Far-red pentamethine cyanine dyes as fluorescent probes for the detection of serum albumins

Benzothiazole based cyanine dyes with bridged groups in the pentamethine chain were studied as potential far-red fluorescent probes for protein detection. Spectral-luminescent properties were characterized for unbound dyes and in the presence of serum albumins (bovine (BSA), human (HSA), equine (ESA)), and globular proteins (β-lactoglobulin, ovalbumin). We have observed that the addition of albumins leads to a significant increase in dyes fluorescence intensity. However, the fluorescent response of dyes in the presence of other globular proteins was notably lower. The value of fluorescence quantum yield for dye bearing a sulfonate group complexed with HSA amounted to 42% compared with 0.2% for the free dye. The detection limit of HSA by this dye was greater than 0.004 mg ml−1 which indicates the high sensitivity of dye to low HSA concentrations. Modelling of structure of the dyes complexes with albumin molecules was performed by molecular docking. According to these data, dyes could bind to up to five sites on the HSA molecule; the most preferable are the haemin-binding site in subdomain IB and the dye-binding site in the pocket between subdomains IA, IIA and IIIA. This work confirms that pentamethine cyanine dyes could be proposed as powerful far-red fluorescent probes applicable for highly sensitive detection of albumins.

1. The authors should demonstrate the advances made by using the bridged dicarbocyanine dyes in comparison with other far-red to near infra-red cyanine dyes. Enhancement of fluorescence of cyanine dye by serum albumin has been reported, for example: G. Patonay et al. "Spectroscopic Study of a Bis(heptamethine cyanine)dye and Its Interaction with Human Serum Albumin" Appl. Spectrosc. 59, 682-90 (2005) Bridged dicarbocyanine dyes are quite common especially in the field of silver halide photographic materials: US5576173, for example.
2. Structures and fluorescence spectra of albumin-bound dyes should be clarified. As shown in Fig. 5, albumin-bound cyanine dyes in different binding site have different conformation. They must show different fluorescence spectra. The dye molecule in Fig. 5b distorted considerably from a planar all-trans conformation, for example. Equation (3), which assumes that all the bound dyes have the same fluorescence intensity, cannot therefore apply to the present case. Albumin complex with dyes of various fluorescence intensities would make the calibration curve dependent on such factors as temperature, coexisting compounds etc. This dependence would impair the reliability of the quantitative analysis.
3. Purity of Dye 1 should be checked.
Molar extinction coefficient of Dye 1 is exceptionally low even in the methanol solution, in which Dye 1 is probably monomeric. (Table 1). 4. Fig. 1: The chemical formula of 1,8-ANS is incomplete. Probably one proton is missing.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The authors developed a far-red pentamethine cyanine dyes as fluorescent probes for detection of serum albumins. It is interesting. Publication is recommended after minor revisions. 1. The authors have briefly introduced the advantage of fluorescent detection in the introduction. Some recent representative progress should be cited here: Angew. Chem. Int. Ed., 2016, 55, 12751-12754;Angew. Chem. Int. Ed. 2017, 56, 16611-16615 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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When submitting your revised manuscript, you must respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". Please use this to document how you have responded to the comments, and the adjustments you have made. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response. ********************************************** 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) In this work, the authors observed that fluorescence due to bridged thiadicarbocyanine dyes intensified with the amount of co-existing serum albumins. These dicarbocyanine dyes are allegedly promising high-efficient far-red probes for serum albumin detection, especially useful for biological samples. Bridged dicarbocyanine dyes are well known compounds. It is also well known that fluorescence of cyanine dyes intensifies in the presence of serum albumins. This paper therefore presents just common properties of common compounds. The authors should elaborate on the advances made by using bridged dicarbocyanine dyes in comparison with other far-red to near infra read cyanine dyes.
My concerns the authors should address are listed below.
1. The authors should demonstrate the advances made by using the bridged dicarbocyanine dyes in comparison with other far-red to near infra-red cyanine dyes. Bridged dicarbocyanine dyes are quite common especially in the field of silver halide photographic materials: US5576173, for example. Fig. 5, albumin-bound cyanine dyes in different binding site have different conformation. They must show different fluorescence spectra. The dye molecule in Fig. 5b distorted considerably from a planar all-trans conformation, for example. Equation (3), which assumes that all the bound dyes have the same fluorescence intensity, cannot therefore apply to the present case. Albumin complex with dyes of various fluorescence intensities would make the calibration curve dependent on such factors as temperature, coexisting compounds etc. This dependence would impair the reliability of the quantitative analysis.

