HK1234771B - Disposable, integrated microfluidic cartridge and methods of making and using same - Google Patents

Description

Disposable integrated microfluidic cartridge and methods of making and using the same

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application Serial No. 61/951,462, filed March 11, 2014, and U.S. Provisional Application Serial No. 61/987,699, filed May 2, 2014, the contents of which are incorporated herein by reference in their entirety and for all purposes.

Background of the Invention

Integrated microfluidic cartridges with complementary metal oxide semiconductor (CMOS) technology, such as CMOS image sensors, and fluid channels are difficult to manufacture. In most cases, the fluid channels are designed within the CMOS surface, which reduces the active area and leads to complex flow patterns. Therefore, new methods for integrating CMOS technology into multi-compartment microfluidic cartridges are needed. Furthermore, because microbubbles are present in the PCR mix and these microbubbles expand during the PCR process, sealing the polymerase chain reaction (PCR) zone in the microfluidic cartridge is a significant challenge. Therefore, new methods for sealing the PCR zone in the microfluidic cartridge are needed.

SUMMARY OF THE INVENTION

The disclosed embodiments relate to a microfluidic box for detecting biological reactions. In some embodiments, the microfluidic box is configured to perform sequencing operations on nucleic acid samples. In one aspect, the microfluidic box includes a stack of fluid layers defining channels and valves for processing the nucleic acid sample to be sequenced, and a solid-state CMOS biosensor integrated in the stack. The biosensor has an active area configured to detect signals of a biological reaction, wherein substantially all of the active area can be used for reagent delivery and illumination during operation. In another aspect, the microfluidic box includes: (a) a flow cell comprising a reaction site area comprising one or more reaction sites; (b) a fluid channel for delivering reactants to the reaction site area and/or removing reactants from the reaction site area; (c) a biosensor having an active area configured to detect signals of a biological reaction in the reaction site area. The reaction site area is close to the active area of the biosensor and the reaction site area spans substantially all of the active area of the biosensor. In some embodiments, the fluid channel does not substantially overlap with the active area of the biosensor.

In a first general aspect, a microfluidic cartridge is configured to perform a sequencing operation on a nucleic acid sample. The microfluidic cartridge comprises: (a) a bioassay system comprising a stack of fluidic layers defining channels and valves for processing the nucleic acid sample to be sequenced; and (b) a solid-state CMOS biosensor integrated in the stack and fluidically and optically coupled to the bioassay system, the biosensor comprising an active area configured to detect signals of a biological reaction, wherein substantially all of the active area is available for reagent delivery and illumination during operation. In some embodiments, the microfluidic cartridge further comprises a housing that at least partially surrounds the stack of fluidic layers and the CMOS sensor. In some implementations, the bioassay system comprises a flow cell mounted on the biosensor.

In a second general aspect, a microfluidic cartridge for detecting a biological reaction is disclosed. The microfluidic cartridge includes: (a) a flow cell including a reaction site area containing one or more reaction sites; (b) a fluid channel for delivering reactants to the reaction site area and/or removing reactants from the reaction site area; (c) a biosensor having an active area configured to detect signals of a biological reaction in the reaction site area. In some implementations, the reaction site area is proximate to the active area of the biosensor, and the reaction site area spans all or substantially all of the active area of the biosensor. In some implementations, the fluid channel does not substantially or does not overlap with the active area of the biosensor.

In some embodiments of the microfluidic cartridge of the second general aspect, the biosensor comprises a light detector. In some implementations, the light detector is a CMOS or CCD sensor. In some implementations, the CMOS sensor is approximately 9200 μm long, approximately 8000 μm wide, approximately 800-1000 μm thick, and has approximately 50 I/O pads.

In some implementations, the microfluidic box of the second overall aspect is configured to perform sequencing operations on nucleic acid samples. The flow cell includes a sequencing chamber, and the signal of the detected biological reaction indicates the type of nucleotide bases involved in the biological reaction. In some implementations, the sequencing chamber is formed on a sequencing chamber layer, and the biosensor is arranged in an opening on a sequencing chamber bottom layer below the sequencing chamber layer, and the fluid channel is formed on a fluid channel layer below the sequencing chamber bottom layer. In some implementations, the flow cell includes a substrate in a hydrophilic area for nucleic acid attachment and amplification surrounded by a hydrophobic area. In some implementations, the reaction site area spans the entirety of the active area of the biosensor.

In some implementations of the microfluidic cartridge of the first and second general aspects, the cartridge further comprises a PCR zone, a reagent mixing and dispensing zone, and one or more membrane valves configured to reversibly prevent the PCR zone from being in fluid communication with the reagent mixing and dispensing zone or the flow cell comprising the reaction site zone. In some implementations, the microfluidic cartridge further comprises a flexible PCB heater. In some implementations, the PCR zone comprises a plurality of PCR channels. In some implementations, the reagent mixing and dispensing zone comprises a plurality of reagent channels and/or reagent feeders. In some implementations, the cartridge further comprises a rotary valve configured to fluidically connect the PCR zone to the reagent mixing and dispensing zone. In some implementations, the rotary valve is further configured to fluidically connect the reagent mixing and dispensing zone to the flow cell comprising the reaction site zone.

In a third general aspect, a stack of fluidic layers of a microfluidic cartridge for nucleic acid molecule sequencing is disclosed. The stack of fluidic layers includes: (a) a sequencing chamber layer having a sequencing chamber region configured to perform clustering and sequencing reactions; (b) a sequencing chamber bottom layer disposed below the sequencing chamber layer, the sequencing chamber bottom layer having an opening configured to accommodate an image sensor with an active area of the image sensor disposed below the sequencing chamber region; (c) a flexible printed circuit board (PCB) layer below the sequencing chamber bottom layer; and (d) a fluidic channel layer disposed below the flexible printed circuit board (PCB) layer, the fluidic channel layer including fluidic channels configured to deliver reactants to the sequencing chamber region.

In some implementations of the stack of fluid layers, the sequencing chamber area spans substantially all of the active area of the image sensor. In some implementations, the fluid channel does not substantially overlap with the active area of the image sensor. In some implementations, the sequencing chamber layer and the sequencing chamber bottom layer include openings for a plurality of membrane valves. In some implementations, the stack of fluid layers further includes a membrane layer disposed on the sequencing chamber layer. The membrane layer, the openings on the sequencing chamber layer and the sequencing chamber bottom layer, and the flexible PCB layer are configured to form a plurality of membrane valves. In some implementations, at least some of the membrane valves are configured to provide a reversible seal of the PCR zone of the microfluidic box, separated from the reagent mixing and dispensing zone of the microfluidic box.

In a fourth general aspect, a method for operating a microfluidic cartridge is provided. In some implementations, the method involves: (a) performing a polymerase chain reaction on a sample in a PCR zone of the microfluidic cartridge; and/or mixing the sample with one or more reagents in a reagent mixing and dispensing zone of the microfluidic cartridge; (b) transferring the sample to a sequencing chamber via a fluid channel, wherein the sequencing chamber: (1) is located at a different location than the PCR zone and/or the reagent mixing and dispensing zone, and (2) the sequencing chamber does not substantially overlap with the fluid channel; (c) performing a sequencing reaction on the sample; and (d) imaging the sequencing reaction using an image sensor having an active area adjacent to the sequencing chamber. In some implementations, the sequencing chamber spans substantially the entirety of the active area. In some implementations, the method further involves: while performing the polymerase chain reaction, sealing the PCR zone from the reagent mixing and dispensing zone; transferring the sample from the PCR zone to the reagent mixing and dispensing zone, and then mixing the sample with one or more reagents.

In a fifth general aspect, a method of manufacturing a microfluidic cartridge is provided. The method involves: (a) forming a fluidics layer comprising a printed circuit board (PCB); (b) attaching an image sensor to the PCB, wherein the image sensor is positioned such that substantially all of an active area of the image sensor is available for illumination and/or reagent delivery; (c) assembling a stack comprising the fluidics layer and the image sensor, and (d) forming the microfluidics cartridge comprising the fluidics layer and the image sensor. In some implementations, the image sensor is a CMOS image sensor.

In some implementations, the stack of fluidic layers includes: (a) a sequencing chamber layer having a sequencing chamber region configured to perform clustering and sequencing reactions; (b) a sequencing chamber bottom layer disposed below the sequencing chamber layer, the sequencing chamber bottom layer including an opening configured to contain an image sensor with an active area of the image sensor disposed below the sequencing chamber region; (c) a flexible PCB layer including the PCB below the sequencing chamber bottom layer; and (d) a fluidic channel layer disposed below the flexible PCB layer, wherein the fluidic channel layer includes a fluidic channel configured to deliver reactants to the sequencing chamber region. In some implementations, the fluidic channel does not, or at least does not substantially, overlap with the sequencing chamber region.

Join by reference

All patents, patent applications, and other publications mentioned herein (including all sequences disclosed in such references) are expressly incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. All documents cited are, in relevant part, incorporated herein by reference in their entirety for the purposes indicated by the context in which they are cited herein. However, the citation of any document should not be construed as an admission that it is prior art with respect to the present disclosure.

Summary of the Figures

FIG1 depicts a flow chart illustrating an example of a method for monolithic integration of flexible printed circuit boards (PCBs) and roll-to-roll (R2R) printed electronics for CMOS technology and digital fluidics;

FIG2 depicts an exploded view of an example of a fluid stack having certain layers that may be laminated and bonded together using the method of FIG1 ;

FIG3 depicts a perspective view of an example of a CMOS device that can be integrated into the fluidics layer of a microfluidic cartridge using the method of FIG1 ;

4A , 4B, 5 , 6 , and 7 depict side views of a structure and illustrate an example of a method of attaching a CMOS device to a flexible PCB using the method of FIG. 1 ;

FIG8 depicts a side view of an example of a structure formed using the method of FIG1 , wherein the fluidics layer and CMOS devices are integrated together in a microfluidics cartridge;

9A and 9B depict perspective views of examples of membrane valves that may be integrated into the fluidics layer;

10A and 10B depict cross-sectional views of a membrane valve in open and closed states, respectively;

FIG11 depicts a schematic diagram of an example of a microfluidic cartridge including integrated CMOS technology and digital fluidics;

12 and 13 depict perspective views of a microfluidic cartridge assembly (which is one example of a physical embodiment of the integrated microfluidic cartridge shown in FIG. 11 );

14A and 14B depict perspective views of examples of fluidic assemblies installed in the microfluidic cartridge assembly shown in FIGS. 12 and 13 ;

15A and 15B depict plan and cross-sectional views, respectively, of examples of heater traces that can be installed in the fluidic assembly shown in FIGs. 14A and 14B;

16 , 17 , 18 , 19 , 20A and 20B depict various other views of the microfluidic cartridge assembly of FIG. 12 , showing greater detail thereof;

21 to 29 depict a method of deconstructing the microfluidic cartridge assembly of FIG. 12 as a means of showing its internal components;

FIG30 illustrates a transparent perspective view of a portion of the microfluidic cartridge assembly of FIG12 and showing the various reagent fluid reservoirs and their sample loading ports;

FIG31 illustrates another transparent perspective view of a portion of the microfluidic cartridge assembly of FIG12 and further illustrating fluid channels thereof;

FIG32 illustrates a cross-sectional view of the microfluidic cartridge assembly of FIG12 showing further details of the microfluidic cartridge assembly;

33A, 33B, 34A, 34B and 35 illustrate various views of the housing of the microfluidic cartridge assembly of FIG. 12 showing further details of the housing;

36 , 37 , 38A, 38B and 39 illustrate various views of the base plate of the microfluidic cartridge assembly of FIG. 12 , showing greater detail of the base plate;

40A and 40B depict additional perspective views of the fluidics assembly of the microfluidic cartridge assembly showing further details thereof;

41A , 41B and 41C depict additional views showing more detail of the flexible PCB heater of the fluidics assembly of the microfluidic cartridge assembly;

42A and 42B illustrate perspective and plan views, respectively, of the inlet/outlet port layer of the fluidics layer shown in FIG. 2 and FIG. 14 ;

