Apparatus and method for integrated sensor cartridge - Patents.com

Abstract

To provide a method and device for integrating a microfluidic housing with a biological chip, and a substrate, such as a printed circuit board (PCB), to form an integrated microfluidic device with hermetic sealing of a flow cell and reliable performance.SOLUTION: A microfluidic device 100 comprises: a PCB (printed circuit board) 110; a biological chip 120 on the PCB 110; and a microfluidic housing 130 on the biological chip 120 and the PCB 110. The microfluidic device 100 has a first adhesive layer 141 that attaches the microfluidic housing 130 to the biological chip 120, and a second adhesive layer 142 that attaches the microfluidic enclosure 130 to the PCB 110. The second adhesive layer 142 is thicker than the first adhesive layer. The first adhesive layer 141 comprises a first adhesive material, and the second adhesive layer 142 comprises a second adhesive material.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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 by reference.

[0002] The present invention relates generally to biosensors for biological or chemical analysis, and more particularly to a method for integrating a microfluidic housing with a biological chip and a substrate, such as a printed circuit board (PCB), to form an integrated sensor cartridge or microfluidic device. [Background technology]

[0003] High throughput analysis of chemical and / or biological species is an important tool in the fields of diagnostics and therapeutics. Attached chemical and / or biological species arrays are designed to define specific target sequences, analyze gene expression patterns, identify specific allelic variations, determine copy numbers of DNA sequences, and identify binding sites of proteins (e.g., transcription factors and other regulatory molecules) throughout the genome. In a particular example, the advent of the Human Genome Project necessitated improved methods for nucleic acid sequence determination, e.g., DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) development. The determination of the sequence of all 3 billion bases of the haploid human genome provided the basis for identifying the genetic basis of many diseases.

[0004] High throughput analyses, such as massively parallel DNA sequencing, often utilize flow cells that contain an array of chemical and / or biological species available for analysis. Flow cells are often fabricated in microfluidic housings that are integrated with biological chips, e.g., silicon-based sensor chips, to form microfluidic devices, e.g., cartridges. Fabrication and use of many current microfluidic designs is complex, costly, and often unreliable. Summary of the Invention

[0005] Embodiments of the present invention include methods and devices for integrating a biological chip with a substrate, such as a printed circuit board (PCB), and a microfluidic housing to form an integrated microfluidic device with hermetic sealing and reliable performance of the flow cell.

[0006] In some embodiments, in a microfluidic device, a first adhesive is used to bond the biological chip and the microfluidic housing to form a hermetic seal of the flow cell, and a second adhesive is used to bond the PCB and the microfluidic housing to provide mechanical support. The adhesive type and properties and the structure of the microfluidic device are designed to provide a hermetic seal of the flow cell, reliable device structure, and a wide process window. The present invention provides many advantages over conventional methods of assembling microfluidic devices or cartridges. Depending on the particular embodiment, the device provides many advantages. For example, the device accommodates variations in sensor thickness, variations in die attach adhesive thickness, and variations in the PCB front surface. The device minimizes stress on the first adhesive, accommodates mismatched thermal expansion between the sensor and the microfluidic device through thermal cycling during operation, and improves the fluid field uniformity of the flow cell.

[0007] In some embodiments of the above microfluidic device, the first adhesive material is solid before curing and substantially maintains its thickness after curing to provide height accuracy and uniformity of the microfluidic device. The second adhesive material is liquid before curing and accommodates variations in the distance between the lower surface of the outer sidewall and the PCB. In some embodiments, the second adhesive material has a higher cure shrinkage than the first adhesive material. In some embodiments, the first adhesive material comprises a die attach film (DAF) and the second adhesive material comprises a liquid epoxy.

[0008] In some embodiments of the above microfluidic device, the first adhesive material is a compatible adhesive after curing, which accommodates mismatched thermal expansion between the biological chip 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 compatible urethane adhesive material and the second adhesive material comprises a liquid epoxy. In an alternative embodiment, the first adhesive material comprises a pressure sensitive adhesive (PSA) and the second adhesive material comprises a liquid epoxy.

[0009] In some embodiments, the first adhesive material and the second adhesive material are cured by different curing processes, such as, for example, heat, moisture or ultraviolet radiation curing.

[0010] In some embodiments, the biological chip comprises a biological sensor chip.

[0011] In some embodiments, the microfluidic housing comprises one or more second cavities between the inner and outer sidewalls that accommodate bond wires that couple the biological chip to the PCB, hi some embodiments, a die attach adhesive layer is used to attach the biological chip to the PCB.

[0012] In some embodiments, the microfluidic device also has a second biological chip attached to the PCB. In some embodiments, the second biological chip includes a biosensor. In some embodiments, the second biological chip includes a fluidic droplet generator.

[0013] In some embodiments, the microfluidic device also includes an integrated circuit chip attached to the PCB, hi some embodiments, the integrated circuit chip includes a processor.

[0014] In some embodiments, the microfluidic device also includes a microelectromechanical systems (MEMS) chip attached to the PCB, hi some embodiments, the MEMS chip includes an actuator that initiates movement in response to signals detected on the biological chip.

[0015] In some embodiments, the second adhesive layer includes an opening for hermetic testing of the first adhesive layer. In an alternative embodiment, the microfluidic housing includes an opening for hermetic testing of the first adhesive layer.

[0016] According to some alternative embodiments of the present invention, a microfluidic device includes a PCB (printed circuit board), a biological chip on the PCB, and a microfluidic housing on the biological chip and the PCB. The microfluidic device also has a first adhesive layer attaching the microfluidic housing to the biological chip and a second adhesive layer attaching the microfluidic housing to the PCB. The second adhesive layer is thicker than the first adhesive layer. The first adhesive layer includes a first adhesive material and the second adhesive layer includes a second adhesive material.

[0017] In some embodiments of the 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, the inner sidewall being attached to the biological chip with a first adhesive layer to form a flow cell, and the microfluidic housing has an outer sidewall being attached to the PCB with a second adhesive layer.

[0018] According to some embodiments of the present invention, a method of making a microfluidic device includes the steps of providing a PCB (printed circuit board), attaching a biological chip to the PCB, providing a microfluidic housing, and depositing a first and a second adhesive material. The first adhesive material attaches the microfluidic housing to the biological chip, and the second adhesive material attaches the microfluidic housing to the PCB. The second adhesive material is in liquid form before curing, and the second adhesive material has a higher cure shrinkage than the first adhesive material. The method of the present invention includes the steps of attaching the microfluidic housing to the biological chip using the first adhesive material and attaching the microfluidic housing to the PCB using the second adhesive material. The method of the present invention further includes the steps of curing the first and second adhesive materials to form first and second adhesive layers, respectively.

