阅读说明：本技术 微流体装置和制造微流体装置的方法 (Microfluidic device and method of manufacturing a microfluidic device ) 是由 方晔 张锐 于 2019-07-30 设计创作，主要内容包括：一种微流体装置,其包括设置在玻璃基基板中的流动通道；以及与玻璃基基板结合并至少部分覆盖流动通道的盖板,其中盖板的厚度至多为200μm。(A microfluidic device comprising a flow channel disposed in a glass-based substrate; and a cover plate bonded to the glass-based substrate and at least partially covering the flow channel, wherein the cover plate has a thickness of at most 200 μm.)

1. A microfluidic device, comprising:

a flow channel provided in the glass base substrate; and

a cover plate bonded to the glass-based substrate and at least partially covering the flow channel,

wherein the thickness of the cover plate is at most 200 μm.

2. The microfluidic device of claim 1, further comprising:

an inlet opening through at least one of the glass-based substrate or the cover plate and in fluid communication with the flow channel; and

an outlet opening through at least one of the glass-based substrate or the cover plate and in fluid communication with the flow channel.

3. The microfluidic device of claim 1 or claim 2, wherein:

the first glass substrate layer defines a bottom of the flow channel;

the second glass substrate layer defining sidewalls of the flow channel; and

the cover plate defines a top of the flow channel.

4. The microfluidic device of any one of claims 1-3, wherein the thickness of the cover plate is in the range of 100 μm to 180 μm.

5. The microfluidic device of any one of claims 1-4, wherein the cover plate comprises:

56 to 72 mol% SiO₂；

5 to 22 mol% of Al₂O₃；

0 to 15 mol% of B₂O₃；

3 to 25 mol% Na₂O；

0 to 5 mol% of K₂O；

1 to 6 mol% of MgO;

0 to 1 mol% of SnO₂。

6. The microfluidic device of claim 5, wherein the cover plate further comprises:

0 to 7 mol% of Li₂O; and

0 to 10 mol% of P₂O₅。

7. The microfluidic device of claim 5, wherein the cover plate further comprises:

0 to 3 mol% CaO; and

0 to 2 mol% of ZrO₂。

8. The microfluidic device of claim 7, wherein the cover plate further comprises:

0 to 6 mol% ZnO.

9. The microfluidic device of any one of claims 1-8, wherein the cover plate is configured to have an autofluorescence as low as that of a pure silica substrate in a wavelength range of 400nm to 750 nm.

10. The microfluidic device of any one of claims 1-9, wherein the cover plate is configured to have an average surface inclination or slope of at most about 100nm/mm, the average surface inclination or slope being measured with a laser interferometer.

11. The microfluidic device of claim 10, wherein the average surface slope or slope is at most about 50 nm/mm.

12. The microfluidic device of any one of claims 1-11, wherein the cover plate is configured to have at most about 10nm/μ ι η²The surface roughness of (2).

13. The microfluidic device of claim 12, wherein the surface roughness is at most about 5nm/μ ι η²。

14. The microfluidic device of any one of claims 1-13, wherein the cover plate is bonded to the glass base substrate with a bonding volume comprising a bonding material diffused into each of the glass base substrate and the cover plate.

15. The microfluidic device of any one of claims 1-14, comprising a bonding layer disposed between the glass base substrate and the cover plate.

16. The microfluidic device of claim 15, wherein the binding layer comprises a metal.

17. The microfluidic device of claim 16, wherein the metal comprises one or more of gold, chromium, titanium, nickel, copper, zinc, cerium, lead, iron, vanadium, manganese, magnesium, germanium, aluminum, tantalum, niobium, tin, indium, cobalt, tungsten, ytterbium, zirconium, or oxides thereof, or combinations thereof.

18. The microfluidic device of claim 15, wherein the bonding layer comprises a polymer-carbon black composite film.

19. The microfluidic device of any one of claims 1-18, wherein the microfluidic device is a flow cell for DNA sequencing.

20. The microfluidic device of any one of claims 1-19, wherein a surface of the bottom channel, a surface of the cover plate, or both comprise an array of patterned nanostructures.

21. A glass composition comprising:

56 to 72 mol% SiO₂；

5 to 22 mol% of Al₂O₃；

0 to 15 mol% of B₂O₃；

3 to 25 mol% Na₂O；

0 to 5 mol% of K₂O；

1 to 6 mol% of MgO;

0 to 1 mol% of SnO₂。

22. The glass composition of claim 21, further comprising:

0 to 7 mol% of Li₂O; and

0 to 10 mol% of P₂O₅。

23. The glass composition of claim 21 or claim 22, further comprising:

0 to 3 mol% CaO; and

0 to 2 mol% of ZrO₂。

24. The glass composition of claim 23, further comprising:

0 to 6 mol% ZnO.

25. The glass composition of any of claims 21-24, configured to have a strength of at least 600 MPa.

26. The glass composition of any of claims 21-25, configured to have a refractive index of at least 1.50.

27. A method of strengthening a glass composition according to any of claims 21-26, comprising: replacing a first alkali metal cation having a first size with a second alkali metal cation having a second size,

wherein the second size is greater than the first size, an

Wherein the glass composition is configured to have a strength of 100MPa to 200MPa prior to the displacing and a strength of at least 600MPa after the displacing.

28. The method of claim 27, wherein the first alkali metal cation is at least one of a lithium cation or a sodium cation, and wherein the second alkali metal cation is at least one of a sodium cation or a potassium cation.

1. Field of the invention

The present disclosure relates to microfluidic devices and methods of manufacturing microfluidic devices.

