Micro-fluidic chip and tissue dyeing device and method comprising micro-fluidic chip

Abstract

The invention discloses a micro-fluidic chip, a tissue dyeing device comprising the micro-fluidic chip and a tissue dyeing method. The micro-fluidic chip comprises an upper slide, wherein the upper slide is provided with an inlet hole and an outlet hole; the double-faced adhesive tape layer is used for removing part of double-faced adhesive tape through cutting so as to present an inflow channel, a dyeing cavity and an outflow channel of the corresponding micro-fluidic chip, the inflow channel is communicated with the inlet hole, and the outflow channel is communicated with the outlet hole; a to-be-dyed tissue is arranged on the lower slide; wherein the upper slide and the lower slide are adhered into a whole through the double-sided adhesive layer. A reagent bin is arranged at an inlet of the micro-fluidic chip, the reagent bin is designed to be open, and an outlet of the micro-fluidic chip is connected with a pressure pump. A reagent in the reagent bin is pumped into the dyeing cavity of the micro-fluidic chip through the pressure pump for dyeing, and is discharged after dyeing. According to the invention, the complexity and cost of the system are reduced, and meanwhile, the waste of reagents is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tissue staining, more particularly to a microfluidic chip and a tissue staining device and method comprising the same. BACKGROUND

[0002] Tissue staining is an important technique in biomedical field for detecting and labeling specific structures or molecules in tissues or cells. It is widely used in pathology, cell biology, molecular biology and other research fields. There are various methods of tissue staining, mainly including routine tissue staining, immunohistochemistry (IHC), immunofluorescence (IF) and fluorescence in situ hybridization (FISH) staining, etc.

[0003] 1. Routine tissue staining

[0004] Routine tissue staining (e.g., H&E staining) is the most basic and widely used method of tissue staining. Its main role is to stain different structures in tissue sections with different dyes, thereby visualizing the morphology, structure and cell types of the tissue. Hematoxylin staining is used to label cell nuclei, while Eosin stains the cytoplasm and extracellular matrix, forming a sharp contrast. This method is widely used in clinical pathology, cancer diagnosis, etc.

[0005] 2. Immunohistochemistry (IHC)

[0006] Immunohistochemistry (IHC) is a staining method based on the specific reaction of antigen-antibody, mainly used to detect the expression of specific proteins or antigens in tissue sections. This technology uses antibodies labeled with fluorescence or enzymes to target specific molecules in tissues, thereby visualizing the distribution and localization of proteins through color development reaction. IHC is widely used in oncology, immunology and neuroscience, helping to diagnose various diseases, study the function and localization of proteins.

[0007] 3. Immunofluorescence (IF)

[0008] Immunofluorescence (IF) is a technique that uses fluorescently labeled antibodies to detect specific antigens in tissues. Compared with IHC, IF uses fluorescent dyes instead of enzymes, allowing the location of antigens to be observed under a fluorescence microscope. IF technology can be multiplexed, allowing researchers to detect multiple proteins or cell markers simultaneously.

[0009] 4. Fluorescence in situ hybridization (FISH)

[0010] Fluorescence in situ hybridization (FISH) is a technique used to detect DNA, RNA, or other nucleic acid molecules in cells or tissue samples. By using fluorescently labeled probes to hybridize with target nucleic acid sequences, FISH enables quantitative and localization analysis of genes or specific RNAs. FISH is widely applied in genomics, cytogenetics, cancer diagnosis, and other fields, especially in the detection of chromosomal abnormalities and gene mutations.

[0011] 5. Microfluidics

[0012] Microfluidics is a technology that uses tiny channels and micro devices to control and manipulate liquid flow, usually on the scale of microns or nanometers. It is widely used in biomedical, chemical analysis, environmental monitoring, and other fields, and can achieve efficient liquid mixing, separation, analysis, and reaction. Microfluidic systems can simulate the microenvironment of biological reactions by precisely controlling the flow rate, direction, and distribution of fluids, and can be used for cell culture, disease detection, gene analysis, and other experiments. The advantages of this technology include small sample size, high throughput, low cost, and fast response.

[0013] Tissue staining based on microfluidics includes but is not limited to IHC, FISH, and routine pathological staining, which is a highly precise and automated technology widely used in biomedical and clinical research. This technology combines microfluidic chips and the above-mentioned pathological staining techniques, and realizes precise distribution of reagents and rapid detection of samples through microfluidic channels.

