A bedside diagnosis microfluidic chip

Abstract

This invention discloses a bedside diagnostic microfluidic chip, comprising a chip substrate. The chip substrate has a reaction cell, a buffer pool, a mixing channel communicating with the reaction cell, a detection channel communicating with the buffer pool and the mixing channel, and a waste liquid pool communicating with the detection channel. The reaction cell stores the test sample and provides a reaction site for the test sample and reagents. A superhydrophobic channel is disposed on the chip substrate between the detection channel and the mixing channel and / or between the detection channel and the buffer pool. The superhydrophobic channel is composed of a grooved microstructure and a superhydrophobic reagent covering the surface of the grooved microstructure. The superhydrophobic reagent and the superhydrophobic surface formed by the grooved microstructure are used to achieve passive flow blocking. The superhydrophobic channel design of this invention forms a passive flow blocking valve, which is simple to package, greatly simplifies the chip manufacturing process, and increases chip yield.

Description

[0001] This application is a divisional application of the invention patent application filed on December 13, 2018, with application number 201811523063X and invention title "Bedside Diagnostic Microfluidic Chip and its Preparation and Detection Method". Technical Field

[0002] This invention belongs to the field of in vitro diagnostic technology for medical devices, and specifically relates to a microfluidic chip for point-of-care testing (POCT). Background Technology

[0003] Point-of-care testing (POCT) involves providing rapid on-site diagnosis to patients using miniaturized or moderately sized desktop devices or reagents. Due to its ease of operation, simple system maintenance, ability to be performed anytime and anywhere, and low cost, POCT testing products are increasingly accepted and promoted in the market.

[0004] In the existing technology, POCT testing products used for disease diagnosis can be roughly divided into two categories according to the testing methodology or product materials: chromatography POCT testing technology and microfluidic POCT testing technology.

[0005] Chromatographic point-of-care testing (POCT) technology has been developing for over thirty years, and the dominant POCT products on the market today are based on this technology. While POCT products based on chromatography are technologically mature and have low technical barriers to entry, their open structure makes them susceptible to external environmental interference, and the uncontrolled sample flow leads to significant individual variability, thus affecting sensitivity and repeatability. Consequently, they fail to meet the clinical diagnostic performance requirements of laboratory physicians.

[0006] Microfluidic chip-based point-of-care testing (POCT) is a rapid detection technology that has developed over the past decade. Due to the characteristics of microfluidic technology, such as sample flow control, chip enclosure, miniature channels, and high controllability, microfluidic chip-based POCT offers improved detection sensitivity and repeatability compared to chromatography-based POCT. Therefore, microfluidic chip-based POCT is increasingly favored by the market.

[0007] Existing microfluidic chip-based point-of-care testing (POCT) products can be broadly categorized into two types based on their sample actuation methods: passive microfluidic chips and passive microfluidic POCT chips. Passive microfluidic chip POCT chips utilize capillary force provided by tiny channels within the chip to drive sample flow. While passive microfluidic chip POCT offers a simple detection method and low system requirements, it suffers from poor control over the overall sample reaction and significant randomness in reagent reactions. The reagent reaction chamber within a passive microfluidic chip is pre-filled with dried reagents. When the sample flows into this chamber, the dried reagents are reconstituted. This reconstitution process is random, and the sample reconstitution volume and process are uncontrollable, resulting in poor repeatability. Compared to chromatography-based POCT technology, its repeatability is not significantly improved.

[0008] Another type of microfluidic chip POCT detection product is the active microfluidic detection chip, which uses external forces such as air pressure, mechanical force, centrifugal force, or electrodynamic force to drive the sample flow within the chip. Active microfluidic chip POCT precisely controls the flow and mixing of the test sample and reagents, thus greatly improving reagent detection repeatability and sensitivity. However, active microfluidic chip POCT integrating magnetic particle detection technology has a major drawback: it can only perform single-item detection because the magnetic particles coated with different antibodies cannot be separated by a magnetic field. Multiple-item detection cannot be performed simultaneously in a single-channel microfluidic chip. Active microfluidic chip POCT integrating pressure valves requires integrating an elastic thin film into the chip, making the entire chip packaging process extremely complex and resulting in low chip manufacturing yield. Summary of the Invention

[0009] In view of this, in order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a bedside diagnostic microfluidic chip that can achieve rapid, accurate, highly sensitive and multi-item quantitative detection on-site.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A point-of-care diagnostic microfluidic chip includes a chip substrate and a chip cover. The chip cover has a first sample inlet for adding a test sample and a second sample inlet for adding a buffer solution. The chip substrate has a reaction cell connected to the first sample inlet, a buffer solution cell connected to the second sample inlet, a mixing channel connected to the reaction cell, a detection channel connected to the buffer solution cell and the mixing channel, and a waste liquid cell connected to the detection channel. The mixing channel is coated with a fluorescent microsphere-labeled reagent for providing a fluorescence detection signal, and the detection channel is coated with at least one capture antibody reagent.

[0012] In practical applications of microfluidic chips, the sample dispensing port can be connected to a driving device. The driving device can be driven by pneumatic, hydraulic, or electric means. This driving device provides the driving force for the added test sample and buffer solution to flow back and forth in the channels of the chip, so that the sample can fully react with the reagents in the channels.

[0013] In some embodiments, the chip substrate has a first sample loading slot located below the first sample loading port, a first sample loading channel connecting the first sample loading slot and the reaction cell, a second sample loading slot located below the second sample loading port, a second sample loading channel connecting the second sample loading slot and the buffer pool, and a transition pipe is also provided between the buffer pool and the detection pipe.

[0014] The reaction chamber can store test samples and provide a reaction site for the test samples and reagents; the buffer chamber can store buffer solutions and enable the washing function of the immune reaction.

