Nanopatterned films with patterned surface chemical composition

Abstract

An article comprising a flexible structured membrane having a first major surface and a second major surface, wherein the first major surface of the flexible structured membrane has a plurality of pillars separated by land areas, and the pillars have an exposed surface. An anti-biofouling layer present in the land areas, and the anti-biofouling layer has a methylated surface. An inorganic layer on the exposed surface of the pillars, wherein the inorganic layer comprises a metal or a metal oxide. An analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from the group consisting of reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and combinations thereof. The exposed surface of the analyte binding layer comprises at least one functional group selected for binding to a biochemical analyte.

Description

Background Technology

[0001] High-throughput chemical and biological assays, such as nucleic acid and peptide sequencing, or protein, gene, and other biochemical and pharmaceutical assays, rely on expensive single-use consumables. For example, in addition to chemical and biological reagents used to probe, detect, or measure unknown samples, high-throughput assays typically require the use of patterned flow cell consumables, in which chemical and biochemical reagents can be selectively reacted with the target analyte.

[0002] Currently, commercially available patterned flow cells comprise glass or silicon substrates with etched nanopores. Each nanopore in the glass substrate contains chemical functional groups selected for binding chosen target analytes and is separated by regions of anti-fouling or non-interacting coatings or surface chemistry. In some examples, the chemical functional groups can be functionalized polymers or oligomers, such as polyacrylamide-containing hydrogels, or directly attached to the substrate via small molecule connectors. These patterned flow cells can be fabricated using complex and expensive wafer-based photolithography processes that include multiple chemical mechanical planarization (CMP), spin-coating, and washing steps to fill the nanopores with the correct chemical functional groups and place an anti-fouling coating between the nanopores.

[0003] To alleviate the financial burden on end users of high-throughput measurements and to make measurements more accessible to new markets, it is necessary to reduce the cost of consumables used, including glass or silicon substrates with etched nanopores. Summary of the Invention

[0004] Generally, this disclosure relates to nanopatterned substrates formed on flexible structured membranes for chemical or biological assays such as, for example, nucleic acids, proteins, and other biochemical screening procedures. In various embodiments, the flexible structured membrane can be structured using nanoscale pillars having functionalized analyte-binding regions selected for binding with target analytes during assays. In some embodiments, the functionalized pillars can be dispersed between anti-biocontamination platform regions.

[0005] Some anti-biofouling layers used in chemical or biological assays can be made of materials such as amorphous fluoropolymer resins, which require baking at high temperatures (above about 50°C) for several 30-minute cycles. In contrast, the anti-biofouling layer of this disclosure is made of a material comprising a non-reactive hydrophobic methylated surface, which eliminates the need for high-temperature baking cycles during production.

[0006] Nanopatterned flexible polymer film substrates can be produced in a continuous manufacturing process, offering higher throughput and lower manufacturing costs compared to wafer-based photolithography methods that produce parts on a foundational basis. These nanopatterned flexible polymer film substrates can be configured for use with a variety of assay reagents and instruments, and can be used for applications such as whole-genome sequencing, microbiome sequencing, gene or gene segment detection, RNA detection, single nucleotide polymorphism detection, mRNA sequencing, non-coding RNA sequencing, DNA methylation sequencing, protein expression, protein sequencing, peptide and other biochemical compound detection, small molecule or biomarker detection, and chemical and environmental pollutant detection. In some embodiments, the flexible organic carrier film substrate can be adhesively mounted on a support layer, enabling its use in screening instruments that currently employ more rigid silicon or glass substrate materials.

[0007] When manufacturing nanostructured substrates, continuous processing (also known in some cases as roll-to-roll processing) offers several advantages and increased design flexibility compared to silicon wafer fabrication techniques. For example, when fabricating nanopatterned substrates using silicon wafers, grafting analyte-bound chemicals onto columnar structures extending away from the wafer surface can be difficult, and therefore, in silicon wafer fabrication, the bound chemicals may be confined to the recessed, porous regions of the wafer. Additionally, because thick inorganic layers take longer to deposit, and due to the necessary flexibility of the polymer carrier web, inorganic layers can be made thin (less than 200 nm). Furthermore, compared to conventional wafer fabrication, amorphous silicon oxide layers deposited via roll-to-roll processing can contain impurities such as aluminum or carbon, allowing for efficient deposition rates on flexible, temperature-sensitive surfaces using processing techniques such as sputtering or plasma-enhanced chemical vapor deposition (PECVD).

[0008] In one aspect, this disclosure relates to an article comprising: a flexible structured membrane having a first main surface and a second main surface, wherein the first main surface of the flexible structured membrane includes a plurality of pillars separated by a platform region, and wherein the pillars include exposed surfaces; an anti-biofouling layer in the platform region, wherein the anti-biofouling layer has methylated surfaces; an inorganic layer on the exposed surfaces of the pillars, wherein the inorganic layer comprises a metal, a metalloid, a metal oxide, or a metalloid oxide; and an analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof, and wherein the exposed surface of the analyte binding layer includes at least one functional group selected for binding to a biochemical analyte.

[0009] In another aspect, this disclosure relates to a method for manufacturing a component of a diagnostic device, the method comprising: providing a flexible structured membrane having a first main surface and a second main surface, wherein the first main surface of the flexible structured membrane includes a patterned surface comprising an arrangement of pillars with dispersed platform regions; applying a release layer to the first main surface of the flexible structured membrane such that the release layer covers the top surface of the pillars and the platform regions, wherein the release layer includes a surface rich in methyl (CH3) groups; applying a planarization layer to the release layer; and etching with an oxygen-containing etching material. A portion of the planarization layer is etched to form a nonmethylated inorganic layer on the top surface of the column, wherein the inorganic layer comprises a metal, a metalloid, a metal oxide, or a metalloid oxide; the planarization layer is removed; and a functionalized silane material is attached to the inorganic layer on the top surface of the column, and the functionalized silane material is polymerized to form an analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalized hydrogels, functionalized polymers, and mixtures and compositions thereof, and wherein the analyte binding layer contains at least one functional group that reacts with a biochemical analyte.

[0010] In another aspect, this disclosure relates to a diagnostic device for detecting a biochemical analyte, the device comprising a flow cell with a patterned arrangement of fluid channels configured to provide flow conduits for a sample fluid containing the biochemical analyte, wherein at least some of the fluid channels in the flow cell are lined on their surfaces with: a flexible structured membrane having a first primary surface and a second primary surface, wherein the first primary surface of the flexible structured membrane includes a plurality of pillars having exposed surfaces extending into the fluid channels of the flow cell, wherein the pillars are distributed with plateau regions; and resistance to biofouling. The platform region comprises: a staining layer, the anti-biofouling layer comprising a methylated surface; a non-methylated inorganic layer, the non-methylated inorganic layer comprising a metal, a metalloid, a metal oxide, or a metalloid oxide; and an analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof, and wherein the exposed surface of the analyte binding layer comprises at least one functional group selected for binding with a biochemical analyte in the sample fluid.

[0011] These and other implementation schemes are included in the following list of exemplary implementation schemes.

[0012] List of exemplary implementation schemes:

[0013] Implementation Scheme A. An article comprising: a flexible structured membrane having a first main surface and a second main surface, wherein the first main surface of the flexible structured membrane includes a plurality of pillars separated by a platform region, and wherein the pillars include exposed surfaces; an anti-biofouling layer in the platform region, wherein the anti-biofouling layer has methylated surfaces; an inorganic layer on the exposed surfaces of the pillars, wherein the inorganic layer comprises a metal or a metal oxide; and an analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof, and wherein the exposed surface of the analyte binding layer includes at least one functional group selected for binding to a biochemical analyte.

[0014] Implementation Scheme B. The article according to Implementation Scheme A, wherein the inorganic layer comprises at least one of Si, Ti or Al or oxides thereof, and wherein the thickness of the inorganic layer is less than about 200 nm.

[0015] Implementation Scheme C. The article according to Implementation Scheme A, wherein the thickness of the inorganic layer is less than 50 nm.

[0016] Implementation Scheme D. The article according to any one of Implementation Schemes A to C, wherein the oxide of the inorganic layer is selected from SiO2, SiC x O y SiAl x O y TiO, AlO x And their mixtures and compositions.

[0017] Implementation Scheme E. The article according to Implementation Scheme D, wherein the oxide is SiC x O y .

[0018] Implementation Scheme F. The article according to any one of Implementation Schemes A to E, wherein the inorganic layer comprises SiC x O y Furthermore, the biofouling-resistant layer comprises methyl-terminated SiC. x H y .

[0019] Implementation scheme G. The article according to any one of implementation schemes A to F, wherein the analyte binding layer comprises an acrylamide copolymer, a condensed silane, and mixtures and compositions thereof.

[0020] Implementation scheme H. The article according to any one of implementation schemes A to G, wherein the column has a diameter of 100 nm to 1500 nm.

[0021] Implementation Scheme I. The article according to any one of Implementation Schemes A to H, wherein the column has a height greater than 0 nm and at most 1000 nm.

[0022] Implementation Scheme J. The article according to any one of Implementation Schemes A to I, wherein the flexible structured membrane is a polymer with low autofluorescence.

[0023] Implementation scheme K. The article according to any one of implementation schemes A to J, wherein the flexible structured membrane is (meth)acrylic resin.

[0024] Implementation Scheme L. The article according to any one of Implementation Schemes A to K, the article further comprising a polymer support layer having a first main surface and a second main surface, wherein the first main surface of the polymer support layer is on the second main surface of the structured membrane.

[0025] Embodiment M. The article according to Embodiment L, wherein the polymer support layer comprises a polymer selected from cyclic olefin copolymers (COP), polypropylene, hydrogenated styrene, poly(meth)acrylate, polycarbonate, and mixtures and compositions thereof.

[0026] Implementation Scheme N. The article according to Implementation Scheme L further includes an adhesive layer on the second main surface of the polymer support layer.

[0027] Implementation scheme O. The article according to implementation scheme N, wherein the adhesive layer is optically transparent.

[0028] According to embodiment P, the article of embodiment N further includes a support layer on the adhesive layer, wherein the support layer is selected from release liner and rigid substrate.

[0029] Implementation Scheme Q. A method for manufacturing a component of a diagnostic device, the method comprising providing a flexible structured membrane having a first main surface and a second main surface, wherein the first main surface of the flexible structured membrane includes a patterned surface comprising an arrangement of pillars with dispersed platform regions; applying a release layer to the first main surface of the flexible structured membrane such that the release layer covers the top surface of the pillars and the platform regions, wherein the release layer includes a surface rich in methyl (CH3) groups; applying a planarization layer to the release layer; etching a portion of the planarization layer with an oxygen-containing etchant to form a non-methylated inorganic layer on the top surface of the pillars, wherein the inorganic layer comprises a metal or a metal oxide; removing the planarization layer; and attaching a functionalized silane material to the inorganic layer on the top surface of the pillars, and polymerizing the functionalized silane material to form an analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalized hydrogels, functionalized polymers, and mixtures and compositions thereof, and wherein the analyte binding layer contains at least one functional group that reacts with a biochemical analyte.

