Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media
Abstract
:1. Introduction
2. Materials and Methods
2.1. Three-Dimensional Design and Printing of PLA Models
2.2. Surface Modification and Enzyme Immobilization on PLA Models
2.3. Activity of Immobilized CALB on PLA Well plates
2.4. Preparation of Deep Eutectic Solvents (DESs)
2.5. Effect of DESs on the Activity of Immobilized CALB
2.6. Effect of DESs on the Stability of Immobilized CALB
2.7. Storage Stability of Immobilized CALB on PLA Well plates
2.8. Spectroscopic and Morphological Characterization
2.9. Assays in PLA Microreactors
2.9.1. Activity Measurement of Immobilized CALB in PLA Microreactors
2.9.2. Effect of Enzyme Concentration
2.9.3. Determination of the Immobilization Yield
2.9.4. Effect of Flow Rate in the Presence or Absence of DES
2.9.5. Operational Stability in the Presence or Absence of DES
2.9.6. Thermal Stability of Immobilized CALB in 100% DES
2.9.7. Transesterification Reactions in Microreactor and Batch Systems
2.9.8. HPLC Analysis
3. Results
3.1. Characterization Studies of Modified 3D-Printed PLA Scaffolds
3.1.1. X-ray Photoelectron Spectroscopy (XPS)
3.1.2. Scanning Electron Microscopy (SEM)
3.2. Enzyme Catalytic Performance in Well plates
3.2.1. Effect of DESs on the Activity of Immobilized CALB
3.2.2. Effect of DESs on the Stability of Immobilized CALB
3.2.3. Storage Stability of Immobilized CALB on PLA Well plates
3.3. Enzyme Performance in PLA Microreactors
3.3.1. Effect of Enzyme Concentration
3.3.2. Operational Stability in the Presence or Absence of DES
3.3.3. Thermal Stability of Immobilized CALB in Microreactor
3.3.4. Continuous Flow Kinetics of Immobilized Lipase in the Presence or Absence of DES
3.4. Implementation of the Enzyme Microreactor in a Transesterification Reaction
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
- Dixit, C.K.; Kadimisetty, K.; Rusling, J. 3D-Printed Miniaturized Fluidic Tools in Chemistry and Biology. TrAC Trends Anal. Chem. 2018, 106, 37–52. [Google Scholar] [CrossRef] [PubMed]
- Kong, Y.L.; Gupta, M.K.; Johnson, B.N.; McAlpine, M.C. 3D Printed Bionic Nanodevices. Nano Today 2016, 11, 330–350. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhou, Y. The Recent Development and Applications of Fluidic Channels by 3D Printing. J. Biomed. Sci. 2017, 24, 80. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Basso, A.; Serban, S. Industrial Applications of Immobilized Enzymes—A Review. Mol. Catal. 2019, 479, 110607. [Google Scholar] [CrossRef]
- Gkantzou, E.; Chatzikonstantinou, A.V.; Fotiadou, R.; Giannakopoulou, A.; Patila, M.; Stamatis, H. Trends in the Development of Innovative Nanobiocatalysts and Their Application in Biocatalytic Transformations. Biotechnol. Adv. 2021, 51, 107738. [Google Scholar] [CrossRef]
- Lyu, X.; Gonzalez, R.; Horton, A.; Li, T. Immobilization of Enzymes by Polymeric Materials. Catalysts 2021, 11, 1211. [Google Scholar] [CrossRef]
- Pose-Boirazian, T.; Martínez-Costas, J.; Eibes, G. 3D Printing: An Emerging Technology for Biocatalyst Immobilization. Macromol. Biosci. 2022, 22, 2200110. [Google Scholar] [CrossRef]