[0015] According to some preferred aspects of the present invention, one end of the detection pipe is connected to the waste liquid pool, and the other end of the detection pipe is connected to a superhydrophobic pipe. The superhydrophobic pipe includes a first superhydrophobic pipe disposed between the detection pipe and the mixing pipe, and a second superhydrophobic pipe disposed between the detection pipe and the buffer solution pool. The first superhydrophobic pipe, the second superhydrophobic pipe, and the detection pipe form a Y-shaped structure. The sample enters the detection pipe through the first superhydrophobic pipe, and the buffer solution enters the detection pipe through the second superhydrophobic pipe.

[0016] More preferably, the superhydrophobic channel includes a grooved microstructure, the surface of which is coated with a superhydrophobic reagent. Each groove in the grooved microstructure has a depth of 50-200 μm and a width of 100-2000 μm; the superhydrophobic reagent is a fluorosilane dissolved in an electronic fluorination solution.

[0017] The superhydrophobic channel consists of a grooved microstructure and a superhydrophobic reagent covering the surface of the grooved microstructure. The superhydrophobic reagent and the superhydrophobic surface formed by the grooved microstructure can realize a passive flow blocking valve. When the test sample or buffer solution is driven to flow to the superhydrophobic surface, it can effectively block the liquid from continuing to flow. The liquid will remain in the groove and will not spread out. The driving force must be increased to drive the liquid to continue to flow forward, which further enhances the controllability of the liquid flow.

[0018] According to some preferred aspects of the present invention, a first micron-structured column is disposed within the mixing channel, and the fluorescent microsphere-labeled reagent is coated on the first micron-structured column. The diameter of the first micron-structure is 20-500 μm, and the height is 10-2000 μm.

[0019] The mixing pipe is connected to the reaction cell and provides space for the sample to flow back and forth. The sample is reconstituted with the fluorescent microsphere-labeled reagent while being uniformly mixed, allowing a homogeneous liquid immunoreaction to form a reaction complex. After the sample enters the mixing pipe, the fluorescent microsphere-labeled reagent is first reconstituted, then the sample and the reagent are mixed, and finally, an incubation reaction occurs between them. Reconstitution and mixing primarily occur in the mixing pipe, while incubation mainly takes place in the reaction cell.

[0020] More preferably, the fluorescent microsphere labeling reagent is a detection antibody labeled with fluorescent microspheres.

[0021] According to some preferred aspects of the present invention, a second micron-structured column is disposed within the detection channel, and the capture antibody reagent is coated on the second micron-structured column. The diameter of the second micron-structure is 10-200 μm, and the height is 10-200 μm.

[0022] If the detection channel is coated with only one type of capture antibody, that capture antibody can be dispersed freely within the channel. However, if the detection channel is coated with two or more types of capture antibody, it needs to be divided into several regions, with different capture antibody reagents coated in different regions. This is necessary to distinguish the fluorescence signals generated by the complexes in the corresponding regions during the final detection.

[0023] More preferably, the capture antibody reagent is a capture antibody labeled with polystyrene nanospheres.

[0024] The present invention also provides a method for fabricating the bedside diagnostic microfluidic chip as described above, specifically including the following steps:

[0025] Step 1) Preparation of fluorescent microsphere labeling reagent: The purified detection antibody raw material was labeled using time-resolved fluorescent microsphere analysis, and the fluorescent microsphere label was collected as the fluorescent microsphere labeling reagent.

[0026] Step 2) Preparation of capture antibody reagent: The purified capture antibody raw material is diluted with diluent, and the diluted capture antibody raw material is labeled on polystyrene nanospheres to prepare capture antibody reagent;

[0027] Step 3) Superhydrophilic modification of chip surface materials: Superhydrophilic modification of chip surface materials is performed by vacuum plasma bombardment or atmospheric plasma bombardment.

[0028] Step 4) Spraying and drying of sealing liquid: After the superhydrophilic modification of the chip surface in step 3) is completed, a layer of sealing liquid is sprayed onto the surface of the microfluidic chip for surface sealing, and then the chip is dried.

[0029] Step 5) Preparation of superhydrophobic channels: After the chip is dried in step 4), a precision spotting instrument and a superhydrophobic reagent are used to modify the surface of specific channels in the chip, i.e. the surface of the groove microstructure, to be superhydrophobic. Then the chip is dried to obtain superhydrophobic channels.

[0030] Step 6) Drying of fluorescent microsphere labeling reagent: After the chip is dried in step 5), the fluorescent microsphere labeling collected in step 1) is added to the mixing channel in the chip substrate and then dried to dry the fluorescent microsphere labeling in the chip.

[0031] Step 7) Immobilization of capture antibody: After the chip is dried in step 6), the capture antibody reagent prepared in step 2) is coated onto the micron column detection channel in the chip substrate using a precision spotting instrument, and then dried to immobilize the polystyrene microsphere-labeled capture antibody on the second micron column.

[0032] If the detection tube is coated with only one type of capture antibody reagent, the capture antibody reagent can be dispersed freely in the detection tube; if the detection tube is coated with two or more types of capture antibody reagents, the detection tube needs to be divided into several regions, with different capture antibody reagents coated in different regions. In this way, the fluorescence signal generated by the complex in the corresponding region can be distinguished in the final detection.

[0033] Step 8) Microfluidic chip packaging: Assemble and bond the chip cover plate to the chip substrate to obtain a bedside diagnostic microfluidic chip. The chip substrate and the cover plate are bonded together by pressure bonding, ultrasonic waves, lasers, or other methods to form a closed bedside diagnostic microfluidic chip.

[0034] Preferably, the blocking solution comprises 0.05%-0.5% BSA, 0.01%-0.5% Tween 20, and 10mM-200mM PBS; the diluent is 10mM PBS. BSA is bovine serum albumin, preferably with a mass-to-volume ratio of 0.1% to the total blocking solution; Tween 20 is a surfactant, preferably with a volume-to-volume ratio of 0.01% to the total blocking solution; and PBS is a buffer solution, preferably with a concentration of 10mM.

