Integrated microfluidic cancer instant detection device and use method

Abstract

The invention relates to a medical detection device, in particular to an integrated microfluidic cancer instant detection device and a use method. The invention relates to an integrated micro-fluidic cancer instant detection device and a use method thereof, and aims to provide the integrated micro-fluidic cancer instant detection device and the use method thereof, so as to solve the problems of high cost, time consumption and invasiveness of the traditional cancer detection method at the present stage, and can quickly detect multiple indexes at low cost and realize early cancer screening while being used for home self-detection. According to the technical scheme, the integrated microfluidic cancer instant detection device is characterized by comprising a separation layer, a reaction layer, a detection layer and a waste liquid layer which are sequentially communicated from top to bottom, the separation layer is provided with an IPMC driver serving as a micro pump and a plasma separation spiral channel for a blood sample; the sample reaction area of the reaction layer is coated with an antigen or a first antibody capable of reacting with a blood sample; the waste liquid collection area of the waste liquid layer is used for temporarily storing all to-be-treated waste liquid; the blood sample may be a whole blood sample, a serum sample, or a plasma sample.

Description

Technical Field

[0001] The present invention relates to a medical detection device, in particular to an integrated microfluidic cancer point-of-care testing device and its usage method. Background Art

[0002] There are many deficiencies in traditional cancer detection methods; for example, imaging examinations and pathological examinations among them have defects such as invasiveness, radiation risk, long detection cycles, and high costs; and the content of cancer cells in the early stage of cancer is relatively low, and the detection sensitivity of traditional tumor markers is limited, which affects the detection effect. In addition, traditional tumor marker detection methods require a large amount of long-term processing of samples step by step, with complex and cumbersome operations and low efficiency.

[0003] Microfluidics refers to the science and technology involved in systems that use microchannels to process or manipulate tiny fluids, and it has currently been widely applied in detection fields such as biology and medicine; its development advantages are reflected in: extremely low sample requirements, only requiring a small amount of sample (such as a few microliters of liquid or body fluid) to complete the detection, especially suitable for early cancer screening; enabling rapid detection and point-of-care diagnosis, integrating sample pretreatment, reaction, and detection on a single chip, greatly shortening the detection time; having the characteristics of high throughput and automation, through multi-channel parallel processing, a large number of samples can be analyzed at one time and the automated process can reduce human interference, etc., being trace, fast, highly sensitive, and low-cost.

[0004] Exosomes are nanoscale vesicles secreted by cells, which are rich in various biofunctional molecules such as lipids, proteins, and nucleic acids. A large number of research results show that various cancers can be diagnosed using protein or nucleic acid markers in exosomes, such as breast cancer, ovarian cancer, prostate cancer, colorectal cancer, etc., providing a theoretical basis for the application of exosome biopsy in cancer diagnosis. Summary of the Invention

[0005] The present invention provides an integrated microfluidic cancer point-of-care testing device and its usage method to solve the problems of the current traditional cancer detection methods being expensive, time-consuming, and invasive, and while being available for home self-testing, it can detect multiple indicators at low cost and quickly, and achieve early cancer screening.

[0006] The technical solution provided by the present invention is:

[0007] Integrated microfluidic cancer point-of-care testing device, characterized in that: the point-of-care testing device includes a separation layer, a reaction layer, a detection layer, and a waste liquid layer that are sequentially connected and communicated from top to bottom; an IPMC driver serving as a micropump is provided on the separation layer, and a spiral channel for plasma separation of blood samples is provided; an antigen or a first antibody capable of reacting with a blood sample is coated in the sample reaction area of the reaction layer; an antigen or a second antibody labeled with signal particles is coated in the sample reaction area of the detection layer, and a corresponding capture antigen or capture antibody is preset in the sample detection area of the detection layer; the waste liquid collection area of the waste liquid layer is used for temporarily storing all waste liquid to be processed;

[0008] The blood sample can be a whole blood sample, a serum sample, or a plasma sample.

[0009] The separation layer, the reaction layer, the detection layer, the waste liquid layer, and the bottom plate are detachably connected into one body from top to bottom; the spiral channel, the sample reaction area of the reaction layer, the sample reaction area and the sample detection area of the detection layer, and the waste liquid collection area of the waste liquid layer are all cavities provided on the bottom surface of the plate and the openings penetrate the bottom surface, and the bottom plate located at the bottommost layer is used to close the cavity opening of the waste liquid layer.

[0010] The separation layer is provided with a sample input port, a micropump cavity installed with a micropump, and a spiral channel that are sequentially connected and communicated. The sample input port and the micropump cavity both penetrate the upper surface of the separation layer plate; the spiral channel is a spiral structure, the spiral center of the spiral channel is connected to the lower end of the micropump cavity, and the waste liquid outlet at the outermost end of the spiral channel is connected to the waste liquid collection area through a downward waste liquid channel; the separation layer sample outlet at the second outermost end of the spiral channel is connected to the reaction layer.

[0011] The reaction layer sample inlet channel of the reaction layer is connected to the separation layer sample outlet at the end of the spiral channel, and is connected to a plurality of microchannels with the same flow rate through a one-to-many channel. Each microchannel is respectively connected to its own first sample reaction area and then connected to the detection layer through the reaction layer sample outlet; a number of turbulence columns are regularly arranged inside the cavity to enable the sample to react fully.

