Next Article in Journal
Optoelectronic Properties of MAPbBr3 Perovskite Light-Emitting Diodes Using Anti-Solvent and PEDOT:PSS/PVK Double-Layer Hole Transport Layers
Next Article in Special Issue
Liposome Deformation Induced by Membrane-Binding Peptides
Previous Article in Journal
Radial Error Motion Measurement and Its Uncertainty Estimation of Ultra Precision Axes of Rotation with Nanometer Level Precision
Previous Article in Special Issue
Double-Sided Sapphire Optrodes with Conductive Shielding Layers to Reduce Optogenetic Stimulation Artifacts
 
 
Article
Peer-Review Record

Potential Universal Engineering Component: Tetracycline Response Nanoswitch Based on Triple Helix-Graphene Oxide

Micromachines 2022, 13(12), 2119; https://doi.org/10.3390/mi13122119
by Luhui Wang 1,†, Yue Wang 2,†, Mengyang Hu 2, Sunfan Xi 1, Rong Liu 2, Meng Cheng 1 and Yafei Dong 1,2,*
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Micromachines 2022, 13(12), 2119; https://doi.org/10.3390/mi13122119
Submission received: 28 September 2022 / Revised: 19 November 2022 / Accepted: 28 November 2022 / Published: 30 November 2022
(This article belongs to the Special Issue Bioinspired Materials and Microdevices: Fabrications and Applications)

Round 1

Reviewer 1 Report

The submitted article contains valuable results and has a potential for the scientific community. Nevertheless, for publishing this manuscript, the following has to be optimized:

Further questions to be answered are:

 1. Real-time monitoring of antibody fragment (Fabs) production processes in bioreactors is highly relevant.

 2. Mechanistic and data-driven models are the basis for better understanding and mapping interactions between process operation and cell leakiness. Please explain how you deal with this?

 3. What methods are used to clean the cell products.

Author Response

Dear Editor and reviewers,

Thanks for your constructive comments and helpful suggestions, and we appreciate the overall enthusiasm you showed for our review manuscript, which have significantly raised the quality of the manuscript and enable us to improve the manuscript.

 

Every revision suggestion and comment put forward by the reviewers are accurately incorporated and considered. As a result of these changes, the revised manuscript is substantially improved and now provides more extensive information.

 

Reviewer 1

Thanks very much for your kind work and comments. Your comments are very helpful for revising and improving our paper. According to your comments, we have made the following modifications and replies:

 

  1. Real-time monitoring of antibody fragment (Fabs) production processes in bioreactors is highly relevant.

In this design, the real-time monitoring is completed by dry experiment and wet experiment. Specifically, the dry experiment is used to simulate the experiment process, determine the approximate temperature and time, and define the scope of real-time monitoring; The wet experiment is more close to the actual situation, and can achieve the purpose of real-time monitoring within the range by means of continuous detection.

 

  1. Mechanistic and data-driven models are the basis for better understanding and mapping interactions between process operation and cell leakiness. Please explain how you deal with this?

Different from the dry experiment in which there is little leakage due to complete reaction of substances, the small difference in operation during the wet experiment may lead to errors and leakage. Therefore, when improving the mechanism, we chose to increase the concentration of protective substances to avoid early exposure of key substances that cause system differences, which may increase the detection probability of false positives. (The specific process is added in PART 2 Materials and Methods.)

 

  1. What methods are used to clean the cell products.

In our method, the required S1-S2 triple helix structure, P1/GO composite platform and targets with different concentrations are configured separately and mixed in proportion during the experiment, so the cleaning step is not involved. Of course, your suggestions will be taken into consideration in the team's subsequent research, and we will try to design an energy-saving platform that can be cleaned and reused. Thank you for providing us with new ideas for our design.

Reviewer 2 Report

This manuscript by Wang et al. demonstrates a tetracycline sensor by relying on strand displacement reactions. Here, the authors utilize the preference for ssDNA to adsorb to graphene oxide surfaces as well as the ability for graphene oxide to quench the fluorescence of nearby dyes to design a system to detect tetracycline. The authors show simulations describing the strand displacement reactions as well as perform device optimization characterization. Finally, they show they can detect tetracycline at concentrations as low as 590 pM.

