RU2793682C2 - Interposer with the first and the second adhesive layers - Google Patents

Abstract

FIELD: microjet devices.

SUBSTANCE: flow cell interposer comprised of a base layer having a first surface and a second surface opposite the first surface, the base layer comprising black polyethylene terephthalate (PET); the first adhesive layer located on the first surface of the base layer, and the first adhesive layer contains an acrylic adhesive; the second adhesive layer located on the second surface of the base layer, and the second adhesive layer contains an acrylic adhesive; and a plurality of microfluidic channels extending through each of the base layer, the first adhesive layer, and the second adhesive layer.

EFFECT: ensuring low autofluorescence, high peel and shear strength, and the ability to withstand exposure to corrosive chemicals and cyclic changes in pressure and temperature.

21 cl, 12 dwg, 5 tbl

Description

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority under U.S. Provisional Application No. 62/693,762, filed July 3, 2018, and Dutch Patent Application No. NL 2021377, filed July 23, 2018, the contents of which are incorporated herein by reference in their entirety.

PRIOR ART

Various biological or chemical research protocols involve conducting a large number of controlled reactions on local support surfaces or within predetermined reaction chambers. The desired reactions can then be observed or detected, and subsequent analysis can help determine or reveal the properties of the chemicals involved in the reaction. For example, in some multiplex assays, an unknown analyte having an identifiable label (eg, a fluorescent label) can be exposed to thousands of known probes under controlled conditions. Each known probe can be placed in the appropriate well of the microplate. Detection of chemical reactions occurring between known probes and an unknown analyte in wells can help determine or reveal the properties of the analyte. Other examples of such protocols include DNA sequencing methods such as sequencing by synthesis or parallel cycle sequencing. In parallel cycle sequencing, a dense array of DNA fragments (eg, template nucleic acids) is sequenced by repeated cycles of enzymatic manipulations. After each cycle, an image can be captured and then analyzed along with other images to determine the sequence of DNA fragments.

Advances in microfluidic technology have allowed the development of flow cells that can provide rapid gene sequencing or chemical analysis using nanoliter or even smaller sample volumes. Desirably, such microfluidic devices can withstand multiple cycles of high and low pressure, exposure to corrosive chemicals, fluctuations in temperature and humidity, and provide a high signal-to-noise ratio (SNR; from English: signal-to-noise ratio).

SUMMARY OF THE INVENTION

Some embodiments of the present invention presented in this publication relate primarily to microfluidic devices. An example of a microfluidic device is a flow cell. Some embodiments of the present invention presented in this publication relate primarily to microfluidic devices containing an interposer and, in particular, to a flow cell containing an interposer formed from black polyethylene terephthalate (PET; from English: polyethylene terephthalate) and a double-sided acrylic adhesive, which contains microfluidic channels passing through these layers. The interposer can be configured to have low autofluorescence, high peel and shear strength, and can withstand exposure to corrosive chemicals and pressure and temperature cycling.

In the first series of embodiments of the present invention, the interposer comprises a base layer having a first surface and a second surface opposite the first surface. The base layer contains black polyethylene terephthalate (PET). On the first surface of the base layer is the first adhesive layer. The first adhesive layer contains an acrylic adhesive. The second adhesive layer is located on the second surface of the base layer. The second adhesive layer contains an acrylic adhesive. A plurality of microfluidic channels extend through the base layer, the first adhesive layer and the second adhesive layer.

In some embodiments of the interposer, the total thickness of the base layer, the first adhesive layer, and the second adhesive layer is in the range of about 50 microns to about 200 microns.

In some interposer embodiments, the base layer has a thickness ranging from about 30 microns to about 100 microns, and each of the first adhesive layer and the second adhesive layer has a thickness ranging from about 10 microns to about 50 microns.

In some embodiments of the interposer, each of the first adhesive layer and the second adhesive layer has autofluorescence in response to an excitation wavelength of 532 nm, less than about 0.25 conventional units relative to the fluorescence standard at 532 nm.

In some embodiments of the interposer, each of the first adhesive layer and the second adhesive layer has autofluorescence in response to an excitation wavelength of 635 nm, less than about 0.15 arbitrary units relative to the fluorescence standard at 635 nm.

In some interposer embodiments, the base layer contains at least about 50% black PET. In some embodiments, the implementation of the base layer essentially consists of black PET.

In some embodiments of the interposer, each of the first adhesive layer and the second adhesive layer contains at least about 10% acrylic adhesive.

In some embodiments of the present invention, the flow cell comprises a first substrate, a second substrate, and any of the interposers described above.

In some flow cell embodiments, each of the first and second substrates comprises glass such that the bond between each of the first and second adhesive layers and the respective surfaces of the first and second substrates can withstand a shear stress greater than about 50 N/cm ² and a shear force under an angle of 180 degrees greater than about 1 N/cm.

In some flow cell embodiments, each of the first and second substrates comprises a resin layer that is less than 1 µm thick and has a surface associated with the respective first and second adhesive layers such that the connection between each of the resin layers and the respective first and second adhesive layers can withstand a shear stress greater than about 50 N/cm ² and a 180 degree shear force greater than about 1 N/cm.

In some flow cell embodiments, a plurality of wells are embossed in the resin layer of at least one of the first substrate or the second substrate. A biological probe is placed in each well, and the microfluidic channels of the interposer are configured to deliver fluid to a plurality of wells.

In another series of embodiments of the present invention, the interposer comprises a base layer having a first surface and a second surface opposite the first surface. On the first surface of the base layer is the first adhesive layer. The first detachable patch is located on the first adhesive layer. The second adhesive layer is located on the second surface of the base layer. The second detachable patch is located on the second adhesive layer. The plurality of microjet channels extend through the base layer, the first adhesive layer, the second adhesive layer, and the second release patch, but not through the first release patch.

In some embodiments of the interposer, the first release patch has a thickness ranging from about 50 µm to about 300 µm, and the second release patch has a thickness ranging from about 25 µm to about 50 µm.

In some embodiments of the interposer, the base layer contains black polyethylene terephthalate (PET) and each of the first and second adhesive layers contains an acrylic adhesive.

In some embodiments of the interposer, the first release patch is at least substantially optically opaque and the second release patch is at least substantially optically clear.

The interposers and flow cells described above and below in this publication can be implemented in any combination to provide the advantages described later in this publication.

In another series of embodiments of the present invention, a method for forming microfluidic channels includes obtaining an interposer containing a base layer having a first surface and a second surface opposite the first surface. The base layer contains black polyethylene terephthalate (PET). The first adhesive layer is located on the first surface of the base layer, the first adhesive layer contains an acrylic adhesive, and the second adhesive layer is located on the second surface of the base layer, the second adhesive layer contains an acrylic adhesive. Microjet channels are formed that pass at least through the base layer, the first adhesive layer and the second adhesive layer.

In some embodiments, the implementation of the method of formation of microfluidic channels includes the use of CO ₂ laser.

In some embodiments, the interposer further comprises a first release patch located on the first adhesive layer and a second release patch located on the second adhesive layer.

In some embodiments, during the microfluidic channeling step, the microfluidic channels are formed using a CO ₂ laser to pass through the second release patch, but not formed in the first release patch.

In some embodiments of the method, the CO ₂ laser has a wavelength ranging from about 5000 nm to about 15000 nm and a beam size ranging from about 50 µm to about 150 µm.

The methods described above and below in this publication can be carried out in any combination to provide the benefits described later in this publication.

All of the embodiments described above, including those of interposers, flow cells, and methods, can be combined in any configuration to provide the benefits described later in this publication. In addition, the embodiments described above and additional embodiments discussed in more detail below (provided that these ideas are not mutually incompatible) should be considered as parts of the subject matter disclosed in this publication, and they can be combined in any configuration.

Although this publication contains many specific details of embodiments of the present invention, they should not be considered as limiting the scope of the present invention or the claims, but as descriptions of features specific to particular embodiments of a particular invention. Some of the features described in this publication in the context of individual embodiments of the present invention can also be implemented in combination in one embodiment of the present invention. In contrast, the various features described in the context of one embodiment of the present invention may also be implemented in a plurality of embodiments individually or in any suitable subcombination. In addition, although the features may be described above as operating in certain combinations, or even be claimed as such, one or more features from the claimed combination may in some cases be excluded from the combination, and the claimed combination may refer to a subcombination or variation of the subcombination.

BRIEF DESCRIPTION OF GRAPHICS

The above and other features of the present invention will become more apparent from the following description and claims of the present invention in conjunction with the accompanying drawings. Given that these drawings depict only a few embodiments of the present invention as described herein, and therefore cannot be considered limiting, the present invention will be described more specifically and in detail using the accompanying drawings.

Fig. 1 is a schematic representation of an example of a flow cell according to an embodiment of the present invention.

Fig. 2 is a schematic representation of an example of an interposer for use in a flow cell according to an embodiment of the present invention.

Fig. 3 is a schematic representation of an example of a flow cell according to another embodiment of the present invention.