- Shao, Y.; Liao, Z.; Gao, B.; He, B. Emerging 3D Printing Strategies for Enzyme Immobilization: Materials, Methods, and Applications. ACS Omega 2022, 7, 11530–11543. [Google Scholar] [CrossRef]
- Ma, Z.; Mi, Y.; Han, X.; Li, H.; Tian, M.; Duan, Z.; Fan, D.; Ma, P. Transformation of Ginsenoside via Deep Eutectic Solvents Based on Choline Chloride as an Enzymatic Reaction Medium. Bioprocess. Biosyst. Eng. 2020, 43, 1195–1208. [Google Scholar] [CrossRef]
- Fotiadou, R.; Bellou, M.G.; Spyrou, K.; Yan, F.; Rudolf, P.; Gournis, D.; Stamatis, H. Effect of Deep Eutectic Solvents on the Biocatalytic Properties of β-Glucosidase@ZnOFe Nano-Biocatalyst. Sustain Chem. Pharm. 2022, 30, 100886. [Google Scholar] [CrossRef]
- Sun, H.; Xin, R.; Qu, D.; Yao, F. Mechanism of Deep Eutectic Solvents Enhancing Catalytic Function of Cytochrome P450 Enzymes in Biosynthesis and Organic Synthesis. J. Biotechnol. 2020, 323, 264–273. [Google Scholar] [CrossRef] [PubMed]
- Chan, J.C.; Zhang, B.; Martinez, M.; Kuruba, B.; Brozik, J.; Kang, C.; Zhang, X. Structural Studies of Myceliophthora Thermophila Laccase in the Presence of Deep Eutectic Solvents. Enzyme Microb. Technol. 2021, 150, 109890. [Google Scholar] [CrossRef] [PubMed]
- Toledo, M.L.; Pereira, M.M.; Freire, M.G.; Silva, J.P.A.; Coutinho, J.A.P.; Tavares, A.P.M. Laccase Activation in Deep Eutectic Solvents. ACS Sustain. Chem. Eng. 2019, 7, 11806–11814. [Google Scholar] [CrossRef]
- Ribeiro, B.D.; de Carvalho Iff, L.; Coelho, M.A.Z.; Marrucho, I.M. Influence of Betaine- and Choline-Based Eutectic Solvents on Lipase Activity. Curr. Biochem. Eng. 2019, 5, 57–68. [Google Scholar] [CrossRef]
- Domínguez de María, P.; Guajardo, N.; Kara, S. Enzyme Catalysis: In DES, with DES, and in the Presence of DES. In Deep Eutectic Solvents: Synthesis, Properties, and Applications; Wiley: New York, NY, USA, 2019; pp. 257–271. ISBN 9783527818471. [Google Scholar]
- Pätzold, M.; Siebenhaller, S.; Kara, S.; Liese, A.; Syldatk, C.; Holtmann, D. Deep Eutectic Solvents as Efficient Solvents in Biocatalysis. Trends Biotechnol. 2019, 37, 943–959. [Google Scholar] [CrossRef]
- Guajardo, N.; Schrebler, R.A.; Domínguez de María, P. From Batch to Fed-Batch and to Continuous Packed-Bed Reactors: Lipase-Catalyzed Esterifications in Low Viscous Deep-Eutectic-Solvents with Buffer as Cosolvent. Bioresour. Technol. 2019, 273, 320–325. [Google Scholar] [CrossRef]
- Guajardo, N.; Domínguez de María, P.; Canales, R. Integrating Biocatalysis with Viscous Deep Eutectic Solvents in Lab-On-A-Chip Microreactors. ChemSusChem 2022, 15, 1–8. [Google Scholar] [CrossRef]
- Zeng, C.-X.; Qi, S.-J.; Xin, R.-P.; Yang, B.; Wang, Y.-H. Enzymatic Selective Synthesis of 1,3-DAG Based on Deep Eutectic Solvent Acting as Substrate and Solvent. Bioprocess. Biosyst. Eng. 2015, 38, 2053–2061. [Google Scholar] [CrossRef]
- Hümmer, M.; Kara, S.; Liese, A.; Huth, I.; Schrader, J.; Holtmann, D. Synthesis of (-)-Menthol Fatty Acid Esters in and from (-)-Menthol and Fatty Acids—Novel Concept for Lipase Catalyzed Esterification Based on Eutectic Solvents. Mol. Catal. 2018, 458, 67–72. [Google Scholar] [CrossRef]
