MICROFLUIDIC DEVICE FOR THE SYNTHESIS OF MICRO AND NANOPRODUCTS AND METHOD OF MANUFACTURING SAID MICROFLUIDIC DEVICE

Abstract

A microfluidic device for the synthesis of micro- and nano-products and a method for manufacturing said microfluidic device. The microfluidic device comprises an upper part made of photopolymerizable resin, which in turn comprises at least two inlet holes and at least one outlet hole; one or more lower parts made of photopolymerizable resin, which in turn comprise microchannels; and means for securing the upper part to the lower part(s). The manufacturing method comprises a step of modeling the parts of the microfluidic device; a step of 3D printing the parts of the microfluidic device; a step of post-processing the parts of the microfluidic device; and a step of assembling the microfluidic device.

Description

MICROFLUIDIC DEVICE FOR THE SYNTHESIS OF MICRO AND NANOPRODUCTS AND METHOD OF MANUFACTURING SAID MICROFLUIDIC DEVICE [1] The present invention relates to a microfluidic device for the synthesis of micro and nanoproducts and to a method of manufacturing the same. TECHNICAL FIELD OF THE INVENTION [2] The technical field to which the present invention belongs is that of physical apparatus for mixing fluids for the manufacture of micro and nanoproducts of interest in the chemical, cosmetic, food and pharmaceutical industries. STATE OF THE ART [3] Microfluidics is a constantly evolving scientific and technological field. In recent years, microfluidic devices, also known as chips, have shown unique advantages in the synthesis of micro- and nanoproducts, the latter being efficient vehicles for drug encapsulation and delivery [Liu, D., Zhang, H., Fontana, F., Hirvonen, JT & Santos, HA. Current developments and applications of microfluidic technology toward clinical translation of nanomedicines. Adv. Drug Deliv. Rev. 128, 54-83 (2018)]. Compared to traditional methods, microfluidic technology can be relevant in the pharmaceutical field for producing micro- and nanoproducts in a scalable and reproducible manner. [4] Key aspects of microfluidic devices are the small dimensions of the channels that allow a laminar flow regime [Jakub Novotny, FF Fluid manipulation on the micro-scale: Basics of fluid behavior in microfluidics. J. Sep. Sci. 40, 1-44 (2016)] and the use of software to control fluid dynamics, allowing precise mixing of reagents [Sackmann, EK, Fulton, AL & Beebe, DJ The present and future role of microfluidics in 1531567 of 16 biomedical research. Nature 507, 181-189 (2014)]. Several considerations must be taken into account to obtain prototypes of highly effective microfluidic devices [Zhang, H., Zhu, Y. & Shen, Y. Microfluidics for Cancer Nanomedicine: From Fabrication to Evaluation. 1800360, 1-25 (2018)]: the dimensions and geometries of the microchannels, the number of inlets, and the materials used to fabricate the devices. The aforementioned parameters determine the quality of the products.[5] The most commonly used materials for manufacturing microfluidic devices include elastomeric and thermoplastic materials. Although these materials are readily available to laboratories as raw materials due to their low cost, the manufacturing methods associated with obtaining microfluidic devices, such as lithography and / or CNC machining, have the significant disadvantage of being expensive and / or time-consuming processes. [Damiati, S., Kompella, UB, Damiati, SA & Kodzius, R. Microfluidic devices for drug delivery systems and drug screening. Genes (Basel). 9, (2018)]. Furthermore, these materials are not suitable for the use of organic solvents, reagents that are essential in the synthesis of micro- and nanoproducts.For example, among the most commonly used materials for manufacturing devices is polydimethylsiloxane (PDMS), which has a high susceptibility to swelling or deformation when exposed to organic solvents such as acetone [Baroud, CN Microchannel deformations due to solvent induced PDMS swelling. 2972-2978 (2010) doi:10.1039 / c003504a]; [Genzer, J., Park, I., Efimenko, K. & Sjo, J. Rapid Removal of Organics and Oil Spills from Waters Using Silicone Rubber “Sponges”. 318-327 (2009) doi:10.1080 / 01932690802540384]; [Lee, JN, Park, C. & Whitesides, GM Solvent Compatibility of Poly (dimethylsiloxane) -Based Microfluidic Devices. 75, 6544-6554 (2003)]. [6] Swelling of microfluidic channels affects fluid flow and leads to uncontrolled product generation [Damiati, S., Kompella, UB, Damiati, SA & Kodzius, R. Microfluidic devices for drug delivery systems and drug screening. Genes (Basel). 9, (2018); Communication, S. Predictive model on micro droplet generation through mechanical cutting. 431-438 (2009) doi:10.1007 / s104042 1531567 of 16 009-0412-y], In addition, PDMS can contaminate solutions flowing through the channels with unreacted oligomers or absorb organic molecules from the solution [Regehr, KJ et al. Biological implications of polydimethylsiloxane-based microfluidic cell culture. 2132–2139 (2009) doi:10.1039 / b903043c; Toepke, MW & Beebe, DJ PDMS absorption of small molecules and consequences in microfluidic applications. 1484–1486 (2006) doi:10.1039 / b612140c], [7] Although these problems have been partially solved by the use of other materials such as glass or polytetrafluoroethylene (PTFE) and cyclic olefin copolymer (COC), the complexity of the method for fabricating microfluidic devices using these materials persists [Damiati, S., Kompella, UB, Damiati, SA & Kodzius, R. Microfluidic devices for drug delivery systems and drug screening. Genes (Basel). 9, (2018); Nguyen, NT, Shaegh, SAM, Kashaninejad, N. & Phan, DT Design, fabrication and characterization of drug delivery systems based on lab-on-a-chip technology. Adv. Drug Deliv. Rev. 65, 1403-1419 (2013)]. [8] 3D printing technology represents a set of powerful techniques under precise digital control that enables the manufacture of cost-effective, time-efficient, easy-to-manufacture, and highly efficient objects [Bhattacharjee, N., Limos, A., Kang, S. & Folch, A. The upcoming 3D-printing revolution in microfluidics. Lab Chip 16, 1720-1742 (2016)]. Therefore, based on 3D printing technology, different types of prototypes can be developed as an opportunity to address a variety of health problems [Hiroyuki Tetsuka and Su Ryon Shin. Materials and Technical Innovations in 3D Printing in Biomedical Applications. (2020) doi:10.1039 / D0TB00034E: Manuscript, A. Point-of-Care Testing: Applications of 3D Printing. (2017) doi:10.1039 / C7LC00397Hl. [9] Among 3D printing technologies, stereolithography (or “SLA”, for “Stereo Lithography Apparatus”), fused deposition modeling (or “FDM”, for “Fused Deposition Modeling”) and 1531567 of 16 photopolymer inkjet printing (PolyJet®), are the most relevant techniques for the development of different types of devices.