Fig. 4A is a top perspective view of an example of a sandwich assembly comprising a plurality of flow cells according to an embodiment of the present invention; Fig. 4B is a side cross-sectional view of a multi-layer assembly made of. Fig. 4A taken along the line A-A shown in FIG. 4.

Fig. 5 is a flow chart of an example method for manufacturing a flow cell interposer according to an embodiment of the present invention.

Fig. 6A is a schematic cross-sectional view of an example of a bonded and patterned flow cell, and FIG. 6B is a schematic cross-sectional view of an example of a bonded and unstructured flow cell used to test the performance of various base layers and adhesives.

Fig. 7 is a histogram of the red fluorescence intensity of various adhesives and flow cell materials.

Fig. 8 is a histogram of green fluorescence intensity of various adhesives and flow cell materials from FIG. 7.

Fig. 9A and 9B show schematic illustrations of an example bond shear test circuit and an example peel test circuit, respectively, for determining bond shear strength and peel strength of various adhesives disposed on a glass base layer.

Fig. 10 is an example of Fourier Transform Infrared (FTIR) of an acrylic adhesive and tape.

Fig. 11 is an example of a gas chromatographic (GC; from English: gas chromatography) spectrum of an acrylic adhesive and Black Kapton.

Fig. 12 is an example of a mass spectroscopic (MS; from English: mass spectroscopy) spectrum of a gaseous compound released from an acrylic adhesive, and a possible chemical structure of the released gaseous compounds.

Throughout the following description, reference is made to the accompanying drawings. In the drawings, like symbols typically designate like components unless the context dictates otherwise. The exemplary embodiments of the present invention described in the detailed description, drawings and claims are not to be considered as limiting the present invention. You can use other embodiments of the present invention and make other changes without deviating from the essence and scope of the subject matter of the present invention presented in this publication. It can be readily understood that the aspects of the present invention outlined in this publication and illustrated in the drawings can be assembled, replaced, combined and designed in a wide variety of different configurations, which are fully contemplated by and are part of the present invention.

INFORMATION CONFIRMING THE POSSIBILITY OF IMPLEMENTING THE INVENTION

This publication provides examples of microfluidic devices. The embodiments of the present invention described in this publication mainly relate to microfluidic devices containing an interposer, in particular to a flow cell that contains an interposer consisting of black polyethylene terephthalate (PET) and double-sided acrylic adhesive and containing microfluidic channels passing through it. through. The interposer is configured to provide relatively low autofluorescence, relatively high peel strength, and relatively high shear bond strength, and can withstand corrosive chemicals and pressure and temperature cycling.

Advances in microfluidic technology have allowed the development of flow cells that can provide rapid gene sequencing or chemical analysis using nanoliter or even smaller sample volumes. Such microfluidic devices must be able to withstand multiple cycles of high and low pressure, exposure to corrosive chemicals, fluctuations in temperature and humidity, and provide a high signal-to-noise ratio (SNR). For example, flow cells may contain different layers connected to each other with adhesives. It is desirable to structure the various layers so that they can be fabricated and connected to one another to form a flow cell in a high throughput manufacturing process. In addition, the various layers must be able to withstand temperature and pressure cycling, corrosive chemicals, and not increase noise too much.

Embodiments of the flow cells described in this publication that include an interposer containing an adhesive deposited on it on both sides and microfluidic channels passing through it provide advantages, including, for example: (1) the ability to form a plurality of flow cells on a single plate, which ensures high-performance production; (2) provide low autofluorescence, high shear strength, peel strength, and corrosion resistance that can be maintained for 300 or more thermal cycles at high pH, providing test results with a high signal-to-noise ratio; (3) enabling the production of flat optically readable microfluidic devices by using a flat interposer containing microfluidic channels passing through it; (4) enabling two resin-coated substrates to be bonded by an interposer with a double-sided adhesive coating; and (5) allowing adhesion of the microfluidic device having one or more opaque surfaces.

Fig. 1 is a schematic representation of a flow cell [100] according to an embodiment of the present invention. The flow cell [100] can be used for any suitable biological, biochemical or chemo-analytical application. For example, the flow cell [100] may contain microarrays for gene sequencing (e.g., DNA or RNA) or epigenetic studies, or they may be configured for high-throughput drug screening, DNA or RNA fingerprinting, proteomic analysis, chemical detection, any other suitable application or combination thereof.

The flow cell [100] contains the first substrate [110], the second substrate [120], and the interposer [130] located between the first substrate [110] and the second substrate [120]. The first and second substrates [110] and [120] may contain any suitable material, for example, silicon dioxide, glass, quartz, pyrex, fused silica, plastics (for example, polyethylene terephthalate (PET), high density polyethylene (HDPE); from English: high density polyethylene), low density polyethylene (LDPE; from English: low density polyethylene), polyvinyl chloride (PVC; from English: polyvinyl chloride), polypropylene (PP; from English: polypropylene), polyvinylidene fluoride (PVDF; from English. : polyvinylidene fluoride) etc.), polymers, TEFLON®, Kapton (i.e. polyimide), paper based materials (e.g. cellulose, cardboard, etc.), ceramic materials (e.g. silicon carbide, alumina , aluminum nitride, etc.), complementary metal oxide semiconductor (CMOS; complementary metal-oxide semiconductor) materials (eg, silicon, germanium, etc.) or any other suitable material. In some embodiments of the present invention, the first and/or second substrates [110] and [120] may be optically transparent. In other embodiments of the present invention, the first and/or second substrates [110] and [120] may be optically opaque. Although not shown in the diagram, the first and/or second substrates [110] and [120] may contain inlets or outlets for pumping fluid to and/or from the microfluidic channels [138] contained in the interposer [130 ]. When used in the context of the present invention, the term "microfluidic channel" means that at least one dimension of the fluidic channel (for example, length, width, height, radius or cross section) is less than 1000 microns.

In various embodiments of the present invention, a plurality of biological probes can be placed on the surface [111] of the first substrate [110] and/or on the surface [121] of the second substrate [120] located after the interposer [130]. Biological probes may be placed in any suitable matrix on the [111] and/or [121] surfaces and may include, for example, DNA probes, RNA probes, antibodies, antigens, enzymes, or cells. In some embodiments of the present invention, chemical or biochemical analytes can be placed on the [111] or [121] surfaces. Biological probes can be covalently bound or immobilized in a gel (eg, hydrogel) on the [111] and/or [121] surfaces of the first and second [110] and [120] substrates, respectively. Biological probes can be labeled with fluorescent molecules (for example, green fluorescent protein (GFP; from English: green fluorescent protein), eosin yellow, luminol, fluoresceins, red and orange fluorescent labels, rhodamine derivatives, metal complex compounds, or any other fluorescent molecules) , or they bind to target biological substances that are labeled with fluorescent molecules, so that optical fluorescence can be used to detect (i.e. determine the presence or absence) or determine (e.g., quantify) biological substances, for example - for DNA sequencing.

The interposer [130] comprises a base layer [132] having a first surface [133] facing the first substrate [110] and a second surface [135] opposite the first surface [133] and facing the second substrate [120]. The base layer [132] contains black polyethylene terephthalate (PET). In some embodiments of the present invention, the base layer [132] may contain at least about 50% black PET or at least 80% black PET, and the remainder is transparent PET or any other plastic or polymeric material. In other embodiments of the present invention, the base layer [132] may essentially consist of black PET. In other embodiments of the present invention, the base layer [132] may consist entirely of black PET. Black PET may have a low level of autofluorescence so that noise is reduced and high contrast is provided, allowing fluorescent imaging of the flow cell with a high signal-to-noise ratio (SNR).

The first adhesive layer [134] is located on the first surface [133] of the base layer [132]. The first adhesive layer [134] contains an acrylic adhesive (eg methacrylic or methacrylate adhesive). In addition, the second adhesive layer [136] is located on the second surface [135] of the base layer [132]. The second adhesive layer [136] also contains an acrylic adhesive (eg methacrylic or methacrylate adhesive). For example, each of the first adhesive layer [134] and the second adhesive layer [136] may contain at least about 10% acrylic adhesive, or at least about 50% acrylic adhesive, or at least about 80% acrylic adhesive. In some embodiments of the present invention, the first and second adhesive layers [134] and [136] may essentially consist of an acrylic adhesive. In some embodiments of the present invention, the first and second adhesive layers [134] and [136] may consist entirely of acrylic adhesive. In specific embodiments, the implementation of the present invention, the acrylic adhesive may contain an adhesive commercially available under the trade name MA-61A ™, which can be purchased from ADHESIVES RESEARCH®. The acrylic adhesive included in the first and second adhesive layers [134] and [136] may be a pressure sensitive adhesive so that it ensures that the base layer [132] of the interposer [130] bonds to the substrates [110] and [120] when applied appropriate pressure. In other embodiments of the present invention, the first and second adhesive layers [134] and [136] may have a composition that is activated by heat, ultraviolet (UV; from English: ultra violet) radiation, or other activating stimuli. In other embodiments of the present invention, the first adhesive layer [134] and the second adhesive layer [136] may contain butyl rubber.

