Microfluidic Cartridge

Abstract

To provide a microfluidic cartridge for nucleic acid analysis for analyzing biological samples.SOLUTION: A microfluidic cartridge comprises a lower cartridge body, a biochip, and an upper cartridge body. The lower cartridge body includes a first substrate and an inlet column. The inlet column protrudes above the substrate and is hollow. The biochip has a plurality of micro wells and is attached to the first substrate of the lower cartridge body. An upper cartridge is disposed over the lower cartridge body and includes a second substrate, a first opening, and a first O-ring. The first opening extends through the second substrate, the inlet column of the lower cartridge body is inserted into the first opening, and the inlet column and the first opening are integrated into the inlet port. The first O-ring is disposed within the first opening. The inlet port and the biochip are connected by an inflow channel.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 309,662, filed February 14, 2022, U.S. Provisional Application No. 63 / 343,066, filed May 17, 2022, and U.S. Patent Application No. 18 / 162,211, filed January 31, 2023, which are incorporated by reference in their entireties.

[0002] The present disclosure relates to a nucleic acid analysis microfluidic cartridge for analyzing biological samples. [Background technology]

[0003] Genetic analysis has been widely used for biomedical research and disease diagnosis to date, and precision medicine is gradually becoming a reality worldwide, especially with the increasing availability of targeted therapies as part of routine medical care. To further facilitate the routine use of genetic analysis in clinical practice, digital PCR-based assays offer advantages over traditional PCR and sequencing-based techniques in terms of sensitivity, quantitative capabilities, and ease of use. Digital PCR has great potential for liquid biopsy in early disease screening or diagnosis, especially when sample volume is limited. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Recent Advances of Fluid Manipulation Technologies in Microfluidic Paper-Based Analytical Devices (μPADs) toward Multi-Step Assays, Micromachines 2020, 11, 269 by TH Kim et al. Summary of the Invention [Problem to be solved by the invention]

[0005] However, currently, fluid flow control in automated digital PCR instruments is still less than ideal: for example, there are fluid dead volumes within the device, and only one sample can be run at a time per cartridge. [Means for solving the problem]

[0006] Some embodiments of the present disclosure provide a microfluidic cartridge that can be used on an automated digital PCR analyzer. Due to the arrangement of the upper cartridge body and the lower cartridge body, the fluid channels in the microfluidic cartridge are more clearly defined, and the fluid flow in the microfluidic cartridge can be more precisely automatically controlled.

[0007] Some embodiments of the present disclosure provide a microfluidic cartridge including a lower cartridge body part, a biochip, and an upper cartridge body part. The lower cartridge body part includes a first substrate and an inlet column. The inlet column protrudes above the first substrate and is hollow. The biochip is attached to the first substrate of the lower cartridge body part. The upper cartridge body part is disposed on the lower cartridge body part and includes a second substrate, a first opening, and a first O-ring. The first opening penetrates the second substrate, the inlet column of the lower cartridge body part is inserted into the first opening, and the inlet column and the first opening are assembled into the inlet port. The first O-ring is disposed in the first opening and abuts against the inlet column of the lower cartridge body part.

[0008] In some embodiments, the first O-ring abuts the inlet column of the lower cartridge body portion.

[0009] In some embodiments, the microfluidic cartridge further comprises a reaction channel, the reaction channel being defined by the bottom surface of the second substrate and the biochip.

[0010] In some embodiments, the microfluidic cartridge further comprises an inflow channel, the fluid inflow channel connecting the inlet port and the reaction channel, the fluid inflow channel being defined by the top surface of the first substrate and the bottom surface of the second substrate.

[0011] In some embodiments, the first O-ring abuts the inlet column of the lower cartridge body portion.

[0012] In some embodiments, the first O-ring directly contacts the first inner wall within the first opening.

[0013] In some embodiments, the first O-ring is secured within the first opening via a mechanical restraint or an adhesive.

[0014] In some embodiments, the lower cartridge body portion further comprises an entry trench and a biochip slot. The entry trench is connected to the inlet column. The biochip slot is connected to the entry trench, and the biochip is disposed within the biochip slot.

[0015] In some embodiments, the entry trench has a first width and the biochip slot has a second width, the first width being less than the second width.

[0016] In some embodiments, the top surface of the biochip is lower than the bottom surface of the inlet trench.

[0017] In some embodiments, the inlet trench is defined by a weld line.

[0018] In some embodiments, the biochip comprises a plurality of microwells, and the number of microwells is at least 1,000.

[0019] In some embodiments, the second substrate of the upper cartridge body portion has a first thickness D1, and the depth of the inlet column inserted into the first opening is between about ⅓D1 and D1.

[0020] In some embodiments, the lower cartridge body portion and the upper cartridge body portion are assembled by adhesive, ultrasonic welding, or mechanical fasteners.

[0021] Some embodiments of the present disclosure provide a microfluidic cartridge including a lower cartridge body part, a biochip, and an upper cartridge body part. The lower cartridge body part includes a first substrate, an inflow trench, and a biochip slot. The inflow trench is disposed in the first substrate and extends in a first direction. The inflow trench and the biochip slot are connected. The biochip is disposed in the biochip slot. The upper cartridge body part is disposed above the lower cartridge body part and includes a second substrate, a first opening, an optical window, a plurality of sample injection ports, and at least one reaction channel spacer. The inflow trench and a bottom surface of the second substrate define a fluid inflow channel. The first opening penetrates the second substrate and connects to the inflow channel. The optical window is disposed above the biochip, and a lower surface of the optical window and the biochip define a reaction channel. The plurality of sample injection ports are disposed between the first opening and the optical window in the first direction. At least one reaction channel spacer is disposed on the lower surface of the upper cartridge body portion, the at least one reaction channel spacer extending in a first direction and dividing the reaction channel into a plurality of reaction sub-channels.

