Advances in Droplet-Based Microfluidic High-Throughput Screening of Engineered Strains and Enzymes Based on Ultraviolet, Visible, and Fluorescent Spectroscopy
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThere are many review articles on analytical methods of microfluidic droplets. This manuscript centers on spectroscopic methods but lacks an in-depth summary of all the prior work. First, Fig.2 is so confusing that the top row shows droplet analysis but the bottom row shows droplet sorting. They are two different droplet microfluidic techniques.
I suggest selecting representative works in each category of the spectrometric methods and putting them in figures. All figures need detailed captions.
Section 5 is not necessary and electrochemical detection does not belong to spectroscopy.
The authors should be more clear on what message they would like to convey and focus on, not blur techniques and confuse readers.
As the title points out "advance", it was expected in this review more advanced methods incorporating spectroscopic detection (i.e. machine learning, statistics tool, AI etl) be introduced but not just list all the traditional methods that were developed two decades ago.
Comments on the Quality of English LanguageNone
Author Response
Reviewer #1:
Comment 1: There are many review articles on analytical methods of microfluidic droplets. This manuscript centers on spectroscopic methods but lacks an in-depth summary of all the prior work. First, Fig.2 is so confusing that the top row shows droplet analysis but the bottom row shows droplet sorting. They are two different droplet microfluidic techniques. I suggest selecting representative works in each category of the spectrometric methods and putting them in figures. All figures need detailed captions.
Response: Thank you for your comments. In lines 308-321 on page 8, droplet analysis and droplet sorting are closely related in droplet microfluidics. Droplet analysis is the expressed principle of signal construction, while droplet sorting relies on signals for sorting. The reader can see more clearly how droplet analysis and droplet sorting are interrelated and applied to droplet microfluidics. The legend has been modified and relevant literature has been inserted as shown below:
In the screening system of droplet microfluidics, microdroplets of picoliter (pL) volume as reactors are used to encapsulate enzyme genes, artificially constructed metabolic pathways, gene expression products, etc., which are coloured using specific fluorescent probes (Fig. 2 top row). Microfluidic detection/sorting chip for efficient analysis and sorting of these microdroplets (Fig. 2 bottom row).
Figure 2A: Screening based on fluorescence spectroscopy is dependent on enzyme reactions. The enzymatic reaction products have a strong fluorescent signal, which is used to quantify the enzyme activity based on the change in fluorescence intensity before and after the reaction (1).
Figure 2B: Small molecules can be indirectly converted to fluorescent signals using an enzyme coupling reaction strategy (2).
Figure 2C: Assay screening based on transcription factors (TFs) (3). TFs-based biosensors, which can respond to different types of effectors, are widely used for HTS of metabolites, including amino acids, organic acids, flavonoids, sugars and lipids.
Figure 2D: Biosensors based on RNA Spinach (53). RNA Spinach is a newly discovered special structure that generates stable signals when combined with fluorescent groups.
Comment 2: Section 5 is not necessary and electrochemical detection does not belong to spectroscopy.
Response: Thank you for your comments. We have deleted this section based on your comments.
Comment 3: The authors should be more clear on what message they would like to convey and focus on, not blur techniques and confuse readers.
Response: This article mainly describes high-throughput screening research based on UV, visible, and fluorescence spectroscopy in droplet microfluidics. However, as the potential of droplet microfluidics high-throughput screening technology is being explored, researchers are attracted to applying it to more targets, but it has been found that some target screening requires the establishment of complex coupling reactions that cannot achieve the goal of simplicity, speed, and convenience. There are also problems with some target molecules that cannot generate detectable signals. Incorporated with other detection methods, label-free detection strategies have been established, including electrochemical detection, mass spectrometry, Raman spectroscopy, nuclear magnetic resonance, and Fourier transform infrared spectroscopy. These label-free detection techniques serve as a complement to droplet microfluidics technology. We have made revisions based on your recommendation.
Conment 4: As the title points out "advance", it was expected in this review more advanced methods incorporating spectroscopic detection (i.e. machine learning, statistics tool, AI etl) be introduced but not just list all the traditional methods that were developed two decades ago.
