CN114829628A

CN114829628A - System and method for fluorescence imaging

Info

Publication number: CN114829628A
Application number: CN202180007252.5A
Authority: CN
Inventors: 羌梁梁
Original assignee: Aisi Biological Laboratory Co ltd
Current assignee: Aisi Biological Laboratory Co ltd
Priority date: 2020-04-01
Filing date: 2021-04-01
Publication date: 2022-07-29
Also published as: WO2021202843A1

Abstract

A microfluidic device for analyzing fluorescence of a sample, comprising a first substrate; and a second substrate, wherein the second substrate is optically transparent; an adhesive layer provided between the first substrate and the second substrate; and a microfluidic chamber defined between the first substrate and the second substrate. The first and second substrates and the bonding layer form walls of the microfluidic chamber. The microfluidic device includes an oligonucleotide bound to at least one of the walls of the microfluidic chamber, and a fluorescent material disposed on at least one of the walls of the microfluidic chamber and forming an alignment pattern.

Description

System and method for fluorescence imaging

Priority declaration

This application claims priority to U.S. patent application serial No. 63/003,383, filed on 1/4/2020, the contents of which are incorporated herein by reference in their entirety.

Background

Image processing, including image alignment and luminescence intensity correction, is one of the data processing steps for several procedures, such as light-based deoxyribonucleic acid (DNA) sequencing, immunohistochemistry with multiple dyes, and other image-based procedures. Image processing may include image movement, rotation, stretching, and intensity correction in the X and Y directions. Such image processing is performed to align multiple sequencing images in different sequencing cycles to determine the sequence of a DNA cluster.

Disclosure of Invention

We describe herein alignment systems and methods for use in the context of fluorescence imaging for microfluidic devices (e.g., microfluidic chambers or flow cells). The alignment marks may be formed of a fluorescent material, a light reflecting material, or a material that appears darker in the fluorescent image. These alignment marks may form an alignment pattern, which may greatly facilitate alignment of multiple images, improving computational efficiency and processing time. For example, processing (e.g., alignment) of multiple images is associated with a sequencing-by-synthesis (SBS) process, wherein multiple images are captured at multiple emission wavelengths in multiple cycles of the SBS process. In combination with these images, a map of the locations and identities of the DNA clusters on the substrate of the microfluidic chamber can be generated. These multiple images can be aligned using a fixed alignment pattern, greatly facilitating alignment and reducing the computational burden of the SBS process.

In one aspect, a microfluidic device for analyzing fluorescence of a sample includes a first substrate; and a second substrate, wherein the second substrate is optically transparent; an adhesive layer provided between the first substrate and the second substrate; and a microfluidic chamber defined between the first substrate and the second substrate. The first and second substrates and the bonding layer form walls of the microfluidic chamber. The microfluidic device comprises an oligonucleotide bound to at least one of the walls of the microfluidic chamber; and a fluorescent material is disposed on at least one of the walls of the microfluidic chamber and forms an alignment pattern.

Embodiments may include one of the following features or any combination of two or more of the following features.

The fluorescent material comprises a photoresist disposed on at least one wall of the microfluidic channel.

The fluorescent material comprises a fluorescent molecule bound or adhered to at least one wall of the microfluidic channel.

The fluorescent material is disposed on a first wall of the microfluidic channel formed by the first substrate, a second wall of the microfluidic channel formed by a second substrate, or both.

The fluorescent material is disposed on a side wall of the microfluidic channel formed by the adhesive layer.

The fluorescent material is partially embedded in at least one wall of the microfluidic channel.

The fluorescent material comprises a reference oligonucleotide labeled with a fluorophore-labeled oligonucleotide. Each reference oligonucleotide is labeled with two or more fluorophore-labeled oligonucleotides.

The alignment pattern includes a predetermined pattern or a random pattern.

Fluorescence of the sample is detected at a plurality of emission wavelengths, and wherein the fluorescent material fluoresces at least at one of the plurality of emission wavelengths.

In one aspect, a system for analyzing sample fluorescence includes a microfluidic device and a fluorescence microscope configured to capture fluorescence images of a sequencing reaction in the microfluidic chamber of the microfluidic device. The microfluidic device includes a first substrate; and a second substrate, wherein the second substrate is optically transparent; an adhesive layer provided between the first substrate and the second substrate; and a microfluidic chamber defined between the first substrate and the second substrate. The first and second substrates and the bonding layer form walls of the microfluidic chamber. The microfluidic device comprises an oligonucleotide bound to at least one of the walls of the microfluidic chamber; and a fluorescent material disposed on at least one of the walls of the microfluidic chamber and forming an alignment pattern. Aspects of the microfluidic device may include one of the foregoing features or any combination of two or more of the foregoing features.

In one aspect, a method of manufacturing a microfluidic device for analyzing fluorescence of a sample includes disposing a fluorescent material on a surface of a first substrate in an alignment pattern; binding oligonucleotides to a surface of the first substrate or on a second substrate; and attaching the first substrate to the second substrate with an adhesive layer. A microfluidic chamber is defined between the first substrate and the second substrate, and a surface of the first substrate, a surface of the second substrate, and an adhesive layer form a wall of the microfluidic chamber. At least one of the first substrate or the second substrate is optically transparent.

Disposing the fluorescent material on a surface of the first substrate includes disposing a photoresist on the first substrate.

Disposing the fluorescent material on a surface of the first substrate includes disposing fluorescent molecules on the first substrate.

The method includes defining the alignment pattern in the layer of fluorescent material using photolithography.

The method includes printing the fluorescent material in an alignment pattern on a surface of the first substrate.

The method includes defining the alignment pattern in the first substrate; and filling the defined pattern at least partially with the fluorescent material. The method includes etching the alignment pattern in the first substrate.

Disposing the oligonucleotide on a surface of the first substrate or a surface of the second substrate comprises: binding the oligonucleotide to a surface of the first substrate or to a surface of the second substrate.

In one aspect, a microfluidic device for analyzing a sample includes a first substrate; a second substrate, wherein the second substrate is optically transparent; an adhesive layer provided between the first substrate and the second substrate; and a microfluidic chamber defined between the first substrate and the second substrate. The first and second substrates and the bonding layer form walls of the microfluidic chamber. The microfluidic device comprises a set of oligonucleotides bound to at least one of the walls of the microfluidic chamber, wherein each strand of the set of oligonucleotides comprises a primer for cluster generation (e.g., bridge amplification); and a set of reference oligonucleotides bound to at least one of the walls of the microfluidic chamber, wherein the set of reference oligonucleotides is configured to generate an alignment mark. In some embodiments, the first substrate and/or the second substrate is optically transparent at each of a plurality of emission wavelengths.

Each strand of the set of reference oligonucleotides includes two or more read primers for sequencing by synthesis.

Each strand of the set of reference oligonucleotides comprises a read primer for sequencing by synthesis, wherein the read primer is different from the read primer of the first set of oligonucleotides.