43A and 43B illustrate a perspective view and a plan view, respectively, of the fluidic channel layer of the fluidic layer shown in FIG. 2 and FIG. 14 ;

44A and 44B illustrate perspective and plan views, respectively, of the flexible PCB layer of the fluidic layer shown in FIG. 2 and FIG. 14 ;

45A and 45B illustrate perspective and plan views, respectively, of the bottom layer of the sequencing chamber of the fluidics layer shown in FIG. 2 and FIG. 14 ;

46A and 46B illustrate a perspective view and a plan view, respectively, of the sequencing chamber layer of the fluidics layer shown in FIG. 2 and FIG. 14 ;

47A and 47B show perspective and plan views, respectively, of the membrane layer of the fluidics layer and the top layer of the sequencing chamber shown in FIG. 2 and FIG. 14 ;

48A and 48B show a flow chart of an example of a method for using the microfluidic cartridge assembly to perform multiplex PCR and downstream mixing required for sequencing;

FIG49 depicts a side view of an example of a CMOS flow cell in which up to approximately 100% of the biosensor active area can be used for reagent delivery and illumination;

FIG50 depicts an exploded view of an example of one embodiment of the CMOS flow cell shown in FIG49;

51 and 52 depict perspective and side views, respectively, of the CMOS flow cell shown in FIG. 50 when fully assembled;

FIG53 depicts a perspective view of an example of a flow cell cover for the CMOS flow cell shown in FIG50 , 51 and 52 ;

Figures 54, 55, 56 and 57 depict examples of methods for providing an extended planar surface in the CMOS flow cell onto which the flow cell cover can be mounted;

Figures 58A, 58B, 58C and 58D depict another example of a method of providing an extended planar surface in the CMOS flow cell onto which the flow cell cover can be mounted;

Figures 59, 60, 61 and 62 depict yet another example of a method of providing an extended planar surface in the CMOS flow cell onto which the flow cell cover can be mounted.

Detailed Description of the Invention

Unless otherwise indicated, the practice of the methods and systems disclosed herein relates to conventional techniques and apparatus commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and the recombinant DNA field, which are within the skill of the art. Such techniques and apparatus are known to those skilled in the art and are described in numerous texts and references (see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," Third Edition (Cold Spring Harbor), [2001]); and Ausubel et al., "Current Protocols in Molecular Biology" [1987]).

Numerical ranges are inclusive of the numbers defining the range. It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein.

The headings provided herein are not intended to limit this disclosure.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Various scientific dictionaries containing the terms included herein are well known and available to those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments disclosed herein, some methods and materials are described.

The terms defined immediately below are described in more detail by reference to the specification as a whole.It will be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by one skilled in the art.

introduction

Sequencing methods

Method described herein can be used in conjunction with various nucleic acid sequencing technologies. Particularly suitable technologies are those in which nucleic acids are attached to fixed positions by arrays so that their relative positions do not change and wherein the arrays are imaged repeatedly. In embodiments in which images are obtained in different color channels, for example, overlapping with different labels for distinguishing a nucleotide base type from another is particularly suitable. In some embodiments, the process of determining the nucleotide sequence of a target nucleic acid can be a kind of automated process. Preferred embodiments include sequencing by synthesis ("SBS") technology.

Sequencing by synthesis ("SBS") techniques generally involve enzymatic extension of nascent nucleic acid chains by iterative addition of nucleotides against a template strand. In traditional SBS methods, a single nucleomonomer can be provided to a target nucleic acid in the presence of a polymerase in each delivery. However, in the methods described herein, more than one type of nucleomonomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.

SBS can utilize nucleotide monomers with terminator moieties or those lacking any terminator moieties. Methods utilizing nucleotide monomers lacking terminators include, for example, pyrophosphate sequencing and sequencing using γ-phosphate-labeled nucleotides, as described in further detail below. In methods using nucleotide monomers lacking terminators, the number of nucleotides added in each cycle is generally variable and depends on the template sequence and the mode of nucleotide delivery. For SBS techniques utilizing nucleotide monomers with terminator moieties, the terminator can be effectively irreversible under the sequencing conditions used in the case of traditional Sanger sequencing using dideoxynucleotides; or the terminator can be reversible in the case of sequencing methods developed by Solexa (now Illumina, Inc.).

SBS technology can utilize nucleotide monomers with a labeling moiety or those lacking a labeling moiety. Thus, incorporation events can be detected based on properties of the label, such as the fluorescence of the label; properties of the nucleotide monomer, such as molecular weight or charge; byproducts of the incorporation of the nucleotide, such as the release of pyrophosphate; or the like. In embodiments where two or more different nucleotides are present in the sequencing reagent, the different nucleotides may be distinguishable from each other; or alternatively, the two or more different labels may be indistinguishable under the detection technology being used. For example, the different nucleotides present in the sequencing reagent may have different labels and they may be distinguished using appropriate optics, as demonstrated by the sequencing method developed by Solexa (now Illumina, Inc.).

Preferred embodiments include pyrophosphate sequencing. Pyrophosphate sequencing detects the release of inorganic pyrophosphate (PPi) as specific nucleotides are incorporated into nascent chains (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing." Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) "A sequencing method based on real-time pyrophosphate." Science 281(5375),363; U.S. Patent No. 6,210,891; U.S. Patent No. 6,258,568 and U.S. Patent No. 6,274,320, the disclosures of which are incorporated herein by reference in their entireties). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfatase, and the level of generated ATP is detected via photons produced by luciferase. The nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminescent signal generated by the incorporation of nucleotides at the features of the array. After treating the array with a specific nucleotide type (e.g., A, T, C, or G), an image can be obtained. The image obtained after the addition of each nucleotide type is different with respect to which features in the array are detected. These differences in the image reflect the different sequence content of the feature on the array. However, the relative position of each feature in the image will remain unchanged. The image can be stored, processed, and analyzed using the methods described herein. For example, images obtained after treating the array with each different nucleotide type can be processed in the same manner as images obtained from different detection channels for a reversible terminator-based sequencing method, as demonstrated herein.

In another exemplary type of SBS, cycle sequencing is accomplished by the gradual addition of reversible terminator nucleotides containing, for example, cleavable or photobleachable dyes as described in International Patent Publication No. WO 04/018497 and U.S. Patent No. 7,057,026 (the disclosures of which are incorporated herein by reference). This method is being commercialized by Solexa (now Illumina Inc.) and is described in International Patent Publication No. WO 91/06678 and International Patent Publication No. WO 07/123,744, each of which is incorporated herein by reference. The availability of fluorescently labeled terminators (wherein the termination is reversible and the fluorescent label is cleavable) facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

Preferably, in a sequencing embodiment based on a reversible terminator, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label can be, for example, removable by cutting or degradation. Following the label incorporation into the nucleic acid features of the array, an image can be captured. In a specific embodiment, each cycle involves the simultaneous delivery of four different nucleotide types to the array and each nucleotide type has a spectrally different label. Four images can then be obtained, each using a detection channel that is selective for one of the four different labels. Alternatively, different nucleotide types can be added in sequence and an image of the array can be obtained between each addition step. In this embodiment, each image will show the nucleic acid features that have been incorporated with a specific type of nucleotide. Due to the different sequence content of each feature, different features will be present or absent in different images. However, the relative position of the feature in the image will remain unchanged. The images obtained from such a reversible terminator-SBS method can be stored, processed, and analyzed as described herein. After the image capture step, the label can be removed and the reversible terminator portion can be removed for subsequent cycles of nucleotide addition and detection. Removal of the label after detection of the label in a particular cycle and before subsequent cycles can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are set forth below.

In a specific embodiment, part or all of nucleotide monomers can include reversible terminators. In such an embodiment, reversible terminator/cleavable fluorescent label can include a fluorescent label (Metzker, Genome Res.15:1767-1776 (2005) connected to a ribose moiety via a 3' ester bond. Other methods have isolated terminator chemistry (Ruparel et al., Proc Natl Acad Sci USA 102:5932-7 (2005), which are incorporated herein by reference in their entirety) from the cutting of fluorescent labels. Ruparel et al. have described the development of reversible terminators, which use little 3' allyl groups to block extension, but can be easily unsealed by utilizing the short time processing of palladium catalysts. Fluorophore is attached to base via a photodegradable joint, which can be easily cut by being exposed to long wavelength UV light for 30 seconds. Therefore, disulfide reduction or photo-cleavage can be used as a cleavable joint. Another method of reversible termination is to use natural termination that occurs after a bulky dye is placed on the dNTP. The presence of a charged bulky dye on the dNTP can act as an effective terminator through steric hindrance and/or electrostatic hindrance. Unless the dye is removed, the presence of an incorporation event prevents further incorporation. Cleavage of the dye removes the fluorescent label and effectively reverses the termination. Examples of modified nucleotides are also described in U.S. Patent No. 7,427,673 and U.S. Patent No. 7,057,026, the disclosures of which are incorporated herein by reference in their entirety.

Additional exemplary SBS systems and methods that can be used with the methods and systems described herein are described in U.S. Patent Publication No. 2007/0166705, U.S. Patent Publication No. 2006/0188901, U.S. Patent 7,057,026, U.S. Patent Publication No. 2006/0240439, U.S. Patent Publication No. 2006/0281109, International Patent Publication No. WO 05/065814, U.S. Patent Publication No. 2005/0100900, International Patent Publication No. WO 06/064199, International Patent Publication No. WO 07/010,251, U.S. Patent Publication No. 2012/0270305, and U.S. Patent Publication No. 2013/0260372, the disclosures of which are incorporated herein by reference in their entireties.

Some embodiments can utilize detection of four different nucleotides using fewer than four different tags. For example, SBS can be performed using the methods and systems described in the incorporation materials of U.S. Patent Publication No. 2013/0079232 (which is incorporated herein by reference in its entirety for the purposes indicated by the context of the citation herein).

As a first example, a pair of nucleotide types can be detected at the same wavelength, but based on the intensity difference of one member of the pair compared to the other member; or based on the change of one member of the pair (such as via chemical modification, photochemical modification or physical modification) that causes the signal to appear or disappear compared to the signal detected for the other member of the pair and distinguished. As a second example, three of four different nucleotide types can be detected under specific conditions, while the fourth nucleotide type lacks a label that is detectable under these conditions or is detected to a minimum extent under these conditions (for example, due to minimum detection of background fluorescence, etc.). The incorporation of the first three nucleotide types into nucleic acids can be determined based on the presence of their respective signals and the incorporation of the fourth nucleotide type into nucleic acids can be determined based on the absence or minimum detection of any signal. As a third example, a nucleotide type can include one (or more) labels detected in two different channels, while other nucleotide types are detected in no more than one channel of the channel. The aforementioned three exemplary configurations are not considered to be mutually exclusive and can be used in various combinations. An exemplary embodiment combining all three examples is a fluorescence-based SBS method using a first nucleotide type detected in a first channel (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type detected in a second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type detected in both the first and second channels (e.g., dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelengths), and a fourth nucleotide type lacking a label that is not or minimally detected in either channel (e.g., unlabeled dGTP).

In addition, as described in the introduction materials of U.S. Patent Publication No. 2013/0079232 (which is incorporated herein by reference in its entirety for the purposes indicated in the context of the citation herein), a single channel can be used to obtain sequencing data. In this so-called single-dye sequencing method, the first nucleotide type is labeled, but the label is removed after the first image is generated, and the second nucleotide type is labeled only after the first image is generated. The third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type is unlabeled in both images.

Some embodiments can utilize sequencing by connecting technology. Such technology utilizes DNA ligase to mix oligonucleotide and determine the mixing of such oligonucleotide. The different marks that this oligonucleotide usually has are relevant to the identity of specific nucleotide in the sequence of this oligonucleotide hybridization. Similar to other SBS methods, image can be obtained after processing the array of nucleic acid feature with labeled sequencing reagent. Each image will show the nucleic acid feature of the mark that has been mixed with a specific type. Because the sequence content of each feature is different, different features will exist or not in different images, but the relative position of this feature in this image will remain unchanged. The image obtained from the sequencing method based on connection can be stored, processed and analyzed as described herein. The exemplary SBS system and method that can be used together with the method and system described herein are described in U.S. Patent No. 6,969,488, U.S. Patent No. 6,172,218 and U.S. Patent No. 6,306,597 (its disclosure content is fully incorporated into this paper by reference).