Response: This article provides a review of recent literature on microfluidic droplet-based technologies, specifically droplet fluorescence-activated sorting (FADS) and absorbance-activated droplet sorting (AADS), which are relevant to high-throughput screening research based on spectral droplet microfluidics. Other droplet microfluidic techniques are also integrated into the discussion.
Reviewer 2 Report
Comments and Suggestions for AuthorsGeneral Comments
- More focus must be given to the biological applications of newly discovered enzyme variants in the cited works and their importance. Many of the citations show that the use of microfluidics/ microsystems with integrated sensors has enabled the discovery of improved variants of the following enzymes: enzymes such as glycosidase, lipase, peroxidase, protease, amylase, oxidase, and transaminase. However, there is insignificant discussion of the usefulness and biological/ industrial/ chemical/ commercial relevance of such (improved variants of) enzymes and their applications.
- The figure legends are, in most cases, extremely brief. Some text legends with fuller details would really help to improve the impactfulness of these figures.
- The outlook section at the end of the conclusions is too focused on the potential impact of technological advancements and lacks sufficient depth. The authors should clearly define why AI/ML will help to make such systems more widespread, as there is also the argument that it increases the start up cost and initial development complexity for such systems. In addition, the authors should look at cited works to help complete a small paragraph of potential enzymes which may be of interest in to the directed evolution and microfluidic community and why.
Specific Comments
- There is some recent literature that is not included in this review article, for example, a 2023 paper by the Hollfelder group (first author - Elliot Medcalf) is not included or discussed. I suggest doing a further literature search to find new articles in the last few years and discussing these in depth within the review.
- Line 296: "However, the throughput was only 2500 droplets/s, which was 1000 times slower than FADS". To my knowledge, no FADS system has a throughput of 2.5M droplets per seconds (1000 x 2500).
Author Response
Reviewer #2:Comment 1: More focus must be given to the biological applications of newly discovered enzyme variants in the cited works and their importance. Many of the citations show that the use of microfluidics/ microsystems with integrated sensors has enabled the discovery of improved variants of the following enzymes: enzymes such as glycosidase, lipase, peroxidase, protease, amylase, oxidase, and transaminase. However, there is insignificant discussion of the usefulness and biological/ industrial/ chemical/ commercial relevance of such (improved variants of) enzymes and their applications.
Response: Thank you for your comments. In lines 209-215 on page 6, we have summarized the effects of β-glucosidase and protease.β-glucosidase can cleave the cellulose disaccharide formed by the endoglucanase hydrolysis of cellulose. It can be used in industry for the hydrolysis of cellulose and lactose (4, 5). Protease is an important enzyme in industry. Although many microorganisms are used for producing protease, only a few proteases are commercially applied and usually require extensive protein engineering, especially directed evolution. Tu et al. (6) were the first to use flow cytometry to screen protease and analyze its resistance improvement. In addition, it was found that the targeted evolution of protease can enhance its thermal stability and resistance to antioxidants.
Comment 2: The figure legends are, in most cases, extremely brief. Some text legends with fuller details would really help to improve the impactfulness of these figures.
Response: Thank you for your comments. In lines 308-321 on page 8, droplet analysis and droplet sorting are closely related in droplet microfluidics. Droplet analysis is the expressed principle of signal construction, while droplet sorting relies on signals for sorting. The reader can see more clearly how droplet analysis and droplet sorting are interrelated and applied to droplet microfluidics. The legend has been modified and relevant literature has been inserted as shown below:
In the screening system of droplet microfluidics, microdroplets of picoliter (pL) volume as reactors are used to encapsulate enzyme genes, artificially constructed metabolic pathways, gene expression products, etc., which are coloured using specific fluorescent probes (Fig. 2 top row). Microfluidic detection/sorting chip for efficient analysis and sorting of these microdroplets (Fig. 2 bottom row).
Figure 2A: Screening based on fluorescence spectroscopy is dependent on enzyme reactions.
The enzymatic reaction products have a strong fluorescent signal, which is used to quantify the enzyme activity based on the change in fluorescence intensity before and after the reaction (1).
Figure 2B: Small molecules can be indirectly converted to fluorescent signals using an enzyme coupling reaction strategy (2).