[0035] The present invention also provides a detection method for the bedside diagnostic microfluidic chip as described above, specifically including the following steps:

[0036] Step A. Sample addition and mixing: Use a pipette to add the test sample and buffer solution into the first and second sample addition wells of the bonded microfluidic chip. Then, place the microfluidic chip into the slot of the detection instrument. The first and second sample addition wells of the microfluidic chip are connected to the gas path drive device of the instrument. Control the gas path drive device to generate alternating positive and negative pressure in the first sample addition well, drive the test sample to flow back and forth in the reaction cell and mixing channel in the chip, and simultaneously re-dissolve the fluorescent microsphere labeled reagent in the mixing channel and mix it evenly. After mixing, stop generating positive or negative pressure, and incubate the mixed solution to allow an immune complex reaction to occur, forming a fluorescent microsphere labeled antibody / antigen complex.

[0037] Step B. Capture of antibody / antigen complex: After step A is completed, the gas path driving device generates a high positive pressure in the first sample loading port, driving the mixed sample containing antibody / antigen complex to flow through the first superhydrophobic channel. Then, the high positive pressure is reduced to a low positive pressure, driving the mixed sample to flow into the detection channel of the chip. At the same time, the capture antibody reagent coated in the channel reacts with the antibody / antigen complex in the mixed sample to form a capture antibody-antigen-labeled antibody complex. Other substances that are not captured flow into the waste liquid pool with the liquid.

[0038] Step C. Buffer Washing: After step B is completed, the gas path driving device generates a high positive pressure in the second sample well, driving the buffer solution to flow through the second superhydrophobic channel. Then, the high positive pressure is reduced to a low positive pressure, driving the buffer solution to flow into the chip's detection channel and waste liquid pool, washing the detection channel and improving detection sensitivity and specificity. Preferably, the buffer solution includes 1% BSA, 0.5% Tween 20, and 10mM PBS.

[0039] Step D. Data Reading: After step C is completed, the optical device of the detection instrument reads the fluorescence intensity on the detection channel of the microfluidic chip, calculates the concentration of capture antibody-antigen-labeled antibody, and generates the detection result.

[0040] The magnitude and duration of the positive pressure, negative pressure, high positive pressure, and low positive pressure generated by the drive device in the above detection steps are set according to actual needs.

[0041] Due to the implementation of the above technical solutions, the bedside diagnostic microfluidic chip of the present invention has the following advantages compared with the prior art:

[0042] 1. The bedside diagnostic microfluidic chip of the present invention is designed with a reaction cell and a mixing channel with a fixed reaction volume. Combined with an active driving mode, it drives the test sample and reagent to flow in the channel, and precisely controls the reconstitution and mixing process of the sample and reagent, thereby achieving strict control of the reagent reconstitution volume and reaction time, which greatly improves the repeatability of the test.

[0043] 2. The superhydrophobic channel of the bedside diagnostic microfluidic chip design of the present invention forms a passive flow blocking valve, which is simple to package, greatly simplifies the chip manufacturing process, and increases chip yield.

[0044] 3. The bedside diagnostic microfluidic chip of the present invention is designed with a detection channel with micron-sized columns, and more than one capture antibody can be coated in the detection channel to realize the joint detection of multiple items;

[0045] 4. The bedside diagnostic microfluidic chip of the present invention has a buffer washing structure design to wash the immune reaction, which further improves the detection sensitivity and detection specificity. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 This is a perspective view of the bedside diagnostic microfluidic chip in preferred embodiment 1 of the present invention;

[0048] Figure 2 This is a perspective view of the chip substrate in the bedside diagnostic microfluidic chip of the preferred embodiment of the present invention;

[0049] Figure 3 for Figure 2 Enlarged view of section I;

[0050] In the attached diagram: chip cover plate-1, chip substrate-2, first sample inlet-31, second sample inlet-32, first sample inlet-41, second sample inlet-42, first sample channel-51, second sample channel-52, reaction cell-61, buffer solution cell-62, mixing channel-71, transition channel-72, first superhydrophobic channel-81, second superhydrophobic channel-82, first micron column-91, second micron column-92, groove microstructure-10, detection channel-11, waste liquid cell-12. Detailed Implementation

[0051] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0052] It should be noted that the terms "first" and "second" used in this document are intended to facilitate the distinction between multiple objects and have no limiting effect. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such process, method, product, or device.

[0053] Example 1: Bedside Diagnostic Microfluidic Chip

[0054] like Figure 1-3 As shown, a point-of-care diagnostic microfluidic chip of this embodiment includes a chip substrate 2 and a chip cover plate 1. The chip cover plate 1 has a first sample inlet 31 for adding a test sample and a second sample inlet 32 ​​for adding a buffer solution. The chip substrate 2 has a reaction cell 61 connected to the first sample inlet 31, a buffer solution cell 62 connected to the second sample inlet 32, a mixing channel 71 connected to the reaction cell 61, a detection channel 11 connected to the buffer solution cell 62 and the mixing channel 71, and a waste liquid cell 12 connected to the detection channel 11. The mixing channel 71 is coated with a fluorescent microsphere-labeled reagent for providing a fluorescence detection signal, and the detection channel 11 is coated with at least one capture antibody reagent. The fluorescent microsphere-labeled reagent is a detection antibody labeled with fluorescent microspheres, and the capture antibody reagent is a capture antibody labeled with nano-polystyrene microspheres.

[0055] The mixing pipe 71 is connected to the reaction chamber 61, providing space for the sample to flow back and forth. The sample is reconstituted with the fluorescent microsphere-labeled reagent while being uniformly mixed, allowing a homogeneous liquid immunoreaction to form a reaction complex. After the sample enters the mixing pipe 71, the fluorescent microsphere-labeled reagent is first reconstituted, then the sample and the reagent are mixed, and finally, an incubation reaction occurs between them. Reconstitution and mixing primarily occur within the mixing pipe 71, while incubation mainly takes place within the reaction chamber 61.