[0022] In some embodiments, each of the plurality of sample injection ports is disposed within a respective one of the plurality of reaction subchannels.

[0023] In some embodiments, the inlet trench is triangular in top view.

[0024] In some embodiments, the microfluidic cartridge for digital PCR further comprises at least one inflow channel spacer configured to divide the fluid inflow channel into a plurality of fluid inflow sub-channels.

[0025] In some embodiments, the microfluidic cartridge for digital PCR further comprises a first O-ring disposed in the first opening and abutting against the inlet column of the lower cartridge body portion.

[0026] In some embodiments, each of the plurality of sample injection ports is sealable by a sealing element.

[0027] In some embodiments, the microfluidic cartridge for digital PCR further comprises a plurality of injection port O-rings disposed within the plurality of sample injection ports, respectively.

[0028] Some embodiments of the present disclosure provide a microfluidic cartridge including a lower cartridge body portion, an upper cartridge body portion, and a biochip. The lower cartridge body portion includes a first substrate, an inflow trench, and a biochip slot. The inflow trench is disposed in the first substrate. The inflow trench and the biochip slot are connected. The upper cartridge body portion is disposed above the lower cartridge body portion and includes a second substrate, a first opening, an optical window, and a second opening. The inflow trench and a bottom surface of the second substrate define a fluid inflow channel. The first opening penetrates the second substrate and connects to the fluid inflow channel. The biochip is disposed within the biochip slot and below the optical window, the biochip has a plurality of microwells, a lower surface of the optical window and the biochip define a reaction channel, and an upper surface of the biochip is lower than a bottom surface of the inflow trench.

[0029] In some embodiments, each of the bottom surfaces of the microwells is hydrophilic.

[0030] In some embodiments, the biochip comprises a silicon layer, and the plurality of microwells are recesses in the silicon layer.

[0031] In some embodiments, the biochip comprises a thermally conductive layer and a patterned layer, the patterned layer being disposed on the thermally conductive layer, the patterned layer comprising a plurality of through holes.

[0032] In some embodiments, the thermally conductive layer comprises gold, copper, aluminum, iron, steel, silicon, graphite, or graphene, and the patterned layer comprises glass, silicon, or a polymer.

[0033] The disclosure of the present application can be more fully understood by reading the following detailed description of the embodiments, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0034] [Figure 1]FIG. 1 is a perspective view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 2A] 1 is a cross-sectional view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 2B] 1 is a cross-sectional view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 3A] FIG. 13 is a top view of a lower cartridge body according to some embodiments of the present disclosure. [Figure 3B] 1A-1C are schematic top views of a lower cartridge body portion according to some embodiments of the present disclosure. [Figure 4A] 1 is a cross-sectional view of a portion of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 4B] 1 is a cross-sectional view of a portion of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 5A] FIG. 1 is a perspective view of a biochip according to some embodiments of the present disclosure. [Figure 5B] 1 is a cross-sectional view of a biochip according to some embodiments of the present disclosure. [Figure 5C] 1 is a cross-sectional view of a biochip according to some embodiments of the present disclosure. [Figure 6] FIG. 1 is a perspective view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 7] 1 is a cross-sectional view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 8] 1 is a cross-sectional view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 9A] FIG. 1 is a top view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 9B] FIG. 1 is a top view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 9C] FIG. 1 is a top view of a microfluidic cartridge according to some embodiments of the present disclosure. [Figure 10]1 is a cross-sectional view of a microfluidic cartridge according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Reference will now be made in detail to the present embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.

[0036] In some embodiments, the microfluidic cartridge is used for digital PCR analysis of genetic variations in a biological sample, where the sample is split into multiple individual PCR reactions to accurately quantitate low abundance target variants.

[0037] Figures 1, 2A, and 2B illustrate a microfluidic cartridge according to some embodiments of the present disclosure, with Figure 1 being a perspective view and Figures 2A and 2B being cross-sectional views taken along line AA of Figure 1.

[0038] 1, the microfluidic cartridge 100 includes a lower cartridge body portion 102 and an upper cartridge body portion 104. An inlet port 170 is disposed near one side of the microfluidic cartridge 100 and configured to transport fluids related to digital PCR to the interior of the microfluidic cartridge 100, and an outlet port 178 is disposed near the opposite side of the microfluidic cartridge 100 and configured to eject fluids from the microfluidic cartridge 100. An optical window 144 is disposed between the inlet port 170 and the outlet port 178 over a biochip (which will be described in more detail below). The optical window 144 is configured to transmit reaction signals emanating from microwells of the biochip so that the reaction signals can be detected.

[0039] 2A, the lower cartridge body part 102, the upper cartridge body part 104, and the biochip 160 are assembled to form the microfluidic cartridge 100. After assembly, a fluid inflow channel 172 and a fluid outflow channel 176 are formed between the lower cartridge body part 102 and the upper cartridge body part 104, and a reaction channel 174 is formed between the biochip 160 and the optical window 144 of the upper cartridge body part 104. In other words, when the lower and upper cartridge body parts 102, 104 are assembled together with the biochip 160 to produce a functional microfluidic cartridge 100, fluid channels (including the fluid inflow channel 172, the reaction channel 174, and the fluid outflow channel 176) are formed, and the fluid channels can be connected to an external fluid control mechanism for material exchange of samples and reaction reagents, which can be liquids, particles, gases, or other forms of matter. The fluid channels and ports, including inlet port 170 and outlet port 178, provide fluid communication conduits with external devices used to affect biological reactions occurring on the biochip mounted within the cartridge. Changes in the optical signal of such biological reactions are detected through the optical window 144 in the upper cartridge body portion 104.

[0040] 3A illustrates a top view of the lower cartridge body portion 102. As shown in FIGs. 2A and 3A, the lower cartridge body portion 102 includes a first substrate 110, an inlet column 112, an inlet trench 116, a biochip slot 118, an outlet trench 120, and an outlet column 122.

