Systems and methods for integrated sensor cartridge

Abstract

The present invention relates to a system and method for an integrated sensor cartridge. A microfluidic device (100) can include a PCB (110), a biochip (120) covering the PCB (110), and a microfluidic housing (130) covering the biochip (120) and the PCB (110). The microfluidic device (100) also has a first adhesive layer (141) attaching the microfluidic housing (130) to the biochip (120) and a second adhesive layer (142) attaching the microfluidic housing (130) to the PCB (110). The second adhesive layer (142) is thicker than the first adhesive layer (141). The first adhesive layer (141) comprises a first adhesive material, while the second adhesive layer (142) comprises a second adhesive material.

Description

[0001] This application is a divisional application of patent application No. 201980075347.3, filed on November 15, 2019, by Shenzhen BGI Genomics Co., Ltd., entitled "System and Method for Integrated Sensor Box".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 767,998, filed November 15, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present invention generally relates to biosensors for biological or chemical analysis, and more specifically, to a method of integrating a microfluidic housing with a biochip and a substrate such as a printed circuit board (PCB) to form an integrated sensor housing or microfluidic device. Background Technology

[0005] High-throughput analysis of chemical and / or biological substances is a vital tool in diagnostics and therapeutics. It allows for the design of specific target sequences from attached chemical and / or biological substances, analysis of gene expression patterns, identification of specific allelic variations, determination of DNA copy numbers, and recognition of protein binding sites (e.g., transcription factors and other regulatory molecules) across the entire genome. In a concrete example, the advent of the Human Genome Project necessitated the development of improved nucleic acid sequencing methods, such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) sequencing. The determination of the entire 3,000,000,000-base sequence of the haploid human genome provided a foundation for identifying the genetic basis of a wide range of diseases.

[0006] High-throughput analysis, such as massively parallel DNA sequencing, often utilizes flow cells, which contain a range of chemicals and / or biological substances that can be analyzed. Flow cells are typically made from microfluidic housings integrated with biochips (e.g., silicon-based sensor chips) to form microfluidic devices (e.g., cartridges). Many current microfluidic designs are complex and expensive to manufacture and use, and are often unreliable. Summary of the Invention

[0007] Embodiments of the present invention include methods and apparatus for integrating a microfluidic housing with a biochip and a substrate (e.g., a printed circuit board (PCB)) to form an integrated microfluidic device with a hermetically sealed flow cell and reliable performance.

[0008] In some implementations, within a microfluidic device, a first adhesive is used to bond the microfluidic housing to the biochip to form a hermetically sealed flow cell, while a second adhesive is used to bond the microfluidic housing to a PCB to provide mechanical support. The type and properties of the adhesives, as well as the structure of the microfluidic device, are designed to provide a hermetically sealed flow cell, a reliable device structure, and a wide process window. Many advantages can be achieved compared to conventional methods used to assemble microfluidic devices or housings. Depending on the specific implementation, the device can offer numerous advantages. For example, the device can accommodate variations in sensor thickness, die-attach adhesive thickness, and the front surface of the PCB. The device can minimize stress on the first adhesive, accommodate thermal expansion mismatches between the sensor and the microfluidic device through thermal cycling during operation, and improve the flow field uniformity of the flow cell.

[0009] In some embodiments of the aforementioned microfluidic device, the first adhesive material is solid before curing and substantially retains its thickness after curing to provide a high degree of precision and uniformity for the microfluidic device. The second adhesive material is liquid before curing to be adjusted according to changes in the distance between the bottom surface of the outer wall and the PCB. In some embodiments, the second adhesive material has a higher curing shrinkage rate than the first adhesive material. In some embodiments, the first adhesive material comprises a die attachment film (DAF), while the second adhesive material comprises a liquid epoxy resin.

[0010] In some embodiments of the aforementioned microfluidic device, the first adhesive material, after curing, is a flexible adhesive to accommodate the mismatch in thermal expansion between the biochip and the microfluidic housing through thermal cycling during operation. The second adhesive material is in liquid form before curing. In some embodiments, the first adhesive material comprises a flexible polyurethane adhesive material, while the second adhesive material comprises a liquid epoxy resin. In alternative embodiments, the first adhesive material comprises a pressure-sensitive adhesive (PSA), while the second adhesive material comprises a liquid epoxy resin.

[0011] In some implementations, the first adhesive material and the second adhesive material are cured by different curing processes such as heat curing, moisture curing or ultraviolet curing.

[0012] In some implementations, a biochip may include a biosensor chip.

[0013] In some embodiments, the microfluidic housing includes one or more second cavities between the inner and outer sidewalls for accommodating bonding wires that couple the biochip to the PCB. In some embodiments, a die attachment adhesive layer is used to attach the biochip to the PCB.

[0014] In some embodiments, the microfluidic device also has a second biochip attached to the PCB. In some embodiments, the second biochip includes a biosensor. In some embodiments, the second biochip includes a fluid droplet generating device.

[0015] In some implementations, the microfluidic device also has an integrated circuit chip attached to a PCB. In some implementations, the integrated circuit chip includes a processor.

[0016] In some embodiments, the microfluidic device also includes a microelectromechanical system (MEMS) chip attached to a PCB. In some embodiments, the MEMS chip includes actuators for initiating actions in response to signals detected in the biochip.

[0017] In some embodiments, the second adhesive layer includes an opening for testing the airtightness of the first adhesive layer. In alternative embodiments, the microfluidic housing includes an opening for testing the airtightness of the first adhesive layer.

[0018] According to some alternative embodiments of the present invention, the microfluidic device may include a PCB (printed circuit board), a biochip covering the PCB, and a microfluidic housing covering the biochip and the PCB. The microfluidic device may also have a first adhesive layer for attaching the microfluidic housing to the biochip and a second adhesive layer for attaching the microfluidic housing to the PCB. The second adhesive layer may be thicker than the first adhesive layer. The first adhesive layer contains a first adhesive material, while the second adhesive layer contains a second adhesive material.

[0019] In some embodiments of the aforementioned microfluidic device, the microfluidic housing includes an inlet, an outlet, and a first cavity. The microfluidic housing has an inner sidewall adjacent to the first cavity, and this inner sidewall is attached to the biochip using a first adhesive layer to form a flow cell. The microfluidic housing also has an outer sidewall attached to a PCB using a second adhesive layer.

[0020] According to some embodiments of the present invention, a method for manufacturing a microfluidic device includes providing a PCB (printed circuit board), attaching a biochip to the PCB, providing a microfluidic housing, and providing first and second adhesive materials. The first adhesive material is used to attach the microfluidic housing to the biochip, and the second adhesive material is used to attach the microfluidic housing to the PCB. The second adhesive material is in liquid form prior to curing and has a higher curing shrinkage rate than the first adhesive material. The method further includes attaching the microfluidic housing to the biochip using the first adhesive material and attaching the microfluidic housing to the PCB using the second adhesive material. The method further includes curing the first adhesive material and the second adhesive material to form a first adhesive layer and a second adhesive layer, respectively.

[0021] In some embodiments of the above method, the microfluidic housing includes an inlet, an outlet, and a first cavity. The microfluidic housing has an inner sidewall adjacent to the first cavity, and this inner sidewall is attached to the biochip using a first adhesive layer to form a flow cell. The microfluidic housing also has an outer sidewall attached to the PCB using a second adhesive layer.