[0056] like Figure 2 As shown, in this embodiment, in order to better control the reaction volume, a first sample loading groove 41 located below the first sample loading port 31, a first sample loading channel 51 connecting the first sample loading groove 41 and the reaction pool 61, and a second sample loading groove 42 located below the second sample loading port 32, and a second sample loading channel 52 connecting the second sample loading groove 42 and the buffer pool 62 are provided on the chip substrate 2. A transition channel 72 is also provided between the buffer pool 62 and the detection channel 11.

[0057] The reaction chamber 61 can store test samples and provide a reaction site for test samples and reagents; the buffer chamber 62 can store buffer solution and realize the washing function of immune reaction.

[0058] like Figure 2 As shown, in this embodiment, one end of the detection pipe 11 is connected to the waste liquid tank 12, and the other end of the detection pipe 11 is connected to a superhydrophobic pipe. The superhydrophobic pipe includes a first superhydrophobic pipe 81 disposed between the detection pipe 11 and the mixing pipe 71, and a second superhydrophobic pipe 82 disposed between the detection pipe 11 and the transition pipe 72. The first superhydrophobic pipe 81, the second superhydrophobic pipe 82 and the detection pipe 11 form a Y-shaped structure. The sample enters the detection pipe 11 through the first superhydrophobic pipe 81, and the buffer solution enters the detection pipe 11 through the second superhydrophobic pipe 82.

[0059] like Figure 2-3 As shown, the superhydrophobic channel includes a grooved microstructure 10, the surface of which is coated with a superhydrophobic reagent. In this embodiment, each groove in the grooved microstructure 10 has a depth of 50 μm and a width of 100 μm; the superhydrophobic reagent is a fluorosilane dissolved in an electronic fluorinated liquid. In other embodiments, the depth of each groove in the grooved microstructure 10 ranges from 50 to 200 μm, and the width ranges from 100 to 2000 μm.

[0060] The superhydrophobic reagent and the superhydrophobic surface formed by the groove microstructure 10 can realize a passive flow blocking valve. When the test sample or buffer solution is driven to flow to the superhydrophobic surface, it can effectively block the liquid from continuing to flow. The liquid will remain in the groove and will not spread out. The driving force must be increased to drive the liquid to continue to flow forward, which further enhances the controllability of the liquid flow.

[0061] like Figure 3 As shown, a first micron-structured column 91 with fluorescent microsphere-labeled reagent is disposed within the mixing channel 71; a second micron-structured column 92 with micron-structured reagent is disposed within the detection channel 11, and a capture antibody reagent is disposed within the second micron-structured column 92. The first micron-structured column 91 has a diameter of 200 μm and a height of 200 μm; the second micron-structured column 92 has a diameter of 100 μm and a height of 200 μm. The density of the first micron-structured column 91 is sparser than that of the second micron-structured column 92, which can be set according to actual needs. In other embodiments, the diameter of the first micron-structured column 91 ranges from 20 to 500 μm, and the height ranges from 10 to 2000 μm; the diameter of the second micron-structured column 92 ranges from 10 to 200 μm, and the height ranges from 10 to 200 μm.

[0062] If the detection channel 11 is coated with only one type of capture antibody reagent, then the capture antibody reagent can be randomly dispersed within the detection channel 11. If the detection channel 11 is coated with two or more types of capture antibody reagents, then the detection channel 11 needs to be divided into several regions, with different capture antibody reagents coated in different regions. In this way, the fluorescence signals generated by the complexes corresponding to different regions can be accurately distinguished in the final detection.

[0063] In the specific use of the microfluidic chip in this embodiment, the sample dispensing port can be connected to a driving device. The driving device can be driven by pneumatic, hydraulic or electric means. The driving device provides the driving force for the added test sample and buffer solution to flow back and forth in the channel of the chip, so that the test sample can fully react with the reagent in the channel.

[0064] The point-of-care diagnostic microfluidic chip in this embodiment features a reaction cell with a fixed reaction volume and a mixing channel. Combined with an active driving method, it drives the flow of test samples and reagents in the channel, precisely controlling the reconstitution and mixing processes of samples and reagents. This ensures strict control over the reagent reconstitution volume and reaction time, greatly improving test repeatability. A superhydrophobic channel is designed to form a passive flow-blocking valve, simplifying the encapsulation process and significantly increasing chip yield. A detection channel with micron-sized columns is designed, allowing different regions within the channel to be coated with more than one capture antibody, enabling multi-item combined detection. A buffer washing structure is incorporated to clean the immune reaction, further improving detection sensitivity and specificity.

[0065] Example 2: Preparation method of bedside diagnostic microfluidic chip

[0066] This embodiment provides a method for fabricating a bedside diagnostic microfluidic chip based on the one described in Embodiment 1, specifically including the following steps:

[0067] Step 1) Preparation of fluorescent microsphere labeling reagent

[0068] The purified detection antibody raw material was labeled using time-resolved fluorescence microsphere analysis, and the fluorescent microsphere labels were collected to form the fluorescent microsphere labeling reagent.

[0069] Step 2) Preparation of capture antibody reagent

[0070] The purified capture antibody raw material was diluted with a diluent, and the diluted capture antibody raw material was labeled onto polystyrene nanospheres to prepare a capture antibody reagent. In this example, the diluent was 10 mM PBS.

[0071] Step 3) Superhydrophilic modification of chip surface materials

[0072] Superhydrophilic modification of chip surface materials is achieved by using vacuum plasma bombardment or atmospheric plasma bombardment.

[0073] Step 4) Spraying and drying of the sealing liquid

[0074] After the superhydrophilic modification of the chip surface in step 3) is completed, a layer of sealing liquid is sprayed onto the surface of the microfluidic chip for surface sealing, and then the chip is dried.