2. Background of the invention

Microfluidic devices have wide application in biomolecule analysis (e.g., nucleic acid sequencing, single molecule analysis, etc.) because they are capable of spatially and/or temporally controlling biological reactions, which is critical for many biomolecule analyses. For example, in parallel gene sequencing technologies based on optical detection, such as Next Generation Sequencing (NGS), millions of short DNA fragments generated from a genomic DNA sample can be immobilized and distributed onto the surface of a microfluidic device such that the DNA fragments are spatially separated from each other to facilitate sequencing by, for example, synthesis, ligation, or single molecule real-time imaging. Glass-based microfluidic devices employing cover glasses are commonly used for NGS or single molecule analysis based on optical detection.

However, difficulties still exist in the manufacture of microfluidic devices having thin cover glass structures. For example, cover glass can be brittle and can break during handling, assembly, packaging, shipping, or use. High resolution imaging tends to be extremely difficult with current microfluidic devices or flow cells that employ thick cover glasses (e.g., 230 μm-700 μm).

The present disclosure presents improved microfluidic devices with thin and enhanced cover glasses for biomolecule analysis, particularly gene sequencing, and methods of making the same.

SUMMARY

In some embodiments, a microfluidic device comprises: a flow channel provided in the glass base substrate; and a cover plate bonded to the glass-based substrate and at least partially covering the flow channel, wherein the cover plate has a thickness of at most 200 μm.

In one aspect combinable with any other aspect or embodiment, the microfluidic device further comprises: an inlet opening through at least one of the glass-based substrate or the cover plate and in fluid communication with the flow channel; an outlet opening through at least one of the glass-based substrate or the cover plate and in fluid communication with the flow channel.

In one aspect which may be combined with any other aspect or embodiment, the first glass-based layer defines a bottom of the flow channel; the second glass substrate layer defining sidewalls of the flow channel; the cover plate defines a top of the flow channel.

In one aspect combinable with any other aspect or embodiment, the cover plate has a thickness in a range of 100 μm to 180 μm.

In one aspect combinable with any other aspect or embodiment, the cover plate comprises: 56 to 72 mol% SiO₂(ii) a 5 to 22 mol% of Al₂O₃(ii) a 0 to 15 mol% of B₂O₃(ii) a 3 to 25 mol% Na₂O; 0 to 5 mol% of K₂O; 1 to 6 mol% of MgO; 0 to 1 mol% of SnO₂。

In one aspect combinable with any other aspect or embodiment, the cover plate further comprises: 0 to 7 mol% of Li₂O; and 0 to 10 mol% of P₂O₅。

In one aspect combinable with any other aspect or embodiment, the cover plate further comprises: 0 to 3 mol% CaO; and 0 to 2 mol% of ZrO₂。

In one aspect combinable with any other aspect or embodiment, the cover plate further comprises: 0 to 6 mol% ZnO.

In one aspect combinable with any other aspect or embodiment, the cover plate is configured to have an autofluorescence as low as that of a pure silica substrate in a wavelength range of 400nm to 750 nm.

In one aspect combinable with any other aspect or embodiment, the cover plate is configured to have an average surface inclination or slope of at most about 100nm/mm as measured with a laser interferometer.

In one aspect combinable with any other aspect or embodiment, the average surface flatness is at most about 50 nm/mm.

In one aspect combinable with any other aspect or embodiment, the cover plate is configured to have at most about 10nm/μm²The surface roughness of (2).

In one aspect combinable with any other aspect or embodiment, the surface roughness is at most about 5nm/μm²。

In one aspect combinable with any other aspect or embodiment, the cover plate is bonded to the glass base substrate with a bonding volume that includes a bonding material diffused into each of the glass base substrate and the cover plate.

In one aspect that may be combined with any of the other aspects or embodiments, the microfluidic device further includes a bonding layer disposed between the glass base substrate and the cover plate.

In one aspect combinable with any other aspect or embodiment, the bonding layer comprises a metal.

In one aspect that may be combined with any other aspect or embodiment, the metal includes one or more of gold, chromium, titanium, nickel, copper, zinc, cerium, lead, iron, vanadium, manganese, magnesium, germanium, aluminum, tantalum, niobium, tin, indium, cobalt, tungsten, ytterbium, zirconium, or oxides or combinations thereof.

In one aspect that may be combined with any of the other aspects or embodiments, the bonding layer includes a polymer-carbon black composite film.

In one aspect which may be combined with any of the other aspects or embodiments, the microfluidic device is a flow cell for DNA sequencing.

In one aspect combinable with any other aspect or embodiment, the surface of the bottom channel, the surface of the cover plate, or both, comprises an array of patterned nanostructures.

In some embodiments, the glass composition comprises: 56 to 72 mol% SiO₂(ii) a 5 to 22 mol% of Al₂O₃(ii) a 0 to 15 mol% of B₂O₃(ii) a 3 to 25 mol% Na₂O; 0 to 5 mol% of K₂O; 1 to 6 mol% of MgO; 0 to 1 mol% of SnO₂。

In one aspect combinable with any other aspect or embodiment, the glass composition further comprises: 0 to 7 mol% of Li₂O; and 0 to 10 mol% of P₂O₅。

In one aspect combinable with any other aspect or embodiment, the glass composition further comprises: 0 to 3 mol% CaO; and 0 to 2 mol% of ZrO₂。

In one aspect combinable with any other aspect or embodiment, the glass composition further comprises: 0 to 6 mol% ZnO.

In one aspect combinable with any other aspect or embodiment, the glass composition is configured to have a strength of at least 600 MPa.

In one aspect combinable with any other aspect or embodiment, the glass composition is configured to have a refractive index of at least 1.50.

In some embodiments, a method of strengthening a glass composition comprises: replacing a first alkali cation having a first size with a second alkali cation having a second size, wherein the second size is larger than the first size, and wherein the glass composition is configured to have a strength of 100MPa to 200MPa prior to the replacing and a strength of at least 600MPa after the replacing.

