CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application No. 63/418,080 filed October 21, 2022, the content of which is incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates to microfluidic devices and methods for manufacturing microfluidic devices.

BACKGROUND

[0003] Microfluidic devices, sometimes referred to as flow cells or flow cell devices, can be useful for a variety of applications including bio-analysis (e.g., nucleic acid sequencing, single molecule analysis, etc.). In one example, the analysis is conducted by employing high resolution fluorescence imaging techniques to identify and quantify specific molecules at a surface of a substrate that is exposed to the microfluidic channel of the device. For instance, in optical detection based parallel gene sequencing techniques, millions of DNA fragments generated from a genomic DNA sample can be immobilized and partitioned onto the substrate surface of the microfluidic device such that the fragments are spatially separated from each other to facilitate sequencing by, for example, synthesis, ligation, or single-molecule real-time imaging.

[0004] Conventional bio-analysis techniques employing these nucleic acid sequencing and single molecule assays often suffer from prolonged collection times for collecting thousands of images to achieve adequate identification and quantification. Accordingly, a need exists for improved flow cell devices which can aid in reduced image collection time.

SUMMARY

[0005] According to aspect (1), a microfluidic device is provided. The microfluidic device comprises: a glass-based substrate comprising a first glass-based layer and a second glass- based layer fused to the first glass-based layer; a flow channel disposed in the glass-based substrate, the flow channel comprising (i) an upper channel portion delimited by opposed first and second upper sidewalls and opening to an outer surface of the glass-based substrate, and (ii) a lower channel portion delimited by opposed first and second lower sidewalls and extending from the upper channel portion to a floor of the flow channel, the first and second lower sidewalls continuous with the first and second upper sidewalls, respectively; and a cover bonded to the glass-based substrate and at least partially covering the flow channel, the cover defining a ceiling of the flow channel, wherein, when viewed in respective orthogonal cross sections of the flow channel, (i) a first angle formed between the floor and at least one of the first and second upper sidewalls is substantially orthogonal and (ii) a second angle formed between the floor and at least one of the first and second lower sidewalls is less than the first angle.

[0006] According to aspect (2), the microfluidic device of aspect (1) is provided, wherein the first glass-based layer defines the floor of the flow channel, and the second glass-based layer defines the first and second upper sidewalls of the flow channel and the first and second lower sidewalls of the flow channel.

[0007] According to aspect (3), the microfluidic device of any one of the proceeding aspects is provided, wherein the first and second upper sidewalls are substantially parallel to one another.

[0008] According to aspect (4), the microfluidic device of any one of the proceeding aspects is provided, wherein the first angle is in a range of from about 85° to about 91°.

[0009] According to aspect (5), the microfluidic device of any one of the proceeding aspects is provided, wherein each of the first and second upper sidewalls forms the first angle with the floor.

[0010] According to aspect (6), the microfluidic device of any one of the proceeding aspects is provided, wherein the second angle is in a range of from about 60° to about 90°.

[0011] According to aspect (7), the microfluidic device of any one of the proceeding aspects is provided, wherein each of the first and second lower sidewalls forms the second angle with the floor.

[0012] According to aspect (8), the microfluidic device of any one of the proceeding aspects is provided, wherein, when viewed in the respective orthogonal cross sections, a first effective angle formed between (i) the floor and (ii) a line extending between (a) an intersection of the outer surface and the first upper sidewall and (b) an intersection of the floor and the first lower sidewall, when measured within the glass-based substrate, is in a range of from about 75° to about 90°.

[0013] According to aspect (9), the microfluidic device of any one of the proceeding aspects is provided, wherein, when viewed in the respective orthogonal cross sections, a second effective angle formed between (i) the floor and (ii) a line extending between (a) an intersection of the outer surface and the second upper sidewall and (b) an intersection of the floor and the second lower sidewall, when measured within the glass-based substrate, is in a range of from about 75° to about 90°.

[0014] According to aspect (10), the microfluidic device of any one of the proceeding aspects is provided, wherein a depth of the upper channel portion from the outer surface corresponds to at least about 60% of a thickness of the second glass-based layer.

[0015] According to aspect (11), the microfluidic device of aspect (10) is provided, wherein the depth of the upper channel portion is in a range of from about 60% to about 98% of the thickness of the second glass-based layer.

[0016] According to aspect (12), the microfluidic device of any one of the proceeding aspects is provided, wherein at least 80% of a total area of at least one of the floor and the ceiling has a surface flatness in a range of from about 50 nm/mm ² to about 1000 nm/mm ² , measured along a length and width of the at least one of the floor and the ceiling.

[0017] According to aspect (13), the microfluidic device of aspect (12) is provided, wherein each of the floor and the ceiling has the local surface flatness.

[0018] According to aspect (14), the microfluidic device of any one of the proceeding aspects is provided, wherein at least one of the floor and the ceiling has a surface roughness in a range of from about 1 nm to about 15 nm.

[0019] According to aspect (15), the microfluidic device of aspect (14) is provided, wherein each of the floor and the ceiling has the surface roughness.

[0020] According to aspect (16), the microfluidic device of any one of the proceeding aspects is provided, further comprising: an inlet opening through at least one of the glass-based substrate or the cover and in fluid communication with the flow channel; and an outlet opening through at least one of the glass-based substrate or the cover and in fluid communication with the flow channel. [0021] According to aspect (17), the microfluidic device of any one of the proceeding aspects is provided, wherein a width of the flow channel is at least about 1 mm.

[0022] According to aspect (18), the microfluidic device of any one of the proceeding aspects is provided, wherein a height of the flow channel is at least about 25 pm.

[0023] According to aspect (19), the microfluidic device of any one of the proceeding aspects is provided, wherein a length of the flow channel along a longitudinal axis thereof is at least about 10 mm.

[0024] According to aspect (20), the microfluidic device of any one of the proceeding aspects is provided, wherein the cover is bonded directly to the glass-based substrate such that an interface between the cover and the glass-based substrate is a glass-glass interface that is free of bonding material.

[0025] According to aspect (21), the microfluidic device of any one of aspects (1) to (19) is provided, wherein the cover is bonded to the glass-based substrate at a bonded volume comprising a bonding material diffused into each of the glass-based substrate and the cover.

[0026] According to aspect (22), the microfluidic device of any one of aspects (1) to (19) is provided, further comprising a bonding layer disposed between the glass-based substrate and the cover.

[0027] According to aspect (23), the microfluidic device of any one of the proceeding aspects is provided, wherein an interface between the first glass-based layer and the second glass-based layer is a glass-glass interface that is free of bonding material.

[0028] According to aspect (24), the microfluidic device of any one of the proceeding aspects is provided, wherein the first glass-based layer has a first glass composition, and the second glass-based layer has a second glass composition that is different than the first glass composition.

[0029] According to aspect (25), the microfluidic device of aspect (24) is provided, wherein the first glass composition has a first degradation rate when exposed to an etchant, and the second glass composition has a second degradation rate that is higher than the first degradation rate when exposed to the etchant.

[0030] According to aspect (26), the microfluidic device of aspect (25) is provided, wherein the second degradation rate is 10 times greater than the first degradation rate. [0031] According to aspect (27), the microfluidic device of any one of aspects (24) to (26) is provided, wherein the second glass composition comprises: from about 45 mol%to about 60 mol% SiC , from about 8 mol% to about 19 mol% AI2O3, from about 5 mol% to about 23 mol% B2O3, and from about 3 mol% to about 21 mol% Na2O.

[0032] According to aspect (28), the microfluidic device of aspect (27) is provided, wherein the second glass composition is substantially free of As and Cd.

[0033] According to aspect (29), the microfluidic device of any one of the proceeding aspects is provided, wherein an auto-fluorescence in a wavelength range of 450 nm to 750 nm of each of the first glass-based layer and the cover is as low as an auto-fluorescence of a quartz substrate or a pure silica substrate.

[0034] According to aspect (30), the microfluidic device of any one of the proceeding aspects is provided, wherein the microfluidic device is a flow cell for high resolution optical fluorescence imaging.

[0035] According to aspect (31), a method for manufacturing a microfluidic device is provided. The method comprises: removing, via a first technique, a first portion of a second glass-based layer of a glass-based substrate to form an upper channel portion in the glass-based substrate, the glass-based substrate comprising a first glass-based layer and the second glassbased layer fused to the first glass-based layer, the upper channel portion opening at an outer surface of the second glass-based layer and extending to a first floor defined by the second glass-based layer; removing, via a second technique different from the first technique, a second portion of the second glass-based layer to form a lower channel portion extending from the upper channel portion to a second floor defined by the first glass-based layer, the upper and lower channel portions forming a flow channel with sidewalls defined by the second glassbased layer; and bonding a cover to the glass-based substrate such that the cover at least partially covers the flow channel.