[0075] The blocking solution in this embodiment includes 0.1% BSA, 0.01% Tween 20, and 10mM PBS. BSA is bovine serum albumin, Tween 20 is a surfactant, and PBS is a buffer solution.

[0076] Step 5) Fabrication of superhydrophobic channels

[0077] After the chip is dried in step 4), a precision spotting instrument and a superhydrophobic reagent are used to modify the surface of specific channels, i.e. the surface of the groove microstructure, in the chip to be superhydrophobic. Then the chip is dried to obtain superhydrophobic channels.

[0078] Step 6) Drying of fluorescent microsphere labeling reagent

[0079] After the chip is dried in step 5), the fluorescent microsphere label collected in step 1) is added to the mixing channel in the chip substrate and then dried to dry the fluorescent microsphere label in the chip.

[0080] Step 7) Immobilization of the captured antibody

[0081] After the chip is dried in step 6), the capture antibody reagent prepared in step 2) is coated onto the micron column detection channel in the chip substrate using a precision spotting instrument, and then dried to fix the capture antibody labeled with polystyrene microspheres onto the second micron column.

[0082] If the detection channel is coated with only one type of capture antibody reagent, the capture antibody reagent can be dispersed freely within the detection channel. If the detection channel is coated with two or more types of capture antibody reagents, the detection channel needs to be divided into several regions, with different capture antibody reagents coated in different regions. This way, in the final detection, the fluorescence signals generated by the complexes in the corresponding regions can be distinguished.

[0083] Step 8) Microfluidic chip packaging

[0084] The chip cover plate and the chip substrate are assembled and bonded to obtain a bedside diagnostic microfluidic chip.

[0085] The chip substrate and cover plate are bonded together by pressure bonding, ultrasonic waves, lasers, etc., to form a closed microfluidic chip for bedside diagnostics.

[0086] Example 3: Detection method for bedside diagnostic microfluidic chips

[0087] This embodiment provides a detection method based on the bedside diagnostic microfluidic chip described in Embodiment 1, specifically including the following steps:

[0088] Step A. Add sample and mix well

[0089] Using a pipette, the test sample and buffer solution are respectively added to the first and second sample wells of the bonded microfluidic chip. The microfluidic chip with the samples added is then placed into the slot of the detection instrument. The first and second sample wells of the microfluidic chip are connected to the gas path drive device of the instrument. The gas path drive device is controlled to generate alternating positive and negative pressure in the first sample well, driving the test sample to flow back and forth in the reaction cell and mixing channel in the chip. While re-dissolving the fluorescent microsphere-labeled reagent in the mixing channel, it is also uniformly mixed with it. After mixing is completed, the generation of positive or negative pressure is stopped, and the mixed solution is incubated to allow an immune complex reaction to occur, forming a fluorescent microsphere-labeled antibody / antigen complex.

[0090] Step B. Capture of antibody / antigen complexes

[0091] After step A is completed, the gas path driving device generates a high positive pressure in the first sample loading port, driving the mixed sample containing the antibody / antigen complex to flow through the first superhydrophobic channel. Then, the high positive pressure is reduced to a low positive pressure, driving the mixed sample to flow into the detection channel of the chip. At the same time, the capture antibody reagent coated in the channel reacts with the antibody / antigen complex in the mixed sample to form a capture antibody-antigen-labeled antibody complex. Other substances that are not captured flow into the waste liquid pool with the liquid.

[0092] Step C. Buffer washing

[0093] After step B is completed, the gas path driving device generates a high positive pressure in the second sample loading well. This pressure drives the buffer solution to flow through the second superhydrophobic channel, then reduces the high positive pressure to a low positive pressure, causing the buffer solution to flow into the chip's detection channels and waste reservoir. This washes the detection channels, improving detection sensitivity and specificity. In this embodiment, the buffer solution includes 1% BSA, 0.5% Tween 20, and 10mM PBS.

[0094] Step D. Data Reading

[0095] After step C is completed, the optical device of the detection instrument reads the fluorescence intensity on the detection channel of the microfluidic chip, calculates the concentration of the capture antibody-antigen-labeled antibody, and generates the detection result.

[0096] The magnitude and duration of the positive pressure, negative pressure, high positive pressure, and low positive pressure generated by the drive device in the above detection steps are set according to actual needs.

[0097] Example 4: Bedside Diagnostic Microfluidic Chip Measurement of Procalcitonin (PCT)

[0098] (I) Antibody labeling

[0099] A. Detection of antibodies using fluorescent microsphere labeling

[0100] 1. Prepare a solution of 20 mg / ml EDC and 10 mM PBS.

[0101] 2. Take 90 μL of 10 mM PBS buffer, add 10 μL of 300 nm fluorescent microspheres, and shake to mix.

[0102] 3. Add 5 μL of EDC activation solution to the buffer solution containing fluorescent microspheres prepared in step 2, shake to mix, and place on a shaker for 15 min (room temperature or 20°C, 250 rpm).

[0103] 4. Prepare 100 μL labeled antibody: Prepare PCT detection monoclonal antibody and rabbit IgG antibody solution with 10 mM PBS buffer to a final concentration of 0.5 mg / mL.

[0104] 5. Place the activated fluorescent microsphere solution obtained in step 3 into a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0105] 6. Add the 0.5 mg / ml PCT antibody and rabbit IgG antibody solution prepared in step 4 to the centrifuged activated fluorescent microsphere solution obtained in step 5, and vortex to mix. Sonicate for 2-3 min, vortex to mix, and then incubate on a shaker for 2 hours. Incubation conditions: room temperature, 250 rpm.

[0106] 7. Take 20 μL of blocking solution (1% bovine serum albumin) and add it to the solution obtained in step 6. Place it on a shaker for 2 hours. Blocking conditions: room temperature, 250 rpm.