In one aspect that may be combined with any other aspect or embodiment, the first alkali metal cation is at least one of a lithium cation or a sodium cation, and wherein the second alkali metal cation is at least one of a sodium cation or a potassium cation.

Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the various embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiments and, together with the description, serve to explain the principles and operations of the various embodiments.

Brief Description of Drawings

Fig. 1A-1D depict a process flow for fabricating a microfluidic device according to some embodiments.

Figure 2 illustrates a schematic plan view of a two-channel microfluidic device according to some embodiments.

Fig. 3 shows a schematic cross-sectional view along the channel direction of a flow cell according to some embodiments.

Fig. 4 shows a schematic cross-sectional view along the channel direction of a single-sided patterned flow-through cell according to some embodiments.

Fig. 5 shows a schematic cross-sectional view along the channel direction of a double-sided patterned flow-through cell according to some embodiments.

FIG. 6 illustrates a box plot of minimum, maximum, mean, and standard deviation of the slope or slope of the cover glass surface according to some embodiments.

Fig. 7A and 7B illustrate autofluorescence of a strengthened thin cover glass substrate according to some embodiments.

FIG. 8 is 156mm according to some embodiments²Illumination of glass-based substrate sheetsA sheet having fourteen independent channels disposed in a glass-based substrate.

Fig. 9A-9C illustrate data for two channels disposed in a glass-based substrate as shown in fig. 8 using laser interferometer imaging, according to some embodiments. In particular, FIG. 9A shows a pseudo-color image, showing the depths of two channels; FIG. 9B is a scatter plot of the channel bottom surface slope or grade of channel A of FIG. 9A; fig. 9C is a box line graph showing the minimum, maximum, mean and standard deviation of the inclination or slope of the channel bottom surface.

FIG. 10 is 156mm as in FIG. 8 according to some embodiments²A photograph of a glass-based substrate sheet, each channel bottom surface having an array of patterned nanopores.

Fig. 11 illustrates a Scanning Electron Microscope (SEM) image of a patterned nanopore array located on one of the channel bottom surfaces of the glass-based substrate shown in fig. 10, according to some embodiments.

Detailed Description

The exemplary embodiments shown in the drawings are described in detail below. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the exemplary embodiments. It is to be understood that the application is not limited to the details or methodology set forth in the description or illustrated in the drawings. It is also to be understood that the terminology is for the purpose of description and should not be regarded as limiting.

Additionally, any examples set forth in this specification are intended to be illustrative, not limiting, and merely set forth some of the many possible embodiments for the claimed invention. Other suitable modifications and adaptations of various conditions and parameters commonly encountered in the art and apparent to those skilled in the art are within the spirit and scope of the present disclosure.

The term "surface roughness" refers to the surface texture as ISO 25178 "Geometric Product Specification (GPS) — surface texture: area "the measured Ra surface roughness, unless otherwise stated, was filtered at 25 μm. The surface roughness values reported herein were obtained using Atomic Force Microscopy (AFM).

The present disclosure provides methods of making and using glass-based microfluidic devices having thin strengthened cover glass structures for NGS or single molecule analysis based on optical detection.

Fig. 1A-1D depict a process flow 100 for fabricating a microfluidic device according to some embodiments.

In a first step, as shown in fig. 1A, a three-layer substrate is provided, which includes a core layer 102 interposed between a first cladding layer 104a and a second cladding layer 104 b. Core layer 102, first cladding layer 104a, and second cladding layer 104b independently comprise a glass-based material (e.g., a glass material, a glass-ceramic material, a ceramic material, or a combination thereof). In some embodiments, the core layer 102 comprises a glass composition that is different from the glass composition of the first cladding layer 104a and the second cladding layer 104 b. First cladding layer 104a and second cladding layer 104b may be formed from a first cladding glass composition and a second cladding glass composition, respectively. In some embodiments, the first cladding glass composition and the second cladding glass composition may be the same material. In other embodiments, the first cladding glass composition and the second cladding glass composition may be different materials.

Fig. 1A shows a core layer 102 having a first surface 102a and a second surface 102b opposite the first surface 102 a. The first cladding layer 104a is fused directly to the first surface 102a of the core layer 102 and the second cladding layer 104b is fused directly to the second surface 102b of the core layer 102. The glass cladding layers 104a and 104b may be fused to the core layer 102 without any additional materials, such as adhesives, polymer layers, coatings, etc., disposed between the core layer 102 and the cladding layers 104a and 104 b. Thus, in this case, the first surface 102a of the core layer 102 is directly adjacent to the first cladding layer 104a and the second surface 102b of the core layer 102 is directly adjacent to the second cladding layer 104 b. In some embodiments, the core layer 102 and the glass cladding layers 104a and 104b are formed by a fusion lamination process (e.g., a fusion draw process). A diffusion layer (not shown) may be formed between the core layer 102 and the cladding layer 104a, or between the core layer 102 and the cladding layer 104b, or both.

The first cladding layer and the second cladding layer may be formed from a composition comprising: silicon dioxide (SiO) in a concentration of 45 to 60 mol%₂) Alumina (Al) in a concentration of 8 to 19 mol%₂O₃) Boron trioxide (B) at a concentration of 5 to 23 mol%₂O₃) And sodium oxide (Na) at a concentration of 3 to 21 mol%₂O). The cladding layer may be substantially free of arsenic (As) and cadmium (Cd) such that the degradation rate of the cladding layer is at least ten times greater than the degradation rate of the core layer when a high concentration of acid (e.g., 10% hydrofluoric acid, HF) is used As an etchant, or at least twenty times greater than the degradation rate of the core layer when a low concentration of acid (e.g., 1% or 0.1% HF solution) is used As an etchant.