[10] SLA 3D printing technology allows the production of 3D pieces from a photoresin precursor using a photopolymerized light source [Bhattacharjee, N., Urrios, A., Kang, S. & Folch, A. The upcoming 3D-phnting revolution in microfluidics. Lab Chip 16, 1720-1742 (2016); Cabot, J.M., Fuguet, E., Roses, M., Smejkal, P. & Breadmore, M.C. Novel instrument for automated pKa determination by Internal Standard Capillary Electrophoresis Novel instrument for automated pK a determination by Internal Standard Capillary Electrophoresis. (2015) doi:10.1021 / acs.analchem.5b00845; Kotz, F. et al. Three-dimensional printing of transparent fused silica glass. Nat. Publ. Gr. 544, 337-339 (2017); Shallan, A.I., Smejkal, P., Corban, M., Guijt, R.M. & Breadmore, M.C. Cost-Effective Three-Dimensional Printing of Visibly Transparent Microchips within Minutes. (2014); Li, F., Macdonald, N.P., Guijt, R.M. & Breadmore, M.CIncreasing the functionalities of 3D printed microchemical devices by single material, multimaterial, and phnt-pause-phnt 3D printing. Lab Chip 19, 35-49 (2019); Anciaux, SK, Geiger, M. & Bowser, Μ. T. 3D Printed Micro Free-Flow Electrophoresis Device.(2016) doi:10.1021 Zacs.analchem.6b01573].

[11] Currently, commercially available resins open the possibility of designing biomedical devices and creating 3D objects with precise, small-scale architectures. Since there is direct or indirect contact between the materials used in 3D-printed microfluidic devices and biological samples, the biocompatibility of the materials is of paramount importance. Among the components of photoresins, the polymerized materials are generally not cytotoxic. However, toxicities can arise from unreacted monomer, as well as from the photoinitiator or adsorbent that leaks out of the part and interacts with the sample.