In some embodiments of the present invention, each of the first and second adhesive layers [134] and [136] has an autofluorescence in response to excitation at a wavelength of 532 nm (for example, from a red excitation laser) of less than about 0.25 arbitrary units (au; from English: arbitrary units) relative to the fluorescence standard at an excitation wavelength of 532 nm. In addition, each of the first and second adhesive layers [134] and [136] has an autofluorescence in response to excitation at a wavelength of 635 nm (for example, from a green excitation laser) of less than about 0.15 conventional units relative to the fluorescence standard at an excitation wavelength of 635 nm. Accordingly, the first and second adhesive layers [134] and [136] also have low autofluorescence, so that the combination of the base layer [132] of black PET and the first and second adhesive layers [134] and [136] containing an acrylic adhesive contributes negligibly small contribution to the fluorescent signal generated at the sites of interaction with biological probes, and therefore provides a high signal-to-noise ratio (SNR).

A plurality of microfluidic channels [138] pass through each of the first adhesion layer {134], the base layer [132], and the second adhesion layer [136]. Microfluidic channels [138] can be formed using any suitable method, for example - laser cutting (for example, using an ultraviolet nanosecond pulsed laser, ultraviolet picosecond pulsed laser, ultraviolet femtosecond pulsed laser, CO ₂ laser or any other suitable laser), stamping, die-cutting stamping, water jet cutting, physical or chemical etching, or any other suitable method.

In some embodiments of the present invention, microfluidic channels [138] can be obtained by a method that does not significantly increase the autofluorescence of the first and second adhesive layers [134] and [136] and the base layer [132], but provides a suitable surface quality. For example, an ultraviolet nanosecond, femtosecond, or picosecond pulsed laser can provide fast cutting, smooth edges and corners, thereby providing the excellent surface quality that is desirable, but it can also modify the surface chemistry of acrylic adhesive layers [134] and [136] and /or base layer [132] of black PET, which can cause autofluorescence of these layers.

In contrast, the CO ₂ laser can provide a surface quality that, although in some cases may be considered worse than when using UV lasers, remains within the design parameters, but does not change the chemical properties of the surfaces of the adhesive layers [134] and [136] and/or the base layer [132], so that there is no significant increase in the autofluorescence of these layers. In particular embodiments of the present invention, a CO ₂ laser having a wavelength ranging from about 5000 nm to about 15000 nm (i.e., about 5000 nm, about 6000 nm, about 7000 nm, about 8000 nm, about 9000 nm, about 10,000 nm, about 11,000 nm, about 12,000 nm, about 13,000 nm, about 14,000 nm, or about 15,000 nm, including all ranges and values in between), and a beam size ranging from about 50 µm to about 150 µm (e.g., equal to about 50 µm, about 60 µm, about 70 µm, about 80 µm, about 90 µm, about 100 µm, about 110 µm, about 120 µm, about 130 µm, about 140 µm, or about 150 µm, including all ranges in between and values) can be used to form microfluidic channels [138] passing through the first adhesive layer [134], the base layer [132], and the second adhesive layer [136].

As shown in FIG. 1, the first adhesive layer [134] links the first surface [133] of the base layer [132] with the surface [111] of the first substrate [110]. In addition, the second adhesive layer [136] bonds the second surface [135] of the base layer [132] to the surface [121] of the second substrate [120]. In various embodiments of the present invention, the first and second substrates [110] and [120] may contain glass. The bond between each of the first and second adhesive layers [134] and [136] and the respective surfaces [111] and [121] of the first and second substrates [110] and [120] may be configured to withstand a shear stress greater than about 50 N/cm ² , and a 180° pull-off force greater than about 1 N/cm. In various embodiments of the present invention, the binder can withstand pressures in microjet channels [138] up to about 15 psi. inch (approximately 103500 Pa).

For example, the shear strength and peel strength of adhesive layers [134] and [136] may be a function of their chemical compositions and their thickness relative to the base layer [132]. The acrylic adhesive included in the first and second adhesive layers [134] and [136] provides strong adhesion to the first and second surfaces [133] and [135] of the base layer [132] and the surfaces [111] and [121] of the first and second substrates [110] and [120], respectively. In addition, to obtain strong adhesion between the substrates [110] and [120] and the base layer [132], the thickness of the adhesive layers [134] and [136] relative to the base layer [132] can be chosen such that it transfers most of the applied peel and /or shear stress applied to the substrates [110] and [120], to the base layer [132].

If the adhesive layers [134] and [136] are too thin, they may not provide sufficient peel and shear strength to withstand the many pressure cycles that the flow cell [100] may be subjected to due to fluid flow under pressure through microjet channels [138]. On the other hand, too thick adhesive layers [134] and [136] can lead to the formation of voids or bubbles in the adhesive layers [134] and [136], which will reduce the adhesive strength. In addition, a large proportion of detachment and shear stress can act on the adhesive layers [134] and [136] and not be transferred to the base layer [132]. This can lead to destruction of the flow cell due to rupture of the adhesive layers [134] and/or [136].

In various embodiments of the present invention, the base layer [132] may have a thickness ranging from about 25 µm to about 100 µm, and each of the first adhesive layer [134] and the second adhesive layer [136] may have a thickness ranging from 5 µm to 50 µm (eg, about 5 µm, about 10 µm, about 20 µm, about 30 µm, about 40 µm, or about 50 µm, including all ranges and values in between). Such structures can provide sufficient peel and shear strength, such as the ability to withstand shear stress in excess of about 50 N/cm ² and shear force in excess of about 1 N/cm, which is sufficient to withstand multiple pressure cycles, eg 100 pressure cycles, 200 pressure cycles, 300 pressure cycles or even more. In specific embodiments of the present invention, the total thickness of the base layer [132], the first adhesive layer [134], and the second adhesive layer [136] may range from about 50 µm to about 200 µm (for example, be equal to about 50 µm, about 100 µm, about 150 µm, or about 200 µm, including all ranges and values in between).

In various embodiments of the present invention, adhesion promoters can also be included in the first and second adhesive layers [134] and [136], and/or adhesion promoters can be applied in the form of coatings on the surfaces of the [111] and [121] substrates [110] and [ 120], for example - to enhance the adhesion between the adhesive layers [134] and [136] and the corresponding surfaces [111] and [121]. Suitable adhesion promoters may include, for example, silanes, titanates, isocyanates, any other adhesion promoters, or combinations thereof.

The first and second adhesive layers [134] and [136] can be designed to withstand multiple pressure cycles and low autofluorescence, as described above in this publication. During operation, the flow cell may also be subjected to temperature cycling (e.g., from about -80°C to about 100°C), high pH values (e.g., pH values up to about 11), vacuum, and corrosive reagents (e.g., formamide, buffer solutions and salts). In various embodiments of the present invention, the first and second adhesive layers [134] and [136] can be configured to withstand temperature cycling in the range from about -80°C to about 100°C, to be resistant to voiding even in a vacuum, and to be stable to corrosion when exposed to pH up to about 11 or corrosive reagents such as formamide.

Fig. 2 is a schematic representation of an interposer [230] according to an embodiment of the present invention. The interposer can be used in the flow cell [100] or in any other flow cell described in this publication. The interposer [230] contains a base layer [132], a first adhesive layer [134], and a second adhesive layer [136], which have been described in detail in relation to the interposer [130] included in the flow cell [100]. The first adhesive layer [134] is located on the first surface [133] of the base layer [132], and the second adhesive layer [136] is located on the second surface [135] of the base layer [132] opposite the first surface [133]. The base layer [132] may contain black PET, and each of the first and second adhesive layers [134] and [136] may contain an acrylic adhesive, as described above. In addition, the base layer [132] may have a thickness B ranging from about 30 µm to about 100 µm (i.e., about 30 µm, about 50 µm, about 70 µm, about 90 µm, or about 100 µm, including all intermediate ranges and values), and each of the first and second adhesive layers [134] and [136] may have a thickness A ranging from about 5 µm to about 50 µm (for example, equal to about 5 µm, about 10 µm, about 20 µm, about 30 µm, about 40 µm, or about 50 µm, including all ranges and values in between).

On the first adhesive layer [134] the first detachable patch [237] can be placed. In addition, a second detachable patch [239] can be placed on the second adhesive layer [136]. The first release patch [237] and the second release patch [239] may serve as protective layers for the first and second adhesive layers [134] and [136] and they may be selectively peelable or otherwise mechanically removed to expose the first and the second adhesive layers [134] and [136], for example, for bonding the base layer [132] with the first and second substrates [110] and [120], respectively.

The first and second release patches [237] and [239] can be made of paper (e.g. super calendared kraft paper, SCK paper coated with polyvinyl alcohol, kraft paper coated paper, machine-smooth kraft paper, single-side glazed paper, polyolefin-coated kraft paper, etc.), resin material (e.g. biaxially oriented PET film, biaxially oriented polypropylene film, polyolefins, high density polyethylene, low density polyethylene, polypropylene resins, etc.), fabrics (eg polyester), nylon, teflon, or any other suitable material. In some embodiments of the present invention, the [237] and [239] release pads may be made from a low surface energy material (e.g., any of the materials described in this publication) to facilitate separation of the [237] and [239] release films from their respective adhesives. layers [134] and [136]. In other embodiments of the present invention, a low surface energy material (e.g., silicone, wax, polyolefin, etc.) can be applied to at least that surface of the release films [237] and [239] that is placed on the respective adhesive layers. [134] and [136], to facilitate detachment of detachable films from them [237] and [239].