[0022] In some embodiments of the above method, the first adhesive material is solid before curing and substantially retains its thickness after curing to provide a high degree of precision and uniformity for the microfluidic device. The second adhesive material is liquid before curing to be adjusted according to changes in the distance between the bottom surface of the outer wall and the PCB. In some embodiments, the second adhesive material has a higher curing shrinkage rate than the first adhesive material. In some embodiments, the first adhesive material comprises a die attachment film (DAF), while the second adhesive material comprises a liquid epoxy resin.

[0023] In some embodiments of the above method, the first adhesive material, after curing, is a flexible adhesive to accommodate the mismatch in thermal expansion between the biochip and the microfluidic housing through thermal cycling during operation. The second adhesive material is in liquid form prior to curing. In some embodiments, the first adhesive material comprises a flexible polyurethane adhesive material, while the second adhesive material comprises a liquid epoxy resin. In some embodiments, the first adhesive material comprises a pressure-sensitive adhesive (PSA), while the second adhesive material comprises a liquid epoxy resin.

[0024] A further understanding of the nature and advantages of the invention can be achieved by referring to the remainder of the specification and the accompanying drawings. Attached Figure Description

[0025] Figure 1 This is a simplified cross-sectional view of a microfluidic device according to some embodiments of the present invention;

[0026] Figure 2 This is a flowchart illustrating a method for forming a microfluidic device according to some embodiments of the present invention;

[0027] Figure 3A-3I Some embodiments of the present invention are shown in Figure 2 A cross-sectional view of the method summarized in the flowchart;

[0028] Figure 3A It shows that it can be shown Figure 2 A cross-sectional view of the printed circuit board (PCB) used in the method;

[0029] Figure 3B It shows that it can be used Figure 2 A cross-sectional view of the die bonding adhesive formed on the PCB used in the method;

[0030] Figure 3C It shows that it can be used Figure 2 A cross-sectional view of the biochip attached to the PCB used in the method;

[0031] Figure 3D This is a cross-sectional view showing the bonding wires formed to electrically connect the biochip and the PCB;

[0032] Figure 3E This is a cross-sectional view of a microfluidic housing according to some embodiments of the present invention;

[0033] Figure 3F This is a cross-sectional view showing the relative dimensions of the microfluidic housing and the PCB according to some embodiments of the present invention;

[0034] Figure 3G This is a cross-sectional view of an adhesive layer disposed on a biochip and a PCB according to some embodiments of the present invention;

[0035] Figure 3H This is a cross-sectional view of an adhesive layer disposed on a microfluidic housing according to some embodiments of the present invention;

[0036] Figure 3I This is a cross-sectional view of a microfluidic housing attached to a biochip and a PCB, according to some embodiments of the present invention;

[0037] Figure 3J A simplified cross-sectional view of a microfluidic device is shown according to an alternative embodiment of the invention;

[0038] Figure 3K A simplified cross-sectional view of a microfluidic device is shown according to another embodiment of the present invention;

[0039] Figure 4 This is a cross-sectional view of a biological sample introduced into a microfluidic device for analysis, according to some embodiments of the present invention;

[0040] Figure 5 This is a simplified diagram illustrating a microfluidic device having multiple devices attached to a PCB, according to some embodiments of the present invention;

[0041] Figure 6 This is a simplified diagram illustrating a microfluidic device formed by alternative methods according to some embodiments of the invention; and

[0042] Figure 7 This is a simplified diagram illustrating a microfluidic device formed by another method according to some embodiments of the present invention. Detailed Implementation

[0043] Figure 1This is a simplified cross-sectional view of a microfluidic device according to some embodiments of the present invention. Figure 1 As shown, the microfluidic device 100 includes a substrate 110, such as a PCB (printed circuit board), a biochip 120 covering the PCB 110, and a microfluidic housing 130 covering the biochip 120 and the PCB 110. The microfluidic housing 130 is attached to the biochip 120 using a first adhesive layer 141 to form a flow cell, and the microfluidic housing is attached to the PCB 110 using a second adhesive layer 142 to provide mechanical support. For example, the biochip 120 can be attached to the PCB 110 using a die-attachment adhesive layer 113. It should be understood that the substrate described herein is not limited to a PCB, and other substrates may also be used, such as semiconductor (e.g., silicon) substrates, glass substrates, ceramic substrates, etc.

[0044] The biochip 120 may include devices for manipulating or analyzing biological or chemical samples, such as sensors, actuators, etc. In some cases, the sensors and actuators may include one or more MEMS devices. For example, the biochip may be configured to detect signals from a biological sample, and a processor may process the detected signals and respond to the signals by triggering an actuator. According to an embodiment, the PCB 110 may be used to connect multiple biochips to other circuit components, such as processors, control devices, storage devices, I / O devices, and communication devices.

[0045] exist Figure 1 In this embodiment, the microfluidic housing 130 has an inlet 131, an outlet 132, and a first cavity 133. The microfluidic housing 130 may have an inner sidewall 135 adjacent to the cavity 133, and this inner sidewall is attached to the biochip 120 using a first adhesive layer 141 to form a flow cell 140 with a hermetically sealed design. As used herein, a hermetically sealed design refers to a seal that is both airtight and liquid-tight, eliminating the passage of air, gas, and liquid. The flow cell 140 includes a channel formed by the cavity 133 between the microfluidic housing 130, the inner sidewall 135 of the microfluidic housing, and the biochip 120. The flow cell 140 also has an inlet 131 and an outlet 132. As an example of this application, a biological sample 137 can be introduced into the cavity 133 through the inlet 131, where sensors in the biochip 120 can determine the characteristics of the biological sample 137. The biological sample 137 can then be removed from the cavity 133 through the outlet 132.

[0046] like Figure 1 As shown, the microfluidic housing 130 may also have an outer sidewall 136 attached to the PCB 110 using a second adhesive layer 142 to provide mechanical support. In some embodiments, the biochip 120 is electrically coupled to the PCB 110 using wire bonding. Figure 1In this embodiment, contact pads 122 on the biochip are connected to contact pads 112 on the PCB via bonding wires 114. Contact pads 122, 112, and bonding wires 114 can be encapsulated within a bonding wire protection structure 115. In this regard, the microfluidic housing 130 may also include a second cavity 138 for accommodating the bonding wires. The gap 148 between the microfluidic housing 130 and the PCB 110 can be filled with an underfill material 149, which may be an epoxy material to provide a flexible layer between the package and the PCB.

[0047] A first adhesive layer 141 forms a hermetically and liquidally sealed gasket between the microfluidic housing 130 and the biochip 120. Furthermore, the first adhesive layer 141 is compatible with the materials used in the flow cell. On the other hand, a second adhesive layer 142 is configured to provide mechanical strength at the junction between the microfluidic housing 130 and the PCB 110. In some examples, the second adhesive layer 142 is thicker than the first adhesive layer 141. The distance 144 between the bottom surface of the outer sidewall 136 of the microfluidic housing and the PCB 110 is greater than the distance 145 between the bottom surface of the inner sidewall 135 of the microfluidic housing and the biochip 120.