[0014] Unlike traditional staining which mainly relies on the slow and inefficient passive diffusion of reagents, microfluidic technology can greatly increase the liquid exchange between reagents and tissues. Microfluidic staining reduces the time of conventional immunohistochemical staining from several hours to about 10 minutes. Kim et al. generated bidirectional flow in a microfluidic device for 2 minutes by hand pipette, which effectively accelerated the antibody-antigen reaction and reduced the waste of antibodies, significantly improved the speed of immunohistochemical staining, and achieved the staining intensity comparable to traditional methods in 1 hour (Kim, Segi, et al. "Pipetting-driven microfluidic immunohistochemistry to facilitate enhanced immunoreaction and effective use of antibodies." Lab on a Chip 17.4 (2017): 702-709.). Brajkovic et al. successfully used microfluidics for rapid immunohistochemical staining of cytokeratin in frozen sections, which compressed the entire staining process from 3 hours to 10 minutes from the inclusion of primary antibody, secondary antibody incubation to DAB development without sacrificing the quality of staining (Brajkovic, S., Dupouy, D., de Leval, L. et al. Microfluidics for rapid cytokeratin immunohistochemical staining in frozen sections. Lab Invest 97, 983-991 (2017). https: / / doi.org / 10.1038 / labinvest.2017.49.). Nguyen et al. used microfluidics to evaluate the human epidermal growth factor receptor 2 (HER2) status of cancer tissue (Nguyen, H., Trouillon, R., Matsuoka, S. et al. Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer. Lab Invest 97, 93-103 (2017).). Compared with conventional FISH technology, microfluidic chip-based FISH technology reduces the consumption of probe solution (5-fold reduction), shortens the hybridization time to 4 hours, 4-fold time reduction compared with conventional FISH, and verifies its effectiveness and accuracy on 51 formalin-fixed paraffin-embedded tissue sections of 17 breast cancer samples.

[0015] There is a strong demand for rapid reporting of clinical pathology diagnoses, such as rapid frozen H&E staining, analysis, and rapid tissue immunohistochemical diagnosis. The multi-immunofluorescence staining technology (mIF) that has emerged in recent years also has an urgent need to reduce time. The circulating staining technology in mIF uses fluorescently labeled antibodies to gradually label multiple targets on the same tissue section through repeated staining, imaging, and elution processes. An image is taken after each staining, and then the fluorescent label is removed by elution to prepare for the next round of staining. This can achieve the detection of 20-60 markers and fuse the images to show the complete tissue immunological information. However, the detection of 20-60 markers is achieved through several rounds of staining cycles, each of which takes 3-5 hours, and the entire staining process is time-consuming and labor-intensive. Microfluidic technology can greatly accelerate the mIF experimental process and reduce the mIF staining time based on conventional immunohistochemistry from several days to several hours.

[0016] However, the existing microfluidic tissue staining has problems such as complex process, tedious operation, high cost, low throughput, and high cost of microfluidic chips themselves in the process of delivering staining reagents to the tissue to be stained.

[0017] The existing microfluidic chip tissue staining method represented by COMET TM The existing microfluidic chip tissue staining method represented by COMET TM Although these methods achieve a certain degree of automation and standardization of staining, there are some shortcomings.

[0018] First, the reaction reagents are put into a closed microfluidic chip, and except for a part of the reagents that can reach the microfluidic channel, the lumen occupies a considerable part of the volume. As a result, many reagents do not participate in the reaction of the chip and are ultimately wasted, increasing the waste and cost of reagents.

[0019] Especially at the microfluidic reagent import end, since multiple reagent sources need to be connected, the reagents enter the microfluidic through a computer-controlled reagent delivery system, which includes multiple reagent delivery pipelines, computer-controlled rotary valves, and computer-controlled pumps. The rotary valve is usually used to switch the reagent source and its channel, and the rotary valve is opened or closed at different positions to achieve the functions of delivery, distribution, or sealing of different reagent sources. However, this solution also increases the complexity and cost of the system. Due to the complexity, physical space, and cost constraints, the staining throughput of such a system is very low, and generally only 1-4 microfluidic staining chips can be accommodated.

[0020] In this scheme, rotary valves are involved. Due to the physical space limitation, rotary valves usually have only a limited number of inlet ports, typically between 1 and 16. However, in practical applications, the number of reagents is often from several to dozens, especially in multiple immunofluorescence (mIF) staining, the number of antibodies used can be as many as dozens, and each reagent usually requires an inlet port. In this case, the increase in the number of reagents and the need for different dilution ratios further increases the demand for inlet ports, resulting in further increase in system complexity and cost.