The set of reference oligonucleotides is configured to have a signal intensity that is higher than the average signal intensity of the DNA cluster.

The set of reference oligonucleotides is labeled with a fluorescent dye.

The set of reference oligonucleotides is labeled with a fluorescence quencher.

In one aspect, a method of manufacturing a microfluidic device for analyzing fluorescence of a sample includes: a first set of oligonucleotides is bound to a surface of a first substrate, wherein the first set of oligonucleotides includes primers for cluster generation (e.g., bridge amplification). The method includes binding a second set of oligonucleotides to a surface of the first substrate or a surface of a second substrate, wherein the second set of oligonucleotides is configured to generate an alignment pattern. The method includes attaching the first substrate to the second substrate with a bonding layer such that a microfluidic chamber is defined between the first substrate and the second substrate, and such that a surface of the first substrate, a surface of the second substrate, and the bonding layer form a wall of the microfluidic chamber. At least one of the first substrate or the second substrate is optically transparent at each of a plurality of emission wavelengths.

In one aspect, a microfluidic device includes a first substrate; a second substrate; a first adhesive layer provided between the first substrate and the second substrate; and a first microfluidic channel defined between the first substrate and the second substrate. The first and second substrates and the first adhesive layer form walls of the first microfluidic channel. The microfluidic device comprises a third substrate; a second adhesive layer provided between the second substrate and the third substrate; and a second microfluidic channel defined between the second substrate and the third substrate. The second and third substrates and the second adhesive layer form walls of the second microfluidic channel.

The microfluidic device comprises oligonucleotides bound to at least one of the walls of the first microfluidic channel and at least one of the walls of the second microfluidic channel, wherein the first set of oligonucleotides comprises primers for bridging for amplification.

An inlet and an outlet are defined in the first substrate.

An inlet is defined in the first substrate and an outlet is defined in the third substrate.

The first microfluidic channel and the second microfluidic channel are fluidically connected in parallel.

The first microfluidic channel and the second microfluidic channel are fluidically connected in series.

The microfluidic device comprises a fourth substrate; a third adhesive layer provided between the third substrate and the fourth substrate; and a third microfluidic channel defined between the third substrate and the fourth substrate. The third and fourth substrates and the third adhesive layer form walls of the third microfluidic channel. An outlet is defined in the fourth substrate. The first, second and third microfluidic channels are fluidically in series.

The first, second and third microfluidic channels are fluidically connected in series.

The first substrate, the second substrate, the third substrate, and/or the fourth substrate are optically transparent.

In one aspect, a microfluidic device for analyzing fluorescence of a sample comprises: a microfluidic chamber defined by a first inner surface and a second inner surface; a first set of oligonucleotides bound to the first inner surface of the microfluidic chamber; and one or more alignment marks fixed to the microfluidic chamber forming an alignment pattern for the first inner surface.

The alignment mark is a fluorescent mark, a reflective mark or a dark mark. The fluorescent label is formed by a fluorophore. The reflective marker is formed of a reflective material. The dark mark is formed by a fluorescence quencher. The dark mark is formed by removing one or more oligonucleotides located at the dark mark.

The alignment mark is formed by a set of reference oligonucleotides. Each strand of the reference oligonucleotide has two or more read primers for sequencing-by-synthesis. Each strand of the reference oligonucleotide has a read primer for sequencing by synthesis. The read primer in each strand of the reference oligonucleotide is different from the read primer used for sample analysis. The reference oligonucleotide is labeled with a fluorescence quencher. The reference oligonucleotide is labeled with a fluorophore.

The alignment mark is deposited on or embedded in the first inner surface.

The microfluidic chamber comprises a second inner surface and a second set of oligonucleotides is bound to the second inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the second inner surface. The microfluidic chamber comprises a third inner surface and a third set of oligonucleotides is bound to the third inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the third inner surface. The microfluidic chamber comprises a fourth inner surface and a fourth set of oligonucleotides is bound to the fourth inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the fourth inner surface. The microfluidic chamber comprises a fifth inner surface and a fifth set of oligonucleotides is bound to the fifth inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern of the fifth inner surface. The microfluidic chamber comprises a sixth inner surface and a sixth set of oligonucleotides is bound to the sixth inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the sixth inner surface.

In one aspect, a method of sequencing a set of nucleic acids of interest includes disposing the set of nucleic acids of interest to a microfluidic device and allowing the set of nucleic acids to randomly hybridize to the oligonucleotides in the microfluidic chamber. The method comprises the following steps: amplifying the nucleic acid of interest, thereby forming a DNA cluster; providing read primers and labeled dntps to initiate sequencing-by-synthesis; taking images of fluorescently labeled DNA clusters for multiple cycles of sequencing while synthesizing; aligning the images through the alignment marks, thereby identifying the positions of the DNA clusters on different images; and determining the sequence of the nucleic acid of interest. The microfluidic device comprises a microfluidic chamber defined by a first inner surface and a second inner surface; a first set of oligonucleotides is bound to the first inner surface of the microfluidic chamber; and one or more alignment marks fixed on the microfluidic chamber, forming an alignment pattern of the first inner surface.

In one embodiment, the method includes adjusting the focal plane of the fluorescence microscope to capture images of the DNA clusters on different interior surfaces.

The details of one or more implementations are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description and drawings, and from the claims.

Drawings

Fig. 1A is a schematic illustration of a substrate having DNC clusters thereon.

FIGS. 1B-1D are schematic illustrations of fluorescence images of the substrate of FIG. 1A at different sequencing cycles.

FIG. 1E is a schematic of a combined fluorescence image.

Fig. 2A is a schematic view of a substrate with fluorescent molecules and an alignment pattern.

FIGS. 2B-2D are schematic illustrations of fluorescence images of the substrate of FIG. 2A at different sequencing cycles.

Fig. 2E is a schematic of a combined fluorescence image.

Fig. 3 is a diagram of an exemplary alignment pattern.

Fig. 4 is a diagram of a microfluidic system for sequencing-by-synthesis.

Fig. 5A-5E are cross-sectional views of a microfluidic device.

Fig. 6 is a flow chart for manufacturing a microfluidic device.

Fig. 7A and 7B are schematic diagrams of sequencing-by-synthesis (SBS) processes.

FIGS. 8A and 8B are diagrams of DNA clusters.

FIGS. 9A and 9B are diagrams of reference oligonucleotides.

FIG. 10 is a fluorescent image with dark marks.

Fig. 11A-11C are diagrams of microfluidic devices.

Detailed Description

We describe herein an alignment system and method for use in a fluorescence imaging environment. The alignment pattern may be formed of a fluorescent material, a light reflecting material, or a material that appears dark in the fluorescent image of the device. The alignment pattern may facilitate alignment of multiple images, enabling computational efficiency and fast processing of multiple images. For example, processing (e.g., alignment) of multiple images is associated with a sequencing-by-synthesis (SBS) process, wherein images are captured at two or more (e.g., 2, 3, or 4) emission wavelengths, throughout a cycle of a plurality of the SBS processes. Combining the images produces a map of the locations and identifications of the DNA clusters on the substrate of the microfluidic device. These multiple images are aligned using a fixed alignment pattern, facilitating alignment and reducing the computational burden of the SBS process.