Figure 2C: Assay screening based on transcription factors (TFs) (3). TFs-based biosensors, which can respond to different types of effectors, are widely used for HTS of metabolites, including amino acids, organic acids, flavonoids, sugars and lipids.
Figure 2D: Biosensors based on RNA Spinach (53). RNA Spinach is a newly discovered special structure that generates stable signals when combined with fluorescent groups.
Comment 3: The outlook section at the end of the conclusions is too focused on the potential impact of technological advancements and lacks sufficient depth. The authors should clearly define why AI/ML will help to make such systems more widespread, as there is also the argument that it increases the start up cost and initial development complexity for such systems. In addition, the authors should look at cited works to help complete a small paragraph of potential enzymes which may be of interest in to the directed evolution and microfluidic community and why?
Response: Thank you for your comments. In lines 417-438 on page 15, Changes have been made to your suggestions, as follows: Enzymes are the most important biocatalysts in nature and have been applied in various fields after thousands of years of natural evolution. However, natural enzymes of-ten cannot meet the requirements of industry, making artificial selection and screening increasingly important. Directed evolution has been a key strategy for generating enzymes with desired characteristics such as high selectivity, but experimental barriers and the cost of analyzing large mutant libraries have limited these efforts. However, the biggest problem faced by directed evolution is that traditional microplate screening methods cannot meet the demands of high-capacity library screening. Meanwhile, the industrial and pharmaceutical sectors continue to have a fast-growing demand for novel and improved microbial catalysts. Therefore, directed evolution of enzymes and the discovery of new enzymes have directly benefited from the development of droplet microfluidics, which provide individual reaction environments and larger screening capacities for enzyme reactions. Enzymes that may require directed evolution in the future include esterases, cellulases, glucose dehydrogenases, and plastic-degrading enzymes. Carboxylesterases, for example, are detoxifying enzymes that can hydrolyze cocaine, chlorpyrifos, and other compounds. Cellulases find applications in the food and beverage industry where they can hydrolyze cell walls and lignocellulosic substrates. Glucose dehydrogenase converts glucose into gluconic acid. Plastic waste management has been one of the major ecological challenges in our society, and plastic-degrading enzymes can offer a solution to this problem. With the development of more new technologies, such as artificial intelligence and bioprinting, they will also provide more applications for microfluidic sorting devices.
Comment 4: - There is some recent literature that is not included in this review article, for example, a 2023 paper by the Hollfelder group (first author - Elliot Medcalf) is not included or discussed. I suggest doing a further literature search to find new articles in the last few years and discussing these in depth within the review.
Response: Thank you for your comments. We have added new recent literatures in the revised manuscrpt, including “Ultra-High-Throughput Absorbance-Activated Droplet Sorting for Enzyme Screening at Kilohertz Frequencies,”, “Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction” and “Research and application progress of microdroplets high-throughput screening methods”.
Comment 5:- Line 296: "However, the throughput was only 2500 droplets/s, which was 1000 times slower than FADS". To my knowledge, no FADS system has a throughput of 2.5M droplets per seconds (1000 x 2500).
Response: Page 9, line 344 Thank you for your comments. I'm sorry, there was a slight mistake. The original text of the article is: "Throughput of 0.7 samples s-1 is achieved with 98 % accuracy using a self-correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro", I apologize for the confusion. The necessary changes have been made.
Reviewer 3 Report
Comments and Suggestions for Authors
The authors have created a good compilation of ultraviolet, visual and fluorescence spectroscopic techniques applied to microfluidics based screening strategies. However it is unclear what the background and current state of art are. It would help to know the caveats and shortcomings of previous and current techniques, especially in the context of microfluidics.
Lastly, it is not clear how this review is pertinent to the general topic area for this journal.
L35-45: Please add citations to the statements about the requirements to evolve or modify. probability of beneficial mutations. The paragraph needs more citations in general.
L46: Consider rephrasing to not suggest that “lab on a chip” and microfluidics are synonymous. A clear distinction of the applications is made in Reference 1.
L47-48: The language is vague and uninformative.
L49: Statement about nanometer-scale channels needs citations.
L57: “Its greatest advantage…” is an opinion and subjective. If it truly is an advantage, there should be extensive citations added to reflect the widespread use of this advantage.