[0036] According to aspect (32), the method of aspect (31) is provided, wherein the first technique comprises directing a laser beam at the outer surface of the second glass-based layer, the laser beam configured to ablate the first portion of the second glass-based layer to form the upper channel portion.

[0037] According to aspect (33), the method of aspect (31) or aspect (32) is provided, wherein the first technique is configured to remove from about 60% to about 98% of a thickness of the second glass-base layer from the outer surface thereof to form the upper channel portion. [0038] According to aspect (34), the method of any one of aspects (31) to (33) is provided, wherein the second technique comprises contacting the first floor of the upper channel portion with an etchant.

[0039] According to aspect (35), the method of aspect (34) is provided, wherein the first glass-based layer has a first degradation rate when exposed to the etchant, and the second glassbased layer has a second degradation rate that is higher than the first degradation rate when exposed to the etchant.

[0040] According to aspect (36), the method of aspect (34) or aspect (35) is provided, wherein the etchant comprises aqueous hydrofluoric acid (HF) with a concentration of at most about 5 weight percent HF.

[0041] According to aspect (37), the method of aspect (36) is provided, wherein the concentration of the aqueous HF is in a range of from about 0.2 to about 2 weight percent HF.

[0042] According to aspect (38), the method of any one of aspects (31) to (37) is provided, wherein a first volume of the first portion of the second glass-based layer removed via the first technique is at least 2 times greater than a second volume of the second portion of the second glass-based layer removed via the second technique.

[0043] According to aspect (39), the method of any one of aspects (31) to (38) is provided, wherein the sidewalls comprise opposed first and second upper sidewalls delimiting the upper channel portion, and wherein an angle formed between the second floor and at least one of the first and second upper sidewalls is substantially orthogonal.

[0044] According to aspect (40), the method of any one of aspects (31) to (39) is provided, wherein the sidewalls comprise opposed first and second lower sidewalls delimiting the lower channel portion, and wherein an angle formed between the second floor and at least one of the first and second lower sidewalls is in a range of from about 60° to about 90°.

[0045] According to aspect (41), the method of any one of aspects (31) to (40) is provided, wherein the cover defines a ceiling of the flow channel, and wherein each of the floor and the ceiling has a local surface flatness in a range of from about 50 nm/mm ² to about 1000 nm/mm ² , measured along a length and width of each of the floor and the ceiling.

[0046] According to aspect (42), the method of any one of aspects (31) to (41) is provided, wherein the bonding the cover to the glass-based substrate comprises: applying hydrofluoric acid (HF) to the outer surface of the second glass-based layer, and positioning the cover on the outer surface with the HF disposed therebetween.

[0047] According to aspect (43), the method of aspect (42) is provided, wherein the bonding the cover to the glass-based substrate further comprises pressing the cover against the outer surface with a press force and a press time.

[0048] According to aspect (44), the method of any one of aspects (31) to (41) is provided, wherein the bonding the cover to the glass-based substrate comprises: depositing a bonding material on the outer surface of the second glass-based layer, positioning the cover on the bonding material, and irradiating the bonding material with electromagnetic radiation sufficient to diffuse at least a portion of the bonding material into the cover and the glass-based substrate, thereby bonding the cover to the glass-based substrate.

[0049] According to aspect (45), the method of aspect (44) is provided, wherein the depositing the bonding material on the outer surface of the second glass-based layer comprises depositing the bonding material by printing, tape bonding, or vapor deposition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 is a top view of a microfluidic device with a plurality of flow channels according to embodiments;

[0051] FIG. 2 is a cross-sectional view of the microfluidic device of FIG. 1 along line A- A showing an embodiment in which a cover is bonded directly to a glass-based laminate substrate;

[0052] FIG. 3 is a cross-sectional view of the microfluidic device of FIG. 1 along line A- A showing an embodiment in which a cover is bonded via a bonding material to a glass-based laminate substrate;

[0053] FIG. 4 is a cross-sectional view of the microfluidic device of FIG. 1 along a line B- B showing structural aspects of a flow channel in the glass-based laminate substrate according to embodiments; and

[0054] FIG. 5 is a duplicate of the cross-sectional view of FIG. 4 showing dimensional aspects and further structural aspects of the flow channel; and [0055] FIGS. 6-11 are a stepwise series of cross-sectional representations that illustrate a process flow for the manufacture of the microfluidic device of FIG. 1 according to embodiments.

DETAILED DESCRIPTION

[0056] For the purposes of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiments illustrated in the drawings and described in the following written specification. It is understood that no limitation to the scope of the disclosure is thereby intended. It is further understood that the present disclosure includes any alterations and modifications to the illustrated embodiments and includes further applications of the principles disclosed herein as would normally occur to one skilled in the art to which this disclosure pertains

[0057] As used herein, the term “and/or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and/or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0058] In this document, relational terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0059] As used herein, the term “about” means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. When the term “about” is used in describing a value or an end-point of a range, the disclosure should be understood to include the specific value or end-point referred to. Whether or not a numerical value or end-point of a range in the specification recites “about,” the numerical value or end-point of a range is intended to include two embodiments: one modified by “about,” and one not modified by “about.” It will be further understood that the end-points of each of the ranges are significant both in relation to the other end-point, and independently of the other end-point.

[0060] The terms “substantial,” “substantially,” and variations thereof as used herein, unless defined elsewhere in association with specific terms or phrases, are intended to note that a described feature is equal or approximately equal to a value or description. For example, a “substantially planar” surface is intended to denote a surface that is planar or approximately planar. Moreover, “substantially” is intended to denote that two values are equal or approximately equal. In some embodiments, “substantially” may denote values within about 10% of each other, such as within about 5% of each other, or within about 2% of each other.

[0061] Directional terms as used herein — for example up, down, right, left, front, back, top, bottom, above, below, and the like — are made only with reference to the figures as drawn and are not intended to imply absolute orientation.

[0062] As used herein the terms "the," "a," or "an," mean "at least one," and should not be limited to "only one" unless explicitly indicated to the contrary. Thus, for example, reference to "a component" includes embodiments having two or more such components unless the context clearly indicates otherwise.

[0063] The term “surface roughness” means Ra surface roughness determined as described in ISO 25178, Geometric Product Specifications (GPS)-Surface texture: areal, filtered at 25 pm unless otherwise indicated. The surface roughness values reported herein were obtained using a Zygo New View 3000.

[0064] In various embodiments, microfluidic devices with flow channels having ultra-flat floor surfaces and high-angle (e.g., substantially orthogonal) sidewalls relative to the floor surfaces are provided. For high resolution optical fluorescence imaging, high flatness along floor and/or ceiling surfaces of flow channels can reduce optical artefacts and enable highspeed imaging. Sidewalls with high wall angles relative to the floors increase the area of the floor surfaces for effective imaging, thereby reducing the consumption of valuable reagents for assays. Microfluidic devices that include these features, alone or in combination, are desirable for biomolecular analysis, in particular gene and protein sequencing.

[0065] Referring now to FIG. 1, a microfluidic device 100 comprising a glass-based laminate substrate 102 including flow channels 104 in accordance with various embodiments is shown. In particular, FIG. 1 provides a top view of the microfluidic device 100 including a plurality of flow channels 104 disposed in the glass-based laminate substrate 102. In the microfluidic device 100 shown in FIG. 1, two flow channels are included, although it is contemplated that the number of flow channels included in the microfluidic device can vary depending on the particular embodiment. For example, a larger microfluidic device 100 can include more flow channels as compared to the microfluidic device shown in FIG. 1. The flow channels 104 can each extend along a respective longitudinal axis 105, or portions of each flow channel 104 can extend along the respective longitudinal axis 105.

[0066] FIG. 2 and FIG. 3 are cross-sectional views of the microfluidic device 100 ofFIG. 1 along a line A-A, which defines a longitudinal section plane passing through one of the flow channels 104. The microfluidic device 100 includes a cover 114 bonded to the glass-based laminate substrate 102. The cover 114 is positioned on the glass-based substrate so as to cover, at least partially, each of the flow channels 104. FIG. 2 depicts an embodiment in which the cover 114 is bonded directly to the glass-based laminate substrate 102. FIG. 3 depicts an embodiment in which the cover 114 is bonded via a bonding material 115 to the glass-based laminate substrate 102. The bonding of the cover 114 to the glass-based laminate substrate 102 is described later in this disclosure.