In a typical sequencing-by-synthesis (SBS) process, a DNA library is generated for the nucleic acid of interest. The sequence in the DNA library may be a fragment of the nucleic acid of interest. Linkers (e.g., right and/or left linkers) may be added to the sequences in the DNA library.

The oligonucleotides are immobilized on the substrate surface of the microfluidic chamber. The sequences in the DNA library may be hybridized to the oligonucleotides by the linkers. Once ligated, bridge amplification can produce identical DNA strands, forming DNA clusters. Some will become forward chains. The remainder are the reverse strands. In some embodiments, the DNA polymerase moves along the original strand of DNA from the DNA library, producing a complementary strand. The original strand is then washed away, leaving only the reverse strand. At the top of the reverse strand, there is a linker sequence. The DNA strand is bent and attached to the oligonucleotide complementary to the top linker sequence. A polymerase attaches to the reverse strand to form a complementary strand identical to the original strand. The double stranded DNA is then denatured so that each strand can be attached separately to the oligonucleotide sequences anchored to the substrate. Finally, the DNA strands in one cluster (i.e., one spot on the surface) are from a single source (clonal amplification). At the end of clonal amplification, in some embodiments, all reverse strands are removed from the substrate, leaving only the forward strand for sequencing-by-synthesis (SBS).

At the start of sequencing by synthesis, a primer is attached to the forward strand linker primer binding site, and a polymerase adds labeled dntps to the DNA strand. The dntps may be labeled with a fluorophore. Due to blocking groups in the dntps, only one base can be added per reaction cycle. In some embodiments, the fluorophore may act as a blocking group. Fluorescence from fluorescently labeled dntps can provide identification of dnpts newly added to the sequence. In some embodiments, four color chemistry is used. Each of the four bases may have a unique fluorescent label. In some embodiments, a two-color chemistry is used. Both bases have a unique fluorescent label. One base has a combination of two fluorescent labels. The last base is not labeled. An imaging system with an appropriate number of channels (e.g., one channel, three channels, four channels, etc.) can be used to record the fluorescence during each cycle. In some embodiments, the images from each channel may overlap or merge. After each reaction cycle, the device records the fluorescence of each cluster. Once the color is recorded, the fluorophore is removed and another dNTP is added. This cycle may be repeated multiple times until a sequence is determined.

When sequences in a DNA library hybridize to the oligonucleotides deposited on the surface of a substrate, they typically hybridize to the oligonucleotides in a random pattern. During sequencing, each DNA cluster is labeled with a fluorescent label that uniquely identifies the last base in the cluster. The DNA clusters are sequenced by fluorescence imaging of the substrate using different excitation wavelengths. Illuminating the substrate with each excitation wavelength excites a corresponding subset of the DNA clusters based on the identity of the last base in the cluster. Since the DNA clusters are randomly distributed, the images need to be properly aligned in order to process the fluorescence images.

FIG. 1A is a schematic illustration of a substrate 100 having DNA clusters. For example, the substrate 100 is the wall of a chamber of a microfluidic device for SBS analysis of DNA. Each dot represents the physical location of a DNA cluster on the substrate. These DNA clusters can be labeled by fluorescence during sequencing.

Fig. 1B-1D are schematic illustrations of

fluorescence images

110a, 110B, 110c (collectively images 110) of the substrate 100 taken during the SBS process. Each dot represents the location of the fluorescent signal at the same emission wavelength. In each image, a subset of the fluorescent molecules depicted in fig. 1A fluoresce at a particular emission wavelength of image 110, depending on the identity of the bases added to the molecule during the sequencing cycle. The subset of molecules that fluoresce at a particular emission wavelength is different for each image. No other DNA clusters with different bases are visible in the images 110 b-d. Thus, each image 110 shows only a subset of all fluorescent molecules present on the substrate 100.

Referring to fig. 1E, to register the positions of DNA clusters based on imaging, multiple images 110 are aligned and stacked to generate a combined image 130. The ideal combined image 130 would include points representing the location of each DNA cluster 102 on the substrate 100. Under real world conditions, not all DNA clusters are detected, and therefore some molecules are missing from the combined image 130, as can be seen by comparing the combined image 130 to the schematic representation of the substrate 100 and the molecules in fig. 1A. For accuracy and completeness, multiple images 110, for example ten or more images, must be used. Alignment and stacking of multiple images involves applying X and Y direction offsets to one or more images, rotating one or more images, stretching one or more images, or other suitable image processing techniques. In some examples, the intensity of the fluorescence signals in the images 110 and/or any drift between the images 110 may make the image processing technique time consuming and/or affect the accuracy of the technique.

To facilitate alignment of multiple images during fluorescence analysis, such as SBS, processes, a substrate, such as a wall or substrate of a microfluidic device, is marked with one or more alignment marks. These alignment marks may include, for example, fluorescent marks, reflective marks, and/or dark marks. These alignment marks may form an alignment pattern that is visible to the images of each sequencing cycle (e.g., merged images of different channels in the same sequencing cycle), and thus visible in all images to be aligned. In some embodiments, all images of each channel are first aligned by the alignment marks. The aligned images of each channel are then aligned to generate a map of the DNA clusters. Images with alignment marks can be aligned by analyzing fewer pixels than images without alignment marks, thereby reducing the computational resources expended in the alignment process and speeding up the process.

Fig. 2A is a schematic illustration of a substrate 200 having fluorescent molecules with alignment marks. For example, the substrate 200 is the wall of a chamber of a microfluidic device for SBS analysis of DNA. Each dot represents the physical location of a DNA cluster on the substrate. The alignment marks form a cross-shaped alignment pattern 204 on the substrate 200. In some embodiments, the alignment marks may be formed of a material that fluoresces or reflects light at all wavelengths at which the substrate 200 is to be imaged. In some examples, the alignment marks are formed of a material that appears darker than the surrounding substrate at all wavelengths of the captured image. In some embodiments, each alignment mark includes only one fluorophore. In some embodiments, each alignment mark includes two or more fluorophores. As used herein, "fluorophore" refers to a molecule comprising or consisting of a functional group that absorbs energy within a particular absorption spectrum and re-emits energy (e.g., such as light) in a different (but also particular) emission spectrum. In some embodiments, fluorophores used as labels include, but are not limited to, fluorescein, cascade blue, hexachlorofluorescein, tetrachlorofluorescein, TAMRA, ROX, FAM, Cy3, Cy3.5, Cy5, Cy5.5, Texas Red (Texas Red), Eosin (Eosin), the Dylight Fluor family available from Thermo Fisher Scientific, Walsh, Mass, and the Alexa Fluor family from Molecular Probes, Yougu, Oreg. In some embodiments, the fluorophore is a fluorescent protein (e.g., a green fluorescent protein, a red fluorescent protein, or a blue fluorescent protein). In some embodiments, the alignment mark is a dark spot where the oligonucleotide is removed, and therefore labeled dntps cannot be added to the dark spot.

