Biomarkers Determination Based on Surface-Enhanced Raman Scattering

Round 1

Reviewer 1 Report

The authors reviewed several progress on biomarkers determination using surface enhanced raman scattering with a reasonable amount of information. 

Some specific recommendations to authors:

1. The written english is not flawed but certain sentence structures and assembly created some difficulties for readers. I recommend authors to polish the manuscript with an academic editor. 
2. Due to the low signal of raman scattering, especially the anti-stokes raman, the research required specified optical configuration and certain precision equipment until surface-enhanced raman scattering. On line 62 in introduction, if authors can add a brief paragraph to introduce to readers why SERS is advantageous over other far field Raman techniques will help the manuscript. 
3. specific points:
   1. line 39, "level of single molecules, genes, and single cells" will have a better logical order
   2. line 42, I feel subcells is rarely used in biomedical context insteadof subcellular components. 
   3. line 49 early diagnosis by definition is disease identification.
   4. line 56, in my opinion, AFP-L3 is the biomarker, and the AFP-L3% will be the functional metric or index used for diagnosis. line 56 may better be rewritten to avoid ambiguity 
   5. line 59 the molecular fingerprint of AFP-L3 can be accurately measured by SERS technology therefore AFP-L3% can be measured and used as a strong basis for diagnosis. 
   6. line 139 picogram 
   7. line 172 missing reference 46?
   8. line 177 Helicobacter pylori should be italic 
   9. line 184 found that human breaths contain more than 3000 species of volatile organic compounds. 
   10. line 327 has been rapidly developed 
   11. line 335 I think perhaps effective protein bioconjugation methods will be a more appropriate description
   12. line 349 i think the authors mean declare no conflict of interests?

Author Response

All the replies are written in boldface.

We appreciate the Reviewer’s comments on this manuscript, which helped us significantly revise the manuscript.

The authors reviewed several progress on biomarkers determination using surface enhanced raman scattering with a reasonable amount of information.

Some specific recommendations to authors:

1. The written english is not flawed but certain sentence structures and assembly created some difficulties for readers. I recommend authors to polish the manuscript with an academic editor. Responses: Based on the Reviewer’s comment, the revised manuscript has been edited for proper English language, grammar, punctuation, spelling, and overall style by two of the highly qualified native English-speaking editors at American Journal Experts (www.aje.com).The editorial certificate for the revised manuscript is attached as supporting information for the Editor.
2. Due to the low signal of raman scattering, especially the anti-stokes raman, the research required specified optical configuration and certain precision equipment until surface-enhanced raman scattering. On line 62 in introduction, if authors can add a brief paragraph to introduce to readers why SERS is advantageous over other far field Raman techniques will help the manuscript. Responses: According to the Reviewer’s comment, we have added sentences to introduce the advantage of SERS. The following sentences were added to the Introduction section of the revised manuscript.

“Raman spectroscopy is a very useful chemical and biological analysis tool. It is based on the Raman effect and has been widely used in many research fields [14,15]. The Raman effect is caused by the inelastic scattering of light by molecules. The intensity of Rayleigh scattering is only approximately 10^-3 of the scattering of the incident light, and the intensity of Raman scattering is only 10^-3-10^-6 of the Rayleigh intensity. Therefore, the normal Raman effect is inherently weak. For bulk samples, a high-energy-density laser light source and a large number of sampled molecules facilitate easy acquisition of Raman spectra. However, the sensitivity of Raman spectroscopy cannot meet the requirements for studying ultrathin films, especially surface molecules. This is mainly due to the small number of molecules: Raman scattering by molecules is a secondary photon process, and the Raman scattering cross section of each molecule is approximately 10^-30/cm². Therefore, a large number of molecules must be sampled to obtain a sufficient rate of conversion of laser photons into Raman photons. To compensate for these shortcomings, Raman derivative techniques are gradually being developed. Surface-enhanced Raman scattering (SERS) was first observed by the British scientist Fleischmann on an electrochemically rough metal electrode surface, which significantly enhanced the Raman signal of a pyridine molecule [16]. Van Duyne [17] and Creighton [18] et al. studied the same system in detail, excluding an increase in the number of molecules and the influence of resonance, and pointed out that the 5-6 orders of magnitude enhancement of the Raman signal from pyridine molecules is caused by the rough electrode surface. This discovery aroused widespread interest in the scientific community, and this phenomenon was named SERS. SERS eliminates the above issues and has provided an excellent tool for studying surface molecules because it can detect monolayer or even submonolayer materials and provide rich information about the structure of matter at the molecular level. The SERS effect has a large enhancement factor. Coin metals such as Ag, Au and Cu are the most common SERS enhancers, among which Ag exhibits the best enhancement effect. Other metals, such as Li, Na, K, and transition metals (i.e., Fe, Co, Ni, etc.) can also produce SERS effects [19]. Recently, semiconductors have been shown to exhibit excellent SERS enhancement and broadened the applicability of SERS-active substrates [20].”