[0030] Implementation scheme R. The method according to implementation scheme Q, wherein the planarization layer is removed before the functional silane is attached.

[0031] Implementation S. The method according to implementation Q, wherein the functional layer is attached before the planarization layer is removed.

[0032] Implementation scheme T. The method according to any one of implementation schemes Q to S, wherein the analyte binding layer is attached to the inorganic layer by reacting the inorganic layer with one of an aminosilane or an acrylamide silane to form an acrylamide.

[0033] Implementation scheme U. The method according to implementation scheme T, wherein the acrylamide is polymerized to form poly(acrylamide).

[0034] Implementation scheme V. The method according to any one of implementation schemes Q to U, wherein the inorganic layer comprises at least one of Si, Ti or Al or oxides thereof, and wherein the thickness of the inorganic layer is less than about 200 nm.

[0035] Implementation scheme W. The method according to any one of implementation schemes Q to V, wherein the oxide of the inorganic layer is selected from SiO2, SiC x O y SiAl x O y TiO, AlO x And their mixtures and compositions.

[0036] Implementation Scheme X. The method according to Implementation Scheme W, wherein the oxide is SiCx O y .

[0037] Implementation scheme Y. The method according to any one of implementation schemes Q to X, wherein the inorganic layer comprises SiC x O y Furthermore, the release layer comprises methyl-terminated SiC. x H y .

[0038] Implementation Scheme Z. The method according to any one of Implementation Schemes Q to Y, wherein the analyte binding layer comprises an acrylamide copolymer, a condensed silane, and mixtures and compositions thereof.

[0039] Implementation scheme AA. The method according to any one of implementation schemes Q to Z, wherein the column has a diameter of 100 nm to 1500 nm.

[0040] Implementation scheme BB. The method according to any one of implementation schemes Q to AA, wherein the column has a height greater than 0 nm and at most 1000 nm.

[0041] Implementation scheme CC. The article according to any one of implementation schemes Q to BB, wherein the flexible structured membrane is (meth)acrylic resin.

[0042] Implementation Scheme DD. The method according to any one of Implementation Schemes Q to CC, wherein the polymer support layer comprises a cyclic olefin copolymer (COP).

[0043] Implementation EE. The method according to any one of implementations Q to DD, further comprising roughening a first primary surface of the polymer support layer prior to adhering the flexible structured membrane.

[0044] Implementation scheme FF. The method according to any one of implementation schemes Q to EE, wherein the planarization layer is a resin selected from PVB, PVA, (meth)acrylates and mixtures and compositions thereof.

[0045] Implementation scheme GG. The method according to any one of implementation schemes Q to FF, wherein the planarization layer is removed by attaching an adhesive layer thereto to form a laminate and then peeling off the laminate.

[0046] Implementation Scheme HH. The method according to any one of Implementation Schemes Q to GG, wherein the planarization layer is removed by coating a UV-curable (meth)acrylate layer thereon, laminating the UV-curable (meth)acrylate layer onto a carrier film, curing the UV-curable (meth)acrylate to form a cured structure, and peeling off the cured structure.

[0047] Implementation Scheme II. The method according to any one of Implementation Schemes Q to HH, wherein the planarization layer is removed by applying a solvent to the planarization layer.

[0048] Implementation Scheme JJ. The method according to any one of Implementation Schemes Q to II, the method further comprising applying an adhesive layer to the second main surface of the polymer support layer.

[0049] Implementation scheme KK. The method according to implementation scheme JJ, wherein the adhesive layer is optically transparent.

[0050] Implementation Scheme LL. The method according to any one of Implementation Schemes JJ to KK, the method further includes applying a support layer on the adhesive layer, wherein the support layer is selected from release liner and rigid substrate.

[0051] Implementation Scheme MM. A diagnostic device for detecting a biochemical analyte, the diagnostic device comprising a flow cell with a patterned arrangement of fluid channels configured to provide flow conduits for a sample fluid containing the biochemical analyte, wherein at least some of the fluid channels in the flow cell are lined on their surfaces with: a flexible structured membrane having a first primary surface and a second primary surface, wherein the first primary surface of the flexible structured membrane includes a plurality of pillars having exposed surfaces extending into the fluid channels of the flow cell, wherein the pillars are distributed with plateau regions; antimicrobial agents; The platform region includes a contamination layer, the anti-biocontamination layer comprising a methylated surface; a non-methylated inorganic layer on the exposed surface of the column, the inorganic layer comprising oxides of Si, Ti, or Al; and an analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof, and wherein the exposed surface of the analyte binding layer comprises at least one functional group selected for binding with a biochemical analyte in the sample fluid.

[0052] Implementation scheme NN. A DNA sequencing kit comprising a diagnostic device according to implementation scheme MM, a fluorescent reagent for DNA sequencing, and instructions.

[0053] Implementation Scheme OO. A method for DNA sequencing, the method comprising: in a diagnostic device, the diagnostic device including a flow cell with a patterned arrangement of fluid channels configured to provide flow conduits for a sample fluid containing a target analyte comprising polynucleotides and nucleic acids, wherein at least some of the fluid channels in the flow cell are lined on their surfaces with: a flexible structured membrane having a first primary surface and a second primary surface, wherein the first primary surface of the flexible structured membrane includes an array of pillars, and wherein each pillar includes an exposed surface extending into the fluid channels of the flow cell and a plurality of plateau regions between the pillars; an anti-biocontamination layer in the plateau regions, wherein the anti-biocontamination layer includes a methylated surface; and non- A methylated inorganic layer, the unmethylated inorganic layer being on the exposed surface of the column, wherein the inorganic layer comprises oxides of Si, Ti, or Al; and an analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof, and wherein the exposed surface of the analyte binding layer contains at least one functional group selected for binding to a biochemical analyte in the sample fluid; binding the target analyte in the sample fluid to the analyte binding layer; exposing the target analyte bound to the analyte binding layer to a fluorescent reagent and an enzyme for detection of the analyte using energy dispersive spectroscopy; and cleaving the fluorescent reagent to allow further exploration of the target analyte.

[0054] Various aspects and advantages of the exemplary embodiments of this disclosure have been summarized. The above summary is not intended to describe every illustrative embodiment or every implementation of the presently available exemplary embodiments of this disclosure. The following drawings and detailed descriptions illustrate more specifically certain preferred embodiments using the principles disclosed herein. Attached Figure Description

[0055] The following detailed description of various embodiments of this disclosure is taken into consideration in conjunction with the accompanying drawings.

[0056] This disclosure can be understood more fully in the accompanying drawings:

[0057] Figure 1 This is a schematic cross-sectional view of one embodiment of a component article on a nanostructured flexible polymer carrier substrate according to the present disclosure, the component article comprising functionalized pillars.

[0058] Figure 2 It is used for manufacturing Figure 1 A schematic cross-sectional view of an exemplary embodiment of the method for producing the article.

[0059] Figure 3 It can be used Figure 2A schematic cross-sectional view of one implementation of the low-platform transfer method in the method.

[0060] Figure 4 These are the fluorescence spectra of the membrane sample and standard from Example 2.

[0061] Figure 5 This is a graph showing XPS data of the selective bonding of the materials in Example 3.

[0062] Figure 6 This is a scanning electron microscope (SEM) image of a 400 nm diameter nanostructured film with release treatment manufactured according to Example 4.

[0063] Figure 7 This is an SEM image of the etched sample film from Example 4.

[0064] Figure 8 yes Figures 6 to 7 SEM image of the peeling membrane.

[0065] Figure 9 This is a contact force microscopy (CFM) image of a 4 μm × 4 μm sample of a nanostructured membrane with release treatment manufactured according to Example 4.

[0066] Figure 10 This is a 4μm × 4μm force volume adhesion map obtained using an atomic force microscope (AFM) probe with CH3 end caps on the membrane of Example 4.

[0067] Figure 11 This is an SEM image of the 200nm diameter structured film after etching in Example 5.

[0068] Figure 12 This is an SEM image of the release film of Example 5.

[0069] Figure 13 This is a TOF-SIMS image of the 1500 nm diameter column from Example 6 after the stripping step. The scale bar in the image is 10 micrometers (μm).

[0070] Figure 14 This is an SEM image of a 400 nm diameter column on the roughened COP film of Example 7.

[0071] In the accompanying drawings, similar reference numerals indicate similar elements. While the above drawings, which may not be drawn to scale, illustrate various embodiments of the present disclosure, other embodiments are contemplated, as indicated in the detailed description. This detailed description depicts representative exemplary and currently preferred embodiments. It should be understood that many other modifications and embodiments will be conceived by those skilled in the art, falling within the scope and spirit of this disclosure and the claims. Detailed Implementation

[0072] For terms defined below, unless a different definition is provided elsewhere in the claims or specification, the entire application shall be governed by these definitions.

[0073] By using orientation terms such as “on top of,” “on,” “above,” “cover,” “topmost,” “below,” “stacked,” etc., to describe the position of various elements in the disclosed multilayer articles, we are referring to the relative position of the elements with respect to a horizontally arranged, upward-facing substrate. However, unless otherwise specified, this invention is not intended for the substrate or article to have any particular spatial orientation during or after manufacturing.

[0074] When using the term "overlay" to describe the position of a layer relative to a substrate or other layer of the article of manufacture of this disclosure, we refer to the layer as being on top of the substrate or other element, but not necessarily in contact with or adjacent to the substrate or other layer.

[0075] By using the term "separated from" to describe the position of a layer relative to other layers, we say that the layer is positioned between two other layers, but not necessarily adjacent to or adjacent to any of them.

[0076] The terms “about” or “approximately” regarding numerical values ​​or shapes mean + / - 5% of that value, property, or characteristic, but explicitly include exact numerical values. For example, “about” a viscosity of 1 Pa-sec refers to a viscosity from 0.95 Pa-sec to 1.05 Pa-sec, but also explicitly includes a viscosity of exactly 1 Pa-sec. Similarly, the perimeter of “essentially square” is intended to describe a geometry with four lateral edges, where the length of each lateral edge is 95% to 105% of the length of any other lateral edge, but also includes geometries where each lateral edge has exactly the same length.

[0077] The term "substantially" in relation to a property or feature means that the property or feature is exhibited to a greater degree than its opposite surface. For example, a "substantially" transparent substrate is one that transmits more radiation (e.g., visible light) than a substrate that does not transmit (e.g., absorbs and reflects). Therefore, a substrate that transmits more than 50% of the visible light incident on its surface is substantially transparent, but a substrate that transmits 50% or less of the visible light incident on its surface is not substantially transparent.

[0078] As used in this specification and the accompanying embodiments, unless the content clearly indicates otherwise, the singular forms “an,” “a,” and “the / described” include multiple referents. Thus, for example, a reference to fine fibers containing “a compound” includes a mixture of two or more compounds. As used in this specification and the accompanying embodiments, unless the content expressly specifies otherwise, the term “or” is generally used in its meaning including “and / or.”

[0079] As used in this specification, a range of values ​​expressed by endpoints includes all values ​​included in that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.8, 4, and 5).