[0041] In some embodiments, the first substrate 110 of the lower cartridge body portion 102 comprises plastic, glass, carbon, or metal. Fabrication of the lower cartridge body portion 102 can be accomplished by a variety of means, such as 3D printing, plastic injection molding, or computer numerically controlled (CNC) machined parts.

[0042] 3B is another top view of the lower cartridge body portion 102. In some embodiments, the lower cartridge body portion 102 can be fabricated with features, such as ribs 190, to increase its mechanical strength and minimize distortion of the assembled cartridge during use.

[0043] 2A, the inlet column 112 protrudes above the first substrate 110. The inlet column 112 is a hollow structure. In addition, the outlet column 122 also protrudes above the first substrate 110. The outlet column 122 is a hollow structure.

[0044] 2A and 3A, the inlet trench 116 is connected to the inlet column 112 and the biochip slot 118. In some embodiments, the inlet trench 116 is defined by two protruding lines on the first substrate 110. As shown in FIG. 2A, the bottom surface 116BS of the inlet trench 116 is higher than the top surface of the biochip 160.

[0045] 2A and 3A, biochip 160 is disposed in biochip slot 118 of lower cartridge body portion 102. Referring to FIG 5A, which shows a perspective view of biochip 160. Biochip 160 includes a plurality of microwells, which may be at least 1,000, e.g., 1,000, 5,000, 10,000, 50,000, 100,000, 200,000, 500,000, or the like.

[0046] 2A and 3A, the outflow trench 120 is connected with the biochip slot 118 and the outlet column 122. In some embodiments, the outflow trench 120 is defined by two protruding lines on the first substrate 110. As shown in FIG. 2A, the bottom surface 120BS of the outflow trench 120 is higher than the top surface of the biochip 160.

[0047] With reference to FIG. 2A, the upper cartridge body portion 104 includes a second substrate 140, a first opening 142, an optical window 144, and a second opening 146.

[0048] In some embodiments, the second substrate 140 of the upper cartridge body portion 104 comprises plastic, glass, carbon, or metal. Fabrication of the upper cartridge body portion 104 may be accomplished by a variety of means, such as 3D printing, plastic injection molding, or CNC machined parts. In some embodiments, the upper cartridge body portion 104 may be fabricated with features, such as ribs, to increase its mechanical strength and minimize distortion of the assembled cartridge during use.

[0049] As shown in FIG. 2A, the first opening 142 corresponds to the inlet column 112 of the lower cartridge body part 102. The inlet column 112 is inserted into the first opening 142. The inlet column 112 of the lower cartridge body part 102 and the first opening 142 of the upper cartridge body part 104 are assembled into an inlet port 170. The inlet port 170 is configured to accommodate a pipette tip or needle for transporting fluids related to digital PCR. As shown in FIG. 2A, the second substrate 140 of the upper cartridge body part 104 has a first thickness D1. In some embodiments, the depth of the inlet column 112 that is inserted into the first opening 142 is between about 1 / 3 D1 and D1.

[0050] The optical window 144 is disposed over the biochip 160. The optical window 144 is made of a transparent material.

[0051] 2A, the second opening 146 corresponds to the outlet column 122 of the lower cartridge body part 102. The outlet column 122 is inserted into the second opening 146. The outlet column 122 of the lower cartridge body part 102 and the second opening 146 of the upper cartridge body part 104 are assembled into an outlet port 178. The outlet port 178 is configured to receive a pipette tip or needle for transporting fluids related to digital PCR.

[0052] As shown in FIG. 2A, after the lower cartridge body portion 102, the biochip 160, and the upper cartridge body portion 104 are assembled, a fluid inflow channel 172 is defined by the inflow trench 116 and the bottom surface 140BS of the second substrate 140, and a fluid outflow channel 176 is defined by the outflow trench 120 and the bottom surface 140BS of the second substrate 140.

[0053] In some embodiments, assembly of the lower cartridge body portion 102 and the upper cartridge body portion 104 may be achieved through the use of adhesives, ultrasonic welding, or mechanical fasteners.

[0054] Ultrasonic welding is a means often used to bond parts of compatible materials together and is well established in the industry. Weld lines are used to bond the upper and lower cartridge body parts together during assembly of the microfluidic cartridge 100. In some embodiments, the plastic upper and lower cartridge body parts are bonded together by ultrasonic welding. Weld lines can be designed into both the lower cartridge body part 102 and the upper cartridge body part 104 to form a tight fit. In some embodiments, as shown in FIG. 3B, the weld line 126 also defines the inflow and outflow channels in the lower cartridge body part 102. The weld line 126 can be a single weld line as shown in FIGS. 2A and 2B, or a double weld line as shown in FIGS. 4A and 4B, improving the air and liquid sealing quality of the fluid channels.

[0055] In some embodiments, the dimensions of the fluidic channels (which include the fluidic inlet channel 172, the reaction channel 174, and the fluidic outlet channel 176) that define the flow rate of fluid along the channel are kept constant along the channel. In some embodiments, the characteristics and dimensions of the fluidic channels that define the flow rate of fluid along the channel vary depending on the need for flow control. The variation in dimensions of different sections of the fluidic channel depends on the intended use, particularly small volume biological samples. In some embodiments, the width of the channel connecting to the fluidic port is significantly different from the channel on the biochip.

[0056] As shown in FIG. 3A, the inlet trench 116 has a first width W1 and the biochip slot 118 has a second width W2, where the first width W1 is less than the second width W2.

[0057] In some embodiments, as illustrated in Figures 3A and 3B, a fluidic channel defined by weld lines 126, 1 or 2 mm wide and 500 micrometers (μm) high, is connected to fluidic ports of similar diameter at connection joints on either side of the cartridge body, with the dimensions of the channel being, for example, on the order of 10 mm wide and less than 500 micrometers (μm) high across the entire surface area of ​​the attached biochip when the cartridge is assembled. The different sections of the channel are tightly connected for continuous liquid flow from inlet port 170 at one end of the microfluidic cartridge 100 to outlet port 178 at the other end. In some embodiments, it may be contemplated that there may be more than two fluidic ports connected to a fluidic channel in the microfluidic cartridge 100.