1. specific points:

1. line 39, "level of single molecules, genes, and single cells" will have a better logical order
2. line 42, I feel subcells is rarely used in biomedical context insteadof subcellular components.
3. line 49 early diagnosis by definition is disease identification.
4. line 56, in my opinion, AFP-L3 is the biomarker, and the AFP-L3% will be the functional metric or index used for diagnosis. line 56 may better be rewritten to avoid ambiguity
5. line 59 the molecular fingerprint of AFP-L3 can be accurately measured by SERS technology therefore AFP-L3% can be measured and used as a strong basis for diagnosis.
6. line 139 picogram
7. line 172 missing reference 46?
8. line 177 Helicobacter pylori should be italic
9. line 184 found that human breaths contain more than 3000 species of volatile organic compounds.
10. line 327 has been rapidly developed
11. line 335 I think perhaps effective protein bioconjugation methods will be a more appropriate description
12. line 349 i think the authors mean declare no conflict of interests?

Responses: According to the Reviewer’s comments, we have revised the manuscript and checked the revised manuscript carefully.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors 

The abstract it's adequate and the introduction shows the importance of the SERS technique. In general, the manuscript is well written with scientific soundness and no English grammar issues were detected. This review used adequate quality and a number of references.

I recommend the publication of this review after the following minor recommendation are made:

(a) use italics for the expression "in situ" (page 2, line 88)

(b) Can you increase the quality of the letters in figure 1? they look blurry

(c) A typing error was found "46" on page 4, line 173

(d) Please, use italics for species names such as "Helicobacter pylori", page 4, line 177

(e) Please, describe all abbreviations of figure 2 (in figure caption)

(f) This is just a recommendation, in the discussion of the importance of the SERS technique, you mentioned some advantages of the SERS method and some disadvantages of the traditional methods. Your review will be more readable and visible if you include a comparative table of advantages and disadvantages between SERS and the traditional one's techniques. Specifying only biomolecules detection

(g) Would be a good idea to explain more in detail the procedure of the SERS technique, of course explaining its good characteristics.

Author Response

All the replies are written in boldface.

We appreciate the Reviewer’s comments on this manuscript, which helped us significantly revise the manuscript.

The abstract it's adequate and the introduction shows the importance of the SERS technique. In general, the manuscript is well written with scientific soundness and no English grammar issues were detected. This review used adequate quality and a number of references.

I recommend the publication of this review after the following minor recommendation are made:

1. use italics for the expression "in situ" (page 2, line 88)

Responses: According to the Reviewer’s comment, we have used italics for the expression “in situ” in the revised manuscript.

(b) Can you increase the quality of the letters in figure 1? they look blurry

Responses: According to the Reviewer’s comment, we have increased the quality of all figures in the revised manuscript.

(c) A typing error was found "46" on page 4, line 173

Responses: According to the Reviewer’s comment, we have corrected this typing error on page 4, line 173, in the revised manuscript.

