Microfluidic device for protein crystallization and method of fabricating the microfluidic device

Abstract

A microfluidic device for crystallization based on gradient mixing and a method of fabricating the microfluidic device with a rigid polymer, such as cyclic olefin copolymer (COC) and a flexible polymer, such as polydimethylsiloxane (PDMS).

Description

Attorney Docket No.208192-0022-WO01 MICROFLUIDIC DEVICE FOR PROTEIN CRYSTALLIZATION AND METHOD OF FABRICATING THE MICROFLUIDIC DEVICE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a non-provisional of and claims the benefit of U.S. Provisional Application No.63 / 664,660, filed on June 26, 2024, the entire contents of which are incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under 2153503 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND

[0003] Macromolecular X-ray crystallography (MX) stands as a cornerstone technique in the field of structural biology, providing invaluable insights into the three-dimensional structures of proteins with atomic resolution. MX relies on the principle of X-ray diffraction originating from atoms within the crystal lattice generating a diffraction pattern. By analyzing this diffraction pattern, the spatial arrangement of atoms within the protein reveals its structural details. MX has revolutionized our understanding of protein structure and function, playing a central role in drug discovery, rational protein engineering, and the elucidation of biochemical mechanisms. By providing detailed insights into the atomic-level organization of proteins, MX continues to drive advances in biochemistry, molecular biology, and drug design, shaping our understanding of life at the molecular level. SUMMARY

[0004] In another embodiment, the present disclosure provides a method of fabricating a microfluidic device. The method comprises preparing a mixture comprising PDMS to curing agent at a 10:1 ratio, spin coating the mixture on a glass slide to form a membrane layer, providing an imprinted first layer comprising a polymer, treating the first layer with a solution of (3-Aminopropyl) triethoxysilane (APTES), applying the membrane layer onto the treated firstAttorney Docket No.208192-0022-WO01 layer to form a control layer, providing an imprinted mask with same structure as the first layer, treating the imprinted mask with a solution of APTES, providing an imprinted second layer comprising the polymer, applying the treated mask onto the second layer to form a fluid layer, and aligning the control layer and the fluid layer.

[0005] In one embodiment, the present disclosure provides a microfluidic device comprising a first layer, a second layer, a membrane layer, and a plurality of inlets. The first layer comprises a polymer material and includes a plurality of first channels and a plurality of chambers, wherein the plurality of chambers are in communication with at least one of the plurality of first channels when vacuum is applied to at least one of the plurality of chambers. The second layer comprises the polymer material and includes a plurality of second channels and a plurality of wells, wherein the plurality of wells are coupled to at least one of the plurality of second channels. The membrane layer comprises an elastomer material and is positioned between the first layer and the second layer. The plurality of inlets are in fluid communication with the plurality of second channels, and the plurality of inlets are configured to deliver a precipitant solution and an analyte solution through the plurality of second channels and the plurality of wells where the precipitant solution and the analyte solution mix to generate a crystallization sample.

[0006] Other aspects of the present disclosure will become apparent by consideration of the detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The features and advantages of the present disclosure, and the manner of attaining them, will become more apparent and the present disclosure will be better understood by reference to the description of the present disclosure taken in conjunction with the accompanying drawings, wherein:

[0008] FIG.1 illustrates a microfluidic device for screening protein crystallization conditions.

[0009] FIG.2A illustrates a cross-section of the microfluidic device of FIG.1 showing a second channel in an empty condition and a chamber in a closed and sealed position.Attorney Docket No.208192-0022-WO01

[0010] FIG.2B illustrates a cross-section of the microfluidic device of FIG.1 showing the second channel in a partially-filled condition and a chamber with a membrane in an open position.

[0011] FIG.2C illustrates a cross-section of the microfluidic device of FIG.1 showing the second channel in a filled condition and a chamber with a membrane in a closed and sealed position.

[0012] FIG.3 illustrates a method of fabricating a microfluidic device of FIG.1 according to an embodiment.

[0013] Those of ordinary skill in the art will understand that the devices and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the various embodiments of the present disclosure is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure. DETAILED DESCRIPTION

[0014] Before any embodiments of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in practice or testing of the disclosed invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.Attorney Docket No.208192-0022-WO01

[0016] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0017] For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.