[0058] 3B, the outflow trench 120 includes passive valves 192 along the trench. The passive valves 192 are configured to regulate the flow rate of fluids. Varying the channel dimensions in some configurations facilitates regulating the flow rates of different fluids inside the fluid channels.

[0059] 2A, a first O-ring 150 is disposed within the first opening 142 of the upper cartridge body portion 104 and abuts against the inlet column 112 of the lower cartridge body portion 102. A second O-ring 152 is disposed within the second opening 146 of the upper cartridge body portion 104 and abuts against the outlet column 122 of the lower cartridge body portion 102. These elastic O-rings act as sealing elements to maintain flow control of fluid or air through the channels when the microfluidic cartridge 100 is coupled to a connecting device, for example, an automated pipette tip or needle. Flow control through the fluid ports can affect biological reactions on the biochip by pressure control, exchange of reagents, and other means.

[0060] 2B illustrates a cross-sectional view of the microfluidic cartridge 100 with a first pipette tip 180 inserted into the inlet port 170 and a second pipette tip 182 inserted into the outlet port 178. First and second elastic O-rings 150, 152 act as sealing elements around the first and second pipette tips 180, 182, respectively.

[0061] 4A, which illustrates a cross-sectional view of the inlet port 170. In some embodiments, the first O-ring 150 is an integral part of the upper cartridge body portion 104. In other words, the first O-ring 150 directly contacts the first inner wall 142i in the first opening 142. The first O-ring 150 is machined as a part of the upper cartridge body portion 104. For example, multiple materials may be used in the plastic injection molding of the upper cartridge body portion 104.

[0062] 4B, which illustrates a cross-sectional view of the inlet port 170. In some embodiments, the first O-ring 150 is secured within the first opening via a mechanical constraint or adhesive. The first O-ring 150 is resilient and secured within the first opening 142 of the upper cartridge body portion 104 by an additional fastener 194. In some embodiments, the fastener of the first O-ring is attached to the upper cartridge body portion 104 by adhesive, screws, or other means.

[0063] The fluid ports, which have O-rings secured to the upper cartridge body portion 104, function independently of the lower cartridge body portion 102. As shown in Figures 4A and 4B, the fluid ports can reduce residual dead volume when biological samples and other reagents are introduced into the fluid channels by an external instrument, such as an automated pipette tip or needle.

[0064] Reference is now made to FIG. 5A, which shows a perspective view of the biochip 160. In some embodiments, the biochip 160 is adhered to the lower cartridge body portion 102 by an adhesive. The choice of adhesive for this purpose may be determined by its compatibility with the materials of the biochip and the cartridge body portion. The adhesive is necessary to form a liquid-tight or air-tight seal around the biochip in the sample analysis process. The biochip 160 may be of a variety of materials and may include a variety of features to suit a particular application.

[0065] 5B is a partial cross-sectional view of the biochip. In some embodiments, the biochip 160 comprises a silicon layer 164, with the plurality of microwells 162 being recesses 164R in the silicon layer 164. In some embodiments, the biochip 160 is silicon-based and includes a matrix of hexagonally shaped reaction microwells between 10-200 μm in size etched into the silicon substrate by semiconductor processing techniques, such as wet or dry etching. In some embodiments, the surfaces of the microwells may also be derivatized by various means to render them hydrophilic, while keeping the surfaces of the walls between the microwells hydrophobic.

[0066] FIG. 5C illustrates a partial cross-sectional view of a biochip. In some embodiments, the biochip 160 comprises a thermally conductive layer 166 and a patterned layer 168 disposed on the thermally conductive layer 166. The patterned layer 168 comprises a number of through-holes 168H. The thermally conductive layer 166 is attached to one surface of the patterned layer 168, thereby generating microwells 162 originating from the through-holes 168H. Each of the microwells 162 is sealed at one end by the thermally conductive layer 166. The attachment of the thermally conductive layer 166 to the bottom surface of the patterned layer 168 forms an air-tight and liquid-tight seal at the bottom surface of each of the through-holes 168H, and the other end of each of the through-holes 168H is open for liquid communication. The shape and size of the through-holes 168H are compatible with liquid distribution and facilitate polymerase chain reaction.

[0067] In some embodiments, the dimensions of the through-holes 168H can range from 10 μm to 10,000 μm in width (labeled Da in FIG. 5B) and 10 μm to 10,000 μm in depth (labeled Db in FIG. 5B). The thickness of the thermally conductive layer 166 can vary significantly depending on the mechanical strength and thermal conductivity of the material, for example, in the range of 10 μm to 1000 μm (labeled Dc in FIG. 5B). The shape of the through-holes 168H can vary from round, square, hexagonal, etc., with various wall angles α with respect to the bottom of the microwell. In some embodiments, the shape of the microwell is hexagonal, with the angle α being 90°, as illustrated in FIG. 5B. Other geometric variations of the microwells are also possible.

[0068] The material of the pattern layer 168 may be selected from various materials known in the art for biological sample analysis, such as glass, silicon, polymeric materials such as PMMA or polypropylene, etc. It may be a good or bad conductor of heat. The processing method for making the patterned through-holes 168H in the pattern layer may be injection molding, CNC machining, 3D printing, etc. The thermally conductive layer 166 includes a good conductor of heat known in the art, such as metals such as gold, copper, aluminum, iron, stainless steel, silicon, carbon such as graphene or graphite, etc. Various methods known in the art may be used to attach the thermally conductive layer 166 to the pattern layer 168 of the biochip 160, such as adhesive or ultrasonic welding.