FIGS. 2B-2D are schematic illustrations of

fluorescence images

210a, 210B, 210c (collectively images 210) of the substrate 200 taken during the sequencing cycle. Each dot represents the location of the fluorescent signal at the emission wavelength. For each sequencing cycle, the alignment pattern 204 may be visible in all images 210.

Referring to fig. 2E, to register the positions of all fluorescent molecules based on imaging, multiple images 210 are aligned and stacked to generate a combined image 230. The alignment pattern 204, which is visible in all images 210, facilitates alignment, reduces the computational power involved in alignment and enables faster image processing.

In one specific example, FIGS. 1B-1D and 2B-2D are 3000x3000 pixel sizes. To align the image 110 of FIGS. 1B-1D, the alignment algorithm searches for almost all 3000x3000 pixels. In contrast, to align the images 210 of fig. 2B-2D, the alignment algorithm may search only a portion of each image, for example, only two rows (3000 pixels +3000 pixels), to identify the location of the alignment pattern 204. This reduction in the magnitude of the search achieved by the presence of the alignment pattern 204 may result in a reduction in computational power and results in a computation speed that is faster than the image alignment without the alignment pattern, e.g., at least or about 1000, 1500, 2000, or 3000 times faster.

An image with an alignment pattern may have other advantages. For example, the image may be processed even if the fluorescence intensity is low (e.g., as depicted in image 210 c) or contaminated fluorescent particles are introduced onto the substrate. The intensities can be normalized across the image for ease of image comparison and combination. There is no need to determine the absolute position of the fluorescent molecules, since the alignment pattern 204 has a known position and the molecule position relative to the alignment pattern 204 can be calculated after image processing. Further, fewer images may be used to determine the location of the molecules, for example, about or less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 images.

Furthermore, in conventional methods, the imaging process relies on fluorescent molecules detected on the substrate to align the images. There is an upper limit to the density of the fluorescent molecules detected. Aligning the images can be very challenging if the density of detected fluorescent molecules is too high. The density of the DNA clusters in the conventional method is usually as high as 100 ten thousand/mm ² . Since the methods described herein rely on the alignment marks, the methods described herein can further increase the density of the DNA clusters to at least or about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 million/mm ² . This can be greatly improvedHigh sequencing efficiency.

Other configurations (e.g., shapes) of the alignment pattern may also be used. Fig. 3 shows an example configuration of an alignment pattern. The alignment pattern may be a rectilinear pattern, such as solid or dotted lines or dashed lines, in various configurations and arranged at different locations on the substrate. The alignment pattern may be individual dots. The alignment pattern may be a curve. The alignment pattern of fig. 3 is a predefined pattern formed on the substrate, for example, using deposition and/or patterning techniques described herein. In some examples, the alignment pattern is a random arrangement of fluorescent material, as also described herein. In some embodiments, the alignment pattern may appear circular, annular, elliptical, rectangular, square, symmetrical, asymmetrical, triangular, polygonal, and the like. The alignment marks may be arranged in a regular repeating pattern, including, for example, a hexagonal or rectilinear pattern. In some embodiments, the pattern may be triangular, hexagonal, or even irregular. In some embodiments, the alignment mark needs to have an appropriate width (e.g., the width of a line, the width of a square, or the diameter of a dot). In some embodiments, the width is about 0.5 to about 10 μm, about 0.5 to about 5 μm, about 0.5 to about 2 μm, about 1 to about 10 μm, about 1 to about 5 μm, or about 1 to about 2 μm. In some embodiments, the width is about or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm.

Fig. 4 is an example of a microfluidic system 400 for performing fluorescence analysis on a sample in a microfluidic device 402. The microfluidic device 402 includes a substrate 404 in which the microfluidic chamber 406 is defined. In some examples, the substrate 404 includes a plurality of components, e.g., a top substrate, a bottom substrate, and an adhesive layer, as shown in fig. 5A-5E, as discussed herein.

Microfluidic channels

408a, 408b (collectively channels 408) are fluidly connected to the microfluidic chamber 406, e.g., operable as inlets and/or outlets for fluid flow into or out of the microfluidic chamber 406. The microfluidic chamber 406 is wider than the channel 408; in some examples, the microfluidic chamber 406 is an elongated channel having a width similar to the width of the inlet and/or outlet channel. In some examples, the microfluidic chamber 404 is a branched channel. In some examples, the microfluidic device 402 includes a plurality of inlet channels, a plurality of outlet channels, or both; or only a single channel that can serve as both an inlet and an outlet.

The fluorescence microscope 450 is positioned to capture an image of at least a portion of the microfluidic chamber 406. The fluorescence microscope 450 illuminates the microfluidic chamber 406 with light at one or more excitation wavelengths and captures an image at each of a plurality of emission wavelengths. The excitation wavelength is selected to excite fluorescent molecules, e.g., fluorophore-labeled DNA, in the microfluidic chamber 406, and the emission wavelength is the wavelength at which the fluorophore emits fluorescence. The substrate 404 is optically transparent at the excitation and emission wavelengths.

The microfluidic device 402 includes an alignment pattern 420 that is visible at all emission wavelengths of the captured molecular image. The consistent presence of the alignment pattern 420 in all fluorescence images enables image alignment based on the pattern 420. In some examples, the alignment pattern 420 is formed of a material that is excited by at least one excitation wavelength used for illumination (referred to herein as a "fluorescent material"). In some examples, the alignment pattern 420 is formed from a material, such as a metal, that is reflective at all of the multiple emission wavelengths (referred to herein as a "light reflective material"). When using a light reflecting material, a separate background light source of much lower intensity may be used to illuminate the alignment marks during imaging. For example, a weak red LED may be placed at an appropriate angle and the light reflected and captured by the camera. At the same time, the red fluorescence from the DNA cluster will be excited by another light source with a different wavelength.

In some examples, the alignment pattern 420 is formed of a material that appears darker than the surrounding substrate 402 in the image of all of the plurality of emission wavelengths (referred to herein as "dark material"). Further details regarding the materials used for the alignment pattern 420 are provided in connection with fig. 5A-5E. As used herein, the term "dark" is intended to refer to an amount of desired signal detected by a detector that is insignificant compared to background signal detected by the detector. For example, a feature of an object may be considered dark when its signal-to-noise ratio is very low, e.g., less than 1. In some embodiments, a dark spot does not produce any number of desired signals (i.e., no signal is produced or detected). In some embodiments, a very low amount of signal relative to background may be considered dark.