[0048] In some embodiments, to bond the microfluidic housing 130 to the biochip 120 and PCB 110, a first adhesive layer and a second adhesive layer are first formed, and then the microfluidic housing 130 is selected and positioned to contact the biochip 120 and PCB 110. In some embodiments, the microfluidic device is designed such that the first adhesive layer can be in solid form and have a well-defined thickness. On the other hand, the second adhesive layer is thick enough and in liquid form before curing, such that the adhesive line thickness of the second adhesive layer is self-adjustable. In other words, it can fill the space required for the structure of the microfluidic device, which can be affected by the thickness of the first adhesive layer in the microfluidic device, the sensor thickness, the die adhesive thickness, PCB surface roughness, and steps in the bonding cavity, etc. Here, the adhesive line thickness refers to the thickness of the adhesive layer between the bottom surface of the device structure above the adhesive layer and the top surface of the device structure below the adhesive layer. Depending on the context, the term "adhesive line thickness" can refer to the adhesive line thickness before or after curing.

[0049] In some embodiments, the first adhesive material substantially maintains its thickness through a curing process to maintain the high uniformity of the microfluidic device and ensure the uniformity of the fluid field in the flow cell. In a particular embodiment, the first adhesive material may be a die-attachment film (DAF). The die-attachment film is a solid adhesive before curing and substantially maintains its thickness after curing.

[0050] In some embodiments, the first adhesive material, after curing, can be a flexible adhesive, also known as a stress-flexible adhesive. In these cases, a constant thickness of the first adhesive material is not required to maintain the structural uniformity of the microfluidic device. The flexible adhesive can be used to accommodate the mismatched thermal expansion between the biochip and the microfluidic housing through thermal cycling during operation. Therefore, it is desirable for the first adhesive to undergo elastic elongation without irreversible deformation or creep. In some embodiments, the elongation of the first adhesive material can be greater than 0.5% of its thickness before creep. For example, the flexible first adhesive material can be a polyurethane adhesive material manufactured by Bostik. As another example, the flexible first adhesive can be a pressure-sensitive adhesive (PSA).

[0051] As mentioned above, Figure 1 An example of a microfluidic device with a single biochip bonded to a PCB is shown. However, the features described above are not limited to microfluidic devices with a single chip, and the chip is not necessarily a biochip, as explained further below.

[0052] Figure 2 This is a flowchart illustrating a method for forming a microfluidic device according to some embodiments of the present invention. Figure 3A-3K This is illustrated by some embodiments of the present invention. Figure 2 A cross-sectional diagram summarizing the methods in the flowchart. Used to form microfluidic devices (such as...) Figure 1 The method of the microfluidic device 100 is now based on Figure 2 Flowcharts and Figure 3A-3K The cross-sectional diagram is described in the text.

[0053] like Figure 2 As shown, the method 200 for forming a microfluidic device can be summarized as follows.

[0054] 210—Provides printed circuit boards (PCBs);

[0055] In step 220—attach the biochip to the PCB;

[0056] 230—Form bonding wires between the biochip and the PCB;

[0057] 240—Provides a microfluidic housing;

[0058] At 250—a first adhesive layer and a second adhesive layer are provided:

[0059] In 260—the microfluidic shell is attached to the biochip and PCB; and

[0060] The adhesive layer is cured at 270°C.

[0061] These processes will be described in detail below.

[0062] In process 210, method 200 begins with providing a printed circuit board (PCB) 110. Figure 3A It shows that it can be used Figure 2 The method uses a cross-sectional view of a printed circuit board (PCB). The PCB may include pads for electrical connections to electronic components on the board. For example, Figure 3A Contact pad 112 is shown, which can be used as a pad to couple to a biochip.

[0063] In process 220, method 200 includes attaching the biochip to the PCB. Figure 3B It shows that it can be shown Figure 2 The method uses a cross-sectional view of the die adhesive formed on a printed circuit board (PCB). Figure 3C It shows that it can be shown Figure 2 A cross-sectional view of the biochip attached to a printed circuit board (PCB) used in the method. Figure 3B In this context, adhesive layer 113 or die bonding adhesive can be an epoxy-based adhesive used to attach the chip to the PCB. Next, as... Figure 3C As shown, the biochip 120 is attached to the PCB 110 using adhesive layer 113.

[0064] Biochip 120 may include a device for processing or analyzing biological or chemical samples. As used herein, “biochip” refers to a structure that associates, immobilizes, or captures biomolecules for analysis. Typically, a biochip includes a series of binding sites, each of which can be independently occupied by a biomolecule such as a protein, nucleic acid, antibody, or polysaccharide. Typically, a detectable signal generated at one or more binding sites can be detected. For example, an enzymatic, binding, or chemical reaction at a binding site can produce a detectable signal, such as fluorescence or chemiluminescence emission, which is detected and identifies a characteristic or property of the biomolecule at that site. As described below, a biochip can be used for nucleic acid sequencing. In some cases, a biochip may include a sensor (i.e., a biosensor). As used herein, the term “biosensor” or “biological sensor” can be used to refer to a device for identifying luminescent material within or attached to a biomolecule (particularly nucleic acid macromolecules exemplified by DNA and branched or otherwise derived nucleic acids). In one example, the biochip can detect a signal, such as a fluorescence or chemiluminescence signal, from a biological sample, and a processor can process the detected signal and respond to it by triggering an actuator. Examples of biochips may include the CMOS biosensor described in U.S. Patent Application No. 16 / 128,120, filed September 11, 2018, the entire contents of which are incorporated herein by reference. For example, a biosensor may include a flow cell covering a complementary metal-oxide-semiconductor (CMOS) layer. The CMOS layer may include: a photosensing layer having a plurality of photodiodes; and an electronic circuitry layer coupled to the photosensing layer to process the sensed signal. Other examples of biochips may include microdroplet processing chips, such as the integrated lab-on-a-chip described in U.S. Patent Application No. 12 / 513,157, filed November 1, 2007, and U.S. Patent Publication No. 20100096266, published April 22, 2010, the entire contents of which are incorporated herein by reference. It should be understood that the microfluidic devices described herein can be used to detect signal generation events unrelated to biological responses (e.g., signals generated by chemical transformations not involving biomolecules).

[0065] In process 230, bonding wires are formed between the biochip and the PCB, and a protective package is formed to protect the bonding wires. Figure 3D This is a cross-sectional view showing the bonding wires formed for electrically connecting the biochip and the printed circuit board (PCB). Figure 3D In the process, the contact pad 122 on the biochip is connected to the contact pad 112 on the PCB via a bonding wire 114. The contact pad 122, the contact pad 112, and the bonding wire 114 can be encapsulated in a bonding wire protection structure 115.

[0066] In process 250 of method 200, a microfluidic housing is provided. Figure 3EThis is a cross-sectional view of a microfluidic housing according to some embodiments of the present invention. For example... Figure 1 As shown, the microfluidic housing 130 is used to form a flow cell with the biochip 120, and the microfluidic housing 130 is also attached to the PCB 110 to provide mechanical structure for the microfluidic device.

[0067] The material used for the microfluidic housing is compatible with the function of the flow cell (e.g., for processing biological samples). This material is compatible with biochips and PCBs and is expected to have a compatible coefficient of thermal expansion. In some embodiments, the microfluidic housing may be made of glass or plastic materials or other suitable materials. For example, the microfluidic housing may be made of molded plastic. The microfluidic housing can be formed separately from the processes described above for chip bonding and PCB fabrication, and does not need to follow the sequence of steps described above.