[0019] 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, the inner sidewall attached to the biological chip with a first adhesive layer to form a flow cell, and the microfluidic housing has an outer sidewall attached to the PCB with a second adhesive layer.

[0020] In some embodiments of the above method, the first adhesive material is solid before curing and substantially maintains its thickness after curing, providing height accuracy and uniformity of the microfluidic device. The second adhesive material is liquid before curing and accommodates variations in the distance between the lower surface of the outer sidewall and the PCB. In some embodiments, the second adhesive material has a higher cure shrinkage than the first adhesive material. In some embodiments, the first adhesive material comprises a die attach film (DAF) and the second adhesive material comprises a liquid epoxy.

[0021] In some embodiments of the above method, the first adhesive material is a compatible adhesive after curing and accommodates mismatched thermal expansion between the biological chip 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 compatible urethane adhesive material and the second adhesive material comprises a liquid epoxy. In some embodiments, the first adhesive material comprises a pressure sensitive adhesive (PSA) and the second adhesive material comprises a liquid epoxy.

[0022] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and drawings. [Brief description of the drawings]

[0023] [Figure 1] 1 is a simplified cross-sectional view illustrating a microfluidic device according to some embodiments of the present invention. [Diagram 2] 1 is a flow chart illustrating a method of forming a microfluidic device according to some embodiments of the present invention. [Figure 3A] 3A-3I are cross-sectional views illustrating the method summarized in the flowchart of Fig. 2 according to some embodiments of the present invention. Fig. 3A is a cross-sectional view illustrating a printed circuit board (PCB) used in the method of Fig. 2. [Figure 3B] 3 is a cross-sectional view illustrating a die attach adhesive formed on a PCB used in the method of FIG. 2. [Figure 3C] 3 is a cross-sectional view illustrating a biological chip attached to a PCB used in the method of FIG. 2. [Figure 3D] FIG. 11 is a cross-sectional view illustrating wire bonds formed to electrically connect the biological chip and the PCB. [Figure 3E] 1 is a cross-sectional view illustrating a microfluidic housing according to some embodiments of the present invention. [Figure 3F] 1 is a cross-sectional view depicting the relative dimensions of a microfluidic housing and a PCB according to some embodiments of the present invention. [Figure 3G]1 is a cross-sectional view illustrating an adhesive layer disposed on a biological chip and a PCB according to some embodiments of the present invention. [Figure 3H] 1 is a cross-sectional view depicting an adhesive layer disposed in a microfluidic housing according to some embodiments of the present invention. [Figure 3I] 1 is a cross-sectional view illustrating a microfluidic housing attached to a biological chip and a PCB according to some embodiments of the present invention. [Figure 3J] 1 is a simplified cross-sectional view illustrating a microfluidic device according to an alternative embodiment of the present invention. [Figure 3K] 1 is a simplified cross-sectional view illustrating a microfluidic device according to another embodiment of the present invention. [Figure 4] 1 is a cross-sectional view depicting a biological sample introduced into a microfluidic device for analysis according to some embodiments of the present invention. [Diagram 5] FIG. 1 is a simplified diagram illustrating a microfluidic device having multiple devices mounted on a PCB according to some embodiments of the present invention. [Figure 6] 1A-1C are simplified diagrams illustrating microfluidic devices formed by alternative methods according to some embodiments of the present invention. [Figure 7] 1 is a simplified diagram illustrating a microfluidic device formed by another method according to some embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] FIG. 1 is a simplified cross-sectional view illustrating a microfluidic device according to some embodiments of the present invention. As shown in FIG. 1, the microfluidic device 100 includes a substrate 110, such as a PCB (printed circuit board), a biological chip 120 on the PCB 110, and a microfluidic housing 130 on the biological chip 120 and the PCB 110. The microfluidic housing 130 is attached to the biological chip 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. The biological chip 120 is attached to the PCB 110 using, for example, a die attach adhesive layer 113 that attaches the biological chip 120 to the PCB 110. It will be understood that the substrate described herein is not limited to a PCB, and other substrates, such as semiconductor (e.g., silicon) substrates, glass substrates, ceramic substrates, etc., may be used.

[0025] The biological chip 120 includes devices, such as sensors, actuators, etc., that manipulate or analyze biological or chemical samples. In some cases, the sensors and actuators include one or more MEMS devices. As an example, the biological chip detects a signal from the biological sample, and a processor processes the detected signal and responds to the signal by initiating operation of the actuator. Depending on the embodiment, the PCB 110 is used to connect multiple biological chips with other circuit components, such as processors, controllers, storage devices, I / O devices, and communication devices.

[0026] In FIG. 1, the microfluidic housing 130 has an inlet 131, an outlet 132, and a first cavity 133. The microfluidic housing 130 has an inner sidewall 135 adjacent to the cavity 133, which is attached to the biological chip 120 using a first adhesive layer 141 to form a flow cell 140 with an airtight seal. As used herein, airtight seal refers to a seal that is airtight and liquid tight, precluding the passage of air, gas, and liquid. The flow cell 140 includes a channel formed by the microfluidic housing 130, the inner sidewall 135 of the microfluidic housing, and the cavity 133 between the biological chip 120. The flow cell 140 also has an inlet 131 and an outlet 132. As an example of an application, a biological sample 137 is introduced through the inlet 131 into the cavity 133 where a sensor of the biological chip 120 determines a characteristic of the biological sample 137. The biological sample 137 is then removed from the cavity 133 through the outlet 132 .

[0027] As shown in FIG. 1, the microfluidic housing 130 also has an outer sidewall 136 that is attached to the PCB 110 using a second adhesive layer 142 to provide mechanical support. In some embodiments, the biological chip 120 is electrically coupled to the PCB 110 using wire bonds. In FIG. 1, the contact pads 122 of the biological chip are connected to the contact pads 112 on the PCB with bond wires 114. The contact pads 122, the contact pads 112 and the bond wires 114 are encapsulated in a wire bond protective structure 115. In this regard, the microfluidic housing 130 also includes a second cavity 138 that accommodates the wire bonds. A gap 148 between the microfluidic housing 130 and the PCB 110 is filled with an underfill material 149, which is an epoxy material and provides a conformal layer between the package and the PCB.

[0028] The first adhesive layer 141 forms an airtight seal between the microfluidic housing 130 and the biological chip 120 that is airtight and liquidtight. Moreover, the first adhesive layer 141 is compatible with the material used in the flow cell. On the other hand, the second adhesive layer 142 is configured to provide mechanical strength to the bond 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. A distance 144 between a lower surface of the outer sidewall 136 of the microfluidic housing and the PCB 110 is greater than a distance 145 between a lower surface of the inner sidewall 135 of the microfluidic housing and the biological chip 120.