[0080] Unless otherwise specified, all figures for expressions or measurements of components, properties, etc., used in this specification and embodiments should in all cases be understood to be modified by the term "about". Therefore, unless stated to the contrary, the numerical parameters shown in the foregoing specification and the appended list of embodiments may vary according to the desired properties sought by those skilled in the art using the teachings of this disclosure. At a minimum, and without attempting to limit the application of the doctrine of equivalence to the embodiments protected by the claims, each numerical parameter should be interpreted at least according to the number of significant digits reported and by applying customary rounding.

[0081] Various exemplary embodiments of the present disclosure will now be described with specific reference to the accompanying drawings. Various modifications and alterations may be made to the exemplary embodiments of the present disclosure without departing from the spirit and scope of the disclosure. Therefore, it should be understood that the embodiments of the present disclosure are not limited to those described below, but are subject to the limiting factors shown in the claims and any equivalents.

[0082] Now for reference Figure 1 A schematic diagram of a portion of the component article 10 (not drawn to scale) includes a flexible structured membrane 16 having a first structured main surface 19 and an opposing second main surface 17. The flexible structured membrane 16 may contain any polymer material suitable for or applicable to roll-to-roll processes.

[0083] In some embodiments, the material and thickness of the flexible structured membrane 16 should be selected to achieve low autofluorescence to provide a low-noise background for bioassays, where analysis is performed using, for example, fluorescent biological structures, fluorescent markers, or fluorophores. For example, fluorescent molecules can be used to detect DNA or RNA nucleotide sequences. In other examples not intended to be limiting, the fluorescent molecules can be labeled nucleotides, such as reversible terminators, or labeled oligonucleotide probes. In some cases, different labeled nucleotides or probes in the kit are labeled with different fluorophores that emit different wavelengths according to a specific sequence, enabling the recall of multiple bases in a single scan. The autofluorescent flexible structured membrane 16 can potentially mask signals from these fluorescent sequencing reagents. In some examples, the polymer resin can optionally be modified to reduce fluorescence, which allows for the use of a wider variety of polymer materials in the flexible structured membrane 16. In some examples not intended to be limiting, the flexible structured film 16 should have autofluorescence measured between 400 nm and 800 nm, or between 450 nm and 650 nm, similar to the autofluorescence of borosilicate glass or other substrates commonly used in bioassays.

[0084] Autofluorescence is not a single figure, as the emission spectrum depends on the excitation wavelength, and a particular polymer material may have high or low autofluorescence depending on the wavelength. In some examples, to provide low autofluorescence for the detection of a variety of biodetector molecules, cyclic olefin copolymers (COPs) or biaxially oriented polypropylene (BOPPs) can be used, each exhibiting low autofluorescence over a broad spectral range. Other examples of suitable low-autofluorescence polymer materials include, but are not limited to, poly(meth)acrylates and their copolymers (wherein the (meth)acrylate comprises acrylates and methacrylates), polyamides, polyesters, polycarbonates (such as, for example, those from Covestro AG (Pittsburgh, PA) under the trade name Makrolon), hydrogenated styrene derivatives (such as, for example, cyclic block copolymers from Vivion, Inc., San Carlos, CA), and mixtures and compositions thereof. In some embodiments, (meth)acrylates can be cured by ultraviolet (UV) radiation.

[0085] In various embodiments, the flexible structured membrane 16 may comprise a single layer or multiple layers of any of these polymers, and the total thickness of the flexible structured membrane may be from about 25 nm to about 1000 μm. In some examples, the structured membrane 16 may be made of a more autofluorescent material and then made thinner to reduce the autofluorescence of layer 16.

[0086] The second primary surface 19 of the flexible polymer carrier membrane 16 includes a plurality of structures 20 extending therefrom. Platform regions 22 are scattered among the structures 20. In various embodiments, not intended to be limiting and provided as examples, the structures 20 may be arranged in a regular or irregular array on the surface 19, and the structures 20 may be present in all or part of the surface 19. Figure 1 In the implementation scheme, structure 20 is typically a cylindrical column or pillar, but structure 20 may also have shapes such as spheres, cones, cubes, etc. Structure 20 may include a wide variety of cross-sectional shapes, such as, for example, approximately rectangular, arcuate, trapezoidal, cube, etc.

[0087] In various embodiments, the surface 19 of the flexible polymer carrier film 16 can be structured by a variety of methods, including but not limited to microreplication for structuring tools, casting, microcontact or inkjet printing, chemical treatment, laser patterning, and combinations thereof. In some embodiments provided as examples, the plurality of structures 20 comprise a regular array of cylindrical or cubic pillars with diameters d of about 100 nm to about 1500 nm, or about 200 nm and 500 nm, and a height h above the surface 19 greater than 0 nm and at most about 1000 nm, or about 50 nm to about 200 nm. In some exemplary embodiments, the pillars have an aspect ratio (height:diameter) of about 5:1 to about 1:70, or about 5:1 to 1:5, or about 2:1 to 1:1. In some embodiments (not shown in…), Figure 1 As shown in the figure, the column 20 may optionally taper with a taper angle of greater than 0° and less than about 25° or about 2° to about 10°, measured relative to the plane of surface 19.

[0088] Structure 20 includes an inorganic layer 24 having a first main surface 25 and a second main surface 27. In some exemplary embodiments, the thickness of the inorganic layer 24 is less than about 200 nm, or less than about 100 nm, less than about 50 nm, or even less than about 20 nm. The composition of the inorganic layer 24 can vary widely, but in some examples not intended to be limiting, it contains silicon oxides such as SiO2 and SiC. x O y or SiAl x O y as well as TiO, aluminum oxide AlO x Oxides of other metals such as Au, Sn, Ge, Ga, Zn, and In, as well as mixtures and compositions thereof. Compared to conventional wafer processing, amorphous silicon oxides deposited by roll-to-roll processing can contain impurities such as aluminum or carbon, which can achieve more efficient deposition rates on flexible, temperature-sensitive surfaces using techniques such as sputtering or PECVD.

[0089] In some embodiments, a silane having a reactive functional group is condensed on at least a second master surface 27 of the inorganic layer 24. The reactive functional group is selected to grow an analyte binding layer 26 on at least a second master surface 27 of the inorganic layer 24, or to graft the analyte binding layer 26 onto at least a second master surface 27 of the inorganic layer 24. Suitable reactive functional groups for silanes include, but are not limited to, epoxides, ethylene oxides, aziridines, isocyanates, alcohols, thiols, amines, chloromethylbenzyl, bromomethylbenzyl, iodomethylbenzyl, alkyl halides, vinyl groups, carbonyl groups such as aldehydes and ketones, carboxylic acids, esters, azides, sulfates, phosphates, alkenes, alkynes, (meth)acrylates, (meth)acrylamide, norbornene, diazonium salts, hydrazines, hydrazones, oximes, halogens, hydroxyl groups, tetrazolium, tetrazines, and mixtures and compositions thereof. In some embodiments, the functional group is a photoreactive functional group, such as benzophenone, aryl azide, haloaryl azide, diazo, or azo, which can be used to grow or graft analyte-binding layers using free radical chemistry. In some embodiments, norbornene silane has been found to be particularly useful.

[0090] In some examples, condensed reactive silane functional groups are selected to provide covalent bonds at the interface between at least the second primary surface 27 of the inorganic layer 24 and the first primary surface 29 of the analyte-binding layer 26. For example, the analyte-binding layer 26 is covalently bonded to the inorganic layer 24 by reacting with a condensed functional silane having any of the reactive functional groups listed above. Suitable examples of functional silanes include, but are not limited to, acrylate silanes, amino silanes, acrylamide silanes, norbornene silanes, and mixtures and compositions thereof.

[0091] The reactive functional group derived from the functional silane is separated from the inorganic layer 24 via a hydrocarbon linking group, which more effectively binds the analyte binding layer 26 and the inorganic layer 24. The hydrocarbon linking group is at least one methylene unit long and, in various embodiments, may contain about 1 to about 20 carbon atoms, or about 2 to about 15 carbon atoms. In various embodiments, the hydrocarbon linking group may be straight-chain, cyclic, branched, or aromatic, and may optionally contain heteroatoms such as, for example, oxygen, nitrogen, sulfur, phosphorus, and combinations thereof.

[0092] The analyte binding layer 26 contains reactive functional groups selected for binding with the target analyte. In some cases, these reactive functional groups may be the same as or different from those used for covalent binding to the inorganic layer 24. In various embodiments not intended to be limiting, reactive functional groups are selected within or on the second master surface 31 of the analyte binding layer to bind biomolecules selected from amino acids, nucleosides, nucleotides, peptides, oligonucleotides, polynucleotides, nucleic acids, proteins, carbohydrates, secondary metabolites, drug molecules, and mixtures and compositions thereof, as well as undesirable chemical contaminants found in liquid aqueous streams and water sources.

[0093] In some cases, biomolecules are chemically modified to facilitate covalent bonding with the analyte binding layer. In other cases, biomolecules can be used to bind additional analytes. For example, but not limited to, the molecule may be an antibody, a carbohydrate that can bind lectins, or an oligonucleotide primer or mixture of oligonucleotide primers that can bind complementary DNA or RNA molecules.

[0094] In various embodiments, the analyte binding layer 26 is made of a functionalized material selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof. Suitable reactive functional groups for these functionalized materials include, but are not limited to, substituted and unsubstituted alkenes, azides, alkynes, substituted and substituted amines, carboxylic acids, substituted and unsubstituted hydrazones, halogens, hydroxyl groups, substituted and unsubstituted tetrazolium, substituted and unsubstituted tetrazines, thiols, epoxides, carbonyl groups (including aldehydes and ketones), aziridine, ethylene oxide, and combinations thereof. In one example, which is not intended to be limiting, a DNA primer oligomer having an alkyne that can be conjugated to an azide-functionalized hydrogel may be used.

[0095] In some embodiments, the analyte binding layer 26 comprises a polymer or hydrogel of formula (Ia) or (Ib):

[0096]

[0097] In equations 1a and 1b, R 1 It is an H or optionally substituted alkyl group; functional group R A Choose from the group consisting of: azides, optionally substituted amines, optionally substituted alkenes, optionally substituted hydrazones, carboxylic acids, halogens, hydroxyl groups, optionally substituted tetrazoliums, optionally substituted tetrazides, and thiols; R 5The alkyl group is selected from H or optionally substituted alkyl groups; each of —(CH2)-p may optionally be substituted; p is an integer in the range of 1 to 50; n is an integer in the range of 1 to 50,000; and m is an integer in the range of 1 to 100,000. In some such embodiments, the functional group comprises an azide. In some embodiments, each R 1 and R 5 All are hydrogen. In some implementations, the functional group R A It is an azide. In some embodiments, p is 5.

[0098] In one embodiment, the polymer or hydrogel contained in the functionalizable layer is PAZAM. Methods for preparing and using PAZAM, as well as other functionalizable materials that can be used in layers of the substrate of this disclosure, are described in U.S. Patent No. 9,012,022, the entire contents of which are incorporated herein by reference.