[0068] like Figure 3E As shown, the microfluidic housing 130 has an inlet 131, an outlet 132, and a first cavity 133. The microfluidic housing 130 also has an inner sidewall 135 adjacent to the cavity 133, and this inner sidewall is used for attachment to the biochip to form a flow cell 140 with a hermetically sealed structure. The flow cell 140 includes a channel formed by the cavity 133 between the microfluidic housing 130, the inner sidewall 135 of the microfluidic housing, and the biochip 120.

[0069] like Figure 3E As shown, the microfluidic housing 130 may also have an outer sidewall 136 for attachment to the PCB to provide mechanical support.

[0070] like Figure 3F As shown, the shape and dimensions of the microfluidic housing 130 are designed for assembly with the biochip 120 and the PCB 110. To demonstrate the design considerations of the microfluidic housing, Figure 3F A microfluidic housing 130 is shown disposed on a biochip 120, with the bottom surface of the inner sidewall 135 contacting the top surface of the biochip 120, and no adhesive material at interface 147. In this configuration, a gap 148 exists between the bottom surface of the outer sidewall 136 of the microfluidic device 130 and the top surface of the PCB 110. The height of the gap can be selected to accommodate variations in the chip and PCB and manufacturing errors. As described below, this configuration and selection of adhesive materials can facilitate the formation of a hermetic bond between the microfluidic housing 130 and the biochip 120, and a bond between the microfluidic housing 130 and the PCB 110 to provide mechanical strength to the device. In some embodiments, the height of the gap 148 can be about 10 μm, for example, 5 to 30 μm. In other embodiments, the height of the gap can be, for example, 30 to 50 μm or greater. The lateral dimension of the cavity 133 in the microfluidic housing 130 is determined by the width of the biochip 120 and the desired size of the flow cell.

[0071] In process 250 of method 200, an adhesive layer is applied to attach the microfluidic housing 130 to the biochip 120 and the PCB 110. In one embodiment, the adhesive layer may be applied to the top surfaces of the biochip and the PCB. Alternatively, the adhesive layer may be disposed at the bottom of the microfluidic housing 130, at the bottom surface. In a first embodiment, as... Figure 3G As shown, a first adhesive layer 141 is disposed on the top surface of the biochip 120, while a second adhesive layer 142 is disposed on the top surface of the PCB 110. In the second embodiment, as... Figure 3H As shown, a first adhesive layer 141 and a second adhesive layer 142 are disposed on the lower surface of the microfluidic housing 130. For example, the first adhesive layer 141 is disposed on the bottom surface of the inner sidewall 135 of the biochip 120, and the second adhesive layer 142 is disposed on the bottom surface of the outer sidewall 136 of the biochip 120.

[0072] In another embodiment, an adhesive layer 141 is disposed on the biochip 120 or the microfluidic housing 130, and then the microfluidic housing 130 is attached to the biochip 120, forming an adhesive bond between the microfluidic housing 130 and the biochip 120. Then, a second adhesive layer 142 can be disposed in the gap 148 between the microfluidic housing 130 and the PCB 110, as described above. Figure 3F The method described above is to form a second bond between the microfluidic housing 130 and the PCB 110. An adhesive dispensing device, such as an automated adhesive dispensing device, can be used to place the adhesive layer in the desired location.

[0073] In process 260 of method 200, the microfluidic housing 130 is attached to the biochip 120 and the PCB 110, as follows: Figure 3I As shown. In some embodiments, both adhesive layers are disposed on the microfluidic housing 130 (e.g. Figure 3H (as shown), or two adhesive layers are respectively disposed on the biochip 120 and the PCB 110 (as shown). Figure 3G (As shown). In these embodiments, the microfluidic housing 130 is picked up, placed on the biochip 120 and the PCB 110, and the microfluidic housing 130 is pressed onto the biochip 120 and the PCB 110 to form an adhesive. Subsequently, a curing process is performed to harden the adhesive layers 141 and 142.

[0074] In another embodiment, a bond can be formed between the microfluidic housing 130 and the biochip 120 by depositing a first adhesive layer 141 onto the biochip 120 and then attaching the microfluidic housing 130 to the biochip 120 via pick-and-press steps. A curing process can be performed to cure the first adhesive layer 141. A second adhesive layer 142 can then be deposited in the gap 148 between the microfluidic housing 130 and the PCB 110 to form an adhesive bond after the curing process.

[0075] In embodiments of the invention, the material, thickness, and volume of the adhesive layer are selected to form an hermetically tight seal for the flow cell and to provide mechanical strength to the packaging. A first adhesive layer 141 forms a hermetically and liquid-tight seal between the microfluidic housing 130 and the biochip 120. Furthermore, the first adhesive layer 141 is compatible with the materials used in the flow cell. On the other hand, a second adhesive layer 142 is configured to provide mechanical strength at the junction between the microfluidic housing 130 and the PCB 110.

[0076] In some implementations, the first adhesive layer may be in solid form and have a sufficiently defined thickness that is substantially maintained throughout the curing process. On the other hand, the second adhesive layer is thick enough and in liquid form before curing, such that the adhesive line thickness of the second adhesive layer is self-adjustable. In other words, it fills the space required for the structure of the microfluidic device, which may include the thickness of the first adhesive layer, sensor thickness, die adhesive thickness, PCB surface roughness, and steps in the bonding cavities of the microfluidic device, etc. Here, adhesive line thickness refers to the thickness of the adhesive layer between the bottom surface of the device structure above the adhesive layer and the top surface of the device structure below the adhesive layer. Depending on the context, the term "adhesive line thickness" can refer to the adhesive line thickness before curing or the adhesive line thickness after curing.

[0077] In embodiments of the invention, the first adhesive layer 141 and the second adhesive layer 142 provide different functions. The first adhesive layer 141 may be made of a first material or a first adhesive, and the second adhesive layer 142 may be made of a second material or a second adhesive. In some embodiments, the first adhesive may be a solid adhesive before curing and substantially maintain its thickness after curing to ensure the uniformity of the microfluidic device. In other embodiments, the first adhesive is a flexible adhesive material after curing to accommodate thermal expansion mismatch during thermal cycling in operation of the microfluidic device. The second adhesive is stretchable and capable of changing shape upon pressing during the assembly step of attaching the microfluidic chip.

[0078] In some embodiments, the first adhesive layer 141 may be a flexible material or a stress-flexible adhesive. In other words, the first adhesive remains elastic after curing to accommodate the mismatch in thermal expansion between the biochip and the microfluidic housing during thermal cycling during operation. For example, in some biological applications, the device undergoes thermal cycling, for example, between room temperature and higher processing temperatures (e.g., 65°C or higher). Components of the microfluidic device may have different materials with different coefficients of thermal expansion. Therefore, in some embodiments, a flexible adhesive is used to form the first adhesive layer 141 between the microfluidic housing 130 and the biochip 120. The flexible adhesive can absorb thermally induced dimensional changes between the microfluidic housing 130 and the biochip 120. An example of a flexible adhesive material is a polyurethane adhesive material made of d. Polyurethane adhesives have low moisture permeability and elastomeric properties and provide a strong, flexible, and tough bond once cured. For example, Bostik 1100FS adhesive is used in some embodiments. Of course, in embodiments of the invention, the first adhesive layer is not limited to polyurethane adhesives. Other suitable flexible adhesive materials may also be used. Another example of flexible adhesives includes epoxy resins, as described in U.S. Patent Publication No. US20070081317 to Choi (published April 12, 2017, entitled “Circuit board mounting for temperature stress reduction,” the entire contents of which are incorporated herein by reference.