[0029] In some embodiments, to bond the microfluidic housing 130 to the biological chip 120 and the PCB 110, first the first and second adhesive layers are formed, and then the microfluidic housing 130 is lifted and placed to contact the biological chip 120 and the PCB 110. In some embodiments, the microfluidic device is designed in such a way that the first adhesive layer is in solid form and has a well-defined thickness. On the other hand, the second adhesive layer is thick enough and in liquid form before curing, and the bond line thickness of the second adhesive layer is self-adjusting. In other words, it fills the space required by the structure of the microfluidic device, which is influenced by the first adhesive layer thickness, the sensor thickness, the die attach adhesive thickness, the PCB surface unevenness, and the step of the wire bond cavity of the microfluidic device. Here, the bond line thickness refers to the thickness of the adhesive layer between the lower surface of the device structure above the adhesive layer and the upper surface of the device structure below the adhesive layer. Depending on the context, the term "bond line thickness" refers to the pre-cure bond line thickness or the post-cure bond line thickness of the adhesive layer.

[0030] In some embodiments, the first adhesive material substantially maintains its thickness throughout the curing process, maintaining the height uniformity of the microfluidic device and ensuring the fluid field uniformity of the flow cell. In certain embodiments, the first adhesive material is a die attach film (DAF). The die attach film is a solid adhesive before curing and substantially maintains its thickness after curing.

[0031] In some embodiments, the first adhesive material is a compatible adhesive, also referred to as a stress-responsive adhesive after curing. In these cases, the thickness of the first adhesive material does not need to be constant to maintain the structural uniformity of the microfluidic device. The compatible adhesive is used to accommodate the mismatched thermal expansion between the biological chip 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 first adhesive material has an elongation of more than 0.5% of its thickness before creep. For example, the compatible first adhesive material is a urethane adhesive material made by Bostik. As another example, the compatible first adhesive is a pressure-sensitive adhesive (PSA).

[0032] As noted above, Figure 1 depicts an example of a microfluidic device having a single biological chip bonded to a PCB, however, the features described above are not limited to microfluidic devices having a single chip, and the chip is not necessarily a biological chip, as described further below.

[0033] Figure 2 is a flow chart illustrating a method of forming a microfluidic device according to some embodiments of the present invention. Figures 3A-3K are cross-sectional views illustrating the method summarized in the flow chart of Figure 2 according to some embodiments of the present invention. A method of forming a microfluidic device, such as the microfluidic device 100 of Figure 1, will now be described with reference to the flow chart of Figure 2 and the cross-sectional views of 3A-3K.

[0034] As shown in FIG. 2, a method 200 for forming a microfluidic device is summarized as follows. At 210, a printed circuit board (PCB) is provided. At 220, the biological chip is attached to the PCB. At 230, wire bonds between the biological chip and the PCB are formed. At 240, a microfluidic housing is provided. At 250, first and second adhesive layers are disposed. At 260, a microfluidic housing is attached onto the biological chip and PCB. At 270, the adhesive layer is cured. These processes are described in detail below.

[0035] In process 210, method 200 begins with providing a printed circuit board (PCB) 110. Figure 3A is a cross-sectional view illustrating a printed circuit board (PCB) used in the method of Figure 2. The PCB includes bond pads that provide electrical connection with electronic components on the circuit board. For example, Figure 3A shows contact pads 112 that are used as bond pads for bonding to a biological chip.

[0036] In process 220, method 200 includes attaching a biological chip to a PCB. FIG. 3B is a cross-sectional view illustrating a die attach adhesive formed on a printed circuit board (PCB) used in the method of FIG. 2. FIG. 3C is a cross-sectional view illustrating a biological chip attached to a printed circuit board (PCB) used in the method of FIG. 2. In FIG. 3B, adhesive layer 113 or die attach adhesive is an epoxy-based adhesive to attach the chip to the PCB. Next, as shown in FIG. 3C, biological chip 120 is attached to PCB 110 using adhesive layer 113.

[0037] The biological chip 120 includes devices for processing or analyzing biological or chemical samples. As used herein, "biological chip" refers to a structure in which biological molecules are associated, immobilized, or captured for analysis. Typically, biological chips include an array of binding sites, each of which is independently occupied by a biological molecule, such as a protein, nucleic acid, antibody, polysaccharide, etc. Typically, a detectable signal generated at one or many binding sites is detected. For example, an enzyme, binding, or chemical reaction at one binding site generates a detectable signal, such as fluorescence or chemiluminescence, that is detected and identifies a characteristic or property of the biological molecule at that site. As described herein below, biological chips are used for nucleic acid sequencing. In some cases, biological chips include a sensor (i.e., a biological sensor). As used herein, the term "biosensor" or "biosensor" refers to a device for determining luminescent substances in or attached to biological molecules, such as nucleic acid polymers, particularly exemplified by DNA, and branched or otherwise derivatized nucleic acids. In an example, the biological chip detects a signal, such as a fluorescent or chemiluminescent signal, from the biological sample, and the processor processes the detected signal and responds to the signal by initiating the operation of the actuator. An example of a biological chip includes a CMOS biological sensor described in U.S. Patent Application No. 16 / 128,120, filed September 11, 2018, which is incorporated herein in its entirety. For example, the biological sensor includes a flow cell on a complementary metal oxide semiconductor (CMOS) layer. The CMOS layer includes a light sensing layer having a plurality of photodiodes and an electronic circuit layer coupled to the light sensing layer that processes the sensed signal. Other examples of biological chips include micro-droplet processing chips, such as integrated lab-on-a-chip cartridges, described in U.S. Patent Application No. 12 / 513,157, filed November 1, 2007, and U.S. Patent Publication No. 2010 / 0096266, published April 22, 2010, which are also incorporated herein in their entirety. It will be understood that the microfluidic devices described herein can be used to detect signal generating events that are not associated with biological reactions (e.g., signals generated by chemical transformations that do not involve biomolecules).

[0038] In process 230, wire bonds are formed between the biological chip and the PCB, and a protective encapsulation is formed to protect the wire bonds. Figure 3D is a cross-sectional view illustrating the wire bonds formed to electrically connect the biological chip and the printed circuit board (PCB). In Figure 3D, contact pads 122 on the biological chip are connected to contact pads 112 on the PCB with bond wires 114. Contact pads 122, contact pads 112, and bond wires 114 are encapsulated in a wire bond protection structure 115.

[0039] In process 250 of method 200, a microfluidic housing is provided. Figure 3E is a cross-sectional view illustrating a microfluidic housing according to some embodiments of the present invention. As shown in Figure 1, the microfluidic housing 130 is used to form a flow cell with the biological chip 120, and the microfluidic housing 130 is also bonded to the PCB 110 to provide the mechanical structure for the microfluidic device.

