Carbocyanine-Based Fluorescent and Colorimetric Sensor Array for the Discrimination of Medicinal Compounds
Round 1
Reviewer 1 Report
This work developed a combined fluorescent and colorimetric sensor for medicinal compounds discrimination based on aggregation and oxidation response mechanisms of the carbocyanine dyes. The manuscript is overall in good condition; I have a few questions:
1. What’s the reason for using CTAB or DDS as the surfactant in this work rather than other surfactants?
2. What’s the principle of the selectivity of this work?
3. Could the authors provide any TEM or SEM images to characterize the nanoparticles w/ and w/o analytes?
Author Response
Rev 1
The manuscript is overall in good condition; I have a few questions:
1. What’s the reason for using CTAB or DDS as the surfactant in this work rather than other surfactants?
Reply. We did not make a selection of surfactants in this work but our previous experience [19] shows that CTAB and DDS are most efficient among other surfactants in aggregation-based fluorimetry. As for the surfactants in redox reactions, they were added to adjust the initial fluorescent signal of carbocyanine at a convenient level (very low signal is observed without surfactant in these systems). The concrete reason why particularly these compounds, CTAB and DDS, are most efficient in aggregation, is unknown. We added the following phrase to the Introduction:
These surfactants proved to be the most efficient in aggregation-based determinations [19].
- What’s the principle of the selectivity of this work?
Reply. 1/ Selectivity of aggregation-based sensing: this was already described under Introduction as follows: A selective response is observed to large hydrophilic ions forming several ionic or hydrogen bonds. We observed fluorescence enhancement of carbocyanine dyes with aminoglycosides, cephalosporins, penicillins and other compounds. - We just added a phrase: The signal intensity differs with the particular analyte. 2/ Selectivity of redox indicator reactions: we added the following words to Introduction after the explanation of catalytic reactions principle: The selectivity of detection can be governed by various complexation constants of analytes with the metal ions in catalytic indicator reactions and different catalytic activity of these complexes.
Could the authors provide any TEM or SEM images to characterize the nanoparticles w/ and w/o analytes?
Reply. – We did not perform the TEM studies for particularly these systems but we published the TEM images of the dye nanoparticles and aggregation products in our paper [19]. This reference is placed in text near each mention of nanoparticles, so we believe that no question can arise, where such information can be found.
Reviewer 2 Report
The manuscript by Anna V. Shik et al., entitled "Carbocyanine-Based Fluorescent and Colorimetric Sensor Array for the Discrimination of Medicinal Compounds", presents an Array-based optical sensing for obtaining an optical response from a wide range of low-molecular-weight organic compounds.
The authors demonstrated the potential of the suggested methodology by discrimination of several model compounds of various nature, with a simple protocol and without does not involve special biomolecules or complicated instrumentation.
The concept of colorimetric sensor is interesting and the platform should be useful for rapid tests. The manuscript is clearly written and easy to read. I recommend the manuscript for publication after the following minor revisions are made.
- How a fluorescence and absorbance properties of compounds to be tested (analytes)? When an analyte has strong fluorescence emission in the NIR region, the measurement should be biased. Also, contaminants from samples (e.g. turkey meat extract) may hamper the measurement. I think some discussion regarding a limitation of the measurement system should be needed.
- In Figure 2b, absorbance around 550 nm and 800 nm increases by adding analyte, it seems to be background signal, and the background is changing by oxidation. I am curious about the absorbance spectra of analyte before and after oxidation.
- The authors should discuss a concentration dependency, limit of detection of target analyte, and
- In 2.4, PCA should be an abbreviation for principal component analysis. I suggest adding full form. Also, inserting brief explanation of PC1, PC2, and PC3 may be helpful for an out-of-the-field readers.
Author Response
Rev 2
The manuscript is clearly written and easy to read. I recommend the manuscript for publication after the following minor revisions are made.
- How a fluorescence and absorbance properties of compounds to be tested (analytes)? When an analyte has strong fluorescence emission in the NIR region, the measurement should be biased.
Reply. We could say quite the opposite: if an analyte has strong NIR fluorescence, it would be easily detected directly, without using any indicator reactions. (However, NIR fluorescence is an exotic thing among common analytes.) The same pertains to the analytes strongly absorbing in the visible region, as they would have intense color.
- Also, contaminants from samples (e.g. turkey meat extract) may hamper the measurement. I think some discussion regarding a limitation of the measurement system should be needed.
Reply. Of course, turkey extract was supposed to affect the detection. We added the following phrases to the beginning and the end of section 3.3:
The components of turkey extract were supposed to affect the detection. Our goal was to demonstrate the discriminating ability of the method in the presence of a complicated matrix.