Although the authors show that they can detect tetracycline, I have several major concerns with this paper. Most importantly, I would strongly suggest the authors to get their manuscript edited or proof-read by a fluent or native English speaker. The writing of this paper is riddled with mistakes which make it difficult to read. Furthermore, not only does this paper have idiomatic and grammatical mistakes, there are several oversights in the paper where the wrong figure is referenced, data in figures are not labeled correctly, or more egregiously, parts of the paper template are still left in the body of the manuscript.

The organization of the paper also requires restructuring. There are very few details as to how the device actually works until page 3, so the entire methods/materials section has no context and cannot be understood without reading section 3.1 first. Even after reading the paper, there are many questions about the device that are not answered by the methods section. How long are the strands used in the device? What buffers were the strands used in? All that is said is that the strands were originally dissolved in water… does the reaction also just take place in water? If I were to try to replicate this experiment, I would not be able to easily because there are simply just not enough details as to how to make this system. What are the initial concentrations of all the parts? What’s the concentration of triple-helix devices used in each tetracycline detection experiment?

Finally, there is very little context for this device. The authors frame the work through the lens that tetracycline poisoning could be problematic, and that traditional methods of detecting tetracycline use resource-intensive equipment/methods, but the authors never return to this point. There are many ways to detect tetracycline that utilize nanomaterials; some of these methods were determined as early as 2010. What makes this method better than existing nanomaterial methods? What are the flaws in existing nanomaterial methods? How are these devices typically used? The authors do not specify any potential drawbacks or future work other than stating “In the follow-up study, we need to explore how to reduce the detection limit as much as possible.” How will the authors do this? Do they really need to reduce the detection limit? What’s considered to be dangerous?

The manuscript does show that they can detect tetracycline, which may be of value, but this manuscript requires major changes before it can be published. My specific comments are below.

Pg. 1 ln 10: What is meant by “the special structure”? Secondary structure?

Pg 1 ln 17: All acronyms must be defined before use

Pg 1 ln 38-40: Please proofread your paper before submission.

Pg 2 ln 50: Quench fluorescence of what?

Pg. 2 ln 89: What is S1 and S2? I assume they stand for strand 1 and strand 2. Again, all acronyms must be defined before use.

Pg 2 ln 93: Why is there a fluorescent signal? Where does it come from?

Pg 3 ln 103: Section 3.1 needs to be moved earlier in the paper – if I do not know how the device operates, I cannot understand the methods/materials.

Pg 3 Ln 121-137: It is unnecessary to justify why simulation is desired, please remove this section.

Pg 4 ln 138: The original NUPACK paper should be cited

Pg 4 ln 143: How is tetracycline simulated in NUPACK? Tetracycline is not a nucleic acid. Is tetracycline being simulated as a strand complementary to the hairpin region? If so, is that a valid comparison? I would imagine the binding affinities of the DNA-DNA interaction vs the real tetracyline-DNA interaction are not the same.

 Pg 4 Fig 3B: This figure shows two curves but has three legend entries. The P1 and S2 legend entries seem to be plotted on top of each other? What’s going on in this figure?

Pg 5 ln 162: Please cite the original visual DSD paper

Pg 5 Fig 4D: What is ST?

Pg 6 ln 197/Fig6: The claim that P1-S2 production is higher in lane 7 than in lane 6 is actually somewhat subtle. I would suggest performing gel analysis to compare the intensity of the bands.

Pg 6 ln 213: Given that SYBR green is an intercalator, have the authors performed a control examining differences in SYBR green signal from the unquenched, single-stranded P1 vs the hybridized P1 + S2 strands? While I’m sure that there is some change in fluorescent signal when P1 is no longer adsorbed to GO, I wonder if the double stranded P1-S2 complex could have increased SYBR-green signal since it will have the conventional double-stranded helix architecture.

Pg 7 ln 225: The abscissa and ordinate of what?

Pg 7 Ln 233: This should say figure 8A (same issue on ln 246 and 254)

Pg 8 Fig 9C: This figure is too small (especially the inset) to read. Please increase the font

Author Response

Dear Editor and reviewers,

Thanks for your constructive comments and helpful suggestions, and we appreciate the overall enthusiasm you showed for our review manuscript, which have significantly raised the quality of the manuscript and enable us to improve the manuscript.

Every revision suggestion and comment put forward by the reviewers are accurately incorporated and considered. As a result of these changes, the revised manuscript is substantially improved and now provides more extensive information.

 

Reviewer 2

Thanks very much for your kind work and comments. Your comments are very helpful for revising and improving our paper. According to your comments, we have made the following modifications and replies.

