer

the term “aggregation”. I prefer to talk about aggregation, except if the authors can

provide some specific sites of interaction, or reaction. The fig SI 1 should also provide

a larger abscise scale to make sure that no aggregation occurs at larger size.

Response: We thank the Reviewer for this excellent suggestion, and we agree with the

Reviewer. We have changed the term “oligomerize” to “aggregate” in line 114 and the

term “oligomerization” to “aggregation” in lines 121, 123 and 144 of the revised

manuscript.

We have also double-checked to confirm that there is no aggregation at larger sizes for all

BSA samples according to DLS measurements. We have appended below the DLS

measurement figure with a larger scale, for review purposes only.

Review-Only Figure. DLS characterization of BSA 5 protein size distribution with a

larger size scale.

DLS measurement of BSA 5 at 25 °C (n=5 technical replicates). The mean hydrodynamic

diameter (D) and polydispersity index (PDI) are indicated.

10) The authors claim that DLS measurements provided in Figure 2 SI provide an

indication of conformational stability of fatted-BSA. This measurement is providing

only the information that aggregation occurs at 60°C for the unfatted BSA. How

could the authors discriminate the conformational stability from a polyelectrolytic

issue provided by a charge effect that is different for unfatted and fatted-BSA.

Response: We thank the Reviewer for this excellent comment and agree with the Reviewer

that this data set alone cannot distinguish between these two possibilities. In the revised

manuscript, we have modified the sentence beginning at line 124 as follows:

10

“Kinetic experiments further verified that defatted BSA proteins underwent more rapid

and extensive aggregation, which points to fatty acid molecules conferring BSA proteins

with greater conformational stability and/or colloidal stability (Supplementary Fig. 3).”

We have also added “and/or colloidal” in line 115 of the revised manuscript.

11) Fig.5 SI is providing the thickness of the layers as a function of the used BSA. It

is measured that the thickness of the BSA layer is higher for the unfatted-BSA. It is

mentioned that monolayers of BSA are adsorbed on the surface, and that unfatted

BSA can spread more easily on the surface thanks to its conformational lower

stability. If these two last claims hold, the unfatted-BSA should have a lower thickness

than fatted one, that could not be easily spread on the surface. The authors should

comment this observation.

Response: We agree with the Reviewer and we have carefully reviewed the Voigt-based

QCM-D modeling. Upon further evaluation of the Voigt-based modeling, we have decided

to forgo this modeling approach for analyzing the protein adlayers in this case because it

requires assumptions about thin film properties that are not applicable in this case, such as

the formation of a homogenous protein film with uniform density. Since there is a

significant difference in the density of protein molecules vs. buffer solvent, variations in

packing density would have a significant effect on the assumed uniform density. On the

other hand, our direct experimental evidence supports that fatted and defatted BSA proteins

adsorb with different packing densities as the result of varying degrees of surface-induced

deformation and electrostatic charge repulsion, as supported by multiple lines of evidence

from adsorption characterization experiments (i.e., via QCM-D, LSPR, ATR-FTIR).

As such, we have decided to focus our supporting analysis of the QCM-D data on the time-

independent frequency-energy dissipation (F-D) curves (as shown below), which is an

empirical method to evaluate the relative degree of adsorbate deformation (extent of

protein denaturation and thus indirectly related to film thickness; see also adsorbed vesicle

examples in Refs. 8,9) and does not require assumptions about film density. These curves

were included in the original version of the manuscript as well and show that the adsorption

of defatted BSA proteins generally led to lower energy dissipation shifts per change in

frequency compared to fatted BSA proteins. This is clear evidence that defatted BSA

underwent greater deformation/denaturation and formed more rigid adlayers.

11

Supplementary Figure 5. QCM-D frequency-energy dissipation curves for BSA

adsorption onto silica surfaces.

Time-independent frequency-energy dissipation (F-D) curves derived from QCM-D

frequency and energy dissipation shifts related to the adsorption of BSA proteins 1-6 onto

silica surfaces at 25°C.