[0079] In some embodiments, because the first adhesive layer 141 is used to form a flow cell chamber between the microfluidic housing 130 and the biochip 120, the first adhesive layer 141 can be used to maintain a defined thickness before curing to maximize the fluid field uniformity of the flow cell. In these modifications, dry film adhesives, such as die-attachment films (DAF), can be used. Die-attachment films comprise an epoxy adhesive that is film-based rather than paste-based and is typically attached to the back side of the wafer before dicing. A chip with DAF on the back side can be attached to a PCB. DAF has advantages over adhesive pastes because the adhesive material does not reform from a droplet shape into a thin two-dimensional layer. Furthermore, the processing window of a film can be wider than that of a paste material, and the assembly process is simplified by using a film.

[0080] Other examples of die attachment films may include materials such as epoxy resins, phenolic resins, acrylic rubbers, silica fillers, or combinations thereof, and can be applied using lamination techniques. One example of a die attachment adhesive is manufactured by Henkel Chemicals Company, Düsseldorf, Germany. 789-3 TM However, any other suitable alternative materials and forming techniques can be used instead.

[0081] Examples of die attachment films are described in U.S. Patent Application Publication No. 20060154078, entitled "Curing Resin Composition, Adhesive Epoxy Resin Paste, Adhesive Epoxy Resin Sheet, Conductive Connection Paste, Conductive Connection Sheet, And Electronic Component Joined Body," published July 13, 2006, by Watanabe, and U.S. Patent Application Publication No. US20080318364, entitled "Process Applying Die Attach Film To Singulated Die," published December 15, 2008, both of which are incorporated herein by reference in their entirety. An example process flow for DAF assembly involves an adhesive step followed by oven curing, for example, at 130°C for 60 minutes. Alternatively, a UV curing process may be used.

[0082] In some embodiments, the second adhesive may be a liquid epoxy resin, which is in liquid form after being distributed on the PCB 110 and before curing. Examples of liquid epoxy resins are described in U.S. Patent Application Publication No. 2018 / 0213635, entitled “Resin Composition And Multilayer Substrate,” published July 26, 2018 by Baba; U.S. Patent Application Publication No. US2018 / 0258325, entitled “Adhesive Layer And Adhesive Sheet,” published September 13, 2018 by Taniquichi; and U.S. Patent Application Publication No. US2018 / 0291164, entitled “Fast Cure Epoxy Composition For Use In High Throughput Manufacturing Processes,” published October 11, 2018 by Bank, the entire contents of which are incorporated herein by reference. A specific example of a liquid epoxy resin is diglycidyl ether of bisphenol A. Other examples may include liquid epoxy materials manufactured by Bostik, Inc. (Wauwatosa, WI, USA), which are moisture-cured. Other suppliers of liquid epoxy include Norland NEA 123S or 123T manufactured by Norland Products, Inc. (Cranbury, New Jersey, USA), Dymax Corporation (Torrignton, CT.), and Electronic Materials Inc. (Breckenridge, CO.).

[0083] Another example of a flexible first adhesive is a pressure-sensitive adhesive (PSA). A PSA is an adhesive that forms a bond when pressure is applied. Some PSAs are based on elastomers, such as acrylic-based elastomers. PSAs exhibit viscoelastic (sticky and elastic) properties, both of which are used for proper bonding.

[0084] As mentioned above Figure 3F As described above, when the microfluidic housing 130 is disposed on the biochip 120, with the bottom surface of the inner sidewall 135 contacting the top surface of the biochip 120, and no adhesive material is present at the interface 147, a gap 148 exists between the bottom surface of the outer sidewall 136 of the microfluidic device 130 and the top surface of the PCB 110. Therefore, when the microfluidic housing 130 is attached to the biochip 120 and the PCB 110, a curing process is performed, such as... Figure 3IAs shown, the thickness of the second adhesive layer 142 is substantially equal to the thickness of the first adhesive layer 141 plus... Figure 3F The height of the gap 148 described herein. In some embodiments, the thickness of the first adhesive layer 141 after curing can be in the range of 10 to 100 μm. This thickness can vary depending on the design of the flow cell. As mentioned above, the height of the gap 148 can be in the range of about 5 to 50 μm. Therefore, the thickness of the second adhesive layer 142 after curing can be in the range of 15 to 150 μm.

[0085] In some embodiments, the microfluidic housing 130 can be attached to the biochip 120 without the use of external adhesive. For example, in some cases, the body material of the microfluidic housing or the embedded material within the microfluidic housing can be used as an adhesive material. For example, the microfluidic housing can be made of a plastic material that has a low melting temperature and is meltable to form a seal with the biochip. In some embodiments, the microfluidic housing 130 may have an embedded adhesive material. For example, grooves can be formed in the sidewalls of the microfluidic housing 130, and an adhesive material such as DAF or other adhesive materials can be inserted into these grooves.

[0086] In process 260 of method 200, the microfluidic shell is attached to the biochip and the PCB. Figure 3I This is a cross-sectional view of a microfluidic housing attached to a biochip and a PCB according to some embodiments of the present invention. Here, standard pick-and-place and press procedures can be used to attach the microfluidic housing to the biochip and the PCB.

[0087] In process 270 of method 200, the assembled microfluidic device is subjected to a curing process to harden the adhesive layer. For polymer-based adhesive materials, curing refers to toughening or hardening the polymer material through cross-linking of polymer chains. Curing can be performed by applying external energy, such as electron beams, heat, or ultraviolet (UV) radiation.

[0088] In some embodiments, to minimize stress on the first adhesive layer, the curing shrinkage rate of the first adhesive layer should not exceed that of the second adhesive layer. Furthermore, it may be desirable for the curing shrinkage rate of the second adhesive layer to be greater than that of the first adhesive layer to ensure that the first adhesive layer can form a hermetically tight seal between the microfluidic housing and the biochip. In some embodiments, the additional curing shrinkage rate of the second adhesive layer may be about 10% of the thickness of the first adhesive layer. In other embodiments, the additional curing shrinkage rate of the second adhesive layer may be about 5-15% of the thickness of the first adhesive layer.

[0089] Figure 3JThis is a simplified cross-sectional view of a microfluidic device according to an alternative embodiment of the invention. Figure 3J As shown, except for the opening 119 in the second adhesive layer 142, the microfluidic device 170 is similar to Figure 1 and Figure 3I The microfluidic device 100 is shown. The opening 119 is designed to allow testing of the airtightness of the first adhesive layer 141. For example, compressed air can be applied between the inlet 131 and outlet 132 of the microfluidic device 170. Any leaks in the first adhesive layer 141 can be detected through the opening 119 in the second adhesive layer 142.

[0090] Figure 3K This is a simplified cross-sectional view of a microfluidic device according to another embodiment of the present invention. Figure 3K As shown, except for the opening 139 in the microfluidic housing 130, the microfluidic device 180 is similar to Figure 1 and Figure 3I The microfluidic device 100 shown above. Figure 3J Similar to the opening 119 described above, Figure 3K The opening 139 in the microfluidic housing 130 is designed to allow testing of the airtightness of the first adhesive layer. For example, compressed air can be applied between the inlet 131 and the outlet 132 of the microfluidic device 180. Any leaks in the first adhesive layer 141 can be detected through the opening 139 in the microfluidic housing 130.