- Ilyas, R.A.; Sapuan, S.M.; Harussani, M.M.; Hakimi, M.Y.A.Y.; Haziq, M.Z.M.; Atikah, M.S.N.; Asyraf, M.R.M.; Ishak, M.R.; Razman, M.R.; Nurazzi, N.M.; et al. Polylactic Acid (PLA) Biocomposite: Processing, Additive Manufacturing and Advanced Applications. Polymers 2021, 13, 1326. [Google Scholar] [CrossRef]
- Gkantzou, E.; Skonta, A.; Tsakni, A.; Polydera, A.; Moschovas, D.; Spyrou, K.; Avgeropoulos, A.; Gournis, D.; Houhoula, D.; Stamatis, H. 3D Printed PLA Enzyme Microreactors: Characterization and Application for the Modification of Bioactive Compounds. J. Biotechnol. 2022, 350, 75–85. [Google Scholar] [CrossRef] [PubMed]
- Ye, J.; Chu, T.; Chu, J.; Gao, B.; He, B. A Versatile Approach for Enzyme Immobilization Using Chemically Modified 3D-Printed Scaffolds. ACS Sustain. Chem. Eng. 2019, 7, 18048–18054. [Google Scholar] [CrossRef]
- Kumar, A.; Dhar, K.; Kanwar, S.S.; Arora, P.K. Lipase Catalysis in Organic Solvents: Advantages and Applications. Biol. Proced. Online 2016, 18, 2. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Erokhin, K.S.; Gordeev, E.G.; Ananikov, V.P. Revealing Interactions of Layered Polymeric Materials at Solid-Liquid Interface for Building Solvent Compatibility Charts for 3D Printing Applications. Sci. Rep. 2019, 9, 20177. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dudu, A.I.; Bencze, L.C.; Paizs, C.; Toşa, M.I. Deep Eutectic Solvents—a New Additive in the Encapsulation of Lipase B from Candida Antarctica: Biocatalytic Applications. React. Chem. Eng. 2022, 7, 442–449. [Google Scholar] [CrossRef]
- Guajardo, N.; Domínguez de María, P. Continuous Biocatalysis in Environmentally-Friendly Media: A Triple Synergy for Future Sustainable Processes. ChemCatChem 2019, 11, 3128–3137. [Google Scholar] [CrossRef]
- Gkantzou, E.; Skonta, A.; Vasios, A.-G.; Stamatis, H. 3D Printed Polylactic Acid Well-Plate for Multi-Enzyme Immobilization. In Methods in Molecular Biology; Stamatis, H., Ed.; Springer US: New York, NY, USA, 2022; Volume 2487, pp. 163–175. [Google Scholar]
- Baran, E.; Erbil, H. Surface Modification of 3D Printed PLA Objects by Fused Deposition Modeling: A Review. Colloids Interfaces 2019, 3, 43. [Google Scholar] [CrossRef] [Green Version]
- Jaidev, L.R.; Chatterjee, K. Surface Functionalization of 3D Printed Polymer Scaffolds to Augment Stem Cell Response. Mater. Des. 2019, 161, 44–54. [Google Scholar] [CrossRef]
- Hildebrand, H.F.; Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F. Surface Coatings for Biological Activation and Functionalization of Medical Devices. Surf. Coat. Technol. 2006, 200, 6318–6324. [Google Scholar] [CrossRef]
- Douglas, J.F. How Does Surface Roughness Affect Polymer-Surface Interactions? Macromolecules 1989, 22, 3707–3716. [Google Scholar] [CrossRef]
- Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
- Skonta, A.; Gkantzou, E.; Spyrou, K.; Spyrou, S.; Polydera, A.; Gournis, D.; Stamatis, H. 3D Printed Polylactic Acid (PLA) Well Plates for Enzyme Inhibition Studies: The Case of Pancreatic Lipase. Catal. Res. 2022, 2, 1–19. [Google Scholar] [CrossRef]