[0067] As shown in FIG. 2 and FIG. 3, the glass-based laminate substrate 102 comprises a core layer 106 (e.g., a first glass-based layer) and at least one cladding layer (e.g., a second glass-based layer), such as a first cladding layer 108a, on one side of the core layer 106. In embodiments, the glass-based laminate substrate 102, comprises the core layer 106 interposed between the first cladding layer 108a and a second cladding layer 108b (shown in FIG. 6) on an opposite side of the core layer 106. In embodiments, the glass-based laminate substrate 102 includes the second cladding layer 108b for at least some steps of a method for manufacturing the microfluidic device 100. For example, FIG. 6 is a cross-sectional view of a three-layered glass-based laminate substrate 102 at an initial step of the method, showing the core layer 106 interposed between the first cladding layer 108a and the second cladding layer 108b. The method for manufacturing the microfluidic device 100 is described later in this disclosure with reference to FIGS. 6-11.

[0068] In embodiments, each of the glass layers, including the core layer 106, the first cladding layer 108a, and the second cladding layer 108b, comprises, independently, a glass material, including, but not limited to a glass, a glass ceramic, a ceramic, or a combination thereof. In embodiments, the glass material (e.g., the glass composition) of the core layer 106 is different from the glass material of the first cladding layer 108a and the second cladding layer 108b. In embodiments, the glass material of the first cladding layer 108a can be the same as the glass material of the second cladding layer 108b, or the glass material of the first cladding layer 108a can be different from the glass material of the second cladding layer 108b.

[0069] Referring to FIGS. 2, 3, and 6, the core layer 106 has a first surface 106a and a second surface 106b opposed to the first surface 106a. The first cladding layer 108a is fused directly to the first surface 106a of the core layer 106 and the second cladding layer 108b (FIG. 5) is fused directly to the second surface 106b of the core layer 106. The first cladding layer 108a and the second cladding layer 108b can be fused to the core layer 106 without any additional materials, such as adhesives, polymer layers, coating layers, bonding materials or layers, or the like being disposed between the glass layers. Thus, in this instance, the first surface 106a is directly adjacent the first cladding layer 108a and the second surface 106b is directly adjacent the second cladding layer 108b. In embodiments, the glass laminate 102 is formed via a fusion lamination process (e.g., fusion draw process). Diffusive layers (not shown) can form between one or more adjacent glass layers.

[0070] One or both of the first cladding layer 108a and the second cladding layer 108b can be formed from a composition (e.g., second glass composition) comprising silicon dioxide (SiO2) having a concentration in a range of 45 mol% to 84 mol %, alumina (AI2O3) having a concentration in a range of 8 mol% to 19 mol%, boron trioxide (B2O3) having a concentration in a range of 5 mol% to 23 mol%, sodium oxide (Na2O) having a concentration in a range of 3 mol% to 21 mol%, and tin oxide (SnCF) having a concentration in a range of 0 mol% to 0.25 mol%. In embodiments, the one or more of the first cladding layer 108a and the second cladding layer 108b can be formed from a composition comprising silicon dioxide (SiCh) having a concentration in a range of 45 mol% to 60 mol %, alumina (AI2O3) having a concentration in a range of 8 mol% to 19 mol%, boron trioxide (B2O3) having a concentration in a range of 5 mol% to 23 mol%, sodium oxide (Na2O) having a concentration in a range of 3 mol% to 21 mol%, and tin oxide (SnC ) having a concentration in a range of 0 mol% to 0.25 mol%.. The one or more of the first cladding layer 108a and the second cladding layer 108b can be substantially free of arsenic (As) and cadmium (Cd).

[0071] In embodiments, the composition of the cladding layers is configured to enable a difference in degradation rate (e.g., etch rate) between the cladding layers and the core layer 106. For example, the core layer 106 (e.g., first glass-based layer) can have a first composition with a first degradation rate when exposed to an etchant, and the first cladding layer 108a (e.g., second glass-based layer) can have a second composition with a second degradation rate that is higher than the first degradation rate when exposed to the etchant. In embodiments, the degradation rate, or etch rate, of the cladding layers is at least 3 times, or 5 times, or 10 times, or 15 times, or 20 times, or 40 times greater than the degradation rate of the core layer in the etchant.

[0072] One or both of the first cladding layer 108a and the second cladding layer 108b can have a clad thickness t _c iad (shown in FIG. 5) of from 25 pm to 300 pm, from 50 pm to 275 pm, or from 50 pm to 250 pm. In embodiments, one or both of the cladding layers has a clad thickness t _c iad of greater than 25 pm, greater than 30 pm, greater than 40 pm, greater than 50 pm. greater than 60 pm, greater than 70 pm, greater than 80 pm, greater than 90 pm, or greater than 100 pm. In embodiments, one or both of the cladding layers has a clad thickness t _c iad of less than 300 pm, less than 275 pm, less than 250 pm, less than 225 pm, less than 200 pm, less than 175 pm, less than 150 pm, less than 125 pm, or less than 100 pm. The clad thickness t _c iad of one or both of the cladding layers may be within a range formed from any of the aforementioned endpoints. It should be appreciated, however, that the cladding layers 108a, 108b can have other clad thicknesses t _c iad- Without being bound by theory, the minimum clad thickness t _c iad disclosed herein may account for the accuracy of the techniques used to form the flow channels 104, such as the accuracy of the laser ablation technique (e.g., ~ 1 pm) discussed later in this disclosure. The minimum clad thickness t _c iad disclosed herein may also account for typical flow cell heights or depths (e.g., 30 pm, 50 pm, or 100 pm).

[0073] The core layer 106 can be formed from a composition (e.g., first glass composition) comprising at least one of an alkaline earth boro-aluminosilicate glass (e.g., Coming Eagle XG®), Coming FotoForm® Glass, Coming Iris™ Glass, or Coming Gorilla® Glass. In embodiments, the core layer 106 can be formed from a composition comprising SiCh having a concentration in a range of from 60 mol%to 85 mol%, AI2O3 having a concentration in a range of from 1 mol% to 10 mol%, Na2O having a concentration in a range of 3 mol% to 25 mol%, MgO having a concentration in range of from 1 mol% to 15 mol%, SrO having a concentration in a range of from 1 mol% to 10 mol%, and tin oxide (SnCF) having a concentration in a range of 0 mol% to 0.25 mol%.. For example, the core layer 106 can be formed from a glass having a composition of 79.3 wt.% (75 mol%) SiC>2, 1.6 wt.% (1.47 mol%) Na2O, 3.3 wt.% (1.99 mol%) K ₂ O, 0.9 wt.% (0.506 mol%) KNO3, 4.2 wt.% (2.34 mol%) AI2O3, 1.0 wt.% (0.698 mol%) ZnO, 0.0012 wt.% (0.000346 mol%) Au, 0.115 wt.% (0.0606 mol%) Ag, 0.015 wt.% (0.00495 mol%) CeC>2, 0.4 wt.% (0.078 mol%) Sb2C>3, and 9.4 wt.% (17.9 mol%) Li2O. In embodiments, the core layer 106 comprises at least one of Coming Eagle XG® Glass or Coming Iris™ Glass, for example, due to the ultra-low auto-fluorescence of each glass. In embodiments, an auto-fluorescence in a wavelength range of 450 nm to 750 nm of each of the core layer 106 and the cover 114 is at least as low as the auto-fluorescence of one or both of a quartz substrate or a pure silica substrate.

[0074] In embodiments, the core layer 106 has a core thickness Lore (shown in FIG. 5) of from 100 pm to 1200 pm, or from 200 pm to 1100 pm. In embodiments, the core layer 106 has a core thickness Lore of greater than 100 pm, greater than 150 pm, greater than 200 pm, greater than 250 pm, greater than 300 pm, greater than 500 pm, greater than 600 pm, or greater than 700 pm. In embodiments, the core layer 106 has a core thickness Lore of less than 1200 pm, less than 1000 pm, less than 900 pm, less than 800 pm, less than 700 pm, or less than 500 pm. The core thickness Lore of the core layer 106 can be within a range formed from any of the aforementioned endpoints. It should be appreciated, however, that the core layer 106 can have other core thicknesses t _C0K . It should also be appreciated that the relative thicknesses of the core layer 106 and the first cladding layer 108a are not shown to scale in FIG. 5.