  1. The writing of this paper is riddled with mistakes which make it difficult to read. Furthermore, not only does this paper have idiomatic and grammatical mistakes, there are several oversights in the paper where the wrong figure is referenced, data in figures are not labeled correctly, or more egregiously, parts of the paper template are still left in the body of the manuscript.

Thank you for your suggestions on manuscript writing, we have done our best to improve it. Specifically: (1) Check and revise the entire manuscript writing; (2) Check the acronyms used in the manuscript and standardize the writing; (3) Standardized citation of NUPACK and Viusal DSD; (4) The legend of the wrong reference in the optimization experiment part is corrected; (5) The inset of Fig. 9C has been enlarged and numbered Fig. 9D.

 

  1. The organization of the paper also requires restructuring. There are very few details as to how the device actually works until page 3, so the entire methods/materials section has no context and cannot be understood without reading section 3.1 first. Even after reading the paper, there are many questions about the device that are not answered by the methods section.

We thank you for bringing up this important question. First, in order to make it easier to understand the whole work, we will advance the 3.1 Principles section to the last paragraph of the introduction. For the Materials and Methods section, we have refined the description of each experimental step to make it more detailed.

 

  1. What makes this method better than existing nanomaterial methods? What are the flaws in existing nanomaterial methods? How are these devices typically used? The authors do not specify any potential drawbacks or future work other than stating “In the follow-up study, we need to explore how to reduce the detection limit as much as possible.” How will the authors do this? Do they really need to reduce the detection limit? What’s considered to be dangerous?

Thank you for your comment. Firstly, in the sensitivity experiment part, we add the comparison between this method and other nano methods, and analyze the advantages and disadvantages of colorimetric methods, electrochemical methods and fluorescence methods, and finally show that the superiority of this strategy lies in "Our design has reached a lower LOD without enzyme assistance and tedious operations, so it has certain." detection advantages”

 

In terms of reducing LOD, although this strategy for TC detection has demonstrated that LODs meet the detection standards set by the European Union, when changing the model aptamer sequence for other substances, lower LODs may be required to achieve the target microdetection. Considering that this design does not include steps for signal amplification, we guess that adding steps to signal amplification can further reduce LODs if we use this model as a basis. Similarly, in the new edition of the manuscript, we have added a brief explanation of why LOD needs to be further reduced.

 

Some of your specific comments have been comprehensively replied in the above, and the following are the replies and revisions to other comments.

 

  1. What is meant by “the special structure”? Secondary structure?

Thanks for your question. "the special structure" here refers to a special composite platform composed of GO and DNA triple helix structure, the previous use of "special structure" may indeed cause ambiguity, so in the new edition of the manuscript we have corrected the description to "special composite platform".

 

  1. Pg 2 ln 50: Quench fluorescence of what?

Thanks for your question. GO usually quenches single-stranded DNA markers or carried fluorescence, which was too brief in the original manuscript, and the description has been corrected in the new manuscript to "GO and its derivatives can effectively adsorb single strand DNA(ssDNA) and quench the fluorescence it labels or carries by fluorescence resonance energy transfer (FRET)”

 

  1. Pg 2 ln 93: Why is there a fluorescent signal? Where does it come from?

Thanks for your question. The fluorescence signal here is generated by the P1-labeled fluorophore FAM, for ease of understanding we have advanced the schematic to the last paragraph of the introduction and updated the description "quenching of P1-labeled FAM fluorescence" in the experimental section.

 

  1. It is unnecessary to justify why simulation is desired, please remove this section.

We strongly agree with your opinion and have deleted this part.

 

  1. How is tetracycline simulated in NUPACK? Tetracycline is not a nucleic acid. Is tetracycline being simulated as a strand complementary to the hairpin region? If so, is that a valid comparison? I would imagine the binding affinities of the DNA-DNA interaction vs the real tetracyline-DNA interaction are not the same.

Thanks for your question. Since NUPACK can only mimic nucleic acid sequences, we use complementary sequences of tetracycline aptamers as substitutes for tetracycline. In the new edition of the manuscript, we discuss the effectiveness of this type of substitution based mainly on free energy and dissociation constant in support materials. In simple terms, we compare the dissociation constants of aptamer-TC, aptamer-complementary sequence, and S1-aptamer complementary sequence, and the results show that the dissociation constants of the three are in the same order of magnitude (0.609nM~1.067nM), so it is proved that the aptamer complementary sequence can be used instead of TC to react in NUPACK.