Some embodiments can utilize nanopore sequencing (Deamer, D.W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis". Acc. Chem. Res. 35: 817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J.A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope." Nat. Mater. 2: 611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or a biological membrane protein, such as α-hemolysin. As the target nucleic acid passes through the nanopore, each base pair can be determined by measuring the fluctuations in the electrical conductance in the pore. (U.S. Patent 7,001,792; Soni, G.V. & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, S.L., Chu, J., Amorin, M. & Ghadiri, M.R. "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution." J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties.) Data obtained from nanopore sequencing can be stored, processed, and analyzed as described herein. In particular, the data may be processed into images according to the exemplary processing of light images and other images set forth herein.

Some embodiments can utilize the method for real-time monitoring related to DNA polymerase activity.Nucleotide incorporation can, for example, as described in U.S. Patent No. 7,329,492 and U.S. Patent No. 7,211,414 (each of which is incorporated herein by reference), by fluorescence resonance energy transfer (FRET) interaction between fluorophore-loaded polymerase and gamma-phosphate-labeled nucleotide to detect or nucleotide incorporation can, for example, as described in U.S. Patent No. 7,315,019 (each of which is incorporated herein by reference), utilize zero-mode waveguide and for example, as described in U.S. Patent No. 7,405,281 and U.S. Patent Publication No. 2008/0108082 (each of which is incorporated herein by reference), use fluorescent nucleotide analogs and engineered polymerase to detect. Illumination can be confined to a zeptoliter-scale volume surrounding the surface-tethered polymerase, allowing incorporation of fluorescently labeled nucleotides to be observed with low background (Levene, M.J. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations." Science 299, 682-686 (2003); Lundquist, P.M. et al. "Parallel confocal detection of single molecules in realtime." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties). Images obtained from such methods can be stored, processed, and analyzed as described herein.

Some SBS embodiments include the detection of protons released after nucleotides are incorporated into the extension products. For example, sequencing based on the detection of the released protons can use electrical detectors and the associated technology commercially available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies) or U.S. Patent Publication No. 2009/0026082; U.S. Patent Publication No. 2009/0127589; U.S. Patent Publication No. 2010/0137143; or the sequencing method and system described in U.S. Patent Publication No. 2010/0282617 (each of which is incorporated herein by reference). The method for amplifying target nucleic acids using kinetic exclusion as set forth herein can be easily applied to substrates for detecting protons. More specifically, the method set forth herein can be used to produce clonal populations of amplicons that are used to detect protons.

The above SBS method can be advantageously carried out in a multiplexed format so that a plurality of different target nucleic acids are manipulated simultaneously. In a specific embodiment, different target nucleic acids can be processed in a common reaction vessel or on the surface of a specific substrate. This allows for the convenient delivery of sequencing reagents, the removal of unreacted reagents and the detection of incorporation events in a multiplex manner. In an embodiment using surface-bound target nucleic acids, the target nucleic acid can be in an array format. In an array format, the target nucleic acid can typically be bound to the surface in a spatially distinguishable manner. The target nucleic acid can be bound by direct covalent binding, attached to beads or other particles or bound to a polymerase or other molecules attached to the surface. The array can include a single copy (also referred to as a feature) of the target nucleic acid at each site or multiple copies with the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as bridge amplification or emulsion PCR, as described in further detail below.

The methods described herein can use arrays having various densities of features including, for example, at least about ¹⁰ features/ ^cm2 , 100 features/cm2, 500 features/ ^cm2 , 1,000 features/ ^cm2 , 5,000 features/ ^cm2 , 10,000 features/ ^cm2 , 50,000 features/ ^cm2 , 100,000 features/ ^cm2 , 1,000,000 features/ ^cm2 , 5,000,000 features/ ^cm2 , or more.

An advantage of the method set forth herein is that they provide rapid and effective detection of multiple target nucleic acids in parallel. Therefore, the present disclosure provides an integrated system that can use those technologies as above-mentioned demonstrations known in the art to prepare and detect nucleic acid. Therefore, the integrated system of the present disclosure can include a fluid assembly that can deliver amplification reagent and/or sequencing reagent to one or more fixed DNA fragments, and this system includes components such as pumps, valves, reservoirs, fluid lines, etc. Flow cell can be configured and/or used in an integrated system for detecting target nucleic acid. Exemplary flow cell is described in, for example, U.S. Patent Publication No. 2010/0111768 A1 and U.S. Patent Application No. 13/273,666 (each of which is incorporated herein by reference). As demonstrated for flow cell, one or more of this fluid assembly of integrated system can be used for amplification method and for detection method. Using nucleic acid sequencing embodiment as an example, one or more of this fluid assembly of integrated system can be used for amplification method set forth herein and for those as demonstrated above, the delivery of sequencing reagents in sequencing method. Alternatively, integrated system can include independent fluid system to perform amplification method and perform detection method. Examples of integrated sequencing systems that can both create amplified nucleic acids and determine the sequence of nucleic acids include, but are not limited to, the MiSeq ^™ platform (Illumina, San Diego, CA) and the devices described in U.S. Patent Application No. 13/273,666, which is incorporated herein by reference.

CMOS technology

Complementary metal oxide semiconductor (CMOS) is a technology used to manufacture integrated circuits, including digital logic circuits (such as microprocessors) and analog circuits (such as CMOS image sensors).

"Activity detector" refers to any device or component capable of detecting activity indicative of a desired reaction. An activity detector may be capable of detecting a predetermined event, attribute, quality, or characteristic within a predetermined volume or region. For example, an activity detector may be capable of capturing an image of the predetermined volume or region. An activity detector may be capable of detecting ion concentrations within a predetermined volume of solution or along a predetermined region. Exemplary activity detectors include charge coupled devices (CCDs) (such as CCD cameras); photomultiplier tubes (PMTs); molecular characterization devices or detectors, such as those used with nanopores; microcircuit assemblies, such as those described in U.S. Patent No. 7,595,883 (which is incorporated herein by reference in its entirety); and sensors made of CMOS with field effect transistors (FETs), including chemically sensitive FETs (chemFETs), ion-sensitive FETs (ISFETs), and/or metal oxide semiconductor field effect transistors (MOSFETs). Exemplary activity detectors are described, for example, in International Patent Publication No. WO2012/058095 (which is incorporated herein by reference in its entirety for the purposes indicated by the context of the citation herein).

"Biosensor" refers to any structure with multiple reaction sites. A biosensor can include a solid-state imaging device (such as a CCD or CMOS imager) and, optionally, a flow cell mounted thereon. The flow cell can include at least one flow channel in fluid communication with the reaction site. As a specific example, the biosensor is configured to be fluidically and electrically coupled to a bioassay system. The bioassay system can deliver reactants to the reaction site and perform multiple imaging events according to a predetermined protocol (such as sequencing by synthesis). The area surrounding the reaction site is referred to as a "reaction site zone." For example, the bioassay system can guide a solution to flow along the reaction site in the reaction site zone. In some embodiments of the present disclosure, the reaction site zone is different from and independent of the fluid channel that guides the solution into and from the reaction site zone. In some applications, at least one of the solutions can include four types of nucleotides having the same or different fluorescent labels. The nucleotides can be bound to corresponding oligonucleotides located at the reaction site. The bioassay system can then illuminate the reaction site using an excitation light source (e.g., a solid-state light source, such as a light emitting diode or LED). The excitation light can have one or more predetermined wavelengths, including a range of wavelengths. The excited fluorescent label provides an emission signal that can be detected by a photodetector.

In one aspect, the solid-state imager comprises a CMOS image sensor comprising an array of photodetectors configured to detect the emission signal. In some embodiments, each of the photodetectors has only a single pixel, and wherein the ratio of pixels to detection paths defined by the filter walls is substantially one to one. Exemplary biosensors are described, for example, in U.S. Patent Application No. 13/833,619 (which is incorporated herein by reference in its entirety for the purposes indicated by the context of the citation herein).

"Detection surface" refers to any surface including an optical detector. The detector can be based on any suitable technology, such as those including a charge coupled device (CCD) or a complementary metal oxide semiconductor (CMOS). In a specific embodiment, a CMOS imager with a single photon avalanche diode (CMOS-SPAD) can be used, for example, to distinguish fluorophores using fluorescence lifetime imaging (FLIM). Exemplary CMOS-based systems that can be used for FLIM are described in U.S. Patent Publication No. 2008/0037008A1; Giraud et al., Biomedical Optics Express 1: 1302-1308 (2010); or Stoppa et al., IEEE European Solid-State Device Conference (ESSCIRC), Athens, Greece, IEEE, pp. 204-207 (2009), each of which is incorporated herein by reference in its entirety. Other useful detection devices that may be used include, for example, those described in US Patent 7,329,860 and US Patent Publication No. 2010/0111768 (each of which is herein incorporated by reference in its entirety).

In addition, it will be appreciated that other signal detection devices as known in the art can be used to detect the signal produced in the method set forth herein. For example, it is particularly useful to detect a detector for pyrophosphate or protons. Pyrophosphate release can be detected using detectors such as those available commercially from 454 Life Sciences (Roche, Branford, Connecticut) or described in U.S. Patent Publication No. 2005/0244870 (which is incorporated herein by reference in its entirety). Exemplary systems for detecting primer extension based on proton release include those available commercially from Ion Torrent (Guilford, Connecticut, a subsidiary of Life Technologies) or described in U.S. Patent Publication No. 2009/0026082; 2009/0127589; 2010/0137143; and those of 10/0282617 (which are each incorporated herein by reference in their entirety). Exemplary detection surfaces and detectors are described, for example, in U.S. Patent Publication No. 20130116128, entitled “Integrated Sequencing Apparatuses and Methods of Use,” published by Min-Jui Richard et al. on May 9, 2013, each of which is incorporated herein by reference in its entirety for the purposes indicated by the context of the citation herein.

"Sequencing module" refers to a CMOS chip that has been adapted for sequencing applications. In some embodiments, the module may include a surface comprising a substrate comprising a hydrophilic region surrounded by a hydrophobic region for nucleic acid attachment and amplification. For example, dynamic pads with hydrophilic patches, such as those described above, may be used. Alternatively or additionally, a collection of dynamic pads (including some in a hydrophilic state and surrounding pads in a hydrophobic state) may form a hydrophilic region surrounded by a hydrophobic region. The surface for nucleic acid attachment will optionally include a plurality of isolation regions such that each isolation region contains a plurality of nucleic acid molecules preferably derived from a nucleic acid molecule for sequencing. For example, the hydrophilic region may comprise a gel. The hydrophilic region may be smooth, textured, porous, non-porous, and the like. The hydrophobic region is preferably located between the hydrophilic regions. The reagents are moved on the surface by means of any number of forces.

Disposable integrated microfluidics cartridge

The present disclosure provides a disposable integrated microfluidic box and methods for its preparation and use. The method for making the disposable integrated microfluidic box uses flexible printed circuit boards (PCBs) and roll-to-roll (R2R) printed electronics for monolithic integration of CMOS technology and digital fluids. That is, the disposable integrated microfluidic box includes a stack of fluid layers, in which CMOS sensors are integrated, all mounted in a housing. Therefore, traditional injection molded fluids can be integrated with flexible PCB technology. The fluid layer is formed using materials suitable for use in R2R printed electronics methods for creating electronic devices on rollers made of flexible plastic or metal foil. Further, the fluid layer includes a polymerase chain reaction (PCR) zone and a reagent mixing and distribution zone. The fluid layer also includes a set of membrane valves, by which the PCR zone can be completely enclosed.

Methods of using the disposable integrated microfluidics cartridge include performing multiplex PCR on the cartridge and downstream mixing required for sequencing.