[0021] For example, Huang et al. integrated micro-rotary valves into microfluidic chips, but this only shifted the control of liquid flow from before entering the microfluidic chip to the microfluidic chip, increasing the complexity of the microfluidic chip. (Huang, S. P., Chuang, Y. J., Lee, W. B., Tsai, Y. C., Lin, C. N., Hsu, K. F., & Lee, G. B. (2020). An integrated microfluidic system for rapid, automatic and high-throughput staining of clinical tissue samples for diagnosis of ovarian cancer. Lab on a Chip, 20(6), 1103-1109.)

[0022] Therefore, there is still a lack of a microfluidic chip with simple structure, reagent saving, and low energy consumption, as well as a tissue staining device and method comprising the same in the art. SUMMARY

[0023] The purpose of the present application is to provide a microfluidic chip and a tissue staining device and method comprising the same, so as to reduce the complexity and cost of the system, while reducing the waste of reagents.

[0024] In a first aspect of the present application, a microfluidic chip is provided, comprising:

[0025] an upper glass slide provided with an inlet hole and an outlet hole;

[0026] a double-sided adhesive layer, the double-sided adhesive layer being cut to remove part of the double-sided adhesive layer to present a corresponding flow-in channel, a staining cavity and a flow-out channel of the microfluidic chip, wherein the flow-in channel is in communication with the inlet hole, and the flow-out channel is in communication with the outlet hole; and

[0027] a lower glass slide on which a tissue to be stained is placed;

[0028] wherein the upper glass slide and the lower glass slide are adhered as a whole by the double-sided adhesive layer.

[0029] In another preferred embodiment, the lower glass slide is a cover glass or a cover slip.

[0030] In another preferred embodiment, the inlet hole is configured to communicate with a reagent cartridge; and the outlet hole is configured to communicate with a pressure pump.

[0031] In another preferred embodiment, no valve is provided in the microfluidic chip.

[0032] In another preferred embodiment, the double-sided adhesive layer is selected from Advanced Research or 3M double-sided adhesive.

[0033] In another preferred embodiment, the thickness of the double-sided adhesive layer is in the range of 10-1000 um; preferably, 50-500 um; more preferably, 50-100 um.

[0034] In another preferred embodiment, in the un-assembled state, the double-sided adhesive layer is isolated from the outside environment by a release paper on both sides.

[0035] In a second aspect of the present application, there is provided a microfluidic chip tissue staining device, the device comprising:

[0036] a reagent station configured to hold a plurality of reagents;

[0037] a microfluidic chip as described above, wherein the inlet of the microfluidic chip is provided with a reagent cartridge, the reagent cartridge is open-ended, the outlet of the microfluidic chip is connected with a pressure pump, the staining chamber, the reagent cartridge and the pressure pump are in fluid communication; and

[0038] an automated pipetting system configured to aspirate the reagents from the reagent station and dispense them above the reagent cartridge;

[0039] wherein the reagents in the reagent cartridge are aspirated by the pressure pump into the staining chamber of the microfluidic chip for staining, and are discharged after staining.

[0040] In another preferred embodiment, the automated pipetting system can be a Thermo Scientific F Series pipette. Automated pipettes such as Opentron OT-2, Opentron Flex are suitable for use as the automated pipetting system of the present application (e.g. Systems and methods for pipette robots, US12013408B2).

[0041] In another preferred embodiment, the reagent station is in any one of the following forms or a combination thereof: a microplate, a test tube, a centrifuge tube.

[0042] In another preferred embodiment, the microplate includes but is not limited to 384-well microplate, 96-well microplate, 24-well microplate, 6-well microplate, which can be selected according to the actual needs.

[0043] In another preferred embodiment, the pressure pump is a peristaltic pump or a syringe pump.

[0044] In another preferred embodiment, the microfluidic chip is connected to the pressure pump through a closed capillary tube.

[0045] In another preferred embodiment, the pressure pump controls the reciprocating movement of the reagent in the staining cavity to accelerate the staining.

[0046] In another preferred embodiment, the excess dyeing agent or sample is pumped into a waste liquid tank, for example, by a pressure pump.

[0047] In another preferred embodiment, the excess waste liquid after staining is pumped into a waste liquid tank.

[0048] In another preferred embodiment, the automatic pipetting system includes a mechanical arm and a controller, and under the control of the controller, the free end of the mechanical arm can suck the reagent from the reagent station and move to above the reagent bin for discharge to discharge the reagent into the reagent bin.