[0040] The material of the microfluidic housing is compatible with the function of the flow cell, for example to process biological samples. The material is preferably compatible with the biological chip and PCB and has a compatible thermal expansion coefficient. In some embodiments, the microfluidic housing is made of glass or plastic material, or other suitable material. As an example, the microfluidic housing is made of molded plastic. The microfluidic housing is formed separately from the above processes for chip bonding and PCB, and does not have to follow the above order of method description.

[0041] 3E, 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, which is used to mount the biological chip and form a flow cell 140 with an airtight seal. 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 biological chip 120.

[0042] As shown in FIG. 3E, the microfluidic housing 130 also has outer sidewalls 136 for attachment to a PCB to provide mechanical support.

[0043] The shape and size of the microfluidic housing 130 is designed to be assembled with the biological chip 120 and PCB 110 as depicted in FIG. 3F. To illustrate the design considerations of the microfluidic housing, FIG. 3F shows the microfluidic housing 130 placed on the biological chip 120 with the underside of the inner sidewall 135 contacting the upper surface of the biological chip 120 without any adhesive material at joint 147. In this configuration, there is a gap 148 between the underside of the outer sidewall 136 of the microfluidic device 130 and the upper surface of the PCB 110. The height of the gap is selected to account for chip and PCB variations and manufacturing tolerances. As described below, this configuration and the selection of adhesive material facilitate the formation of an airtight bond between the microfluidic housing 130 and the biological chip 120 and the formation of a bond between the microfluidic housing 130 and the PCB 110 to provide mechanical strength of the device. In some embodiments, the height of the gap 148 is about 10 μm, for example 5 to 30 μm. In other embodiments, the gap height is, for example, 30 to 50 μm or more. The lateral dimensions of the cavity 133 of the microfluidic housing 130 are determined by the width of the biological chip 120 and the desired size of the flow cell.

[0044] In process 250 of method 200, an adhesive layer is disposed to attach the microfluidic housing 130 to the biological chip 120 and PCB 110. In one embodiment, the adhesive layer is applied to the upper surface of the biological chip and PCB. Alternatively, the adhesive layer is disposed on the lower part of the microfluidic housing 130 on the lower surface. In a first embodiment, as shown in FIG. 3G, the first adhesive layer 141 is disposed on the upper surface of the biological chip 120, and the second adhesive layer 142 is disposed on the upper surface of the PCB 110. In a second embodiment, as shown in FIG. 3H, the first adhesive layer 141 and the 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 lower surface of the inner sidewall 135 of the biological chip 120, and the second adhesive layer 142 is disposed on the lower surface of the outer sidewall 136 of the biological chip 120.

[0045] In yet another embodiment, a bond is formed between the microfluidic housing 130 and the biological chip 120 by placing a first adhesive layer 141 on the biological chip 120 or the microfluidic housing 130 and then attaching the microfluidic housing 130 to the biological chip 120. A second adhesive layer 142 is then placed in the gap 148 between the microfluidic housing 130 and the PCB 110, as described above in connection with FIG. 3F, to form a second bond between the microfluidic housing 130 and the PCB 110. The adhesive layer is placed in the desired location using an adhesive dispensing device, such as an automated adhesive dispensing device.

[0046] In process 260 of method 200, the microfluidic housing 130 is attached to the biological chip 120 and PCB 110, as depicted in FIG. 3I. In some embodiments, both adhesive layers are disposed on the microfluidic housing 130 (as shown in FIG. H) or two adhesive layers are disposed on the biological chip 120 and PCB 110, respectively (as shown in FIG. G). In these embodiments, the microfluidic housing 130 is lifted and placed on the biological chip 120 and PCB 110, and the microfluidic housing 130 is pressed against the biological chip 120 and PCB 110 to form a bond. A curing process is then performed to cure the adhesive layers 141 and 142.

[0047] In yet another embodiment, a bond is formed between the microfluidic housing 130 and the biological chip 120 by placing a first adhesive layer 141 on the biological chip 120 or the microfluidic housing 130 followed by a pick and press process to attach the microfluidic housing 130 to the biological chip 120. A curing process is performed to solidify the first adhesive layer 141. Then, a second adhesive layer 142 is placed in the gap 148 between the microfluidic housing 130 and the PCB 110 to form a bond after the curing process.

[0048] In an embodiment of the present invention, the material, thickness and volume of the adhesive layer are selected to form a hermetic seal of the flow cell and to provide mechanical strength of the package. The first adhesive layer 141 forms a hermetic seal between the microfluidic housing 130 and the biological chip 120 that is air-tight and liquid-tight. Moreover, the first adhesive layer 141 is compatible with the materials used in the flow cell. On the other hand, the second adhesive layer 142 is configured to provide mechanical strength for the bond between the microfluidic housing 130 and the PCB 110.

[0049] In some embodiments, the first adhesive layer is in solid form and has a well-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, and the bond line thickness of the second adhesive layer is self-regulating. In other words, it fills the space required by the structure of the microfluidic device, including the first adhesive layer thickness, the sensor thickness, the die attach adhesive thickness, the PCB surface unevenness, and the step of the wire bond cavity of the microfluidic device. Here, the bond 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 "bond line thickness" refers to the pre-cure bond line thickness or the post-cure bond line thickness.

[0050] In embodiments of the present invention, the first adhesive layer 141 and the second adhesive layer 142 provide different functions. The first adhesive layer 141 is made of a first material or a first adhesive, and the second adhesive layer 142 is made of a second material or a second adhesive. In some embodiments, the first adhesive is a solid adhesive before curing and substantially maintains its thickness after curing, ensuring uniformity of the microfluidic device. In other embodiments, the first adhesive is a conformable adhesive material after curing, accommodating thermal expansion non-uniformities during thermal cycles of operation of the microfluidic device. The second adhesive is malleable and can change shape upon compression of the assembly step to attach the microfluidic chip.

[0051] In some embodiments, the first adhesive layer 141 is a compatible material or a stress-responsive adhesive. In other words, the first adhesive remains elastic after curing and accommodates the non-uniform thermal expansion between the biological chip and the microfluidic housing through thermal cycling during operation. For example, in certain biological applications, the device undergoes thermal cycling between, for example, room temperature and high process temperatures, for example, 65° C. or higher. The components of the microfluidic device have different materials with different thermal expansion coefficients. Thus, in some embodiments, a compatible adhesive is used to form the first adhesive layer 141 between the microfluidic housing 130 and the biological chip 120. The compatible adhesive absorbs the thermally induced dimensional variations between the microfluidic housing 130 and the biological chip 120. An example of a compatible adhesive material is a urethane adhesive material made of d. The urethane adhesive has low water vapor permeability, elastic properties, and when cured, provides a strong, flexible, and strong adhesive bond. For example, Bostik 1100FS adhesive is used in some embodiments. Of course, in embodiments of the present invention, the first adhesive layer is not limited to a urethane adhesive. Other suitable compatible adhesive materials may also be used. Another example of a compatible adhesive includes the epoxy resins described in U.S. Patent Publication No. 2007 / 0081317 to Choi, entitled "Circuit Board Mounting for Reducing Temperature Stress," published April 12, 2017, which is incorporated herein by reference in its entirety.