In the example of fig. 4, the alignment pattern 420 is defined on a bottom wall of the microfluidic chamber 406 formed by the bottom of the substrate 404. In some examples, the alignment pattern 420 may be defined on a top or a sidewall of the microfluidic chamber 406, or on an outer surface of the substrate 404 in which the microfluidic chamber 406 is defined. Example locations of the alignment patterns are shown in fig. 5A-5E, as discussed herein.

In some examples, the microfluidic system 400 is used for SBS analysis of DNA. In these examples, the oligonucleotides are disposed on one or more walls of the microfluidic chamber 406, e.g., immobilized on one or more walls by covalent bonds. In some examples, the oligonucleotide is covalently bound to a reactive silane moiety on the substrate.

Fig. 5A-5E show cross-sectional views of a portion of a microfluidic device 500. Each of fig. 5A-5E shows a different position for an alignment mark in the microfluidic device 500. The alignment mark may form various alignment patterns.

The microfluidic device 500 has a first substrate 502 and a second substrate 504. An adhesive layer 508 is disposed between the first substrate 502 and the second substrate 504 and adhered to both

substrates

502, 504. A microfluidic chamber 506 is defined between the

substrates

502, 504 such that the first substrate 502 and the second substrate 504 form a top wall 510 and a bottom wall 512, respectively, of the microfluidic chamber 506, and the bonding layer 508 forms a side wall 514 of the microfluidic chamber 506. The microfluidic chamber 508 is fluidly connected to one or more inlet or outlet channels, not shown. One or both of the

substrates

502, 504 are optically transparent at the excitation and emission wavelengths used for fluorescence imaging of the microfluidic device 500. For example, one or both of the

substrates

502, 504 are formed of glass or transparent plastic.

Oligonucleotides (not shown) are disposed on the bottom wall 510 of the microfluidic chamber 506, for example by covalent bonding. In some examples, oligonucleotides may be disposed on other walls of the microfluidic chamber 506, such as the top wall 512. Due to the limited focal depth of the fluorescence microscope, the images of the bottom wall 510 and the top wall 512 can be taken separately, doubling the number of DNA clusters that can be sequenced by one microfluidic device. In some embodiments, the distance between the bottom wall 510 and the top wall 512 is at least or about 1, 2, 3, 4, or 5 μm.

In the specific example of fig. 5A, the first alignment mark 520 may be a fluorescent mark, a reflective mark, or a dark mark. In some embodiments, the alignment marks may be formed of a fluorescent material, a reflective material, or a dark material, and are fixed to the bottom wall 510 of the microfluidic chamber 506.

Similarly, the second alignment marks 520 may be fluorescent marks, reflective marks, or dark marks. In some embodiments, the alignment marks may be formed of a fluorescent material, a reflective material, or a dark material, and are fixed to the upper wall 512 of the microfluidic chamber 506. The alignment marks 520, 522 may form the same or different alignment patterns (e.g., shapes).

In some examples, the fluorescent material used for the alignment pattern includes a photoresist, such as an epoxy-based photoresist, such as SU-8, AZ series positive photoresists, Shipley 1800 series photoresists, photoimageable Printed Circuit Board (PCB) solder marks, or other suitable photoimageable photoresists. The alignment pattern may be formed by photolithographic patterning of a photoresist layer, screen or laser printing of a photoresist material, or by other suitable deposition and/or patterning methods.

In some examples, the fluorescent material used for the alignment pattern includes, for example, fluorophores and/or fluorophore-labeled DNA or proteins. Fluorescent molecules may also be disposed on the walls of the microfluidic chamber 506 by screen printing, laser printing, or other suitable deposition techniques.

The light reflecting material used for the alignment pattern includes, for example, a patterned thin film of a metal such as chromium, gold, or platinum. The alignment pattern may be formed by photolithographic patterning of a thin metal film, screen printing, laser printing, three-dimensional (3D) printing, or other suitable methods for forming metal patterns.

The alignment marks may also be formed from dark materials (e.g., carbon nanotubes or fluorescence quenchers) or dark spots. Dark spots for the alignment pattern can be generated by removing or destroying the oligonucleotides at the spots. For example, the oligonucleotides on the surface may be burned or removed in some way, e.g., by a laser, to create regions where no DNA molecules can bind. The regions will always appear dark during the sequencing process and they can be easily identified for alignment.

In the specific example of fig. 5B, the first alignment marks 530 are defined by a material, such as a fluorescent material, a reflective material, or a dark material, for example, disposed on and partially embedded in the top wall 512 of the microfluidic chamber 506. The second alignment marks 532 are defined by a material, such as a fluorescent material, a reflective material or a dark material, for example, which is arranged on the bottom wall 510 of the microfluidic chamber 506 and partially embedded therein. The alignment marks 530,532 may form the same or different alignment patterns.

To form the alignment marks 530 disposed on the top wall 512 of the microfluidic chamber 506 and partially embedded therein, a recess is defined in the top wall 512, for example by photolithography and etching, laser etching or another suitable process for removing material. The material of the alignment pattern is deposited into the recesses, for example by screen printing, laser printing, photolithography and etching of a uniform thin film material or another suitable process for depositing a pattern.

In the specific example of fig. 5C, the first alignment marks 540 are defined by a material, such as a fluorescent material or a light reflecting material, for example, disposed on an outer surface of the first substrate 502. The second alignment marks 542 are defined by a material, such as a fluorescent material or a light-reflecting material, for example, provided on the bottom wall 510 of the microfluidic chamber 506 and partially embedded therein. The alignment marks 540, 542 may form the same or different alignment patterns. Furthermore, in some embodiments, the alignment marks 540 and the top surface 512 need to be small enough so that they can conform to the same fluorescence microscope focal depth.

In the specific example of fig. 5D, the alignment marks 550 are defined by a material, such as a fluorescent material or a light-reflective material, for example, disposed on the sidewalls 514 of the microfluidic chamber 506. In the example of fig. 5D, the material is disposed on all of the sidewalls 514. In some examples, the material may be disposed on less than all of the sidewalls. The fluorescent material may be incorporated into the sidewall 514, for example, by incorporating a fluorophore-labeled adhesive (e.g., a pressure sensitive adhesive or an epoxy adhesive) into the bonding material. In some examples, the fluorescent material may be incorporated into the binding material, for example, by using a laser, such as an ultraviolet laser, to transfer the binding material to the fluorescent material.

In the specific example of fig. 5E, the first alignment marks 560 are defined by a material, such as a fluorescent material or a light reflecting material, for example, completely embedded in the first substrate 502. The second alignment marks 562 are defined by, for example, a material completely embedded in the second substrate 504, such as a fluorescent material or a light reflecting material. The alignment marks may have the same configuration or may be of different configurations.