[0069] The bottom surfaces of the microwells 162 and the inner surfaces of the through-holes 168H of the biochip 160 that come into contact with the reaction mixture during biological sample analysis are preferably hydrophilic. These surfaces can be modified or derivatized to increase hydrophilicity by various chemical modifications. Techniques for creating the surface can be, for example, plasma surface treatment, attachment of hydroxyl or carboxyl groups, hydrogel deposition on the surface, etc.

[0070] FIG. 6 illustrates a perspective view of a microfluidic cartridge according to some embodiments of the present disclosure. The microfluidic cartridge 200 is used for simultaneous analysis of multiple samples, particularly in digital PCR amplification and quantification of targets of interest in these samples on an automated instrument. The disclosed microfluidic cartridge 200 has multiple sample injection ports 248, multiple physically separated individual microfluidic channels (see FIGS. 9A-9C), an inlet port 270, and an outlet port 278, as well as a biochip 260 with multiple microwells where sample splitting takes place (see FIGS. 9A-9C). The microfluidic cartridge 200 is assembled from a lower cartridge body portion 202, a biochip 260, and an upper cartridge body portion 204. Multiple subchannels in the microfluidic cartridge 200 are formed when the upper cartridge body portion 204 and the biochip 260 are attached together to form enclosed subchannels, each of which connects with the inlet port 270, the outlet port 278, the respective sample injection port 248, the fluidic channels, and the microwells of the portion of the biochip 260. Multiple subchannels can be used for different samples, or different targets of interest of the same sample, in the assay process.

[0071] 7 is a cross-sectional view taken along line CC in FIG. 6. As shown in FIG. 7, the microfluidic cartridge 200 includes a lower cartridge body part 202, an upper cartridge body part 204, and a biochip 260. The lower cartridge body part 202 includes a first substrate 210, an inlet column 212, an inflow trench 216, a biochip slot 218, an outflow trench 220, and an outlet column 222. The inlet column 212 protrudes above the first substrate 210. The inlet column 212 is a hollow structure. In addition, the outlet column 222 also protrudes above the first substrate 210. The outlet column 222 is a hollow structure. The inlet column 212, the inflow trench 216, the biochip slot 218, the outflow trench 220, and the outlet column 222 are connected in sequence. The biochip 260 is disposed in the biochip slot 218 of the lower cartridge body portion 202 .

[0072] The upper cartridge body part 204 includes a second substrate 240, a first opening 242, an optical window 244, and a second opening 246. As shown in FIG. 7, the first opening 242 corresponds to the inlet column 212 of the lower cartridge body part 202. The inlet column 212 is inserted into the first opening 242. The inlet column 212 of the lower cartridge body part 202 and the first opening 242 of the upper cartridge body part 204 are assembled into an inlet port 270. The inlet port 270 is configured to accommodate a pipette tip or needle for transporting fluids related to digital PCR. The optical window 244 is disposed above the biochip 260. The optical window 244 is made of a transparent material.

[0073] 7, the second opening 246 corresponds to the outlet column 222 of the lower cartridge body portion 202. The outlet column 222 is inserted into the second opening 246. The outlet column 222 of the lower cartridge body portion 202 and the second opening 246 of the upper cartridge body portion 204 are assembled into an outlet port 278. The outlet port 278 is configured to receive a pipette tip or needle for transporting fluids related to digital PCR.

[0074] As shown in FIG. 7, the fluid channels of the microfluidic cartridge 200 are defined by the assembly of a lower cartridge body portion 202, an upper cartridge body portion 204, and a biochip 260.

[0075] As shown in FIG. 7, a first O-ring 250 is disposed within a first opening 242 of the upper cartridge body portion 204 and abuts against the inlet column 212 of the lower cartridge body portion 202. A second O-ring 252 is disposed within a second opening 246 of the upper cartridge body portion 204 and abuts against the outlet column 222 of the lower cartridge body portion 202. These elastic O-rings act as sealing elements to maintain flow control of fluid or air through the channels when the microfluidic cartridge 200 is coupled to a connecting device, for example, an automated pipette tip or needle. Flow control through the fluid ports can affect biological reactions on the biochip by pressure control, exchange of reagents, and other means.

[0076] After the lower cartridge body portion 202, the biochip 260, and the upper cartridge body portion 204 are assembled into the microfluidic cartridge 200, a fluid inlet channel 272 is defined by the inlet trench 216 and the lower surface of the second substrate 240, a reaction channel 274 is defined by the biochip 260 and the lower surface of the optical window 244, and a fluid outlet channel 276 is defined by the outlet trench 220 and the lower surface of the second substrate 240.

[0077] FIG. 8 is a cross-sectional view taken along line DD in FIG. 6. The upper cartridge body portion 204 further includes a plurality of reaction channel spacers 284. Although FIG. 8 shows three reaction channel spacers 284, more or less reaction channel spacers 284 may be implemented. The reaction channel spacers 284 are disposed on a lower surface of the upper cartridge body portion 204, and after the microfluidic cartridge 200 is assembled, the reaction channel 274 is physically divided into a plurality of reaction sub-channels 274a, 274b, 274c, and 274d by the reaction channel spacers 284. Such physical separation of adjacent individual microfluidic sub-channels ensures that sample analysis in each individual sub-channel proceeds independently and without interference from other channels. In some embodiments, the cross dimensions of the disclosed microfluidic subchannels may be as small as 5 μm, or 10 μm, or 100 μm, or as large as 1000 μm, or even greater than 10,000 μm, and the cross-sectional profile of the channel may be rectangular, square, round, or other shapes. The number, cross dimensions, or shapes of the individual microfluidic subchannels may be varied as desired, and one of ordinary skill in the art would be able to form design variations based on the disclosed design principles.

[0078] As shown in FIG. 9A, in some embodiments, the inlet trench 216, the outlet trench 220, and the reaction channel spacer 284 extend in a first direction (e.g., the X direction), and the multiple sample injection ports 248 are arranged in a second direction (e.g., the Y direction).