The core layer may be formed from an alkaline earth boroaluminosilicate glass (e.g., corning Eagle)) "Kangning" medicine for curing diabetesGlass and corning Iris^TMGlass or kang ningAt least one of the glasses. For example, the core layer may be formed of a glass having the following composition: 79.3 wt.% SiO₂1.6% by weight of Na₂O, 3.3 wt.% K₂O, 0.9 wt.% KNO₃4.2% by weight of Al₂O₃1.0% by weight of ZnO, 0.0012% by weight of Au, 0.115% by weight of Ag, 0.015% by weight of CeO₂0.4% by weight of Sb₂O₃And 9.4 wt% Li₂And O. In some embodiments, the core layer comprises corning EagleGlass or corning Iris^TMAt least one of the glasses because they have ultra-low autofluorescence.

Fig. 1B illustrates a coating and patterning process whereby a glass-glass bonding material 106 (e.g., a bonding layer) is deposited on the surface of the first cladding layer 104 a. For example, the glass-glass bonding material 106 includes 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., Al₂O₃、ZnO₂、Ta₂O₅、Nb₂O₅、SnO₂MgO, Indium Tin Oxide (ITO), CeO₂、CoO、Co₃O₄、Cr₂O₃、Fe₂O₃、Fe₃O₄、In₂O₃、Mn₂O₃、NiO、a-TiO₂(anatase), r-TiO₂(rutile), WO₃、Y₂O₃、ZrO₂) An adhesive (e.g., uv curable), an adhesive tape (e.g., double-sided pressure tape, double-sided polyimide tape), or a polymer-carbon black composite film (e.g., polyimide-carbon black film).

The bonding material 106 of the composite structure of FIG. 1B may be formed using at least one of the following methods: spin coating, dip coating, Chemical Vapor Deposition (CVD) (e.g., plasma assisted, Atomic Layer Deposition (ALD), Vapor Phase Epitaxy (VPE), etc.), Physical Vapor Deposition (PVD) (e.g., sputtering, evaporation, electron beam, etc.), laser assisted deposition, and the like.

Fig. 1C illustrates a wet chemical etching process in which, after patterning the glass-glass bonding material 106 (as shown in fig. 1B), the triple-layer glass substrate is selectively chemically etched to remove a portion of the first cladding layer 104a and the second cladding layer 104B not protected by the patterned glass-glass bonding material 106 until the glass core layer 102 is exposed and its surface becomes one surface of the microfluidic channel (e.g., for immobilizing biomolecules). In a wet chemical etching process, the patterned glass-glass bonding material 106 is used as an etch mask to prevent the masked regions of the first cladding layer 104a from contacting the etchant. The etching rate of the first cladding layer 104a and the second cladding layer 104b in the etchant may be higher than that of the glass core layer 102, so that the glass core layer 102 functions as an etch stop layer to control the depth of the microfluidic channel. In some embodiments, a polymer layer is deposited on the glass-glass bonding material 106 prior to the wet chemical etching process.

Alternatively, an etch-resistant polymer sheet layer may be formed on the etchant-contacting surface of the second cladding layer 104b and/or on the region of the first cladding layer 104a containing the patterned glass-glass bonding material 106 prior to etching, such that after etching, the second cladding layer 104b remains intact, while the exposed region of the first cladding layer 104a is removed to form the channel.

Patterning of the glass-glass bonding material 106 may be performed using additive or subtractive patterning techniques (e.g., ink printing, tape bonding, vapor deposition, plasma etching, wet etching, etc.).

The wet etch chemistry includes suitable components that can degrade or dissolve the glass article. Suitable wet etch chemistries include acids (e.g., HCl, HNO)₃、H₂SO₄、H₃PO₄、H₃BO₃、HBr、HClO₄HF, acetic acid), bases (e.g., LiOH, NaOH, KOH, RbOH, CsOH, Ca (OH)₂、Sr(OH)₂、Ba(OH)₂) Or a combination thereof.

Fig. 1D shows the final assembly of the microfluidic device after applying a glass cover plate 108 (having a first surface 108a and a second surface 108b) over the glass-glass bonding material 106. The glass cover plate 108 includes a glass-based material (e.g., a glass material, a glass-ceramic material, a ceramic material, or a combination thereof).

In some examples, the thickness of the cover plate is at most 200 μm. In some examples, the thickness of the cover plate is in the range of 10 μm to 200 μm, or in the range of 50 μm to 200 μm, or in the range of 75 μm to 200 μm, or in the range of 100 μm to 180 μm, or in the range of 125 μm to 160 μm, or in the range of 150 μm to 175 μm.

In some examples, the composition of the cover sheet includes: silica (SiO) at a concentration in the range of 56 to 72 mole%₂) (ii) a The concentration is 5 molAlumina (Al) in the range of mol% to 22 mol%₂O₃) (ii) a Boron trioxide (B) at a concentration in the range of 0 to 15 mol%₂O₃) (ii) a Sodium oxide (Na) at a concentration in the range of 3 to 25 mole%₂O); potassium oxide (K) at a concentration in the range of 0 to 5 mol%₂O); magnesium oxide (MgO) at a concentration in the range of 1 mol% to 6 mol%; and tin oxide (SnO) in a concentration ranging from 0 to 1 mol%₂)。

In some examples, the cover plate may further include lithium oxide (Li) at a concentration in a range of 0 mol% to 7 mol%₂O) and phosphorus pentoxide (P) in a concentration in the range of 0 to 10 mol%₂O₅). In some examples, the cover plate may further include calcium oxide (CaO) at a concentration ranging from 0 mol% to 3 mol% and zirconium dioxide (ZrO) at a concentration ranging from 0 mol% to 2 mol%₂). In some examples, the cover plate may further include zinc oxide (ZnO) at a concentration in a range of 0 mol% to 6 mol%.