A plurality of microfluidic channels [238] pass through the base layer [132], the first adhesive layer [134], the second adhesive layer [136], and the second release patch [239], but not through the first release patch [237]. For example, the second detachable pad [239] could be the interposer's upper detachable pad [230], and the passage of microfluidic channels [238] through the second detachable pad [239] but not through the first detachable pad [237] could be an indicator of interposer orientation [230]. ] for the user, which facilitates fabrication of the flow cell by the user (eg, flow cell [100]). In addition, the manufacturing process of the flow cell (e.g. flow cell [100]) can be carried out so that first the second detachable patch [239] is torn off from the second adhesive layer [136] to bond to the substrate (e.g. the second substrate [220]) . The first release liner can then be removed and the first adhesive layer [134] bonded to another substrate (e.g. to substrate [110].

The first and second detachable pads [237] and [239] may have the same or different thickness. In some embodiments of the present invention, the first release patch [237] may be significantly thicker than the second release patch [239] (e.g., about 2 times thicker, about 4 times thicker, about 6 times thicker, about 8 times thicker or about 10 times thicker), for example, to provide structural rigidity to the interposer [230], and it can serve as an auxiliary layer to facilitate user handling of the interposer [230]. In specific embodiments of the present invention, the first detachable patch [237] may have a first thickness L1 ranging from about 50 µm to about 300 µm (e.g., about 50 µm, about 100 µm, about 150 µm, about 200 µm, about 250 µm or about 300 µm, including all ranges and values in between), and the second detachable patch [239] may have a second thickness L2 ranging from about 25 µm to about 50 µm (e.g., about 25 µm, about 30 µm , about 35 µm, about 40 µm, about 45 µm, or about 50 µm, including all ranges and values in between).

The first and second detachable patches [237] and [239] may be optically opaque, transparent or translucent, and they may be of any suitable color. In some embodiments of the present invention, the first detachable patch [237] may be at least substantially optically opaque (including completely opaque), and the second detachable patch [239] may be at least substantially optically transparent (including completely opaque). transparent). As described above in this publication, the second detachable patch [239] can be first removed from the second adhesive layer [136] to bond to the appropriate substrate (eg, to the second substrate [120]). Making the second release patch [239] optically transparent can make it easy to distinguish the second release patch [239] from the opaque first release patch [237]. In addition, a substantially optically opaque second detachable patch [239] can provide suitable contrast to facilitate optical alignment of the substrate (eg the second substrate [120]) with the microfluidic channels [238] located in the interposer [230]. In addition, having a second release patch [239] that is thinner than the first release patch [237] may allow the second release patch [239] to be preferentially detached over the first release patch [237], thereby preventing accidental separation of the first release patch [237]. release patch [237] during separation of the second release patch [239] from the second adhesive layer [136].

In some embodiments of the present invention, one or more flow cell substrates may comprise a plurality of wells disposed thereon, with each well containing a biological probe (eg, an array consisting of a single biological probe or different biological probes). In some embodiments of the present invention, a plurality of wells may be etched into one or more substrates. For example, the substrate (e.g., substrate [110] or [120]) may contain glass, and a plurality of wells are etched into the substrate using wet etching (e.g., hydrofluoric acid buffered etching) or dry etching (e.g., using reactive ion etching (RIE). ; from English: reactive ion etching) or deep RIE).

In other embodiments of the present invention, a plurality of wells may be formed in the resin layer located on the surface of the substrate. For example, Fig. 3 is a schematic representation of a flow cell [300] according to an embodiment of the present invention. The flow cell [300] includes an interposer [130] containing a base layer [132], a first adhesive layer [134], and a second adhesive layer [136] and having a plurality of microfluidic channels [138] passing through them, as described in detail earlier in this publication. .

The flow cell [300] also contains the first substrate [310] and the second substrate [320] with an interposer [132] located between them. The first and second substrates [310] and [320] can be made from any suitable material, e.g. silica, glassa, quartz, pyrex, plastics (e.g. polyethylene terephthalate (PET), high density polyethylene (HDPE), low density polyethylene). density (LDPE), polyvinyl chloride (PVC), polypropylene (PP), etc.), polymeric materials, TEFLON®, Kapton or any other suitable material. In some embodiments of the present invention, the first and/or second substrates [310] and [320] may be optically transparent. In other embodiments of the present invention, the first and/or second substrates [310] and [320] may be opaque. As shown in FIG. 3, the second substrate [320] (for example, the top substrate) comprises a fluid inlet [323] for communicating with the microfluidic channels [138] and a fluid outlet [325] for removing fluid from the microfluidic channels [138] . Although the figure shows one fluid inlet [323] and one fluid outlet [325], in various embodiments of the present invention, the second substrate [320] may have a plurality of fluid inlets and/or outlets. In addition, inlet and/or outlet channels for the fluid may also be provided in the first substrate [310] (for example, in the lower substrate). In specific embodiments of the present invention, the first substrate [310] may be significantly thicker than the second substrate [320]. For example, the first substrate [310] may have a thickness ranging from about 350 µm to about 500 µm (e.g., about 350 µm, about 400 µm, about 450 µm, or about 500 µm, including all ranges and values in between), and the second substrate [320] may have a thickness ranging from about 50 µm to about 200 µm (e.g., about 50 µm, about 100 µm, about 150 µm, or about 200 µm, including all ranges and values in between).

The first substrate [310] contains the first layer [312] of resin located on its surface [311] facing the interposer [130]. In addition, the second resin layer [322] is located on the surface [321] of the second substrate [320] facing the interposer [130]. The first and second layers [312] and [322] of the resin may contain, for example, polymethyl methacrylate (PMMA; from English: polymethyl methacrylate), polystyrene, glycerol-1,3-diglycerolate diacrylate (GDD; from English: glycerol 1,3 -diglycerolate diacrylate), Ingacure 907, rhodamine 6G tetrafluoroborate, UV curable resin (eg novolac epoxy, PAK-01, etc.), any other suitable resin, or a combination thereof. In specific embodiments, implementation of the present invention layers [312] and [322] resin may contain a resin for nanoimprint lithography (NIL; from English: nanoimprint lithography) (for example, PMMA).

In various embodiments of the present invention, the [312] and [322] resin layers may be less than 1 µm thick and are bonded to the respective first and second adhesive layers [134] and [136]. The first and second adhesive layers [134] and [136] have a composition such that the binder between each of the layers [312] and [322] of the resin and the respective first and second adhesive layers [134] and [136] can withstand a shear stress exceeding about 50 N/cm ² , and a tear force greater than about 1 N/cm. Accordingly, the adhesive layers [134] and [136] form a sufficiently strong direct connection with the corresponding substrates [310] and [320] or with the corresponding layers [312] and [322] of the resin located on them.

A plurality of wells [314] are formed in the first resin layer [312] by nanoimprint lithography (NIL). A plurality of wells [324] can also be formed in the second layer [322] of the resin by nanoimprint lithography (NIL). In other embodiments of the present invention, a plurality of holes [314] can be formed in the first layer [312] of the resin, in the second layer [322] of the resin, or in both layers. The plurality of wells may have a diameter or cross section of about 50 microns or less. A biological probe (not shown in the figure) can be placed in each well of the set of wells [314] and [324]. The biological probe may include, for example, DNA probes, RNA probes, antibodies, antigens, enzymes, or cells. In some embodiments of the present invention, chemical or biochemical analytes can additionally or alternatively be placed in a plurality of wells [314] and [324].

In some embodiments of the present invention, the first and/or second layers [312] and [322] of the resin may comprise a first region and a second region. The first region may comprise a first polymeric layer containing a first plurality of functional groups providing reactive sites for covalently linking a functionalized molecule (eg, a biological probe such as an oligonucleotide). The first and/or second [312] and [322] resin layers may also comprise a second region that includes a first polymer layer and a second polymer layer, the second polymer layer being on, immediately adjacent to, or adjacent to the first polymer layer. layer. The second polymeric layer may completely cover the underlying first polymeric layer and may optionally contain a second plurality of functional groups. It should also be understood that in some embodiments of the present invention, the second polymer layer of the polymer may cover only a portion of the first polymer layer. In some embodiments, the implementation of the present invention, the second polymer layer covers a significant part of the first polymer layer, and this significant part provides coverage of the first polymer layer by more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, or more than about 99 % or provides coverage between any two of the above values. In some embodiments, implementation of the present invention, the first and second polymeric layers do not contain silicon or silicon oxide.