The present disclosure provides CMOS flow cells in which a majority, or up to about 100%, of the biosensor active area can be used for reagent delivery and illumination.

FIG1 depicts a flow chart illustrating an example of a method 100 for monolithic integration of a flexible printed circuit board (PCB) and roll-to-roll (R2R) printed electronics for CMOS technology and digital fluidics. That is, using method 100, multi-layer laminated fluidics can be integrated with flexible PCB technology (see FIG2 ). Furthermore, using the structure formed from applying method 100, conventional injection molded fluidics can be integrated with flexible PCB technology (see FIGS. 13 through 32 ). Method 100 may include, but is not limited to, the following steps.

In step 110, fluidic layers are formed and then laminated and bonded together. For example, FIG2 depicts an exploded view of a set of fluidic layers 200 that may be laminated and bonded together in this step. In this example, the fluidic layers 200 include, in order, an inlet/outlet port layer 210, a fluidic channel layer 220, a flexible PCB layer 260, a sequencing chamber bottom layer 280, a sequencing chamber layer 250, and a membrane layer 240 coplanar with a sequencing chamber top layer 290. The inlet/outlet port layer 210, the fluidic channel layer 220, the flexible PCB layer 260, the sequencing chamber bottom layer 280, the sequencing chamber layer 250, the membrane layer 240, and the sequencing chamber top layer 290 are suitable for formation using a R2R printed electronics process. In some implementations, other layers may also be formed using the R2R process. In addition, suitable processes for forming layers on PCBs other than R2R may be used to form the fluidic layers in some implementations.

The inlet/outlet port layer 210 can be formed from, for example, polycarbonate, poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), and/or polyimide. The inlet/outlet port layer 210 can be about 25 μm to about 1000 μm thick in one example; or about 250 μm thick in another example. An opening (or hole) is provided in the inlet/outlet port layer 210. The opening (or hole) provides a fluid path that can serve as an inlet port and/or outlet port for, for example, various liquid supply reservoirs (not shown). More details of the inlet/outlet port layer 210 are shown and described herein below with reference to Figures 42A and 42B.

The fluid channel layer 220 can be formed of, for example, polycarbonate, PMMA, COC and/or polyimide. The fluid channel layer 220 can be about 25 μm to about 1000 μm thick in one example; or about 250 μm thick in another example. The fluid channel arrangement is provided in the fluid channel layer 220. The fluid channel provides a fluid path from one destination to another along the fluid layer 200. Because the fluid channel layer 220 is sandwiched between the inlet/outlet port layer 210 and the flexible PCB layer 260, the fluid can be confined within the fluid channel by the inlet/outlet port layer 210 at the bottom and the flexible PCB layer 260 at the top. In one example, the fluid channel layer 220 is used to perform PCR and downstream mixing required for sequencing. More details of the fluid channel layer 220 are shown and described herein below with reference to Figures 43A and 43B.

The flexible PCB layer 260 can be formed from, for example, polycarbonate, PMMA, COC, and/or polyimide. The flexible PCB layer 260 can be about 30 μm to about 300 μm thick in one example; or about 200 μm thick in another example. An opening (or hole) is provided in the flexible PCB layer 260. The opening (or hole) provides a fluid path that can serve as an inlet and/or outlet for a membrane valve used to control the flow of liquid in the fluid channel of the fluid channel layer 220. More details of the flexible PCB layer 260 are shown and described herein below with reference to Figures 44A and 44B.

The sequencing chamber bottom layer 280 can be formed of, for example, polycarbonate, PMMA, COC and/or polyimide. The sequencing chamber bottom layer 280 can be about 25 μm to about 1000 μm thick in one example; or about 250 μm thick in another example. An opening is provided in the sequencing chamber bottom layer 280 for forming the membrane valve within the stack of fluidic layers 200. The sequencing chamber bottom layer 280 also includes a CMOS device, such as a CMOS image sensor 262, placed proximate to the sequencing chamber of the sequencing chamber layer 250. The sequencing chamber bottom layer 280 is coplanar with the CMOS device and serves as a fluid connection layer for the inlet/outlet of the sequencing chamber of the sequencing chamber layer 250. Further details of the sequencing chamber bottom layer 280 are shown and described herein below with reference to Figures 45A and 45B.

Sequencing chamber layer 250 can be formed from, for example, polycarbonate, PMMA, COC, and/or polyimide. Sequencing chamber layer 250 can be about 50 μm to about 300 μm thick in one example, or about 100 μm thick in another example. An opening is provided in sequencing chamber layer 250 for forming the membrane valve within the stack of fluidic layers 200. Sequencing chamber layer 250 also includes a sequencing chamber. Further details of sequencing chamber layer 250 are shown and described herein below with reference to Figures 46A and 46B.

Membrane layer 240 can be formed from, for example, a silicone elastomer. Membrane layer 240 can be about 25 μm to about 1000 μm thick in one example, or about 250 μm thick in another example. Membrane layer 240 acts as an elastic membrane for opening and closing the membrane valve within the stack of fluidic layers 200, wherein the membrane valve is created by sequentially combining flexible PCB layer 260, sequencing chamber bottom layer 280, sequencing chamber layer 250, and membrane layer 240. Further details of the membrane valve are shown and described herein below with reference to Figures 9A, 9B, 10A, and 10B. Further details of membrane layer 240 are shown and described herein below with reference to Figures 47A and 47B.

Sequencing chamber top layer 290 is formed from a low-autofluorescence material with good optical properties, such as COC. Sequencing chamber top layer 290 can be approximately 25 μm to approximately 1000 μm thick in one example, or approximately 250 μm thick in another example. Sequencing chamber top layer 290 is used to cover the sequencing chambers in sequencing chamber layer 250. Further details of sequencing chamber top layer 290 are shown and described herein below with reference to Figures 47A and 47B.

Referring now again to FIG. 1 , in step 115 , a CMOS device is attached to the flexible PCB. For example, a CMOS image sensor 262 (see FIG. 2 ) is attached to the sequencing chamber bottom layer 280 of the fluidics layer 200 . FIG. 3 depicts a perspective view of an example of a CMOS image sensor 262 . In one example, the CMOS image sensor 262 is approximately 9200 μm long, approximately 8000 μm wide, and approximately 800-1000 μm thick; and may have approximately 50 I/O pads. The CMOS image sensor 262 may include a pixel array. In one example, the pixel array is 4384 x 3292 pixels, with an overall size of 7272 μm x 5761 μm.

Continuing with step 115, Figures 4A, 4B, 5, 6, and 7 depict side views of structure 400, illustrating an example of a process for attaching a CMOS device to a flexible PCB. Structure 400 is a multi-layer structure. Referring now to Figure 4A, the initial formation of structure 400 begins with a flexible PCB. For example, the flexible PCB includes, in order, a polyimide layer 410, a PCB heating layer 412, a polyimide layer 414, a PCB routing layer 416, and a polyimide layer 418. In other words, Figure 4 illustrates a flexible PCB with a PCB heating layer and a PCB routing layer, also known as a coupon foil.

Next, referring now to FIG. 4B , a low temperature isotropic conductive adhesive (low temperature ICA) 420 is dispensed atop the polyimide layer 418 .

5 , a CMOS device, such as CMOS image sensor 262, is placed on the voucher foil; that is, atop low-temperature ICA 420. In one example, CMOS image sensor 262 is placed atop low-temperature ICA 420 using a well-known pick and place process. FIG5 shows that I/O pads 422 of CMOS image sensor 262 are in contact with low-temperature ICA 420, thereby being electrically connected to PCB wiring layer 416. FIG5 also shows that CMOS image sensor 262 includes a bio-layer 424 facing away from polyimide layer 418. A protective film 426 may be placed atop bio-layer 424 until ready for use.

Next and now referring to FIG6 , a set of fluid layers 428 are provided on top of the polyimide layer 418 of the flexible PCB. That is, a laminated polycarbonate film is provided coplanar with the CMOS surface. An example of the fluid layer 428 is the fluid layer 200 shown in FIG2 .

Next, referring now to FIG. 7 , flip-chip bonding of the CMOS image sensor 262 on the foil is completed by dispensing an under-fill epoxy adhesive 430 in the gap around the CMOS image sensor 262 .

Referring again to FIG. 1 , in step 120 , the final assembly of the microfluidic cartridge, including the integrated fluidics layer and one or more CMOS devices, is performed. For example, FIG. 8 depicts a side view of an example of a microfluidic cartridge 800 . The microfluidic cartridge 800 includes a fluidics portion 810 and a CMOS portion 812 based on the structure 400 shown in FIG. 7 . The final assembly steps may include, for example, dispensing (printing) the underfill epoxy adhesive 430 , removing the protective film 426 , laminating a low-temperature non-conductive adhesive 814 (e.g., a UV or thermal non-conductive adhesive) to the CMOS portion 812 , laminating a low-autofluorescence cyclic olefin copolymer (COC) layer 816 to the CMOS portion 812 of the microfluidic cartridge 800 , and laminating a flexible PCB heater 818 to both sides of the fluidics portion 810 . During the formation of the microfluidic cartridge 800 , a self-aligned process flow is typically used so that the surfaces of the CMOS device and the fluidics layer are flush with each other.

A fluid path is formed in the microfluidic cartridge 800. That is, a sample inlet 820 is provided at the input of the fluidics section 810, and an outlet 822 is provided downstream of the CMOS section 812. The sample inlet 820 provides a PCR chamber 824. The PCR chamber 824 then provides a reagent dispensing area 826. The reagent dispensing area 826 then provides a sequencing chamber 828. The biolayer 424 of the CMOS image sensor 262 faces the sequencing chamber 828. The sequencing chamber 828 then provides an outlet 822. Furthermore, the microfluidic cartridge 800 includes certain membrane valves 830 that control the flow of liquid into and out of the PCR chamber 824.

9A and 9B depict perspective views of an example of a membrane valve 830 that can be integrated into, for example, the fluidic layer 200. Referring now to FIG. 9A , which is a perspective view of the membrane valve 830. In this example, the membrane valve 830 includes, in order, a base layer 910, a fluidic channel layer 912, and a reservoir layer 914. The base layer 910, the fluidic channel layer 912, and the reservoir layer 914 can be formed from, for example, polycarbonate, PMMA, COC, and/or polyimide. The reservoir layer 914 has a recessed area that creates a small reservoir 916 in the reservoir layer 914. A membrane layer 918 extends across the reservoir 916. The reservoir layer 916 has an inlet 920 and an outlet 922 that provide a flow path for each fluidic channel 924. To better illustrate the function of the reservoir 916 and the inlet 920 and outlet 922, Figure 9B shows the membrane valve 830 without the membrane layer 918 covering the reservoir 916. The membrane layer 918 is formed of a flexible and stretchable elastomeric membrane material, such as a silicone elastomer.

Figures 10A and 10B respectively show cross-sectional views of the membrane valve 830 taken along line AA of Figure 9A. An actuator, such as actuator 1010, can be used to open and close the membrane valve 830. For example, Figure 10A shows the membrane valve 830 in an open state, with the actuator 1010 not engaged with the membrane layer 918. In contrast, Figure 10B shows the membrane valve 830 in a closed state, with the actuator 1010 engaged with the membrane layer 918. In other words, the front end of the actuator 1010 is used to push the central portion of the membrane layer 918 against the outlet 922 and thereby block the flow of liquid therethrough. The membrane valve 830 (i.e., the membrane valves 242, 244, and 246) can be actuated using, for example, mechanical or pneumatic actuation, such as a solenoid or a pneumatic pump.