[0049] In a third aspect of the present application, a microfluidic chip tissue staining method is provided, which comprises:

[0050] (1) providing a microfluidic chip tissue staining device as described above;

[0051] (2) adding the reagent to the reagent bin: adding the staining reagent to the reagent bin in sequence according to the staining procedure by the automatic pipetting system;

[0052] (3) sucking the reagent: sucking the reagent in the reagent bin into the microfluidic chip by the pressure pump;

[0053] (4) staining process: accelerating the liquid exchange between the reagent liquid phase and the stained tissue solid phase, accelerating the staining reaction by controlling the flow rate or pressure and direction of the pressure pump, and by controlling the frequency to change the flow direction of the reagent in the microfluidic chip; and

[0054] (5) discharging the reagent: discharging the reagent in the microfluidic chip by the pressure pump.

[0055] In another preferred embodiment, the operation of sucking and discharging the reagent is repeated to ensure the sufficiency and consistency of the staining.

[0056] In another preferred embodiment, in the above-mentioned staining process, the pressure pump first guides the reagent in the staining cavity of the microfluidic chip to flow towards the reagent container, and then guides the reagent to flow towards the pressure pump, and so on.

[0057] In another preferred embodiment, the amount M of the reagent added into the reagent container is greater than the amount m of the reagent sucked from the reagent container into the microfluidic chip (the staining cavity) by the pressure pump.

[0058] In another preferred embodiment, m is 80%-98% of M; preferably, 85%-95%; more preferably, 90%-92%.

[0059] In another preferred embodiment, the reagent remaining in the reagent container plays a sealing role to separate the microfluidic chip (the staining cavity) from the outside world, so as to avoid external gas from being sucked into the staining cavity to affect the staining effect.

[0060] In another preferred embodiment, the amount of the reagent sucked is determined by controlling the number of suction times of the pressure pump on the basis that the single suction amount of the pressure pump itself is fixed.

[0061] It should be understood that, within the scope of the present application, each of the above technical features of the present application and each of the technical features specifically described below (such as in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0062] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0063] Figure 1 is a schematic diagram of a microfluidic chip tissue staining device in an example of the present application;

[0064] Figure 2 is a schematic diagram of a microfluidic chip in an example of the present application;

[0065] Figure 3 is a rapid immunohistochemical CK staining diagram in an example of the present application;

[0066] Figure 4 is a multiplex immunofluorescence staining diagram with FoxP3 as the staining marker in an example of the present application;

[0067] Figure 5Figure 4 is a multiplex immunofluorescence staining chart in which the staining marker is CK in one example of the present application;

[0068] Figure 6 Figure 5 is a multiplex immunofluorescence staining chart in which the staining marker is CD8 in one example of the present application;

[0069] Figure 7 Figure 6 is a multiplex immunofluorescence staining chart in which the staining marker is PD-1 in one example of the present application;

[0070] Figure 8 Figure 7 is a multiplex immunofluorescence staining chart in which the staining marker is FoxP3+CK+CD8+PD-1 fusion in one example of the present application.

[0071] In each figure, each mark shows as follows:

[0072] 1-microfluidic chip;

[0073] 2-upper glass slide;

[0074] 3-double-sided adhesive layer;

[0075] 4-lower glass slide;

[0076] 5-inlet hole;

[0077] 6-outlet hole;

[0078] 7-reagent bin;

[0079] 8-outlet;

[0080] 9-staining cavity;

[0081] 10-pressure pump;

[0082] 11-reagent station;

[0083] 12-waste liquid barrel. DETAILED DESCRIPTION

[0084] The present inventors have made extensive and in-depth research, and through a large number of screening, for the first time developed a microfluidic chip and a tissue staining device and method comprising the same. Compared with the prior art, the present application combines a sealed pressure pump system with an open reagent bin, controls the reagent to enter and exit the chip through the pressure pump, avoids the complex pipeline system in the traditional method, the open reagent bin design close to the chip port eliminates the connecting pipe, reduces the dead volume caused by the pipeline system and rotary valve in the traditional method, and reduces the reagent waste. Compared with the limited number of inlet ports of the rotary valve, the reagent station of the present application has an order of magnitude improvement, and the type of reagent that can be delivered has an order of magnitude improvement. On this basis, the present application is completed.

[0085] TERMS

[0086] As used herein, the term "microfluidic chip technology" is a technology that manipulates microfluids (usually in nanoliters to microliters) on a micrometer scale, and realizes precise control, mixing, separation and detection of fluids through structures such as microchannels, micropumps, microvalves, etc. It has wide application in the fields of biomedical science, chemical analysis, environmental monitoring, etc., and has advantages such as high throughput, low power consumption and portability.