In some embodiments, glass cover plate 108 is bonded to first cladding layer 104a using a laser assisted radiation bonding process using glass-glass bonding material 106. Without being bound by any particular theory or method, it is believed that the bonding of the glass-glass bonding material 106 to the first cladding layer 104a and the glass cover plate 108, respectively, is a result of the partial diffusion of the glass-glass bonding material 106 into the first cladding layer 104a and the glass cover plate 108, such that the portions of the first cladding layer 104a and the glass cover plate 108 that each include the diffused glass-glass bonding material 106 are bonded volume layers (not shown). As oriented, the glass-glass bonding material 106 may be opaque to the wavelength of the laser radiation, while the first cladding layer 104a and the glass cover plate 108 may be transparent to the wavelength of the laser radiation. In such embodiments, the laser radiation may pass through the glass cover plate 108 and/or the tri-layer substrate and be absorbed by the glass-glass bonding material 106. In some embodiments, the glass-glass bonding material 106 diffuses into the first cladding layer 104a and the glass cover plate 108, respectively, such that the bonded stack is transparent to the wavelength of the laser radiation.

In some embodiments, the bonding of glass-glass bonding material 106 to first cladding layer 104a and glass cover plate 108, respectively, is accomplished using a laser having a wavelength such that at least one substrate (e.g., first cladding layer 104a and/or glass cover plate 108) is transparent to the wavelength. The interface between the layers causes a change in the propagation index or optical transmission, which results in absorption and localized heating of the laser energy at the interface, thereby forming a bond.

In some embodiments, where the glass-glass bonding material 106 is Cr/CrON, the Cr component may act as a heat sink layer that is opaque or blocks the laser wavelength and has an affinity to diffuse into the first cladding layer 104a and/or the glass cover plate 108. In alternative embodiments, other materials with appropriate wavelength absorption and diffusion affinity properties may be used as the heat absorbing layer. The thickness of the heat sink layer may be as thick as desired to compensate for surface roughness or to control timing and temperature of the process.

Additionally and/or alternatively, bonding of the glass-glass bonding material 106 to the first cladding layer 104a and the glass cover plate 108 throughout the bonded build-up layer may include melting at least one of the glass-glass bonding material 106, the first cladding layer 104a, and/or the glass cover plate 108 (e.g., localized melting at laser radiation absorption sites). Further, the bonding may also include fusing the glass-glass bonding material 106 to at least one of the first cladding layer 104a or the glass cover plate 108. In some embodiments, the bonding volume layer is transparent to the wavelength of the laser radiation.

In some embodiments, the bonding may be achieved by separate laser radiation (not shown), as described in U.S. patent nos. 9,492,990, 9,515,286, and/or 9,120,287, the entire contents of which are incorporated herein by reference.

In other words, after the glass cover plate 108 is placed over the etched structure of fig. 1C (as described above) and in intimate contact with the glass-glass bonding material 106, the combination is exposed to radiation (e.g., laser treatment) to bond each of the first cladding layer 104a and the glass cover plate 108, respectively, to the glass-glass bonding material 106 through the bonded laminate layer. Creating the structure of fig. 1D may include positioning the cover substrate 108 on the glass-glass bonding material 106 and irradiating the bonding material 106 with electromagnetic radiation sufficient to diffuse at least a portion of the bonding material 106 into the cover substrate 108 and the first cladding layer 104 a.

As a result, the second surface 108b of the glass cover plate 108 faces and is directly opposite the first surface 102a of the core layer 102, the second surface 108b being a top surface of the microfluidic channel 112 and the first surface 102a being a bottom surface of the microfluidic channel 112. The top surface 108b and the bottom surface 102a of the channel 112 may be highly parallel due to the precise bonding and ultra-flatness of the channel surfaces. A fluid (e.g., a DNA sample) is controllably introduced into and removed from the glass cover plate 108 through a hole 110 (e.g., a through hole) extending from the first surface 108a to the second surface 108 b. Microfluidic channel 112 provides a flow path (dashed line) for fluids passing through the microfluidic device. For example, when used for DNA sequencing, microfluidic channel 112 provides a flow path for a DNA sample such that DNA fragments can be immobilized and distributed on top surface 108b and/or bottom surface 102a of channel 112 to facilitate sequencing. The top surface 108b and/or the bottom surface 102a of the channel 112 may be treated, e.g., chemically functionalized or physically structured (e.g., with an array of nanopores), to help perform a desired function (e.g., capture desired fragments).

In some embodiments, although the substrate is described as a three-layer substrate (see fig. 1A), a two-layer substrate including a glass core layer and a cladding layer as described above is also contemplated. In this case, the wet chemical etching process of fig. 1C will result in a portion of the cladding layer not protected by the patterned glass-glass bonding material being removed until the glass core layer is exposed. Thus, in this case, the glass-glass bonding material is patterned on top of the cladding.

Fig. 2 is a schematic plan view of a two-channel microfluidic device 200 that includes a thin, strengthened, and substantially flat cover glass and is manufactured by the methods disclosed herein, according to some embodiments. In this example, the microfluidic device 200 includes a microfluidic channel 202 as a flow path for the test sample, which connects an inlet 204 and an outlet 206, respectively, for controlled entry and exit of the test sample. In other words, each of the inlet 204 and the outlet 206 is in fluid communication with the microfluidic channel 202. As described above, the microfluidic channel 202 has a bottom surface that is a surface of the core layer 102, a top surface that is a surface of the glass cover plate 108, and the first cladding layer 104a is at least a portion of a sidewall of the microfluidic channel 202.

Microfluidic channels, inlets and outlets may be formed on a glass cover plate or a base substrate. In some examples, the inlet and outlet are formed on a base substrate made of glass, glass ceramic, silicon, pure silicon dioxide, or other substrate.