[0099] Examples of usable reactive silanes include, but are not limited to, (meth)acrylate-functionalized silanes, (meth)acrylamide-functionalized silanes, aldehyde-functionalized silanes, amino-functionalized silanes, acid anhydride-functionalized silanes, azide-functionalized silanes, carboxylic acid ester-functionalized silanes, phosphonate-functionalized silanes, sulfonate-functionalized silanes, epoxy-functionalized silanes, ester-functionalized silanes, vinyl-functionalized silanes, olefin-functionalized silanes, halogen-functionalized silanes, and bimodal silanes having any of the above functional groups or not having the above functional groups. Norbornene silanes have been found to be particularly useful.

[0100] The choice of silane functional groups can be based on the reactivity of the materials with which they will react. For example, acrylamide- or norbornene-functionalized silanes can react with azide-functionalized polymers. Amino-functionalized silanes can react with carbonyl-functionalized polymers, where the carbonyl group is a carboxylic acid, ester, aldehyde, ketone, or activated ester, or combinations thereof. Silanes with photoactive functional groups (such as benzophenone, diazo, or azidobenzyl) can be used to graft any polymer with hydrocarbon linkages via hydrogen abstraction.

[0101] In some embodiments, the analyte binding layer 26 may comprise a hydrogel. Non-limiting examples of hydrogels are described in U.S. Patent No. 9,012,022 and include polyacrylamide hydrogels and arrays based on polyacrylamide hydrogels. Other hydrogels are poly(meth)acrylate hydrogels and arrays based on poly(meth)acrylate. Once the hydrogel has been formed, biomolecules can then be attached to the hydrogel to generate a molecular array. The hydrogel can be chemically modified after formation. For example, the hydrogel can be polymerized with a comonomer having initiating or pre-activated functional groups to react with the biomolecules to be arranged. In some examples, the array is formed simultaneously with the direct copolymerization of polynucleotides derived from acrylamide. In one example, acrylamide phosphoridamide, traded under the name ACRYDITE from Mosaic Technologies, Boston, MA, can be reacted with the polynucleotide prior to copolymerization of the resulting monomer with acrylamide.

[0102] In some embodiments, the analyte binding layer 26 comprises a polymer having one or more functional groups capable of reacting with the biomolecule of interest. In some such embodiments, the functional groups may be selected from substituted and unsubstituted alkenes, azides, substituted or unsubstituted amines, carboxylic acids, substituted or unsubstituted hydrazones, halogens, hydroxyl groups, substituted or unsubstituted tetrazolium, substituted or unsubstituted tetrazine, thiols, and combinations thereof.

[0103] In some embodiments, polymers of formula (Ia) or (Ib) are also represented by formula (IIa) or (IIb):

[0104]

[0105] Where n is an integer in the range of 1 to 20,000, and m is an integer in the range of 1 to 100,000.

[0106] In some embodiments, the functionalizing molecule used for direct conjugation is poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM). PAZAM can be prepared by polymerizing acrylamide and azido(N-(5-(2-azidoacetamido)pentyl)acrylamide in any ratio. In various embodiments, PAZAM is a linear polymer, a mildly crosslinked polymer, which is available in an aqueous solution or can be supplied as an aqueous solution with one or more solvent additives. For example, in some embodiments, a polymer containing an aryl azide as described in US20190232890 can be used.

[0107] Refer again Figure 1In some embodiments, the analyte binding layer 26 may comprise at least one photocurable polymer selected from polyurethanes, acrylates, silicones, epoxy resins, polyacrylic acid, polyacrylates, epoxy silicones, epoxy resins, polydimethylsiloxane (PDMS), silsesquioxanes, acyloxysilanes, maleate polyesters, vinyl ethers, monomers having vinyl or ethynyl groups, or copolymers and compositions thereof.

[0108] like Figure 1 As schematically shown, the structured surface 19 of the flexible polymer carrier membrane 16 in the platform region 22 between structures 20 is free of the analyte binding layer 26 and includes an anti-biofouling layer 32 thereon. For example, the anti-biofouling layer 32 may include a first primary surface 33 in contact with the surface 19 of the carrier membrane 16 and a second primary surface 35 exposed in the platform region 22. In some embodiments, the anti-biofouling layer 32 may have the same bulk composition as the inorganic layer 24 and a different surface composition on the exposed surface 35. In some embodiments, the anti-biofouling layer 32 may also be present on all or part of the wall 37 of structure 20 in some cases.

[0109] The exposed surface of the anticontamination layer 35 may contain any material that resists or prevents the accumulation or formation of biological species (such as, for example, microorganisms) or biomolecules (such as nucleic acids and proteins). Therefore, the exposed surface of the anticontamination layer 35 prevents the nonspecific adhesion of target analytes, sequencing reagents, or fluorophores to at least a portion of the gap regions or platform regions 22 between structures 20. If the anticontamination layer 32 is applied to a specific region of the article 10, other regions not coated with the anticontamination layer 32 can bind to biological samples. Because the anticontamination surface 35 is absent in the platform region 22 and on the exposed surface of the top surface 27 of the inorganic layer 24, biological material can bind to the analyte-binding material 26 on top of the structure 20. Thus, the surface 35 of the anticontamination layer 32 provides specific placement of analyte-binding material (and the biological material bound thereto) in one or more regions of the article 10.

[0110] The exposed surface 35 of the anti-biofouling layer 32 contains a plurality of methyl (CH3) groups. The exposed surface 35 of the anti-biofouling layer 32 (which is hydrophobic and relatively non-reactive, such as a silicone material) can comprise any composition with a surface rich in methyl groups. The anti-biofouling layer 32 thus forms a release layer in the platform region 22. In an example not intended to be limiting, the methyl groups are formed by plasma-enhanced chemical vapor deposition (PECVD) of hexamethyldisiloxane, forming a thin surface 35 with a thickness of about 1 nm to about 10 nm or about 2 nm to about 8 nm. The methyl groups in the film are attached to Si atoms in the anti-biofouling layer 32 and provide a hydrophobic, non-reactive layer in the platform region 22.

[0111] In some examples, the methyl-terminated surface 35 of the anti-biofouling layer 32 is sufficiently enriched with methyl groups to provide a water contact angle greater than 100 degrees. In some examples, the methyl groups can be formed by plasma dissociation of molecular fragments of hexamethyldisiloxane, although any method of producing a methyl-terminated surface on a metal, metalloid, metal oxide, or metalloid oxide can provide similar functionality.

[0112] Another example of forming a biofouling-resistant surface on an inorganic layer involves the reaction of silanes with hydrolysis-sensitive centers having organic substitutions containing methyl groups. Examples of hydrolysis-reactive groups are chlorine, methoxy, ethoxy, propoxy, methoxyalkoxy, acetoxy, amines such as dimethylamine, silazane, or oxime. Examples of organic substitutions include methyl, straight-chain alkyl, branched-chain alkyl, aryl, and biarm alkyl. In various examples, silanes can be applied by vapor deposition, spraying, or solvent coating.

[0113] Other chemical components (such as tetraethyl orthosilicate, tetramethylsilane, hexamethyldisilane, bis(trimethylsilyl)amine, trimethylamine, or tetramethyltin and other similar metallic alkyl compounds) can be deposited using plasma-enhanced chemical vapor deposition to create methyl-terminated surfaces. Alternatively, precursors such as trimethylamine can be deposited on suitable surfaces using atomic layer deposition to form a monolayer of methyl groups.

[0114] The optional polymer support membrane 12 lies beneath the flexible structured membrane 16, and... Figure 1 The embodiments include a first main surface 13 and a second main surface 15. The first main surface 13 of the support film 12 is beneath the second main surface 17 of the carrier film 16. In some embodiments, the first main surface 13 of the support film 12 may optionally be roughened, chemically treated, corona-treated, etc., to enhance adhesion to the carrier film 16. Suitable surface modification techniques for the first main surface 13 of the support film 12 include, for example, plasma-enhanced chemical vapor deposition (PECVD).

[0115] The composition of the support membrane 12 can vary widely and, in various embodiments, may include any low autofluorescence polymer material for the flexible structured membrane 16. In some embodiments, which are not intended to be limiting, the support membrane 12 may be selected from poly(meth)acrylates and copolymers thereof, wherein (meth)acrylates include acrylates and methacrylates, polypropylene, hydrogenated styrene, poly(meth)acrylates, polycarbonate, and mixtures and compositions thereof.

[0116] In some implementations, (meth)acrylate can be cured by ultraviolet (UV) radiation. The thickness of the support film 12 can be adjusted as needed to control the overall autofluorescence of layers 12 and 16.

[0117] In some embodiments, the second primary surface 15 of the polymer support membrane 12 includes an optional adhesive layer 40. Any adhesive can be used in the adhesive layer 40, but materials with low autofluorescence have been found particularly suitable for analytical devices containing biochemical analytes. In some examples, not intended to be limiting, the adhesive layer 40 includes an optically clear adhesive, such as the adhesive available from 3M Corporation (3M) under the trade name 3M Optical Clear Adhesive 8171, and a polyisobutylene polymer adhesive. Suitable isobutylene adhesives may contain styrene-isobutylene copolymers or have multifunctional components such as (meth)acryloyl and vinyl ether groups.

[0118] In some examples, not intended to be limiting, the thickness of the adhesive layer 40 is from about 1 μm to about 50 μm or from about 5 μm to about 15 μm. In some embodiments, the adhesive layer 40 should be sufficiently uniform such that the exposed surface of the analyte bonding layer 26 ( Figure 1 The variation of the focal plane of the second primary surface 31) in the focal plane is no more than about 5 μm, or no more than about 2 μm, or no more than about 1 μm, 500 nm, 250 nm or 100 nm.

[0119] The surfaces 41 and / or 43 of the adhesive layer 40 may optionally be structured, for example, with a network of venting channels to reduce trapped air when the adhesive layer 40 is applied to a flat surface of a rigid substrate, such as a glass plate. In some embodiments, the adhesive layer 40 may be a repositionable adhesive and may optionally include glass beads, adhesives with low green strength, vacuum lamination, etc.

[0120] Various techniques can be used to apply the adhesive layer 40 to the second main surface 15 of the polymer support film 12, including direct coating on the surface 15 or by laminating the transfer adhesive onto the flexible substrate 12.

[0121] In some examples, the adhesive layer 40 is attached to an optional reinforcing layer or rigid substrate 42, which provides increased rigidity, making the article of manufacture 10 more readily usable in devices typically used to perform biochemical assays. The reinforcing layer 42 can vary widely and includes silicon, glass, plastic, metal, metal oxide, paper, and combinations thereof in various embodiments. In various embodiments, the reinforcing layer 42 may comprise a single layer or multiple layers. In some embodiments, the main surface 45 of the rigid substrate 42 may optionally be treated to enhance the removal of the adhesive layer 40.

[0122] In another embodiment, the reinforcing layer 42 may be a release liner protecting the adhesive layer 40 and may be peeled off from the adhesive layer 40, allowing the article of manufacture to be applied to a selected substrate prior to use in an apparatus for performing biochemical assays. Suitable release liners 42 include, but are not limited to, polymer films, paper, metals, metal oxides, and combinations thereof. Release liners 42 may comprise a single layer or multiple layers.

[0123] Figure 2 It is used to form by Figure 1 The diagram illustrates one embodiment of a method 100 for producing component articles having functionalized analyte-binding columns. In various embodiments, method 100 can be performed on a production line in a roll-to-roll process to manufacture component articles on a polymer support layer, or individual articles can be produced individually.