[0087] As used herein, the term "immunohistochemistry" refers to the use of antigen-antibody reaction principle to determine the antigen in the tissue cells by labeling the antibody to develop color, and to conduct localization, qualitative and relative quantitative research. It has important significance in the diagnosis of tumors, inflammatory diseases and autoimmune diseases.

[0088] As used herein, the term "fluorescence in situ hybridization (FISH)" is an important technology combining non-radioactive molecular biology and cytogenetics. It uses fluorescently labeled nucleic acid probes to hybridize with target DNA on chromosome or DNA fiber sections, and observes the hybridization signal by fluorescence microscope to realize qualitative, quantitative or relative positioning analysis of the DNA to be tested, and is widely used in the fields of genetic disease diagnosis and tumor research.

[0089] As used herein, the term "multiplexed immunofluorescence staining" is an advanced biological technology that uses the principle of antigen-antibody reaction to label different antigens with multiple different colored fluorescent dyes, thereby simultaneously detecting multiple target molecules on one tissue section. This technology has the characteristics of high sensitivity, high resolution and high throughput, and is widely used in the fields of oncology, immunology, neuroscience, etc.

[0090] The simplified microfluidic chip tissue staining device of the present application combined with the closed pump system and the open reagent warehouse design is used to solve the following problems in the prior art:

[0091] 1. High complexity of reagent delivery: The present application eliminates the need for a complex and precise reagent delivery system to pump multiple reagents into the microfluidic staining chamber, which includes multiple reagent delivery pipelines, computer-controlled rotary valves and computer-controlled pumps. That is, the present application eliminates the complexity of reagents before entering the microfluidic.

[0092] 2. Compared with the current solution, the present application can accommodate multiple microfluidic devices on a single device, and can perform more microfluidic staining, with an order of magnitude improvement in the number of stained specimens.

[0093] 3. Compared with the current solution, the present application has an order of magnitude improvement in the number or type of reagents.

[0094] 2. Low cost of processing: reducing the complexity of reagent into microfluidic, directly reducing the cost.

[0095] 3. Poor flexibility: also solves the problem of poor flexibility of the system caused by multi-pipeline closed system.

[0096] 4. Reagent waste: because the reagent is directly added to the development reagent warehouse of the microfluidic device through the automatic pipetting device, there is no pipeline for the reagent to enter the microfluidic device, eliminating the loss of reagent in the fine tube and reducing the cost of reagent.

[0097] The main advantages of the present application include:

[0098] (a) greatly shorten the staining time: the traditional immunohistochemical staining needs 3.5-5.5 hours, and the method of the present application can shorten the immunohistochemical staining time to 10 minutes through controllable and rapid liquid exchange, greatly improving the experimental efficiency;

[0099] (b) ensure the consistency of staining: through the pressure pump system to accurately control the flow rate and pressure, ensure the consistency of staining between different experimental samples;

[0100] (c) reduce the complexity and cost of the system: the open reagent warehouse design reduces the use of pipeline system, reduces the complexity and cost of the system, and reduces the waste of reagent;

[0101] (d) flexible and dead volume-free reagent in and out: the open design of the reagent warehouse ensures the flexible and dead volume-free reagent in and out of the microfluidic chip;

[0102] (e) low cost: the present application saves the cost by abandoning the complex pipeline, rotary valve and pressure pump of the front-end reagent delivery system, and the microfluidic based on double-sided adhesive further reduces the cost of the system.

[0103] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In addition, the drawings are schematic drawings, therefore the size or proportion of the device and equipment of the present application is not limited by the schematic drawings.

[0104] It is to be noted that the relational terms herein, such as first and second, and the like, are used solely to distinguish one from another entity or action without necessarily requiring or implying any actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "comprises... a" does not, without more constraints, exclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0105] Embodiment

[0106] The microfluidic chip tissue staining device of the present embodiment is shown in Figure 1 The microfluidic chip tissue staining device includes a reagent station 11, an automatic pipetting system, and a microfluidic chip 1, wherein the microfluidic chip 1 has a specially designed reagent bin 7 at the inlet for the automatic pipetting system to add samples, and the outlet 8 is connected to a pump system.

[0107] The reagent station 11 supports standard size microplates (384, 96, 24, 6 well microplates), test tubes, centrifuge tubes, and other forms to meet the storage needs of different volumes of reagents.

[0108] The automatic pipetting system sucks reagents from the test tube or microplate in the reagent station 11 and pipettes them to the reagent bin 7 of the microfluidic chip 1. The dashed line in Figure 1 shows a pipetting track of the automatic pipetting system. The automatic pipetting system can be a Thermo Scientific F series pipette. Automatic pipettes such as Opentron OT-2, Opentron Flex are suitable for use as the automatic pipetting system of the present embodiment (e.g. Systems and methods for pipette robots, US12013408B2).