(d) Please, use italics for species names such as "Helicobacter pylori", page 4, line 177

Responses: According to the Reviewer’s comments, we have used italics for species names in the revised manuscript.

(e) Please, describe all abbreviations of figure 2 (in figure caption)

Responses: According to the Reviewer’s comment, we have defined all abbreviations in Figure 2 in the revised manuscript.

The following sentence has been added to the caption of Figure 2 in the revised manuscript.

“AuNPs, gold nanoparticles; VOCs, volatile organic compounds; RGO, reduced graphene oxide; GO, graphene oxide; and PC, principal component analysis.”

(f) This is just a recommendation, in the discussion of the importance of the SERS technique, you mentioned some advantages of the SERS method and some disadvantages of the traditional methods. Your review will be more readable and visible if you include a comparative table of advantages and disadvantages between SERS and the traditional one's techniques. Specifying only biomolecules detection.

Responses: According to the Reviewer’s comment, we have added the advantages and disadvantages of SERS and other techniques.

The following sentences were added to the revised manuscript.

“Generally, nuclear magnetic resonance, mass spectrometry, electrochemistry, and cryoelectron microscopy are used for biomedical and bioanalytical studies and provide rich and powerful information. However, these methods are limited in spatial resolution and in in vivo single-cell studies. A SERS spectrum contains native fingerprint vibrational information from a sample, which has several advantages: (1) SERS-active substrates can be designed with different sizes, shapes, and coatings for different purposes. (2) SERS is suitable for long-term monitoring. (3) SERS can realize single-molecule detection. (4) SERS provides fingerprint information for biological systems and produces bands with a very narrow bandwidth.”

(g) Would be a good idea to explain more in detail the procedure of the SERS technique, of course explaining its good characteristics.

Responses: According to the Reviewer’s comment, we have added sentences to introduce the advantage of SERS.

The following sentences were added to the Introduction part of the revised manuscript.

“Raman spectroscopy is a very useful chemical and biological analysis tool. It is based on the Raman effect and has been widely used in many research fields [14,15]. The Raman effect is caused by the inelastic scattering of light by molecules. The intensity of Rayleigh scattering is only approximately 10^-3 of the scattering of the incident light, and the intensity of Raman scattering is only 10^-3-10^-6 of the Rayleigh intensity. Therefore, the normal Raman effect is inherently weak. For bulk samples, a high-energy-density laser light source and a large number of sampled molecules facilitate easy acquisition of Raman spectra. However, the sensitivity of Raman spectroscopy cannot meet the requirements for studying ultrathin films, especially surface molecules. This is mainly due to the small number of molecules: Raman scattering by molecules is a secondary photon process, and the Raman scattering cross section of each molecule is approximately 10^-30/cm². Therefore, a large number of molecules must be sampled to obtain a sufficient rate of conversion of laser photons into Raman photons. To compensate for these shortcomings, Raman derivative techniques are gradually being developed. Surface-enhanced Raman scattering (SERS) was first observed by the British scientist Fleischmann on an electrochemically rough metal electrode surface, which significantly enhanced the Raman signal of a pyridine molecule [16]. Van Duyne [17] and Creighton [18] et al. studied the same system in detail, excluding an increase in the number of molecules and the influence of resonance, and pointed out that the 5-6 orders of magnitude enhancement of the Raman signal from pyridine molecules is caused by the rough electrode surface. This discovery aroused widespread interest in the scientific community, and this phenomenon was named SERS. SERS eliminates the above issues and has provided an excellent tool for studying surface molecules because it can detect monolayer or even submonolayer materials and provide rich information about the structure of matter at the molecular level. The SERS effect has a large enhancement factor. Coin metals such as Ag, Au and Cu are the most common SERS enhancers, among which Ag exhibits the best enhancement effect. Other metals, such as Li, Na, K, and transition metals (i.e., Fe, Co, Ni, etc.) can also produce SERS effects [19]. Recently, semiconductors have been shown to exhibit excellent SERS enhancement and broadened the applicability of SERS-active substrates [20].”