[0124] In step 160, a polymer support film 112 is used as a low autofluorescence backing substrate for the component article, the polymer support film having a first main surface 113 and a second main surface 115. As described above, a suitable support film 112 contains a low autofluorescence material, such as, for example, COP, PET, etc.

[0125] In some embodiments, the first primary surface 113 of the support film 112 may optionally be roughened to enhance adhesion to subsequent layers. In one example, surface 113 may be roughened by plasma-enhanced chemical vapor deposition (PECVD), but it may also be roughened by contacting the surface with a tool or with another structured film. In another example, which is not intended to be limiting, surface 113 may be modified by adding random nanostructures by depositing a silicon-containing discontinuous layer using plasma-enhanced chemical vapor deposition (PECVD) while or sequentially etching the surface with a reactive material, as described in U.S. Patent Nos. 10,134,566 and 8,634,146, respectively, which are incorporated herein by reference in their entirety.

[0126] In step 162, the flexible structured membrane 116 is transferred onto the surface 113 of the carrier membrane 112. The structured membrane 116 includes a structured surface 119.

[0127] In some embodiments, the flexible structured membrane 116 is prepared by casting and curing a polymerizable resin onto a support membrane 112. As described in U.S. Patent Nos. 5,175,030 to Lu et al. and 5,183,597 to Lu, the article can be prepared by a method comprising the steps of: depositing a polymerizable composition in an amount sufficient to fill the structure of a host microstructured or nanostructured molded surface, or filling the structure by moving beads of the polymerizable composition between the support membrane 112 and the host molded surface, wherein at least one of the support membrane and the host molded surface is flexible; curing the composition; and separating the structured membrane 116 from the host molded surface. In other examples, the surface 119 of the flexible structured membrane 116 can be structured by a variety of methods, including but not limited to microreplication for structured tools, microcontact or inkjet printing, chemical treatment, laser patterning, and combinations thereof.

[0128] In another embodiment, the structured membrane 116 is transferred onto the surface 113 of the support membrane 112. Figure 3 The implementation of method 200 is described. The steps of method 200 can be performed in a number of different sequences, and Figure 3 The order of steps in the process is not intended to be restrictive.

[0129] like Figure 3 As shown in step 202, the optional support layer 280 includes a patterned layer 282. In some embodiments, the patterned layer 282 can be structured by a variety of methods, including but not limited to micro-replication for structured tools, casting, micro-contact or inkjet printing, chemical processing, laser patterning, and combinations thereof. The patterned layer 282 includes a patterned surface 283, which includes one or more recessed features 284, each recessed feature adjacent to at least one platform feature 286.

[0130] In step 204, a structured layer 288 is applied to the patterned surface 283 of the patterned layer 282 to form a transfer structure 289.

[0131] In step 206, the transfer structure 289 formed in step 204 is laminated onto... Figure 2 On the first main surface 113 of the polymer support film 112 shown in step 160.

[0132] In step 208, the structured layer 288 of the transfer structure 289 separates from the patterned layer 282, leaving a flexible structured film 116 with a patterned surface 119. Figure 2The patterned surface 119 includes an arrangement of columnar structures 120 scattered with plateau regions 122. The patterned surface 119 includes a pattern of protrusions 146 and plateau regions 148, which is an inversion of the pattern of recessed features 284 and plateau features 286 in surface 283. Method 200 thus provides a low-plateau transfer of the structured surface layer 116 to the polymer support film 112, the result of which is shown in… Figure 2 In step 162.

[0133] In one exemplary embodiment, the structured film 116 is a UV-curable (meth)acrylate having a patterned surface 119 with an arrangement of pillars 146 having diameters of about 100 nm to about 1500 nm or about 200 nm and about 500 nm, the pillars being scattered with an arrangement of plateau regions 148. In various embodiments not intended to be limiting, the aspect ratio of the pillars 146 in the patterned surface 119 is about 5:1 to about 1:5 (height:diameter), or about 2:1 and 1:1 (height:diameter). In some embodiments (not shown in...) Figures 2 to 3 As shown in the figure, column 120 may optionally taper at a taper angle of more than 0° and less than about 25° or about 2° to about 10°.

[0134] Refer again Figure 2 In step 164 of method 100, an anti-biofouling layer 132 having a methyl-rich surface 135 is applied to the surface 119 of the carrier membrane 116 to form a release coating thereon. In some exemplary embodiments, not intended to be limiting, layer 132 comprises having SiC x H y The composition of the plasma polymerized material is applied to the surface 119 of the carrier film 116. In some examples not intended to be limiting, layer 132 may be deposited on surface 119 by plasma enhanced chemical vapor deposition (PECVD) or sputtering of hexamethyldisiloxane in a roll-to-roll manner.

[0135] Layer 132 forms a release coating on the pillars 120 and plateau regions 122 of the surface 119 of the carrier film 116 (the release coating comprises amorphous SiC with methylated Si surfaces 135). x H y This is commonly referred to as HMDSO treatment. The methylated Si surface 135 is rich in methyl (CH3) groups attached to Si atoms and includes a non-reactive hydrophobic surface that, in some examples, is reactively similar to silicone. Other examples of materials used for layer 132 are mixtures and compositions of silicon, carbon, hydrogen, aluminum, tin, germanium, gallium, zinc, and indium, wherein surface 135 is methyl or other hydrocarbons.

[0136] In various embodiments, the thickness of layer 132 is about 1 nm to about 200 nm, about 1 nm to about 50 nm, about 2 nm to about 10 nm, or about 2 nm to about 8 nm.

[0137] In step 166, a planarization layer 190 is applied to the inorganic layer 132 to cover the pillar 120 and the platform region 122. In various embodiments, not intended to be limiting, the planarization layer 190 is a polymer resin, such as, for example, polyvinyl butyral (PVB), polyvinyl alcohol (PVA), (meth)acrylate, etc.

[0138] In step 168, an oxygen-rich reactive ion etching (RIE) step is used to remove a portion of the planarization layer 190 and the methylated surface 135 to form an inorganic layer 124 with a non-methylated inorganic surface 127 on the pillar 120, while leaving the planarization layer covering the plateau region 122. In some embodiments, if the planarization layer 190 contains Si, fluorine may optionally be added to the etchant. The etching depth can be controlled based on the etching duration and selectivity to remove the desired portion of the planarization layer 190.

[0139] The addition of oxygen from the etchant provides a wide range of possible compositions for the inorganic layer 124, but in some examples not intended to be limited, it includes materials such as SiO2 and SiC. x O y or SiAl x O y Silicon oxides, as well as TiO and aluminum oxides AlO x Oxides of other metals such as Au, Sn, Ge, Ga, Zn, and In, as well as mixtures and compositions thereof. In some cases, the remainder of layer 132 may be retained beneath layer 124. In some embodiments, the exposed surface of planarization layer 190 may optionally be structured to enhance adhesion to or release from subsequently applied layers.

[0140] The etching step alters the chemical composition of the methyl-rich surface 135 to form a non-methylated inorganic surface 127. An oxygen-containing etchant removes substantially all methyl groups from surface 127, resulting in a more reactive silica-like surface containing exposed silanol groups, which is useful for subsequent bonding steps.

[0141] In step 170, the planarization layer 190 is removed using any suitable technique to expose the methylated surface 135 in the platform region 122. In various embodiments, the planarization layer 190 can be removed by applying an adhesive and peeling it off, or by coating a UV-curable acrylate, laminating it with a carrier film, curing it, and then peeling it off. In another embodiment, the planarization layer 190 can be removed by washing with a solvent, mask removal agent solution, or water, optionally using agitation such as ultrasonication or spraying. In some embodiments, the surface of the planarization layer 190 can optionally be roughened or structured to facilitate the peeling process.

[0142] In step 172, an analyte binding layer 126, for example a functionalized alkoxysilane, is applied over the inorganic layer 124 and bonded to its exposed surface 127 to form a component structure 194 for, for example, biochemical assays. The analyte binding layer 126 does not react with or bind to the methylated surface 135 within the platform region 122.

[0143] In some exemplary embodiments, the silane in the analyte binding layer 126 contains reactive groups that can be used to form a hydrogel polymer on the column 120. In one example, which is not intended to be limiting, the alkoxysilane contains an acrylamide functional group. Following column functionalization, the acrylamide in the analyte binding layer 126 polymerizes on the surface, resulting in the growth of poly(acrylamide) on the column 120. In another embodiment, the silane in the analyte binding layer 126 reacts directly with an acrylamide silane.

[0144] exist Figure 2 In another embodiment not shown, the analyte binding layer 126 may optionally be applied to the surface 127 in step 168 before the planarization layer 190 is removed in step 170.

[0145] Although not in Figure 2 As shown in the diagram, but as discussed above, in some embodiments, an optional adhesive layer may be applied to the second primary surface 115 of the support membrane 112 of the component construction 194. The adhesive layer may include an optional protective release liner that can be removed, allowing the adhesive-backed component construction 194 to adhere to a reinforcing layer such as glass, paper, or a polymer film.

[0146] The aforementioned components and devices can be used in a variety of biochemical analysis procedures, including but not limited to DNA sequencing tests. For example, a diagnostic device for DNA sequencing may include a flow cell with a patterned arrangement of fluid channels configured to provide flow conduits for sample fluid containing target analytes of polynucleotides and nucleic acids.

[0147] At least some of the fluid channels in the flow cell may be lined with pillars on their surfaces, the pillars comprising an analyte-binding layer bonded to an underlying Si oxide layer via a network of methylene groups. Target analytes in the sample fluid bind to the analyte-binding layer, and the bound analytes are exposed to a fluorescent reagent, enabling the analytes to be detected using spectroscopy.

[0148] In another example, the diagnostic device may be included in a DNA sequencing kit, a kit for detecting environmental pollutants, a kit for detecting specific viral or bacterial pathogens, etc. The kit may contain the diagnostic device, reagents selected for the specific assay to be performed using the diagnostic device (such as fluorescent reagents), and appropriate instructions for using the diagnostic device to perform the specific assay or assay set.

[0149] The operation of this disclosure will be further described with reference to the embodiments detailed below. These embodiments are provided to further illustrate various specific and preferred implementations and techniques. However, it should be understood that many variations and modifications can be made while still falling within the scope of this disclosure.

[0150] Example

[0151] These embodiments are for illustrative purposes only and are not intended to unduly limit the scope of the appended claims. While the numerical ranges and parameters illustrating the broad scope of this disclosure are approximations, the values ​​shown in the specific examples are recorded as precisely as possible. However, any numerical value inherently contains some error, which is necessarily caused by the standard deviation present in the respective test measurements. At a minimum, and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should at least be interpreted according to the number of significant digits reported and by applying customary rounding.

[0152] Material

[0153] Unless otherwise specified or readily apparent from the context, all parts, percentages, ratios, etc., in the examples and the remainder of the specification are by weight. The materials and reagents used in the examples are shown in Table 1 below.

[0154] Table 1: Materials and Sources

[0155]

[0156]

[0157]

[0158] Test methods

[0159] The following test methods are used to evaluate some of the embodiments of this disclosure.