[0018] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0019] As used herein, the term “analyte solution” may include any suitable particle or analyte including, but not limited to, proteins, such as membrane proteins such as photosystem I (PSI) and pigment protein complexes like photoactive yellow protein (PYP) and phycocyanin, as well as enzymes like lysozymes, and other substances such as nucleic acids, microparticles, nanoparticles, biological cells, viruses, biomolecules, nanocrystals, cancer cells, mitochondria or other cell organelles.

[0020] As used herein, the term “precipitant” may include solutions containing a salt such as sodium chloride (NaCl), lithium chloride (LiCl), magnesium sulfate (MgSO4), potassiumAttorney Docket No.208192-0022-WO01 phosphate (KH2PO4), polyethylene glycol (PEG), and ammonium sulfate ((NH4)2SO4) or other constituents such as buffering agents, detergents, organic solvents or polymers, for example.

[0021] The present disclosure provides a microfluidic device and method of fabricating the microfluidic device for protein growth and crystallization analysis based on gradient mixing. The microfluidic device can facilitate the study of several hundreds of individual solution combinations in a microfluidic nanowell array for crystal growth, as well as the characterization of the resulting crystals in the nanoliter wells. The microfluidic device can permit parallel high throughput batch crystallization experiments in nanoliter wells arranged in arrays that allow analyte and precipitant solutions to mix, split, and recombine as the nanowells are filled. The array design and integrated valves further permit filling and mixing of analyte and precipitant solutions under laminar flow conditions initiated through suction or pressure pumping, for example. The arrangement of channels and nanowells in the array, in conjunction with controlled flow rates, can facilitate the generation of hundreds of unique concentrations in the plurality of nanowells. Once filled, the nanowells may be advantageously sealed via valves thereby allowing crystallization to occur therein in parallel with each other. Still further, filling of the nanowells may be accomplished using laminar flow that may permit straightforward estimation of concentrations of analyte and precipitant in the nanowell array. The arrangement provides for numerical simulations based on analyte and precipitant concentration, flow parameters and device geometry, for example, may allow phase diagrams to be constructed to identify nanocrystal growth.

[0022] The present disclosure provides a microfluidic device 100, as illustrated in FIG.1, for screening protein crystallization conditions. The microfluidic device 100 can provide rapid screening of protein crystallization conditions when a small amount of sample is available. In some embodiments, the microfluidic device 100 can screen or test various conditions to find the optimal environment for growing protein crystals. This can involve systematically varying parameters like protein concentration, temperature, pH, and precipitant type and concentration to identify conditions that favor crystal formation. In an example, the device 100 can provide screening of G-protein coupled receptor proteins (GPCR) when a suitable precipitant solution is employed. The device 100 also provides a plurality of wells for generating unique samples forAttorney Docket No.208192-0022-WO01 sample analysis and use with X-ray sources such as the compact X-ray light source (CXLS) or X-ray free electron lasers (XFELs).

[0023] The microfluidic device 100 includes a first layer 104, a membrane 108, and a second layer 112. In one embodiment, the first layer 104 and the second layer 112 can comprise a first material (e.g., one or more polymer materials). For example, the first material may have rigid properties, such as, cyclic olefin copolymer (COC). In other examples, the first material can be polyethylene therephthalate (PET), cyclic olefin polymer (COP), Kapton, Mylar, polycarbonate (PC), poly(methyl methacrylate) (PMMA), SU-8, glass, quartz, or fused silica). The membrane 108 can comprise a second material (e.g., one or more polymer materials, rubber, or elastomer). The second material may be the same as or different than the first material. For example, the second material may have flexible properties, such as polydimethylsiloxane (PDMS). In other examples, the second material can be polyethylene therephthalate (PET), cyclic olefin polymer (COP), Kapton, Mylar, polycarbonate (PC), poly(methyl methacrylate) (PMMA), SU-8, glass, quartz, or fused silica). The membrane 108 is configured to deflect or flex under vacuum and to return to its original position when vacuum is removed. FIG.2A shows the membrane 108 in a closed, unflexed position, and FIG.2B shows the membrane 108 in an open, flexed position under vacuum. In one embodiment, the membrane 108 may be non-permeable. The membrane 108 has a first surface and a second surface and is arranged such that the first surface of the membrane 108 is coupled to the first layer 104, and the second surface of the membrane 108 is coupled to the second layer 112. The device 100 can be fabricated using microfabrication techniques in glass and silicon or other solid materials. In one embodiment, the first layer 104 and the second layer 112 may be translucent, X-Ray transparent, or have low X-Ray absorption.