[0052] In some embodiments, the first adhesive layer 141 is used to maintain a defined thickness before curing and maximize the flow cell fluid field uniformity since the first adhesive layer 141 is used to form the flow cell chamber between the microfluidic housing 130 and the biological chip 120. In these modifications, a dry film adhesive is used, for example a die attach film (DAF). Die attach films are film-based instead of paste-based and often contain an epoxy adhesive that is attached to the backside of the wafer before dicing. The chip with the DAF on the backside is attached to a PCB. DAF has an advantage over adhesive pastes since there is no reformation of the adhesive material from a drop shape to a thin two-dimensional layer. Furthermore, the process window of the film is wider than the paste material and the assembly process is simplified by the use of the film.

[0053] Other examples of die attach films include materials such as epoxy resins, phenolic resins, acrylic rubbers, silica fillers, or combinations thereof, and are applied using lamination techniques. An example of a die attach adhesive is ABLEBOND 789-3® from Henkel Chemicals, Dusseldorf, Germany. However, other suitable alternative materials and formation techniques may be used instead.

[0054] Examples of die attach films are described in U.S. Patent Publication No. 2006 / 0154078 to Watanabe, entitled "Curable Resin Composition, Adhesive Epoxy Resin Paste, Adhesive Epoxy Resin Sheet, Conductive Connection Paste, Conductive Connection Sheet, and Electronic Components Bonded to Body," published on July 13, 2006, and U.S. Patent Publication No. 2008 / 0318364 to Foong, entitled "Process for Applying Die Attach Film to Singulated Die," both of which are incorporated herein by reference in their entirety. An exemplary process flow for the DAF assembly process includes a bonding step followed by oven curing, for example, at 130° C. for 60 minutes. Alternatively, a UV curing process may be used.

[0055] In some embodiments, the second adhesive is a liquid epoxy that is in liquid form after being dispensed onto the PCB 110 and before curing. Examples of liquid epoxies are described in U.S. Patent Publication No. 2018 / 0213635 to Baba, entitled "Resin Composition and Multilayer Substrate," published on July 26, 2018; U.S. Patent Publication No. 2018 / 0258325 to Taniquichi, entitled "Adhesive Layer and Adhesive Sheet," published on September 13, 2018; and U.S. Patent Publication No. 2018 / 0291164 to Bank, entitled "Fast Curing Epoxy Resin for Use in High Throughput Manufacturing Processes," published on October 11, 2018, all of which are incorporated herein by reference. A specific example of a liquid epoxy is a diglycidyl ether of bisphenol A. Other examples include liquid epoxy materials made by Bostik, Inc., Wauwatosa, Wisconsin, USA, that are moisture cured. Other providers of liquid epoxies include Norland NEA 123S or 123T, made by Norland Corporation of Cranberry, New Jersey, USA, Dymax Corporation of Trigton, Connecticut, and Electronic Materials, Inc. of Breckenridge, Colorado.

[0056] Another example of a suitable first adhesive is a pressure sensitive adhesive (PSA). A pressure sensitive adhesive is an adhesive that forms a bond when pressure is applied. Some PSAs are elastomer-based, for example acrylic-based elastomers. PSAs exhibit viscoelastic (viscous and elastic) properties, both of which are used for suitable bonding.

[0057] As noted above in connection with FIG. 3F, when the microfluidic housing 130 is placed on the biological chip 120 without any adhesive material at the mating surface 147 and with the lower surface of the inner sidewall 135 contacting the upper surface of the biological chip 120, there is a gap 148 between the lower surface of the outer sidewall 136 of the microfluidic device 130 and the upper surface of the PCB 110. Thus, when the microfluidic housing 130 is attached to the biological chip 120 and the PCB 110 and the curing process is performed as depicted in FIG. 3I, the thickness of the second adhesive layer 142 is substantially equal to the thickness of the first adhesive layer 141 plus the height of the gap 148 described in FIG. 3F. In some embodiments, the thickness of the first adhesive layer 141 after curing ranges from 10 to 100 μm. The thickness varies depending on the fluidic cell design. As noted above, the height of the gap 148 ranges from about 5 to 50 μm. Thus, after curing, the thickness of the second adhesive layer 142 ranges from 15 to 150 μm.

[0058] In some embodiments, the microfluidic housing 130 is attached to the biological chip 120 without the use of an external adhesive. For example, in some cases, the body material of the microfluidic housing or a material embedded in the microfluidic housing is used as an adhesive material. For example, the microfluidic housing is made of a plastic material that has a low melting temperature and is melted to form a seal with the biological chip. In some embodiments, the microfluidic housing 130 has an embedded adhesive material. For example, a groove is formed in the sidewall of the microfluidic housing 130 and an adhesive material, such as DAF or other adhesive material, is inserted into the groove.

[0059] In process 260 of method 200, the microfluidic housing is attached to the biology chip and PCB. Figure 3I is a cross-sectional view illustrating the microfluidic housing attached to the biology chip and PCB according to some embodiments of the present invention, where a standard pick, place and press process is used to attach the microfluidic housing to the biology chip and PCB.

[0060] In process 270 of method 200, the assembled microfluidic device undergoes a curing process to harden the adhesive layer. In polymer-based adhesive materials, curing refers to the strengthening or hardening of the polymer material by cross-linking of the polymer chains. Curing occurs with the application of external energy, such as, for example, an electron beam, heat, or ultraviolet (UV) radiation.

[0061] In some embodiments, to minimize stress in the first adhesive layer, the cure shrinkage of the first adhesive layer should not exceed the cure shrinkage of the second adhesive layer. Furthermore, it is desirable that the cure shrinkage of the second adhesive layer is greater than the cure shrinkage of the first adhesive layer to ensure that the first adhesive layer forms an airtight seal between the microfluidic housing and the biological chip. In some embodiments, the additional cure shrinkage of the second adhesive layer is about 10% of the thickness of the first adhesive layer. In other embodiments, in some embodiments, the additional cure shrinkage of the second adhesive layer is about 5-15% of the thickness of the first adhesive layer.

[0062] FIG. 3J is a simplified cross-sectional view depicting a microfluidic device according to an alternative embodiment of the present invention. As shown in FIG. 3J, the microfluidic device 170 is similar to the microfluidic device 100 depicted in FIGS. 1 and 3I, except for the opening 119 in the second adhesive layer 142. The opening 119 is designed to allow testability of the airtightness of the first adhesive layer 141. For example, compressed air is applied between the inlet 131 and the outlet 132 of the microfluidic device 170. Any leakage of the first adhesive layer 141 is detected through the opening 119 in the second adhesive layer 142.