[0160] Test Method 1: (DNA Adhesion)

[0161] Step 1: Cut a portion of the test sample and apply one side of the double-sided adhesive to the untreated side of the sample.

[0162] Step 2: Obtain the punch (5mm) of the sample and remove the release liner from the back of the adhesive using fine-tipped tweezers.

[0163] Step 3: Attach the punches from each sample to the bottom of the 96-hole transparent base plate with the treated side facing up.

[0164] Step 4: Prepare DNA mixture for sample processing. Denature 500 μL of undiluted pooled sequencing library (average concentration of 15 nM per sample) generated using the Illumina Nextera DNA flex library preparation kit (Illumina 20018704) with 50 μL of 1N NaOH for 5 minutes.

[0165] Add 50 μL of 1M Tris-HCl pH 7.5 to the DNA to neutralize it, and briefly vortex the mixture. Further denature the DNA library by heating at 95°C for 3 minutes, then rapidly cool it on ice. Add the denatured, merged DNA library to 4.5 mL of HT1 hybridization buffer, and vortex the mixture at high speed for 30 seconds.

[0166] Step 5: Transfer the mixture from Step 4 to the reagent reservoir and use a multichannel pipette to transfer 75 μl to each well of a 96-well plate. 75 μl allows for complete flooding of the sample. Materials + dye control or materials control only accepts 75 μl of DNA-free HTI hybridization buffer.

[0167] Step 6: Securely cover the 96-well plate with a sealing film and incubate at room temperature for 1 hour.

[0168] Step 7: Remove the liquid using a multichannel pipette.

[0169] Step 8: Add 100 μL of 20 mM Tris-HCl (pH 7.5) containing 1X SYBR gold dye to well n=4 ( Figure 9 A to Figure 9 (AD row in B) and material + dye control n = 2 / sample ( Figure 9 A to Figure 9 In rows E and F of B).

[0170] Step 9: Add 100 μL of dye-free 20 mM Tris-HCl pH 7.5 to the material in each sample with only n=2 wells.

[0171] Step 10: Obtain fluorescence readings from the top of the plate using excitation / emission at 495nm / 537nm, with 80% gain obtained using a Synergy Neo2BioTek microplate reader, to obtain the first measurement designated as reading 1.

[0172] Remove the liquid from the well and add fresh 20 mM Tris pH 7.5 without dye, and read the plate again to obtain a second measurement designated as reading 2.

[0173] Remove the liquid from the well and add fresh 20 mM Tris pH 7.5 without dye, and read the plate again to obtain a third measurement designated as reading 3.

[0174] Remove the liquid from the well and add fresh 20 mM Tris pH 7.5 without dye, and read the plate again to obtain the fourth measurement designated as reading 4.

[0175] Remove the liquid from the well and add fresh 20 mM Tris pH 7.5 without dye, and read the plate again to obtain the fifth measurement designated as reading 5.

[0176] Remove the liquid from the well and add fresh 20 mM Tris pH 7.5 without dye, and read the plate again to obtain the sixth measurement designated as reading 6.

[0177] Remove the liquid from the well and add fresh 20 mM Tris pH 7.5 without dye, and read the plate again to obtain the seventh measurement designated as reading 7.

[0178] Step 11: To determine the relative adhesion of DNA to the test material, subtract the final fluorescence reading obtained from the material + dye sample from the final fluorescence reading obtained from the material + DNA + dye sample. The error is propagated orthogonally.

[0179] Test Method 2: Autofluorescence

[0180] The sample was measured while the instrument was standing freely in the front sample position (sample perpendicular to the incident direction at a 30-degree angle, detector optics perpendicular to the incident direction at a 10-degree angle) on a Perkin Elmer Lambda 1050 spectrophotometer equipped with a PELA 1002 integrating sphere attachment. The scan rate was set to 102 nm / min, UV-Vis integration to 0.56 sec / pt, data interval to 1 nm, and slit width to 5 nm. The instrument was set to "% transmittance" and "% reflectance" modes.

[0181] To compare with known references, a 10 ppm quinine solution was prepared from quinine hemisulfate monohydrate in 0.5 N sulfuric acid and placed in a 10 mm quartz cell.

[0182] Test Method 3: (X-ray photoelectron spectroscopy)

[0183] Samples were examined using X-ray photoelectron spectroscopy (XPS) (also known as analytical chemical electron spectroscopy (ESCA)). This technique provides analysis of the outermost 3 to 10 nanometers (nm) of the sample surface. Photoelectron spectroscopy provides information on the concentration of elements and chemicals (oxidation states and / or functional groups) present on the solid surface. XPS is sensitive to the detection of all elements in the periodic table except hydrogen and helium, up to detection limits for most substances in the concentration range of 0.1 atomic percent to 1 atomic percent. XPS concentrations should be considered semi-quantitative unless standards are included in the dataset. XPS instrument setup is described in Table 2.

[0184] Table 2: XPS Instrument Settings

[0185] instrument NEXSA, Thermo Fisher Scientific Analysis area >>400μm Photoelectron fly-off angle 90°±30° Acceptable solid angle X-ray source Monochrome Al Ka ​​(1486.6eV) 72W Charge neutralization <![CDATA[Low-energy e - and Ar + immersion electron source]]> Charge correction none Analysis chamber pressure <![CDATA[<5x10 -7 [millbar]]>

[0186] Test Method 4: (Time-of-Flight Secondary Ion Mass Spectrometry)

[0187] Surface analysis is performed using time-of-flight secondary ion mass spectrometry (TOF-SIMS). TOF-SIMS offers monolayer sensitivity for analyzing atoms and molecules at depths ranging from 1 nm to 2 nm. SIMS uses a liquid metal ion gun to bombard the surface with high-energy ionized particles, inducing a collisional cascade on the sample surface. Fragment ions are ejected during the SIMS process; these fragment ions are extracted and transmitted through a time-of-flight (TOF) mass analyzer, which determines the mass of the ion with high resolution and thus its structure.

[0188] Positive and negative secondary ions are collected in separate analyses. External 1nm-2nm imaging is achieved by rapidly scanning the ion beam across the analytical region and recording the positions of secondary ions detected from each primary ion bombardment to determine the surface distribution of chemical composition. The TOF-SIMS instrument setup is shown in Table 3 below.

[0189] Table 3: TOF-SIMS Instrument Settings

[0190]

[0191] Test Method 5: Chemical Force Microscopy

[0192] Atomic force microscopy (AFM) is an imaging technique consisting of a flexible cantilever and a sharp tip attached to the free end of the cantilever. The cantilever-tip assembly performs a raster scan across a surface to produce a topographic image. The interaction force between the tip and the sample causes the cantilever to deflect as it scans the surface. The cantilever bending force is described by Hooke's Law: F c =-kδ c Where k is the cantilever spring constant, F c It is the force of the cantilever, and δ c It is cantilever deflection.

[0193] The simplest implementation of AFM is called contact mode, where feedback control is used to maintain a constant force (fixed cantilever deflection) between the probe and the sample to produce a topographic image. At each xy position, the cantilever deflection is measured via a laser beam reflected from the back side of the cantilever and detected by a photodiode. A three-dimensional topographic map of the surface is constructed using the z(x,y) data. In addition to topographic imaging, AFM can also obtain the force-distance profile between the tip and the sample.

[0194] Force-distance curves are obtained as the tip approaches and withdraws from the surface. These force curves contain a wealth of information about the sample's properties, including adhesion between the tip and the sample. Adhesion refers to the force that causes the tip to detach from the surface as it is withdrawn.

[0195] Chemical force microscopy (CFM) is an aerodynamic force microscopy technique that uses chemically functionalized probes to measure force profiles. CFM measurements were performed in an aqueous environment using a Dimension ICON AFM system (Nanoscope V, Bruker, Santa Barbara, CA, USA) in force-volume mapping mode. Adhesion force maps were obtained using a CH3-terminated tip (Novascan CT.AU.CH3, Au coating 30 nm, SiN probe, nominal spring constant 0.32 N / m). It was expected that the CH3 group would exhibit high adhesion to hydrophobic groups and low adhesion to hydrophilic groups.

[0196] The spring constant was determined by thermal tuning, and deflection sensitivity was established using a sapphire sample in air. Typical force volume plots were obtained at 4 μm × 4 μm with 64 × 64 data points. The ramp size was set to 535 nm, and the typical probe forward / reverse velocity was 2.18 μm / s. The trigger force threshold was set to 4 nN.

[0197] Test Method 6: Angle-Resolved X-ray Photoelectron Spectroscopy (ARXPS)

[0198] In angle-resolved X-ray photoelectron spectroscopy (ARXPS), a sample is tilted continuously at multiple angles while the same area of ​​the sample is simultaneously irradiated. ARXPS can produce non-destructive depth profiles of the sample. This is particularly advantageous here because information about the chemical state relative to depth is important.

[0199] Measurements were performed using a Thermo Fisher Scientific K-α instrument, which employs focused monochromatic Al K-α radiation as a probe beam, achieving surface neutralization via low-energy electron and Ar+ ion beams. The X-ray beam size on the sample surface was approximately 400 μm. Photoelectron detection was performed using an input lens with a ±30° acceptance solid angle. Spectral data were obtained with a 25 eV pass and analyzed using Thermo Avantage v5.9915 software.

[0200] Test Method 7: Confocal Microscopy

[0201] The fluorescently labeled sample was imaged using a confocal microscope (Zeiss Axioplan 2, with LSM 510 laser module, Zeiss, Thornwood NJ) equipped with an Achroplan 63× / 1.4 Oil DIC M27 (FWD = 0.19mm) objective. A single drop of Resolver was used. TM Microscope oil immersion (Corver Corporation, Riverdale, New Jersey): The membrane sample was adhered to a 1-inch × 3-inch microscope slide, covered with a glass coverslip, and another drop of microscope oil was added. Fluorescence images were then captured using a 488 nm laser at 60% to 80% power and a 505 nm long-pass filter. Scanning parameters were set to define a field of view of 23.88 μm × 23.88 μm or 40.93 μm × 40.93 μm.

[0202] Preparation Examples

[0203] Preparation Example 1

[0204] An acrylate mixture was prepared by first adding 75 wt% PHOTOMER 6210 with 25 wt% SR238 and 0.5% TPO to produce acrylate resin A. A second acrylate mixture was then produced by adding 93 wt% acrylate resin A to a 7 wt% HFPO-UA solution. An acrylate solution was then produced by manually mixing 14 wt% of the second acrylate mixture with 43 wt% PGME and 43 wt% MEK.

[0205] Preparation Example 2:

[0206] Resin D was prepared by combining and mixing PHOTOMER 6210, SR238, SR351, and TPO in a weight ratio of 60 / 20 / 20 / 0.5. After adding all components, the mixture was blended by heating to approximately 50°C and mixing on a roller mixer for 12 hours until the mixture appeared homogeneous.

[0207] Preparation Example 3: 10mM potassium phosphate buffer at pH 7.0

[0208] First, a 0.1M potassium phosphate buffer was prepared by combining 38.5g of 1M KH₂PO₄ and 61.5g of 1M dipotassium hydrogen phosphate trihydrate. Then, a 10mM phosphate buffer with pH 7.0 was prepared by mixing 10g of the 0.1M potassium phosphate buffer with 90g of deionized water.