Figure 11 depicts a schematic diagram of an example of a microfluidic cartridge 1100 comprising integrated CMOS technology and digital fluidics. That is, the microfluidic cartridge 1100 includes a fluidic layer 200 fluidically and operably connected to four sample feeders 1110 (e.g., sample feeders 1110a, 1110b, 1110c, 1110d), thirteen reagent feeders 1112 (e.g., reagent feeders 1112a-1112m), and an outlet pump 1114. The fluidic layer 200 includes a PCR zone 270 and a reagent mixing and distribution zone 275. The PCR zone 270 includes, for example, four PCR channels 222 (e.g., PCR channels 222a, 222b, 222c, 222d). The inlets of the PCR channels 222a, 222b, 222c, and 222d are supplied by the sample feeders 1110a, 1110b, 1110c, and 1110d, respectively. Because the microfluidic cartridge 1100 includes four PCR channels 222 fed by four sample feeders 1110 , the microfluidic cartridge 1100 is configured for 4X sample multiplexing.

The input ends of the four PCR channels 222 are controlled by four membrane valves 242. That is, the input ends of PCR channels 222a, 222b, 222c and 222d are controlled by membrane valves 242a, 242b, 242c and 242d respectively. Similarly, the output ends of the four PCR channels 222 are controlled by four membrane valves 244. That is, the output ends of PCR channels 222a, 222b, 222c and 222d are controlled by membrane valves 242a, 242b, 242c and 242d respectively. The output ends of the four PCR channels 222 provide a common PCR output channel 224, which then provides reagent mixing and distribution area 275. The presence of membrane valves 242 and membrane valves 244 in the fluid layer 200 allows the PCR zone 270 to be completely enclosed.

Reagent mixing and distribution area 275 includes the settings of thirteen reagent channels 226 (such as reagent channels 226a-226m). In addition, the thirteen reagent channels 226a-226m are supplied by the thirteen reagent feeders 1112a-1112m respectively. The rotary valve assembly (not shown) is used to connect a certain PCR channel 222 fluidically to a certain reagent feeder 1112. When doing so, a certain PCR Mix can be created. The rotary valve assembly (not shown) can also be used to connect a certain PCR Mix fluidically to a sequencing feed channel 228, which provides the entrance to the sequencing chamber 258. Further, a CMOS image sensor 262 is positioned at the sequencing chamber 258.

A sequencing outlet channel 230 is provided at the outlet of the sequencing chamber 258. An outlet pump 1114 is fluidically and operatively connected to the sequencing outlet channel 230. The outlet pump 1114 is used to provide positive or negative pressure to move liquid in any direction along the flow path of the fluidic layer 200. Further, a series of three membrane valves 246 are provided along the length of the sequencing outlet channel 230. The membrane valves 242, 244, and 246 can be implemented according to the membrane valve 830 shown and described in Figures 9A, 9B, 10A, and 10C.

The three membrane valves 246 at the sequencing outlet channel 230 can be used as pumps to replace or combine the outlet pump 1114. Therefore, in one embodiment, the microfluidic cartridge 1100 includes only the outlet pump 1114 and omits the three membrane valves 246. In another embodiment, the microfluidic cartridge 1100 includes only the three membrane valves 246 and omits the outlet pump 1114. In yet another embodiment, the microfluidic cartridge 1100 includes both the outlet pump 1114 and the three membrane valves 246. In another embodiment, the microfluidic cartridge 1100 includes any other type of pumping mechanism instead of the outlet pump 1114 and/or the three membrane valves 246. More details of examples of implementing the microfluidic cartridge 1100 are shown and described herein below with reference to Figures 12 to 47B.

Figures 12 and 13 depict perspective views of a microfluidic cartridge assembly 1200 (which is an example of a physical embodiment of the integrated microfluidic cartridge 1100 shown in Figure 11). The microfluidic cartridge assembly 1200 is an example of a conventional injection-molded fluidic cartridge integrated with flexible PCB technology. In this example, the microfluidic cartridge assembly 1200 is a multi-compartment microfluidic cartridge comprising a housing 1210 fastened to a top of a substrate 1212. The housing 1210 and substrate 1212 can be formed of, for example, molded plastic and fastened together via screws (see Figure 19). The overall height of the microfluidic cartridge assembly 1200 can be, for example, from about 12 mm to about 100 mm. The total length of the microfluidic cartridge assembly 1200 can be, for example, from about 100 mm to about 200 mm. The total width of the microfluidic cartridge assembly 1200 can be, for example, from about 100 mm to about 200 mm.

The inside of the housing 1210 is a fluid assembly 1400, as shown in Figures 14A and 14B. That is, Figures 14A and 14B depict perspective views of an example of a fluid assembly 1400 installed in the microfluidic cartridge assembly 1200 shown in Figures 12 and 13. The fluid assembly 1400 is based on the integrated microfluidic cartridge 1100 shown in Figure 11. That is, the fluid assembly 1400 includes the fluid layer 200 shown and described in Figures 2 and 11. The fluid assembly 1400 also includes a rotary valve assembly 1410, which is provided with respect to the reagent mixing and distribution area 275 of the fluid layer 200. The length of the fluid layer 200 can be, for example, about 100mm to about 200mm. The width of the fluid layer 200 can be, for example, about 100mm to about 200mm.

Further, fluidics assembly 1400 includes a flexible PCB heater 1412 that wraps around both sides of PCR zone 270 of fluidics layer 200. Two independently controlled heater traces are provided in flexible PCB heater 1412, such that one heater trace is present on one side of PCR zone 270 and another heater trace is present on the other side of PCR zone 270. Flexible PCB heater 1412 is an example of flexible PCB heater 818 of microfluidics cartridge 800 shown in FIG8 . Examples of heater tracers are shown and described in more detail herein below with reference to FIG15A and FIG15B . Examples of flexible PCB heater 1412 are shown and described in more detail herein below with reference to FIG41A , 41B , and 41C .

12 and 13, the housing 1210 of the microfluidic cartridge assembly 1200 further includes four sample loading ports 1214 (e.g., sample loading ports 1214a, 1214b, 1214c, 1214d) that are substantially aligned with the input ends of the four PCR channels 222 (e.g., PCR channels 222a, 222b, 222c, 222d) of the fluidic layer 200. The housing 1210 of the microfluidic cartridge assembly 1200 further includes thirteen reagent reservoirs 1216 that provide the thirteen channel reagents 226 (e.g., reagent channels 226a-226m) of the fluidic layer 200. The thirteen reagent reservoirs 1216 can be the same or different sizes. For example, the reagent reservoirs 1216 can accommodate a liquid volume ranging from about 0.001 ml to about 0.150 ml.

The housing 1210 of the microfluidic cartridge assembly 1200 also includes a waste reservoir 1218 provided by the sequencing outlet channel 230. The waste reservoir 1218 can accommodate a range of liquid volumes, for example, from about 25 ml to about 100 ml. FIG13 shows that the reagent reservoir 1216 and the waste reservoir 1218 can be covered and sealed with, for example, a foil seal 1220.

Figures 15A and 15B illustrate a plan view and a cross-sectional view, respectively, of an example of a heater trace 1500 that can be installed in the fluid assembly 1400 shown in Figures 14A and 14B. Specifically, Figure 15A shows a plan view of an example of a heater trace 1500 having a serpentine layout. Figure 15B shows a cross-sectional view of one side of a flexible PCB heater 1412 of the fluid assembly 1400 including the heater trace 1500. The flexible PCB heater 1412 is a multi-layer structure that includes, in order, for example, a single-sided flexible copper layer 1510, an adhesive layer 1512, a dielectric layer 1514, a copper heating layer 1516 in which the heater trace 1500 is patterned, and a layer 1518. The copper heating layer 1516 shows a cross-section of the heater trace 1500 taken along line AA of Figure 15A.

Figures 16, 17, 18, 19, 20A, and 20B depict various other views, showing more details, of the microfluidic cartridge assembly 1200 of Figure 12. Specifically, Figure 16 shows a perspective view of the housing 1210 side of the microfluidic cartridge assembly 1200, while Figure 17 shows a plan view thereof, both of which show more details of the configuration of the thirteen reagent reservoirs 1216 and the waste reservoir 1218.

18 shows a plan view of the housing 1210 side of the microfluidic cartridge assembly 1200 with the foil seal 1220 installed. The foil seal 1220 has openings so that the four sample loading ports 1214 remain exposed and accessible.

FIG19 shows a perspective view of the substrate 1212 side of the microfluidic cartridge assembly 1200. FIG20 shows a plan view of the substrate 1212 side of the microfluidic cartridge assembly 1200. FIG20B shows a side view of the microfluidic cartridge assembly 1200. FIG19, 20A, and 20B show further details of the substrate 1212. Specifically, the substrate 1212 includes an opening 1222 and an opening 1224 for exposing a portion of the PCR region 270 of the fluidics layer 200 of the fluidics assembly 1400. Illustrated through the opening 1224 are a set of I/O pads 1226 for contacting the flexible PCB heater 1412 of the fluidics assembly 1400.

Along one edge of opening 1222 are four openings 1228 for accessing and actuating the four membrane valves 242 of the fluidic layer 200 of fluidic assembly 1400. That is, opening 1228a is substantially aligned with membrane valve 242a. Opening 1228b is substantially aligned with membrane valve 242b. Opening 1228c is substantially aligned with membrane valve 242c. Opening 1228d is substantially aligned with membrane valve 242d.

Along opposite edges of opening 1222 are four openings 1230 for accessing and actuating the four membrane valves 244 of the fluidic layer 200 of fluidic assembly 1400. That is, opening 1230a is substantially aligned with membrane valve 244a. Opening 1230b is substantially aligned with membrane valve 244b. Opening 1230c is substantially aligned with membrane valve 244c. Opening 1230d is substantially aligned with membrane valve 244d.

Additionally, the substrate 1212 includes an opening 1232 for accessing and actuating the membrane valve 246 of the fluidic layer 200 of the fluidic assembly 1400. The substrate 1212 also includes an opening 1234 at the sequencing chamber 258. One corner of the substrate 1212 has a bevel 1236, which is used to orient the microfluidic cartridge assembly 1200 in, for example, an instrument platform (not shown) of a microfluidic system. Figures 19 and 20A also show four screws 1238 used to secure the substrate 1212 to the housing 1210. Further, the reagent mixing and dispensing area 275 of the fluidic layer 200 of the fluidic assembly 1400 is shown in the rotary valve assembly 1410. The rotary valve assembly 1410 includes a handle having a grip portion 1240 through which a user or device can rotate a flow controller portion 1242 (see Figure 22).

Starting with the microfluidic cartridge assembly 1200 facing upward with the substrate 1212 side facing upward, Figures 21 through 29 generally illustrate a step-by-step deconstruction of the microfluidic cartridge assembly 1200 as a means of illustrating the arrangement and installation of its internal components. First, Figure 21 shows the microfluidic cartridge assembly 1200 with the substrate 1212 removed to expose the fluidics assembly 1400. In doing so, the flexible PCB layer 260 side of the fluidics layer 200 is visible. Furthermore, one side of the flexible PCB heater 1412 is visible. Also exposed is the spacer 1244 between the fluidics layer 200 and the substrate 1212. In Figure 21, membrane valves 242, 244, and 246 are visible.

22 , the grip portion 1240 of the rotary valve assembly 1410 has been removed, making visible the flow controller portion 1242. The underside (not shown) of the grip portion 1240 is designed to engage the flow controller portion 1242 so that the flow controller portion 1242 can be rotated to direct liquid flow through one of the thirteen reagent channels 226.

Referring now to FIG. 23 , the flow controller portion 1242 of the rotary valve assembly 1410 has been removed so that the fluid paths associated with the PCR output channel 224 , the reagent channel 226 , and the sequencing feed channel 228 of the fluidics layer 200 are visible.

Referring now to FIG. 24 , the fluidics layer 200 is shown having transparency such that the fluidic paths within the microfluidics cartridge assembly 1200 are visible.

25 , the fluidic layer 200 has been removed and the flexible PCB heater 1412 is shown alone within the housing 1210. Referring now to FIG26 , the flexible PCB heater 1412 has been removed and the fluidic layer 200 is shown alone within the housing 1210.