[0091] The microfluidic device is now ready to receive biological samples for analysis. Figure 4 This is a cross-sectional view illustrating the introduction of biological samples into a microfluidic device for analysis, according to some embodiments of the present invention. Figure 4 The microfluidic device 400 in the middle is similar to Figure 1 The microfluidic device 100 is shown, with a biological sample 137 in the cavity 133 of the microfluidic device.

[0092] For example, a biochip can detect signals, and a processor can process the detected signals and determine which actuator to trigger. The microfluidic device may include two adhesive layers. A first adhesive layer is used to attach the microfluidic housing to the IC chip, and a second adhesive layer is used to attach the microfluidic housing to the PCB.

[0093] As mentioned above, Figure 1 An example of a microfluidic device with a single biochip bonded to a PCB is shown. However, the features described above are not limited to microfluidic devices with a single chip, and the chip is not limited to a biochip. An example is described below.

[0094] Figure 5This is a simplified diagram illustrating a microfluidic device having multiple chips attached to a PCB, according to some embodiments of the present invention. Figure 5 As shown, the microfluidic device 500 includes a PCB (printed circuit board) 510, a first chip 520 and a second chip 525 covering the PCB, and a microfluidic housing 530 covering the chips and the PCB. The first chip 520 can be a biochip, similar to... Figure 1 The microfluidic device 100 is depicted in the image. The second chip 525 can be a second biochip, similar to... Figure 1 The microfluidic device 100 is depicted in the figure. Alternatively, the second chip 525 may be an integrated circuit (IC) chip, such as a processor chip, or a microelectromechanical system (MEMS) chip, such as an actuator.

[0095] The microfluidic housing 530 is attached to chips 520 and 525 using a first adhesive layer 541 to form a flow cell, and the microfluidic housing 530 is attached to the PCB 510 using a second adhesive layer 542 to provide mechanical support. Chips 520 and 525 can be attached to the PCB 510, for example, using a die-attachment adhesive layer 513 for attaching chips to a PCB. Furthermore, a filling portion 527 can be formed to fill the space between chips 520 and 525.

[0096] exist Figure 5 In this design, the microfluidic housing 530 has an inlet 531, an outlet 532, and a first cavity 533. The microfluidic housing 530 may have an inner sidewall 535 adjacent to the cavity 533, and this inner sidewall is attached to the chips 520 and 525 using a first adhesive layer 541 to form a flow cell 540 with a hermetically sealed structure. The flow cell 540 includes a channel formed by the microfluidic housing 530, the inner sidewall 535 of the microfluidic housing, and the cavity 533 between the chips 520 and 525. A biological sample 537 can be introduced into the cavity 533 through the inlet 531, where the chips 520 and 525 can determine the characteristics of the biological sample 537. The biological sample 537 can then be removed from the cavity 533 through the outlet 532.

[0097] like Figure 5 As shown, the microfluidic housing 530 may also have an outer sidewall 536 attached to the PCB 510 using a second adhesive layer 542 to provide mechanical support. In some embodiments, similar to... Figure 1 As described above, chips 520 and 525 are electrically coupled to PCB 510 using wire bonding. In this respect, the microfluidic housing 530 may also include a second cavity for accommodating the wire bonding.

[0098] The first adhesive layer 541 forms a hermetically and liquidally sealed environment between the microfluidic housing 530 and the chips 520 and 525. Furthermore, the first adhesive layer 541 is compatible with the materials used in the flow cell and its operation. On the other hand, the second adhesive layer 542 is configured to provide mechanical strength at the junction between the microfluidic housing 530 and the PCB 510.

[0099] Figure 6 This is a simplified diagram illustrating a microfluidic device formed by an alternative method according to some embodiments of the present invention. Figure 6 As shown, the microfluidic device 600 includes a PCB (printed circuit board) 610, a CMOS image sensor (CIS) 620 covering the PCB 610, and a microfluidic housing 630 covering the CMOS image sensor (CIS) 620 and the PCB 610. The microfluidic housing 630 is attached to the CMOS image sensor (CIS) 620 using adhesive 641 to form a flow cell, and the microfluidic housing 630 is attached to the PCB 610 using latches 642 to provide mechanical support. The CMOS image sensor 620 can be attached to the PCB 610 using solder 612.

[0100] exist Figure 6 In the flow cell 640, the microfluidic housing 630 has an inlet 631 and an outlet 632. A cavity 633 is formed between the microfluidic housing 630 and the CMOS image sensor 620. The flow cell 640 includes a channel formed by the cavity 633 between the microfluidic housing 630, the inlet 631, and the outlet 632. A biological sample 637 can be introduced into the cavity 633 through the inlet 631, where the CMOS image sensor 620 can determine the characteristics of the biological sample 637. Subsequently, the biological sample 637 can be removed from the cavity 633 through the outlet 632.

[0101] In the microfluidic device 600, adhesive 641 is used to form an hermetically sealed flow cell and comes into contact with the biological sample. Therefore, the material of adhesive 641 is compatible with the biological sample, the fluid used to process the biological sample, and the operating conditions. For example, in some embodiments, a polyurethane adhesive compatible with the biological sample can be used as adhesive 641 for forming the flow cell.

[0102] In the microfluidic device 600, a latch 642 is used to attach the microfluidic housing 630 to the PCB 610 to provide mechanical support. In some embodiments, the latch may include a snap-fit ​​feature that passes through a hole in the PCB board. In this way, the snap-fit ​​feature can be used to provide or maintain compressive force between the microfluidic device 600 and the PCB board 610, which in turn helps to provide a seal between the microfluidic housing 630 (bonded using adhesive 641) and the CMOS image sensor (CIS) 620.

[0103] Furthermore, the description of the microfluidic device 600 above is not limited to CMOS sensors. Other types of chips, such as sensor chips or biological sample processing chips, can also be used to implement microfluidic devices. Similar to... Figure 5 The microfluidic device 500 and microfluidic device 600 may also include multiple devices attached to the PCB.

[0104] Figure 7 This is a simplified diagram illustrating a microfluidic device formed by another method according to some embodiments of the present invention. Figure 7 The microfluidic device 700 in the middle is similar to Figure 6 The microfluidic device 600 is depicted in the image. One difference between the microfluidic device 700 and the microfluidic device 600 is that in the microfluidic device 700, O-rings are used instead of the adhesive used in the microfluidic device 600 to attach the microfluidic housing to the CMOS image sensor. (See image for details.) Figure 7 As shown, the microfluidic device 700 includes a PCB (printed circuit board) 710, a CMOS image sensor (CIS) 720 covering the PCB 710, and a microfluidic housing 730 covering the CMOS image sensor (CIS) 720 and the PCB 710. The microfluidic housing 730 is attached to the CMOS image sensor (CIS) 720 using an O-ring 741 to form a flow cell, and the microfluidic housing 730 is attached to the PCB 710 using a latch 742 to provide mechanical support. The CMOS image sensor 720 can be attached to the PCB 710 using solder 712.

[0105] exist Figure 7 In the flow cell 740, the microfluidic housing 730 has an inlet 731 and an outlet 732. A cavity 733 is formed between the microfluidic housing 730 and the CMOS image sensor 720. The flow cell 740 includes a channel formed by the cavity 733 between the microfluidic housing 730, the inlet 731, and the outlet 732. A biological sample 737 can be introduced into the cavity 733 through the inlet 731, where the CMOS image sensor 720 can determine the characteristics of the biological sample 737. Subsequently, the biological sample 737 can be removed from the cavity 733 through the outlet 732.