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript entitled " Biomarkers Detection Based on Surface-Enhanced Raman Scattering". The topic is scientifically important. The authors have described the importance of such a review clearly. however, there are several points I would like the authors to address before accepting this manuscript:

1- The authors did not review enough amount of articles to represents the overall purpose of the review, for example with simple google research on Alzheimer's biomarkers using SERS you will find many articles, despite of that authors only cited 4 articles. That is also true for other diseases that they mentioned in this manuscript.

2- I am suggesting that authors make more effort to collect more related articles and add/analyze the data to have more comprehensive conclusions. 

3- The conclusions on such a vital topic need to reflect on what is currently available using this technology (SERS) and what it needs to be improved in the short and long future.

4- Also some of the figures are fuzzy with low resolution need to be improved.

5- authors also need to add one paragraph explaining what is SERS phenomenon is, and why it is possible to detect one single molecule using SERS technology as of the current manuscript I did not notice one single sentence explaining what SERS is.

Best Regards

Author Response

All the replies are written in boldface.

We appreciate the Reviewer’s comments on this manuscript, which helped us significantly revise the manuscript.

The manuscript entitled " Biomarkers Detection Based on Surface-Enhanced Raman Scattering". The topic is scientifically important. The authors have described the importance of such a review clearly. however, there are several points I would like the authors to address before accepting this manuscript:

1. The authors did not review enough amount of articles to represents the overall purpose of the review, for example with simple google research on Alzheimer's biomarkers using SERS you will find many articles, despite of that authors only cited 4 articles. That is also true for other diseases that they mentioned in this manuscript.

Responses: According to the Reviewer’s comment, we have reviewed more references in the revised manuscript.

The following sentences were added in the revised manuscript.

“Ganesan et al. used an ultrashort pulsed laser to fabricate a 3D biocompatible aluminum-based quantum structure for CEA determination [52]. In addition, molecularly imprinted techniques and sandwich immunoassay techniques have been designed for the quantitative evaluation of CEA, and the detection limit is as low as the nanogram scale [53,54].”

“Beyene et al. developed a gold nanoflower (AuNF)-based SERS method for the detection of MUC4 and demonstrated approaches to improve the reliability of the method. The proposed method is very sensitive, and the detection limit is at the picogram level [64]. Pang et al. designed a magnetic SERS-active substrate for the one-step detection of microRNAs in exosomes and residual plasma from blood, which are biomarkers for the early detection of pancreatic cancer. The proposed method is fast and sensitive and can be used to test ultramicro-scale samples, which is helpful for the early detection of pancreatic cancer [65].”

“Monolayer graphene, which has an electronic structure and interface properties that are highly sensitive to PSA, is employed as a Raman substrate. During specific binding to PSA and its aptamer, the G band of graphene undergoes a frequency shift, the extent of which is dependent on the PSA concentration [74]. Ag-decorated electrospun fibers and magnetic beads were employed for the recyclable determination of PSA with high sensitivity. The determination method produced uniform and reproducible SERS signals and exhibited excellent sensitivity (picogram scale) [75,76].”

“Ma et al. designed an Ag film to discriminate single-site phosphorylated S396 in a Tau410 protein, which is an important biomarker in Alzheimer’s disease [83]. Park et al. employed a sequential nanotransfer printing technique to fabricate a graphitic nanolayer-coated three-dimensional SERS substrate that successfully detected the two abnormally folded proteins and analyzed the secondary structural changes quantitatively [84].”

1. I am suggesting that authors make more effort to collect more related articles and add/analyze the data to have more comprehensive conclusions.

Responses: According to the Reviewer’s comment, we have reviewed more related references in the revised manuscript.

The following references were cited in the revised manuscript.

• “Ganesan, S.; Venkatakrishnan, K.; Tan, B. Wrinkled Metal Based Quantum Sensor for In Vitro Cancer Diagnosis. Biosens. Bioelectron. 2020, 151, 111967.