[12] The amount of leachable products is greatly affected by factors including the geometry and formulation of the resin, among others. Various strategies have been employed to reduce the cytotoxicity of printed parts. For the most part, 1531567 of 16 The potentially cytotoxic components are removed by washing the 3D printed parts afterward with a suitable solvent. Post-treatment with UV light is also commonly used to increase monomer-to-polymer conversion, since polymerization never reaches complete conversion during printing.

[13] In the prior art, there are various microfluidic devices for the production of nanoparticles, such as the invention proposed in US patent 10843194B2 entitled “Microfluidic mixing devices and systems,” where the microfluidic device is made of PDMS material using positive molds that were manufactured by lithography. Likewise, US patent 9381477B2 entitled “Microfluidic synthesis of organic nanoparticles” describes chips manufactured using various techniques such as lithography, etching, stamping, or molding of a polymeric surface.

[14] Elastomeric materials such as PDMS are polymers widely used in microfluidic chips using techniques such as lithography. However, despite their advantage of optical transparency, these materials are unsuitable for use with organic solvents, as they deform in their presence. The use of organic solvents is essential for most micro- and nanoproduct syntheses. The deformation of the microchannels affects the flow of the liquids. Thus, these materials have limitations in chemical synthesis applications.

[15] On the other hand, microfluidic devices made of materials resistant to organic solvents, such as glass, certain metals, or silicones, overcome this limitation. In this regard, the previously mentioned patent US9381477B2 refers to glass, metals, or silicones as possible chip materials. Another patent that mentions chips made of glass and / or silicone is WQ2020115178A2, titled “Microfluidic devices.” However, it should be noted that microfluidic devices made of these materials are difficult to prototype because the manufacturing methods are complex, requiring, among other things, cleanroom environments. 1531567 of 16

[16] Compared to patent CN105727857A entitled “Microfluidic apparatus produced by 3D printing,” the latter concerns a microfluidic device manufactured by 3D printing and uses photopolymerizable resins, but the technology used differs from that of the present invention because it employs digital light processing (DLP) technology. Furthermore, it proposes its use for the production of water / oil / water (W / O / W) double emulsions, without mentioning the use of organic solvents, an advantage that is demonstrated in the present invention through the use of organic solvent-resistant resins for the synthesis of micro- and nanoproducts.

[17] Finally, it should be noted that none of the aforementioned background information describes a microfluidic device resistant to organic solvents that allows the synthesis of micro- and nanoproducts and that comprises two or more parts joined together by clamping means. This has the advantage of being easy to manufacture in less complex laboratories, using, for example, stereolithography 3D printing. OBJECT OF THE INVENTION

[18] The object of the present invention is a microfluidic device, made of photopolymerizable resin, resistant to organic solvents, for use in the synthesis of micro and nanoproducts, and the method of manufacturing said microfluidic device.

[19] The microfluidic device comprises an upper part, which in turn comprises at least two inlet ports and at least one outlet port; one or more lower parts, which in turn comprise microchannels; and means for securing the upper and lower parts together. Its manufacturing method comprises a modeling step for the microfluidic device parts; a 3D printing step for the microfluidic device parts; a post-processing step for the parts; and an assembly step for the microfluidic device. 1531567 of 16

[20] The use of photopolymerizable resin offers the advantage of resistance to organic solvents frequently used in organic chemistry, such as ethanol, methanol, acetone, and chloroform, among others. Furthermore, this resin allows for the rapid and simple fabrication of the microfluidic device using 3D printing. DESCRIPTION OF THE FIGURES

[21] For greater clarity and understanding of the object of the present invention, the following figures of a variant of the microfluidic device and its method of manufacture are presented:

[22] Figure 1: A) Top view of the upper part (10) of the embodiment; B) Top view of the lower part (20) of the embodiment; C) Top view of the microfluidic device of the embodiment.

[23] Figure 2: Schematic of the zigzag microchannels (21) (213) of the microfluidic device of the embodiment example.

[24] Figure 3: Schematic of an example of the stereolithography 3D printing manufacturing method of the microfluidic device of the example embodiment.