- Zaak, H.; Fernandez-Lopez, L.; Otero, C.; Sassi, M.; Fernandez-Lafuente, R. Improved Stability of Immobilized Lipases via Modification with Polyethylenimine and Glutaraldehyde. Enzyme Microb. Technol. 2017, 106, 67–74. [Google Scholar] [CrossRef] [PubMed]
- Rios, N.S.; Arana-Peña, S.; Mendez-Sanchez, C.; Lokha, Y.; Cortes-Corberan, V.; Gonçalves, L.R.B.; Fernandez-Lafuente, R. Increasing the Enzyme Loading Capacity of Porous Supports by a Layer-by-Layer Immobilization Strategy Using PEI as Glue. Catalysts 2019, 9, 576. [Google Scholar] [CrossRef] [Green Version]
- Patel, V.; Shah, C.; Deshpande, M.; Madamwar, D. Zinc Oxide Nanoparticles Supported Lipase Immobilization for Biotransformation in Organic Solvents: A Facile Synthesis of Geranyl Acetate, Effect of Operative Variables and Kinetic Study. Appl. Biochem. Biotechnol. 2016, 178, 1630–1651. [Google Scholar] [CrossRef]
- Chalmpes, N.; Patila, M.; Kouloumpis, A.; Alatzoglou, C.; Spyrou, K.; Subrati, M.; Polydera, A.C.; Bourlinos, A.B.; Stamatis, H.; Gournis, D. Graphene Oxide–Cytochrome c Multilayered Structures for Biocatalytic Applications: Decrypting the Role of Surfactant in Langmuir–Schaefer Layer Deposition. ACS Appl. Mater. Interfaces 2022, 14, 26204–26215. [Google Scholar] [CrossRef]
- Valerga, A.; Batista, M.; Fernandez-Vidal, S.; Gamez, A. Impact of Chemical Post-Processing in Fused Deposition Modelling (FDM) on Polylactic Acid (PLA) Surface Quality and Structure. Polymers 2019, 11, 566. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Valerga, A.P.; Fernandez-Vidal, S.R.; Girot, F.; Gamez, A.J. On the Relationship between Mechanical Properties and Crystallisation of Chemically Post-Processed Additive Manufactured Polylactic Acid Pieces. Polymers 2020, 12, 941. [Google Scholar] [CrossRef] [Green Version]
- Elgharbawy, A.A. Shedding Light on Lipase Stability in Natural Deep Eutectic Solvents. Chem. Biochem. Eng. Q. 2018, 32, 359–370. [Google Scholar] [CrossRef]
- Kim, S.H.; Park, S.; Yu, H.; Kim, J.H.; Kim, H.J.; Yang, Y.-H.; Kim, Y.H.; Kim, K.J.; Kan, E.; Lee, S.H. Effect of Deep Eutectic Solvent Mixtures on Lipase Activity and Stability. J. Mol. Catal. B Enzym. 2016, 128, 65–72. [Google Scholar] [CrossRef]
- Nian, B.; Cao, C.; Liu, Y. How Candida Antarctica Lipase B Can Be Activated in Natural Deep Eutectic Solvents: Experimental and Molecular Dynamics Studies. J. Chem. Technol. Biotechnol. 2020, 95, 86–93. [Google Scholar] [CrossRef]
- Papadopoulou, A.A.; Tzani, A.; Polydera, A.C.; Katapodis, P.; Voutsas, E.; Detsi, A.; Stamatis, H. Green Biotransformations Catalysed by Enzyme-Inorganic Hybrid Nanoflowers in Environmentally Friendly Ionic Solvents. Environ. Sci. Pollut. Res. 2018, 25, 26707–26714. [Google Scholar] [CrossRef] [PubMed]
- Álvarez, M.S.; Longo, M.A.; Deive, F.J.; Rodríguez, A. Synthesis and Characterization of a Lipase-Friendly DES Based on Cholinium Dihydrogen Phosphate. J. Mol. Liq. 2021, 340, 117230. [Google Scholar] [CrossRef]