[0075] FIG. 4 is a cross-sectional view of the microfluidic device of FIG. 1 along line B-B to illustrate structural aspects of the flow channels 104. Line B-B passes through a portion of the glass-based laminate substrate 102, as shown in FIG. 1, to illustrate an orthogonal cross section of one of the flow channels 104, as shown in FIG. 4. As used herein, the term “orthogonal cross section” refers to any cross section taken along a length of the flow channel 104 that is orthogonal to the longitudinal axis 105 of the flow channel 104. In other words, the orthogonal cross section can be taken at any position along the longitudinal axis 105 and is not limited only to the position of line B-B shown in FIG. 4. In embodiments, the flow channel 104 has the orthogonal cross section of FIG. 4 for an entire length of the flow channel 104 along the longitudinal axis 105. The flow channel 104 in embodiments can have the orthogonal cross section of FIG. 4 for less than the entire length of the flow channel, such as for equal to or less than 90%, 80%, 70%, 60%, 50%, or 40% of the entire length of the flow channel 104. In embodiments, the flow channel 104 has the orthogonal cross section of FIG. 4 between certain features of the flow channel 104, such as between holes 118 in the cover 114. In embodiments, the longitudinal axis of the flow channel can curve within a plane such as U- shape or circular shape (not shown) and the flow channel can have the orthogonal cross section of FIG. 4 for the entire length or less than the entire length of the flow channel along the curved longitudinal axis.

[0076] As shown in FIG. 4, each flow channel 104 is defined by two sidewalls 110 and a floor 112. In embodiments, the first cladding layer 108a (e.g., the second glass-based layer) defines the sidewalls 110 of each of the flow channels 104, and the core layer 106 (e.g., the first glass-based layer) defines the floor 112 of each of the flow channels 104. In embodiments, different portions of the same flow channel 104 can have different attributes due to the process for manufacturing the microfluidic device 100, which is described later in this disclosure. For example, the flow channel 104 can include an upper channel portion 120 that opens to an outer surface 122 of the glass-based laminate substrate 102 and a lower channel portion 124 that extends from the upper channel portion 120 to the floor 112 of the flow channel 104. For ease of understanding only, a boundary plane 126 (depicted as a line in the cross-sectional view of FIG. 4) is used to delineate a theoretical boundary between the upper channel portion 120 and the lower channel portion 124 of the flow channel 104.

[0077] The upper channel portion 120 is delimited by a first upper sidewall 128a and a second upper sidewall 128b that is spaced apart from and positioned opposite the first upper sidewall 128a (e.g., the first and second upper sidewalls 128a, 128b are opposed). The lower channel portion 124 is delimited by a first lower sidewall 130a and a second lower sidewall 130b spaced apart from and positioned opposite the first lower sidewall 130a (e.g., the first and second lower sidewalls 130a, 130b are opposed). The first lower sidewall 130a is continuous with the first upper sidewall 128a such that there are no breaks or discontinuities at a transition of the first lower sidewall 130a to the first upper sidewall 128a proximate the boundary plane 126. Similarly, the second lower sidewall 130b is continuous with the second upper sidewall 128b such that there are no breaks or discontinuities at a transition of the first lower sidewall 130a to the first upper sidewall 128b proximate the boundary plane 126. In other words, the first and second lower sidewalls 130a, 130b are continuous with the first and second upper sidewalls 128a, 128b, respectively. In embodiments in which the sidewalls 110 of the same flow channel 104 have different attributes due to the process for manufacturing the microfluidic device 100, such as shown in FIG. 4, the first cladding layer 108a (e.g., the second glass-based layer) defines the first upper sidewall 128a, the second upper sidewall 128b, the first lower sidewall 130a, and the second lower sidewall 130b of each flow channel 104.

[0078] FIG. 5 is a duplicate of the cross-sectional view of FIG. 4 to illustrate dimensional aspects and further structural aspects of the flow channels 104. When viewed in respective orthogonal cross sections of the flow channel 104, such as the orthogonal cross section of the flow channel 104 shown in FIG. 5, at least one of the first upper sidewall 128a and the second upper sidewall 128b forms a first angle al with the floor 112 of the flow channel 104. In embodiments, the first angle al is substantially orthogonal (i.e., approximately 90°). The first upper sidewall 128a and the second upper sidewall 128b in embodiments are substantially parallel to one another. In embodiments, the first angle al is in a range of from about 85° to about 91°, or from about 85° to about 90°, or from about 86° to about 91°, or from about 86° to about 90°, or from about 87° to about 91°, or from about 87° to about 90°, and also comprising all sub-ranges and sub-values between these range endpoints, when measured within the first cladding layer 108a (e.g., the second glass-based layer). Unless otherwise indicated, angles or angle values disclosed herein are measured within the first cladding layer 108a (e.g., the second glass-based layer). As used herein, the term “measured within” means that the angle or angle value is measured at a location where the material of the indicated portion of the glass-based laminate substrate 102 exists (e.g., first cladding layer 108a) as opposed a location where the material of the indicated portion of the glass-based laminate substate 102 does not exists (e.g., the empty volume of the flow channel 104). In embodiments, each of the first upper sidewall 128a and the second upper sidewall 128b forms the first angle al with the floor 112.

[0079] When viewed in respective orthogonal cross sections of the flow channel 104, such as the orthogonal cross section of the flow channel 104 shown in FIG. 5, at least one of the first lower sidewall 130a and the second lower sidewall 130b forms a second angle a2 with the floor 112 of the flow channel 104. In embodiments, the second angle a2 is less than the first angle al . As such, the different attributes of the different portions of the same sidewall (e.g., the first upper sidewall 128a and the first lower sidewall 130a or the second upper sidewall 128b and the second lower sidewall 130b) can comprise different angles. In embodiments, the second angle a2 is in a range of from about 60° to about 90°, or from about 62° to about 90°, or from about 64° to about 90°, or from about 66° to about 90°, or from about 68° to about 90°, or from about 70° to about 90°, or from about 75° to about 90°, or from about 60° to about 80°, and also comprising all sub-ranges and sub-values between these range endpoints, when measured within the first cladding layer 108a (e.g., the second glass-based layer). In embodiments, each of the first lower sidewall 130a and the second lower sidewall 130b forms the second angle a2 with the floor 112. In one example, the first angle al formed between the floor 112 and the first upper sidewall 128a is approximately 90° and the second angle a2 formed between the floor 112 and the first lower sidewall 130a is approximately 88° when measured within the first cladding layer 108a (e.g., the second glass-based layer). In another example, the first angle al formed between the floor 112 and the second upper sidewall 128b is approximately 89° and the second angle a2 formed between the floor 112 and the second lower sidewall 130b is approximately 75° when measured within the first cladding layer 108a (e.g., the second glassbased layer).

[0080] When viewed in respective orthogonal cross sections of the flow channel 104, such as the orthogonal cross section of the flow channel 104 shown in FIG. 5, a first effective angle aleff. is formed between the floor 112 of the flow channel 104 and a first line 132 (shown in dashed line type) that extends between an intersection of the outer surface 122 and the first upper sidewall 128a (shown at point Pl in FIG. 5) and an intersection of the floor 112 and the first lower sidewall 130a (shown at point P2 in FIG. 5). In embodiments, the first effective angle is in a range of from about 75° to about 90°, or from about 76° to about 90°, or from about 76° to about 89°, or from about 77° to about 90°, or from about 78° to about 90°, or from about 79° to about 90°, or from about 80° to about 90°, and also comprising all sub-ranges and sub-values between these range endpoints, when measured within the glass-based laminate substrate 102. In the same respective orthogonal cross sections of the flow channel 104, a second effective angle (not shown) is formed between the floor 112 of the flow channel 104 and a second line (not shown) that extends between an intersection of the outer surface 122 and the second upper sidewall 128b and an intersection of the floor 112 and the second lower sidewall 130b. In embodiments, the second effective angle is in a range of from about 75° to about 90°, or from about 76° to about 90°, or from about 76° to about 89°, or from about 77° to about 90°, or from about 78° to about 90°, or from about 79° to about 90°, or from about 80° to about 90°, and also comprising all sub-ranges and sub-values between these range endpoints, when measured within the glass-based laminate substrate 102. The first line 132 and the second line are straight lines in embodiments.

[0081] As shown in FIG. 5, when at least one of the first effective angle al _e ff. and the second effective angle is less than 90° when measured within the glass-based laminate substrate 102, a width at the top of the flow channel Wtop can be greater than a width of at the bottom of the flow channel ^bottom. In embodiments, each flow channel 104 has a width W that corresponds to the width at the bottom of the flow channel JFbottom. The width W in embodiments is greater than 1 mm when measured along the floor 112 between the intersection of the floor 112 and the first lower sidewall 130a and the intersection of the floor 112 and the second lower sidewall 130b. For example, each flow channel 104 can have a width W of 1 mm, 1.25 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.75 mm or greater. In embodiments, the width W of the flow channel 104 is at most about 2 mm, or at most about 5 mm, or at most about 10 mm. In embodiments, each flow channel 104 has a length L (shown in FIG. 1) that is greater than 10 mm when measured at the floor 112 in a longitudinal direction along the longitudinal axis 105 at a central portion of the floor 112. In embodiments, each flow channel 104 has a length L that is greater than 20 mm. For example, each flow channel 104 may have a length L that is 10 mm, 15 mm, 20 mm, 25 mm, 27 mm, 30 mm, or greater. Because the width W and the length L of each flow channel 104 are measured at the floor 112, these values may be referred to herein as “the width of the floor” and “the length of the floor,” respectively.