Accordingly, we have also edited the sentence starting at line 187 of the revised manuscript:

“Further analysis of |ΔFmax/ΔDmax| ratios and time-independent frequency-energy

dissipation (F-D) curves revealed that adsorbed, defatted BSA proteins underwent more

surface-induced denaturation, as indicated by lower energy dissipation shifts relative to

the frequency shift, which is consistent with lower solution-phase conformational stability

(Fig. 3e and Supplementary Fig. 6).”

12) The authors concluded p.14 that close-packed protein layers could be formed.

AFM characterization, for example, should support this conclusion. It will also may

be provide some inside to answer the previous question, i.e. the fatted-BSA could then

be deposited on their flat surface while unfitted-one could be adsorbed as random

coil? This way of adsorption should be clearly addressed in the manuscript.

Response: We thank the Reviewer for this great question. As discussed in our previous

replies, the ATR-FTIR data supports that defatted BSA denatures more than fatted BSA in

the adsorbed state. It is also important to point out that the QCM-D and LSPR data both

indicate greater total adsorption of defatted BSA as opposed to fatted BSA. These findings

support that defatted BSA undergoes greater denaturation in the adsorbed state and forms

more tightly packed adlayers while fatted BSA undergoes less denaturation in the adsorbed

state and forms less tightly packed adlayers. We have also conducted more detailed ATR-

FTIR data analysis in order to evaluate how defatted and fatted BSA proteins adsorb in

12

terms of changes in different secondary structure elements. The data support that both

fatted and defatted BSA underwent a loss in α-helical structure upon adsorption due to

surface-induced denaturation and this structural change is balanced by an increase in

random coil fraction. The addition of fatty acid stabilizers in fatted BSA samples reduces

the extent of this transition from α-helix to random coil. On the other hand, the changes in

fractions of other secondary structure elements were not appreciably different between

fatted and defatted BSA proteins, indicating that fatty acid stabilization mainly conferred

protection to α-helical regions. We believe that the ATR-FTIR method is best suited for

addressing such questions as our data supports. On the other hand, the AFM technique is

more commonly used for measuring film thickness and surface roughness. While it has

been employed to visualize the surface morphology of protein films, it does not readily

offer quantitative insights into the packing of adsorbed proteins, especially when

comparing between a series of protein monolayers at adsorption saturation. With these

considerations in mind, we anticipate that it would be particularly challenging to use the

AFM technique to determine the different conformations of adsorbed BSA in this work

and believe that the ATR-FTIR spectroscopy data together with the QCM-D and LSPR

adsorption data provide strong evidence to compare defatted and fatted BSA adsorbates.

We have also carefully considered the usage of terminology to describe the adsorbate

properties. With respect to the relatively high packing density of adsorbed BSA proteins,

we have reviewed the use of the term “close-packed” to describe adsorbed protein layers.

Upon further evaluation, we have decided to change the term “close-packed” to “tightly

packed” in line 331 of the revised manuscript because the term “close-packed” is more

commonly used to describe specific dense arrangements such as face-centered cubic

lattices, which we do not wish to state. Rather, our intention is to state that the combination

of different measurement techniques support that defatted BSA proteins form denser

adlayers than those of fatted BSA proteins, which is in line with the mass adsorption and

spectroscopic data and also consistent with the likely role of electrostatic forces in

modulating protein-protein interactions between two fatted BSA proteins vs. two defatted

BSA proteins. Accordingly, we have also changed the terms “well-packed” to “tightly

packed” in lines 338 and 340 in the revised manuscript for consistency throughout the

manuscript.

The work provided in the manuscript may interest the community using BSA as an

antifouling material, and the manufacturer who may provide a new reference in its

catalogue… I recommend major modifications before considering this manuscript

for publication.

Response: We sincerely thank the Reviewer for helpful feedback and suggestions on our

manuscript and we have done our best to thoroughly incorporate each suggestion into the

revised manuscript.