[0063] FIG. 3K is a simplified cross-sectional view illustrating a microfluidic device according to another embodiment of the present invention. As shown in FIG. 3K, the microfluidic device 180 is similar to the microfluidic device 100 depicted in FIG. 1 and 3I, except for the opening 139 of the microfluidic housing 130. Similar to the opening 119 of FIG. 3J above, the opening 139 of the microfluidic housing 130 of FIG. 3K is designed to enable testability of the airtightness of the first adhesive layer. For example, compressed air is applied between the inlet 131 and the outlet 132 of the microfluidic device 180. Any leakage of the first adhesive layer 141 is detected through the opening 139 of the microfluidic housing 130.

[0064] The microfluidic device is now ready to accept a biological sample for analysis. Figure 4 is a cross-sectional view depicting a biological sample being introduced into a microfluidic device for analysis according to some embodiments of the present invention. The microfluidic device 400 of Figure 4, similar to the microfluidic device 100 of Figure 1, shows a biological sample 137 in the cavity 133 of the microfluidic device.

[0065] For example, the biochip can detect a signal, and the processor processes the detected signal and decides to initiate the operation of the actuator. The microfluidic device includes two types of adhesive layers. The first adhesive layer is used to attach the microfluidic housing to the IC chip, and the second adhesive layer is used to attach the microfluidic housing to the PCB.

[0066] As mentioned above, Figure 1 depicts an example of a microfluidic device having a single biological chip coupled to a PCB. However, the above features are not limited to microfluidic devices having a single chip, and the chip is not limited to a biological chip. Examples are described below.

[0067] 5 is a simplified diagram illustrating a microfluidic device having multiple chips attached to a PCB according to some embodiments of the present invention. As shown in FIG. 5, the microfluidic device 500 includes a PCB (printed circuit board) 510, a first chip 520 and a second chip 525 on the PCB, and a microfluidic housing 530 on the chips and the PCB. The first chip 520 is a biological chip similar to the microfluidic device 100 shown in FIG. 1. The second chip 525 is a second biological chip similar to the microfluidic device 100 shown in FIG. 1. Alternatively, the second chip 525 is an integrated circuit (IC) chip, such as a processor chip, or a microelectromechanical system (MEMS) chip, such as an actuator.

[0068] Microfluidic housing 530 is attached to chips 520 and 525 using a first adhesive layer 541 to form a flow cell, and microfluidic housing 530 is attached to PCB 510 using a second adhesive layer 542 to provide mechanical support. Chips 520 and 525 are attached to PCB 510 using, for example, a die attach adhesive layer 513 that attaches the chip to the PCB. Additionally, a filler 527 is formed filling the space between chips 520 and 525.

[0069] 5, microfluidic housing 530 has an inlet 531, an outlet 532, and a first cavity 533. Microfluidic housing 530 has an inner sidewall 535 adjacent to cavity 533, which is attached to chips 520 and 525 using a first adhesive layer 541 to form a flow cell 540 with an airtight seal. Flow cell 540 includes a channel formed by microfluidic housing 530, the inner sidewall 535 of the microfluidic housing, and the cavity 533 between chips 520 and 525. A biological sample 537 is introduced through inlet 531 into cavity 533 where chips 520 and 525 determine a characteristic of biological sample 537. Biological sample 537 is then removed from cavity 533 through outlet 532.

[0070] 5, the microfluidic housing 530 also has an outer sidewall 536 that is attached to the PCB 510 using a second adhesive layer 542 to provide mechanical support. In some embodiments, the chips 520 and 525 are electrically coupled to the PCB 510 using wire bonds, similar to the chips described in FIG. 1. In this regard, the microfluidic housing 530 also includes a second cavity that accommodates the wire bonds.

[0071] The first adhesive layer 541 forms a hermetic seal between the microfluidic housing 530 and the chips 520 and 525 that is air-tight and liquid-tight. Moreover, 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 to the bond between the microfluidic housing 530 and the PCB 510.

[0072] Figure 6 is a simplified diagram illustrating a microfluidic device formed by an alternative method according to some embodiments of the present invention. As shown in Figure 6, the microfluidic device 600 includes a PCB (printed circuit board) 610, a CMOS image sensor (CIS) 620 on the PCB 610, and a microfluidic housing 630 on 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 is attached to the PCB 610 using solder 612.

[0073] 6, 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 chip 637 is introduced through the inlet 631 into the cavity 633 where the CMOS image sensor 620 determines the characteristics of the biological sample 637. The biological sample 637 is then removed from the cavity 633 through the outlet 632.

[0074] In the microfluidic device 600, adhesive 641 is used to form an airtight seal of the flow cell and contacts the biological sample. Therefore, the material of adhesive 641 is compatible with the biological sample, the fluids used in manipulating the biological sample, and the conditions under which it can be operated. For example, in some embodiments, a urethane adhesive that is compatible with biological samples is used as adhesive 641 to form the flow cell.

[0075] In the microfluidic device 600, latches 642 are used to attach the microfluidic housing 630 to the PCB 610 and provide mechanical support. In some embodiments, the latches include a snap-click mechanism that fits through an opening in the PCB board 610. In this way, the snap-click mechanism operates to provide or maintain a compressive force between the microfluidic device 600 and the PCB board, helping to provide a seal between the microfluidic housing 630 and the CMOS image sensor (CIS) 620, which are bonded using adhesive 641.

[0076] Furthermore, the above description of microfluidic device 600 is not limited to CMOS sensors. Other types of chips, such as sensor chips or biological sample manipulation chips, may be used to implement the microfluidic device. Similar to microfluidic device 500 of FIG. 5, microfluidic device 600 also includes multiple devices mounted on a PCB.

[0077] FIG. 7 is a simplified diagram illustrating a microfluidic device formed by another method according to some embodiments of the present invention. The microfluidic device 700 of FIG. 7 is similar to the microfluidic device 600 shown in FIG. 6. One difference between the microfluidic device 700 and the microfluidic device 600 is that in the microfluidic device 700, the adhesive used in the microfluidic device 600 to attach the microfluidic housing to the CMOS image sensor is replaced with an O-ring. As shown in FIG. 7, the microfluidic device 700 includes a PCB (printed circuit board) 710, a CMOS image sensor (CIS) 720 on the PCB 710, and a microfluidic housing 730 on 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 is attached to the PCB 710 using solder 712 .

[0078] 7, a 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 is introduced through the inlet 731 into the cavity 733 where the CMOS image sensor 720 determines the characteristics of the biological sample 737. The biological sample 737 is then removed from the cavity 733 through the outlet 732.