[0082] Referring now to FIG. 4 and FIG. 5, each flow channel 104 has a depth or Z-height measured in a direction substantially parallel to the clad thickness Gad and/or the core thickness /core from at least one reference surface, such as the floor 112 or the outer surface 122 of the glass-based laminate substrate 102. In embodiments, the first cladding layer 108a (e.g., the second glass-based layer) defines the sidewalls 1 10, 128a, 128b, 130a, 130b of each flow channel 104 and the core layer 106 (e.g., the first glass-based layer) defines the floor 112 of each flow channel 104 such that the depth of each flow channel 104 corresponds to the clad thickness Gad. The upper channel portion 120 of each flow channel 104 also has a depth or Z- height measured relative to the clad thickness Gad and/or the core thickness >re. In embodiments, the depth of the upper channel portion 120 is measured in the direction substantially parallel to the clad thickness Gad and/or the core thickness from the outer surface 122 to the theoretical boundary between the upper channel portion 120 and the lower channel portion 124 (indicated by boundary plane 126). In such embodiments, the depth of the upper channel portion 120 corresponds to at least about 80% of the clad thickness Gad of the first cladding layer 108a (e.g., the second glass-based layer). The depth of the upper channel portion 120 in embodiments can correspond more closely with the clad thickness Gad, such as at least about 60%, 70%, 75%, 85%, 90%, 95%, or 98% of the clad thickness Gad- In embodiments, the depth of the upper channel portion 120 can be in a range of from about 60% to about 98%, or from about 75% to about 98%, or from about 80% to about 98%, or from about 70% to about 95%, or from about 75% to about 90% of the clad thickness t _c iad of the first cladding layer 108a (e.g., the second glass-based layer). It should be appreciated that the depth of the upper channel portion 120 can vary, depending on the clad thickness Gad of the first cladding layer 108a. The thinner the clad thickness Gad of the first cladding layer 108a, the lower percentage of the depth of the upper channel portion 120 relative to the clad thickness tciad of the first cladding layer 108a. For instance, when the clad thickness Gad of the first cladding layer 108a is about 30 pm, the depth of the upper channel portion 120 can be in a range of from about 60% to about 90% of the clad thickness t _c iad of the first cladding layer 108a.

[0083] In embodiments, the floor 112 of each flow channel 104 is planar and substantially parallel to a plane defined by the outer surface 122 on the first cladding layer 108a. In embodiments, the plane of the floor 112 has an area that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of an area of a plane defined by the perimeter of the flow channel 104 at the outer surface 122 on the first cladding layer 108a.

[0084] In embodiments, at least 80% of the total area of the floor 112 of each of the flow channels 104 has a surface flatness of less than about 700 nm/mm ² , or less than about 500 nm/mm ² , or less than about 300 nm/mm ² , or less than about 200 nm/mm ² , or less than about 150 nm/mm ² , or about 100 nm/mm ² when measured along the length L and the width W of the floor 112 in each of the plurality of flow channels 104. In embodiments, at least 90% of the total area of the floor 112 of each of the flow channels 104 has a surface flatness of less than about 700 nm/mm ² , or less than about 500 nm/mm ² , or less than about 300 nm/mm ² , or less than about 200 nm/mm ² , or less than about 150 nm/mm ² , or about 100 nm/mm ² . In embodiments, at least 95% of the total area of the floor 112 of each of the flow channels 104 has a surface flatness of less than about 700 nm/mm ² , or less than about 500 nm/mm ² , or less than about 300 nm/mm ² , or less than about 200 nm/mm ² , or less than about 150 nm/mm ² , or about 100 nm/mm ² . For example, in embodiments, from 80% to 100%, from 85% to 100%, from 90% to 100%, from 95% to 100%, from 80% to 99%, from 85% to 99%, from 90% to 99%, from 95% to 99%, from 80% to 98%, from 85% to 98%, from 90% to 98%, or from 95% to 98% of the total area of the floor 112 of each of the flow channels 104 has a surface flatness of less than about 700 nm/mm ² , or less than about 500 nm/mm ² , or less than about 300 nm/mm ² , or less than about 200 nm/mm ² , or less than about 150 nm/mm ² , or about 100 nm/mm ² . It should be appreciated that the percentage of the total area of the floor 112 of each of the flow channel 104 having a highly flat surface is dependent on the total area. The greater the total area, the higher the percentage.

[0085] The surface flatness can be measured using a laser interferometer (e.g., Zygo New View 7300), which measures differences in shape and tilt between a test sample surface and reference surfaces of the interferometer. For flow channels produced by the methods disclosed herein, the flatness of the floor 112 of each flow channel 104 is measured relative to an outer surface 134 (as shown in FIG. 8) of the second cladding layer 108b (e.g., in embodiments in which the second cladding layer 108b is present), or a reference substrate when the test sample is placed against the reference substrate, or an outer surface 122 of the first cladding layer 108a. For bonded microfluidic devices or flow cells, the flatness of the floor 112 of each flow channel 104 is measured relative to a surface of the reference substrate, such as when the device or flow cell is placed atop the reference substrate. As used herein, “surface flatness” is defined as the peak to valley flatness, which reports the absolute difference between the highest and lowest points on a surface relative to the ideal reference surface in a specific area.

[0086] In embodiments, the local surface flatness is measured by first defining a local flatness zone. For example, the local surface flatness of the floors of the flow channels can be measured over an area of 35 mm x 35 mm, and sites are scanned every 1 mm in each of the X and Y directions with a 0.5 mm increment. The local flatness zone includes all scans that include any part of the flow channel within a plane defined by an outer surface of the cladding layer. In other words, if a particular scan would include the sidewall, a portion of the floor, and a portion that is not formed by the methods disclosed herein (e.g., via laser ablation and/or etching), the scan site is included, and the entire area of that scan site is included in the calculation of the area of the flatness zone. The laser interferometer can be used to measure the surface flatness over each of the local flatness zones and identify zones that meet a local flatness target. In embodiments, the local flatness target is 500 nm/mm ² or less. A percentage within interval (PWI) can be calculated to determine the area of the floor of each of the flow channels that has a local surface flatness that meets or is less than the local flatness target. The PWI can be calculated according to the following equation: where Atarget is the area of the floor of a flow channel that has a local surface flatness that is less than or equal to the local flatness target and A _0V eraii is a total area of the floor of the flow channel.

[0087] Referring still to FIG. 4 and FIG. 5, the floor 112 and the sidewalls 110 can have respective surface roughness values that are similar to one another or that differ from one another due to the process for manufacturing the microfluidic device 100, which is described later in this disclosure. The first and second upper sidewalls 128a, 128b can have an upper sidewall surface roughness Ra _up per, the first and second lower sidewalls 130a, 130b can have a lower sidewall surface roughness Raiower, and the floor 112 can have a floor surface roughness Ranoor. In embodiments, the upper surface roughness Ra _up per of the first and second sidewalls 128a, 128b is greater than the lower surface roughness Raiower of the first and second lower sidewalls 130a, 130b. For example, the upper surface roughness Ra _up per can be greater than about 15 nm (e.g., 20 nm, 25 nm, 30 nm, or 50 nm) and the lower surface roughness Raiower can be less than or equal to about 15 nm (e.g., 10 nm, 8 nm, 6 nm, 4 nm, 2 nm, or 1 nm). In embodiments, the lower surface roughness Raiower is greater than or equal to the floor surface roughness Rarioor. For example, the floor surface roughness Rarioor can be less than or equal to about 10 nm. The floor surface roughness Rarioor in embodiments is in a range of from about 1 nm to about 15 nm.