Fig. 6 illustrates an example method of manufacturing a microfluidic device for analyzing fluorescence of a sample, such as for SBS analysis process. The microfluidic device includes an alignment pattern to facilitate alignment of the plurality of images. Fluorescent material is disposed on a surface of a first substrate in an alignment pattern (600). In some examples, a photoresist is deposited on the surface of the first substrate by, for example, spin coating, and patterned into the alignment pattern using photolithography. In some examples, the photoresist or fluorescent molecules are deposited onto the surface using screen printing, laser printing, 3D printing, or another suitable deposition and/or patterning method. The fluorescent molecule may be a fluorophore or some other molecule (e.g. nucleic acid, protein) labelled with a fluorophore. In some examples, a recess is defined in the first substrate, for example using photolithography and etching, and the recess is filled with a fluorescent material, for example photoresist or fluorescent molecules. In some embodiments, the fluorescent material fluoresces at one or more wavelengths at which the sample is to be imaged. In some embodiments, the fluorescent material fluoresces at all of the plurality of wavelengths at which the sample is to be imaged.

In some examples, the light reflecting material is disposed in an alignment pattern rather than the fluorescent material. The light reflecting material may be a patterned thin metal film formed, for example, by photolithography and etching, screen printing, laser printing, 3D printing, or any suitable deposition and/or patterning method.

Oligonucleotides are disposed on the same surface as the alignment pattern or on a different surface of the substrate (602). In some embodiments, the oligonucleotide is immobilized on the surface by covalent bonding. In some embodiments, the oligonucleotide is immobilized on a surface forming the microfluidic chamber.

The first and second substrates may be attached (604) to each other by an adhesive layer such that a microfluidic chamber is defined between the first and second substrates. The surface of the first substrate, the surface of the second substrate, and the bonding layer on which the alignment pattern is defined form the walls of the microfluidic chamber.

Referring to fig. 7A and 7B, in some examples, oligonucleotides (e.g., ssDNA) are used both for SBS analysis and as alignment markers. Fig. 7A depicts a typical SBS process. The oligonucleotide 702 is immobilized on (e.g., bound to) a wall 704 of the microfluidic chamber 700. The oligonucleotides include forward and reverse primers (depicted in black and grey, respectively). A nucleic acid of interest (e.g., DNA) is flowed through the microfluidic chamber 700 and hybridized to the oligonucleotides on the substrate. In some embodiments, the DNA clusters are generated by bridge amplification. The wavelength at which a given cluster fluoresces is controlled by the identity of the last nucleotide of the cluster. Fluorescence images 708 of the microfluidic chamber 700 are captured at multiple wavelengths, showing the DNA clusters that fluoresce at each wavelength.

Fig. 7B depicts an SBS process in which an immobilized reference oligonucleotide (e.g., single-stranded dna (ssdna)) is used as an alignment marker. In this process, the oligonucleotide 752 is immobilized on the wall 754 of the microfluidic chamber 750. The oligonucleotides include both forward and reverse primers (depicted in black and grey, respectively).

A reference oligonucleotide 760 is also immobilized on the wall 754 of the microfluidic chamber 750. The reference oligonucleotide 760 forms a DNA cluster after bridge amplification. In some embodiments, the reference oligonucleotide has two or more read primer target sites. During the sequencing reaction, two or more nucleic acids are synthesized for one DNA strand. As a result, the DNA cluster formed by the reference oligonucleotide 760 can produce a fluorescent signal of higher intensity than the surrounding DNA cluster formed by the nucleic acid of interest 756. Capturing a fluorescent image 758 of the microfluidic chamber 700 shows that the DNA cluster formed by the reference oligonucleotide 760 appears as a lighter spot in the image 758. These bright spots can be used as alignment marks.

Referring to fig. 8A, the reference oligonucleotide may have two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) read primer target sites for sequencing. The sequencing region after each read primer target site may have the same or different sequence. During the sequencing reaction, two or more fluorophore-labeled nucleotides are combined. Therefore, the luminance in the image is much higher. These bright spots can be used as alignment marks.

Referring to fig. 8B, the reference oligonucleotide (e.g., ssDNA) has a target site for a different reading primer for sequencing. In the presence of the read primer for the reference cluster, several sequencing cycles (e.g., 2-5 bases) can be performed on the reference cluster. The position of the reference cluster can be determined based on the base combination. The primers can then be denatured and conventional sequencing cycles can be performed using standard reading primers and reading primers of the reference cluster. The images may be aligned by reference cluster positions.

In some embodiments, these reference clusters can also be darkened by performing a sequencing reaction in the absence of a reading primer for the reference cluster. In some embodiments, the reference cluster can be labeled with a fluorescence quencher, such as a fluorescence quencher-labeled oligonucleotide that can hybridize to the reference cluster (fig. 9A).

Referring to fig. 9A-9B, the reference oligonucleotide (e.g., ssDNA) may have a unique sequence. The labeled oligonucleotide can hybridize to a single-stranded DNA in a DNA cluster formed by the reference oligonucleotide. In some embodiments, the oligonucleotide is labeled with a fluorescence Quencher (e.g., BHQ-1(Black Hole Quencher 1, 3')). In some embodiments, the fluorescence Quencher is 3'-BBQ-650(Black Berry Quencher 650), BHQ-0(Black Hole Quencher 0, 3'), BHQ-1(Black Hole Quencher 1,3'), BHQ-2(Black Hole Quencher 2, 3'), BHQ-3(Black Hole Quencher 3, 3'), BHQ-1(Black Hole Quencher-1, 5'), BHQ-2(Black Hole Quencher-2,5'), BHQ-3(Black Hole Quencher-3,5'), Atto 540Q, Atto 575Q, Atto 612Q, BBQ-650NHS (Black Berry Quencher 650NHS), BHQ-650-dT (Black Berry Quencher 650-650), BHQ-650-dT (BHQ-1-BHS), BHQ-650-dT (Black Berry Quencher 650-1), BHQ-2-dT (Black Hole Quencher 2dT), Dabcyl Quencher deoxythymidine dT, Dabcyl Quencher-3', Dabcyl-5', MGB 3' CDPI3, MGB-5' CDPI3, Tamra NHS, TAMRA-3' (carboxytetramethylrhodamine) or Tamra-dT (carboxytetramethylrhodamine-dT). As these labeled oligonucleotides bind to the reference DNA cluster, the fluorescence quencher can form alignment marks (e.g., dark motion) on the image where no fluorescence can be detected at or near these alignment marks.

Similarly, a labeled oligonucleotide may be labeled with one or more fluorophores (e.g., fluorescent dyes). In some embodiments, a labeled oligonucleotide may have about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10 fluorophore molecules. The fluorophore molecules may be different or identical. In some embodiments, a labeled oligonucleotide may have at least 2, 3, or 4 different fluorophore molecules. These labeled oligonucleotides can form bright spots in the image. These bright spots can be used for alignment. In some embodiments, the labeled oligonucleotide comprises a fluorophore molecule for a sequencing reaction in a labeled dNTP. As a result, the reference cluster will always form a bright spot in the image.