[0079] An O-ring is a ring of elastomeric material with a round cross section, often designed to be mounted in a groove and compressed during assembly between two or more components to create a seal at the mating surface. Elastomer refers to an elastic polymer or a rubber-like solid with elastic properties. In the microfluidic device 700, the O-ring contacts the biological sample to be analyzed by the CMOS sensor. Thus, the material of the O-ring is compatible with the biological sample, the fluids used in manipulating the biological sample, and the conditions under which it can be operated. Furthermore, the above description of the microfluidic device 700 is not limited to the CMOS sensor 720. Other types of chips, such as other types of sensors or biological sample manipulation chips, can also be used to implement the microfluidic device 700. Similar to the microfluidic device 500 of FIG. 5, the microfluidic device 700 also includes multiple devices mounted on a PCB.

[0080] Some embodiments of the present invention are used to analyze biological or chemical samples. The biological or chemical samples may include any of a number of components. For example, the samples may include nucleic acid macromolecules (e.g., templates, DNA, RNA, etc.), proteins, etc. The samples may be analyzed to determine gene sequences, DNA-DNA hybridization, single nucleotide polymorphisms, protein interactions, peptide interactions, antigen-antibody interactions, glucose monitoring, cholesterol monitoring, etc.

[0081] In some embodiments, the biological sample is 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 U.S. Patent Publication No. 2016 / 0237488, all of which are incorporated herein by reference. Without limitation, the DNA biological molecule is a DNA nanoball (single-stranded concatemer) hybridized to a labeled probe (e.g., in ligation or cPAL-based DNB sequencing) or to a complementary growing strand (e.g., in synthesis-based DNB sequencing) or to both, or to a single DNA molecule (e.g., single molecule sequencing), or to a clonal population of DNA molecules, such as those generated in bridge PCR-based sequencing. Thus, reference to a "biomolecule," "DNA polymer," or "nucleic acid polymer" encompasses a plurality of molecules (e.g., a DNB associated with a plurality of growing complementary strands or a DNA cluster comprising a clonal population of hundreds or thousands of DNA molecules). Exemplary methods for making DNBs (e.g., a DNB library) or making an array of discrete spaced regions separated by inter-region 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, all of which are incorporated herein by reference. In some embodiments, DNBs or other polymers are immobilized at discrete spaced 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 regions comprise functional moieties (e.g., amines). In some embodiments, the discrete spaced regions comprise capture oligonucleotides attached thereto for binding template DNA (e.g., DNBs). Typically, the discrete spaced regions are arranged in a rectilinear pattern, although regular arrays with other configurations (eg, concentric circles of regions, spiral patterns, hexagonal patterns, etc.) may be used.

[0082] In some embodiments, the nucleic acid polymer is an amplicon of a genomic DNA fragment or a cDNA library. As used herein, an "amplicon" is generally the product of amplification of a nucleic acid molecule, such as a fragment of genomic DNA or 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 (hereby incorporated in its entirety by reference), or bridge polymerase chain reaction (PCR), as described, for example, in U.S. Patent No. 7,972,820 (hereby incorporated in its entirety by reference). Amplification is performed before the nucleic acid contacts the biosensor, or in situ, as described, for example, in U.S. Patent No. 7,910,354 (hereby incorporated in its entirety by reference).

[0083] A biological sample, such as a DNA polymer, oligonucleotide, or nucleotide associated with a fluorescent or chemiluminescent dye, is placed on the photodiode 117. In the case of fluorescence, the dye is illuminated with excitation light from an excitation light source. The excitation light corresponds to any suitable type or intensity of light, including, for example, visible light, infrared (IR), ultraviolet (UV), and the like. The excitation light may also come from any suitable source, such as, for example, a light emitting diode (LED), a lamp, a laser, or a combination thereof. When the dye is illuminated with excitation light of a particular wavelength, the biological sample absorbs the light and then emits light with a different wavelength. For example, a biological sample may absorb excitation light having a wavelength of 450 nm and emit light with a wavelength of 550 nm. In other words, fluorescence of a characteristic wavelength is emitted when the dye is illuminated by light of a characteristic different wavelength (i.e., the excitation light source). The excitation light is used to illuminate the dye resulting in fluorescence, but must be filtered out in order to take an accurate measurement of the fluorescence with the photodiode.

[0084] In the case of chemiluminescence, an excitation light source is not required for a photodiode to detect the emitted light. Instead, the biological sample emits light due to a chemical or enzymatic reaction that occurs between the biological sample and the chemiluminescent dye (or other solution) and causes light to be emitted by breaking or forming chemical bonds (e.g., the action of a luminescent enzyme protein on a luciferin substrate).

[0085] In both fluorescence and chemiluminescence, a photodiode detects the intensity of the emitted light and converts it into an electronic signal based on the light intensity that is provided by metal wiring to an external device that correlates the electronic signal to a particular wavelength and brightness based on the electronic signal.

[0086] In some embodiments, the active spots or wells on the surface of the biosensor and the nucleic acid polymers are configured such that each spot binds only one nucleic acid polymer. This is accomplished, for example, by contacting the surface with an amplicon whose size corresponds to the active spot (e.g., an amplicon having a diameter that is substantially equal to or larger than the diameter of the active spot). See U.S. Patent No. 8,445,194, which is incorporated herein in its entirety. Alternatively, the active spots are configured to bind a single DNA fragment, which is then amplified to fill a large area at or around the original binding site.

[0087] Some embodiments of the invention are used to determine different labels corresponding to different wavelengths of light. The labels are, for example, fluorescent, chemiluminescent or bioluminescent labels. For example, in gene sequencing (or DNA sequencing), embodiments of the invention are used to determine the exact order of nucleotide bases within a nucleic acid polymer (e.g., a strand of DNA). The nucleotide bases (e.g., adenine (A), guanine (G), cytosine (C) or thymine (T)) are labeled with specific fluorescent labels. Alternatively, for example, one-, two- or three-color sequencing methods are used.

[0088] For fluorescence, each of the nucleotide bases is determined in turn by sequentially exciting the nucleic acid polymer with an excitation light. The nucleic acid polymer absorbs the excitation light and transmits emitted light of different wavelengths onto a biosensor as described herein. The biosensor measures the wavelength of the emitted light and the intensity received by a photodiode. Each nucleotide (e.g., a fluorescently labeled nucleotide), when excited by an excitation light of a particular wavelength and / or intensity, emits a particular wavelength of light and / or photodiode intensity, allowing identification of the presence of a particular nucleotide base at a particular location in the nucleic acid polymer. Once a particular nucleotide base is determined, it is removed from the nucleic acid polymer and the next successive nucleotide base is determined following a similar process.