[0109] The microfluidic chip 1 has an inlet hole 5 and an outlet hole 6. The inlet hole 5 is connected to a reagent reservoir 7. The reagent needed for staining is introduced into the microfluidic chip 1 through the reagent reservoir 7 and enters the staining cavity 9, and reacts with the tissue in the staining cavity 9 to stain the tissue. The reagent reservoir 7 is in communication with the microfluidic staining cavity 9 through the inlet hole 5. The outlet hole 6 of the microfluidic chip 1 is connected to a closed tube at the outlet 8. The tube is connected to a pressure pump 10 (peristaltic pump or injection pump) to drive the reagent to enter and exit the microfluidic chip 1. The present application is not connected to a closed pipeline system at the reagent inlet of the microfluidic device, but an open reagent reservoir 7 which can be designed to have different sizes according to the needs.

[0110] The microfluidic chip 1 of the embodiment is shown in Figure 2 The microfluidic chip 1 includes an upper glass slide 2, a double-sided adhesive layer 3 and a lower glass slide 4.

[0111] The upper glass slide 2 is provided with an inlet hole 5 and an outlet hole 6. The double-sided adhesive layer 3 is cut to remove part of the double-sided adhesive to present the corresponding microfluidic chip 1 flow-in channel, staining cavity 9 and flow-out channel, wherein the flow-in channel is in communication with the inlet hole 5, and the flow-out channel is in communication with the outlet hole 6. The lower glass slide 4 is provided with the tissue to be stained. The upper glass slide 2 and the lower glass slide 4 are adhered together by the double-sided adhesive layer 3. The lower glass slide 4 can be a glass slide or a cover glass. The inlet hole 5 is used to communicate with the reagent reservoir 7; the outlet hole 6 is used to communicate with the pressure pump 10.

[0112] The double-sided adhesive layer 3 is selected from Advanced Research or 3M. The thickness of the double-sided adhesive layer 3 is in the range of 10-1000 um; preferably, in the range of 50-500 um; more preferably, in the range of 50-100 um. In the un-assembled state, the double-sided adhesive layer 3 is isolated from the outside by release paper on both sides.

[0113] It should be noted that the microfluidic chip 1 in the embodiment does not need to be provided with a valve.

[0114] Manufacture of the microfluidic chip 1:

[0115] 1. Manufacture of the upper glass slide 2: punch two holes on a glass substrate with appropriate size (for example, 25mmx75mmx1.1mm), the positions are respectively at the upper half and the lower half. The reagent reservoir 7 is a cylinder with both ends open. Smear the edge of the lower end of the cylinder with glue, and then align the small hole at the bottom of the cylinder with the small hole on the glass substrate and paste them together to ensure that the liquid in the reagent reservoir 7 can pass through the small hole on the glass substrate without obstruction. Align another cylinder with the other hole on the glass slide and paste them together in the same way.

[0116] 2. Cut the flow-in channel, the staining cavity 9 and the flow-out channel of the microfluidic chip 1 on the double-sided adhesive.

[0117] 3. Place the tissue to be stained on the lower glass slide 4, which can be a cover glass or a cover slip

[0118] 4. Assemble the upper glass slide 2, the cut double-sided adhesive tape, and the other glass slide together in a sandwich, forming a closed flow-in channel, a staining chamber 9, and a flow-out channel between the upper glass slide 2 and the lower glass slide 4, completing the assembly of the microfluidic chip.

[0119] The microfluidic chip tissue staining method of the embodiment can be implemented by the following steps:

[0120] 1. After placing the tissue to be stained on the lower glass slide 4, assemble the closed microfluidic chip 1

[0121] 2. Connect the system: connect the outlet 8 of the microfluidic chip 1 to a closed pressure pump system, and the other end is an open reagent tank 7.

[0122] 3. Add reagents to the reagent tank 7: add staining reagents to the reagent tank 7 according to the order of the staining process.

[0123] 4. Suck in the reagent: use the pressure pump 10 to suck the reagent in the reagent tank 7 into the microfluidic chip 1.

[0124] 5. Staining process: control the flow rate or pressure and direction of the pressure pump 10, change the flow direction of the reagent in the microfluidic chip 1 at a set frequency, accelerate the liquid exchange between the reagent liquid phase and the staining tissue solid phase, and accelerate the staining reaction.

[0125] 6. Discharge the reagent: use the pressure pump 10 to discharge the reagent in the chip.

