From the journal Digital Discovery Peer review history

12-Mar-2024

Dear Dr Quijano Velasco:

Manuscript ID: DD-ART-12-2023-000255
TITLE: Optimization of Liquid Handling Parameters for Viscous Liquid Transfers with Pipetting Robots, a “Sticky Situation”

Thank you for your submission to Digital Discovery, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Professor Jason Hein
Associate Editor, Digital Discovery

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Reviewer 1

I am responsible for reviewing the data and code aspects of this manuscript. I appreciate the authors' effort in sharing their data and code via a public repository. I only have a few minor comments:
1. Please create a release for the GitHub repository and archive it (using ex. Zenodo) such that the code is persistent.
2. Please include a document describing versioned software dependencies (ex. requirements.txt). This is particularly important for vendor APIs and external optimizers.

Reviewer 2

Comments on “Optimization of Liquid Handling Parameters for Viscous Liquid Transfers with Pipetting Robots, a “Sticky Situation””

The idea in this article is attractive, however, it requires improvement before it can be accepted for publication in this esteemed journal.

1) A fluid with low viscosity can be considered as a Newtonian fluid, which is easy to be modelled, however, for high viscosity， a rheological model has to be considered. For the present study, Zuo’s Fractal Rheological model is extremely useful, see Zuo, YT. EFFECT OF SiC PARTICLES ON VISCOSITY OF 3-D PRINT PASTE A Fractal Rheological Model and Experimental Verification, Thermal Science, 25 (3)(2021) , pp.2405-2409

2) The author uses “Optimization” in the title, but no Optimization model is developed, so it is not suitable for this article entitled as “Optimization”

3) Commercial Pipetting Robots were already appeared, the author should point out the present bottle problems and future research frontiers.

Reviewer 3

The manuscript presents a detailed study on the semi-automated optimization of liquid handling parameters using a MOBO (Multi-Objective Bayesian Optimization) algorithm. The authors compare human intuition-driven optimization with MOBO algorithm-driven optimization using two different pipettes across various viscosity standards. Furthermore, the manuscript discusses the incorporation of fully automated methods for the initialization step and gravimetric testing, leading to a closed-loop optimization process. The findings indicate that both human-driven and MOBO-driven optimization methods are effective, with the fully automated approach demonstrating promising results.

The manuscript provides a comprehensive description of the methodology. This includes detailed descriptions of experimental setups and algorithmic procedures, enhancing the reproducibility and clarity of the study. The results are well-organized and presented with clarity. Figures and tables effectively illustrate key findings. The discussion section offers valuable insights into the observed trends, such as the impact of liquid viscosity on transfer errors and the comparative performance of human intuition-driven versus MOBO-driven optimization.
It would be beneficial to expand the discussion section to include practical implications and potential applications of the optimized liquid handling parameters beyond the scope of the current study. This could involve discussing how the findings contribute to enabling high-throughput screening in various research domains. While the manuscript is generally well-written, ensuring consistent terminology usage and providing concise explanations of technical concepts would facilitate understanding for readers across diverse backgrounds. For example, if terms like "liquid handling parameters" are used frequently, make sure they are consistently defined.
With minor revisions addressing the suggestions outlined above, the manuscript has the potential to make a significant contribution to the field of laboratory automation and liquid handling optimization.

Pablo Quijano Velasco
Institute of Materials Research and Engineering
2 Fusionopolis Way, Singapore 138634



22nd March 2024



Dear Professor Hein and Reviewers,

Thank you for the time you’ve spent reading this manuscript and providing suggestions to improve our article. We have taken into consideration each of the comments provided by the reviewers.
On behalf of the authors, I submit our point-by point response to the comments we received. These responses can be found attached at the end of this letter. We copied the original comments provided by the reviewers (shown in black font) and we added our response after each of the comments (shown in blue font).