The top and bottom surfaces of each channel 202 may be used to immobilize biomolecules. Each individual channel may be separated by a bonding region 208, where in the bonding region 208 the first cladding layer 104a and the glass cover plate 108 are bonded to the glass-glass bonding material 106 as described above. In other words, the bonding region 208 depicts a region where a hermetic seal is formed via the bonding layer. In some examples, the bonding layer may be formed by: patterning is first performed on the base substrate and then protected with a photoresist or etch resistant polymer tape. After the chemical etching, the photoresist protectant or polymer strip is removed to expose the bonding layer. In some examples, a laser assisted radiation bonding process may also be utilized to bond the glass cover plate to the bottom substrate.

In some examples, the cover plate has an average surface flatness of at most about 100nm/mm, measured longitudinally at a central portion of the flow channel. In some examples, the average surface flatness of the cover plate is in the range of 10nm/mm to 90nm/mm, or in the range of 20nm/mm to 80nm/mm, or in the range of 40nm/mm to 60nm/mm, measured in the longitudinal direction at the central portion of the flow channel. In some examples, the average surface flatness of the cover plate is at most about 75nm/mm, or at most about 50nm/mm, or at most about 25nm/mm, measured longitudinally at the central portion of the flow channel.

Surface flatness can be measured using a laser interferometer (e.g., Zygo New View 3000, Zygo Z-mapper, Tropel FlatMaster) that can measure shape and tilt differences between the sample surface and the interferometer reference surface. For the etched channel, the flatness of the microfluidic channel bottom surface is measured relative to the top surface of the glass-glass bonding material 106, or when the coupon is placed against the reference substrate, the flatness of the microfluidic channel bottom surface is measured relative to the top surface of the reference substrate. For a bonded microfluidic device or flow cell, the flatness of the bottom surface of the microfluidic channel is measured relative to the surface of the reference substrate, thereby placing the device or flow cell on top of the reference substrate.

In some examples, the cover plate has a surface roughness of at most about 10nm/μm². In some examples, the surface roughness of the cover plate is at 1nm/μm²To 9 nm/. mu.m²In the range of 2 nm/. mu.m, or²To 8 nm/. mu.m²In the range of 3 nm/. mu.m, or²To 7 nm/. mu.m²Within the range of (1). In some examples, the surface roughness of the cover plate is at most about 7.5nm/μm²Or up to about 5 nm/. mu.m²At most about 2.5 nm/. mu.m². Surface roughness can be measured using an Atomic Force Microscope (AFM), which uses force between a probe (e.g., a pyramidal tip) and a sample to measure surface topography, including surface roughness.

In some embodiments, a microfluidic device may comprise a thin, strengthened, and substantially flat cover glass, the bottom substrate of which is triple glazing, comprising a core layer sandwiched between two cladding layers and having pre-etched channels, the channel bottom surfaces of which are also substantially flat. Since the core layer has a different composition than the cladding layer and has a much lower etch rate for the etchant, the core layer can act as an etch stop layer, resulting in a substantially planar channel bottom surface. In some examples, the flatness in the central region of the bottom surface of the channel is less than 100nm/mm, or less than 75nm/mm, or less than 50nm/mm, or less than 25 nm/mm.

For example, fig. 3 shows a schematic cross-sectional view along the channel direction of a flow-through cell according to some embodiments. Specifically, the unpatterned microfluidic device 300 includes a thin, strengthened, and substantially planar cover glass 310 and a three-layer bottom glass substrate 320 having a core layer 330 sandwiched between two cladding layers 340. The base substrate 320 includes pre-etched channels 380 on the side facing the cover glass 310, the end faces of the channel side walls being formed with a bonding layer 350. In some examples, the bonding layer 350 may be a metal or polymer-carbon black composite film or an adhesive or tape. The bottom substrate 320 also includes an inlet 360 and an outlet 370. The inlet is connected to an external solution for introducing the solution into the microfluidic channel 380, while the outlet is connected to an external waste container for discharging the solution from the microfluidic channel 380.

In some examples, a surface of the bottom channel, a surface of the cover plate, or both, includes an array of patterned nanostructures.

Fig. 4 shows a schematic cross-sectional view along the channel direction of a single-sided patterned flow-through cell according to some embodiments. In particular, the single-sided patterned microfluidic device 400 includes a thin, strengthened, and substantially flat cover glass 410. The components 410 and 480 in fig. 4 are similar to the components 310 and 380 described above with respect to fig. 3. The bottom glass substrate 420 includes pre-etched channels 480 whose channel bottom surfaces can be modified by chemical or physical means to form the nanopatterned features 490. In some examples, the nanopatterned features may be deposited chemical moieties (chemical entities). In some examples, the nanopatterned features may be a predetermined surface roughness. In some examples, the nanopatterned features (e.g., nanopores) may be formed using a lithographic technique (e.g., photolithography, nanoimprint, nanosphere etching, etc.) that enables nanopatterning inside the pre-etched deep channels.

Fig. 5 shows a schematic cross-sectional view along the channel direction of a double patterned flow cell according to some embodiments. In particular, the double-sided patterned microfluidic device 500 includes a thin, strengthened, and substantially flat cover glass 510. The components 510 and 580 in fig. 5 are similar to the components 310 and 380 described above with respect to fig. 3. The bottom glass substrate 520 includes pre-etched channels 580 whose channel bottom surfaces can be modified by chemical or physical means to form the nanopatterned features 590. In addition, the cover glass 510, whose bottom surface may serve as the top of the channel 580, may also be modified by similar means (as described above with respect to fig. 4) to form the nanopatterned features 595.