 

  1. Fig 3B: This figure shows two curves but has three legend entries. The P1 and S2 legend entries seem to be plotted on top of each other? What’s going on in this figure?

Thank you for your question. The dry experiment is performed before the wet experiment, and the leakage generated in the dry experiment is small, so the approximate reaction trend can be obtained when the simulated chain is input at the same proportional concentration. The P1-S2 double-stranded structure is generated by the proportional consumption of single-stranded P1 and S2, so the P1 and S2 legend entries overlap each other. In the manuscript, we added some instructions before and after the simulation experiment such as: "In addition, because the above two complexes are synthesized from two substrates, the trend of substrate concentration is similar and overlapping."

 

  1. Fig 4D: What is ST?

Thanks for your reminder, ST is the abbreviation of S1+TC, and we have corrected the legend to ensure consistency in context.

 

  1. Fig6: The claim that P1-S2 production is higher in lane 7 than in lane 6 is actually somewhat subtle. I would suggest performing gel analysis to compare the intensity of the bands.

Thank you very much for your suggestion, we have added grayscale analysis of electropherogram using Image J in the new manuscript, and displaying the results with numerical values instead of the naked eye really makes the results more objective.

 

  1. Given that SYBR green is an intercalator, have the authors performed a control examining differences in SYBR green signal from the unquenched, single-stranded P1 vs the hybridized P1 + S2 strands? While I’m sure that there is some change in fluorescent signal when P1 is no longer adsorbed to GO, I wonder if the double stranded P1-S2 complex could have increased SYBR-green signal since it will have the conventional double-stranded helix architecture.

Thank you very much for your suggestion, we added the fluorescence experiments of P1 and P1+S2, and found that the fluorescence value of P1 was extremely low, which could be used as the lower limit value of fluorescence standardization treatment; P1+S2, on the other hand, has a high fluorescence value and can be used as the upper limit of fluorescence normalization. Normalizing the fluorescence values of systems with or without TC makes the feasibility results more intuitive.

 

  1. Pg 7 ln 225: The abscissa and ordinate of what?

Thanks for your reminder, the abscissa and ordinate here should be corrected to the abscissa and ordinate of the optimization experiment; We have refined the narrative "In the optimization experiment, the abscissa marks the different levels of each experimental factor, and the ordinate represents the fluorescence intensity (FI)" in the new version of the manuscript.

Reviewer 3 Report

The article describes the development of nanosensor based system to detect tetracycline. The topic of article is new and promising. The authors presented a big massive of data to support their invention. The results are correctly done and well described. The data are supported by corresponding references and well discussed. The conclusions are correctly made. As for English, I am not native speaker, for me English is good to read and understand the article. I did not find any serious mistakes to mention.

However, I should notice that the specificity of this nanosensor is a weak point of this study. In future studies (1) the number of antibiotics to study specificity should be increased including tetracycline analogs , (2) the chemical substances with similar to tetracycline structures should be included in the experiments to study specificity.

But, finally, the article can be published.

Author Response

Dear Editor and reviewers,

Thanks for your constructive comments and helpful suggestions, and we appreciate the overall enthusiasm you showed for our review manuscript, which have significantly raised the quality of the manuscript and enable us to improve the manuscript.

 

Every revision suggestion and comment put forward by the reviewers are accurately incorporated and considered. As a result of these changes, the revised manuscript is substantially improved and now provides more extensive information.

 

Reviewer 3

Thanks very much for your kind work and comments. Your comments are very helpful for revising and improving our paper. According to your comments, we have made the following modifications and replies.

 

However, I should notice that the specificity of this nanosensor is a weak point of this study. In future studies (1) the number of antibiotics to study specificity should be increased including tetracycline analogs , (2) the chemical substances with similar to tetracycline structures should be included in the experiments to study specificity.