- Xu, P.; Zheng, G.-W.; Zong, M.-H.; Li, N.; Lou, W.-Y. Recent Progress on Deep Eutectic Solvents in Biocatalysis. Bioresour. Bioprocess. 2017, 4, 34. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fredes, Y.; Chamorro, L.; Cabrera, Z. Increased Selectivity of Novozym 435 in the Asymmetric Hydrolysis of a Substrate with High Hydrophobicity Through the Use of Deep Eutectic Solvents and High Substrate Concentrations. Molecules 2019, 24, 792. [Google Scholar] [CrossRef] [Green Version]
- Nicolás, P.; Lassalle, V.; Ferreira, M.L. Immobilization of CALB on Lysine-Modified Magnetic Nanoparticles: Influence of the Immobilization Protocol. Bioprocess. Biosyst. Eng. 2018, 41, 171–184. [Google Scholar] [CrossRef]
- Yu, D.; Zhang, X.; Wang, T.; Geng, H.; Wang, L.; Jiang, L.; Elfalleh, W. Immobilized Candida Antarctica Lipase B (CALB) on Functionalized MCM-41: Stability and Catalysis of Transesterification of Soybean Oil and Phytosterol. Food Biosci. 2021, 40, 100906. [Google Scholar] [CrossRef]
- Gkantzou, E.; Govatsi, K.; Chatzikonstantinou, A.V.; Yannopoulos, S.N.; Stamatis, H. Development of a ZnO Nanowire Continuous Flow Microreactor with β-Glucosidase Activity: Characterization and Application for the Glycosylation of Natural Products. ACS Sustain. Chem. Eng. 2021, 9, 7658–7667. [Google Scholar] [CrossRef]
- Gumel, A.M.; Annuar, M.S.M. Thermomyces Lanuginosus Lipase-Catalyzed Synthesis of Natural Flavor Esters in a Continuous Flow Microreactor. 3 Biotech 2016, 6, 24. [Google Scholar] [CrossRef] [Green Version]
- Liu, X.; Meng, X.-Y.; Xu, Y.; Dong, T.; Zhang, D.-Y.; Guan, H.-X.; Zhuang, Y.; Wang, J. Enzymatic Synthesis of 1-Caffeoylglycerol with Deep Eutectic Solvent under Continuous Microflow Conditions. Biochem. Eng. J. 2019, 142, 41–49. [Google Scholar] [CrossRef]
- Carvalho, F.; Marques, M.; Fernandes, P. Sucrose Hydrolysis in a Bespoke Capillary Wall-Coated Microreactor. Catalysts 2017, 7, 42. [Google Scholar] [CrossRef] [Green Version]
- Hafeez, S.; Aristodemou, E.; Manos, G.; Al-Salem, S.M.; Constantinou, A. Modelling of Packed Bed and Coated Wall Microreactors for Methanol Steam Reforming for Hydrogen Production. RSC Adv. 2020, 10, 41680–41692. [Google Scholar] [CrossRef] [PubMed]
- Matosevic, S.; Lye, G.J.; Baganz, F. Immobilised Enzyme Microreactor for Screening of Multi-Step Bioconversions: Characterisation of a de Novo Transketolase-ω-Transaminase Pathway to Synthesise Chiral Amino Alcohols. J. Biotechnol. 2011, 155, 320–329. [Google Scholar] [CrossRef]
- Compton, D.L.; Laszlo, J.A.; Evans, K.O. Antioxidant Properties of Feruloyl Glycerol Derivatives. Ind. Crops Prod. 2012, 36, 217–221. [Google Scholar] [CrossRef]
- Yao, N.; Sun, S. Hydrophilic Glyceryl Ferulates Preparation Catalyzed by Free Lipase B from Candida Antartica. J. Oleo. Sci. 2020, 69, 43–53. [Google Scholar] [CrossRef] [Green Version]
- Sun, S.; Chen, X. Kinetics of Enzymatic Synthesis of Monoferuloyl Glycerol and Diferuloyl Glycerol by Transesterification in [BMIM]PF6. Biochem. Eng. J. 2015, 97, 25–31. [Google Scholar] [CrossRef]