Overall, the discrimination of the model analytes was still feasible, notwithstanding the matrix effect.
- In Figure 2b, absorbance around 550 nm and 800 nm increases by adding analyte, it seems to be background signal, and the background is changing by oxidation. I am curious about the absorbance spectra of analyte before and after oxidation.
Reply. Yes, the said small increase in intensity can be associated with the oxidation of penicillin. We have studied this process experimentally and found that the oxidation of penicillin under the same conditions but in the absence of the dye gives a different kinetic spectral pattern. We have to conclude that the pattern observed in Fig. 2b is due to the products of joint oxidation of the dye and penicillin. We are not sure if these details are worth to be included in the text of the article.
- The authors should discuss a concentration dependency, limit of detection of target analyte, and
Reply. Quantitative determination of analytes is quite a separate task which will be the subject of a different study. We have some preliminary results showing that submicromolar concentrations of benzylpenicillin or chlorpromazine can change the discoloration time of dye 2. More concrete information on the sensitivity can be obtained in further studies.
- In 2.4, PCA should be an abbreviation for principal component analysis. I suggest adding full form. Also, inserting brief explanation of PC1, PC2, and PC3 may be helpful for an out-of-the-field readers.
Reply. We have added the following passage:
Principal component analysis (PCA) reduces the dimensionality of initial data: the intensities measured for every indicator process (up to 16 data columns) for each analyte in several parallel runs are substituted for the 3 columns of the first three principal components (PC); a greater number of PCs is usually meaningless. The data then can be conveniently viewed in the scores plots as PC2 vs. PC1 and PC3 vs. PC1.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
I really enjoyed reading this manuscript consisting on two analytical approaches (colorimetric/fluorimetric and redox-based) for probing different pharmaceutical compounds in various chemical environments.
The paper is well written, scientifically sound and it would certainly be interesting to the readers of Chemosensors. Below are some comments to be considered by the authors before accepting for publication.
Line 125-127: “The selected model analytes included 4 cephalosporins (ceftriaxone, cefazolin, cefotaxime, cefotaxime), 3 phenothiazines (promethazine, prom-126 azine, chlorpromazine), and 2 penicillins (benzylpenicillin, ampicillin).” Please provide a few (chemical) characteristics that differentiate each of these classes. In what cephalosporines are different from phenothiazine and in what phenothiazines are different from penicillins.
Line 135-137: “Carbocyanine dyes 1–4 (Scheme 2) were synthesized by us. Dye 1 was obtained according to paper [19] and dye 4 following protocol [57]. Dyes 2 and 3 were not reported previously; the routes of their syntheses are depicted in Scheme 3”. Please revise as: “Carbocyanine dyes 1 and 4 were obtained following previously reported procedures [19], [57] whereas the synthesis route for dyes 2 and 3 is depicted in Scheme 3.”
Line 142: 0.1M HCl revise as 0.1 M HCl
Line 149: Please revise the scheme capture as: The chemical structure of dyes 1-4 used in this study, and the chemical structure of nine pharmaceutical compounds used as analytes.
In scheme 2, the chemical structures look different, some have thicker lines, and the three last compounds have shaded box. Please revise this figure so all the chemical structures look consistent.
Figure 1 and Figure 2 look a bit blurry. If possible, please provide figures with improved resolution. Additionally, given that reader community is international, there is a common consent to use the English enumeration system. In the figure axis, please replace 0,1; 0,2 etc etc as 0.1, 0.2 and so on (a point instead of period).
Line 214: 3.1 Selection of Indicator Systems and Reaction Conditions. All other paragraph titles are all lower case. Please revise to be consistent with all other paragraph titles (all lower case).
Line: 226-227: “It can be seen that spectral differences are caused by сcefazolin and benzylpenicillin in 0.1–0.2 mM CTAB, which is typical for the aggregational mechanism 227 of ‘turn-on’ fluorescence in these systems [19].” Can you comment on why is there a fluorescence turn-on? The photophysical properties of cyanines strongly depend on their photoisomerization in various microenvironments. (Maybe the authors could add a line or two on this also in the introduction).
Why was it important to realize these experiments also in turkey meat extract? And why not testing in wastewater for example? Why is this relevant to this study?
Author Response
Line 125-127: “The selected model analytes included 4 cephalosporins (ceftriaxone, cefazolin, cefotaxime, cefotaxime), 3 phenothiazines (promethazine, prom-126 azine, chlorpromazine), and 2 penicillins (benzylpenicillin, ampicillin).” Please provide a few (chemical) characteristics that differentiate each of these classes. In what cephalosporines are different from phenothiazine and in what phenothiazines are different from penicillins.