In some embodiments of the present invention, the first region is structured. In some embodiments, implementation of the present invention, the first region may contain structures of micrometer or nanometer size. In some other embodiments of the present invention, the micrometer-sized or nanometer-sized structures in the first and/or second layers [312] and [322] of the resin are channels, grooves, rods, holes, or combinations thereof. For example, the structure may include a plurality of wells or other elements that form a matrix. High-density matrices are characterized in that they contain elements separated by a distance of less than about 15 microns. Medium density matrices contain elements separated by a distance ranging from about 15 microns to about 30 microns, while low density arrays have regions separated by a distance greater than about 30 microns. An array suitable for the present invention may, for example, have elements separated by a spacing that is less than about 100 µm, less than about 50 µm, less than about 10 µm, less than about 5 µm, or less than about 0.5 µm, or between any two of the above values.

In some embodiments of the present invention, each element present in the first and/or second layers [312] and [322] of the resin may have an area greater than about 100 nm ² greater than about 250 nm ² greater than about 500 nm ² greater than about 1 μm ² greater than about 2.5 μm ² , greater than about 5 μm ² , greater than about 10 μm ² , greater than about 100 μm ² , or greater than about 500 μm ² , or between any two of the above values. Alternatively or additionally, each of the elements may have an area of less than about 1 mm ² , less than about 500 μm ² , less than about 100 μm ² , less than about 25 μm ² , less than about 10 μm ² , less than about 5 μm ² , less than about 1 µm ² less than about 500 nm ² or less than about 100 nm ² or lying in the range between any two of the above values.

As shown in FIG. 3 the first and/or second layers [312] and [322] of the resin contain a plurality of holes [314] and [324], but they may also contain other elements or matrices that contain at least about 10 elements, about 100 elements, about 1 x 10 ³ elements, about 1 x 10 ⁴ elements, about 1 x 10 ⁵ elements, about 1 x 10 ⁶ elements, about 1 x 10 ⁷ elements, about 1 x 10 ⁸ elements, about 1 x 10 ⁹ elements or more , or the number of elements lies between any two of the above values. Alternatively or additionally, the first and/or second layers [312] and [322] of the resin may contain no more than about 1 x 10 ⁹ elements, no more than about 1 x 10 ⁸ elements, no more than about 1 x 10 ⁷ elements, no more than about 1 × 10 ⁶ elements, not more than about 1 × 10 ⁵ elements, not more than about 1 × 10 ⁴ elements, not more than about 1 × 10 ³ elements, not more than about 100 elements, not more than about 10 elements or less, or the number of elements is between any two of the above values. In some embodiments of the present invention, the average distance between the elements in the first and/or second layers [312] and [322] of the resin may be equal to, for example, at least about 10 nm, at least about 0.1 μm, at least about 0.5 µm, at least about 1 µm, at least about 5 µm, at least about 10 µm, at least about 100 µm or more, or it may be between any two of the above values. Alternatively or additionally, the average distance may be, for example, no more than about 100 µm, no more than about 10 µm, no more than about 5 µm, no more than about 1 µm, no more than about 0.5 µm, no more than about 0.1 µm, or less, or it may be in the range between any two of the above values.

In some embodiments of the present invention, the first region is hydrophilic. In some other embodiments of the present invention, the first region is hydrophobic. In specific cases, the first and second regions are opposite in terms of their hydrophobicity and hydrophilicity. In some embodiments of the present invention, the first plurality of functional groups of the first polymeric layer is selected from C _8-14 cycloalkenes, 8-14 membered heterocycloalkenes, C _8-14 cycloalkynes, 8-14 membered heterocycloalkynes, alkynyl groups, vinyl groups, halogen atoms, azido groups, amino groups, amido groups, epoxy groups, glycidyl groups, carboxyl groups, hydrazonyl groups, hydrazinyl groups, hydroxyl groups, tetrazolyl groups, tetrazinyl groups, nitrile oxide groups, nitrene groups, nitrone groups or thiol groups, or from optionally substituted variants and combinations of these groups. In some of such embodiments of the present invention, the first plurality of functional groups is selected from halogen atoms, azido groups, alkynyl groups, carboxyl groups, epoxy groups, glycidyl groups, norbornene groups, or amino groups, or from optionally substituted variants and combinations of these groups.

In some embodiments of the present invention, the first and/or second layers of [312] and [322] resin may contain a photocurable polymer composition containing a silsesquioxane backbone (also known as "POSS; from English: polyhedric oligomeric silsesquioxane"). An example of a POSS would be the POSS described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, the contents of which are incorporated herein by reference in their entirety. In some cases, a silane may be used to enhance adhesion between the [310] and [320] substrates and the corresponding [312] and [322] resin layers. The ratio of monomers in the final polymer (p:q:n:m) may depend on the stoichiometric ratio of monomers in the initial polymer composite mixture. The silane molecule contains an epoxy fragment, which can be covalently included in the first (lower) polymer layer in contact with [310] or [320] substrates. The second (top) polymeric layer included in the first and/or second layers of [312] and [322] resin can be deposited on the semi-cured first polymeric layer, which can provide sufficient adhesion without the use of silane. The first polymeric layer will naturally propagate polymerization to the monomeric elements of the second polymeric layer, covalently bonding them to each other.

The alkylene bromide groups in the walls of the [314] and [324] wells can act as attachment points for further spatially selective functionalization. For example, alkylene bromide groups can react with sodium azide to form an azide-coated well surface [314] and [324]. This azide-coated surface can then be used directly to capture alkyne-terminated oligomers, for example using copper-catalyzed click chemistry, or bicyclo[6.1.0]non-4-yne (BCN) terminated oligomers. 6.1.0]non-4-yne), using non-catalyst click chemistry. Alternatively, sodium azide can be replaced by a norbornene-functionalized amine or a similar ring-deformed alkene or alkyne, such as dibenzocyclooctyne-functionalized amine (DIBCO; from English: dibenzocyclooctyne), with the addition of a ring-deforming element to the polymer, which can subsequently participate in a catalyst-free click. ring-strain-stimulated reaction with tetrazine-functionalized oligomers to graft the primers to the surface.

The addition of glycidol to the second photocurable resin composition can provide a polymer surface with multiple hydroxyl groups. In other embodiments of the present invention, alkylene bromide groups can be used to provide a bromide-functionalized surface, which can then react with 5-norbornene-2-methanamine to form a norbornene-coated well surface. Then, an azide-containing polymer, for example, a copolymer of N-(5-azidoacetamidylpentyl)acrylamide and acrylamide (PAZAM; poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide), can be selectively attached to this norbornene-coated surface localized in wells [314] and [324], and then grafted with alkyne terminated oligomers. Ring-deformed alkynes such as BCN or DIBCO terminated oligomers can also be used in place of alkyne terminated oligomers in a catalyst free, voltage promoted cycloaddition reaction. In the case of an inert second polymer layer, covering intermediate regions of the support, PAZAM attachment and grafting are localized in the wells.314 and 324. Alternatively, tetrazine-terminated oligomers can be grafted directly to the polymer by reaction with a norbornene moiety, thus eliminating the PAZAM attachment step.

In some embodiments of the present invention, the first photocurable polymer included in the first and/or second layers [312] and [322] of the resin may contain an auxiliary substance. Various non-limiting examples of excipients that can be used in the photocurable resin composition included in the first and/or second layers of [312] and [322] resins include epibromohydrin, glycidol, glycidylpropargyl ether, methyl-5-norbornene-2, 3-dicarboxylic anhydride, 3-azido-1-propanol, tert-butyl-N-(2-oxiranylmethyl)carbamate, propiolic acid, 11-azido-3,6,9-trioxaundecan-1-amine, cis-epoxysuccinic acid, 5-nonbornene-2-methylamine, 4-(2-oxiranylmethyl)morpholine, glycidyltrimethylammonium chloride, fosfomycin disodium salt, polyglycidyl methacrylate, polypropylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, dimethylsiloxane copolymer and (2-(3,4-epoxycyclohexyl )ethyl)methylsiloxane, copolymer of propylmethacryl-heptaiisobutyl-PSS and hydroxyethyl methacrylate, copolymer of propylmethacryl-heptaiisobutyl-PSS and tert-butyl methacrylate, [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane, trans-cyclohexanediolisobutyl-POSS , aminopropylisobutyl-POSS, octatetramethylammonium-POSS, polyethylene glycol-POSS, octadimethylsilane-POSS, octaammonium-POSS, octamaleamic acid-POSS, trisnorbornenylisobutyl-POSS, pyrogenic silica, surfactants or combinations and derivatives thereof.

As for the interposer [130] of FIG. 3, the microfluidic channels [138] of the interposer are configured to deliver fluid to a plurality of wells [314] and [324]. For example, the interposer [130] can be connected to the substrates [310] and [320] so that the microfluidic channels [138] are matched with the corresponding wells [314] and [324]. In some embodiments of the present invention, microfluidic channels [138] may have a structure that provides the delivery of a fluid medium (for example, blood, plasma, plant extract, cell lysate, saliva, urine, etc.), chemically active substances, buffer solutions, solvents , fluorescent labels or any other solution to each of the many wells [314] and [324] in series or in parallel.