[0089] The nucleic acid polymer is labeled with one or more different fluorescent, chemiluminescent, or bioluminescent labels before or after attachment to a biosensor for any purpose. For example, the nucleic acid polymer is hybridized with a labeled oligonucleotide probe or amplification primer. Alternatively, the nucleic acid polymer is hybridized with an unlabeled oligonucleotide that is then coupled with a labeled probe and extended with a labeled nucleotide analog. By way of illustration, labeling can be done for the purpose of characterizing the nucleic acid polymer (e.g., the presence of a single nucleotide polymorphism (SNP) associated with a disease) or for nucleic acid sequencing of all or part of the nucleic acid polymer as described above. DNA sequencing by probe hybridization is described, for example, in U.S. Pat. No. 8,105,771, which is incorporated herein in its entirety. Sequencing by anchor probe ligation is described, for example, in U.S. Pat. No. 8,592,150, which is incorporated herein in its entirety. Sequencing by synthesis is described, for example, in U.S. Pat. No. 7,883,869, which is incorporated herein in its entirety. In general, sequencing by synthesis is a method in which nucleotides are added sequentially to free 3' hydroxyl groups provided by a sequencing primer hybridized to a template sequence, resulting in the synthesis of a nucleic acid strand in the 5' to 3' direction. In one approach, another exemplary type of SBS, pyrosequencing technology, is used (Ronaghi et al., 1998, Science 281:363).

[0090] In some embodiments, the biosensor is reversibly coupled to a flow cell (not shown). The nucleic acid polymer is attached to the biosensor by contacting the biosensor with a liquid sample in the flow cell. The flow cell includes one or more flow channels in fluid communication with the reaction site. In one example, the biosensor is fluidly and electrically coupled to a biological assay system. The biological assay system delivers reagents to the reaction site according to a predetermined protocol and performs an imaging event. For example, the biological assay system directs a solution to flow along the reaction site. The solution includes four types of nucleotides with the same or different fluorescent labels. In some embodiments, the biological assay system then illuminates the reaction site with an excitation light source. The excitation light has a predetermined wavelength or wavelengths. The exciting fluorescent labels provide an emission signal that is detected by a photodiode.

[0091] The user prepares for sequencing by contacting a biosensor according to the described embodiments with a nucleic acid amplicon, or a nucleic acid to be subsequently amplified; the nucleic acid polymer binds and is retained by the active spot or well, and excess nucleic acid polymer is washed away. The nucleic acid polymer is contacted with a labeling reagent, either beforehand or in situ. The biosensor then operates as described herein to measure the light emitted on or around the nucleic acid polymer on the array. The light is quantified, or it is sufficient to determine in a binary fashion which nucleic acid polymers on the surface are labeled with a label that emits at a particular wavelength. Different probes or different nucleic acid analogs, bearing labels that emit light at different wavelengths, can be used simultaneously to determine, for example, various bases at a particular position in a sequence or to sequence multiple positions.

[0092] Although described herein with respect to back-illuminated CMOS sensors, it is expected that embodiments of the present invention may be applied to front-illuminated CMOS sensors as well. Furthermore, embodiments of the present invention may be applied to any suitable biosensors, such as those biosensors described in U.S. Patent Application No. 15 / 803,077, filed November 3, 2017, which is incorporated herein in its entirety by reference.

[0093] The above description includes exemplary aspects of the presently described technology, including methods, systems and / or structures and uses thereof. Although various aspects of the present technology have been described above with a certain degree of particularity or with reference to one or more individual aspects, those skilled in the art can make numerous modifications to the disclosed aspects without departing from the spirit or scope of the technology herein. Since many aspects can be made without departing from the spirit and scope of the presently described technology, appropriate scope exists within the following appended claims. Other aspects are therefore anticipated. Furthermore, it should be understood that any operations may be performed in any order unless expressly claimed otherwise or a particular order is inherently required by the language of the claims. All matters contained in the above description and shown in the accompanying drawings are intended to be construed as only illustrative of particular aspects and not limited to the embodiments shown. Unless otherwise clear from the context or expressly stated, concentration values ​​provided herein are generally given in terms of mixture values ​​or percentages, regardless of any transformations that occur upon or after the addition of a particular component of the mixture. To the extent not already expressly incorporated herein, all publications and patent documents referenced in this disclosure are hereby incorporated by reference in their entirety for all purposes. Changes in detail or structure may be made without departing from the fundamental elements of the present technology as defined in the following claims.

Claims

1. 1. A method for making a microfluidic device comprising a PCB (printed circuit board), a biological chip attached to the PCB, and a microfluidic housing, comprising: adhering an inner sidewall of the microfluidic housing to the biological chip using a first adhesive material to form a flow cell; attaching an outer portion of the microfluidic housing to the PCB using a second adhesive material; The method of claim 1, wherein the second adhesive material is thicker than the first adhesive material at least after bonding the inner sidewall of the microfluidic housing to the biological chip and after attachment of the outer portion of the microfluidic housing to the PCB.

2. The method of claim 1 , wherein after fabricating the microfluidic device, the biological chip is between the PCB and the microfluidic housing, and the biological chip is attached to the PCB.

3. The method of claim 2 , wherein the biological chip is attached to the PCB with a third adhesive material.

4. The method of claim 2 , wherein the first adhesive material is elastic after the inner sidewall of the microfluidic housing is attached to the biological chip.

5. The method of claim 2 , 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 prior to attaching the outer portion of the microfluidic housing to the PCB.

6. the first adhesive material is solid before curing and maintains a thickness during and after curing; The method of claim 2 , wherein the second adhesive material is liquid before curing and adjusts for distance variations between a bottom surface of the microfluidic housing and the PCB.

7. the first adhesive material comprises a die attach film (DAF); The method of claim 6 , wherein the second adhesive material comprises a liquid epoxy.

8. The method of claim 6 , wherein the second adhesive material has a greater cure shrinkage than the first adhesive material.

9. the first adhesive material comprises a urethane adhesive material; The method of claim 6 , wherein the second adhesive material comprises a liquid epoxy.

10. the first adhesive material comprises a pressure sensitive adhesive (PSA); The method of claim 6 , wherein the second adhesive material comprises a liquid epoxy.

11. the microfluidic housing having an inlet, an outlet, the inner sidewall, an outer sidewall, and a first cavity; the inner sidewall of the microfluidic housing is adjacent to the first cavity; The method of claim 2 , wherein the microfluidic housing is attached to the PCB at the outer sidewall.

12. The method of claim 2 , further comprising attaching a second biological chip to the PCB prior to attaching the microfluidic housing.

13. The method of claim 2 , further comprising attaching an integrated circuit chip to the PCB prior to attaching the microfluidic housing.

14. The method of claim 2 , further comprising attaching a micro-electromechanical system (MEMS) chip to the PCB prior to attaching the microfluidic housing.

15. The method of claim 2 including providing an opening in the second layer of adhesive material for hermetic testing of the first layer of adhesive material.

16. The method of claim 2 , further comprising an opening in the microfluidic housing for hermetic testing of the first layer of adhesive material.

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