Referring to fig. 10, in some examples, the alignment pattern is formed of dark dots or material darker than the surrounding image. FIG. 10 shows a fluorescence image with fluorescence from a DNA cluster that fluoresces at the wavelength of the captured image. The gray dots (identified by arrows) represent dark alignment marks. In some examples, the dark alignment mark is formed by labeling a reference oligonucleotide with a fluorescence quenching moiety, e.g., in a predetermined pattern or a random pattern. In some examples, the dark alignment mark is formed by denaturing or destroying a portion of the oligonucleotides on the substrate, e.g., by irradiating the oligonucleotides with a laser to burn the oligonucleotides, e.g., in a predetermined pattern or in a random pattern. In some examples, the dark alignment marks are formed by etching recesses into the substrate such that the recesses will appear dark in the fluorescence image. In some examples, the dark alignment marks are formed of a patterned material that appears dark, such as a material that absorbs light of the wavelength used to excite the sample.

Fig. 11A-11C are schematic diagrams of multi-channel microfluidic devices. In these devices, multiple channels are vertically stacked such that, for example, a substrate forming a top wall of one channel of the device also forms a bottom wall of an adjacent channel. Having multiple vertically stacked channels in a single microfluidic device increases the number of surfaces on which DNA clusters can grow during SBS, thereby increasing the amount of data that can be collected from a single microfluidic device. For microfluidic cells with more than one surface for data generation, in some embodiments, the depth of focus of the fluorescence microscope allows images of these surfaces to be taken individually without interference from fluorescence on other surfaces. Thus, the fluorescence microscope can be adjusted for the appropriate focal plane and/or focal depth, and images can be taken for different surfaces.

With particular reference to fig. 11A, the multi-channel microfluidic device 10 includes two parallel

microfluidic channels

12, 14. Both channels are connected to a common inlet 16 and a common outlet 18, such that fluid flows from the inlet 16 out of the outlet 18 in parallel along both

channels

12, 14. The microfluidic device 10 is formed by three

substrates

20, 22, 24, each pair of substrates being attached to each other by a

bonding layer

26, 28, respectively.

Referring to fig. 11B, the multi-channel microfluidic device 30 comprises two

microfluidic channels

32, 34 connected in series. Fluid enters through inlet 36, flows along the channel 32 and then along the channel 34, and exits through outlet 38. The microfluidic device 30 is formed by three

substrates

40, 42, 44, each pair of substrates being attached to each other by a

bonding layer

46, 48, respectively.

Referring to fig. 11C, the multi-channel microfluidic device 50 comprises three

microfluidic channels

52, 53, 54 in series. Fluid enters through inlet 56, flows along the channel 52, the channel 53, and then the channel 54, and exits through outlet 58. The microfluidic device 50 is formed by four

substrates

60, 62, 64, 65, each pair of substrates being attached to each other by a bonding layer 66, 67, 68, respectively. The microfluidic device 50 has six surfaces on which SBS can be performed.

Alignment marks may be added to any surface of the substrate as shown in fig. 11A-11C.

Specific embodiments of the present subject matter have been described. Other embodiments are within the scope of the following claims.

Claims

1. A microfluidic device for analyzing fluorescence of a sample, the microfluidic device comprising:

a first substrate;

a second substrate, wherein the second substrate is optically transparent;

an adhesive layer disposed between the first substrate and the second substrate;

a microfluidic chamber defined between the first and second substrates such that the first and second substrates and the bonding layer form a wall of the microfluidic chamber; and

an oligonucleotide bound to at least one of the walls of the microfluidic chamber; and

a fluorescent material disposed on at least one of the walls of the microfluidic chamber and forming an alignment pattern.

2. The microfluidic device according to claim 1, wherein the fluorescent material comprises a photoresist disposed on at least one wall of the microfluidic channel.

3. The microfluidic device according to claim 1 or 2, wherein the fluorescent material comprises a fluorescent molecule bound or adhered to at least one wall of the microfluidic channel.

4. The microfluidic device according to any one of the preceding claims, wherein the fluorescent material is disposed on a first wall of the microfluidic channel formed by the first substrate, a second wall of the microfluidic channel formed by the second substrate, or both.

5. The microfluidic device according to any one of the preceding claims, wherein the fluorescent material is disposed on a sidewall of the microfluidic channel formed by the adhesive layer.

6. The microfluidic device according to any one of the preceding claims, wherein the fluorescent material is partially embedded in at least one wall of the microfluidic channel.

7. The microfluidic device according to any one of the preceding claims, wherein the fluorescent material comprises an oligonucleotide-labeled reference oligonucleotide labeled with a fluorophore.

8. The microfluidic device according to claim 7, wherein each reference oligonucleotide is labeled with two or more fluorophore-labeled oligonucleotides.

9. The microfluidic device according to any one of the preceding claims, wherein the alignment pattern comprises a predetermined pattern or a random pattern.

10. The microfluidic device according to any one of the preceding claims, wherein the fluorescence of the sample is detected at a plurality of emission wavelengths, and wherein the fluorescent material fluoresces at least at one of the plurality of emission wavelengths.

11. A system for analyzing fluorescence of a sample, the system comprising:

the microfluidic device of any one of the preceding claims; and

a fluorescence microscope configured to capture fluorescence images of a sequencing reaction in the microfluidic chamber of the microfluidic device.

12. A method of manufacturing a microfluidic device for analyzing fluorescence of a sample, the method comprising:

disposing a fluorescent material on a surface of a first substrate in an alignment pattern;

binding the oligonucleotide to the surface of the first substrate or the surface of the second substrate: and

attaching the first substrate to the second substrate with an adhesive layer such that a microfluidic chamber is defined between the first substrate and the second substrate and such that the surface of the first substrate, the surface of the second substrate and the adhesive layer form walls of the microfluidic chamber,

wherein at least one of the first substrate or the second substrate is optically transparent.

13. The method of claim 12, wherein disposing the fluorescent material on a surface of a first substrate comprises disposing a photoresist on the first substrate.

14. The method of claim 12 or 13, wherein disposing the fluorescent material on a surface of a first substrate comprises disposing fluorescent molecules on the first substrate.

15. The method of any one of claims 12 to 14, comprising defining the alignment pattern in the layer of fluorescent material using photolithography.

16. The method of any one of claims 12 to 15, comprising printing the fluorescent material in the alignment pattern on the surface of the first substrate.

17. The method according to any one of claims 12 to 16, comprising:

defining the alignment pattern in the first substrate; and

the defined pattern is at least partially filled with the fluorescent material.

18. The method of claim 17, comprising etching the alignment pattern in the first substrate.

19. The method of any one of claims 12 to 18, wherein disposing the oligonucleotide on the surface of the first substrate or the surface of the second substrate comprises: binding the oligonucleotide to the surface of the first substrate or the surface of the second substrate.

20. A microfluidic device for analyzing a sample, the microfluidic device comprising:

a first substrate;

a second substrate, wherein the second substrate is optically transparent;

a microfluidic chamber defined between the first and second substrates such that the first and second substrates and the bonding layer form a wall of the microfluidic chamber;

a set of oligonucleotides bound to at least one of the walls of the microfluidic chamber, wherein each strand of the set of oligonucleotides comprises a primer for cluster generation;

a set of reference oligonucleotides bound to at least one of the walls of the microfluidic chamber, wherein the set of reference oligonucleotides is configured to generate an alignment mark.