Reviewer 2

Remarks to the Author:

This communication reports interesting studies on the effect of fatty acids on BSA

stability, with important results showing that extensive defatting leads to less

13

conformational stability upon adsorption, a strategy with impact on its use for

antifouling processes. The work is well conducted to reveal the effect of fatty acids on

BSA stability by using varied and complimentary techniques. My comments are

minor and, overall, I think this work deserves to the published after clarification of

the below:

Response: We sincerely thank the Reviewer for the positive evaluation of our manuscript

and for expert feedback to help us improve the manuscript. Below, we have provided point-

by-point responses describing how we have addressed each point made by the Reviewer

and how we have improved the manuscript accordingly.

1) It is not clear to me whether commercially available fat-free BSA was studied as it

was provided, or after purification. If not, it would be important to test this widely

used sample and to compare its behavior with the purified BSA.

Response: We thank the Reviewer for this excellent comment. We believe the Reviewer

is asking whether the defatted BSA proteins in this study were used as provided or not; and

if we did use them as provided, whether we can confirm if they are fatty acid free.

In our study, all fatty acid-containing and fatty acid-free BSA proteins used were obtained

from a high-quality commercial source (i.e., Sigma-Aldrich) and used as provided. All

processing steps, including the fatty acid removal step for defatted BSA proteins had

already been performed by the manufacturer and quality control was evaluated by gas

chromatography experiments according to the manufacturer. To clarify this point in the

revised manuscript, we have added a sentence beginning at line 368 in the Methods section:

“All six BSA proteins were used as provided and the fatty acid-free versions were

confirmed to have ≤ 0.01% fatty acid residues by gas chromatography according to the

manufacturer (Supplementary Table 1).”

In addition, we performed additional quality control experiments by doping fatty acid-free

BSA 5 with caprylic acid to convert it into a fatty acid-containing BSA (CA-BSA 5) as

detailed in Supplementary Note 4 in the revised Supplementary Information. Solution-

phase and adsorption characterization revealed that CA-BSA 5 had increased

conformational stability and decreased adsorption uptake compared to fatty acid-free BSA

5, displaying a similar trend as that between the commercially available fatty acid-

containing and fatty acid-free BSA proteins. This evidence further verifies the absence of

fatty acids in the commercially available fatty acid-free BSA.

2) In this respect, I found two earlier reports on BSA adsorption onto inorganic

nanoparticles that display CD curves agreeing with those shown here for fatted BSA.

(Rial et al, Langmuir 2018 and Galdino et al, Colloids Surf B, 2020 – the latter using

Aldrich s fat free BSA). For the benefit of the community using these commercial

samples, this clarification would be most helpful.

Response: We thank the Reviewer for this insightful comment, and we have thoroughly

read both works that were mentioned. We have added their citations in the text at relevant

locations and would also like to point out a few details.

14

1. In the work presented by Rial et al., it was not specified whether a fatted or defatted

BSA sample was used. The CD data reported in that work suggested that BSA

underwent conformational changes upon adsorption onto hydroxyapatite nanorods, as

indicated by a reduction in percentage helicity. This trend agrees well with our ATR-

FTIR spectroscopy measurements, which showed that all BSA proteins underwent

adsorption-related denaturation, although defatted BSA proteins in our case had lower

conformational stability and underwent more extensive denaturation.

2. Likewise, in the work by Galdino et al., the CD data revealed slight differences

between free BSA incubated with spherical silica nanoparticles (~100 nm in diameter)

in solution (i.e., BSA in non-adsorbed state) and BSA after adsorption onto the

nanoparticles. However, the conclusions regarding the conformational states of BSA

based on the CD curves are largely qualitative since the difference in percentage

helicity was not quantified in that work.