The use of strengthened glass allows patterning to be performed directly on thin flat glass using a variety of etching techniques, including photolithography, nanoimprint, and nanosphere etching. Nanopatterning is typically only available for thick glass substrates (e.g., 0.7 mm and 0.5 mm). For thin glasses (e.g., 0.3 mm, particularly about 0.15 mm) that are highly fragile and extremely difficult to handle without damage, a support is typically required, which increases the cost and complexity of the nanopatterning process.

May ultimately be cut by laser (e.g., CO) in an ablation process₂IRIS laser) to produce a single microfluidic device. In some examples, the microfluidic device is a flow cell for DNA sequencing.

Examples

The embodiments described herein will be further illustrated with reference to the following examples.

Example 1 glass composition

The glass compositions of table 1 may be used as thin, strengthened, and substantially flat cover glasses for the microfluidic devices disclosed herein.

TABLE 1

Series of	1 (mol%)	2 (mol%)	3 (mol%)
				SiO2	64-66	56-72	66-74
Al2O3	9.39-15	5-18	9-22
				B2O3	0-9.0	0-15	3-4.5
Li2O	0-5	0-7	---
				Na2O	6-15	3-25	9-20
K2O	0-4	0-5	0-5
				MgO	1-3	1-6	1-6
CaO	0-3	---	0-2
				ZrO2	0-2	---	0-2
P2O5	0-5	0-10	---
				ZnO	---	---	0-6
SnO2	0-1	0-1	0-1

The glass series of table 1 can be made using a fusion draw process to achieve better scratch resistance, an important attribute of microfluidic devices for optical imaging of biomolecular interactions, compared to existing glasses such as soda lime glass or biophotonic glass (e.g., D263T or D236M). The glass series shown in table 1 can be strengthened using an ion exchange process, which results in a significant improvement in the strengthening properties of the glass, increasing the resistance to damage from, for example, sharp impacts or presses. The alkali and alkaline earth metal cations used as network modifiers may form non-bridging oxygens (i.e., oxygens bonded to only one silicon atom), thereby reducing the glass' resistance to damage from abrasion, scratching, and the like. In the ion exchange process, cations (e.g., monovalent alkali metal cations (e.g., Li, Na, etc.)) present in the glass series shown in table 1 are replaced with larger cations (e.g., larger monovalent alkali metal cations (e.g., Na, K, etc.)). This ion displacement results in the glass surface being in a compressive state and the core being in a compensatory tensile state, increasing the surface compressive stress from about 100MPa to over 600MPa, which gives the glass a higher resistance to damage.

By ion exchange, using 100% KNO₃The depth of layer (DOL) of the glasses described in Table 1 was in the range of 35 μm to 45 μm when the salt bath was heated. The depth of layer may be a measure of the glass compressive strength characteristic of chemically strengthened glass. It is the depth at which compressive stress can be introduced into the surface of the glass, defined as the distance from the physical surface to the point of zero stress inside the glass. The depth of layer can be controlled by the glass composition and the ion exchange protocol (e.g., time, temperature, and salt bath cycling). In some examples, the temperature of the molten salt bath is in a range of 380 ℃ to 450 ℃. In some examples, the immersion time is in a range of 2 hours to 16 hours.

The glasses made by the fusion draw process of fig. 1A-1D with the compositions shown in table 1 may have a strength of 100MPa to 200MPa prior to ion exchange. After ion exchange, the glass composition may have an enhanced strength in excess of 600 MPa. Further, the average surface flatness of the glass compositions in table 1 was in the range of 10nm/mm to 50nm/mm both before and after ion exchange was performed.

For example, FIG. 6 shows two 170 μm thick cover glass sheet samples (approximately 156mm area) according to Table 1²) By using a box plot of the minimum, maximum, mean and standard deviation of the slope or slope of the cover glass surfaceThe laser interferometer measures flatness. The slope calculated as a derivative (i.e., first order fit) of the flatness data indicates that the slope or slope of both samples is very small (less than 50nm/mm), indicating that the thin cover glass is very flat.

For each glass, the transmission of light having a wavelength in the range of 350nm to 2250nm is greater than 90% before and after ion exchange. Further, the cover glass prepared with the glass composition of Table 1 had a refractive index of 1.50 before ion exchange, but after ion exchangeAt the surface compression level after the sub-exchange, the refractive index is 1.51, and therefore has better imaging quality when used for optical imaging. The Coefficient of Thermal Expansion (CTE) of the glasses in Table 1 was 75X 10^-7From/° C to 82X 10^-7In the range/° c.

Example 2 autofluorescence characterization

In a confocal fluorescence scanner, autofluorescence measurements were performed with an excitation wavelength in the range of 450nm to 750nm using a glass cover plate having the composition shown in table 2. The scanner can image the entire surface of a typical slide (1 inch by 3 inches). In addition to the autofluorescence uniformity across the surface, the average autofluorescence level can also be calculated.

TABLE 2

Series of	A (mol%)	B (mol%)	C (mol%)
				SiO2	65.78	64.96	61.85
Al2O3	13.75	16.42	19.68
				B2O3	0	0	3.90
Na2O	13.67	14.77	12.91
				MgO	4.11	3.39	1.43
SnO2	0.46	0.40	0.22

Fig. 7A and 7B illustrate autofluorescence of a strengthened thin cover glass substrate compared to other glass slides, according to some embodiments. Specifically, fig. 7A and 7B show the autofluorescence of a glass cover plate having A, B and C-series composition, respectively, when an excitation wavelength of 550nm (measured emission wavelength of 570nm) and an excitation wavelength of 650nm (measured emission wavelength of 670nm) were used. The results show that the autofluorescence signal of each glass cover plate is similar or comparable to that of a pure silica substrate, but is much lower than that of other widely used biophotonic glasses, such as D263T and D263M (both available from Schott)). Pure silica is generally considered to be the substrate that exhibits the lowest autofluorescence. In some examples, the a-C series autofluorescence may be at most 100RFU, or at most 90RFU, or at most 80RFU, or at most 70RFU, or at most 60RFU, or at most 50 RFU.