The flow cells described in this publication may be particularly well suited for mass production. For example, Fig. 4A is a top perspective view of a sandwich assembly [40] containing a plurality of flow cells [400]. Fig. 4B is a side cross-sectional view of the sandwich assembly [40] taken along the line A-A shown in FIG. 4A. The multilayer prefabricated structure [40] contains the first substrate plate [41], the second substrate plate [42], and an interposer [43] located between the first and second substrate plates [41], [42]. As shown in FIG. 4B, the sandwich assembly [40] contains a plurality of flow cells [400]. The interposer [43] contains a base layer [432] (for example, the base layer [132]), the first adhesive layer [434] (for example, the first adhesive layer [134]), connecting the base layer [432] with the surface of the first substrate plate [ 41], and a second adhesive layer [436] (for example, a second adhesive layer [136]) connecting the base layer [432] to the surface of the second substrate plate [42].

A plurality of microfluidic channels [438] pass through the base layer [432] and the first and second adhesion layers [434] and [436]. A plurality of dimples [414] and [424] can be formed on the first substrate plate [41] and the second substrate plate [42] (for example, etched on the substrate plates [41] and [42]), or formed in the resin layer, located on the surfaces of the substrate plates [41] and [42] facing the interposer [43]. The biological probe can be placed in any of the many wells [414] and [424]. A plurality of wells [414] and [424] are in fluid communication with the respective microfluidic channels [438] of the interposer [43]. The sandwich assembly can then be cut to separate the plurality of flow cells [400] from the sandwich assembly [40]. In various embodiments of the present invention, the multilayer assembly [40] can provide flow cell yields in excess of about 90%.

Fig. 5 is a flow chart of a method [500] for forming microfluidic channels in an interposer (e.g., interposer [130], [230]) of a flow cell (e.g., flow cell [100], [300], [400]) according to an embodiment of the present inventions. Method [500] includes obtaining an interposer [step 502]. An interposer (eg, interposer [130], [230]) contains a base layer (eg, base layer [132]) having a first surface and a second surface opposite the first surface. The base layer contains black polyethylene terephthalate (PET) (eg, contains at least about 50% black PET, consists essentially of black PET, or consists entirely of black PET). On the first surface of the base layer is the first adhesive layer (for example, the first adhesive layer [134]), and the second adhesive layer (for example, the second adhesive layer [136]) is located on the second surface of the base layer. The first and second adhesive layers contain an acrylic adhesive (for example, they may contain at least about 10% acrylic adhesive, contain at least about 50% acrylic adhesive, consist essentially of an acrylic adhesive, or consist entirely of an acrylic adhesive). In some embodiments, implementation of the present invention, the adhesive may contain butyl rubber. The base layer may have a thickness ranging from about 30 μm to about 100 μm, and each of the first and second adhesive layers may have a thickness ranging from about 10 μm to about 50 μm, such that the interposer (e.g., interposer [ 130]) may have a thickness ranging from about 50 µm to about 200 µm.

In step [504], microfluidic channels are formed through at least the base layer, the first adhesive layer, and the second adhesive layer. In some embodiments, implementation of the present invention at the stage of formation of microfluidic channels, microfluidic channels are formed using a CO ₂ laser. In some embodiments of the present invention, also using a CO ₂ laser, microfluidic channels are formed passing through the second detachable patch, but not through the first detachable patch (although in other embodiments of the present invention, the microfluidic channels may partially extend into the first detachable patch). The CO ₂ laser may have a wavelength ranging from about 5000 nm to about 15000 nm and a beam size ranging from about 50 µm to about 150 µm. For example, a CO ₂ laser may have a wavelength ranging from about 3000 nm to about 6000 nm, from about 4000 nm to about 10000 nm, from about 5000 nm to about 12000 nm, from about 6000 nm to about 14000 nm, from about 8000 nm to about 16000 nm, or from about 10000 nm to about 18000 nm. In particular embodiments of the present invention, the CO ₂ laser may have a wavelength of about 5000 nm, about 6000 nm, about 7000 nm, about 8000 nm, about 9000 nm, about 10000 nm, about 11000 nm, about 12000 nm, about 13000 nm, about 14,000 nm, or about 15,000 nm, including all ranges and values in between. In some embodiments of the present invention, the CO ₂ laser may have a beam size ranging from about 40 µm to about 60 µm, from about 60 µm to about 80 µm, from about 80 µm to about 100 µm, from about 100 µm to about 120 microns, from about 120 microns to about 140 microns, or from about 140 microns to about 160 microns, including all ranges and values in between. In particular embodiments, the CO ₂ laser may have a beam size of about 50 µm, about 60 µm, about 70 µm, about 80 µm, about 90 µm, about 100 µm, about 110 µm, about 120 µm, about 130 µm, approx. 140 µm, or approx. 150 µm, including all ranges and values in between

As described earlier in this publication, various lasers can be used to form microfluidic channels in an interposer. Important parameters include cutting speed, which determines the overall production time, edge smoothness, which is a function of beam size and laser wavelength, and the chemical changes caused by the laser in the various layers included in the interposer, which are a function of the type of laser. Pulsed UV lasers can provide a smaller beam size, so they produce smoother edges. However, UV lasers can cause changes in the chemical properties of the edges of the adhesive layers and the base layer, or lead to the appearance of fragments of the second detachable patch, which can cause autofluorescence. Autofluorescence can significantly contribute to the background fluorescence signal during fluorescence imaging of a flow cell that contains the interposer described in this publication, thereby significantly reducing the signal-to-noise ratio (SNR). In contrast, the CO ₂ laser can provide a suitable edge smoothness and is chemically inert, ie does not cause chemical changes in the adhesive layers and the base layer and does not lead to fragmentation generated by the second release patch. Therefore, the formation of microfluidic channels in the interposer using a CO ₂ laser does not lead to a significant increase in autofluorescence and gives a higher signal-to-noise ratio (SNR).

DESCRIPTION OF EXAMPLES OF CARRYING OUT THE INVENTION

This section describes various experiments demonstrating low autofluorescence and excellent adhesiveness of an acrylic adhesive. The experimental examples described in this section are illustrative only and should not be construed as limiting the present invention.

Material Properties: The ability of various materials to bond the flow cell and provide high quality sequencing results at low cost was investigated. The following properties are of particular importance: 1) no or low level of autofluorescence: gene sequencing is based on fluorescent labels attached to nucpeotides, and the signals from these labels are relatively weaker than normal. To improve the signal-to-noise ratio from DNA clusters with fluorophores, the absence of light emitted or scattered by the edges of the binder materials is desirable; 2) connection strength: flow cells are often subjected to high pressure (for example, from 13 psi or even higher). For connections in a flow cell, high bond strengths are desirable, including peel and shear strengths; 3) joint quality: for high-quality bonding of the flow cell, a high quality binder without voids and leaks is desirable; 4) connection strength after stress: gene sequencing involves the use of many buffer solutions (solutions with high pH, high salt content and elevated temperature), and it may also involve the use of organic solvents. Holding the flow cell substrates (eg, top and bottom substrates) in place under such voltage is desirable for a successful sequencing run; 5) chemical stability: it is desirable that the adhesive layers and the base layer are chemically stable and do not release (for example, do not degas) any chemicals into solutions, since the enzymes and high-purity nucleotides used in gene sequencing are very sensitive to any contaminants contained in buffer solution.

Flow Cell Configurations: Pressure sensitive adhesives (PSA) were used in two different flow cell configurations as shown in FIG. 6A and FIG. 6 B. FIG. 6A is a schematic cross-sectional view of a bonded and structured flow cell, i.e. a flow cell containing wells formed in a nanoimprint lithography (NIL) resin deposited on the surface of glass substrates between which an interposer is glued, and FIG. 6A is a schematic cross-sectional view of a bonded and non-structured flow cell containing an interposer bonded directly to glass substrates (ie, no resin on the substrates). Fig. 6A shows a structured flow cell configuration with a 100 µm thick adhesive tape formed from about 25 µm thick layers of pressure sensitive adhesives (PSA) applied to a black PET base layer of about 50 µm thickness. The structured surface contains low surface energy materials that have shown poor bond strength to some PSAs.

Material Screening Method: In the complete material screening process, 48 different screening experiments were performed. In order to screen adhesives and carrier materials with high throughput, the screening methods were divided according to five different levels of priority, as summarized in Table 1. Many adhesives were rejected after stage 1 testing. Early rejection allowed a significant number of materials to be screened ( more than 20) in a few weeks.

Autofluorescence properties: Autofluorescence properties were measured with a confocal fluorescence scanner (Typhoon) with a green (532 nm) and a red (635 nm) laser as the excitation source. In the case of the green laser, a band pass filter with a center wavelength of 570 nm was used, and in the case of the red laser, a long wavelength pass filter was used with a pass wavelength of 665 nm. The excitation and emission settings were similar to those used in the exemplary gene sequencing example. Fig. 7 is a histogram of the fluorescence intensity of various adhesives and flow cell materials in the red signal channel. Fig. 8 is a histogram of the fluorescence intensity of various adhesives and flow cell materials from FIG. 7 in the green signal channel. Table II lists the autofluorescence values for all materials.