[25] Figure 4: Scanning Electron Microscopy (SEM) images of the microfluidic device of the embodiment example, before and after acetone treatments: a) 0 min, b) 2 min, c) 30 min and d) 60 min.

[26] Figure 5: Analysis by gas chromatography coupled to mass spectrometry (GC-MS) of the organic solvent used for the synthesis of the polymeric micelles, before and after coming into contact with the microchannels, of the embodiment example. 1531567 of 16

[27] Figure 6: SEM image of the 3D-printed microfluidic device of the embodiment, after treatment with acetone until evident damage. The treatment consisted of placing the microfluidic device in contact with acetone under mechanical agitation.

[28] References in the Figures: Microfluidic device: (10) Top piece: (11) Inlet hole (12) Outlet hole (20) Lower piece: (21) Microchannels: (211) Input channel (212) Input angle (213) Zigzag (30) Clamping means (40) Computer (50) 3D printer (60) Light source DESCRIPTION OF THE INVENTION

[29] The device of the present invention is described below:

[30] A microfluidic device for the synthesis of micro and nanoproducts, comprising: • an upper piece, comprising at least two inlet holes, which allow the entry of the aqueous and organic phases, and at least one outlet hole, through which the micro and nanoproducts of the synthesis are obtained; • one or more lower pieces, comprising microchannels, which allow mixing and diffusion between the phases; and 1531567 of 16 • fastening means, which allow assembly between the upper piece and the lower pieces; wherein said upper piece and said lower piece(s) are made of photopolymerizable resin, wherein said photopolymerizable resin is resistant to organic solvents; wherein said light-cured resin is selected from the group comprising acrylic and epoxy-acrylic resins; where said upper and lower pieces are joined together by said fastening means; and where said inlet holes and said outlet hole(s) are located coincidentally at the height of the ends of said microchannels.

[31] The method of the present invention is described below:

[32] A method for manufacturing a microfluidic device for the synthesis of micro and nanoproducts, comprising the following steps: a) Model said microfluidic device in 3D using computer-aided design (CAD); b) Export the 3D models from step a) to STL format files; c) Import the files from step b) into 3D printing software, and set in the 3D printing software a printing layer height parameter and a curing time parameter (The printing software divides the structure to be printed according to the thickness of the set printing layer, obtains a series of cuts of equal thickness, determines the precise position, the intensity of the lighting and the projection lighting time of each cut); d) Generate new STL format files with the printing parameters from step c); e) Input the STL format files from step d) into a 3D printer; f) Print the parts from step a) in photopolymerizable resin using the 3D printer from step e); g) Remove the printed parts from the printing support of said 3D printer; 1531567 of 16 h) Wash the printed parts from step g) with a solvent compatible with the photopolymerizable resin used in step f); i) Perform post-processing of said printed parts; and j) Assemble the microfluidic device; • where said step a) comprises modeling in 3D an upper part and one or more lower parts in computer-aided design (CAD); • wherein said step c) comprises importing said files from step b) into the 3D printing software, and setting in said 3D printing software the printing layer height parameter, between 0.025 mm and 0.1 mm, and the curing time parameter, between 3 and 15 seconds; • wherein said step e) comprises entering the STL format files from step d) into a 3D printer with a photopolymerizing light source, comprising a wavelength of between 300 and 410 nm; • wherein said step f) comprises printing with said 3D printer of step e) said parts of step a) in photopolymerizable resin resistant to organic solvents, which is selected from the group comprising acrylic and epoxy-acrylic resins; • wherein said step i) comprises curing said printed and washed parts with a light source having a wavelength between 300 and 410 nm for 1 to 5 minutes; and • wherein said step j) comprises assembling said microfluidic device by assembling said upper part obtained in said step i) to said lower part(s) obtained in said step i), by means of clamping means. EXAMPLE OF IMPLEMENTATION

[33] For the microfluidic device for the synthesis of micro and nanoproducts and the method of manufacturing said microfluidic device described in the present invention, the following embodiment was carried out: 1531567 of 16