[0107] 8. Place the solution obtained in step 7 in a centrifuge and centrifuge at 15,000 rpm for 15 minutes, then discard the supernatant.

[0108] 9. Add 500 μL of 10 mM PBS buffer to the substance obtained in step 8 to reconstitute the fluorescent microspheres. After shaking and mixing, place the mixture in a centrifuge and centrifuge at 15,000 rpm for 15 min. Discard the supernatant.

[0109] 10. Add 200 μL of microsphere preservation solution (0.1% BSA, 5% sucrose, 10 mM PBS) to the substance obtained in step 9, shake to mix, sonicate for 3 min, and store at 2-8℃.

[0110] B. Polystyrene microsphere-labeled capture antibody

[0111] 1. Prepare a solution of 20 mg / ml EDC and 10 mM PBS.

[0112] 2. Take 90 μL of 10 mM PBS buffer, add 10 μL of 150 nm polystyrene microspheres, and vortex to mix.

[0113] 3. Add 5 μL of EDC activation solution to the buffer solution containing polystyrene microspheres prepared in step 2, shake to mix, and place on a shaker for 15 min (room temperature or 20°C, 250 rpm).

[0114] 4. Prepare 100 μL of capture antibody: Prepare PCT-coated monoclonal antibody and goat anti-rabbit IgG antibody solution with 10 mM PBS buffer to a final concentration of 0.5 mg / mL.

[0115] 5. Place the activated polystyrene microsphere solution prepared in step 3 into a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0116] 6. Add the 0.5 mg / ml PCT antibody and goat anti-rabbit IgG antibody solution prepared in step 4 to the substance obtained in step 5, and vortex to mix. Sonicate for 2-3 minutes, vortex to mix, and then incubate on a shaker for 2 hours. Incubation conditions: room temperature, 250 rpm.

[0117] 7. Add 20 μL of blocking solution (1% bovine serum albumin) to the substance prepared in step 6, and place it on a shaker for 2 hours. Blocking conditions: room temperature, 250 rpm.

[0118] 8. Place the substance obtained in step 7 into a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0119] 9. Add 500 μL of 10 mM PBS buffer to the substance prepared in step 8 to reconstitute the polystyrene microspheres. After mixing by shaking, place the mixture in a centrifuge and centrifuge at 15,000 rpm for 15 min. Discard the supernatant.

[0120] 10. Add 200 μL of 10 mM PBS to the substance prepared in step 9, shake to mix, sonicate for 3 min, and store at 2-8°C.

[0121] (II) Microfluidic Chip Assembly

[0122] 1. Superhydrophilic modification of chip substrate surface materials is carried out by vacuum plasma bombardment or atmospheric plasma bombardment.

[0123] 2. After the superhydrophilic modification of the chip surface is completed, a layer of sealing solution is sprayed onto the surface of the microfluidic chip for surface sealing, and then the chip is dried. The sealing solution contains 0.1% BSA, 0.01% Tween 20 and 10mM PBS.

[0124] 3. After the chip in step 2 is dried, a precision spotting instrument and a superhydrophobic reagent are used to modify the surface of specific channels, i.e. the surface of the groove microstructure, in the chip to be superhydrophobic. Then the chip is dried to obtain superhydrophobic channels.

[0125] 4. After the chip in step 3 is dried, the fluorescent microsphere-labeled PCT monoclonal antibody and rabbit IgG antibody collected in step (I) are added to the mixing channel in the chip substrate and then dried so that the fluorescent microsphere labeling is dried on the first micron column of the chip mixing channel.

[0126] 5. After the chip in step 4 is dried, the polystyrene-labeled PCT capture antibody and goat anti-rabbit IgG antibody reagent prepared in step (I) are coated onto the micron column detection channel in the chip substrate using a precision spotting instrument. Then, the chip is dried to fix the polystyrene microsphere-labeled capture antibody onto the second micron column.

[0127] 6. Assemble the chip cover plate and chip substrate and bond them together with pressure adhesive to obtain the bedside diagnostic microfluidic chip.

[0128] (III) Sample Testing

[0129] 1. Apply 100 μL of the serum sample containing PCT antigen to the first and second wells of the microfluidic chip, respectively, using a pipette. Then, place the microfluidic chip into the slot of the detection instrument. The first and second wells of the microfluidic chip are connected to the gas drive device of the instrument. The gas drive device generates alternating positive and negative pressure in the first well, driving the sample to flow back and forth in the reaction cell and mixing channel of the chip. While reconstituted with the fluorescent microsphere-labeled PCT monoclonal antibody and rabbit IgG antibody reagent in the mixing channel, the sample is uniformly mixed with them. After mixing, the positive or negative pressure is stopped, and the mixed solution is incubated to allow an immune complex reaction to occur, forming a fluorescent microsphere-labeled PCT monoclonal antibody / PCT antigen complex.

[0130] 2. After step 1 is completed, the gas path driving device generates a high positive pressure in the first sample loading port, driving the mixed sample containing the PCT antibody / PCT antigen complex to flow through the first superhydrophobic channel. Then, the high positive pressure is reduced to a low positive pressure, driving the mixed sample to flow into the detection channel of the chip. At the same time, the PCT capture antibody and goat anti-rabbit IgG antibody labeled with polystyrene microspheres coated in the channel react with the PCT antibody / PCT antigen complex and rabbit IgG antibody in the mixed sample, respectively, to form PCT capture antibody-PCT antigen-PCT labeled antibody complex and rabbit IgG-goat anti-rabbit IgG antibody complex. Other substances that are not captured flow into the waste liquid pool with the liquid.

[0131] 3. After step 2 is completed, the gas path driving device generates a high positive pressure in the second sample loading port. After the driving buffer flows through the second superhydrophobic channel, the high positive pressure is reduced to a low positive pressure. The driving buffer flows into the chip's detection channel and waste liquid pool to wash the detection channel and improve detection sensitivity and specificity.