Tape samples 1 to 4 and 7 to 8 were adhesives containing thermoset epoxy resins, tape sample 5 was an adhesive containing but and l rubber adhesive, and tape sample 6 contained a silicone acrylate film backing. As seen from FIG. 7, FIG. 8 and Table II black Kapton (polyimide) and glass were used as negative controls. In order to meet the requirement of low fluorescence in this experiment, any test material must emit less light than black Kapton. Only a few adhesives or carriers have passed this screening process, including Methyl Acrylic Adhesive, PET-1, PET-2, PET-3, Tape Sample 7, and Tape Sample 8. Most carrier materials such as Kapton 1, PEEK, and Kapton 2 , failed the test due to high background fluorescence. The acrylic adhesive has an autofluorescence at 532 nm excitation wavelength of less than about 0.25 relative to the 532 nm fluorescence standard (FIG. 7) and an autofluorescence at 532 nm excitation wavelength of less than about 0.15 arbitrary units relative to the standard fluorescence at 635 nm (Fig. 8), which is low enough for use in flow cells.

Adhesion under load and without load: When bonding a flow cell, the quality of the adhesive bond, in particular the adhesion strength, should be evaluated. Bond shear strength and 180° peel strength tests were used to quantify adhesion strength. Fig. 9A and FIG. 9B shows shear strength and peel strength test patterns that were used to investigate shear strength and peel strength of various adhesives. As shown in FIG. 9A and FIG. 9B, the stacks of adhesive wafers were assemblies having a "sandwich" structure. The bottom surface was a glass or NIL surface similar to that of a flow cell. A thick film of Kapton was placed over the adhesive, which transmitted force from the tool to the adhesive during shear or peel strength testing. Table III summarizes the results of the shear strength and peel strength tests.

Table III

Initial adhesion when testing adhesives is shown in Table III. Most of the adhesives met the minimum requirements (i.e. showed a shear stress of more than 50 N/cm ² and a peel force of more than 1 N/cm) on the glass surface, with the exception of PET-1, PET-2 and PET-3, which did not pass the strength test when peeled off and also contained voids after gluing. The adhesive of Tape Sample 1 had a relatively low peel strength on the NIL surface and failed the test. The adhesives were also subjected to a high salt, high pH buffer solution (1 M NaCl, pH 10.6 carbonate buffer and 0.05% Tween 20) at about 60 degrees Celsius for 3 days as a stress test. Tape Sample 5 and Tape Sample 1 lost more than about 50% shear strength and peel strength. After screening for autofluorescence and bond strength, the acrylic adhesive was found to be the lead adhesive showing all the desired characteristics. The next best material was ND-C, but it showed about 30% higher background signal in the red fluorescence channel relative to the acrylic adhesive.

Formamide, High Temperature, and Low Temperature Loading: To further evaluate the performance of the adhesive in a flow cell bonding application, additional experiments were performed with the acrylic adhesive and the Tape Sample 5 and Tape Sample 1 adhesives. Experiments included soaking in formamide at about 60 degrees Celsius for about 24 hours , cold storage at about -20 degrees Celsius and about 4 degrees Celsius for about 24 hours, and vacuum heat treatment at about 60 degrees Celsius for about 24 hours. All results are summarized in Table IV.

Both adhesives passed most tests. However, the tape sample 5 adhesive showed many voids after vacuum drying and lost more than 40% shear strength, which did not meet the minimum requirements. The acrylic adhesive also lost a significant amount of peel strength after the formamide load test, but still met the minimum requirements.

Gaseous Solvent Emission and Transfer: Many reagents used in gene sequencing are very sensitive to contaminants contained in buffers or solutions, which can affect the sequencing matrix. Thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and gas chromatography-mass spectroscopy (GC-MS) were used to determine the underlying chemical structures of adhesives and outgassing from adhesives. As measured by TGA, the dry acrylic adhesive, the ND-C adhesive, and the tape sample adhesive 5 showed very little weight loss (0.5%). Tape 1 sample showed a mass loss in excess of 1%, which may indicate a higher risk of release of harmful materials during a sequencing run.

The weight loss of the adhesives was also determined after loading with formamide and buffer solution. The acrylic adhesive showed a weight loss of about 1.29%, indicating that this adhesive is more sensitive to formamide, and is in line with the previous formamide stress test. Tape sample 5 showed a large weight loss (about 2.6%) after the buffer solution load test, which also explains the poor results of the buffer solution shear strength test. The base polymers of the acrylic adhesive and ND-C were classified as acrylic by FTIR. The biocompatibility of the acrylic polymer is well known and reduces the chance of release of harmful materials during a sequencing run. Fig. 10 is the FTIR spectrum of an acrylic adhesive and duct tape. Table V summarizes the results of measurements by TGA and FTIR.

For additional outgassing testing, the acrylic adhesive and black Kapton were examined by GC-MS. Both samples were incubated at about 60° C. for one hour and the gas released from these materials was collected by means of a cold trap and analyzed by GC-MS. As seen in FIG. 11, there was no measurable outgassing from black Kapton and a total of approximately 137 ng/mg of volatiles were detected from the acrylic adhesive after curing for one hour at 60°C. 0.014% of the total weight of the acrylic adhesive. All evolved gaseous compounds were analyzed by GC-MS, they were very similar to each other and originated from acrylic adhesives containing acrylate/methacrylate monomer and aliphatic side chains and the like. Fig. 12 shows typical MS spectra of these evolved gaseous compounds with an inset showing the likely chemical structure of the evolved gaseous compound. Since acrylic and methacrylic adhesives are generally known to be biocompatible, the small amount of acrylate/methacrylate containing gas released is not expected to have any adverse effect on gene sequencing reagents.

The present invention covers the following embodiments:

1. Interposer, containing: a base layer having a first surface and a second surface opposite the first surface; the first adhesive layer located on the first surface of the base layer; a second adhesive layer located on the second surface of the base layer; and a plurality of microfluidic channels extending through the base layer, the first adhesive layer, and the second adhesive layer.

2. Interposer according to claim 1, characterized in that the base layer contains black polyethylene terephthalate (PET); the first adhesive layer contains an acrylic adhesive; the second adhesive layer contains an acrylic adhesive.

3. Interposer according to claim 2, characterized in that the total thickness of the base layer, the first adhesive layer and the second adhesive layer is in the range from about 1 µm to about 200 µm.

4. An interposer according to claim 2 or claim 3, characterized in that the base layer has a thickness ranging from about 10 microns to about 100 microns, and each of the first adhesive layer and the second adhesive layer has a thickness ranging from about 5 µm to about 50 µm.

5. The interposer according to any one of claims 1 to 4, characterized in that each of the first and second adhesive layers has autofluorescence in response to an excitation wavelength of 532 nm, which is less than about 0.25 conventional units with respect to the fluorescence standard 532 nm.

6. An interposer according to any of the preceding claims, characterized in that each of the first and second adhesive layers has an autofluorescence in response to an excitation wavelength of 635 nm, which is less than about 0.15 conventional units with respect to the fluorescence standard at the excitation wavelength equal to 635 nm.

7. Interposer according to any one of claims 2 to 6, characterized in that the base layer contains at least about 50% black PET.

8. Interposer according to claim 7, characterized in that the base layer consists essentially of black PET.

9. An interposer according to any one of claims 2 to 8, wherein each of the first and second adhesive layers contains at least about 5% acrylic adhesive.

10. Interposer according to claim 9, characterized in that each of the first and second adhesive layers consists essentially of an acrylic adhesive.

11. Interposer according to any of the previous paragraphs, characterized in that it additionally contains: the first detachable pad located on the first adhesive layer; a second release patch located on the second adhesive layer; wherein the plurality of microfluidic channels extend through each of the base layer, the first adhesive layer, and the second adhesive layer and through the second release patch, but not through the first release patch.

12. An interposer according to claim 11, wherein the first release patch has a thickness ranging from about 50 microns to about 300 microns, and the second release patch has a thickness ranging from about 25 microns to about 50 microns.

13. Interposer according to claim 11 or claim 12, characterized in that the base layer contains black polyethylene terephthalate (PET), and each of the first and second adhesive layers contains an acrylic adhesive.

14. An interposer according to any one of claims 11 to 13, characterized in that the first release patch is at least substantially opaque and the second release patch is at least substantially transparent.

15. Flow cell containing the first substrate; a second substrate and an interposer located between the first substrate and the second substrate according to any one of paragraphs 2 to 10, wherein the first adhesive layer connects the first surface of the base layer with the surface of the first substrate, and the second adhesive layer connects the second surface of the base layer with the surface of the second substrate.

16. The flow cell of claim. 15, characterized in that each of the first and second substrates contains glass, and the binder between each of the first and second adhesive layers and the respective surfaces of the first and second substrates is configured to withstand a shear stress in excess of about 50 N /cm ² and a tear force greater than about 1 N/cm.