[0126] 7. Repeat the operation: repeat the operation of sucking in and discharging the reagent as needed to ensure the sufficiency and consistency of the staining.

[0127] 8. The reagent is transferred from the reagent tank 7 to the staining chamber 9 of the microfluidic chip 1, which is completed by an automatic pipetting device controlled by a computer, and the reagent sources include but are not limited to microwell plates, test tubes, centrifuge tubes, etc., to store including but not limited to dewaxing, hydration, antigen repair liquid, coating liquid, 1st antibody, 2nd antibody, washing liquid, color developing liquid, and antibody elution liquid, etc. According to the experimental process, the automatic pipetting device takes the pre-set amount of reagent from the corresponding reagent and transfers it to the reagent tank 7 on the microfluidic chip 1. The outlet 8 of the microfluidic chip 1 is connected to a computer-controlled pressure pump, including but not limited to a syringe pump and a peristaltic pump. The negative pressure applied by the pump sucks the reagent in the reagent tank 7 into the microfluidic chip 1.

[0128] Excessive staining agents or samples can be pumped into the waste liquid tank 12, and excess waste liquid after staining can also be pumped into the waste liquid tank 12.

[0129] The double-sided adhesive tape-based microfluidic chip 1 in the embodiment is simple to manufacture, low in cost, and reliable in performance. In the embodiment, an automatic pipettor and a reagent bin 7 of a pipetting machine are used to replace a complex multi-pipe, a rotary valve, and a pressure pump 10 required for delivery of reagents to the microfluidic chip 1 in the traditional way, thereby reducing the complexity of delivery of reagents to the microfluidic staining cavity 9 and greatly expanding the types of staining reagents. In the embodiment, one pipe is connected to the microfluidic outlet 8 and the pressure pump 10 to complete the liquid transfer control of the system, which is simple and reliable.

[0130] Examples of staining performed by the microfluidic chip tissue staining device and method are as follows:

[0131] Example 1: Rapid immunohistochemical staining (CK staining)

[0132] 1. After the tissue section on the slide is subjected to baking, deparaffinization, hydration, and antigen repair (the slide is referred to as a sample slide), the sample slide is assembled with the double-sided adhesive tape with the microfluidic channel dug out and the slide with a hole connected to a thin pipe for reagent inflow and outflow, that is, a connecting slide, to form a sandwich-like microfluidic structure, in which the sample is in the center of the microfluidic structure. In this way, the assembly of the microfluidic chip system containing the tissue to be stained is completed. The immunohistochemical staining is performed in the microfluidic chip.

[0133] 2. The staining control software sets relevant information according to the pipette tip box station, reagent station, and staining box station, and the staining program automatically obtains the pipette tip from the corresponding station set in the program, sucks a set volume of reagent from the reagent container of the corresponding reagent station, and delivers it to the corresponding staining microfluidic reagent bin. The following staining process is automatically executed according to the staining program setting by the automatic staining machine of the application. The following volumes are the volumes of reagents delivered to each staining microfluidic reagent bin:

[0134] Quenching endogenous peroxidase activity: 45 μl of 0.6% hydrogen peroxide (H2O2) solution, control stepper motor speed 6 μl / s, do suction-pushing cycle incubation, change liquid direction every 5 s, a total of 4 cycles for 20 s;

[0135] PBS washing: (100 μl, one-way suction, speed 20 μl / s, 5 s) x 2 times;

[0136] Blocking: 45 μl of blocking solution, change liquid direction every 5 s, a total of 2 cycles for 20 s;

[0137] Primary antibody incubation: 45 μl - speed 6 μl / s, do suction-pushing cycle incubation, change liquid direction every 5 s, a total of 20 cycles for 200 s;

[0138] PBS-T washing: (100 μl, one-way suction, speed 20 μl / s) x 2 times;

[0139] Secondary antibody-HRP incubation: 45 μΐ speed 6 μΐ / s, pull-push cycle incubation, change liquid direction every 5 s, 20 cycles for 200 s;

[0140] PBS-T wash: (100 μΐ, single direction suction, speed 20 μΐ / s) x 2 times;

[0141] DAB staining: DAB staining solution 45 μΐ - change liquid direction every 5 s, 9 cycles for 90 s;

[0142] H2O or PBS wash, -100 μΐ - single direction suction, speed 20 μΐ / s.

[0143] 3. Total staining time: ~10 min

[0144] The rapid immunohistochemical CK staining picture is shown in Figure 3 .

[0145] Example 2: Multiplexed immunofluorescence staining based on tyramide signal amplification (TSA)

[0146] 1. Sample processing and microfluidic assembly are the same as Example 1, and the washing steps are the same as Example 1.