Referring now to FIG. 27 , both the fluidic layer 200 and the flexible PCB heater 1412 have been removed from the housing 1210. FIG. 27 also illustrates four threaded holes 1252 for receiving screws 1238. Furthermore, FIG. 27 illustrates the CMOS image sensor 262 and a portion of a protective cap 1254 covering the CMOS image sensor 262. Referring now to FIG. 28 , the CMOS image sensor 262 has been removed, allowing the protective cap 1254 to be fully visible. Referring now to FIG. 29 , the protective cap 1254 has been removed, revealing a gap area 1256 in the housing 1210 associated with the CMOS image sensor 262.

30 shows a transparent perspective view of the housing 1210 of the microfluidic cartridge assembly 1200 to illustrate the positions of the openings relative to the sample loading port 1214, the reagent reservoir 1216, and the waste reservoir 1218. That is, the position of the opening 1246 relative to the sample loading port 1214, the position of the opening 1248 relative to the reagent reservoir 1216, and the position of the opening 1250 relative to the waste reservoir 1218 can be seen in this view.

Figure 31 shows a transparent perspective view of the housing 1210 of the microfluidic cartridge assembly 1200 with the various fluid channels covered thereon. That is, in this view, the positions of the various fluid channels relative to the sample loading port 1214, the reagent reservoir 1216, and the waste reservoir 1218 can be seen. Figure 32 shows a cross-sectional view of the microfluidic cartridge assembly of Figure 12, showing more details of the microfluidic cartridge assembly.

Figures 33A, 33B, 34A, 34B and 35 show various views of the housing 1210 of the microfluidic cartridge assembly of Figure 12, which show more details of the housing. That is, Figures 33A and 33B show a plan view and a side view of the housing 1210, respectively. In one example, the housing 1210 is about 12 mm to about 100 mm in height, about 100 mm to about 200 mm in length, and about 100 mm to about 200 mm in width. Figure 34A shows a perspective view of the housing 1210 without the foil seal 1220 installed. Figure 34B shows a perspective view of the housing 1210 with the foil seal 1220 installed. Figures 33A, 33B, 34A and 34B show the exterior of the housing 1210, while Figure 35 shows a plan view of the interior of the housing 1210.

Figures 36, 37, 38A, 38B, and 39 illustrate various views of the substrate 1212 of the microfluidic cartridge assembly of Figure 12, showing further details of the substrate. Specifically, Figures 36 and 37 illustrate perspective views of the exterior and interior of the substrate 1212, respectively. Figure 38A illustrates a plan view of the exterior of the substrate 1212, while Figure 38B illustrates a side view of the substrate 1212. Figures 36, 37, 38A, 38B, and 39 illustrate that the substrate 1212 further includes four holes 1258 for receiving screws 1238, with a recessed area 1260 having a center portion for receiving the grip portion 1240 and the flow controller portion 1242 of the rotary valve assembly 1410.

Figures 40A and 40B depict additional perspective views of the fluidics assembly 1400 of the microfluidic cartridge assembly 1200, showing greater detail. Specifically, Figures 40A and 40B each show a perspective view of the fluidics assembly 1400. Figure 40A shows the fluidics assembly 1400 without the flexible PCB heater 1412, while Figure 40B shows the fluidics assembly 1400 with the flexible PCB heater 1412 installed. Furthermore, a groove 1414 is provided on one edge of the fluidics layer 200 and within the PCR zone 270. The groove 1414 is designed to receive the flexible PCB heater 1412.

Figures 41A, 41B, and 41C depict various views showing more details of the flexible PCB heater 1412 of the fluid assembly 1400 of the microfluidic cartridge assembly 1200. That is, Figures 41A and 41B each show a perspective view of the flexible PCB heater 1412, while Figure 41C shows a side view of the flexible PCB heater 1412. The flexible PCB heater 1412 includes a U-shaped coiled panel 1416 and a side extension panel 1418, all formed using flexible PCB technology. The U-shaped coiled panel 1416 includes a panel 1420 and a panel 1422, each of which has patterned heater traces 1500, such as heater traces 1500a and 1500b. Examples of heater traces 1500 are shown in Figures 15A and 15B. The space between panels 1420 and 1422 is set so that the flexible PCB heater 1412 can be press-fitted over the PCR region 270 of the fluidics layer 200 and assembled into the recess 1414, as shown in Figure 40B. Figures 41B and 41C also show the I/O pads 1226, which provide electrical connections to the two heater traces 1500 and to the CMOS image sensor 262.

Side extension panels 1418 extend from panel 1420 near the bend in the U-shaped wrap panel 1416. Side extension panels 1418 are designed to extend toward the CMOS image sensor 262. As shown in FIG40B , the end of the side extension panel 1418 farthest from the U-shaped wrap panel 1416 is shaped to fit against the CMOS image sensor 262. The purpose of the side extension panel 1418 is to provide electrical connection to the CMOS image sensor 262, which is assembled on top of a rigid or flexible PCB.

Figures 42A and 42B show a perspective view and a plan view of the inlet/outlet port layer 210 of the fluidic layer shown in Figures 2 and 14, respectively. Again, the inlet/outlet port layer 210 can be formed of, for example, polycarbonate or any other material suitable for use with the R2R process. The inlet/outlet port layer 210 provides an interface between the fluidic layer 200 and the housing 1210 of the microfluidic cartridge assembly 1200. In other words, the inlet/outlet port layer 210 provides a fluid path from the sample loading port 1214, the thirteen reagent reservoirs 1216, and the waste reservoir 1218 of the housing 1210 to the fluid channel layer 220 of the fluidic layer 200. For example, the inlet/outlet port layer 210 includes a set of openings 212 that are substantially aligned with the opening 1246 of the sample loading port 1214 in the housing 1210. The inlet/outlet port layer 210 includes a set of openings 214 that are substantially aligned with the opening 1248 of the reagent reservoir 1216 in the housing 1210. The inlet/outlet port layer 210 also includes an opening 216 that is substantially aligned with the opening 1250 of the waste reservoir 1218 in the housing 1210 .

Figures 43A and 43B respectively show a perspective view and a plan view of the fluid channel layer 220 of the fluid layer 200 shown in Figures 2 and 14. Again, the fluid channel layer 220 can be formed of, for example, polycarbonate or any other material suitable for use with the R2R process. The fluid channel layer 220 is the layer that facilitates all liquid flows in the fluid layer 200. In other words, all PCR and sequencing operations occur at the fluid channel layer 220. The PCR operation occurs in the PCR channels 222 at the PCR area 270. The PCR output channel 224 provides a reagent mixing and distribution area 275. Reagent distribution occurs in the reagent mixing and distribution area 275 using reagent channels 226. The thirteen reagent channels 226 are patterned to provide a rotary valve assembly 1410. The sequencing feed channel 228 provides an inlet to the sequencing chamber 258 of the sequencing chamber layer 250 shown in Figures 45A and 45B. Then, the sequencing outlet channel 230 is fluidically connected to the outlet of the sequencing chamber 258.

Figures 44A and 44B show perspective and plan views, respectively, of the flexible PCB layer 260 of the fluidic layer 200 shown in Figures 2 and 14. Again, the flexible PCB layer 260 can be formed, for example, from polyimide or any other material suitable for use with the R2R process. The flexible PCB layer 260 includes a set of openings (or holes) 264 associated with the inlet/outlet of the membrane valve 242. The flexible PCB layer 260 also includes a set of openings (or holes) 266 associated with the inlet/outlet of the membrane valve 244. If the membrane valve 246 is present, the flexible PCB layer 260 includes a set of openings (or holes) 267 associated with the inlet/outlet of the membrane valve 246. Further, the flexible PCB layer 260 includes a set of openings 268 that are substantially aligned with the rotary valve assembly 1410 and provide a fluid path to the rotary valve assembly 1410.

Figures 45A and 45B illustrate perspective and plan views, respectively, of the sequencing chamber bottom layer 280 of the fluidics layer 200 shown in Figures 2 and 14 . Again, the sequencing chamber bottom layer 280 can be formed, for example, from polycarbonate or any other material suitable for use with the R2R process. The sequencing chamber bottom layer 280 includes a set of openings 282 for forming the membrane valves 242 within the stack of fluidics layers 200. The sequencing chamber bottom layer 280 also includes a set of openings 284 for forming the membrane valves 244 within the stack of fluidics layers 200. If present, the sequencing chamber bottom layer 280 includes a set of openings 286 for forming the membrane valves 246 within the stack of fluidics layers 200. Furthermore, the sequencing chamber bottom layer 280 includes a set of openings 288 that are substantially aligned with and provide a fluid path to the rotary valve assembly 1410. Additionally, the sequencing chamber bottom layer 280 includes a pair of openings 289 that are fluidly coupled to the sequencing chambers 258 of the sequencing chamber layer 250 .

Sequencing chamber bottom layer 280 is a fluidic layer in which the CMOS technology is integrated. That is, CMOS image sensor 262 is mounted on sequencing chamber bottom layer 280. The position of CMOS image sensor 262 substantially corresponds to the position of sequencing chamber 258 of sequencing chamber layer 250.

Figures 46A and 46B show perspective and plan views, respectively, of the sequencing chamber layer 250 of the fluidics layer 200 shown in Figures 2 and 14. Again, the sequencing chamber layer 250 can be formed, for example, from polycarbonate or any other material suitable for use with the R2R process. The sequencing chamber layer 250 is the layer of the fluidics layer 200 where sequencing operations occur (i.e., using the sequencing chamber 258).

The sequencing chamber layer 250 includes a set of openings 252 for forming the membrane valve 242 within the stack of fluidics layers 200. The sequencing chamber layer 250 also includes a set of openings 254 for forming the membrane valve 244 within the stack of fluidics layers 200. If the membrane valve 246 is present, the sequencing chamber layer 250 includes a set of openings 255 for forming the membrane valve 246 within the stack of fluidics layers 200. Further, the sequencing chamber layer 250 includes a set of openings 256 that are substantially aligned with the rotary valve assembly 1410 and provide a fluid path to the rotary valve assembly 1410.

Figures 47A and 47B show a perspective view and a plan view of the membrane layer 240 and the sequencing chamber top layer 290 of the fluidic layer 200 shown in Figures 2 and 14, respectively. The membrane layer 240 can be formed of, for example, silicone rubber, while the sequencing chamber top layer 290 can be formed of, for example, COC. The membrane layer 240 acts as an elastic membrane for opening and closing membrane valves 242, 244, and 246 within the stack of fluidic layers 200, wherein the membrane valves 242, 244, and 246 are created by sequentially combining the flexible PCB layer 260, the sequencing chamber bottom layer 280, the sequencing chamber layer 250, and the membrane layer 240. Figures 47A and 47B also show the sequencing chamber top layer 290, which is used to cover the sequencing chamber 258 of the sequencing chamber layer 250.

Figures 48A and 48B depict a flow chart of an example of a method 4800 for using the microfluidic cartridge assembly 1200 to perform multiplex PCR and downstream mixing required for sequencing. Because the microfluidic cartridge assembly 1200 is based on the microfluidic cartridge 1100 shown in Figure 11, the microfluidic cartridge assembly 1200 is configured for 4X sample multiplexing. Further, in method 4800, the thirteen reagent reservoirs 1216 are designated reagent reservoirs 1216a, 1216b, 1216c, 1216d, 1216e, 1216f, 1216g, 1216h, 1216i, 1216j, 1216k, 1216l, and 1216m. Further, method 4800 utilizes an outlet pump 1114, which is fluidically connected to the microfluidic cartridge assembly 1200. The outlet pump 1114 is positioned downstream of the sequencing chamber 258. The outlet pump 1114 is capable of providing both positive pressure and negative pressure (ie, vacuum pressure).The method 4800 includes, but is not limited to, the following steps.

In step 4810, a ready-to-use microfluidic cartridge assembly 1200 is provided. Specifically, the microfluidic cartridge assembly 1200 is provided with one or more reservoirs containing desired liquids. For example, the reagent reservoir 1216 can be filled with the same or different reagent liquids. In one example, all of the reagent reservoirs 1216a-m are filled with hydrogenation buffer (HT1). Method 4800 proceeds to step 4815.