[0106] An O-ring is a ring of elastomeric material with a circular cross-section, typically designed to be seated in a groove and compressed between two or more parts during assembly, thereby forming a seal at the interface. An elastomeric material refers to an elastic polymer or a rubbery solid with elasticity. In the microfluidic device 700, the O-ring comes into contact with a biological sample analyzed by a CMOS sensor. Therefore, the material of the O-ring is compatible with the biological sample, the fluid used to process the biological sample, and the operating conditions. Furthermore, the description of the microfluidic device 700 above is not limited to the CMOS sensor 720. Other types of chips, such as other types of sensors or biological sample processing chips, can also be used to implement the microfluidic device 700. Similar to... Figure 5 The microfluidic device 500 and microfluidic device 700 may also include multiple devices attached to the PCB.

[0107] Some embodiments of the present invention can be used for the analysis of biological or chemical samples. Biological or chemical samples may include any one of a variety of components. For example, the sample may contain nucleic acid macromolecules (e.g., template, DNA, RNA, etc.), proteins, etc. The sample can be analyzed to determine gene sequences, DNA-DNA hybridization, single nucleotide polymorphisms, protein interactions, peptide interactions, antigen-antibody interactions, glucose monitoring, cholesterol monitoring, etc.

[0108] In some implementations, the biological sample may be a nucleic acid, such as DNA. See U.S. Patent Nos. 8,778,849; 8,445,194; 9,671,344; 7,910,354; 9,222,132; 6,210,891; 6,828,100; 6,833,246; 6,911,345 and Patent Application Publication No. 2016 / 0237488, the entire contents of which are incorporated herein by reference. DNA biomolecules can be, but are not limited to, DNA nanospheres (single-stranded multiplys) that hybridize with labeled probes (e.g., in DNB sequencing by attachment or cPAL methods) or with complementary growing strands (e.g., in DNB sequencing by synthetic methods) or both; or with a single DNA molecule (e.g., in single-molecule sequencing); or with a cloned population of DNA molecules, such as those generated in bridge-based PCR sequencing. Therefore, references to “biomolecule,” “DNA macromolecule,” or “nucleic acid macromolecule” can include more than one molecule (e.g., a DNB associated with multiple growing complementary strands or a DNA cluster comprising hundreds or thousands of DNA molecules). Exemplary methods for manufacturing DNBs (e.g., DNB libraries) and for manufacturing arrays of discretely spaced regions separated by inter-regional regions are well known in the art. See, for example, U.S. Patent Nos. 8,133,719; 8,445,196; 8,445,197; and 9,650,673, the entire contents of which are incorporated herein by reference. In some embodiments, DNB or other macromolecules are immobilized on discrete, spaced-apart regions or spots by attractive non-covalent interactions (e.g., van der Waals forces, hydrogen bonds, and ionic interactions). In some embodiments, the discrete, spaced-apart regions contain functional portions (e.g., amines). In some embodiments, the discrete, spaced-apart regions contain capture oligonucleotides attached thereto to bind template DNA (e.g., DNB). Typically, the discrete, spaced-apart regions are arranged in a linear pattern; however, regular arrays with other arrangements (e.g., concentric circles, spiral patterns, hexagonal patterns, etc.) can be used.

[0109] In some implementations, the nucleic acid macromolecule may be an amplicon of a genomic DNA fragment or a cDNA library. As used herein, an "amplicon" may be the product of amplification of a nucleic acid molecule, typically a fragment of genomic DNA or a fragment of a cDNA library. Methods of amplification include, but are not limited to, rolling circle amplification, as described, for example, in U.S. Patent No. 8,445,194 (the entire contents of which are incorporated herein by reference), or bridged polymerase chain reaction (PCR), as described, for example, in U.S. Patent No. 7,972,820 (the entire contents of which are incorporated herein by reference). Amplification may be performed before the nucleic acid is contacted with a biosensor, or in situ, for example, as described in U.S. Patent No. 7,910,354, the entire contents of which are incorporated herein by reference.

[0110] For example, a biological sample (such as a DNA macromolecule, oligonucleotide, or nucleotide) associated with a fluorescent or chemiluminescent dye can be placed above a photodiode 117. In the case of fluorescence, the dye can be irradiated with excitation light from an excitation source. The excitation light can correspond to any suitable type or intensity of light, including, for example, visible light, infrared light (IR), ultraviolet light (UV), etc. The excitation light can also come from any suitable light source, such as a light-emitting diode (LED), a lamp, a laser, a combination thereof, etc. When the dye is irradiated with excitation light of a specific wavelength, the biological sample can absorb the light and then emit light of a different wavelength. For example, a biological sample can absorb excitation light with a wavelength of 450 nm but emit light with a wavelength of 550 nm. In other words, when the dye is irradiated with light (i.e., an excitation source) of a different characteristic wavelength, it can emit fluorescence with a characteristic wavelength. However, because the excitation light is used to irradiate the dye, thereby producing fluorescence, it must be filtered out in order to accurately measure the fluorescence at the photodiode.

[0111] In the case of chemiluminescence, photodiodes detect emitted light without requiring an excitation source. Conversely, biological samples may emit light due to a chemical or enzymatic reaction that may occur between the biological sample and the chemiluminescent dye (or other solution), resulting in luminescence due to the breaking or formation of chemical bonds (e.g., the action of luciferase proteins on luciferin substrates).

[0112] For both fluorescence and chemiluminescence, a photodiode can detect the intensity of the emitted light and convert it into an electronic signal based on that light intensity, which can be provided to an external device via metal wiring. The external device can then correlate the electronic signal with a specific wavelength and brightness.

[0113] In some embodiments, the effective spots or pores on the surface of the biosensor and nucleic acid macromolecules can be configured such that each spot binds only one nucleic acid macromolecule. For example, this can be achieved by contacting the surface with an amplicon whose size corresponds to that of the effective spot (e.g., an amplicon whose diameter is effectively the same as or larger than the diameter of the effective spot). See U.S. Patent No. 8,445,194, the entire contents of which are incorporated herein by reference. Alternatively, the effective spot may be chemically adapted to bind a single DNA fragment, which can then be amplified to fill the original binding site and the larger region surrounding it.

[0114] Some embodiments of the present invention can be used to determine different labels corresponding to different wavelengths of light. The labels can be, for example, fluorescent labels, chemiluminescent labels, or bioluminescent labels. For example, in gene sequencing (or DNA sequencing), embodiments of the present invention can be used to determine the precise sequence of nucleotide bases within a nucleic acid macromolecule (e.g., a DNA strand). Nucleotide bases (e.g., adenine (A), guanine (G), cytosine (C), or thymine (T)) can be labeled with specific fluorescent markers. Alternatively, sequencing methods using, for example, one color, two colors, or three colors can be used.

[0115] Regarding fluorescence, each nucleotide base can be sequentially determined by continuously exciting a nucleic acid macromolecule with excitation light. The nucleic acid macromolecule can absorb excitation light and transmit emitted light of different wavelengths to a biosensor, as described herein. The biosensor can measure the wavelength and intensity of the emitted light received by a photodiode. Each nucleotide (e.g., a fluorescently labeled nucleotide) can emit light of a specific wavelength and / or intensity to a photodiode when excited by excitation light of a specific wavelength and / or intensity, thereby enabling the identification of a specific nucleotide base at a specific position within the nucleic acid macromolecule. Once the specific nucleotide base is determined, it can be removed from the nucleic acid macromolecule, allowing the determination of the next consecutive nucleotide base using a similar method.