[0132] 4. After step 3 is completed, the optical device of the detection instrument reads the fluorescence intensity (T1 value) of the PCT capture antibody-PCT antigen-PCT labeled antibody complex and the fluorescence intensity (C value) of the rabbit IgG-goat anti-rabbit IgG antibody complex on the detection channel of the microfluidic chip, obtains the fluorescence intensity ratio (T / C value), and then calculates the concentration of PCT capture antibody-PCT antigen-PCT labeled antibody through the calibration curve and generates the detection result.

[0133] The magnitude and duration of the positive pressure, negative pressure, high positive pressure, and low positive pressure generated by the drive device in the above detection steps are set according to actual needs.

[0134] Comparative Example 1

[0135] This comparative example provides a conventional passive point-of-care diagnostic microfluidic chip. The microfluidic chip in this comparative example is essentially the same as that in Example 1, except that it lacks the superhydrophobic channel and buffer solution combination, buffer solution loading port, etc. The microfluidic chip in this example includes a chip substrate and a chip cover. The chip cover has a loading port for adding the detection sample. The chip substrate has a reaction cell connected to the loading port, a detection channel connected to the reaction cell, and a waste liquid tank connected to the detection channel. In this comparative example, the reaction cell is coated with fluorescent microsphere-labeled reagent to provide a fluorescence detection signal, and the detection channel is coated with polystyrene-labeled capture antibody reagent.

[0136] (I) Antibody labeling

[0137] A. Detection of antibodies using fluorescent microsphere labeling

[0138] 1. Prepare a solution of 20 mg / ml EDC and 10 mM PBS.

[0139] 2. Take 90 μL of 10 mM PBS buffer, add 10 μL of 300 nm fluorescent microspheres, and shake to mix.

[0140] 3. Add 5 μL of EDC activation solution to the buffer solution containing fluorescent microspheres prepared in step 2, shake to mix, and place on a shaker for 15 min (room temperature or 20°C, 250 rpm).

[0141] 4. Prepare 100 μL labeled antibody: Prepare PCT detection monoclonal antibody and rabbit IgG antibody solution with 10 mM PBS buffer to a final concentration of 0.5 mg / mL.

[0142] 5. Place the activated fluorescent microsphere solution obtained in step 3 into a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0143] 6. Add the 0.5 mg / ml PCT antibody and rabbit IgG antibody solution prepared in step 4 to the centrifuged activated fluorescent microsphere solution obtained in step 5, and vortex to mix. Sonicate for 2-3 min, vortex to mix, and then incubate on a shaker for 2 hours. Incubation conditions: room temperature, 250 rpm.

[0144] 7. Take 20 μL of blocking solution (1% bovine serum albumin) and add it to the solution obtained in step 6. Place it on a shaker for 2 hours. Blocking conditions: room temperature, 250 rpm.

[0145] 8. Place the solution obtained in step 7 in a centrifuge and centrifuge at 15,000 rpm for 15 minutes, then discard the supernatant.

[0146] 9. Add 500 μL of 10 mM PBS buffer to the substance obtained in step 8 to reconstitute the fluorescent microspheres. After shaking and mixing, place the mixture in a centrifuge and centrifuge at 15,000 rpm for 15 min. Discard the supernatant.

[0147] 10. Add 200 μL of microsphere preservation solution (0.1% BSA, 5% sucrose, 10 mM PBS) to the substance obtained in step 9, shake to mix, sonicate for 3 min, and store at 2-8℃.

[0148] B. Polystyrene microsphere-labeled capture antibody

[0149] 1. Prepare a solution of 20 mg / ml EDC and 10 mM PBS.

[0150] 2. Take 90 μL of 10 mM PBS buffer, add 10 μL of 150 nm polystyrene microspheres, and vortex to mix.

[0151] 3. Add 5 μL of EDC activation solution to the buffer solution containing polystyrene microspheres prepared in step 2, shake to mix, and place on a shaker for 15 min (room temperature or 20°C, 250 rpm).

[0152] 4. Prepare 100 μL of capture antibody: Prepare PCT-coated monoclonal antibody and goat anti-rabbit IgG antibody solution with 10 mM PBS buffer to a final concentration of 0.5 mg / mL.

[0153] 5. Place the activated polystyrene microsphere solution prepared in step 3 into a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0154] 6. Add the 0.5 mg / ml PCT antibody and goat anti-rabbit IgG antibody solution prepared in step 4 to the substance obtained in step 5, and vortex to mix. Sonicate for 2-3 minutes, vortex to mix, and then incubate on a shaker for 2 hours. Incubation conditions: room temperature, 250 rpm.

[0155] 7. Add 20 μL of blocking solution (1% bovine serum albumin) to the substance prepared in step 6, and place it on a shaker for 2 hours. Blocking conditions: room temperature, 250 rpm.

[0156] 8. Place the substance obtained in step 7 into a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0157] 9. Add 500 μL of 10 mM PBS buffer to the substance prepared in step 8 to reconstitute the polystyrene microspheres. After mixing by shaking, place the mixture in a centrifuge and centrifuge at 15,000 rpm for 15 min. Discard the supernatant.

[0158] 10. Add 200 μL of 10 mM PBS to the substance prepared in step 9, shake to mix, sonicate for 3 min, and store at 2-8°C.

[0159] (II) Microfluidic Chip Assembly

[0160] 1. Superhydrophilic modification of chip substrate surface materials is carried out by vacuum plasma bombardment or atmospheric plasma bombardment.

[0161] 2. After the superhydrophilic modification of the chip surface is completed, a layer of sealing solution is sprayed onto the surface of the microfluidic chip for surface sealing, and then the chip is dried. The sealing solution contains 0.1% BSA, 0.01% Tween 20 and 10mM PBS.