We appreciate your suggestions. It should be explained that in this strategy we used Kwon et al. truncated 8-mer TC aptamers from 76-mer, and this 8-mer short sequence has a high affinity for four different tetracyclines[tetracycline (TC), oxytetracycline (OTC), doxycycline (DOX) and chlortetracycline (CHLOR)], so our method has a similar response to tetracyclines. Kwon et al. have selected the nonsteroidal anti-inflammatory drugs diclofenac (DIC) and naproxen (NPX), the organic phosphine herbicide glyphosate (GLY), and the antiepileptic drug carbamazepine (CBZ) as counter targets to prove that they will not bind to 8-mer aptamers, so we chose the different types of quinolone antibacterial drug ofloxacin and the β-lactam antibiotic amoxicillinas counter targets. In the latest version of the manuscript, we have added an introduction to the properties of 8-mer aptamers.

Round 2

Reviewer 2 Report

I thank the authors for their detailed responses and revisions to the manuscript. I believe the writing in the manuscript has improved significantly, although some errors remain. 

For the most part, the authors have revised the manuscript to address my concerns. I still have a few remaining questions and suggestions:

1. Could the authors describe the average size of the GO particles? Understandably these particles have some range, but since the authors discuss that different degrees of dispersion can affect the sensitivity of the device, it would be good to explicitly include the size of the particles in this manuscript.

2. Do the authors have any thoughts or suggestions as to why the concentration of GO has a non-monotonic affect on F0? I'm surprised that the highest concentration of GO has a higher F0 than at 25 ug/mL. I am not too surprised that F and dF are non-monotonic since a high concentration of GO might make it difficult for P1 to "find" S2 to hybridize over other GO particles, but it is a bit surprising that more P1 seems to be released with higher GO concentration.

3. I am curious why the authors prefer to use the absolute fluorescence over the change in fluorescence to construct their calibration curve? If dF is used instead of F, then presumably the authors could use the lower GO concentration and get higher sensitivity due to increased signal strength. 

4. Why do the authors show the fluorescence spectra again in figure 9a,b? Do we expect that under different TC concentrations we could get signal from other parts of the spectrum? Does it matter? Ultimately you will just look for the signal corresponding the fluorophore on P1 so showing the entire spectrum seems unnecessary.

5. I am still confused by the data shown in figure 6. In lane 6 (where both TC and P1 are mixed with the S2/S1 complex), we expect that the S2/S1 complex is released and S1 complexes with TC and S2 hybridizes with P1. We see two major features in this lane; a tight band and a faster moving "smear". The smear seems to correspond with the S2/P1 duplex based on the data in lane 5. The slower band seems migrate at a similar rate to S1, which makes sense because if TC is present, it is only ~450 Da so I don't expect it to significantly slow down migration.

What confuses me is that in lane 7, we see the S2/P1 duplex and again, the slower moving S1 band, but we do not see the S2+S1 band at all. I agree based on the gel results it appears that there is slightly less free S1, but then my question is: where is the bound S1? 

 

Again, I applaud the authors for their time in revising the manuscript. The method is interesting and the results confirm that the method works. The manuscript should be considered for publication, but the story itself (i.e. presented data, interpretations, etc.) can be improved.

Author Response

Dear Reviewer,

Thank you for your helpful suggestions on our research and for giving us opportunities to improve our work, we have made some changes to improve the quality of the manuscript. According to your comments, we have made the following modifications and replies.

 

  1. Could the authors describe the average size of the GO particles? Understandably these particles have some range, but since the authors discuss that different degrees of dispersion can affect the sensitivity of the device, it would be good to explicitly include the size of the particles in this manuscript.

Thank you for the kindly reminder. Based on the results of our previous work, the GO dispersion used in this paper has a size of <500 nm, and we have changed the description of GO dispersion selection in the manuscript to "Therefore, in this paper, we continue to use the more uniform crushing GO XF020 with a sheet diameter of <500nm."

 

  1. Do the authors have any thoughts or suggestions as to why the concentration of GO has a non-monotonic affect on F0? I'm surprised that the highest concentration of GO has a higher F0 than at 25 ug/mL. I am not too surprised that F and dF are non-monotonic since a high concentration of GO might make it difficult for P1 to "find" S2 to hybridize over other GO particles, but it is a bit surprising that more P1 seems to be released with higher GO concentration.

