Surface Plasmon Resonance for Protease Detection by Integration of Homogeneous Reaction
Round 1
Reviewer 1 Report
This is an Originality paper, reporting an SPR biosensor for protease detection by integration of homogeneous reaction.
The review paper has been well written and the authors have completed most of the introduction related work to prove the usefulness of their technical methods.
However, the author needs to explain the advantages of using SPR to detect Cas-3 based biosensors.
It is recommended that the author modify Figure 1(A) and re-experiment the Fluorescence microscopy images. The Fluorescence microscopy images in Figure 1(A) are completely black. This image is not scientifically meaningful.
All explain points are clear and the adding results make the scientific proofs stronger.
I would like to recommend the acceptance this manuscript to publish in MDPI Biosensors, but needs to be revised.
Author Response
We thank the reviewer for his/her positive and constructive comments: “This is an Originality paper, reporting an SPR biosensor for protease detection by integration of homogeneous reaction. The review paper has been well written and the authors have completed most of the introduction related work to prove the usefulness of their technical methods.”
Comment 1: However, the author needs to explain the advantages of using SPR to detect Cas-3 based biosensors.
Response: It is a good suggestion. The advantages of SPR biosensors for protease (Cas-3) detection have been added.
Comment 2: It is recommended that the author modify Figure 1(A) and re-experiment the Fluorescence microscopy images. The Fluorescence microscopy images in Figure 1(A) are completely black. This image is not scientifically meaningful.
Response: We have repeated the experiment several times and found that the Figure 1A is light green but the Figure 1B is completely black, which can be observed by amplifying the figures. The result is understandable since that the FITC-NA proteins were attached onto the gold chip surface through the formation of self-assembly monolayer. We have amplified the image and improved the figure quality in the revised manuscript.
Comment 3: All explain points are clear and the adding results make the scientific proofs stronger. I would like to recommend the acceptance this manuscript to publish in MDPI Biosensors, but needs to be revised.
Response: We thank the reviewer for his/her comments. We have revised the manuscript carefully.
Reviewer 2 Report
The authors describe a biosensor for protease detection. I have the following minor comments that needs to be addressed:
- The fluorescence image is not clear 1(A), there is hardy anything visible
- fig 4 (B) which looks more like a bright field image should be a magnified version to show distinct changes in cell density/morphology.
Author Response
We thank the reviewer for his/her positive comments: The authors describe a biosensor for protease detection. I have the following minor comments that needs to be addressed:
Comment 1: The fluorescence image is not clear 1(A), there is hardy anything visible.
Response: We have repeated the experiment several times and found that the Figure 1A is light green but the Figure 1B is completely black, which can be observed by amplifying the figures. The result is understandable since that the FITC-NA proteins were attached onto the gold chip surface through the formation of self-assembly monolayer. We have amplified the image and improved the figure quality in the revised manuscript.
Comment 2: Fig 4 (B) which looks more like a bright field image should be a magnified version to show distinct changes in cell density/morphology.
Response: We have amplified the image and improved the figure quality.
Reviewer 3 Report
In this paper, the author has designed an SPR biosensor for Cas-3 detection. There are some questions regarding this paper.
Q1: For the heterogeneous biosensor, the steric hindrance is not the main issue; the author should discuss many groups are trying to solve the problem. Efforts include changing the functionalized surface structure (using a nanostructure surface) and optimizing the passivation reagent and peptide reporter ratio. Here are some references that should be added.
- Electrochemical Activity Assay for Protease Analysis Using Carbon Nanofiber Nanoelectrode Arrays. Anal. Chem. 2019, 91, 6, 3971–3979
- Simultaneous, multiplex quantification of protease activities using a gold microelectrode array. Biosensors and Bioelectronics. 2020, 165, 112330
- Electrochemical DNA-Based Immunoassay That Employs Steric Hindrance To Detect Small Molecules Directly in Whole Blood. ACS Sens. 2017, 2, 6, 718–723
Q2: Page 2, line 53. Please clarify the advantage, lower detection limit? Faster response? Or higher signal to background ratio?
Q3: Scheme 1, is it possible that both Biotins are functionalized on the surface in the first step? Peptide molecule is "lying on the surface" instead of "standing up." If not, why?
Q4: Page 3, line 103. What is the buffer? Please clarify it.
Q5: Figure 2. The author has fitted the data points. What algorithm was used? What is the purpose? What is the detection limit?
Q6: Figure 4B. I cannot say any difference thin these two figures.
Author Response
We thank the reviewer for his/her comments: “In this paper, the author has designed an SPR biosensor for Cas-3 detection. There are some questions regarding this paper.”
Comment 1: For the heterogeneous biosensor, the steric hindrance is not the main issue; the author should discuss many groups are trying to solve the problem. Efforts include changing the functionalized surface structure (using a nanostructure surface) and optimizing the passivation reagent and peptide reporter ratio. Here are some references that should be added. Electrochemical Activity Assay for Protease Analysis Using Carbon Nanofiber Nanoelectrode Arrays. Anal. Chem. 2019, 91, 6, 3971–3979; Simultaneous, multiplex quantification of protease activities using a gold microelectrode array. Biosensors and Bioelectronics. 2020, 165, 112330; Electrochemical DNA-Based Immunoassay That Employs Steric Hindrance To Detect Small Molecules Directly in Whole Blood. ACS Sens. 2017, 2, 6, 718–723.
Response: We have discussed the strategies to solve of the problem of steric hindrance and highlighted the advantages of this method in Introduction. The references related to the steric hindrance have been cited in the revised manuscript.
Comment 2: Page 2, line 53. Please clarify the advantage, lower detection limit? Faster response? Or higher signal to background ratio?
Response: The advantages of SPR method such as fast response, real-time detection, high signal-to-noise and good compatibility with microfluidic system have been added in the revised manuscript.
Comment 3: Scheme 1, is it possible that both Biotins are functionalized on the surface in the first step? Peptide molecule is "lying on the surface" instead of "standing up." If not, why?
Response: It is a good question. The SPR result indicated that the biotin-GDEVDGK-biotin was not bound to two NA proteins immobilized on the gold surface because the biotin-GDEVDGK-biotin is a small molecule and the distance between two NA proteins is far. Moreover, the unreacted gold surface between two NA proteins was blocked by BSA and GSH, which may prevent the binding of biotin-GDEVDGK-biotin to two NA proteins immobilized on the gold surface. Actually, the previous report also confirmed that the biotin-peptide-biotin did not bind to two NA proteins immobilized on the surface of one gold nanoparticle (Anal. Chem. 2014, 86, 2345.).
Comment 4: Page 3, line 103. What is the buffer? Please clarify it.
Response: The gold chips were modified with NA in carbonate buffer and then washed with the same buffer. It has been clarified in the revised manuscript.
Comment 5: Figure 2. The author has fitted the data points. What algorithm was used? What is the purpose? What is the detection limit?
Response: The data points were interpolated by first order curve in Origin, but not point-to-point. The detection limit was estimated to be 0.5 pg/mL by measuring the sensor response to a dilution series and determining the smallest target concentration at which the signal was clearly distinguishable from the response to the blank solution.
Comment 6: Figure 4B. I cannot say any difference thin these two figures.
Response: We have amplified the image and improved the figure quality.