[0088] Referring again to FIG. 2 and FIG. 3, the cover 114 of the microfluidic device 100 is further described. The cover 114 (sometimes referred to as a glass cover substrate) has a first surface 114a on one side and a second surface 114b on an opposite side. The cover 114 comprises a glass material, such as glass, glass ceramic, ceramic, or combinations thereof. In embodiments, such as the embodiment of FIG. 2, the cover 114 is bonded directly to the first cladding layer 108a (e.g., the second glass-based layer) such that an interface between the cover 114 and the first cladding layer 108a (e.g., an interface between the second surface 114b of the cover 114 and the outer surface 122 of the first cladding layer 108a) is a glass-glass interface that is free of bonding material. In embodiments, such as the embodiment of FIG. 3, a glass-to-glass bonding material, such as the bonding material 115, is disposed on the outer surface 122 of the first cladding layer 108a and the cover 114 is bonded to first cladding layer 108a via the bonding material 115. The bonding material 115 in embodiments comprises at least one of Cr/CrON, metals (e.g., Zn, Ti, Ce, Pb, Fe, Va, Cr, Mn, Mg, Ge, Au, Ni, Cu, Al, Ta, Nb, Sn, In, Co, W, Yb, Zr, etc.), metal oxides thereof (e.g., AI2O3, ZnCh. Ta2Os, Nb2Os, SnC , MgO, indium tin oxide (ITO), CeO2, CoO, CO3O4, CYCf. Fe2O3, FesCfi, I Or. MroCf. NiO, a-TiO2 (anatase), r-TiO2 (rutile), WO3, Y2O3, ZrO2), or polymer-carbon black composite films (e.g., polyimide -carbon black film).

[0089] When the cover 114 is bonded to the first cladding layer 108a, as shown in FIG. 2 and FIG. 3, the second surface 114b of the cover 114 faces and is directly opposed to the first surface 106a of the core layer 106. In this configuration, the second surface 114b of the cover 114 defines a ceiling surface or ceiling of the flow channel 104, and the first surface 106a of the core layer 106 (e.g., the first glass-based layer) defines a floor surface or the floor 112 of the flow channel 104. The surfaces corresponding to the ceiling 114b and the floor 112 of the flow channel 104 can be substantially parallel, for example, due to precision bonding and ultra- flatness of these surfaces. In embodiments, the ceiling 114b has a ceiling surface roughness Ra _C eiiing in a range of from about 1 nm to about 15 nm.

[0090] Controlled entry and exit of a fluid (e.g., test DNA samples) can be conducted through holes 118 in the cover 114 extending from the first surface 114a to the second surface 114b (e.g., through-holes). In embodiments, such as shown in the embodiments of FIG. 2 and FIG. 3, the holes include two holes 118, one of which forms an inlet port opening through the cover 114 and in fluid communication with the flow channel 104 and the other of which forms an outlet port opening through the cover 114 and in fluid communication with the flow channel 104. In embodiments, the holes 118 alternatively extend through the glass-based laminate substrate 102 and fluidically communicate with the flow channel 104.

[0091] The holes 118 and the flow channel 104 are configured to provide a flow path (shown as a dashed line in FIG. 2 and FIG. 3) for the fluid through the microfluidic device 100. For example, when used for DNA sequencing, the flow channel 104 provides a flow path for test DNA samples such that DNA fragments can be immobilized and partitioned onto the ceiling 114b and/or the floor 112 of the flow channel 104 to facilitate sequencing. The ceiling 114b and/or the floor 112 of the flow channel 104 can be treated, for example, chemically functionalized or physically structured (e.g., with nanowell arrays), to aid in performing a desired function (e.g., capture of desired DNA fragments).

[0092] Although the microfluidic device 100 is shown and described comprising a twolayered glass-based laminate substrate 102 (e.g., FIG. 2 and FIG. 3), a microfluidic device comprising a three-layered glass-based laminate substrate is also contemplated. In such embodiments, the three-layered glass-based laminate substrate comprises a core layer (e.g., first glass-based layer) interposed between a first cladding layer and a second cladding layer (e.g., second glass-based layers) with at least one flow channel disposed in one of the cladding layers. The cover in such embodiments is bonded to the one cladding layer with the at least one flow channel and the other cladding layer remains fused to the core layer. Having described embodiments of glass laminates including flow channels and microfluidic devices including the same, methods for making the glass laminates including the flow channels will now be described.

[0093] FIGS. 6-11 illustrate an example of a method for making the microfluidic device 100 including the flow channels 104 described herein. According to embodiments, the plurality of flow channels 104 can be formed in the first cladding layer 108a of the glass-based laminate substrate 102 via a two-step material removal process. FIG. 6 illustrates an example of a glass-based laminate substrate 102 that can be used in the method to make the microfluidic device 100. As described above, the glass-based laminate substrate 102 comprises the core layer 106 (e.g., first glass-based layer) interposed between the first cladding layer 108a and the second cladding layer 108b (e.g., second glass-based layers).

[0094] Various processes can be used to produce the glass-based laminate substrate 102 described herein, including, without limitation, lamination slot draw processes, lamination float processes, or fusion lamination processes. Each of these lamination processes generally involves flowing a first molten glass composition, flowing a second molten glass composition, and contacting the first molten glass composition with the second molten glass composition at a temperature greater than the glass transition temperature of either glass composition to form an interface between the two compositions such that the first and second molten glass compositions fuse together at the interface as the glass cools and solidifies. In embodiments, the glass-based laminate substrates 102 described herein can be formed by a fusion lamination process, such as the process described in U.S. Patent No. 4,214,886, which is incorporated by reference in its entirety.

[0095] FIG. 7 illustrates one step of a method for removing material from the glass-based laminate substrate 102 to form each of the flow channels 104. The method includes using a first technique to remove a first portion of the first cladding layer 108a (e.g., second glassbased layer) to form the upper channel portion 120 in the glass-based laminate substrate 102. The upper channel portion 120, as formed, opens to the outer surface 122 of the first cladding layer 108a (e.g., second glass-based layer) and extends away from the outer surface 122 to a first floor 138 defined by the first cladding layer 108a.

[0096] In embodiments, the first technique comprises a laser ablation process that includes directing a laser beam at the outer surface 122 of the first cladding layer 108a (e.g., second glass-based layer). The laser beam is configured to remove the first portion of the first cladding layer 108a via ablation to form the upper channel portion 120. As used herein “ablation” and “laser ablation” mean the removal of glass material from the glass-based laminate substrate 102 by vaporization due to the energy introduced by the laser beam. In embodiments, the laser ablation follows a top-down ablation approach that uses a laser energy above a threshold to cause the outer surface 122 of the first cladding layer 108a to absorb enough energy, so that the glass is heated to trigger ablation. [0097] Initiating a top-down ablation process with certain lasers, such as a ns-laser, can require extra laser power to adequately heat the outer surface 122 of the first cladding layer 108a, but once a first portion of the first cladding layer 108a at or near the outer surface 122 is ablated, the resultant modified surface texture again acts as an ablation catalyst and allows channel structures to form top-down. With this type of ablation, particles are ejected from the outer surface 122 of the first cladding layer 108a. As such, it can be helpful to capture and/or extract the ejected particles, for example, using a vacuum nozzle or similar collection device. Alternatively, a bottom-up ablation approach can also be used. Here, the bottom-up ablation occurs on the back surface of the substrate. When the laser energy is below a threshold, it will transmit through-the-glass and ablation will be initiated on the back side of the glass.

[0098] In embodiments, the laser ablation process is performed using a laser system configured to direct and focus the laser beam from a laser source onto the outer surface 122 of the first cladding layer 108a. The laser system is configured to create a beam with a small spot size in a focal area with a large depth of focus (DOF). The small beam spot size provides sufficient energy density to initiate glass damage through ablation. The laser system can further comprise a lens system with a focusing lens that focuses laser beam. The laser system can comprise a beam expander in front of the focusing lens so the beam spot size can be varied before the focusing lens. As a result, an effective DOF can be controlled and expanded as desired. The laser source has an output power sufficient to initiate ablation of the glass material on the outer surface 122 and then to propagate a crack or defect within the first cladding layer 108a. In one embodiment, the upper channel portion 120 can be formed using 9W nanosecond (ns) laser with a center wavelength of 532 nm and a pulse width of ~ 6 ns, coupled to optimized linear travel speed of the beam and frequency of the laser. The laser system can include a varioscan to provide depth or Z-height control during formation of the upper channel portion 120.

[0099] The first technique is used to remove a majority (but not all) of the clad thickness tciad of the first cladding layer 108a (e.g., the second glass-based layer) to form the upper channel portion 120 by controlling the laser ablation power and duration. In embodiments, the first technique is configured to remove at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the clad thickness t _c iad of the first cladding layer 108a from the outer surface 122 thereof to form the upper channel portion 120. In embodiments, the first technique is configured to remove from about 60% to about 98% of the clad thickness t _c iad of the first cladding layer 108a from the outer surface 122 thereof to form the upper channel portion 120. [0100] FIG. 8 illustrates another step of the method for removing material from the glassbased laminate substrate 102 to form each of the flow channels 104. The method further includes using a second technique different from the first technique to remove a second portion of the first cladding layer 108a (e.g., second glass-based layer) to form the lower channel portion 124 (FIG. 4) in the glass-based laminate substrate 102. As best shown in FIG. 4, the lower channel portion 124, as formed, extends from the upper channel portion 120 (e.g., the bottom of the upper channel portion 120 at the boundary plane 126) to a second floor (e.g., the floor 112) defined by the core layer 106 (e.g., first glass-based layer). As used herein, a first technique that is “different from” a second technique means that the techniques employ materially different processes or mechanisms to achieve the intended the removal of material from the glass-based laminate substrate. For example, a first etch process that differs via one or more of etchant composition, etchant concentration, etch duration, and etch temperature is not “different from” a second etch process because the first and second processes are nonetheless etch processes that remove material via etching.