Expectedly, the degree of BSA denaturation varies across different works, especially

between those involving planar and non-planar surfaces. Comparing the data presented in

the abovementioned works particularly highlights the complexity associated with protein

adsorption onto nanostructured surfaces, especially when the types of BSA used were not

specified in detail. At the nanoscale, protein adsorption behavior can be significantly

influenced by differences in nanoparticle surface chemistry, structural geometry, and nano-

curvature effects, as reported in Refs. 10-13. Considering these intricacies, our work

primarily focused on characterizing the adsorption uptake and adsorption-related

conformational changes of different types of BSA proteins onto planar silica surfaces,

which allowed a focused approach to directly compare adsorption properties using well-

established, surface-sensitive measurement techniques. Moreover, the adsorption and

conformational trends observed on the flat silica surfaces also agreed well with the trends

observed on the silica-coated nanostructured surfaces used in the LSPR measurements.

Therefore, we believe our present study can greatly benefit the scientific community by

demonstrating that the specific type of commercially available BSA (i.e., fatted vs, defatted)

can significantly influence adsorption behavior across flat and nanostructured surfaces.

In line with the Reviewer’s comments, we have included the following remarks beginning

at line 351 of the revised manuscript:

“Looking forward, these findings will be relevant for studying other fatty acid-binding

proteins such as human serum albumin across various applications, including protein

corona formation, and can also be further explored in the context of nanoparticle

properties (i.e., surface chemistry, shape, and size)22,42,50,51. We anticipate that the rational

selection of BSA protein options without or with fatty acid stabilizers can enable the

fabrication of superior antifouling coatings for a wide range of applications, which can be

readily implemented by researchers across different fields of materials science and

nanobiotechnology.”

We have also included the following references in the revised manuscript:

50 Rial, R. et al. Structural and kinetic visualization of the protein corona on

bioceramic nanoparticles. Langmuir 34, 2471-2480 (2018).

51 Galdino, F. E., Picco, A. S., Sforca, M. L., Cardoso, M. B. & Loh, W. Effect of

particle functionalization and solution properties on the adsorption of bovine serum

15

albumin and lysozyme onto silica nanoparticles. Colloids Surf., B 186, 110677

(2020).

3) The other curiosity is whether these findings could be transferred to HAS, due to

its similarity with BSA and, if possible, whether this would have impact of processes

related to protein corona formation onto surfaces ?

Response: We thank the Reviewer for this great question. We believe that our findings are

likely relevant to HSA, which is another fatty acid-binding serum albumin. Thus, we

expect that fatty acids would affect HSA adsorption processes related to protein corona

formation. Indeed, serum albumin proteins naturally contain fatty acids from serum as part

of its biological function (HSA is a fatty acid carrier) and naturally sourced HSA would

also have to undergo a fatty acid removal step in order to be fatty acid free. The same

would apply to recombinant HSA (rHSA) from transgenic rice and yeast as well since Refs.

2,16,17 have shown that these rHSA can contain varying amounts fatty acids. From an

application perspective, the insights obtained from our present study may serve as a guide

for the design of protein coatings aimed at modulating the process of corona formation in

vivo. From a biology perspective, the work could potentially contribute to insights into

how protein corona formation may be affected by differing levels of free fatty acids in

human serum, brought about by certain health conditions (see Ref. 18).

In line with the Reviewer’s comments, we have added the following remark at line 351 of

the revised manuscript:

“…these findings will be relevant for studying other fatty acid-binding proteins such as

human serum albumin across various applications, including protein corona formation…”

References

1 Chuang, V. T. G., Kragh-Hansen, U. & Otagiri, M. Pharmaceutical strategies

utilizing recombinant human serum albumin. Pharmaceutical research 19, 569-

577 (2002).

2 He, Y. et al. Large-scale production of functional human serum albumin from

transgenic rice seeds. Proc. Natl. Acad. Sci. 108, 19078-19083 (2011).

3 Zhu, T.-T. et al. Difference in binding of long-and medium-chain fatty acids with

serum albumin: The role of macromolecular crowding effect. J. Agric. Food Chem.

66, 1242-1250 (2018).