In step 4815, all membrane valves are closed, and then sample/PCR MIX is loaded. "PCR MIX" refers to a PCR master mix optimized for use in conventional PCR for amplifying DNA templates. In this step, membrane valves 242a and 244a are closed, membrane valves 242b and 244b are closed, membrane valves 242c and 244c are closed, and membrane valves 242d and 244d are closed. In this way, PCR channels 222a, 222b, 222c, and 222d are all completely sealed. Then, the first sample liquid is mixed with PCR MIX (hereinafter referred to as sample/PCR_MIX1) and loaded into sample loading port 1214a. The second sample liquid is mixed with PCR MIX (hereinafter referred to as sample/PCR_MIX2) and loaded into sample loading port 1214b. The third sample liquid is mixed with PCR MIX (hereinafter referred to as sample/PCR_MIX3) and loaded into sample loading port 1214c. The fourth sample solution is mixed with PCR MIX (hereinafter referred to as sample/PCR_MIX4) and loaded into sample loading port 1214d. Upon completion of this step, a volume of sample/PCR MIX is located in each of the sample loading ports 1214 and is ready for processing. Method 4800 proceeds to step 4820.

In step 4820, the membrane valve for the first sample is opened. The first sample is then drawn into the PCR zone. The membrane valve for the first sample is then closed. For example, membrane valves 242a and 244a for PCR channel 222a are opened. Then, using outlet pump 1114, the sample/PCR MIX 1 is drawn into PCR channel 222a. Then, membrane valves 242a and 244a for PCR channel 222a are closed, wherein the volume of sample/PCR MIX 1 is now sealed inside PCR channel 222a. Method 4800 proceeds to step 4825.

In decision step 4825, a determination is made as to whether another sample is waiting to be loaded into the PCR area, i.e., PCR area 270. If so, the method 4800 proceeds to step 4830. If not, the method 4800 proceeds to step 4835.

In step 4830, the membrane valve for the next sample is opened. The next sample is then drawn into the PCR zone. The membrane valve for the next sample is then closed. In one example, membrane valves 242b and 244b for PCR channel 222b are opened. Sample/PCR_MIX2 is then drawn into PCR channel 222b using outlet pump 1114. Membrane valves 242b and 244b for PCR channel 222b are then closed, with the volume of sample/PCR_MIX2 now sealed within PCR channel 222b.

In another example, membrane valves 242c and 244c for PCR channel 222c are opened. Sample/PCR_MIX3 is then drawn into PCR channel 222c using outlet pump 1114. Membrane valves 242c and 244c for PCR channel 222c are then closed, with the volume of sample/PCR_MIX3 now sealed within PCR channel 222c.

In yet another example, membrane valves 242d and 244d for PCR channel 222d are opened. Sample/PCR_MIX4 is then drawn into PCR channel 222d using outlet pump 1114. Membrane valves 242d and 244d for PCR channel 222d are then closed, with the volume of sample/PCR_MIX4 now sealed within PCR channel 222d.

Method 4800 returns to step 4825.

In step 4835, a PCR operation is performed with sample/PCR_MIX1 in PCR lane 222a, sample/PCR_MIX2 in PCR lane 222b, sample/PCR_MIX3 in PCR lane 222c, and sample/PCR_MIX4 in PCR lane 222d. After the PCR operation is complete, sample/PCR_MIX1 is now referred to as PCR_MIX1, sample/PCR_MIX2 is now referred to as PCR_MIX2, sample/PCR_MIX3 is now referred to as PCR_MIX3, and sample/PCR_MIX4 is now referred to as PCR_MIX4. Method 4800 proceeds to step 4840.

In step 4840 , the rotary valve assembly 1410 is rotated to the first PCR MIX position. For example, by rotating the grip 1240 of the rotary valve assembly 1410 , the rotary valve assembly 1410 is positioned to accommodate the PCR channel 222 a of PCR_MIX1. The method 4800 proceeds to step 4845 .

In step 4845, the membrane valve for the first PRC MIX is opened. The first PRC MIX is then pulled through the rotary valve toward the CMOS device. The membrane valve for the first PRC MIX is then closed. For example, membrane valves 242a and 244a for PCR channel 222a are opened. Then, using outlet pump 1114, PCR_MIX1 is pulled out of PCR channel 222a, into PCR output channel 224, and through rotary valve assembly 1410. Membrane valves 242a and 244a are then closed. Method 4800 proceeds to step 4850.

In step 4850, the rotary valve is rotated to the hydrogenation buffer (HT1) position, specifically to the reagent reservoir 1216 containing HT1. In method 4800, at least one reagent reservoir 1216 contains a certain volume of HT1. By way of example, reagent reservoir 1216k contains a certain volume of HT1. Thus, by rotating grip 1240 of rotary valve assembly 1410, rotary valve assembly 1410 is now positioned to accommodate reagent reservoir 1216k containing HT1. Method 4800 proceeds to step 4855.

In step 4855, the first PCR_MIX is pushed into the HT1 reservoir. For example, using the outlet pump 1114, the PCR_MIX1 is pushed through the rotary valve assembly 1410 and into the reagent reservoir 1216k and mixed with the HT1 therein. The method 4800 proceeds to step 4860.

In decision step 4860, it is determined whether there is another PCR MIX waiting to be mixed with HT1. If so, the method 4800 proceeds to step 4865. If not, the method 4800 proceeds to step 4885.

In step 4865, the rotary valve is rotated to the next PRC MIX position. In one example, by rotating the grip portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is set to accommodate the PCR channel 222b of PCR_MIX2. In another example, by rotating the grip portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is set to accommodate the PCR channel 222c of PCR_MIX3. In yet another example, by rotating the grip portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is set to accommodate the PCR channel 222d of PCR_MIX4. Method 4800 proceeds to step 4870.

In step 4870, the membrane valve for the next PRC MIX is opened. The next PCR MIX is then pulled through the rotary valve toward the CMOS device. The membrane valve for the next PRC MIX is then closed. In one example, membrane valves 242b and 244b for PCR channel 222b are opened. Then, using outlet pump 1114, PCR_MIX2 is pulled from PCR channel 222b into PCR output channel 224 and passed through rotary valve assembly 1410. Then, membrane valves 242b and 244b are closed. In another example, membrane valves 242c and 244c for PCR channel 222c are opened. Then, using outlet pump 1114, PCR_MIX3 is pulled from PCR channel 222c into PCR output channel 224 and passed through rotary valve assembly 1410. Then, membrane valves 242c and 244c are closed. In yet another example, membrane valves 242d and 244d for PCR channel 222d are opened. Then, using outlet pump 1114, PCR_MIX4 is pulled out of PCR channel 222d, into PCR output channel 224, and through rotating valve assembly 1410. Then, membrane valves 242D and 244D are closed. Method 4800 proceeds to step 4875.

In step 4875 , the rotary valve assembly 1410 is rotated to the HT1 position. For example, the rotary valve assembly 1410 is rotated to return to the position of the reagent reservoir 1216 k containing HT1 by rotating the grip 1240 of the rotary valve assembly 1410 . The method 4800 proceeds to step 4880 .

In step 4880, the next PCR MIX is pushed into the HT1 reservoir. In one example, outlet pump 1114 is used to push PCR_MIX2 through rotary valve assembly 1410 and into reagent reservoir 1216k and mix with the HT1 therein. In another example, outlet pump 1114 is used to push PCR_MIX3 through rotary valve assembly 1410 and into reagent reservoir 1216k and mix with the HT1 therein. In yet another example, outlet pump 1114 is used to push PCR_MIX4 through rotary valve assembly 1410 and into reagent reservoir 1216k and mix with the HT1 therein. Method 4800 returns to step 4860.

In step 4885, the mixture from the HT1 reservoir is drawn into the sequencing chamber and the clustering/sequencing recipe is executed. For example, reagent reservoir 1216k is now used to hold a mixture of HT1, PCR_MIX1, PCR_MIX2, PCR_MIX3, and PCR_MIX4. This mixture is drawn from reagent reservoir 1216k and then drawn along sequencing feed channel 228 and into sequencing chamber 258. The clustering/sequencing recipe is then executed using CMOS image sensor 262. Method 4800 ends.

CMOS flow cell with available biosensor active area

CMOS flow cells can be designed as single-use consumables. Therefore, it may be beneficial for the CMOS flow cell to be a small and inexpensive device. In a small CMOS flow cell, it is important to use as much as possible of the biosensor active area. However, current CMOS flow cell designs do not allow 100% utilization of the biosensor active area. Therefore, new methods are needed to provide increased utilization of the biosensor active area in the CMOS flow cell. Various implementations of the present disclosure provide CMOS flow cells in which a majority of, or up to about 70%, 80%, 90%, 95%, 98%, 99% or 100% of the biosensor active area can be used for reagent delivery and illumination, as shown and described herein below with reference to Figures 49 to 62.

Figure 49 depicts a side view of an example CMOS flow cell 4900, in which a majority, or up to approximately 100%, of the biosensor active area can be used for reagent delivery and illumination. In some implementations, a CCD or other image sensor can be used in place of or in addition to the CMOS sensor. CMOS flow cell 4900 includes a PCB substrate 4910, which can be, for example, a flexible PCB substrate. As depicted here, atop PCB substrate 4910 is a CMOS sensor device 4920. CMOS biosensor device 4920 is a CMOS image sensor having a biolayer thereon. Furthermore, atop PCB substrate 4910 and surrounding CMOS biosensor device 4920 is a laminate film 4930. Laminate film 4930 can be formed, for example, from epoxy, polyimide or other plastic films, silicon, bismaleimide-triazine (BT) substrate, and the like. PCB substrate 4910 and laminate film 4930 can be formed using flexible PCB technology.

The purpose of the laminating film 4930 is to provide an extended surface around the perimeter of the CMOS biosensor device 4920 that is substantially coplanar with the top of the CMOS biosensor device 4920. In one example, if the die thickness of the CMOS biosensor device 4920 is approximately 100 μm, the thickness of the laminating film 4930 is approximately 100 μm ± approximately 5 μm.

The slight gap between the PCB substrate 4910 and the laminate film 4930 forms a groove or channel 4950 around the perimeter of the CMOS biosensor device 4920. The width of the groove or channel 4950 can be, for example, from about 100 μm to about 1000 μm. The groove or channel 4950 is filled with a filling material 4952 to form a substantially continuous planar surface across both the CMOS biosensor device 4920 and the laminate film 4930. The filling material 4952 is a material that does not interfere with the reactions occurring within the CMOS biosensor device 4920. The filling material 4952 can be, for example, an ultraviolet (UV) cured epoxy, a heat cured epoxy, or the like.

Above the CMOS biosensor device 4920 and the laminate film 4930 is a flow cell cover 4940 over the flow channel 4942. Further, the flow cell cover 4940 includes a first port 4944 and a second port 4946 providing inlet/outlet ports to the flow channel 4942. The flow cell cover 4940 is formed of an optically transparent material (but not limited to cyclic olefin copolymer (COC)) with low or no autofluorescence. The total thickness of the flow cell cover 4940 can be, for example, from about 300 μm to about 1000 μm. There is a bonding area outside the flow channel 4942 for bonding the flow cell cover 4940 to the laminate film 4930. Bonding can be via a low autofluorescence adhesive.

Because there is a substantially continuous planar surface across both the CMOS biosensor device 4920 and the laminate film 4930, the area of the flow channel 4942 within the flow cell cover 4940 can be sized to span the entire CMOS biosensor device 4920; that is, it can span approximately 100% of the biosensor active area. In one example, if the die size of the CMOS biosensor device 4920 is approximately 8 mm x 9 mm, the active area is approximately 7 mm x 8 mm. However, the die size of the CMOS biosensor device 4920 can range, for example, up to approximately 25 mm x 25 mm, with a proportionally larger active area.

FIG49 shows, for example, a reagent fluid 4954 filling a flow channel 4942. A chemical reaction occurs in the reagent fluid 4954 in the flow channel 4942 atop a CMOS biosensor device 4920. When illuminated through the flow cell cover 4940, the CMOS biosensor device 4920 is used to detect the chemical reaction occurring in the flow channel 4942. Electrical connections (not shown) are provided through the PCB substrate 4910 for acquiring signals from the CMOS sensor device 4920. In the CMOS flow cell 4900, approximately 100% of the biosensor active area of the CMOS biosensor device 4920 can be used for reagent delivery and illumination.