21. The microfluidic device according to claim 20, wherein each strand of the set of reference oligonucleotides comprises two or more read primers for sequencing-by-synthesis.

22. The microfluidic device according to claim 20, wherein each strand of the set of reference oligonucleotides comprises a read primer for sequencing by synthesis, wherein the read primer is different from the read primer of the first set of oligonucleotides.

23. The microfluidic device according to claim 21 or 22, wherein the set of reference oligonucleotides is configured to have a signal intensity higher than the average signal intensity of the DNA cluster.

24. The microfluidic device according to any of claims 20 to 23, wherein the set of reference oligonucleotides are labeled with a fluorescent dye.

25. The microfluidic device according to any one of claims 20 to 23, wherein the set of reference oligonucleotides are labeled with a fluorescence quencher.

26. A method of manufacturing a microfluidic device for analyzing fluorescence of a sample, the method comprising:

binding a first set of oligonucleotides to a surface of a first substrate, wherein the first set of oligonucleotides comprises primers for cluster generation;

binding a second set of oligonucleotides to a surface of the first substrate or a surface of a second substrate, wherein the second set of oligonucleotides is configured to generate an alignment pattern; and

wherein at least one of the first substrate or the second substrate is optically transparent at each of the plurality of emission wavelengths.

27. A microfluidic device comprising:

a first substrate;

a second substrate;

a first adhesive layer disposed between the first substrate and the second substrate;

a first microfluidic channel defined between the first substrate and the second substrate such that the first substrate and the second substrate and the first adhesive layer form walls of the first microfluidic channel;

a third substrate;

a second adhesive layer disposed between the second substrate and the third substrate; and

a second microfluidic channel defined between the second substrate and the third substrate such that the second substrate and the third substrate and the second adhesive layer form walls of the second microfluidic channel.

28. The microfluidic device according to claim 27, comprising oligonucleotides bound to at least one of the walls of the first microfluidic channel and at least one of the walls of the second microfluidic channel, wherein the first set of oligonucleotides comprises primers for cluster generation.

29. The microfluidic device according to claim 27 or 28, wherein an inlet and an outlet are defined in the first substrate.

30. The microfluidic device according to any one of claims 27 to 29, wherein an inlet is defined in the first substrate and an outlet is defined in the third substrate.

31. The microfluidic device according to any one of claims 27 to 30, wherein the first and second microfluidic channels are fluidically connected in parallel.

32. The microfluidic device according to any one of claims 27 to 31, wherein the first and second microfluidic channels are fluidically connected in series.

33. The microfluidic device according to any one of claims 27 to 32, comprising:

a fourth substrate;

a third bonding layer provided between the third substrate and the fourth substrate; and

a third microfluidic channel defined between the third and fourth substrates such that the third and fourth substrates and the third bonding layer form walls of the third microfluidic channel.

34. The microfluidic device according to claim 33, wherein an outlet is defined in the fourth substrate.

35. The microfluidic device according to claim 33 or 34, wherein the first, second and third microfluidic channels are fluidically connected in series.

36. The microfluidic device according to any one of claims 27 to 35, wherein the first, second and third microfluidic channels are fluidically connected in series.

37. The microfluidic device according to any one of claims 27 to 36, wherein the first, second, third and/or fourth substrate is optically transparent.

38. A microfluidic device for analyzing fluorescence of a sample, the microfluidic device comprising:

a microfluidic chamber defined by a first inner surface and a second inner surface;

a first set of oligonucleotides bound to the first inner surface of the microfluidic chamber; and

one or more alignment marks secured to the microfluidic chamber forming an alignment pattern for the first inner surface.

39. The microfluidic device according to claim 38, wherein the alignment mark is a fluorescent mark, a reflective mark, or a dark mark.

40. The microfluidic device according to claim 39, wherein the fluorescent label is formed from a fluorophore.

41. The microfluidic device according to claim 39, wherein the reflective marker is formed of a reflective material.

42. The microfluidic device according to claim 39, wherein the dark mark is formed from a fluorescence quencher.

43. The microfluidic device according to claim 39, wherein the dark mark is formed by removing one or more oligonucleotides located at the dark mark.

44. The microfluidic device according to claim 38, wherein the alignment mark is formed by a set of reference oligonucleotides.

45. The microfluidic device according to claim 44, wherein each strand of the reference oligonucleotide has two or more read primers for sequencing-by-synthesis.

46. The microfluidic device according to claim 44, wherein each strand of the reference oligonucleotide has a read primer for sequencing-by-synthesis, wherein the read primer in each strand of the reference oligonucleotide is different from the read primer for sample analysis.

47. The microfluidic device according to claim 44, wherein the reference oligonucleotide is labeled with a fluorescence quencher.

48. The microfluidic device according to claim 44, wherein the reference oligonucleotide is labeled with a fluorophore.

49. The microfluidic device according to any of claims 38-48, wherein the alignment mark is deposited on or embedded in the first inner surface.

50. The microfluidic device according to any of claims 38-49, wherein the microfluidic chamber comprises a second inner surface and a second set of oligonucleotides is bound to the second inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the second inner surface.

51. The microfluidic device according to claim 50, wherein the microfluidic chamber comprises a third inner surface and a third set of oligonucleotides is bonded to the third inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the third inner surface.

52. The microfluidic device according to claim 51, wherein the microfluidic chamber comprises a fourth inner surface and a fourth set of oligonucleotides is bonded to the fourth inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the fourth inner surface.

53. The microfluidic device according to claim 52, wherein the microfluidic chamber comprises a fifth inner surface and a fifth set of oligonucleotides is bonded to the fifth inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the fifth inner surface.

54. The microfluidic device according to claim 53, wherein the microfluidic chamber comprises a sixth inner surface and a sixth set of oligonucleotides is bonded to the sixth inner surface of the microfluidic chamber, wherein one or more alignment marks form an alignment pattern for the sixth inner surface.

55. A method of sequencing a set of nucleic acids of interest, comprising:

disposing the set of nucleic acids of interest to the microfluidic device according to any one of claims 38-54 and allowing the set of nucleic acids to randomly hybridize to the oligonucleotides in the microfluidic chamber;

amplifying the nucleic acid of interest, thereby forming a DNA cluster;

providing read primers and labeled dntps to initiate sequencing-by-synthesis;

capturing images of fluorescently labeled DNA clusters for multiple cycles of sequencing while synthesizing;

aligning the images through the alignment marks, thereby identifying the positions of the DNA clusters on different images; and

determining the sequence of the nucleic acid of interest.

56. The method of claim 55, wherein the method comprises adjusting a focal plane of a fluorescence microscope to capture images of the DNA clusters on different inner surfaces.