[0116] For any purpose, nucleic acid macromolecules may be labeled with one or more different fluorescent, chemiluminescent, or bioluminescent labels before or after attachment to a biosensor. For example, nucleic acid macromolecules may hybridize with labeled oligonucleotide probes or amplification primers. Alternatively, nucleic acid macromolecules may hybridize with unlabeled oligonucleotides, which may then be attached to labeled probes or amplified using labeled nucleotide analogs. Labeling may be used, for example, to characterize nucleic acid macromolecules (e.g., the presence of disease-associated single nucleotide polymorphisms (SNPs)) or for the sequencing of all or part of the nucleic acid macromolecules, as described above. DNA sequencing by probe hybridization is described, for example, in U.S. Patent No. 8,105,771, the entire contents of which are incorporated herein by reference. Sequencing by anchored probe attachment is described, for example, in U.S. Patent No. 8,592,150, the entire contents of which are incorporated herein by reference. Sequencing by synthesis is described, for example, in U.S. Patent No. 7,883,869, the entire contents of which are incorporated herein by reference. Typically, sequencing by synthesis is a method in which nucleotides are sequentially added to a free 3' hydroxyl group provided by a sequencing primer that hybridizes with a template sequence, resulting in the synthesis of a nucleic acid chain in the 5' to 3' direction. In one approach, another exemplary type of SBS, pyrosequencing, can be used (Ronaghi et al., 1998, Science 281:363).

[0117] In some embodiments, the biosensor can be reversibly coupled to a flow cell (not shown). Nucleic acid macromolecules can be attached to the biosensor by contacting it with a liquid sample in the flow cell. The flow cell may include one or more flow channels in fluid communication with the reaction site. In one example, the biosensor may be fluidly and electrically coupled to a bioassay system. The bioassay system may deliver reagents to the reaction site and perform imaging events according to a predetermined protocol. For example, the bioassay system may guide a solution to flow along the reaction site. The solution may include four types of nucleotides with the same or different fluorescent labels. In some embodiments, the bioassay system may then illuminate the reaction site using an excitation light source. The excitation light may have one or more predetermined wavelengths. The excited fluorescent label can provide an emission signal that can be detected by a photodiode.

[0118] Users can prepare for sequencing by contacting a biosensor according to the described embodiment with a nucleic acid amplicon or subsequently amplified nucleic acid, such that nucleic acid macromolecules bind to and are retained by effective spots or wells, and excess nucleic acid macromolecules can be washed away. The nucleic acid macromolecules can be pre- or in situ contacted with labeling reagents. The biosensor can then be manipulated as described herein to determine light emitted on or around the nucleic acid macromolecules on the array. The light can be quantified, or it can be sufficiently determined in a binary manner which nucleic acid macromolecules on the surface have been labeled with a label that emits light at a specific wavelength. Different probes or different nucleic acid analogs with labels emitting light at different wavelengths can be used simultaneously, for example, to determine different bases at specific positions in a sequence, or to sequence multiple positions.

[0119] Although this document describes back-illuminated CMOS sensors, embodiments of the invention are contemplated to be similarly applicable to front-illuminated CMOS sensors. Furthermore, embodiments of the invention are contemplated to be similarly applicable to any suitable biosensors, such as those described in U.S. Patent Application No. 15 / 803,077, filed November 3, 2017, the entire contents of which are incorporated herein by reference.

[0120] The above description includes methods, systems, and / or structures and their applications in the exemplary aspects of the technology currently described. Although various aspects of the technology have been described above with some degree of specificity or reference to one or more individual aspects, those skilled in the art can make various changes to the disclosed aspects without departing from the spirit or scope of the technology. Since many aspects can be performed without departing from the spirit and scope of the technology currently described, the appropriate scope lies in the appended claims. Therefore, other aspects are contemplated. Furthermore, it should be understood that any operation can be performed in any order unless otherwise expressly stated or claimed, or the language inherently requires a specific order. It is intended that everything contained in the above description and shown in the accompanying drawings should be interpreted as examples of specific aspects only and not as limited to the embodiments shown. Unless the context clearly indicates otherwise, any concentration values ​​provided herein are generally given as mixture values ​​or percentages, without regard to any transformations that occur at or after the addition of a particular component of the mixture. All published references and patent documents referenced in this disclosure are incorporated herein by reference in their entirety for all purposes, to the extent that they are not explicitly indicated as incorporated herein. Changes in detail or structure may be made without departing from the essential elements of the present technology as defined in the appended claims.

Claims

1. A method for manufacturing a microfluidic device, the microfluidic device comprising a printed circuit board (PCB), a biochip, and a microfluidic housing, the method comprising: The inner wall of the microfluidic shell is attached to the biochip using a first adhesive material to form a flow cell; The outer portion of the microfluidic housing is attached to the PCB using a second adhesive material; Specifically, at least after the microfluidic housing adheres to the inner wall of the biochip and after the outer portion of the microfluidic housing adheres to the PCB, the second adhesive material is thicker than the first adhesive material. In this embodiment, after the microfluidic device is manufactured, the biochip is located between the PCB and the microfluidic housing, the biochip is attached to and covers the PCB, and the microfluidic housing is attached to and covers both the biochip and the PCB.

2. The method of claim 1, wherein the biochip is attached to the PCB using a third adhesive material.

3. The method of claim 1, wherein the first adhesive material is elastic after it has adhered to the inner wall of the microfluidic housing to the biochip.

4. The method of claim 1, wherein the second adhesive material is applied in liquid form to at least one of the outer portion of the microfluidic housing and the PCB before attaching the outer portion of the microfluidic housing to the PCB.

5. The method according to claim 1, wherein: The first adhesive material is solid before curing and essentially maintains its thickness during and after curing. and The second adhesive material is liquid before curing to adjust the distance variation between the bottom surface of the microfluidic housing and the PCB.

6. The method of claim 5, wherein the first adhesive material comprises a die attachment film (DAF); and the second adhesive material comprises liquid epoxy resin.

7. The method of claim 5, wherein the second adhesive material has a higher curing shrinkage rate than the first adhesive material.

8. The method of claim 5, wherein the first adhesive material comprises a polyurethane adhesive material; and the second adhesive material comprises a liquid epoxy resin.

9. The method according to claim 5, wherein: The first adhesive material includes pressure-sensitive adhesive (PSA); and the second adhesive material includes liquid epoxy resin.

10. The method of claim 1, wherein the microfluidic housing includes an inlet, an outlet, an inner sidewall, an outer sidewall, and a first cavity, the inner sidewall of the microfluidic housing being adjacent to the first cavity, and the microfluidic housing being attached to the PCB on the outer sidewall.

11. The method of claim 1, further comprising attaching a second biochip to the PCB prior to attaching the microfluidic housing.

12. The method of claim 1, further comprising attaching the integrated circuit chip to the PCB prior to attaching the microfluidic housing.

13. The method of claim 1, further comprising attaching the MEMS chip to the PCB prior to attaching the microfluidic housing.

14. The method of claim 1, further comprising an opening in the second adhesive layer for airtightness testing of the first adhesive layer.

15. The method of claim 1, further comprising an opening in the microfluidic housing for airtightness testing of the first adhesive layer.

Images