4 Freyer, M. W. & Lewis, E. A. Isothermal titration calorimetry: experimental design,

data analysis, and probing macromolecule/ligand binding and kinetic interactions.

Methods Cell Biol. 84, 79-113 (2008).

5 Wallevik, K. Isoelectric focusing of bovine serum albumin: Influence of binding of

carrier ampholytes. Biochim. Biophys. Acta, Protein Struct. 322, 75-87 (1973).

6 Basu, S. P., Rao, S. N. & Hartsuck, J. A. Influence of fatty acid and time of focusing

on the isoelectric focusing of human plasma albumin. Biochim. Biophys. Acta,

Protein Struct. 533, 66-73 (1978).

7 Product Information, <https://www.sigmaaldrich.com/content/dam/sigma-

aldrich/docs/Sigma/Product_Information_Sheet/a2153pis.pdf> (2000).

16

8 Reimhult, E., Höök, F. & Kasemo, B. Intact vesicle adsorption and supported

biomembrane formation from vesicles in solution: influence of surface chemistry,

vesicle size, temperature, and osmotic pressure. Langmuir 19, 1681-1691 (2003).

9 Reimhult, E., Höök, F. & Kasemo, B. Vesicle adsorption on SiO2 and TiO2:

dependence on vesicle size. J. Chem. Phys. 117, 7401-7404 (2002).

10 Givens, B. E., Xu, Z., Fiegel, J. & Grassian, V. H. Bovine serum albumin

adsorption on SiO2 and TiO2 nanoparticle surfaces at circumneutral and acidic pH:

A tale of two nano-bio surface interactions. J. Colloid Interface Sci. 493, 334-341

(2017).

11 Chakraborty, S. et al. Contrasting effect of gold nanoparticles and nanorods with

different surface modifications on the structure and activity of bovine serum

albumin. Langmuir 27, 7722-7731 (2011).

12 Roach, P., Farrar, D. & Perry, C. C. Surface tailoring for controlled protein

adsorption: effect of topography at the nanometer scale and chemistry. J. Am. Chem.

Soc. 128, 3939-3945 (2006).

13 Satzer, P., Svec, F., Sekot, G. & Jungbauer, A. Protein adsorption onto

nanoparticles induces conformational changes: particle size dependency, kinetics,

and mechanisms. Eng. Life Sci. 16, 238-246 (2016).

14 Rial, R. et al. Structural and kinetic visualization of the protein corona on

bioceramic nanoparticles. Langmuir 34, 2471-2480 (2018).

15 Galdino, F. E., Picco, A. S., Sforca, M. L., Cardoso, M. B. & Loh, W. Effect of

particle functionalization and solution properties on the adsorption of bovine serum

albumin and lysozyme onto silica nanoparticles. Colloids Surf., B 186, 110677

(2020).

16 Lang, B. E. & Cole, K. D. Unfolding properties of recombinant human serum

albumin products are due to bioprocessing steps. Biotechnol. Prog. 31, 62-69

(2015).

17 Ohtani, W. et al. Physicochemical and immunochemical properties of recombinant

human serum albumin from Pichia pastoris. Anal. Biochem. 256, 56-62 (1998).

18 Gonçalves-de-Albuquerque, C. F. et al. Serum albumin saturation test based on

non-esterified fatty acids imbalance for clinical employment. Clin. Chim. Acta 495,

422-428 (2019).

Decision letter and referee reports: second round

10th Jun 20

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Communications Materials

REVIEWERS' COMMENTS:

Reviewer #1 (Remarks to the Author):

The authors responded roughly to the concerns raised by the first review, allowing thus this

manuscript for publication.

Reviewer #2 (Remarks to the Author):

The authors have performed an extensive revision of their original Ms, including new data (on

more hydrophobic nitrocellulose surfaces) and re-analyses of their IR results. Overall, my

impressions is that these have clarified the points raised by the reviewers, and complemented the

work significantly.

My previous evaluation was already positive and now I am even more confident to recommend its

publication.