[34] Figure 1 shows a variant of the microfluidic device for the synthesis of micro and nanoproducts of the present invention, 3D printed, wherein said microfluidic device comprises: • an upper piece (10), comprising two inlet holes (11), one through which the aqueous phase enters and another through which the organic phase enters, and an outlet hole (12), through which the micro and nanoproducts of the synthesis are obtained; • a lower piece (20), comprising a microchannel (21) comprising two inlet channels (211) connected in a Ύ” or “V” shape, forming a 90° angle between them (212), and converging in a zigzag (213); and • fastening means (30), comprising the photopolymerizable acrylic resin 3Dresyn WC Clear®, which allows assembly and fastening between said upper piece (10) and said lower piece (20); wherein said upper piece (10) and said lower piece (20) are made of 3Dresyn WC Clear® photopolymerizable acrylic resin, which is resistant to organic solvents; where said upper piece (10) and said lower piece (20) are joined together by said fastening means (30); where said inlet holes (11) are located coincidentally at the height of the ends of said inlet channels (211) of said microchannel (21); where said outlet orifice (12) is located coincidentally at the height of the end of said zigzag (213) of said microchannel (21); and where said microchannel (21) is 300 pm wide and 300 pm deep.

[35] Figure 2 shows a schematic of the microchannel (21) of the microfluidic device of the embodiment, which enables the generation of micro- and nanoproducts. It comprises two inlets, one for the aqueous phase and the other for the organic phase. The two inlet channels (211) of the microchannel (21) converge in a zigzag (213) where the mixing of the reactants occurs. The geometry of the zigzag channel, with its curves and turns, forces the fluids to generate transverse flows, inducing chaotic advection within a laminar regime. This process favors the mixing of the aqueous and organic solutions, forming the micro- and nanoproducts of interest [Manz, Andreas and 1531567 of 16 Neuzil, Pavel and O'Connor, Jonathan S and Simone, Giuseppina. Microfluidics and Lab-on-a-chip. The Royal Society of Chemistry. 2021],

[36] On the other hand, Figure 3 shows a schematic of an example of the implementation of the stereolithography 3D printing manufacturing method of the microfluidic device.

[37] The microfluidic device was manufactured by 3D printing using stereolithography (SLA) according to the following steps: a) Model in 3D said upper part (10) of said microfluidic device and said lower part (20) of said microfluidic device in computer-aided design (CAD) (40); b) Export the 3D models from step a) to STL format files, a standard file type for 3D printers; c) Import the files from step b) into the 3D printing software, and set the layer height parameter to 0.025 mm and the curing time parameter to 8 seconds in the 3D printing software; d) Generate new STL format files with the printing parameters from step c); e) Input the STL format files from step d) into an LCD-based SLA 3D printer (50) operating with a photopolymerizing light source rated at 405 nm wavelength and a pixel size of 47 pm in the image plane (Anycubic Photon®); f) Print on said 3D printer (50) of step e) said parts (10) (20) of step a) in 3Dresyn WC Clear® photopolymerizable acrylic resin, which is resistant to organic solvents, biocompatible, translucent and washable with water; g) Remove the printed parts from the printing support of said 3D printer (50); h) Wash the printed pieces from step g) with distilled water; i) Curing said printed parts under a 45 W LED source (60) with a wavelength of 405 nm for 1 minute; 1531567 12 of 16 j) Assemble the microfluidic device by gluing said upper piece (10) and said lower piece (20), using as a fastening medium (30) said 3Dresyn WC Clear® light-cured resin, distributing it over the surface of said upper piece (10) and said lower piece (20), in such a way that said resin (30) does not enter the microchannel (21); and k) Cure said microfluidic device under a 45 W LED source (60) with a wavelength of 405 nm for 5 minutes (This further ensures the absence of uncured resin prior to use).

[38] In order to characterize the resistance to organic solvents commonly used in the synthesis of micro- and nanoproducts, the microfluidic device with the exposed microchannel from the example embodiment was agitated under mechanical mixing in acetone for three different times: 2, 30, and 60 minutes. Subsequently, the resistance of the microfluidic device's microchannels to the organic solvent was analyzed using scanning electron microscopy (SEM), as shown in Figure 4. It was observed that the microstructure of the channels was not altered, even after one hour of contact with the pure organic solvent. Notably, neither the walls and edges of the zigzag, nor the bottom of the microchannel showed any cracks or other signs of degradation.