[0101] In embodiments, the second technique comprises a wet etching process that includes contacting the first floor 138 (FIG. 7) of the upper channel portion 120 with an etchant. As described above, the composition of the cladding layers 108a, 108b is configured to enable a difference in the degradation rate (e.g., etch rate) between the cladding layers 108a, 108b and the core layer 106 when the layers are exposed to the etchant. In particular, upon exposure to the etchant, the cladding layers 108a, 108b have a higher etch rate than the core layer 106, which enables portions of the cladding layers 108a, 108b that remain after using the first technique to be selectively removed by the etchant while the core layer 106 serves as an etch stop.

[0102] According to the second technique, the first floor 138 (FIG. 7) is contacted with the etchant at an etch temperature and for an etch time to remove the second portion of the first cladding layer 108a and form the lower channel portion 124 (FIG. 4). In embodiments, the first floor 138 is contacted with the etchant until the core layer 106 is adequately exposed to define the floor 112. It should be appreciated that the wet etching process used to form the lower channel portion 124 does not require any masking and/or patterning prior to performing the etching since the upper channel portion 120 formed by the laser ablation process provides an existing structure configured to contain and guide the etchant during the wet etching process. The lower channel portion 124, as formed, and the upper channel portion 120 collectively form the flow channel 104 with the sidewalls 110, 128a, 128b, 130a, 130b defined by the first cladding layer 108a (e.g., the second glass-based layer) and with the floor 112 defined by the core layer 106 (e.g., the first glass-based layer), as shown in FIG. 4.

[0103] The etchant is a wet etching chemical that comprises a component capable of degrading or dissolving the glass material of the cladding layers 108a, 108b, as described herein. For example, the etchant may include an acid (e.g., HC1, HNOs, H2SO4, H3PO4, H3BO3, HBr, HCIO4, HF, acetic acid, etc ), a base (e g ., LiOH, NaOH, KOH, RbOH, CsOH, Ca(OH) ₂ , Sr(OH)2. Ba(OH)2, etc.) or a combination thereof. In embodiments, the etchant is aqueous hydrofluoric acid (HF) with a concentration of at most about 5 weight percent HF. The concentration of the aqueous HF can be in range of from about 0.1 to about 6 weight percent HF, or from about 0.1 to about 5 weight percent HF, or from about 0.15 to about 4 weight percent HF, or from about 0.15 to about 3.5 weight percent, or from about 0.2 to about 3 weight percent HF, or from about 0.2 to about 2.5 weight percent HF, or from about 0.2 to about 2 weight percent HF in embodiments.

[0104] The etch temperature can be about 20 °C, about 22 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, or about 50 °C, or any ranges defined by any combination of the stated values. In embodiments, a lower etch temperature can enable an increased etch time and/or improved flow channel geometry as described herein. The etch temperature can be room temperature in embodiments.

[0105] The etch time can be from about 2 minutes to about 75 minutes, depending on the embodiment. For example, the etch time can vary depending on the specific glass compositions used, the etchant used, the temperature, and the amount of the first cladding layer 108a to be removed. In embodiments, the etch time is from about 2 minutes to about 50 minutes, or from about 35 minutes to about 45 minutes. In embodiments, the etch time can be about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, or about 90 minutes, or any ranges defined by any combination of the stated values. A relatively longer etch time (e.g., an etch time of greater than 10 minutes), due to a lower concentration of the etchant used, can enable the second angle a2 formed between the floor 112 and each of the first and second lower sidewalls 130a, 130b of each of the flow channels 104 to be closer to 90°, for example, the second angle a2 in a range of from about 70° to about 90°, or from about 75° to about 90°, or from about 80° to about 90°. [0106] As described above, the first portion of the first cladding layer 108a removed via the first technique (e.g., the laser ablation process) corresponds to a majority (but not all) of the clad thickness t _c iad of the first cladding layer 108a. The second portion of the first cladding layer 108a removed via the second technique (e.g., the wet etching process) corresponds to a remainder of the clad thickness t _c iad since the second portion is removed until the core layer is exposed to define the floor 112 of the flow channel. As a result of the first and second portions of the first cladding layer 108a removed at different depths, a first volume of the first portion removed during the first technique is greater than a second volume of the second portion removed during the second technique. In embodiments, the first volume of the first portion is at least 1.5 times greater, 2 times greater, 3 times greater, or at least 3.5 times greater, or at least 4 times greater, or at least 4.5 times greater, or at least 10 times greater.

[0107] As described above, the core layer 106 has a lower etch rate in the etchant than the etch rate of first and second cladding layers 108a, 108b. Accordingly, the core layer 106 serves as an etch stop for the method step comprising the wet etching process. Moreover, in embodiments, the second cladding layer 108b can be at least partially removed, for example when no etch mask, protection tape, or other protectant is applied thereon, during the wet etching process. In embodiments, such as the embodiment shown in FIG. 9, the second cladding layer 108b can be completely removed during the wet etching process, and the resulting glass-based laminate substrate 102 includes the core layer 106 and the first cladding layer 108a having a plurality of flow channels extending through the first cladding layer 108a to the core layer 106. In embodiments, such as the embodiment shown in FIG. 8, the second cladding layer 108b can be partially or fully retained, such as by applying an etch mask, protection tape, or other protectant thereon prior to etching. In such embodiments, the second cladding layer 108b is retained, and the resulting glass-based laminate substate 102 includes the core layer 106, the first cladding layer 108a having a plurality of flow channels extending through the first cladding layer 108a to the core layer 106, and the second cladding layer 108b. Without being bound by theory, the use of a three-layer glass-based laminate substrate, such as shown in FIG. 8, can eliminate the need for the use of a separate carrier to be bound during fabrication of the glass-based laminate substrate 102 and/or the microfluidic device 100.

[0108] FIG. 10 and FIG. 11 illustrate further aspects of the method for making the microfluidic device 100 including bonding the cover 114 to the glass-based laminate substrate 102 once the once the plurality of flow channels 104 are formed therein. FIG. 10 depicts embodiments of the bonding in which the cover 114 is bonded directly to the first cladding layer 108a such that an interface between the second surface 114b of the cover 114 and the outer surface 122 of the first cladding layer 108a is a glass-glass interface that is free of bonding material. In embodiments, the direct bonding depicted in FIG. 10 comprises applying hydrofluoric acid (HF) or a (functionally) similar etchant, such as the etchants listed above, to the outer surface 122 of the first cladding layer 108a (e.g., second glass-based layer) and positioning the cover 114 on the outer surface 122 with the HF disposed therebetween.

[0109] In embodiments, the applying the HF or similar etchant to the outer surface 122 can include selectively positioning aqueous HF solution on the outer surface 122. The amount of the aqueous HF solution to be selectively positioned on the outer surface 122 can depend on the total bonding area. Typically, about 10 pL of the aqueous HF solution is believed to be sufficient to enable 1” x 3” glass-to-glass bonding. The HF solution can be selectively positioned on the outer surface 122 using any equipment and/or technique, such as using an automated or manual dropper to drop the HF solution onto the outer surface 122. The HF solution to be applied to the outer surface 122 can have a HF concentration configured to achieve a sufficient bond between the cover 114 and the glass-based laminate substrate 102 after the direct bonding process. In embodiments, the HF solution has a concentration in a range of from about 0.5% HF to about 1.5% HF, though other concentration ranges and amounts are contemplated.

[0110] In embodiments, the applying the HF or similar etchant to the outer surface 122 can include exposing at least the outer surface 122 to HF steam for an exposure time. The outer surface 122 can be selectively exposed to the HF steam by, for example, applying a mask to surfaces of the glass-based laminate substrate 102 that are not intended to be exposed to the HF steam. In embodiments, the exposing the outer surface 122 to the HF steam comprises placing the glass-based laminate substrate 102 and the cover 114 into a chamber containing about 1 mol L' ¹ HF/NFfiF solution and treating the glass-based laminate substrate 102 and the cover 114 with HF steam for an exposure time in a range of about 10 minutes to about 35 minutes.