Example 3 flatness of etched vias in triple-layer glass-based substrates

Resist material pair 156mm by inkjet printing²The three glass sheets of (a) were patterned and the back side of the sheet was protected with HF resistant polymer tape. After etching with a 10% HF solution at 35 c for about 70 minutes, the exposed top cladding layer was selectively etched away to form channels in the glass substrate, and then the tape was peeled off and subjected to ultrasonic treatment to remove the resist. The glass sheet has two clad layers with a thickness of 0.11mm and a core layer with a thickness of 0.8 mm. FIG. 8 shows a 156mm etch channel comprising fourteen etch channels²Photograph of three glass sheets, each channel having a length of 135mm, a width of 5mm and a depth of 110 μm. Then, useThe laser interferometer detects the depth and bottom surface flatness of channels fabricated on the glass substrate.

Fig. 9A to 9C show depth and bottom surface flatness data of two channels disposed in a glass base substrate as shown in fig. 8, which are imaged using a laser interferometer. In particular, FIG. 9A shows a pseudo-color image, showing the depths of two channels; FIG. 9B is a scatter plot of the channel bottom surface slope or grade of channel A of FIG. 9A; fig. 9C is a box line graph showing the minimum, maximum, mean and standard deviation of the inclination or slope of the channel bottom surface. The results in FIG. 9A show that channels A and B each have a relatively uniform depth of about 110 μm 2.5 μm, defined by the thickness of the cladding. FIGS. 9B and 9C show that the slope or slope of the channel bottom surface is small-below 50nm/mm, indicating that the channel bottom surface is flat.

Example 4 Nanopatterned channels

After characterizing depth and bottom surface flatness, 156mm of FIG. 8²The three glass sheets are patterned to form a patterned nanostructure array. First, the top surface of the sheet was protected with vinyl tape, leaving the channels open and unprotected. Next, the close-packed 600nm polystyrene was treated using Langmuir-Blodgett equipmentThe monolayer of olefinic beads was transferred to the sheet such that the bottom surface of the channel was coated with a monolayer of beads, and then the polymer tape was removed. Thereafter, the sheet with the monolayer of beads on the bottom surface of the channels was exposed to an oxygen plasma to reduce the bead size to about 260nm and to deposit 50nm of Al thereon using electron beam deposition₂O₃And (3) a layer. Finally, all beads were stripped using sonication in a water bath to form a nanopore array of well-defined size, geometry, and depth on the channel bottom surface.

FIG. 10 is 156mm as in FIG. 8 according to some embodiments²A photograph of a glass-based substrate sheet, each channel bottom surface having an array of patterned nanopores. When the sheet is illuminated with intense white light, an angled photograph can be obtained that reveals the interference pattern due to the long-range order of the nanopores on the bottom surface of the channel.

Fig. 11 shows a Scanning Electron Microscope (SEM) image of a patterned nanopore array located on one of the channel bottom surfaces of the glass-based substrate shown in fig. 10. The side wall of the nano-hole is made of Al₂O₃Made with the bottom being the core surface. The average circular diameter of the nanopores is about 256. + -.8 nm, and the average spacing between adjacent pores is about 608. + -.30 nm. Atomic Force Microscopy (AFM) data (not shown) showed that the depth of the nanopore was about 50nm, which is due to Al₂O₃And (4) determining deposition.

Combining the thin, strengthened and substantially flat cover glass structure (as described above), the ultra-flat channel bottom surface (formed from three glass substrates, as described above) and patterning (formed from nanosphere etching, as described above) can provide a high quality microfluidic device that can be used for high quality biomolecular analysis using an optical imaging system.

Thus, as described herein, glass compositions and methods of making glass-based microfluidic devices are provided to form microfluidic devices with thin, strengthened cover glass structures and low autofluorescence for NGS or single molecule analysis based on optical detection.

Since the thin, strengthened, and substantially flat cover glass has low autofluorescence, the device can: (1) detecting biomolecules on the surface of the channel with a high signal-to-noise ratio; (2) allows higher quality optical fluorescence imaging (e.g., with faster scanning and focusing speeds), thereby speeding up sequencing; (3) high dimensional stability is achieved, especially at elevated temperatures, thereby reducing damage events related to handling, processing, assembly, packaging, nanopatterning, shipping, and/or scratching. In addition, the microfluidic devices disclosed herein include a bottom glass substrate with etched channels having substantially flat channel bottom surfaces, allowing for rapid scanning and imaging of the top and bottom surfaces of the channels, increasing the sequencing throughput of such devices. Finally, the manufacturing process disclosed herein is scalable, flexible, and provides high yields. Wafer-level processing and assembly of microfluidic devices is possible.

As used herein, the terms "about," "approximately," "substantially," and similar terms are intended to have a broad meaning, consistent with the commonly accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains. Those skilled in the art who review this disclosure will appreciate that these terms are intended to facilitate description of certain features described and claimed herein, and not to limit the scope of such features to the precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the invention as recited in the claims appended hereto.

As used herein, "optional," "optionally," and the like, are intended to mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. The indefinite articles "a" or "an" and their corresponding definite articles "the" as used herein mean at least one, or one or more, unless otherwise indicated.

References herein to the position of elements (e.g., "top," "bottom," "upper," "lower," etc.) are merely used to describe the orientation of the various elements in the drawings. It should be noted that the orientation of the various components may differ according to other exemplary embodiments, and such variations are intended to be covered by the present disclosure.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. Various singular/plural permutations may be expressly set forth herein for the sake of clarity.

It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the claimed subject matter. Accordingly, the claimed subject matter is not limited, except as by the appended claims and their equivalents.