[0147] 2. This staining procedure sequentially performs multiplexed immunofluorescence staining based on TSA technology, and detects ki67 antigen, Treg cell regulatory factor FoxP3, CD3 antigen, and cytokeratin (Cytokeratin-CK) on the same slice. The first three markers use TSA method, and CK uses conventional indirect immunofluorescence method, that is, Cy7 fluorescently labeled secondary antibody is used instead of HRP labeled secondary antibody in TSA method after using anti-CK primary antibody.

[0148] 3. The following processes are automatically executed by the automatic staining machine of the present application according to the staining program setting, and the following volumes are the volumes of reagents delivered to each staining microfluidic reagent tank:

[0149]

[0150] The staining markers are FoxP3, CK, CD8, PD-1, and FoxP3+CK+CD8+PD-1 fusion based on tyramide signal amplification (TSA) and indirect immunofluorescence technology. The multiplexed immunofluorescence staining pictures are shown in Figures 4-8 .

[0151] In conclusion, the microfluidic chip tissue staining device and method of the embodiment can greatly shorten the staining time and greatly improve the experimental efficiency; and the pressure pump system accurately controls the flow rate and pressure, ensuring the consistency of staining between different experimental samples.

[0152] All documents mentioned in the present application are incorporated herein by reference as if each individual document were specifically and individually incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding benefits depending on the precise placement of the elements, to those skilled in the art, in light of the above teaching, many modifications and changes can be made in the implementation without departing from the scope of the application, which is defined by the scope of the appended claims.

Claims

1. A microfluidic chip, characterized in that, The microfluidic chip includes: An upper glass slide, wherein the upper glass slide is provided with an inlet hole and an outlet hole; A double-sided adhesive layer, wherein a portion of the double-sided adhesive is removed by cutting to reveal the corresponding inflow channel, staining chamber, and outflow channel of the microfluidic chip, wherein the inflow channel is connected to the inlet port, and the outflow channel is connected to the outlet port; and A lower glass slide, on which the tissue to be stained is placed; The upper glass slide and the lower glass slide are bonded together as a whole by the double-sided adhesive layer.

2. The microfluidic chip as described in claim 1, characterized in that, The inlet port is used to connect to the reagent chamber; the outlet port is used to connect to the pressure pump.

3. The microfluidic chip as described in claim 1, characterized in that, The microfluidic chip does not contain valves.

4. The microfluidic chip as described in claim 1, characterized in that, The double-sided adhesive layer is selected from Advanced Research or 3M.

5. The microfluidic chip as described in claim 1, characterized in that, The thickness of the double-sided adhesive layer is in the range of 10-1000um.

6. The microfluidic chip as described in claim 1, characterized in that, In the unassembled state, the double-sided adhesive layer is isolated from the outside world on both sides by release paper.

7. A microfluidic chip tissue staining device, characterized in that, The device includes: A reagent station, which is used to hold various reagents; The microfluidic chip as described in any one of claims 1-6, wherein the inlet of the microfluidic chip is provided with a reagent compartment, the reagent compartment being an open design, and a pressure pump is connected to the outlet of the microfluidic chip; the staining chamber, the reagent compartment, and the pressure pump are in fluid communication; and An automated pipetting system is used to draw the reagent from the reagent station and move it above the reagent compartment for discharge; The reagent in the reagent chamber is drawn into the staining chamber of the microfluidic chip by the pressure pump for staining, and then discharged after staining.

8. The apparatus as claimed in claim 7, characterized in that, The pressure pump controls the reciprocating motion of the reagent in the staining chamber to accelerate staining.

9. A microfluidic chip tissue staining method, characterized in that, The method includes: (1) A microfluidic chip tissue staining apparatus as described in claim 8 is provided; (2) Add the reagent to the reagent compartment: Add the staining reagent to the reagent compartment using the automatic pipetting system in the order of the staining process; (3) Aspiration of the reagent: The reagent in the reagent chamber is aspirated into the microfluidic chip by the pressure pump; (4) Staining process: By controlling the flow rate or pressure and direction of the pressure pump, and by controlling the frequency to change the flow direction of the reagent in the microfluidic chip, the liquid exchange between the reagent liquid phase and the solid phase of the stained tissue is accelerated, thereby accelerating the staining reaction; and (5) Discharge of reagents: The reagents in the microfluidic chip are discharged by the pressure pump.

10. The method as described in claim 9, characterized in that, Repeat the inhalation and exhalation of reagents to ensure adequate and consistent staining.

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