Thank you for your question. Like you said, after the GO concentration is higher than 25 μg/mL, the higher GO concentration actually increases the initial fluorescence value in the system, which is indeed a strange result. We conjecture that when the GO concentration is 25 μg/mL, the adsorption of P1 has basically reached the upper limit. Although the GO concentration is high enough, there will still be a very small amount of P1 free in wet experiments, which also results in the initial fluorescence in the system can only be approached to the minimum, but cannot reach 0. We note that some researchers use guanine to quenching probes to determine specific DNA [1], Therefore, it is speculated that some free P1 in the system may be close to the S1-S2 complex due to the intermolecular movement, and the guanine contained in the complex has a certain degree of inhibition of the FAM fluorophore labeled on P1, so it shows extremely low fluorescence in the system. When the concentration of GO in the system further increases, it may increase the probability of it being close to the loop-part of the S1-S2 complex, so free S1-S2 complex in the system will be decrease, which on the one hand leads to a very limited fluorescence that can be recovered in the presence of TC, and on the other hand, it also makes the free small amount of FAM labeled P1 lack of inhibition, thus causing the initial fluorescence value in the system to rise without falling. Similarly, we have updated this part of the conjecture in the manuscript.

[1] Torimura, M., Kurata, S., Yamada, K., Yokomaku, T., Kamagata, Y., Kanagawa, T., & Kurane, R. (2001). Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base. Analytical sciences : the international journal of the Japan Society for Analytical Chemistry, 17(1), 155–160. https://doi.org/10.2116/analsci.17.155

 

  1. I am curious why the authors prefer to use the absolute fluorescence over the change in fluorescence to construct their calibration curve? If dF is used instead of F, then presumably the authors could use the lower GO concentration and get higher sensitivity due to increased signal strength.

Thank you very much for your advice. We tried to construct the calibration curve using the fluorescence change value, but when the curve was also constructed using the first 5 points, the correlation coefficient R2 = 0.969 was slightly inferior to the situation when using the absolute fluorescence value, so we chose the absolute fluorescence value for linear fitting.

Of course, we strongly agree with your suggestion that lower GO concentrations can maintain high signal intensities and thus increase sensor sensitivity. In fact, we are accustomed to pursuing lower background fluorescence in our research and ignoring other issues. Thank you again for pointing this out and pointing out how to proceed with subsequent experiments that require further reduced detection limits.

 

  1. Why do the authors show the fluorescence spectra again in figure 9a,b? Do we expect that under different TC concentrations we could get signal from other parts of the spectrum? Does it matter? Ultimately you will just look for the signal corresponding the fluorophore on P1 so showing the entire spectrum seems unnecessary.

Thank you for your question. Our linear fit data is derived from the values in the fluorescence spectrum at an emission wavelength of 518 nm, so we chose to retain the entire fluorescence spectrum to better observe the change in fluorescence at different emission wavelengths with different TC concentrations in the system. In addition, in daily experiments, we have found that some instruments for fluorescence detection cannot flexibly adjust the excitation wavelength and emission wavelength according to needs, and can only detect fluorescence signals through filters with a fixed range of excitation wavelength and emission wavelength, so we keep the fluorescence spectrum to investigate whether it can also conform to the linear fit when the emission wavelength is not optimal. Fortunately, in the 500-600 nm wavelength range, when the first five points are linearly fitted, the resulting R2 can remain almost above 0.99, and less than one-tenth is below this threshold. We have added relevant figures in the supporting material and briefly described this part in the manuscript.

 

  1. What confuses me is that in lane 7, we see the S2/P1 duplex and again, the slower moving S1 band, but we do not see the S2+S1 band at all. I agree based on the gel results it appears that there is slightly less free S1, but then my question is: where is the bound S1? 

Thank you for your question. In order to better see the substances represented by each strip in the gel diagram, we further marked the gel diagram. In fact, we found that the S1 and S1+S2 composite strips are very close to each other, after making the auxiliary lines, S1 is located below the auxiliary line, and the S1+S2 composite is located above the auxiliary line, but the bands in Lane 6 and 7 seem to be just on the auxiliary line and do not perfectly coincide with S1 or S2. So we conjecture that this part of the strip may be obtained by mixing S1 and S1+S2 in different proportions. In the presence of TC, the band is roughly located in the middle of the auxiliary line, and the concentration of S1 and S1+S2 complexes may be similar. In the absence of TC, the band is positioned slightly above the auxiliary line, and the concentration of the S1+S2 complex may be greater than that of S1. In addition, unfortunately, due to the long running time, the problem of temperature rise will inevitably occur during the running process, which will lead to the spontaneous opening of a small number of S1+S2 complexes (as discussed in simulation experiments), so P1+S2 double strands also appear in band 7. As an improvement, we have added a similar narrative to the manuscript.

 

Back to TopTop