Logical gates in ensembles of proteinoid microspheres

Panagiotis Mougkogiannis; Andrew Adamatzky

doi:10.1371/journal.pone.0289433

Peer Review History

Original SubmissionJuly 16, 2023
24 Aug 2023 Decision Letter - Jianhui Liu, Editor PONE-D-23-22314Logical gates in ensembles of proteinoid microspheresPLOS ONE Dear Dr. Mougkogiannis, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would be happy to consider a revised version of the manuscript provided that you satisfy the remaining concerns of the reviewers. We understand that some revisions take time, but I should mention that we take into account the published literature available on the day we make our final decision. Please submit your revised manuscript by Oct 08 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 8. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper is indeed excellent !!! It opens completely new aspects of the biomolecular computing. In addition to the fundamental scientific value, the paper offers novel options for practical applications, particularly in the field of biomedical use. It is strongly recommended for urgent publication after some minor (mostly formatting) editing. The following changes are recommended for improving the paper, which is scientifically excellent: 1. All figures must be improved for better visibility (increase the letters/numbers, make lines thicker and points enlarged. In other words, please make all parts of the figures better visible. Do not use light colors like yellow, for example. 2. Do the authors really mean an oscilloscope in Figure 3? Oscilloscopes were used from 1930s till 1980s. They are old instruments. Normally, they are not used anymore being substituted with modern computerized electronics. Potentially, they might be used now, but it looks strange. 3. Do not use the reference to the German version of a paper published in Angew. Chem. Please change it to the English version, which will be more convenient for international readers. If needed, they can find the German version. 4. The technical details on the electrochemical experiments should be provided. They include: a) the kind of the electrochemical instrument (name, company, etc.) b) the reference electrode (e.g., Ag/AgCl or calomel, etc.) c) the initial and final potentials used in the chronoamperometric measurements d) the composition of the electrolyte solution, its pH, etc. e) the kind of the working and counter electrodes (e.g., gold or glassy carbon, etc.) The suggestions above do not compromise the excellent scientific quality of the paper. Reviewer #2: The manuscript by Adamatzky et al. presents a new version of electrochemical logic gates. Previously reported systems by Amatore and Warkocz were based on simple redox-active molecular, whereas current submission used complex biopolymers. Manuscript is technically sound, only minor corrections are necessary. In the introduction, it would be very beneficial to discuss briefly previous accounts of electrochemical logic gases, e.g. based on those papers: 1. AMATORE, C.; BROWN, A. R.; THOUIN, L.; WARKOCZ, J.-S., Mimicking neuronal synaptic behaviour: Processing of information with 'AND' or 'OR' Boolean logic via paired-band microelectrode assemblies. C. R. Acad. Sci. Paris 1998, 509-515. 2. Amatore, C.; Thouin, L.; Warkocz, J.-S., Artificial Neurons with Logical Properties Based on Paired-Band Microelectrode Assemblies. Chem. Eur. J. 1999, 5, 456-465. Some other references to electrochemica logic systems may be also added for completness of the picture. Experimental part misses some precision and crispness. First of all, thermal processing of aminoacids is a polycondensation process, as small molecules (water, ammonia) are side products (page 2). For the experimental part, detailed recipes, including amounts of reagents and detailed reaction conditions should be given. For electrochemical measurement the configuration of the potentiostat should be given (2- or 3-electrode setup, concentration of protenoids, supporting electrolyte...). More detailed information of the experimental setup should be given: types of electrodes, materials of electrodes, electrode spacing, etc. Detailed drawing of photo of the setup would be very beneficial. Applied voltage values should be given for all experiments. Authors present detailed analysis of signal statistical features, but use single current measurements for logic analysis - how the time-dependent variability of the signal (Figure 4 and 9) translate into logic performance. It may not be relevant, but should be briefly discussed, as the relevance of signal variability may potentially influence the performance ,especially when different protenoid will have significantly different time scale of variability. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". 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Revision 1