Reply. We did not find any specific chemical properties of the named pharmaceuticals that would be useful for the purposes of our study except complexation with metal ions. However, we have added a summary of the chemical properties of cephalosporins, phenothiazines, and penicillins after lines 125-127 as follows:
Both penicillins and cephalosporins are prone to oxidation at the sulfur(2-) atom to form the corresponding sulfones [57]. The beta-lactam ring of cephalosporins and penicillins can be cleaved by primary amines. Cephalosporins demonstrate lower reactivity in this reaction, as they have a dihydrothiazine ring rather than a thiazolidine ring in penicillins. Moreover, cephalosporins yield no stable product of amine addition, forming a mixture of degradation products [58]. Similar to beta-lactams, phenothiazines can be oxidized to form sulfoxides, but this process occurs under milder conditions [59]. As organic bases, they form ionic associates with anionic dyes and metal complexes. Phenothiazines exhibit electron-donor properties and can form charge-transfer complexes with electron acceptors [59]. A common property of the compounds belonging to the beta-lactam and phenothiazine groups is their ability to form complexes with transition metal ions [57, 59], which was employed in this study.
Line 135-137: “Carbocyanine dyes 1–4 (Scheme 2) were synthesized by us. Dye 1 was obtained according to paper [19] and dye 4 following protocol [57]. Dyes 2 and 3 were not reported previously; the routes of their syntheses are depicted in Scheme 3”. Please revise as: “Carbocyanine dyes 1 and 4 were obtained following previously reported procedures [19], [57] whereas the synthesis route for dyes 2 and 3 is depicted in Scheme 3.”
Done.
Line 142: 0.1M HCl revise as 0.1 M HCl
Done.
Line 149: Please revise the scheme capture as: The chemical structure of dyes 1-4 used in this study, and the chemical structure of nine pharmaceutical compounds used as analytes.
Done.
In scheme 2, the chemical structures look different, some have thicker lines, and the three last compounds have shaded box. Please revise this figure so all the chemical structures look consistent.
Done.
Figure 1 and Figure 2 look a bit blurry. If possible, please provide figures with improved resolution.
Reply. All original Figures are done in high resolution. We inserted Figs. 1 and 2 with higher resolution in the final text. Besides, we are attaching the MS Word file with the quality images.
Additionally, given that reader community is international, there is a common consent to use the English enumeration system. In the figure axis, please replace 0,1; 0,2 etc etc as 0.1, 0.2 and so on (a point instead of period).
Reply. The decimal comma was replaced for the dot and the images renewed.
Line 214: 3.1 Selection of Indicator Systems and Reaction Conditions. All other paragraph titles are all lower case. Please revise to be consistent with all other paragraph titles (all lower case).
Done.
Line: 226-227: “It can be seen that spectral differences are caused by cefazolin and benzylpenicillin in 0.1–0.2 mM CTAB, which is typical for the aggregational mechanism 227 of ‘turn-on’ fluorescence in these systems [19].” Can you comment on why is there a fluorescence turn-on? The photophysical properties of cyanines strongly depend on their photoisomerization in various microenvironments.
Reply. We added the following explanation:
: the anionic analyte forms an association complex with the cationic surfactant, and the hydrophobic dye enters the hydrocarbon domains of the aggregate with a considerable fluorescence enhancement
(Maybe the authors could add a line or two on this also in the introduction).
Reply. This was already discussed in the Introduction:
“If the analyte can form an aggregate (more specifically, a nanoparticle) with an oppositely charged ion containing hydrocarbon chains (a surfactant), the formed nanoparticle will have hydrophobic domains that are able to solubilize the dye. In the hydrocarbon environment of the domain, fluorescence of the dye is turned on, which serves as the analytical signal. A selective response is observed to large hydrophilic ions forming several ionic or hydrogen bonds.”
Why was it important to realize these experiments also in turkey meat extract? And why not testing in wastewater for example? Why is this relevant to this study?
Reply. On the one hand, we were looking for a model matrix of complex composition to test the selectivity of discrimination. From our viewpoint, biological samples are more complex and therefore present more challenging matrices than waters. On the other hand, the actual concentrations of contaminants in wastewater were expected to be much lower than the present technique could detect. Our goal was to demonstrate selectivity rather than sensitivity. That was the reason to select this matrix.
We added a phrase to section 3.3:
The components of turkey extract were supposed to affect the detection. Our goal was to demonstrate the discriminating ability of the method in the presence of a complicated matrix.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
I recommend this work for publication.