L65: The formatting has changed to add more line spacing. It should be consistent.
L71: Not clear what “enzyme-directed evolution” means. Is it directed evolution of enzymes?
L88/Figure 1: The figure caption needs to adequately describe the figure. It is not clear what the intent with different layers is. The logic in the arrangement and connections among the layers is not clear.
L102-108: This section is not clear. It is also a digression from the main purpose of the review.
L177-179: This is not correct. FRET involves either a donor-quencher or primary-secondary pair of molecules.
L191: Not clear what “indirectly convert” means
L231: RNA spinach needs citation
L300: Not clear how real time analysis is unique to Raman spectroscopy.
None of the sections discuss the caveats and shortcomings of the broad HTS technique categories or specific implementation. The authors are urged to include this information.
Also, there are no quantitative comparisons to non-microfludic methods. Such comparisons would be the basis of statements made by the authors of this review.
Finally, connections to fermentation need to be made for this article to fit with the topic area of the journal.
Author Response
Reviewer #3:
Comment 1: L35-45: Please add citations to the statements about the requirements to evolve or modify. probability of beneficial mutations. The paragraph needs more citations in general.
Response: Thank you for your comments. Thank you for your comments. In lines 38-39 on page 1Seven related references have been added in the revised manuscript.
Comment 2: L46: Consider rephrasing to not suggest that “lab on a chip” and microfluidics are synonymous. A clear distinction of the applications is made in Reference 1.
Response: Thank you for your comments. lines 50 -52 on page 1. The following modifications have been made: Microfluidic technology has emerged as a response to the multidisciplinary convergence.
Comment 3: L47-48: The language is vague and uninformative.
Response: Thank you for your comments. In lines 50 -52 on page 1. The following modifications have been made: Microfluidic technology has emerged as a response to the multidisciplinary convergence. It utilizes chemistry, fluid physics, microelectronic materials, nanotechnology, and biotechnology.
Comment 4: L49: Statement about nanometer-scale channels needs citations
Response: Thank you for your comments. In lines 52 -53 on page 2. Two related references have been inserted
Comment 5: L57: “Its greatest advantage…” is an opinion and subjective. If it truly is an advantage, there should be extensive citations added to reflect the widespread use of this advantage.
Response: Thank you for your comments. In lines 61 -63 on page 2.The following modifications have been made: Its advantage lies in the ability to encapsulate single cells in droplets, where each droplet serves as an independent reaction system capable of culturing cells and producing metabolites or enzymes.
Comment 6: L65: The formatting has changed to add more line spacing. It should be consistent.
Response: Thank you for your comments. In lines 70 -72 on page 2. It has been modified in the revised manuscript.
Comment 7: L71: Not clear what “enzyme-directed evolution” means. Is it directed evolution of enzymes?
Response: Thank you for your comments. In lines 89 -91 on page 2. The two phrases have the same meaning and have been modified on your basis as follows: Through oil encapsulation, the adsorption in the flow channel can be reduced. Droplet encapsulation of individual bacteria allows for the synchronized directional evolution of enzymes during the screening process, making it widely applicable in enzyme directed evolution
Comment 8: L88/Figure 1: The figure caption needs to adequately describe the figure. It is not clear what the intent with different layers is. The logic in the arrangement and connections among the layers is not clear.
Response: Thank you for your comments. In lines 117-127 on page 4. By adding a specific legend, modified as follows: There are a total of 4 layers from the inside to the outside in Fig. 2. The first layer represents microfluidic high-throughput screening. The second layer represents main functions of microfluidic high-throughput screening, including dynamic analysis, detection, screening, and small molecule reactions. The third layer represents the specific application fields of microfluidic high-throughput screening, mainly including food, biologic medicine, textile industry, and chemical industry. The fourth layer represents the specific applications of microfluidic high-throughput screening, mainly including chemical synthesis, nanomaterials, single-cell analysis, antibody screening, drug delivery, strain/enzyme screening, enzyme activity detection, and pathogenic bacteria.
Comment 9: L102-108: This section is not clear. It is also a digression from the main purpose of the review.