[0079] 9A is a top view of the microfluidic cartridge 200. To more clearly illustrate the structure of the microfluidic cartridge 200, the inlet trench 216 and the outlet trench 220 in the lower cartridge body portion 202, and the reaction channel spacer 284 in the upper cartridge body portion 204 are shown.

[0080] As shown in FIG. 9A, each of the individual microfluidic subchannels 274a, 274b, 274c, and 274d includes a sample injection port 248 positioned within the respective subchannel. Through each sample injection port 248, the injected sample is directed toward the fluid outlet channel 276 and away from the fluid inlet channel 272. The solid arrows indicate the direction of the fluid. The hollow arrows indicate the direction of the fluid flow for each of the subchannels. The main function of the sample injection port 248 is to allow the introduction of the sample into the individual microfluidic subchannels, where it affects the directional flow of the sample toward the fluid outlet channel 276. The geometric design of the sample injection port 248 should be compatible with a sample injection device, such as a pipette tip or customized needle, in the device to which the microfluidic cartridge 200 is coupled for smooth and complete sample introduction during use. The aforementioned coupling between the sample injection port 248 and the sample injection device requires a proper seal to overcome any resistance that occurs inside the individual microfluidic subchannels when the sample is introduced. In some embodiments, a plurality of injection port O-rings 254 are disposed within a plurality of sample injection ports 248, respectively, as shown in FIG.

[0081] In some embodiments, the sample injection fixture of the device includes the use of single-use disposable pipette tips routinely used in biological sample analysis to avoid potential sample cross-contamination.

[0082] In some embodiments, as shown in FIG. 7, the individual microfluidic subchannels preferably include fluidics control functions, indicated by fluidics control 296A and fluidics control 296B, that help to direct the liquid flow inside each individual subchannel toward the fluidic outlet channel 276. Various designs of these fluidics control functions include, but are not limited to, passive valves by changing the geometry in the microfluidic channel, and are summarized in Recent Advances of Fluid Manipulation Technologies in Microfluidic Paper-Based Analytical Devices (μPADs) toward Multi-Step Assays, Micromachines 2020, 11, 269, by TH Kim et al., which is incorporated herein by reference. These said fluidics controls 296A and 296B, together with the fluidics control mechanism of the instrument, such as a syringe pump or air compressor, ensure that the parallel liquid flow in all individual subchannels is driven in the same direction as the sample analysis process, i.e., from the fluidic inlet channel 272 to the fluidic outlet channel 276 in the microfluidic cartridge 200.

[0083] As shown in FIG. 9A, in some embodiments, the inlet port 270 and outlet port 278, as well as the fluid inlet channel 272 and the fluid outlet channel 276, are shared among all the individual microfluidic subchannels. When the fluid inlet channel 272 is shared, shown as a triangular region on the left side of the microfluidic cartridge 200 in FIG. 9A, it can include either a smooth or structured shared surface, on which the inlet liquid from the inlet port 270 can freely flow into all connected sub-microfluidic channels. Similarly, when the fluid outlet channel 276 is shared, shown as a triangular region on the right side of the microfluidic cartridge 200 in FIG. 9A, it can include either a smooth or structured surface, on which the liquid outflow from all the microfluidic subchannels join together to form a continuous flow toward the outlet port 278. In other embodiments, the shared fluid inlet channel 272 and the fluid outlet channel 276 can have different designs and features to facilitate uniform fluidics performance among the different individual microfluidic channels.

[0084] 9B is another top view of the microfluidic cartridge 200 according to some embodiments. The microfluidic cartridge 200 further comprises a plurality of inflow channel spacers 286 each connected to a reaction channel spacer 284. The inflow channel spacers 286 are configured to divide the fluid inflow channel 272 into a plurality of fluid inflow sub-channels 272a, 272b, 272c, and 272d. In some embodiments, the plurality of inflow channel spacers 286 are defined in the lower cartridge body portion 202 and disposed on an upper surface of the first substrate 210. In other embodiments, the plurality of inflow channel spacers 286 are defined in the upper cartridge body portion 204 and disposed on a lower surface of the second substrate 240. In some embodiments, each of the plurality of inflow channel spacers 286 is connected to a respective one of the reaction channel spacers 284.

[0085] As shown in FIG. 9B, the microfluidic cartridge 200 further comprises a plurality of outflow channel spacers 288 each connected to a reaction channel spacer 284. The outflow channel spacers 288 are configured to divide the fluid outflow channel 276 into a plurality of fluid outflow sub-channels 276a, 276b, 276c, and 276d. In some embodiments, the plurality of outflow channel spacers 288 are defined in the lower cartridge body portion 202 and disposed on an upper surface of the first substrate 210. In other embodiments, the plurality of outflow channel spacers 288 are defined in the upper cartridge body portion 204 and disposed on a lower surface of the second substrate 240. In some embodiments, each of the plurality of outflow channel spacers 288 is connected to a respective one of the reaction channel spacers 284.

[0086] In some embodiments, the inlet port 270 and outlet port 278 are shared, but the fluid inlet channel 272 and fluid outlet channel 276 of the microfluidic cartridge 200 are not shared between the individual microfluidic subchannels, forming a microfluidic cartridge 200 with individual inlet subchannels and individual outlet subchannels connected to each individual microfluidic channel for liquid communication, resulting in completely physically isolated individual channels between the common inlet port 270 and outlet port 278, as illustrated in Figure 9B. It is also possible for one skilled in the art to fabricate a microfluidic cartridge with multiple subchannels with other design combinations of inlet and outlet channels in terms of common or compartmentalized functionality.