• Carneiro, M.C.C.G.; Sousa-Castillo, A.; Correa-Duarte, M.A.; Sales M.G.F. Dual Biorecognition by Combining Molecularly-Imprinted Polymer and Antibody in SERS Detection. Application to Carcinoembryonic Antigen. Biosens. Bioelectron. 2019, 146, 111761.

• Song, C.; Yang, Y.; Yang, B.; Min, L.; Wang, L. Combination Assay of Lung Cancer Associated Serum Markers Using Surface-enhanced Raman Spectroscopy. J. Mater. Chem. B 2016, 4, 1811-1817.

• Beyene, A.B.; Hwang, B.J.; Tegegne, W.A.; Wang, J.S.; Tsai, H.C.; Su, W.N. Reliable and Sensitive Detection of Pancreatic Cancer Marker by Gold Nanoflower-based SERS Mapping Immunoassay. Microchem. J. 2020, 158, 105099.

• Pang, Y.; Wang, C.; Lu, L.; Wang, C.; Sun, Z.; Xiao, R. Biosens. Bioelectron. 2019, 130, 204-213.

• Gao, R.; Cheng, Z.; Wang, X.; Yu, L.; Guo, Z.; Zhao, G.; Choo, J. Simultaneous Immunoassays of Dual Prostate Cancer Markers Using a SERS-based Microdroplet Channel. Biosens. Bioelectron. 2018, 119, 126-133.

• Liu, S.; Huo, Y.; Bai, J.; Ning, B.; Peng, Y.; Li, S.; Han, D.; Kang, W.; Gao, Z. Rapid and Sensitive Detection of Prostate-specific Antigen via Label-free Frequency Shift Raman of Sensing Graphene. Biosens. Bioelectron. 2020, 158, 112184.

• Du, Y.; Liu, H.; Chen, Y.; Tian, Y.; Zhang, X.; Gu, C.; Jiang, T.; Zhou, J. Recyclable Label-free SERS-based Immunoassay of PSA in Human Serum Mediated by Enhanced Photocatalysis Arising from Ag Nanoparticles and External Magnetic Field. Appl. Surf. Sci. 2020, 528, 146953.

• Yun, B.J.; Koh, W.G. Highly-sensitive SERS-based Immunoassay Platform Prepared on Silver Nanoparticle-decorated Electrospun Polymeric Fibers. J. Ind. Eng. Chem. 2020, 82, 341-348.

• Buividas, R.; Dzingelevičius, N.; Kubiliūtė, R.; Stoddart, P.R.; Truong, V.K.; Ivanova, E.P.; Juodkazis, S. Statistically Quantified Measurement of an Alzheimer’s Marker by Surface-enhanced Raman Scattering. J. Biophotonics, 2015, 8, 567-574.

• Yang, J.K.; Hwang, I.J.; Cha, M.G.; Kim, H.I.; Yim, D.; Jeong, D.H.; Lee, Y.S.; Kim, J.H. Reaction Kinetics-Mediated Control over Silver Nanogap Shells as Surface-Enhanced Raman Scattering Nanoprobes for Detection of Alzheimer’s Disease Biomarkers. Small, 2019, 15, 1900613.

• Ma, H.; Liu, S.; Liu, Y.; Zhu, J.; Han, X.X.; Ozaki, Y.; Zhao, B. In-situ Fingerprinting Phosphorylated Proteins via Surface-enhanced Raman Spectroscopy: Single-site Discrimination of Tau Biomarkers in Alzheimer’s Disease. Biosens. Bioelectron. 2021, 171, 112748.

• Park, H.J.; Cho, S.; Kim, M.; Jung, Y.S. Carboxylic Acid-Functionalized, Graphitic Layer-Coated Three-Dimensional SERS Substrate for Label-Free Analysis of Alzheimer’s Disease Biomarkers. Nano Lett. 2020, 20, 2576-2584.”

1. The conclusions on such a vital topic need to reflect on what is currently available using this technology (SERS) and what it needs to be improved in the short and long future.