[39] On the other hand, in addition to the possible migration of resin components due to printing-related issues, the printed materials could be dissolved by the solvent used as the organic phase in the synthesis of the micro- and nanoproducts. To ensure that the resin components of the present microfluidic device are not removed during synthesis, the organic phase (without any dissolved components) was flowed through the microchannels used for the synthesis of micro- and nanoproducts and, furthermore, through a previously unused microfluidic device. The organic solvent before and after flowing through the zigzag microchannels was analyzed by gas chromatography-mass spectrometry (GC-MS).

[40] While the chemical composition of most resins used in SLA printers remains a trade secret, the data sheets of 1531567 13 of 16 safety data indicate that the resins generally contain acrylate and / or methacrylate monomers. A qualitative analysis was performed using scanning GC-MS to study the probable presence of resin derivatives and methacrylate-type residues. In scanning mode, only the first quadrupole is used; the ions that can be observed in the mass spectrum correspond to the molecular ion and / or fragments generated by the high-energy electrons bombarding the molecule. Figure 5 shows the results of the GC-MS analyses, where it can be seen that no signs of resin component release were observed in the organic solvent circulating through the new or used microfluidic device.

[41] For comparison, a new zigzag microchannel was agitated in acetone until evident damage occurred. The acetone collected after agitation was analyzed by GC-MS under the same conditions as above, and the results are plotted in Figure 5. Six peaks are present in the chromatogram, which are detailed in Table 1. Some of the ions (147, 91) coincide with those reported by Oskui et al. [Oskui, SM et al. Assessing and Reducing the Toxicity of 3D-Printed Parts. Environ. Sci. Technol. Lett. 3, 16 (2016)], where the results showed that at least three different chemical species were present in their leachate. These species have different retention times in gas chromatography (GC) but very similar fragments in mass spectrometry (MS), as in the embodiment of the present invention.This supports the hypothesis that short-chain monomers or polymers are present in the leachate from the damaged microfluidic device, but not in the new and used microchannels. The visibly affected microstructure of the microfluidic device is shown in Figure 6. This image of the 3D-printed microfluidic device from the implementation example was obtained after immersion in acetone under mechanical agitation. This experiment allowed for the evaluation, under extreme conditions, of the damage caused to the microfluidic device by organic solvents such as acetone.

[42] Table 1: Major ions in acetone peaks after shaking a microchannel to evident damage: 1531567 of 16 Retention time [min] m / z ratio fragments Compound returned by the library (% probability) 4.60 40, 81, 65, 109 6-methyl-3,5-heptadien-2-one (“3,5 heptadien-2-one, 6methyl”) (75) 5.20 41, 91, 77, 147, 119 2-methyl-3,5-dodecadiyne (“3,5 dodeadiyne, 2-methyl”) (73) 5.75 91, 146, 164, 117, 39 2,4,6-trimethylbenzoic acid (“Benzoic acid, 2,4,6 trimethyl”) (75) 5.80 40, 78, 142, 105 Phenylphosphonosic acid (“Phenylphosphonosic acid”) (72) 6.20 77, 40, 158, 213, 105 Phosphonic acid, phenyl, diethylester (79) 14.45 147, 77, 91.40, 119 2-oxo-2-(2,4,6-trimethylphenyl) ethyl acetate 2oxo-2-(2,4,6trimethylphenyl)acetate” or “Ethyl mesitylglyoxylate”) (82) The following are 8 demands. 1531567 of 16 National Atomic Energy Commission - 30546660210 Digitally signed by PORTALTRAMITES - INPI Date: 2021.10.06 12:47:46 -03:00 Reason: Digitally Signed by the INPI Location: Buenos Aires, Argentina 1531567

Claims

1. A microfluidic device for the synthesis of micro- and nanoproducts, characterized in that it comprises: • an upper part, comprising at least two inlet holes and at least one outlet hole; • one or more lower parts, comprising microchannels; and • fastening means; wherein said upper part and said lower part(s) are made of photopolymerizable resin, wherein said photopolymerizable resin is resistant to organic solvents; wherein said photopolymerizable resin is selected from the group comprising acrylic and epoxy-acrylic resins; wherein said upper and lower parts are joined together by said fastening means; and wherein said inlet holes and said outlet hole(s) are located coincidentally at the height of the ends of said microchannels. 7 Claims follow