[0087] 9C is another top view of the microfluidic cartridge 200 according to some embodiments. The microfluidic cartridge 200 has a plurality of subchannels and a shared inlet port 270 and a shared outlet port 278. The fluid inlet channel 272 is divided into a plurality of fluid inlet subchannels 272a, 272b, 272c, and 272d. Each of the fluid inlet subchannels 272a, 272b, 272c, 272d is connected to a corresponding one of the reaction subchannels 274a, 274b, 274c, 274d. In some embodiments, the shape (or pattern) of the fluid inlet channel 272 and the fluid inlet subchannels 272a, 272b, 272c, 272d are defined in the lower cartridge body portion 202, which is disposed on the upper surface of the first substrate 210.

[0088] 9C, the plurality of fluid outlet subchannels 276a, 276b, 276c, and 276d converge into a fluid outlet channel 276. Each of the fluid outlet subchannels 276a, 276b, 276c, and 276d is connected to a corresponding one of the reaction subchannels 274a, 274b, 274c, and 274d. In some embodiments, the shape (or pattern) of the fluid outlet subchannels 276a, 276b, 276c, and 276d and the fluid outlet channel 276 are defined in the lower cartridge body portion 202, which is disposed on the upper surface of the first substrate 210.

[0089] In some embodiments, the fabrication methods for making the microfluidic cartridge 200 can be injection molding, CNC machining, 3D printing, etc. In some embodiments, the microfluidic cartridge 200 is fabricated by gluing different parts or sections by techniques known in the art, such as ultrasonic welding or adhesives. The material of choice for the microfluidic cartridge 200 needs to have compatibility with the biological sample under investigation, and preferably a low optical detection background, such as glass, silicon, polymeric materials such as PMMA or polypropylene. The microfluidic cartridge 200 should also be able to withstand the conditions of the analytical process, including chemical and temperature changes.

[0090] The disclosed microfluidic cartridge 200 can be used for assays of multiple samples isolated in their own individual microfluidic subchannels, or multiple assays of the same sample isolated in separate microfluidic channels with a subset of the analytes analyzed in a particular subchannel.

[0091] In some embodiments, the assay is a digital PCR (dPCR) based assay in which the sample and PCR reagent mixture is partitioned into multiple microwells of a biochip in separate microfluidic subchannels of a microfluidic cartridge, each of the microwells is physically isolated from adjacent microwells by a water-immiscible liquid at the top opening, and during the subsequent analysis process, temperature regulation is applied to the biochip 260 of the microfluidic cartridge 200 on an instrument for target amplification and detection. Figure 10 is an illustration showing the sample distribution process in the separate microfluidic subchannels of the microfluidic cartridge in a digital PCR assay. The digital PCR assay in the microfluidic cartridge 200 is performed on an automated instrument and includes the following steps:

[0092] (1) Sample injection: Four aqueous samples 310a, 310b, 310c, 310d are injected through respective sample injection ports 248 into respective individual reaction sub-channels 274a, 274b, 274c, 274d.

[0093] (2) Sealing of the sample injection port: The sample injection port 248 is sealed by a sealing element 330 after the sample is injected into the microfluidic cartridge 200 to prevent liquid and / or air leakage in the subsequent process.

[0094] (3) Sample partitioning within individual reaction subchannels: The aforementioned samples 310a, 310b, 310c, 310d gradually fill the microwells of the biochip 260 as they are pushed along the individual microfluidic channels by the water-immiscible hydrophobic liquid 320 that is introduced into the individual microfluidic channels through a shared inlet on the left side of the cartridge. The hydrophobic liquid 320 seals and isolates all the microwells at the top of the biochip as it flows along the microfluidic channels toward the shared outlet channel and liquid outlet port. As a result of this process, the samples 310a, 310b, 310c, 310d are evenly partitioned within the microwells in the physically separated reaction subchannels 274a, 274b, 274c, 274d and are completely isolated from adjacent microwells by the hydrophobic liquid 320.

[0095] (4) Digital PCR target amplification and analysis: The bottom side of the biochip in the microfluidic cartridge 200 is then exposed to temperature regulation and / or other chemical or physical conditions to enable target amplification and subsequent signal analysis, thereby elucidating the composition and amount of targets of interest in the sample.

[0096] In the sample distribution process, the sample volume gradually decreases as it fills the microwells of the biochip, and any residual volume is flushed out of the microfluidic cartridge 200 through the outflow channel and liquid outlet port. Various methods for sealing the sample inlet port may be used in step (2), including adhesive tape, heat or UV-initiated adhesives, or a mechanical plunger-like device in the instrument in which the microfluidic cartridge 200 is used. Other factors such as the flow rate and pressure at which the hydrophobic liquid is introduced into the cartridge significantly affect the efficiency of sample partitioning in the microwells. Targets of interest in the samples are identified and quantified by statistical analysis of the amplified signals from those microwells of the biochip, resulting in the results of multiple samples in a single assay.

[0097] Although the invention has been described in considerable detail with reference to certain embodiments thereof, other embodiments are possible, and therefore, the spirit and scope of the appended claims should not be limited to the description of the embodiments contained herein.

[0098] It will be apparent to those skilled in the art that various modifications and variations can be made to the structure of the present invention without departing from the scope or spirit of the invention. In view of the foregoing, it is intended that the present invention cover modifications and variations of this invention provided they fall within the scope of the following claims. [Explanation of symbols]