- Wang, J.; Gu, S.-S.; Cui, H.-S.; Wu, X.-Y.; Wu, F.-A. A Novel Continuous Flow Biosynthesis of Caffeic Acid Phenethyl Ester from Alkyl Caffeate and Phenethanol in a Packed Bed Microreactor. Bioresour. Technol. 2014, 158, 39–47. [Google Scholar] [CrossRef]
Parameter | Value |
---|---|
Layer Height | 0.16 mm |
Infill Density | 100% |
Printing Temperature | 200 °C |
Build Plate Temperature | 50 °C |
Print Speed | 80 mm/s |
Nozzle Size | 0.4 mm |
Filament Diameter | 1.75 mm |
DES [HBA: HBD (: HBD)] | Molar Ratio | Name Code |
---|---|---|
Choline Chloride: Glycerol | 1:2 | ChCl:Gly |
Choline Chloride: Butylene Glycol | 1:4 | ChCl:BG |
Choline Chloride: Propylene Glycol | 1:2 | ChCl:PG |
Choline Chloride: Ethylene Glycol | 1:2 | ChCl:EG |
Betaine: Glycerol | 1:3 | Bet:Gly |
Betaine: Ethylene Glycol | 1:3 | Bet:EG |
Choline Chloride: Glycerol: Ethylene Glycol | 1:1:1 | ChCl:Gly:EG |
Choline Chloride: Urea: Ethylene Glycol | 1:1:1 | ChCl:U:EG |
Choline Chloride: U: Glycerol | 1:1:1 | ChCl:U:Gly |
Choline Chloride: Urea | 1:2 | ChCl:U |
Ethylammonium Chloride: Urea | 1:1.5 | EAC:U |
Ethylammonium Chloride: Ethylene Glycol | 1:1.5 | EAC:EG |
Ethylammonium Chloride: Glycerol | 1:1.5 | EAC:Gly |
Choline Dihydrogen Phosphate: Glycerol | 1:3 | Chol DHP:Gly |
Choline Dihydrogen Phosphate: Ethylene Glycol | 1:3 | Chol DHP:EG |
Choline Chloride: Fructose: H2O | 5:2:5 | ChCl:Fru:H2O |
Choline Chloride: Glucose: H2O | 5:2:5 | ChCl:Glc:H2O |
Flow Rate (μL min−1) | Km(app) (mM) | ||
---|---|---|---|
0% v/v DES (R2) | 10% v/v Bet:Gly (R2) | ||
Free CALB | - | 0.35 (0.88) | 0.39 (0.93) |
Microreactor immobilized CALB | 75 | 0.62 (0.99) | 0.30 (0.78) |
150 | 0.59 (0.98) | 0.38 (0.98) | |
300 | 0.60 (0.99) | 0.45 (0.95) | |
600 | 0.50 (0.98) | 0.83 (0.94) |
Biocatalytic System | Amount of Enzyme (μg) | Reaction Volume (mL) | Amount of Product (μg) | Reaction Time (h) | Productivity (μg Product min−1 μg enzyme−1) |
---|---|---|---|---|---|
Microbioreactor | 4.5 | 0.15 | 18.6 | 24 runs × 10 min | 17.20 × 10−3 |
Immobilized CALB (Novozyme 435) | 30 | 1 | 5.4 | 4 | 0.75 × 10−3 |
Free CALB | 30 | 1 | 97.9 | 4 | 13.60 × 10−3 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Bellou, M.G.; Gkantzou, E.; Skonta, A.; Moschovas, D.; Spyrou, K.; Avgeropoulos, A.; Gournis, D.; Stamatis, H. Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media. Micromachines 2022, 13, 1954. https://doi.org/10.3390/mi13111954
Bellou MG, Gkantzou E, Skonta A, Moschovas D, Spyrou K, Avgeropoulos A, Gournis D, Stamatis H. Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media. Micromachines. 2022; 13(11):1954. https://doi.org/10.3390/mi13111954
Chicago/Turabian StyleBellou, Myrto G., Elena Gkantzou, Anastasia Skonta, Dimitrios Moschovas, Konstantinos Spyrou, Apostolos Avgeropoulos, Dimitrios Gournis, and Haralambos Stamatis. 2022. "Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media" Micromachines 13, no. 11: 1954. https://doi.org/10.3390/mi13111954
APA StyleBellou, M. G., Gkantzou, E., Skonta, A., Moschovas, D., Spyrou, K., Avgeropoulos, A., Gournis, D., & Stamatis, H. (2022). Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media. Micromachines, 13(11), 1954. https://doi.org/10.3390/mi13111954