Responses: According to the Reviewer’s comment, we have discussed the currently available SERS technology and how to improve it in the short and long term.

The following sentences were added to the Conclusion of the revised manuscript.

“For example, the poor repeatability of SERS substrate preparation results in poor repeatability of experimental data and affects the application and promotion of SERS. The following issues need to be resolved. (1) The development of the preparation and application of new universal SERS-active nanostructures with high sensitivity, specificity, and biocompatibility and low interference for biomarker determination is urgent. The optimal SERS-active nanostructures do not cause irreproducible changes in the spectrum. In addition to the improvement and development of the original SERS-enhanced substrate preparation method, it is also necessary to develop more kinds of SERS-active substrates and establish new SERS-enhanced substrate preparation technologies. (2) New SERS testing technology suitable for medical testing needs to be developed. Combining SERS technology with biomedical technologies, especially nondestructive detection and analysis technologies, will lead to new SERS detection technologies and the comprehensive analysis of biomarker information and fully utilize the advantages of SERS technology in disease-related biomarker detection with high sensitivity and specificity.”

1. Also some of the figures are fuzzy with low resolution need to be improved.

Responses: According to the Reviewer’s comment, we have improved the quality of the figures.

1. authors also need to add one paragraph explaining what is SERS phenomenon is, and why it is possible to detect one single molecule using SERS technology as of the current manuscript I did not notice one single sentence explaining what SERS is.

Responses: According to the Reviewer’s comment, we have added sentences to introduce the advantages of SERS.

The following sentences were added to the Introduction section of the revised manuscript.

“Raman spectroscopy is a very useful chemical and biological analysis tool. It is based on the Raman effect and has been widely used in many research fields [14,15]. The Raman effect is caused by the inelastic scattering of light by molecules. The intensity of Rayleigh scattering is only approximately 10^-3 of the scattering of the incident light, and the intensity of Raman scattering is only 10^-3-10^-6 of the Rayleigh intensity. Therefore, the normal Raman effect is inherently weak. For bulk samples, a high-energy-density laser light source and a large number of sampled molecules facilitate easy acquisition of Raman spectra. However, the sensitivity of Raman spectroscopy cannot meet the requirements for studying ultrathin films, especially surface molecules. This is mainly due to the small number of molecules: Raman scattering by molecules is a secondary photon process, and the Raman scattering cross section of each molecule is approximately 10^-30/cm². Therefore, a large number of molecules must be sampled to obtain a sufficient rate of conversion of laser photons into Raman photons. To compensate for these shortcomings, Raman derivative techniques are gradually being developed. Surface-enhanced Raman scattering (SERS) was first observed by the British scientist Fleischmann on an electrochemically rough metal electrode surface, which significantly enhanced the Raman signal of a pyridine molecule [16]. Van Duyne [17] and Creighton [18] et al. studied the same system in detail, excluding an increase in the number of molecules and the influence of resonance, and pointed out that the 5-6 orders of magnitude enhancement of the Raman signal from pyridine molecules is caused by the rough electrode surface. This discovery aroused widespread interest in the scientific community, and this phenomenon was named SERS. SERS eliminates the above issues and has provided an excellent tool for studying surface molecules because it can detect monolayer or even submonolayer materials and provide rich information about the structure of matter at the molecular level. The SERS effect has a large enhancement factor. Coin metals such as Ag, Au and Cu are the most common SERS enhancers, among which Ag exhibits the best enhancement effect. Other metals, such as Li, Na, K, and transition metals (i.e., Fe, Co, Ni, etc.) can also produce SERS effects [19]. Recently, semiconductors have been shown to exhibit excellent SERS enhancement and broadened the applicability of SERS-active substrates [20].”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed all my comments to better this manuscript. 

I believe this review article on SERS technology will be beneficial to readers of Chemosensors.

Reviewer 3 Report

After the authors addressed all the points mentioned in my first review the article now is suitable to be published in your prestigious journal.

Best Regards

Zeid