[0162] 3. After the chip in step 2 is dried, the fluorescent microsphere-labeled PCT monoclonal antibody and rabbit IgG antibody collected in step (i) are added to the reaction cell in the chip substrate and then dried to dry the fluorescent microsphere label in the reaction cell of the chip.

[0163] 4. After the chip is dried in step 3, the polystyrene-labeled PCT capture antibody and goat anti-rabbit IgG antibody reagent prepared in step (I) are coated onto the micron column detection channel in the chip substrate using a precision spotting instrument. Then, the chip is dried to fix the polystyrene microsphere-labeled capture antibody onto the micron column detection channel.

[0164] 5. Assemble the chip cover plate and chip substrate and bond them together with pressure adhesive to obtain the bedside diagnostic microfluidic chip.

[0165] (III) Sample Testing

[0166] 1. Apply 100 μL of serum sample containing PCT antigen using a pipette. Add the sample to the well of the microfluidic chip that has been bonded as described above. Then place the microfluidic chip into the slot of the detection instrument. The sample flows into the reaction cell through capillary action. The fluorescent microspheres labeled with PCT monoclonal antibody and rabbit IgG antibody in the reaction cell react with the antibody to form a fluorescent microsphere-labeled PCT monoclonal antibody / PCT antigen complex.

[0167] 2. Subsequently, the mixed sample containing the PCT antibody / PCT antigen complex flows into the detection channel of the chip through capillary self-drive. At the same time, the PCT capture antibody and goat anti-rabbit IgG antibody labeled with polystyrene microspheres coated in the channel react with the PCT antibody / PCT antigen complex and rabbit IgG antibody in the sample, respectively, to form PCT capture antibody-PCT antigen-PCT labeled antibody complex and rabbit IgG-goat anti-rabbit IgG antibody complex. Other substances that are not captured flow into the waste liquid pool with the liquid.

[0168] 3. After step 2 is completed, the optical device of the detection instrument reads the fluorescence intensity (T1 value) of the PCT capture antibody-PCT antigen-PCT labeled antibody complex and the fluorescence intensity (C value) of the rabbit IgG-goat anti-rabbit IgG antibody complex on the detection channel of the microfluidic chip, obtains the fluorescence intensity ratio (T / C value), and then calculates the concentration of PCT capture antibody-PCT antigen-PCT labeled antibody through the calibration curve and generates the detection result.

[0169] Results and Analysis of Example 5

[0170] The microfluidic chips prepared in Example 4 and Comparative Example 1 were used to test the same sample with a PCT concentration of 0.05 ng / mL 10 times. The performance detection and analysis comparison are shown in the table below:

[0171] Table 1 Comparison of Test Results

[0172]

[0173] The experimental results in Table 1 show that when the same test sample is used for testing, the detection results of the active microfluidic chip in Example 1 and the corresponding detection method in Example 4 are compared with those of the passive microfluidic chip and its detection method in Comparative Example 1. The detection results of the active microfluidic chip in Example 1 show a significant improvement in detection sensitivity as can be seen from the T / C value, and the detection repeatability can be greatly improved as can be seen from the detection coefficient of variation.

[0174] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A bedside diagnostic microfluidic chip, comprising a chip substrate, characterized in that, The chip substrate has a reaction cell, a buffer cell, a mixing pipe connected to the reaction cell, a detection pipe connected to the buffer cell and the mixing pipe, and a waste liquid cell connected to the detection pipe. The reaction cell is used to store the test sample and provide a reaction site for the test sample and reagent. The chip substrate is provided with superhydrophobic channels, which are composed of groove microstructures and superhydrophobic reagents covering the surface of the groove microstructures. The superhydrophobic reagents and the superhydrophobic surface formed by the groove microstructures are used to achieve passive flow blocking. The superhydrophobic channel includes a first superhydrophobic channel disposed between the detection channel and the mixing channel, and a second superhydrophobic channel disposed between the detection channel and the buffer pool; the first superhydrophobic channel, the second superhydrophobic channel and the detection channel form a Y-shaped structure; The chip cover includes a first sample inlet for adding a test sample and a second sample inlet for adding a buffer solution. The reaction cell is connected to the first sample inlet, and the buffer solution cell is connected to the second sample inlet. When the bedside diagnostic microfluidic chip is in use, the sample dispensing port is connected to a driving device, which provides the driving force for the added test sample and buffer solution to flow back and forth in the chip's channels, so that the sample can fully react with the reagents in the channels. The mixing channel is coated with fluorescent microsphere-labeled reagents for providing fluorescence detection signals.

2. The bedside diagnostic microfluidic chip according to claim 1, characterized in that, The groove microstructure has a depth of 50-200 μm and a width of 100-2000 μm for each groove; the superhydrophobic reagent is a fluorosilane dissolved in an electronic fluorinated liquid.

3. The bedside diagnostic microfluidic chip according to claim 1, characterized in that, The fluorescent microsphere labeling reagent is a detection antibody labeled with fluorescent microspheres.

4. The bedside diagnostic microfluidic chip according to claim 3, characterized in that, The mixing channel contains a first micron column with a micron structure, and the fluorescent microsphere-labeled reagent is coated on the first micron column; the diameter of the first micron column is 20-500 μm and the height is 10-2000 μm.

5. The bedside diagnostic microfluidic chip according to claim 1, characterized in that, The detection channel is coated with at least one capture antibody reagent, which is a capture antibody labeled with polystyrene microspheres.

6. The bedside diagnostic microfluidic chip according to claim 5, characterized in that, The detection channel is coated with two or more capture antibody reagents, and the detection channel is divided into several regions, with different capture antibody reagents coated in different regions.

7. The bedside diagnostic microfluidic chip according to claim 5, characterized in that, The detection channel is provided with a second micron column with a micron structure, and the capture antibody reagent is coated on the second micron column; the diameter of the second micron column is 10-200 μm and the height is 10-200 μm.

Images