[0209] Example

[0210] Example 1: Biological pollution

[0211] Biocontamination experiments were conducted on the materials prepared in Examples 1 and 2 according to Test Method 1. The image brightness and standard deviation of the two materials + dye control and the four materials + dye + DNA samples are shown in Table 4.

[0212] Table 4: Results of Biocontamination Tests

[0213] Material Image brightness Standard deviation HMDSO on ST505 1,971 2,326 Acrylic resin A on ST505 5,016 1,245 SiCxOy on COP 5 73 COP -10 18 membrane-free -34 29

[0214] Example 2: Fluorescence

[0215] Fluorescence of the 100 nm, 300 nm, and 2 μm planar coating of acrylate resin A on COP was measured according to test method 2. Fluorescence was also measured for COP and BOPP films, as well as controls of quartz, borosilicate, and quinine-doped samples. Intensities are shown as CPS / µampere. Figure 4 middle.

[0216] Example 3: Selectivity of SiCxOy / HMDSO

[0217] A silane coating solution was prepared by mixing 3-aminopropyltrimethoxysilane (1.00 g), anhydrous ethanol (46.3 g), acetic acid (200 mg), and water (2.5 g). The coated membrane was cut into 4-inch squares and immersed in the silane coating solution for 45 minutes. Immediately after removal from the solution, the membrane was rinsed with excess ethanol using a spray bottle and then placed in an oven maintained at 70°C for 30 minutes. The relative concentration of nitrogen on the surface was then determined by XPS analysis before and after silane treatment, as described in Test Method 3. The nitrogen concentrations on the flat HMDSO-treated membrane before and after exposure to aminosilane, and the flat SiOx on the COP, are shown. Figure 5 middle.

[0218] Example 4: 400nm column

[0219] copy

[0220] A nanostructured film was prepared by coating a resin D-mold onto a polycarbonate support layer. The coated film was then pressed onto a nanostructured nickel surface, which was attached to a steel roller controlled at a speed of 15.2 m / min using a rubber-coated roller at 60°C. The nanostructured nickel surface consisted of twelve patterned regions of 6 mm × 6 mm, with pore feature sizes ranging from 75 nm to 500 nm. The features were approximately 200 nm high and had sidewall angles of approximately 4 degrees.

[0221] The resin D coating on the membrane is thick enough to completely wet the nickel surface and forms rolling beads of resin when the coated membrane is pressed onto the nanostructured nickel surface. The membrane is exposed to radiation from two Fusion UV lamp systems (trade name "F600" from Fusion UV Systems, Gaithersburg, MD), both operating at 142 W / cm, simultaneously in contact with the nanostructured nickel surface. After the membrane is peeled off from the nanostructured nickel surface, the nanostructured side of the support layer is again exposed to radiation from the Fusion UV lamp systems equipped with D lamps operating at 142 W / cm.

[0222] Release processing

[0223] In a parallel-plate capacitively coupled plasma reactor, an inorganic layer with a methylated surface, assembled according to the methods described in U.S. Patent Nos. 6,696,157 (David et al.), 8,664,323 (Iyer et al.), and U.S. Patent Publication No. 2013 / 0229378 (Iyer et al.), is applied to a nanostructured membrane to form an unstructured release membrane. The chamber has a central cylindrical energized electrode with a surface area of ​​1.7 m² (18.3 ft²). After the nanostructured membrane and polymer support layer are placed on the energized electrode, the reaction chamber is pumped down to a base pressure less than 1.3 Pa (2 mTorr). O₂ gas is introduced into the chamber at a rate of 1000 SCCM. The reaction is then processed using plasma-enhanced CVD by coupling RF power into the reactor at a frequency of 13.56 MHz and an applied power of 2000 W.

[0224] The treatment time was controlled by moving the nanostructured membrane through the reaction zone at a rate of 9.1 m / min (30 ft / min), resulting in an exposure time of approximately 10 seconds. After deposition was complete, the RF power was turned off and the gas was evacuated from the reactor. A second plasma treatment was performed in the same reactor after the first treatment without returning the chamber to atmospheric pressure. HMDSO gas was introduced into the chamber at approximately 1750 SCCM to achieve a pressure of 9 mTorr. Subsequently, 13.56 MHz RF power was coupled into the reactor at an applied power of 1000 W.

[0225] The membrane was then transported through the reaction zone at a rate of 9.1 m / min (30 ft / min), resulting in an exposure time of approximately 10 seconds. At the end of this treatment time, RF power and gas supply were stopped, and the chamber was returned to atmospheric pressure. SEM images of the nanostructured release membrane are shown below. Figure 6 middle.

[0226] planarization layer

[0227] A 5 wt% PVB 30H solution in IPA was die-coated onto a nanostructured release membrane using a slit die at a rate of 0.025 m / s in a roll-to-roll process. The solution was coated to a width of 15.3 cm and pumped at a rate of 2.23 sccm using a Harvard injection pump. The coating was dried at 65°C for 4 minutes to produce a planarized film.

[0228] Etching

[0229] The planarized membrane was subjected to reactive ion etching in the same homemade reaction chamber used for depositing the PECVD release layer to produce the etched membrane. After placing the coated membrane on the energized electrode, the reaction chamber was pumped down to a base pressure less than 1.3 Pa (1 mTorr). O2 gas was introduced into the chamber at a rate of 100 SCCM. A 13.56 MHz RF power was then coupled into the reactor at an applied power of 7500 W. The membrane was then transported through the reaction zone at a rate of 10 ft / min to achieve an exposure time of approximately 30 seconds. At the end of this processing time, the RF power and gas supply were stopped, and the chamber was returned to atmospheric pressure. SEM images of the etched membrane are shown below. Figure 7 middle.

[0230] peeling

[0231] In a roll-to-roll process, the etched film was laminated with a corona-treated Melinex 454 film at a speed of 0.025 m / s (5 fpm). Acrylic resin A was injected into the roll gap using a syringe to maintain a coating width of 10-12 cm. The roll gap consisted of a rubber roller with a hardness of 90 and a steel roller set at 54°C. The roll gap was joined by two Bimba cylinders pressurized at 0.27 MPa. The films were held in contact for approximately 1.5 m, during which they were cured with molten D bulbs and subsequently peeled off. This method transferred the remaining PVB after etching to Melinex 454, leaving a clean but chemically distinct surface on the nanostructured film. SEM images are shown below. Figure 8 middle.

[0232] Sample Analysis

[0233] The contact force microscopy (CFM) morphology image of the 4μm × 4μm sample according to test method 5 is shown below. Figure 9 The 4 μm × 4 μm force-volume adhesion map obtained using a CH3-terminated AFM probe is shown in the figure. Figure 10 middle.

[0234] Example 5: 200nm diameter column:

[0235] Repeated experiments

[0236] The nanostructured membrane was prepared as in Example 4, except that a Melinex 454 membrane was used instead of a polycarbonate membrane, and a nickel surface consisting of a sheet of about 5 cm × 5 cm with regularly spaced pores of 200 nm diameter was used.

[0237] Release processing

[0238] The nanostructured membrane was released as described in Example 4.

[0239] planarization layer

[0240] As described in Example 4, 4% by weight of PVB 30H solution was coated in IPA.

[0241] Etching

[0242] The etched planarized structured film is as described in Example 4, wherein the speed is changed to 10 ft / min. Partially covered pillars can be... Figure 11 I saw it in the middle.

[0243] peeling

[0244] 3 ml of acrylate resin A was placed between the etched membrane and the Melinex 454 sheet. The droplets were dispersed using a hand roller. The resin was cured by exposure to 385 nm light from a UV-LED system for 30 seconds. The sample was peeled off by hand, and the PVB was transferred from the etched membrane to the Melinex. Different column treatments can be seen in [details omitted]. Figure 12 SEM images of the structured membrane stripped from the membrane.

[0245] Example 6: 1500nm column

[0246] Repeated experiments

[0247] The nanostructured membrane was prepared as in Example 4, except that a conventional 75 μm thick biaxially oriented polyethylene terephthalate (PET) membrane (homemade) was used instead of a polycarbonate membrane. The resin-contacting side of the PET membrane was primed with a thermosetting acrylic polymer (Rhoplex 3208, purchased from Dow Chemical, Midland, MI). A nickel surface consisting of an approximately 15 cm × 15 cm sheet with regularly spaced pores of 1500 nm diameter and 350 nm depth was used.

[0248] Release processing :

[0249] The nanostructured membrane was released as described in Example 4.

[0250] planarization layer

[0251] As described in Example 4, 4% by weight of PVB 30H solution was coated in IPA, and the pump rate was changed to 2.97 sccm.

[0252] Etching

[0253] The film was etched planarized as described in Example 4, wherein the speed was changed to 10 ft / min.

[0254] peeling

[0255] As described in Example 1, PVB was stripped from the etched film, and the sample was analyzed using Test Method 4: TOF-SIMS. Figure 13 Showing the chemically distinct column tops and valley regions.

[0256] Functionalization

[0257] The sample was coated with silane as described in Example 3.

[0258] Example 7: Nanostructured film with low autofluorescence

[0259] Roughened COP

[0260] As described in U.S. Patent No. 10,134,566, the COP film substrate is roughened by depositing a discontinuous silicon-containing layer using PECVD while simultaneously etching the surface with a reactive material. Reactive ion etching is performed on the COP film substrate in the same homemade reaction chamber used for depositing the PECVD release layer. After the film is placed on the energized electrodes, the reaction chamber is pumped down to a base pressure less than 1.3 Pa (1 mTorr). HMDSO and O2 gases are introduced into the chamber at rates of 18 sccm and 750 sccm, respectively. A 13.56 MHz RF power supply is then coupled into the reactor at an applied power of 6000 W. The film is then transported through the reaction zone at a rate of 10 ft / min to achieve an exposure time of approximately 30 seconds. At the end of this processing time, the RF power and gas supply are stopped, and the chamber is returned to atmospheric pressure.

[0261] Low-platform replication

[0262] First, as described in the peeling step of Example 4, a nanostructured porous membrane is generated by obtaining the released nanostructured membrane from Example 4, coating it with acrylate resin A, curing it, and peeling it onto a Melenex 454 membrane. Then, the nanostructured porous membrane is subjected to a release treatment using the procedure described in Example 4.

[0263] Nanostructured pillars were replicated onto roughened COPs by pre-coating acrylate solution C onto a release-treated nanostructured porous membrane using a slit die at a rate of 0.051 m / s. The solution was coated to a width of 15.3 cm and pumped at a rate of 1.8 sccm using a Harvard syringe pump.

[0264] The coating was dried under ambient conditions for 4 minutes and then laminated onto the roughened COP film from the previous step in a roll gap. The roll gap consisted of a rubber roller with a hardness of 90 and a steel roller set at 54°C. The roll gap was engaged by two Bimba cylinders pressurized at 0.27 MPa. The films were held in contact for approximately 1.5 μm, during which they were cured with molten D lamps and subsequently peeled off. Figure 14 The SEM images show that, according to Figure 4 According to the data, the membrane will have low autofluorescence.