FIG50 depicts an exploded view of an example of an embodiment of the CMOS flow cell 4900 shown in FIG49 . FIG50 shows that the CMOS biosensor device 4920 includes an active area 4922. Any portion of the CMOS sensor device 4920 outside the active area 4922 is an inactive area 4924. The CMOS biosensor device 4920 can be attached to the PCB substrate 4910 using, for example, flip-chip technology. Furthermore, the laminating film 4930 includes an opening or window 4932 sized to receive the CMOS biosensor device 4920 when laminated against the PCB substrate 4910. The opening or window 4932 is provided in the laminating film 4930 prior to laminating the laminating film 4930 to the PCB substrate 4910. When the flow cell cover 4940 is bonded to the laminating film 4930, the flow channel 4942 is substantially aligned with the CMOS biosensor device 4920 and its area extends beyond the area of the CMOS biosensor device 4920. In Figure 50, the flow cell cover 4940 is shown as transparent. Figures 51 and 52 depict perspective and side views, respectively, of the CMOS flow cell 4900 shown in Figure 50 when fully assembled.

FIG53 depicts a perspective view of an example of a flow cell cover 4940 of the CMOS flow cell 4900 shown in FIG50, 51, and 52. Specifically, FIG53 shows top and bottom perspective views of the flow cell cover 4940 of the CMOS flow cell 4900 shown in FIG50, 51, and 52. In this example, the diameters of the first port 4944 and the second port 4946 can be approximately 750 μm. Furthermore, the depth or height of the flow channel 4942 can be approximately 100 μm.

Figures 54, 55, 56 and 57 depict examples of methods for providing an extended planar surface in a CMOS flow cell onto which the flow cell cover can be mounted.

54 , a laminate film 4930 and a CMOS sensor device 4920 are provided atop a PCB substrate 4910. A trench or channel 4950 exists around the perimeter of the CMOS biosensor device 4920. The trench or channel 4950 exists because the opening or window 4932 in the laminate film 4930 is slightly larger than the CMOS sensor device 4920.

In the next step and referring now to FIG55 , the upper side of the groove or channel 4950 is sealed with, for example, an optically clear elastomer 4960 having features for a tight fit against the groove or channel 4950. The elastomer 4960 is optically clear, allowing UV light to pass therethrough. The purpose of the elastomer 4960 is to block the top of the groove or channel 4950 in preparation for filling.

In the next step and referring now to FIG. 56 , the trench or channel 4950 is filled with a filler material 4952 , such as a UV cured epoxy, using, for example, a pair of through holes 4916 in the PCB substrate 4910 , which is why the elastomer 4960 is optically transparent.

In the next step and referring now to FIG. 57 , once the filler material 4952 is cured, the elastomer 4960 is removed and a substantially continuous planar surface now exists in the flow cell for receiving a flow cell cover, such as flow cell cover 4940 .

Figures 58A, 58B, 58C and 58D depict another example of a method for providing an extended planar surface in a CMOS flow cell onto which the flow cell cover can be mounted.

In a first step and referring now to FIG. 58A , a CMOS biosensor device 4920 is provided atop a PCB substrate 4910 .

58B , a mold 5510 (e.g., a clamshell mold) is provided around the CMOS biosensor device 4920 and the PCB substrate 4910. The mold 5510 provides a space or gap 5512 atop the PCB substrate 4910 and around the perimeter of the CMOS biosensor device 4920.

In the next step and referring now to FIG. 58C , the space or void 5512 in the mold 5510 is filled with a filler material 4952 , such as a UV-cured or heat-cured epoxy, using, for example, a low pressure injection molding process or a reaction injection molding process.

In the next step and referring now to FIG. 58D , once the fill material is cured, mold 5510 is removed and a substantially continuous planar surface now exists in the flow cell for receiving a flow cell cover, such as flow cell cover 4940 .

Figures 59, 60, 61 and 62 depict yet another example of a method for providing an extended planar surface in a CMOS flow cell onto which the flow cell cover may be mounted.

In the first step, and referring now to FIG. 59 , a CMOS sensor device 4920 is provided atop a PCB substrate 4910. Furthermore, a piece of mechanical material 5910 is provided atop the PCB substrate 4910 and at one end of the CMOS biosensor device 4920. Similarly, a piece of mechanical material 5912 is provided atop the PCB substrate 4910 and at the other end of the CMOS biosensor device 4920. Mechanical material pieces 5910 and 5912 can be, for example, blank silicon, glass, or plastic. A trench or channel 5914 is provided between the mechanical material piece 5910 and the CMOS sensor device 4920. Another trench or channel 5914 is provided between the mechanical material piece 5912 and the CMOS sensor device 4920.

60 , a set of barriers 5916 are provided at the ends of the trenches or channels 5914. For example, barriers 5916a and 5916b block the end of one trench or channel 5914 and barriers 5916c and 5916d block the end of the other trench or channel 5914, ready for filling.

61, the trench or channel 5914 is filled with a filler material 4952, such as a UV cured or thermally cured epoxy. The filler material 4952 is retained between the barriers 5916a and 5916b and between the barriers 5916c and 5916d.

In the next step and referring now to FIG. 62 , once fill material 4952 is cured, a substantially continuous planar surface now exists in the flow cell for receiving a flow cell cover, such as flow cell cover 4940 .

system

It should be understood that various aspects of the present disclosure may be implemented as methods, systems, computer-readable media, and/or computer program products. Various aspects of the present disclosure may take the form of hardware implementations, software implementations (including firmware, resident software, microcode, etc.), or implementations combining software and hardware aspects (all of which may be generally referred to herein as "circuits," "modules," or "systems"). Further, the methods of the present disclosure may take the form of a computer program product on a computer-usable storage medium having computer-usable program code embodied in the medium.

Any suitable computer-usable medium may be used for the software aspects of the present disclosure. A computer-usable or computer-readable medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, device, apparatus, or propagation medium. The computer-readable medium may include temporary and/or non-temporary implementations. More specific examples (non-exhaustive list) of computer-readable media would include some or all of the following: an electrical connection with one or more wires (RAM), a portable computer disk, a hard disk, a random access memory, a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a transmission medium (such as those supporting the Internet or an intranet), or a magnetic storage device. Note that the computer-usable or computer-readable medium may even be paper or another suitable medium with the program printed thereon, as the program can be captured electronically, for example, by optical scanning of paper or other media, and then compiled, interpreted, or, if necessary, processed in an appropriate manner, and then stored in a computer memory. In the context of this document, a computer-usable or computer-readable medium may be any medium that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.

The program code for performing the operation of the method and apparatus set forth herein can be written in an object-oriented programming language, such as Java, Smalltalk, C++ or the like. However, the program code for performing the operation of the method and apparatus set forth herein can also be written in a traditional process programming language, such as "C" programming language or similar programming language. This program code can be performed by a processor, an application specific integrated circuit (ASIC) or other components that perform this program code. This program code can be referred to as the application software stored in the memory (computer-readable medium as discussed above). This program code can cause this processor (or the equipment of any processor control) to produce a graphical user interface ("GUI"). This graphical user interface can be visually generated on a display device, but this graphical user interface also can have an audible function. However, this program code can be operated in the equipment of any processor control, such as a computer, server, personal digital assistant, phone, television or any processor-controlled equipment utilizing this processor and/or digital signal processor.

This program code can be executed locally and/or remotely. This program code, for example, can be stored in whole or in part in the local memory of the equipment of this processor control. However, this program code also can be stored, read and downloaded to the equipment of this processor control at least in part remotely. For example, the user's computer can execute this program code completely or only partially execute this program code. This program code can be at least partially on this user's computer and/or be partially executed on the remote computer or a stand-alone software package fully on the remote computer or server. In the latter case, this remote computer can be connected to this user's computer through a communication network.

The methods and devices described herein can be applied regardless of the network environment. The communication network can be a wired network running in a radio frequency domain and/or an Internet Protocol (IP) domain. However, the communication network can also include a distributed computing network, such as the Internet (sometimes alternatively referred to as the "World Wide Web"), an intranet, a local area network (LAN), and/or a wide area network (WAN). The communication network can include coaxial cables, copper wires, fiber optic lines, and/or hybrid coaxial lines. The communication network can even include a wireless portion that utilizes any portion of the electromagnetic spectrum and any signaling standard (such as the IEEE 802 family of standards, GSM/CDMA/TDMA, or any cellular standard; and/or an ISM band). The communication network can even include a power line portion, in which signals are connected via electrical wiring. The methods and devices described herein can be applied to any wireless/wired communication network, regardless of the physical component parts, physical configuration, or one (or more) communication standards.

Certain aspects of the present disclosure are described with reference to various methods and method steps. It will be understood that each method step can be performed by the program code and/or by machine instructions. The program code and/or the computer instructions can create a device for performing the function/action specified in the method.

The program code may also be stored in a computer-readable memory, which may direct a processor, a computer, or other programmable data processing device to act in a specific manner so that the program code stored in the computer-readable memory generates or converts an article of manufacture including an instruction device for executing various aspects of the method steps.

The program code can also be loaded into a computer or other programmable data processing device to cause a series of operating steps to be executed to produce a processor/computer execution process, so that the program code provides steps for performing various functions/actions specified in the method of the present disclosure.

Claims (14)

1. A microfluidic cartridge for detecting biological reactions, comprising: (a) A flow cell, which includes a reaction site region containing one or more reaction sites; (b) a fluid channel for delivering reactants to and/or removing reactants from the reaction site region; and (c) An image sensor having an active region configured to detect a signal of a biological response in the reaction site region. in The reaction site region is located near the active region of the image sensor. The reaction site region spans substantially the entire active region of the image sensor, and The fluid channel does not substantially overlap with the active region of the image sensor. The flow cell includes a sequencing chamber formed on a sequencing chamber layer. The image sensor is disposed in an opening in a bottom layer of the sequencing chamber below the sequencing chamber layer. The fluid channel is formed on a fluid channel layer below the bottom layer of the sequencing chamber. 2. The microfluidic cell of claim 1, wherein... This microfluidic chamber is configured to perform sequencing operations on nucleic acid samples, and The detected biological response signal indicates the type of nucleotide bases involved in the biological response. 3. The microfluidic cell of claim 1 or 2, wherein the flow cell includes a substrate surrounded by hydrophobic regions and hydrophilic regions for nucleic acid attachment and amplification. 4. The microfluidic cell of claim 1 or 2, wherein the reaction site region spans the entire active region of the image sensor. 5. The microfluidic cell of claim 1 or 2, wherein the fluid channel does not overlap with the active region of the image sensor. 6. The microfluidic cell of claim 1 or 2, wherein the image sensor includes a photodetector. 7. The microfluidic cell of claim 6, wherein the photodetector comprises a CMOS/CCD sensor. 8. The microfluidic cell of claim 7, wherein the CMOS sensor is 9200 μm long, 8000 μm wide, and 800-1000 μm thick. 9. The microfluidic cell of claim 7, wherein the CMOS sensor has 50 I/O pads. 10. The microfluidic cell of claim 1 or 2, further comprising: The PCR region, the reagent mixing and dispensing region, and one or more membrane valves are configured to reversibly prevent fluid communication between the PCR region and the reagent mixing and dispensing region or the flow cell including the reaction site region. 11. The microfluidic cartridge of claim 10, wherein the PCR region comprises a plurality of PCR channels. 12. The microfluidic cartridge of claim 10, wherein the reagent mixing and dispensing zone comprises a plurality of reagent channels and/or reagent dispensers. 13. The microfluidic cartridge of claim 10, further comprising a rotary valve configured to fluidly connect the PCR region to the reagent mixing and dispensing region. 14. The microfluidic cartridge of claim 13, wherein the rotary valve is further configured to fluidly connect the reagent mixing and dispensing zone to the flow cell including the reaction site zone.