[0024] The first layer 104 includes a plurality of first channels 124 and a plurality of chambers 128 both arranged therein. The plurality of chambers 128 are each coupled to at least one of the plurality of first channels 124 when vacuum is applied to the chambers 128. In one embodiment, the first layer 104 may be configured to withstand vacuum pressure applied to the plurality of first channels 124 and to the plurality of chambers 128. The device 100 also includes a plurality of wells 132 and a plurality of second channels 136 both arranged in the second layer 112. The wells 132 are each coupled to at least one of the second channels 136. In some examples, the wells 132 include a depth of about 15-500 μm. In some examples, the depth of theAttorney Docket No.208192-0022-WO01 wells 132 can vary based on a thickness of the membrane 108. In one embodiment, the second layer 112 may be configured to receive fluid samples in the form of analyte and precipitant solutions in the plurality of second channels 136 and the plurality of wells 132. A plurality of barrier walls 140 are disposed in the plurality of second channels 136. Each of these barrier walls 140 is arranged opposite to one of the plurality of chambers 128 such that a portion of the membrane 108 separates the chamber 128 from a corresponding barrier wall 140. In one embodiment, the plurality of first channels 124 and the plurality of chambers 128 may be configured to be subject to vacuum and the plurality of second channels 136 and the plurality of wells 132 may be configured to receive one or more fluids.

[0025] With reference to FIGS.2A-2C, the plurality of first channels 124 and the chambers 128 may both be arranged in a top surface of the first layer 104 such that each of the chambers 128 interface with the portion of the membrane 108 overlying them. In a further embodiment, the plurality of wells 132 and the plurality of second channels 136 are both arranged in a bottom surface of the second layer 112, such that any fluid in the wells 132 and the second channels 136 may interface with the second surface of the membrane 108. This arrangement may permit vacuum V applied to the plurality of first channels 124 and the chambers 128 to act upon the membrane 108, causing the membrane 108 to flex into the chambers 128. Then fluid advancing in the second channels 136 may pass under the barrier walls 140 extending over the flexed membrane 108 (see FIG.2B) and into the wells 132. After filling of the wells 132 with fluid is complete and vacuum is removed, the membrane 108 is biased to return to a closed position adjacent the barrier walls 140, as shown in FIG.2C. Accordingly, the barrier walls 140, the membrane 108 and the chambers 125 act as self-sealing valves.

[0026] In one embodiment, as shown in FIG.1, the plurality of wells 132 may be arranged in a plurality of rows between a first end 144 and a second end 148 of the second layer 112. The arrangement illustrated in FIG.1 may beneficially promote mixing, splitting, and remixing of an analyte solution and a precipitant solution as the fluid moves between wells 132 from the first end 144 to the second 148 of the device 100.

[0027] The device 100 includes a first inlet 160 and a second inlet 164 with each being coupled to one of the plurality of second channels 136 at the first end 144. In operation, anAttorney Docket No.208192-0022-WO01 analyte solution may be advanced into the first inlet 160 and a precipitant solution may be advanced into the second inlet 164 and may mix together within the second channels 136 and wells 132. The device 100 may include an outlet 168 coupled to at least one of the first channels 124. This outlet 168 may be configured to be coupled to a vacuum source that may cause the membrane 108 to deflect during filling of the second channels 136 and wells 132 thereby allowing solution to pass underneath barrier walls 140, as shown in FIG.2B. In some examples, the device 100 may include a second outlet 172 coupled to at least one of the second channels 136 positioned at the second end 148. This second outlet 172 may be coupled to a vacuum source or may alternatively be coupled to a receptacle with ambient conditions to receive any overflow solution during filling of the second channels 136 and wells 132.

[0028] FIG.3 illustrates a fabrication process 300 of the device 100 according to an embodiment. Fabrication of the device 100 includes preparation of a control layer, a fluid layer, and integration of these two layers.