30 Aug 2023 Author Response Response to Reviewer Comments on "Logical Gates in Ensembles of Protenoid Microspheres" Reviewer 1 All figures must be improved for better visibility (increase the letters/numbers, make lines thicker and points enlarged. In other words, please make all parts of the figures better visible. Do not use light colors like yellow, for example. We have improved the figures 1, 2, 3, 7, and 11 as per your suggestion. We have increased the font size, contrast, and resolution of the figures to make them more visible and clear. Do the authors really mean an oscilloscope in Figure 3? Oscilloscopes were used from 1930s till 1980s. They are old instruments. Normally, they are not used anymore being substituted with modern computerized electronics. Potentially, they might be used now, but it looks strange. We have updated the figure and corresponding text to reflect the use of a potentiostat for the chronoamperometry measurements. Do not use the reference to the German version of a paper published in Angew. Chem. Please change it to the English version, which will be more convenient for international readers. If needed, they can find the German version. We have changed the citation to the English version of the paper “Molecules That Make Decisions” by Alberto Credi, which was published in Angewandte Chemie International Edition in 2007 The technical details on the electrochemical experiments should be provided. They include: a) the kind of the electrochemical instrument (name, company, etc.) The Zimmer Peacock potentiostat Anapot EIS ZP1000080 was utilised for conducting chronoamperometry measurements. b) the reference electrode (e.g., Ag/AgCl or calomel, etc.) A two-electrode setup was used for the electrochemical measurements, with iridium-coated stainless steel sub-dermal needle electrodes (Spes Medica S.r.l., Italy) serving as the working and counter/reference electrodes. c) the initial and final potentials used in the chronoamperometric measurements The potentiostat applied a constant dc potential (Edc) and measured the current response at 0.1 s intervals for 25,000 s. For the results shown, Edc was 0.01 V, with no initial equilibration period. d) the composition of the electrolyte solution, its pH, etc. The electrodes were positioned approximately 10 mm apart in a protenoid solution containing 10 mg/100 ml protenoid in water as the supporting electrolyte. e) the kind of the working and counter electrodes (e.g., gold or glassy carbon, etc.) See also b) Reviewer 2 In the introduction, it would be very beneficial to discuss briefly previous accounts of electrochemical logic gases, e.g. based on those papers: 1. AMATORE, C.; BROWN, A. R.; THOUIN, L.; WARKOCZ, J.-S., Mimicking neuronal synaptic behaviour: Processing of information with 'AND' or 'OR' Boolean logic via paired-band microelectrode assemblies. C. R. Acad. Sci. Paris 1998, 509-515. 2. Amatore, C.; Thouin, L.; Warkocz, J.-S., Artificial Neurons with Logical Properties Based on Paired-Band Microelectrode Assemblies. Chem. Eur. J. 1999, 5, 456-465. We have added a paragraph to the paper based on the following papers: 1. AMATORE, C.; BROWN, A. R.; THOUIN, L.; WARKOCZ, J.-S., Mimicking neuronal synaptic behaviour: Processing of information with 'AND' or 'OR' Boolean logic via paired-band microelectrode assemblies. C. R. Acad. Sci. Paris 1998, 509-515. 2. Amatore, C.; Thouin, L.; Warkocz, J.-S., Artificial Neurons with Logical Properties Based on Paired-Band Microelectrode Assemblies. Chem. Eur. J. 1999, 5, 456-465. ‘’The field of electrochemical logic gates has seen significant research activity. Amatore and colleagues were among the early pioneers in this area, showcasing the capabilities of paired-band microelectrode assemblies. These assemblies successfully imitate the behaviour of neuronal synapses and are capable of performing Boolean logic operations. Amatore et al. demonstrated that artificial neurons utilising coupled double-band electrodes have the capability to operate as AND and OR logic gates [1],[2]. This is achieved by leveraging the distinctive diffusional cross-talk effects in close proximity to the electrodes. A detailed investigation was conducted on the time responses and theoretical features of these electrochemical logic gates. Expanding upon the encouraging progress made with paired microband electrodes as electrochemical logic gates, our current research focuses on the development of protenoid microsphere-based logic gates. Protenoid microspheres serve as a biomolecular platform that integrates the propagation, transmission, and detection of signals, similar to how natural neurons function.’’ Some other references to electrochemica logic systems may be also added for completness of the picture. We have added literature from the following papers to our paper: Zhang, L., Wang, H.X., Li, S. and Liu, M., 2020. Supramolecular chiroptical switches. Chemical Society Reviews, 49(24), pp.9095-9120. Willner, I., Willner, B. and Katz, E., 2007. Biomolecule–nanoparticle hybrid systems for bioelectronic applications. Bioelectrochemistry, 70(1), pp.2-11. Valov, I., Waser, R., Jameson, J.R. and Kozicki, M.N., 2011. Electrochemical metallization memories—fundamentals, applications, prospects. Nanotechnology, 22(25), p.254003. Experimental part misses some precision and crispness. First of all, thermal processing of aminoacids is a polycondensation process, as small molecules (water, ammonia) are side products (page 2). We have added a paragraph to page 2 of our paper to clarify this point. The paragraph we added is as follows: ‘’Thermal proteins (proteinoids)~\\cite{fox1992thermal} are synthesized by thermal polycondensation of amino acids. This involves heating a mixture of amino acids to 160-200~\\textsuperscript{o}C under an inert atmosphere, triggering a polycondensation reaction between the amino acids. Rather than a typical polymerization which links monomer units together directly, this is a step-growth polymerization which also generates small molecule byproducts like water and ammonia. The high temperatures cause bifunctional amino acids like glutamic acid to cyclize, which facilitates their role as solvents and initiators for the polycondensation reaction. The end result is a complex mixture of polypeptides with a broad distribution of chain lengths~\\cite{harada1958thermal,fox1992thermal}.’’ For the experimental part, detailed recipes, including amounts of reagents and detailed reaction conditions should be given. We have revised the sentences to specify the amount of amino acids used: "The amino acids weighing 5 g in total were heated to their boiling points and mixed together in equimolar amounts." ‘’The resulting mixture was then dissolved in water at a temperature of 80 degrees Celsius, while continuously mixing, to achieve a concentration of 10 mg/100 ml for each protenoid.’’ For electrochemical measurement the configuration of the potentiostat should be given (2- or 3-electrode setup, concentration of protenoids, supporting electrolyte...). More detailed information of the experimental setup should be given: types of electrodes, materials of electrodes, electrode spacing, etc. Detailed drawing of photo of the setup would be very beneficial. Applied voltage values should be given for all experiments. Here is a revised paragraph that has been expanded and improved for clarity and coherence: ‘’A two-electrode setup was used for the electrochemical measurements, with platinum and iridium-coated stainless steel sub-dermal needle electrodes (Spes Medica S.r.l., Italy) serving as the working and counter/reference electrodes. The electrodes were positioned approximately 10 mm apart in a protenoid solution containing 10 mg/100 ml protenoid in water as the supporting electrolyte. The potentiostat applied a constant dc potential (Edc) and measured the current response at 0.1 s intervals for 25,000 s. For the results shown, Edc was 0.01 V, with no initial equilibration period. The proteinoids exhibited varying spiking frequency and amplitude when exposed to different applied potentials, specifically positive and negative Edc values. Communication with the protenoids was established using this two-electrode electrochemical cell connected to the potentiostat.’’ Authors present detailed analysis of signal statistical features, but use single current measurements for logic analysis - how the time-dependent variability of the signal (Figure 4 and 9) translate into logic performance. It may not be relevant, but should be briefly discussed, as the relevance of signal variability may potentially influence the performance ,especially when different protenoid will have significantly different time scale of variability. We have added the following text in the discussion section: ‘’Although our analysis primarily focuses on using single current measurements for implementing logic gates, we acknowledge that the protenoid signals display time-dependent variability, as demonstrated in Figure~\\ref{savdsbfgsndghmjmk,k}. Distinct time scales are observed for the spiking behaviour and noise characteristics of different proteonid types. The variability in the signals over time has the potential to affect the reliability and precision of logic operations, especially when using proteinoids with significantly different dynamics. Logic gating that relies on transient spikes may be impacted if the timing of the spikes is inconsistent. In order to obtain consistent logic inputs from protenoid currents that have a high degree of variability, it may be necessary to employ suitable signal processing or filtering techniques. Further investigation is needed to understand the impact of signal noise and fluctuations on logic accuracy. The present study showcases the use of simple DC signal levels to perform proof-of-concept logic operations. This serves as a starting point for further investigation into more intricate signal processing methods that can help reduce time-dependent variations.’’ Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0289433.r002
1 Sep 2023 Decision Letter - Jianhui Liu, Editor Logical gates in ensembles of proteinoid microspheres PONE-D-23-22314R1 Dear Dr. Mougkogiannis, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jianhui Liu Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper was well revised and can be published in the present version. All requests made by the reviewers were satisfied. The paper is indeed good and its urgent publication will be beneficial for the experts in this research area. Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0289433.r003
Formally Accepted
7 Sep 2023 Acceptance Letter - Jianhui Liu, Editor PONE-D-23-22314R1 Logical gates in ensembles of proteinoid microspheres Dear Dr. Mougkogiannis: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Jianhui Liu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289433.r004

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