Structures and fluorescence spectra of albumin-bound dyes should be clarified. As shown in
3. Purity of Dye 1 should be checked.
Molar extinction coefficient of Dye 1 is exceptionally low even in the methanol solution, in which Dye 1 is probably monomeric. (Table 1). 4. Fig. 1: The chemical formula of 1,8-ANS is incomplete. Probably one proton is missing.

Reviewer: 2
Comments to the Author(s) The authors developed a far-red pentamethine cyanine dyes as fluorescent probes for detection of serum albumins. It is interesting. Publication is recommended after minor revisions. 1. The authors have briefly introduced the advantage of fluorescent detection in the introduction. Some recent representative progress should be cited here: Angew. Chem. Int. Ed., 2016, 55, 12751-12754;Angew. Chem. Int. Ed. 2017, 56, 16611-16615

Decision letter (RSOS-200453.R1)
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Dear Dr Kovalska:
Title: Far-red pentamethine cyanine dyes as fluorescent probes for detection of serum albumins Manuscript ID: RSOS-200453.R1 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.
Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science and the Royal Society of Chemistry, I look forward to your continued contributions to the Journal. Dear Dr. Laura Smith, Thank you for your kind attention to our manuscript, and I am also thankful to the referees for their valuable remarks. We did our best to account for all of the reviewers' comments. Our answers to the comments of the referees are as follows: Reviewer: 1 1. The authors should demonstrate the advances made by using the bridged dicarbocyanine dyes in comparison with other far-red to near infra-red cyanine dyes. Enhancement of fluorescence of cyanine dye by serum albumin has been reported, for example: G. Patonay et al. "Spectroscopic Study of a Bis(heptamethine cyanine)dye and Its Interaction with Human Serum Albumin" Appl. Spectrosc. 59, 682-90 (2005); M. Saikiran et al. "Photophysical investigation of squaraine and cyanine dyes and their interaction with bovine serum albumin", J. Physics: Conference Series, 2016, 704, 012012, doi:10.1088/1742. Bridged dicarbocyanine dyes are quite common especially in the field of silver halide photographic materials: US5576173, for example.
-We have extended the Introduction, and added some discussion about the used NIR cyanine dyes and pentamethine cyanine dyes (including bridged ones); the articles referred by the Reviewer were discussed. Particularly, based on the literature, the advantages of bridged pentametnine cyanines such as thermostability and higher fluorescence quantum yield were mentioned. Fig. 5, albumin-bound cyanine dyes in different binding site have different conformation. They must show different fluorescence spectra. The dye molecule in Fig. 5b distorted considerably from a planar all-trans conformation, for example. Equation (3), which assumes that all the bound dyes have the same fluorescence intensity, cannot therefore apply to the present case. Albumin complex with dyes of various fluorescence intensities would make the calibration curve dependent on such factors as temperature, coexisting compounds etc. This dependence would impair the reliability of the quantitative analysis.

Structures and fluorescence spectra of albumin-bound dyes should be clarified. As shown in
-We agree with the reviewer in that if a protein globule possesses several (more than one) site for the dye binding, the dyes bound to different sites could give different characteristics of fluorescence spectrum (i.e. maximum wavelength and quantum yield); this could be due to different conformation and degree of internal motions restriction of the dyes bound in different sites. Meanwhile, our measurements showed that the shape and position of maximum of the dye 3 fluorescence spectrum does not depend on HSA concentration; thus, either the protein-bound dye molecules have similar conformation, or the molecules with other conformations make negligible contribution into the fluorescence spectrum. It is still possible that dye molecules bound to different sites have the same conformation but different quantum yield due to different fixation strength. But (having in mind the mentioned complications) for the rough estimation of the binding constant we consider the dye fluorescence intensity to be proportional to the number of the bound dye molecules. The above considerations were added to the Materials and Methods part of the manuscript.