[0029] Preparation of the control layer is shown in FIG.3 (at a-d). First, at 304, a 15 µm thick PDMS membrane (for the membrane 110) was prepared by spin coating a mixture of PDMS to curing agent at a 10:1 ratio on a glass slide. In some examples, the PDMS membrane can be printed onto a glass slide, via, for example, three-dimensional printing techniques. Next, at 308, an imprinted cyclic olefin copolymer (COC) control layer (for the first layer 104) was treated with oxygen plasma and 1% aqueous solution of (3-Aminopropyl) triethoxysilane (APTES) respectively. This step is followed by a thorough drying of the control layer surface with N2. On the next step, at 312, the cured PDMS membrane on glass slide is plasma oxidized for 1 min, and at 316, the membrane is brought into contact with the chemically treated COC surface for 2 min. Under room temperature, the PDMS membrane is cut along the edges of the COC control layer to transfer the membrane from the glass slide to the control layer surface. It is noted that other ratios (e.g., 15:1 or 8:1) of PDMS to curing agent could be implemented by adjusting the elastic modulus of the PDMS layer.

[0030] Preparation of the fluid layer is shown in FIG.3 (at e-f). In the first step, at 320, a PDMS stamp with the same design as of the control layer is inked for 20 min with 1% aqueous solution of APTES. At 324, the stamp is dried with N2and aligned with an imprinted COC fluidAttorney Docket No.208192-0022-WO01 layer (for the second layer 112) so that the APTES molecules are transferred everywhere other than the chamber areas.

[0031] The two layers, control layer and fluid layer, are then integrated. See FIG.3 (at g-h). First, at 328, the control layer is plasma oxidized for 1 min and then aligned at 332 with the stamped fluid layer. The integrated hybrid device is left overnight before use.

[0032] Additional features and advantages of the present disclosure are set forth in the following claims.

Claims

Attorney Docket No.208192-0022-WO01 CLAIMS What is claimed is:

1. A method of fabricating a microfluidic device, the method comprising: (a) preparing a mixture comprising PDMS to curing agent at a 10:1 ratio; (b) spin coating the mixture on a glass slide to form a membrane layer; (c) providing an imprinted first layer comprising a polymer; (d) treating the first layer with a solution of (3-Aminopropyl) triethoxysilane (APTES); (e) applying the membrane layer onto the treated first layer to form a control layer; (f) providing an imprinted mask with same structure as the first layer; (g) treating the imprinted mask with a solution of APTES; (h) providing an imprinted second layer comprising the polymer; (i) applying the treated mask onto the second layer to form a fluid layer; and (j) aligning the control layer and the fluid layer.

2. The method of claim 1, wherein the membrane layer has a thickness of about 15 μm.

3. The method of claim 1 or 2, further comprising treating the first layer with oxygen plasma prior to treating the first layer with APTES.

4. The method of claim 3, wherein the solution is a 1% aqueous solution of APTES.

5. The method of any one of claims 1-4, further comprising drying the control layer with N2.

6. The method of any one of claims 1-5, further comprising applying oxygen plasma for one minute to the membrane layer.Attorney Docket No.208192-0022-WO01 7. The method of any one of claims 1-6, wherein applying the membrane layer onto the treated first layer to form a control layer is completed at room temperature.

8. The method of any one of claims 1-7, wherein aligning the control layer and the fluid layer is completed at room temperature.

9. A microfluidic device comprising: a first layer comprising a polymer material, the first layer including a plurality of first channels and a plurality of chambers, wherein the plurality of chambers are in communication with at least one of the plurality of first channels when vacuum is applied to at least one of the plurality of chambers; a second layer comprising the polymer material, the second layer including a plurality of second channels and a plurality of wells, wherein the plurality of wells are coupled to at least one of the plurality of second channels; a membrane layer comprising an elastomer material, the membrane layer positioned between the first layer and the second layer; and a plurality of inlets in fluid communication with the plurality of second channels, the plurality of inlets configured to deliver a precipitant solution and an analyte solution through the plurality of second channels and the plurality of wells where the precipitant solution and the analyte solution mix to generate a crystallization sample.

10. The microfluidic device of claim 9, wherein the polymer material is cyclic olefin copolymer (COC).

11. The microfluidic device of claim 9 or 10, wherein the elastomer is polydimethylsiloxane (PDMS).

12. The microfluidic device of claim 11, wherein the PDMS comprises a 10:1 ratio of PDMS to curing agent.Attorney Docket No.208192-0022-WO01 13. The microfluidic device of any one of claims 9-12, wherein the membrane layer has a thickness of about 15 μm.

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