[oni] In embodiments, the direct bonding is performed at a relative low temperature, such as at a bonding temperature in a range from about 35 °C to about 120 °C, or from about 40 °C to about 115 °C, or from about 40 °C to about 110 °C, or from about 45 °C to about 110 °C, or from about 50 °C to about 110 °C, from about 50 °C to about 100 °C, or from about 55 °C to about 95 °C, or from about 65 °C to about 90 °C, or from about 75 °C to about 85 °C, and also comprising all sub-ranges and sub-values between these range endpoints. [0112] Subsequent to selectively positioning the HF solution on the outer surface 122 or exposing the outer surface 122 to HF steam, the cover 114 is positioned directly on the first cladding layer 108a such that the cover 114 at least partially covers the plurality of flow channels 104. In embodiments, the direct bonding further comprises pressing the cover 114 against the outer surface 122 with a press force and a press time. The press force in embodiments can be configured to generate a pressure between the bonded surfaces in a range of from about 0.1 MPa to about 1 MPa though other pressures outside of this range are contemplated. In embodiments, the cover 114 can be pressed against the outer surface 122 for a press time in a range of from 1 hour to 48 hours though other press times outside of this range are contemplated. The direct bonding described above can be referred to as HF-assisted low temperature direct glass to glass fusion or HF -assisted bonding. Subsequent to the direct bonding of the cover 114 to the glass-based laminate substrate 102, the HF or similar etchant evaporates and/or reacts with metal oxide in the glass such that it is no longer detectable between the cover 114 and the glass-base laminate substrate 102.

[0113] In embodiments, the direct bonding can take place immediately after the completion of the wet etching process used to remove the second portion of the first cladding layer 108a to form the lower channel portion 124. In such embodiments, the wet etching process not only selectively removes the remaining portion of the first cladding layer 108a to form the flow channel 104 but can also clean the surfaces to be joined by the directed bonding and enable the HF-assisted bonding described above. If the direct bonding does not take place near in time (e.g., less than 1 hour) the wet etching process used to form the lower channel portion 124, the method further comprises a cleaning process prior to the direct bonding. In embodiments, the cleaning process can comprise washing or successively washing the glass-based laminate substrate 102 and the cover 114 in one or more of acetone, ethanol, pure water, a mixed solution of sulfuric acid and hydrogen peroxide (2: 1), 1 MNaOH solution, and pure water.

[0114] FIG. 11 depicts embodiments of the bonding in which the cover 114 is bonded (indirectly) to the first cladding layer 108a via the bonding material 115. In such embodiments, an interface between the second surface 114b of the cover 114 and the outer surface 122 of the first cladding layer 108a comprises the bonding material 115.

[0115] In embodiments, the (indirect) bonding depicted in FIG. 11 can use a laser-assisted radiation bonding process to bond the cover 114 with the first cladding layer 108a using the bonding material 115. Without being bound by theory, it is believed that the bonding of the bonding material 115 to the first cladding layer 108a and the cover 114, respectively, is the result of diffusing a portion of the bonding material 115 into the first cladding layer 108a and the cover 114 such that each portion of the first cladding layer 108a and the cover 114 comprising the diffused glass-to-glass-bonding material is the bonded volume layer (not shown). As oriented, the bonding material 115 may not be transparent to the wavelength of the laser emission while the first cladding layer 108a and the cover 114 may be transparent to the wavelength of the laser emission. In such embodiments, the laser emission can pass through the cover 114 and/or the glass-based laminate substrate 102 and be absorbed by the bonding material 115. In embodiments, the diffusion of the bonding material 115 into the first cladding layer 108a and the cover 114, respectively, renders the bonded volume layer transparent to the wavelength of laser emission.

[0116] In embodiments, the bonding of the bonding material 115 to the first cladding layer 108a and cover 114, respectively, is accomplished using a laser which has a wavelength such that at least one of the substrates (e.g., first cladding layer 108a and/or cover 114) is transparent to that wavelength. An interface between the layers provides a change in the index of transmission or optical transmissivity which results in absorption of laser energy at the interface and localized heating to create a bond.

[0117] In embodiments in which the bonding material 115 is Cr/CrON, the Cr component can function as a heat absorption layer which is opaque or blocking to the laser wavelength and has an affinity for diffusion into the first cladding layer 108a and/or the cover 114. In alternative embodiments, other materials having appropriate wavelength absorption and diffusion affinity characteristics can be employed as the heat absorption layer. The thickness of the heat absorption layer may be as thick as desired to compensate for surface roughness or control timing and temperatures of the process.

[0118] Additionally, and/or alternatively, the bonding of the bonding material 115 to the first cladding layer 108a and the cover 114 throughout the bonded volume layer can include melting at least one of the bonding material 115, first cladding layer 108a, and/or cover 114 (e.g., localized melting at the site of laser emission absorption). Moreover, the bonding can also include fusing the bonding material 115 to at least one of the first cladding layer 108a or cover 114. In embodiments, the bonded volume layer is transparent to the wavelength of the laser emission.

[0119] In embodiments, the bonding can be achieved via separate laser emission (not illustrated), for example, as described in United States Patent Nos. 9,492,990, 9,515,286, and/or 9, 120,287, which are incorporated herein by reference in their entirety. In further embodiments, adhesives or other bonding materials can be employed to bond the first cladding layer 108a and the cover 114 without the use of laser emissions.

[0120] Accordingly, in embodiments, after the cover 114 is placed onto the etched structure of FIG. 11 (as described above) and is in close contact with the bonding material 115, the combination is exposed to radiation (e.g., laser light treatment) to bond each of the first cladding layer 108a and the cover 114 to bonding material 115 through bonded volume layers, respectively. Fabricating the structure of FIG. 11 can include positioning the cover 114 on the bonding material 115 and irradiating the bonding material 115 with electromagnetic radiation sufficient to diffuse at least a portion of the bonding material 115 into the cover 114 and the first cladding layer 108a.

[0121] Referring again to FIG. 2 and FIG. 3, the method further includes forming the holes 118 in microfluidic device 100. In embodiments, such as the embodiments shown in FIG. 2 and FIG. 3, the holes 118 are formed in the cover 114. The holes 118 can be formed in the cover 114 using any technique that enables the positional and dimensional accuracy required for the applications that use the microfluidic device 100. It should also be appreciated that the holes 118 can be formed in the glass-based laminate substrate 102 using the same techniques that form the flow channels 104 (e.g., laser ablation followed by wet etching). Such placement of the holes in the glass-based laminate substrate 102 can reduce the overall cost to manufacture microfluidic devices or flow cells.

[0122] The various embodiments of the microfluidic devices and methods of making the microfluidics devices disclosed herein after numerous advantages. One advantage is high throughout. The methods and devices disclosed herein leverage the high-throughput capability of laser ablation technique, which can be fully automated in embodiments. Another advantage is high channel wall angle. The methods and devices disclosed herein leverage the ability of laser ablation technique to form a channel having straight sidewall (i.e., substantially orthogonal relative to floor) within a glass.

[0123] Another advantage is a highly flat channel floor surface. The methods and devices disclosed herein take advantages of the differentiated etching rate of the clad and core layers of a glass laminate. The glass laminate comprises at least one clad layer and one core layer. Due to differing compositions, the clad layer can be etched away much faster (e.g., 5x, lOx, 15x, 20x) than the core layer. Laser ablation can be controlled such that a majority (but not all) of the clad layer is removed, which leaves a channel having straight sidewalls and a relatively rough channel floor surface. Upon exposure to wet chemical etching, the remaining clad layer can be selectively etched away until the core layer is exposed. In this way, the channel floor surface can be significantly smoothened, leading to a decrease in local flatness of the floor surface. In embodiments, the local flatness decrease is from several to tens microns per mm ² obtained via laser ablation to several hundreds of nanometers per mm ² obtained via wet chemical etching.

[0124] Another advantage is simplified process flow. Pre-cleaning and (mask) patterning of glass substrate are typically required to form a channel in the glass substrate using typical wet chemical etching techniques. In contrast, the disclosed laser ablation technique does not require pre-cleaning and patterning of the glass substrate. Additionally, the follow-up wet chemical etching and smoothening also does not require pre-cleaning and patterning of glass substrate. Furthermore, the selective etching away of the clad layer not only results in clean glass, but also can be used to assist with glass-to-glass direct fusion bonding process, such as via HF-assisted glass-to-glass bonding at relatively low temperatures as disclosed herein.

[0125] While the disclosure has been illustrated and described in detail in the drawings and foregoing description, the same should be considered as illustrative and not restrictive in character. It is understood that only the preferred embodiments have been presented and that all changes, modifications and further applications that come within the spirit of the disclosure are desired to be protected.