With kind regards,
Pablo Quijano Velasco
 
Referee: 1
Comments to the Author
I am responsible for reviewing the data and code aspects of this manuscript. I appreciate the authors' effort in sharing their data and code via a public repository. I only have a few minor comments:
1. Please create a release for the GitHub repository and archive it (using ex. Zenodo) such that the code is persistent.

Authors’ response
We thank the reviewer for pointing out the need for a GitHub repository release and a separate repository to store such a release to record the version of the code used during our study. We have created the release “LiqTransferOptimizer_v1.0.0” and uploaded the release onto Zenodo (DOI: 10.5281/zenodo.10847233).

Comments to the Author
2. Please include a document describing versioned software dependencies (ex. requirements.txt). This is particularly important for vendor APIs and external optimizers.

Authors’ response
We thank the reviewer for noticing the need of a requirement file listing all software dependencies required to run the code. We have added a “requirement.txt” into our Github repository listing all the python packages required to run the code including their versions (this file is included in the release “LiqTransferOptimizer_v1.0.0” uploaded to Zenodo).


Referee: 2
Comments to the Author
Comments on “Optimization of Liquid Handling Parameters for Viscous Liquid Transfers with Pipetting Robots, a “Sticky Situation””

The idea in this article is attractive, however, it requires improvement before it can be accepted for publication in this esteemed journal.

1) A fluid with low viscosity can be considered as a Newtonian fluid, which is easy to be modelled, however, for high viscosity， a rheological model has to be considered. For the present study, Zuo’s Fractal Rheological model is extremely useful, see Zuo, YT. EFFECT OF SiC PARTICLES ON VISCOSITY OF 3-D PRINT PASTE A Fractal Rheological Model and Experimental Verification, Thermal Science, 25 (3)(2021) , pp.2405-2409

Authors’ response
We thank the reviewer for the recommendation and providing interesting literature on rheological models for non-Newtonian fluids. For our studies we chose to focus on Newtonian fluids due to their constant viscosity. It is well pointed by the reviewer that to address the challenge of liquid transfer of non-Newtonian fluids a rheological model may be needed. However, this lies out of the scope of the current study. Nonetheless, we have incorporated a brief discussion about the future opportunities to address the transfer of non-Newtonian fluids in the future.

Changes made to the manuscript (second paragraph, page 9, first column):
“The transfer of non-Newtonian fluids was out of the scope of this study, thus further research is required to verify and improve the applicability of this protocol for the transfer of this kind of liquids. We also believe that the ability of air-displacement pipettes to accurately transfer liquids with high viscosity will be limited and there will exist highly viscous liquids that will not be suited for these tools even after optimization. Further research and development of reliable and accessible automated positive displacement pipettes is still required to offer liquid transfer solutions for this sort of liquids.”

Comments to the Author
2) The author uses “Optimization” in the title, but no Optimization model is developed, so it is not suitable for this article entitled as “Optimization”

Authors’ response
Although no new optimization model or algorithm is reported in the manuscript, we do propose a methodology to optimize the aspiration and dispense rates of air-displacement pipettes to minimize the mass transfer errors and transfer times of viscous liquids. For this reason, we believe that the word “optimization” should be included in the title of the article and hope that the reasoning behind this decision is now clear to the reviewer.

Comments to the Author
3) Commercial Pipetting Robots were already appeared, the author should point out the present bottle problems and future research frontiers.

Authors’ response
We thank the reviewer for pointing out the need to expand our discussion on the future research frontiers and bottlenecks present in the field of automated liquid transfer. We have expanded the discussion section of the manuscript to include a “Prospects and future directions” sub-section.