[0265] Release processing

[0266] As in Example 4, the surface of the nanostructured membrane is subjected to a release treatment.

[0267] planarization layer

[0268] Except for changing the flow rate to 1.5 cc / min and the width to 10.2 cm, the released membrane was coated with PVB as described in Example 4.

[0269] Remaining steps

[0270] The remaining steps of etching, stripping, and functionalization can be performed as described above. The sample can optionally be laminated onto one or more adhesives from adhesive groups A, B, C, or 3M™ Optical Clear Adhesive 8171.

[0271] Example 8: Functionalization of 1500nm column membrane with azide-based silane

[0272] Instead of the final treatment of 3-aminopropyltrimethoxysilane, the 1500 nm column-patterned film of Example 6 was functionalized with a solution of 3-azidopropyltriethoxysilane by means of the same method as the functionalization method defined in Example 3, but with 3-azidopropyltriethoxysilane instead of 3-aminopropyltrimethoxysilane.

[0273] Example 9: Acrylamide-silane functionalization of 1500nm column membrane

[0274] Instead of 3-aminopropyltrimethoxysilane, the final treatment was performed by functionalizing the 1500 nm column-patterned film of Example 6 with an acrylamide silane solution using the same method as defined in Example 3, but with acrylamide silane instead of 3-aminopropyltrimethoxysilane.

[0275] Based on XPS measurements, the nitrogen percentage increased from 0.7% to 3.8% after acrylamide silane functionalization. Angle-resolved XPS analysis of the functionalized substrate was also used, where the sample stage could be tilted from 0° to 75° to selectively analyze the elemental composition of the top portion of the column. As the XPS stage tilted from 0° to 75°, the nitrogen percentage increased from 3.7% to 4.6%, indicating preferential functionalization of the column with acrylamide silane.

[0276] Example 10: Covalently linking fluorescent dyes to 1500nm column membranes using aminosilanes

[0277] The 1500 nm column membrane with aminosilane from Example 6 was then placed in a 12-well plate and washed three times with TE buffer at pH 8.0. Approximately 500 μL of 0.1 mg / mL Alexa Fluor was added. TM A solution of 488 NHS ester (succinimide ester) in TE buffer at pH 8.0 was pipetted onto the surface of an aminosilane-functionalized nanopillar sample. Functionalization was set for one hour, followed by rinsing with TE buffer at pH 8.0, drying with nitrogen, and imaging using a confocal microscope.

[0278] Confocal images of the fluorescent nanopillar structure of Example 10 were obtained using Test Method 7 (confocal microscopy) as described above. The confocal micrographs show that we have achieved selectivity / contrast in fluorescence at the top of the pillars compared to the plateau region treated with HMDSO.

[0279] Example 11: Covalently linking alkyne oligonucleotides to a 1500 nm column membrane using azidosilane

[0280] Then, using Cu-catalyzed azido-alkyne cycloaddition, the 1500 nm column membrane of the azidosilane membrane from Example 8 was functionalized with a fluorescein-containing alkyne oligonucleotide. 10 mM potassium phosphate buffer (pH 7.0) (1.429 mL), alkyne oligonucleotide (3 nmol), N,N,N',N',N"-pentamethyldiethylenetriamine (PMDETA, 13.14 μL), copper sulfate pentahydrate (CuSO4·5H2O, 4 w / v % solution, 7.49 μL), and sodium ascorbate (400 mg / mL, 6 μL) were added one after another to 1.5 mL DNA LoBind tubes, and vortexed after each component was added.

[0281] The membrane was placed in an aluminum weighing pan. 500 μL of oligonucleotide solution was pipetted onto the surface of the nanopillar membrane, and then placed in an oven set to 60°C for 30 minutes. The membrane was removed, rinsed with deionized H₂O, and dried with nitrogen. The presence of the oligonucleotides was confirmed by confocal microscopy. A confocal image of the fluorescent nanopillar structure of Example 11 was obtained using Test Method 7 (confocal microscopy) as described above.

[0282] Confocal micrographs show that we have achieved selectivity / contrast in column-top fluorescence compared to the plateau region treated with HMDSO. In addition to fluorescence, the column top also exhibits additional, coarser features, which can be attributed to oligonucleotide deposition.

[0283] Example 12: Growth of amine-acrylamide brush polymer on micropillar membrane

[0284] Add the solutions shown in Table 5 to 25 mL vials. Place the 1500 nm column-patterned membrane with acrylamide silane from Example 9 into the vial, ensuring it is completely submerged in the solution. Cover the vial with a septum and bubble nitrogen through the solution for 10 minutes using a needle that punctures the septum. Then remove the needle and place the vial in an oven maintained at 70°C for 2 hours. Remove the membrane from the vial and immerse it in water overnight, then air dry.

[0285] Table 5: Solutions for the growth of acrylamide brush polymers on micropillar membranes

[0286]

[0287] Example 13: Covalent bonding of fluorescent dye to a micropillar membrane with an aminoacrylamide brush-coated top

[0288] Using the same method as described for Example 10, the membrane of Example 6 was replaced with the membrane of Example 12, and the fluorescent Alexa Fluor was applied using an amine-containing brush polymer. TM 488 NHS ester dye was covalently linked to the micropillar membrane. The presence of the covalently linked fluorescent label was confirmed by confocal microscopy.

[0289] Throughout this specification, the terms "an embodiment," "certain embodiments," "one or more embodiments," or "implementation," whether or not preceded by the term "exemplary," mean that a particular feature, structure, material, or characteristic described in connection with that embodiment is included in at least one of the exemplary embodiments of this disclosure. Therefore, phrases such as "in one or more embodiments," "in some embodiments," "in one embodiment," or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment of the exemplary embodiments of this disclosure. Furthermore, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0290] While certain exemplary embodiments have been described in detail in this specification, it should be understood that modifications, variations, and equivalents of these embodiments will readily occur to those skilled in the art upon understanding the foregoing. Therefore, it should be understood that this disclosure should not be unduly limited to the exemplary embodiments shown above. In particular, as used herein, numerical ranges expressed in terms of endpoints are intended to include all values ​​contained within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5). Furthermore, all numbers used herein are considered to be modified by the term “about”.

[0291] Furthermore, all publications and patents cited herein are incorporated herein by reference in their entirety, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Various exemplary embodiments have been described. These and other embodiments are within the scope of the following claims.

Claims

1. An article comprising: A flexible structured membrane having a first main surface and a second main surface, wherein the first main surface of the flexible structured membrane includes a plurality of pillars separated by a platform region, and wherein the pillars include exposed surfaces; An anti-biofouling layer is provided in the platform region, wherein the anti-biofouling layer has a methylated surface. An inorganic layer, the inorganic layer being on the exposed surface of the pillar, wherein the inorganic layer comprises a metal, a quasi-metal, a metal oxide, or a quasi-metal oxide; and An analyte binding layer is provided on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof, and wherein the exposed surface of the analyte binding layer contains at least one functional group selected for binding with a biochemical analyte.

2. The article of claim 1, wherein the inorganic layer comprises at least one of Si, Ti or Al or oxides thereof, and wherein the thickness of the inorganic layer is less than 200 nm.

3. The article of claim 1, wherein the inorganic layer comprises SiC x O y Furthermore, the anti-biofouling layer comprises methyl-terminated SiC. x H y .

4. The article of claim 1, wherein the analyte binding layer comprises an acrylamide copolymer, a condensed silane, and mixtures and compositions thereof.

5. The article of any one of claims 1 to 4, wherein the column has a diameter of 100 nm to 1500 nm.

6. The article of any one of claims 1 to 4, wherein the column has a height greater than 0 nm but at most 1000 nm.

7. The article of claim 1 to 4, wherein the flexible structured film has low autofluorescence.

8. The article of claim 1 to 4, further comprising a polymer support layer having a first main surface and a second main surface, wherein the first main surface of the polymer support layer is on the second main surface of the structured membrane.

9. The article of claim 8, further comprising an adhesive layer on the second main surface of the polymer support layer.

10. The article of claim 9, further comprising a support layer on the adhesive layer, wherein the support layer is selected from release liner and rigid substrate.

11. A method for manufacturing a component of a diagnostic device, the method comprising: A flexible structured membrane is provided, the flexible structured membrane having a first main surface and a second main surface, wherein the first main surface of the flexible structured membrane includes a patterned surface, the patterned surface including an arrangement of columns with platform regions scattered throughout; A release layer is applied to the first main surface of the flexible structured membrane such that the release layer covers the top surface of the column and the platform region, wherein the release layer includes a surface rich in methyl (CH3) groups; A planarization layer is applied to the release layer; A portion of the planarization layer is etched with an oxygen-containing etching material to form a non-methylated inorganic layer on the top surface of the pillar, wherein the inorganic layer comprises a metal, a metalloid, a metal oxide, or a metalloid oxide. Remove the planarization layer; as well as A functional silane material is attached to the inorganic layer on the top surface of the column, and the functional silane material is polymerized to form an analyte binding layer on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof, and wherein the analyte binding layer contains at least one functional group that reacts with a biochemical analyte.

12. The method of claim 11, wherein the planarization layer is removed before the functional silane is attached.

13. The method of claim 11, wherein the functional layer is attached before the planarization layer is removed.

14. The method according to any one of claims 11 to 13, wherein the analyte binding layer is attached to the inorganic layer by reacting the inorganic layer with one of an aminosilane or an acrylamide silane to form an acrylamide.

15. The method of claim 14, wherein the acrylamide is polymerized to form polyacrylamide.

16. The method according to any one of claims 11 to 13, wherein the inorganic layer comprises SiC x O y The release layer comprises methyl-terminated SiC. x H y .

17. The method according to any one of claims 11 to 13, wherein the analyte binding layer comprises an acrylamide copolymer, a condensed silane, and mixtures and compositions thereof.

18. The method according to any one of claims 11 to 13, the method further comprising applying an adhesive layer to the second main surface of the polymer support layer.

19. A diagnostic device for detecting a biochemical analyte, the diagnostic device comprising a flow cell with a patterned arrangement of fluid channels configured to provide flow conduits for a sample fluid comprising the biochemical analyte, wherein at least some of the fluid channels in the flow cell are lined on their surfaces with: A flexible structured membrane having a first main surface and a second main surface, wherein the first main surface of the flexible structured membrane includes a plurality of pillars having exposed surfaces extending into the fluid channels of the flow pool, wherein the pillars are distributed with platform regions. An anti-biofouling layer, wherein the anti-biofouling layer is located in the platform region, and wherein the anti-biofouling layer includes a methylated surface; A nonmethylated inorganic layer, said nonmethylated inorganic layer on the exposed surface of the pillar, wherein said inorganic layer comprises a metal, a metalloid, a metal oxide, or a metalloid oxide; and An analyte binding layer is provided on the inorganic layer, wherein the analyte binding layer is selected from reactive silanes, functionalizable hydrogels, functionalizable polymers, and mixtures and compositions thereof, and wherein the exposed surface of the analyte binding layer contains at least one functional group selected for binding with the biochemical analyte in the sample fluid.

20. A DNA sequencing kit comprising the diagnostic device according to claim 19, a fluorescent reagent for DNA sequencing, and instructions.

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