[0099] 100 Microfluidic Cartridges 102 Lower cartridge body part 104 Upper cartridge body part 110 First substrate 112 Injection column 116 Inflow Trench 116BS Bottom 118 Biochip Slot 120 Runoff Trench 120BS bottom 122 Outlet column 126 Welding Line 140 Second Board 140BS bottom 142 First Opening 142i First Inner Wall 144 Optical window 146 Second Opening 150 First O-ring 152 Second O-ring 160 Biochips 162 Microwell 164 Silicon Layer 164R recessed part 166 Thermal Conduction Layer 168 Pattern Layer 168H through hole 170 Inlet Port 172 Fluid Inlet Channel 174 reaction channels 176 Fluid Outlet Channel 178 Exit Port 180 First pipette tip 182 Second Pipette Tip 190 Ribs 192 Passive Valves 194 Fasteners 200 Microfluidic Cartridges 202 Lower cartridge body 204 Upper cartridge body 210 First substrate 212 Injection column 216 Inflow Trench 218 Biochip Slot 220 Runoff Trench 222 Outlet column 240 Second Board 242 First Opening 244 Optical window 246 Second Opening 248 sample injection ports 250 First O-ring 252 Second O-ring 254 Injection port O-ring 260 Biochips 270 Inlet Port 272 Fluid Inlet Channel 272a, 272b, 272c, 272d Fluid inlet subchannels 274 Reaction Channels 274a, 274b, 274c, 274d Reaction subchannels 276 Fluid Outflow Channel 276a, 276b, 276c, 276d Fluid outlet subchannels 278 Exit Port 284 Reaction Channel Spacer 286 Inflow Channel Spacer 288 Outflow Channel Spacer 296A, 296B Fluid Engineering Control 310a, 310b, 310c, 310d Aqueous samples

Claims

1. a first substrate having a protruding inlet trench; and an inlet column extending above the first substrate and being hollow; a lower cartridge body portion comprising: a biochip attached to the first substrate of the lower cartridge body portion; an upper cartridge body portion disposed on the lower cartridge body portion; Equipped with The upper cartridge body portion is A second substrate; a first opening through the second substrate into which the inlet column of the lower cartridge body portion is inserted; and a first O-ring disposed within the first opening; Equipped with the inlet column and the first opening are assembled within an inlet port; by adhering the protruding inlet trench on the first substrate to the second substrate, such that, in a state in which the lower cartridge body portion and the upper cartridge body portion are assembled, a fluid inlet channel is defined by the inlet trench and a portion of the bottom surface of the second substrate, and a reaction channel is defined by the biochip and another portion of the bottom surface of the second substrate, and the fluid inlet channel connects the inlet port and the reaction channel. Microfluidic cartridge.

2. The microfluidic cartridge of claim 1 , wherein the first O-ring directly contacts a first inner wall within the first opening.

3. The microfluidic cartridge of claim 1 , wherein the first O-ring is secured within the first opening via a mechanical restraint or an adhesive.

4. The lower cartridge body portion is A biochip slot connected to the inlet trench. Further equipped with The biochip is disposed in the biochip slot. The microfluidic cartridge of claim 1 .

5. 5. The microfluidic cartridge of claim 4, wherein the inlet trench has a first width and the biochip slot has a second width, the first width being less than the second width.

6. The microfluidic cartridge of claim 4 , wherein a top surface of the biochip is lower than a bottom surface of the inlet trench.

7. The microfluidic cartridge of claim 1 , wherein the biochip comprises a plurality of microwells, the number of the microwells being at least 1,000.

8. A first substrate; an entry trench disposed in the first substrate and extending in a first direction; and A biochip slot to which the inlet trench is connected a lower cartridge body portion comprising: a biochip disposed within the biochip slot, the biochip comprising a plurality of microwells; an upper cartridge body portion disposed on the lower cartridge body portion; Equipped with The upper cartridge body portion is a second substrate, the inlet trench and a bottom surface of the second substrate defining a fluid inlet channel; a first opening through the second substrate and connecting to the fluid inlet channel; an optical window disposed over the biochip, a lower surface of the optical window and the biochip defining a reaction channel; a plurality of sample injection ports disposed between the first opening and the optical window in the first direction; and at least one reaction channel spacer disposed on a lower surface of the upper cartridge body portion, the reaction channel spacer extending in the first direction and dividing the reaction channel into a plurality of reaction subchannels, each of the plurality of sample injection ports being connected to a respective one of the plurality of reaction subchannels; Microfluidic cartridge.

9. The microfluidic cartridge of claim 8 , wherein each of the plurality of sample injection ports is disposed within a respective one of the plurality of reaction subchannels.

10. 10. The microfluidic cartridge of claim 8, further comprising at least one inflow channel spacer, said at least one inflow channel spacer configured to divide said fluid inflow channel into a plurality of fluid inflow sub-channels.

11. The microfluidic cartridge of claim 8 , wherein each of the plurality of sample injection ports is sealable by a sealing element.

12. The microfluidic cartridge of claim 8 , further comprising a plurality of injection port O-rings disposed within the plurality of sample injection ports, respectively.

13. A first substrate; an entry trench disposed in the first substrate; and A biochip slot to which the inlet trench is connected a lower cartridge body portion comprising: a second substrate, the inlet trench and a bottom surface of the second substrate defining a fluid inlet channel; a first opening through the second substrate and connecting to the fluid inlet channel; and Optical Window an upper cartridge body portion disposed on the lower cartridge body portion; a biochip disposed within the biochip slot and below the optical window; Equipped with A microfluidic cartridge, wherein the biochip has a plurality of microwells, a lower surface of the optical window and the biochip define a reaction channel, an upper surface of the biochip is lower than a bottom surface of the inlet trench, and an upper surface of the reaction channel is lower than the bottom surface of the inlet trench.

14. The microfluidic cartridge of claim 13 , wherein each of the bottom surfaces of the microwells is hydrophilic.

15. 14. The microfluidic cartridge of claim 13, wherein the biochip comprises a silicon layer, and the plurality of microwells are recesses in the silicon layer.

16. The biochip comprises: A thermally conductive layer; a pattern layer disposed on the thermally conductive layer and having a plurality of through holes; The microfluidic cartridge of claim 13 , comprising:

17. 17. The microfluidic cartridge of claim 16, wherein the thermally conductive layer comprises gold, copper, aluminum, iron, steel, silicon, graphite, or graphene, and the patterned layer comprises glass, silicon, or a polymer.

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