Changes made to the manuscript (last paragraph, page 8, second column):
“Prospects and future directions
Considering that most automated air-displacement pipettes are manufactured with the intention to transfer aqueous-like liquids, we envision that this protocol could become a common practice in labs using liquid handling automated tools to verify and improve their liquid transfers. This would not only give greater statistical confidence to their results but also ensure the repeatability of their experiments. We believe that fields of formulation, polymer, colloidal and biological sciences would benefit the most by the adoption of this protocol. For example, Chitre et al.5 recently reported the development of an automated tool that adjusts the pH of shampoo-like formulations. However, the platform still requires a liquid handling system that can accurately transfer the viscous reagents to complete a self-optimizing loop. This drawback could be solved by incorporating an automated air-displacement pipette that uses our protocol to optimize the liquid handling parameters for each viscous liquid. Beaucage and Martin14 recently published an autonomous formulation platform to study colloidal and polymeric solutions. For their platform, the authors used an OT2 robot to prepare solutions containing the viscous colloids. However, they limited their reagents to liquids with viscosities in the order of 136 cp due to the loss of pipetting accuracy with liquids with higher viscosities. Implementing our optimization protocol could expand the design space for their colloidal formulations. Finally, biological assays may involve the transfer of viscous fluids (e.g. biological fluids, glycated liquids, surfactants, oils, etc) that currently rely on manual optimization of the liquid handling parameters15,16. Our protocol would benefit by increasing the level of automation of these tasks and improving the repeatability of the assays.
We also envision several avenues that could be explored to further improve our optimization protocol. Future work on the application of MOBO algorithms that can handle categorical variables to add blowout steps could potentially increase the accuracy of the transfers and would be able to obtain liquid handling parameters for accurate transfer of liquids with higher viscosities. The transfer of non-Newtonian fluids was out of the scope of this study, thus further research is required to verify and improve the applicability of this protocol for the transfer of this kind of liquids. We also believe that the ability of air-displacement pipettes to accurately transfer liquids with high viscosity will be limited and there will exist highly viscous liquids that will not be suited for these tools even after optimization. Further research and development of reliable and accessible automated positive displacement pipettes is still required to offer liquid transfer solutions for this sort of liquids.”

Referee: 3
Comments to the Author
The manuscript presents a detailed study on the semi-automated optimization of liquid handling parameters using a MOBO (Multi-Objective Bayesian Optimization) algorithm. The authors compare human intuition-driven optimization with MOBO algorithm-driven optimization using two different pipettes across various viscosity standards. Furthermore, the manuscript discusses the incorporation of fully automated methods for the initialization step and gravimetric testing, leading to a closed-loop optimization process. The findings indicate that both human-driven and MOBO-driven optimization methods are effective, with the fully automated approach demonstrating promising results.

The manuscript provides a comprehensive description of the methodology. This includes detailed descriptions of experimental setups and algorithmic procedures, enhancing the reproducibility and clarity of the study. The results are well-organized and presented with clarity. Figures and tables effectively illustrate key findings. The discussion section offers valuable insights into the observed trends, such as the impact of liquid viscosity on transfer errors and the comparative performance of human intuition-driven versus MOBO-driven optimization.
It would be beneficial to expand the discussion section to include practical implications and potential applications of the optimized liquid handling parameters beyond the scope of the current study. This could involve discussing how the findings contribute to enabling high-throughput screening in various research domains. While the manuscript is generally well-written, ensuring consistent terminology usage and providing concise explanations of technical concepts would facilitate understanding for readers across diverse backgrounds. For example, if terms like "liquid handling parameters" are used frequently, make sure they are consistently defined.
With minor revisions addressing the suggestions outlined above, the manuscript has the potential to make a significant contribution to the field of laboratory automation and liquid handling optimization.