17. The flow cell according to claim 15, characterized in that each of the first and second substrates contains a resin layer having a thickness of less than about 1 micron, and has a surface associated with the respective first and second adhesive layers, and the binder between each of the resin layers and the respective first and second adhesive layers are configured to withstand a shear stress greater than about 50 N/cm ² and a peel force greater than about 1 N/cm.

18. The flow cell according to claim 15, characterized in that a plurality of wells is extruded in the resin layer on at least one of the first substrate or the second substrate, a biological probe is placed in each of the wells, and the microfluidic channels of the interposer are configured to deliver fluid to the plurality of wells .

19. A method for forming microfluidic channels, including: forming an interposer containing a base layer having a first surface and a second surface opposite the first surface, wherein the base layer contains black polyethylene terephthalate (PET), the first adhesive layer is located on the first surface of the base layer, and the first adhesive the layer contains an acrylic adhesive, the second adhesive layer is located on the second surface of the base layer, and the second adhesive layer contains an acrylic adhesive; and forming microfluidic channels passing through at least the base layer, the first adhesive layer and the second adhesive layer.

20. The method according to claim 19, characterized in that the formation of microfluidic channels includes the use of a CO ₂ laser.

21. The method according to claim 20, characterized in that the interposer additionally comprises a first detachable lining located on the first adhesive layer and a second detachable lining located on the second adhesive layer, and at the stage of forming microfluidic channels, microfluidic channels are additionally formed passing through the second detachable pad, but do not form microfluidic channels passing through the first detachable pad.

22. The method of claim 21, wherein the CO ₂ laser has a wavelength ranging from about 5000 nm to about 15000 nm and a beam size ranging from about 50 µm to about 150 µm.

It should be understood that all combinations of the ideas set forth above and additional ideas set forth in more detail below (provided that these ideas are not mutually incompatible) are intended to be part of the subject matter of the present invention disclosed in this publication. In particular, all combinations of the claims at the end of this specification are to be considered as part of the subject matter of the present invention as disclosed in this publication.

When used in the context of the present invention, the singular forms include the plural, unless expressly stated otherwise. Thus, for example, the term "element" means one element or a combination of elements, the term "material" means one or more materials or their combination.

When used in the context of the present invention, the terms "about" and "approximately" usually mean plus or minus 10% of the specified value. For example, "about 0.5" could include 0.45 and 0.55, "about 10" could include 9 to 11, "about 1000" could include 900 to 1100.

When used in the context of the present invention, the term "substantially" and similar terms have a broad meaning, consistent with the standard and conventional use of these terms by experts in the field of technology to which the present invention pertains. Those skilled in the art who will read this specification should understand that these terms are intended to describe individual features specified and claimed without limiting the scope of those features to precise definitions and/or ranges of numerical values given. Accordingly, these terms should be interpreted as indicating that minor or minor modifications or changes to the described and claimed subject matter of the present invention fall within the scope of the present invention as defined in the appended claims.

It should be noted that the term "example" when used in this publication to describe various embodiments of the same indicates that these embodiments are possible examples, representations and/or illustrations of possible embodiments of the present invention (and this term does not mean that such embodiments of the present invention are extraordinary or best examples).

The term "connected" and similar terms when used in the context of the present invention means the direct or indirect connection of two elements to each other. Such a connection may be fixed (eg permanent) or collapsible (ie removable or removable). Such a connection can be made using two elements or two elements and any additional intermediate elements integrally combined into a single body with one or two additional elements or with two elements and any additional intermediate elements attached to each other.

It is important to note that the construction and arrangement of the various exemplary embodiments of the present invention are illustrative only. Although only a few embodiments of the present invention have been described in this specification, those skilled in the art who read this publication will readily appreciate that many modifications are possible (e.g., variations in sizes, measurements, structures, shapes and proportions of various elements, parameter values, circuits arrangement, use of materials, colors, orientations, etc.) without substantially deviating from the new principles and advantages of the present invention described in this publication. Other substitutions, modifications, changes and deletions may also be made in the plan, operating conditions and arrangement of the various embodiments of the present invention without deviating from the scope of the present invention.

Claims (49)

1. Interposer for a flow cell, comprising: a base layer having a first surface and a second surface opposite the first surface, the base layer comprising black polyethylene terephthalate (PET); the first adhesive layer located on the first surface of the base layer, and the first adhesive layer contains an acrylic adhesive; a second adhesive layer located on the second surface of the base layer, and the second adhesive layer contains an acrylic adhesive; And a plurality of microfluidic channels extending through each of the base layer, the first adhesive layer, and the second adhesive layer. 2. The interposer according to claim 1, characterized in that the total thickness of the base layer, the first adhesive layer and the second adhesive layer is in the range from 1 µm to 200 µm. 3. The interposer according to claim 1 or 2, characterized in that the base layer has a thickness in the range from 10 µm to 100 µm, and each of the first adhesive layer and the second adhesive layer has a thickness in the range from 5 µm to 50 µm. 4. Interposer according to any one of paragraphs. 1-3, characterized in that each of the first and second adhesive layers has autofluorescence in response to an excitation wavelength of 532 nm, which is less than 0.25 arbitrary units with respect to the fluorescence standard at 532 nm. 5. Interposer according to any one of the preceding claims, characterized in that each of the first and second adhesive layers has an autofluorescence in response to an excitation wavelength of 635 nm, which is less than 0.15 conventional units with respect to the fluorescence standard at 635 nm. 6. Interposer according to any one of the preceding claims, characterized in that the base layer contains at least 50% black PET. 7. Interposer according to any one of the preceding claims, characterized in that the base layer consists essentially of black PET. 8. Interposer according to any one of the preceding claims, characterized in that each of the first and second adhesive layers contains at least 5% acrylic adhesive. 9. Interposer according to any one of the preceding claims, characterized in that each of the first and second adhesive layers consists essentially of an acrylic adhesive. 10. Interposer according to any of the previous paragraphs, additionally containing: a first release patch located on the first adhesive layer; And the second detachable pad located on the second adhesive layer. 11. Interposer for a flow cell, containing: a base layer having a first surface and a second surface opposite the first surface, the base layer comprising black polyethylene terephthalate (PET); the first adhesive layer located on the first surface of the base layer; a first release patch located on the first adhesive layer; a second adhesive layer located on the second surface of the base layer; a second release patch located on the second adhesive layer; And a plurality of microfluidic channels extending through each of the base layer, the first adhesive layer, the second adhesive layer and the second release patch, but not through the first release patch, wherein each of the first and second adhesive layers contains an acrylic adhesive. 12. Interposer according to claim 11, characterized in that: the first detachable patch has a thickness in the range of 50 µm to 300 µm; and the second release patch has a thickness in the range of 25 µm to 50 µm. 13. Interposer according to any one of paragraphs. 11, 12, characterized in that the first detachable patch is at least substantially optically opaque and the second detachable patch is at least substantially optically transparent. 14. Flow cell containing: the first substrate; a second substrate and an interposer according to any of the preceding claims, located between the first substrate and the second substrate, wherein the first adhesive layer connects the first surface of the base layer with the surface of the first substrate, and the second adhesive layer connects the second surface of the base layer with the surface of the second substrate. 15. The flow cell according to claim. 14, characterized in that each of the first and second substrates contains glass, and the binder between each of the first and second adhesive layers and the respective surfaces of the first and second substrates is configured to withstand a shear stress exceeding 50 N/ cm ² , and a tear force exceeding 1 N/cm. 16. The flow cell according to claim 14 or 15, characterized in that each of the first and second substrates contains a resin layer with a thickness of less than 1 micron, and having a surface associated with the respective first and second adhesive layers, and the binder between each of the resin layers and the respective first and second adhesive layers are configured to withstand a shear stress greater than 50 N/cm ² and a peel force greater than 1 N/cm. 17. Flow cell according to any one of paragraphs. 14-16, characterized in that: a plurality of holes is extruded in the resin layer on at least one of the first substrate or the second substrate, a biological probe is placed in each of the wells, and the microfluidic channels of the interposer are configured to deliver fluid to a plurality of wells. 18. A method for forming an interposer for a flow cell, including the steps in which: form an interposer containing: a base layer having a first surface and a second surface opposite the first surface, the base layer comprising black polyethylene terephthalate (PET), the first adhesive layer located on the first surface of the base layer, and the first adhesive layer contains an acrylic adhesive, a second adhesive layer located on the second surface of the base layer, and the second adhesive layer contains an acrylic adhesive; and forming microfluidic channels passing through at least the base layer, the first adhesive layer and the second adhesive layer. 19. The method according to claim 18, characterized in that the formation of microfluidic channels includes the use of a CO ₂ laser. 20. The method according to claim 19, characterized in that: interposer additionally contains: the first detachable pad located on the first adhesive layer, and a second release pad located on the second adhesive layer, and at the stage of formation of microfluidic channels using a CO ₂ laser, microfluidic channels passing through the second detachable patch are additionally formed, but microfluidic channels passing through the first detachable patch are not formed. 21. The method according to claim 18 or 19, characterized in that the CO ₂ laser has a wavelength in the range from 5000 nm to 15000 nm and a beam size in the range from 50 µm to 150 µm.

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