Response: Thank you for your comments. In lines 142 -151 on page 5. The author believes that this part is still necessary. In the screening discussion based on ultraviolet spectrum, this part takes up a shorter space, although it shows that the detection can be carried out by adding pH indicator, metal ion chelating agent, enzyme reaction or chemical reaction coupling method, which shows that droplet microfluidic technology can achieve the purpose of screening
Comment 10: L177-179: This is not correct. FRET involves either a donor-quencher or primary-secondary pair of molecules
Response: Thank you for your comments. In lines 227 -229 on page 5. Modify as follows: Förster resonance energy transfer (FRET) is a special part of the enzymatic reaction strategy. After the substrate is catalyzed, the fluorescence wavelength will change strongly, which is often called the substrate as a fluorescent probe.
Comment 11: L191: Not clear what “indirectly convert” means.
Response: Thank you for your comments. In lines 238-242 on page 7. Because many small molecule metabolites cannot be directly detected by fluorescence, the enzyme coupling reaction strategy can indirectly convert these small molecule substances into fluorescence signals.
Comment 12: L231: RNA spinach needs citation.
Response: Thank you for your comments. In lines 283-284 on page 7, Thank you for your comments. Two related references have been inserted.
Comment 13: L300: Not clear how real time analysis is unique to Raman spectroscopy.
Response: The author simply wants to demonstrate the rapid and real-time advantages of Raman spectroscopy.
Comment 14: None of the sections discuss the caveats and shortcomings
Response: Thank you for your comments. In lines 97 -104 on page 3.To add the relevant content, as follows: Currently, FADS technology also has some drawbacks, such as the need to develop suitable fluorescent probes based on different enzyme molecules and catalytic reactions. For some enzyme molecules, rare cells, and target analytes that are small molecule drugs, there is a lack of applicable biomarkers and fluorescent probes, which limits its widespread application. To overcome these limitations, other types of droplet sorting technology, such as droplet sorting technology activated by different methods such as absorption, mass, and Raman, have been developed one after another.
Reference
- Hardiman E, Gibbs M, Reeves R, Bergquist P. Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production. Applied Biochemistry and Biotechnology. 2010;161(1):301-12.
- Abalde-Cela S, Gould A, Liu X, Kazamia E, Smith AG, Abell C. High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform. Journal of the Royal Society, Interface. 2015;12(106).
- Siedler S, Stahlhut SG, Malla S, Maury J, Neves AR. Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli. Metabolic Engineering. 2014;21:2-8.
- Turner P, Mamo G, Karlsson EN. Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microbial cell factories. 2007;6:9 -
- Nakkharat P, Haltrich D. Purification and characterisation of an intracellular enzyme with beta-glucosidase and beta-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58. Journal of biotechnology. 2006;123(3):304-13.
- Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U. A Flow Cytometry–Based Screening System for Directed Evolution of Proteases. SLAS Discovery. 2011;16(3):285-94.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThanks for implementing my recommended changes. Congratulations on the nice paper.
Author Response
Reviewer #2:
Comment 1: Thanks for implementing my recommended changes. Congratulations on the nice paper.
Response: Thank you for your affirmation.
Reviewer 3 Report
Comments and Suggestions for AuthorsMany of the comments have been appropriately addressed.
However, the authors are urged to use standard terminology, especially in the context of comment 10. The phenomenon that is described in their response seems like fluorescence activation, fluorogenesis and if the fluorescence spectra are changed, then that sounds like a chromic shift.
Author Response
Reviewer #3: Many of the comments have been appropriately addressed.
Comment 1: However, the authors are urged to use standard terminology, especially in the context of comment 10. The phenomenon that is described in their response seems like fluorescence activation, fluorogenesis and if the fluorescence spectra are changed, then that sounds like a chromic shift.
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In lines 217-219 on page 6, "Förster resonance energy transfer (FRET) is a special part of the enzymatic reaction strategy. After the substrate is catalyzed, the fluorescence wavelength will change strongly, which is often called the substrate as a fluorescent probe." was revised as “Förster resonance energy transfer (FRET) is a special part of the enzymatic reaction strategy. After the substrate is catalyzed, the fluorescence is activated, and this substrate is usually used as a fluorescent probe.”