Authors’ response
We thank the reviewers for his comments and evaluation of our manuscript, we have addressed the minor concerns raised by Reviewer 3 in our manuscript.
We have expanded the discussion section of the manuscript to include a “Prospects and future directions” sub-section.
Changes made to the manuscript (last paragraph, page 8, second column):
“Prospects and future directions
Considering that most automated air-displacement pipettes are manufactured with the intention to transfer aqueous-like liquids, we envision that this protocol could become a common practice in labs using liquid handling automated tools to verify and improve their liquid transfers. This would not only give greater statistical confidence to their results but also ensure the repeatability of their experiments. We believe that fields of formulation, polymer, colloidal and biological sciences would benefit the most by the adoption of this protocol. For example, Chitre et al.5 recently reported the development of an automated tool that adjusts the pH of shampoo-like formulations. However, the platform still requires a liquid handling system that can accurately transfer the viscous reagents to complete a self-optimizing loop. This drawback could be solved by incorporating an automated air-displacement pipette that uses our protocol to optimize the liquid handling parameters for each viscous liquid. Beaucage and Martin14 recently published an autonomous formulation platform to study colloidal and polymeric solutions. For their platform, the authors used an OT2 robot to prepare solutions containing the viscous colloids. However, they limited their reagents to liquids with viscosities in the order of 136 cp due to the loss of pipetting accuracy with liquids with higher viscosities. Implementing our optimization protocol could expand the design space for their colloidal formulations. Finally, biological assays may involve the transfer of viscous fluids (e.g. biological fluids, glycated liquids, surfactants, oils, etc) that currently rely on manual optimization of the liquid handling parameters15,16. Our protocol would benefit by increasing the level of automation of these tasks and improving the repeatability of the assays.
We also envision several avenues that could be explored to further improve our optimization protocol. Future work on the application of MOBO algorithms that can handle categorical variables to add blowout steps could potentially increase the accuracy of the transfers and would be able to obtain liquid handling parameters for accurate transfer of liquids with higher viscosities. The transfer of non-Newtonian fluids was out of the scope of this study, thus further research is required to verify and improve the applicability of this protocol for the transfer of this kind of liquids. We also believe that the ability of air-displacement pipettes to accurately transfer liquids with high viscosity will be limited and there will exist highly viscous liquids that will not be suited for these tools even after optimization. Further research and development of reliable and accessible automated positive displacement pipettes is still required to offer liquid transfer solutions for this sort of liquids.”

Regarding the use of consistent terminology for technical concepts in the manuscript we would like to point out that the definition of liquid handling parameters for our study can be found in the following excerpt located in the last paragraph of the introduction section (last paragraph, first column, page 2):
“Here, we propose a new protocol that allows researchers to obtain optimized liquid handling parameters (defined by aspiration and dispense rates) capable of transferring liquids with viscosities as high as 1275 cP within 5% of percentage transfer error.”
However, we do agree of importance of ensuring consistent terminology and clarity throughout the manuscript for this reason we edited the manuscript to reiterate the definition of liquid handling parameters in the first paragraph of the “Optimization protocol of liquid handling parameters”.
Changes made to the manuscript (last paragraph, page 3, second column):
“Our protocol for the optimization of the liquid handling parameters (defined as a combination of aspiration and dispense rates in this study) for accurate viscous liquid transfer is shown in Fig 2.”
In addition, we found that we used the term “liquid transfer parameters” interchangeably with “liquid handling parameters”. To ensure consistency in the terminology we changed the manuscript to only use the term “liquid handling parameters”.
Changes made to the manuscript (first paragraph, page 3, second column):
“At the beginning of each calibration experiment the pipette would be prewetted with an unrecorded transfer using the optimal liquid handling parameters found during the optimization campaign.”
Changes made to the manuscript (second paragraph, page 5, second column):
“In Fig. 4 we present a summary of the performance of the best optimized liquid handling parameters for each of the tested liquids.”

14-Apr-2024

Dear Dr Quijano Velasco:

Manuscript ID: DD-ART-12-2023-000255.R1
TITLE: Optimization of Liquid Handling Parameters for Viscous Liquid Transfers with Pipetting Robots, a “Sticky Situation”

Thank you for submitting your revised manuscript to Digital Discovery. I am pleased to accept your manuscript for publication in its current form.

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Professor Jason Hein
Associate Editor, Digital Discovery


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