[0012] The detection layer includes a number of detection layer sample inlet channels that are respectively connected to the sample outlets of a number of the first sample reaction areas. Each detection layer sample inlet is sequentially connected to a second sample reaction area and a sample detection area, and then connected to the waste liquid layer inlet channel through the detection layer outlet; among them, the structure of the second sample reaction area is the same as that of the first sample reaction area; the sample detection area receives samples with different labeling effects from the second sample reaction area to complete fluorescence detection.

[0013] The waste liquid layer is provided with a waste liquid collection area and a plurality of waste liquid layer inlet channels connecting the waste liquid collection area and the liquid outlets of a plurality of detection layers, and an air outlet is also provided.

[0014] The sample input port has a tapered structure with a larger upper part and a smaller lower part to accelerate the flow rate of the sample; the micro pump cavity has a tapered shape with a larger upper part and a smaller lower part, and the side is connected to the sample input port, and the lower end is connected to the spiral channel;

[0015] Check valves are respectively arranged at the connection parts of the micro pump cavity with the sample input area and the spiral channel to prevent the backflow of the fluid sample.

[0016] The antigen or the first antibody can specifically react with a specific antibody or antigen in the sample, thereby forming an antigen-antibody complex.

[0017] The antibody is an antibody against a protein marker of cancer cell exosomes, including EDIL3 antibody (breast cancer), Survivin antibody (prostate cancer), MAGE3 / 6 antibody (ovarian cancer), CD63 antibody (melanoma), CD151 antibody (non-small cell lung cancer), GPC1 antibody (pancreatic cancer), CD147 antibody (colorectal cancer).

[0018] The antigen or the second antibody can specifically react with the antigen-antibody complex, thereby forming an antigen-antibody-secondary antibody-fluorescent microsphere complex;

[0019] The signal particles include at least one of organic fluorescent dyes (such as FITC, PE, TRITC, etc.), synthetic fluorescent dyes (such as AlexaFluor, DyLight, etc.), inorganic nanomaterials (QDs, UCNPs, etc.), time-resolved fluorescent markers (such as lanthanide chelates, etc.), near-infrared fluorescent dyes (such as IRDye 800CW, Cy5.5, etc.), metal nanoparticles (AuNPs, AgNPs, etc.), enzyme-linked fluorescence amplification systems (such as HRP, AP, etc.).

[0020] The usage method of the integrated microfluidic cancer point-of-care testing device includes the following steps:

[0021] Step 1: Drop the sample to be detected from the sample input port;

[0022] Step 2: Apply an alternating current with a suitable voltage and frequency to the IPMC driver, so as to drive the sample to flow into the separation area through the driver, thereby centrifugally separating the plasma sample without large-volume cells, and the separated waste liquid containing large cells is directly discharged to the waste liquid layer cavity of the detection layer;

[0023] Step 3: The plasma sample continues to flow into the reaction layer and then undergoes a shunting process, reaching the first sample reaction areas that connect to each microchannel respectively. The exosome protein molecule detection markers secreted by specific cancer cells in the plasma sample specifically react and bind with the coated antigen or the first antibody in the first sample processing area, forming an antigen-antibody complex.

[0024] Step 4: The antigen-antibody complex obtained in Step 3 continues to be transported to the second sample reaction area of the detection layer, and specifically binds again with the antigen coated in the second sample reaction area or the second antibody labeled with signal particles, forming an antigen-antibody-secondary antibody-fluorescent microsphere complex.

[0025] Step 5: The complex sample labeled with signal particles obtained in Step 4 continues to be transported to the sample detection area and is specifically captured by the corresponding preset capture antigen or capture antibody during the flow in the detection area cavity.

[0026] Step 6: The remaining sample continues to flow until it is collected by the waste liquid layer.

[0027] Step 7: After all reactions are completed, remove the fixing bolts to separate each layer of the detection device, take out the detection layer plate and insert it into the fluorescence analysis immunoassay instrument for detection; the antigen-antibody-secondary antibody-fluorescent microsphere complex in the sample detection area will generate a fluorescence signal after being excited by a light source with a specific wavelength from the outside, and provide the fluorescence immunoassay analyzer for detecting the fluorescence signal intensity. Thus, the concentration of the corresponding cancer cell exosomes in the sample is automatically converted with a preset calibration curve and compared with the normal value, so as to judge whether the sample provider has cancer.

[0028] The detection is completed.

[0029] The beneficial effects of the present invention are as follows: The present invention utilizes the advantages of microfluidics, namely microscale, rapidity, high sensitivity, and low cost, and applies them to the field of preliminary cancer detection. By dropping a small amount of blood sample into the sample input hole and using the IPMC driver as a micro pump to pump the sample to flow, the plasma without large-volume cells such as blood cells is efficiently separated based on the principle of inertial focusing at high throughput. The plasma sample continues to flow to the sample reaction area and binds to the corresponding specific antigen or antibody, and the concentration of exosomes secreted by cancer cells is measured through fluorescence immunoassay. Based on this, it is preliminarily determined whether the provider has the corresponding cancer. In this way, pumping the sample to flow is no longer limited by the large-volume pumping device. Only by providing alternating current with a certain voltage and frequency can the entire process of work be completed. The whole process is automated, without the need for manual intervention, reducing human interference and achieving convenient, efficient, and accurate detection functions. At the same time, the present invention aims to detect the concentration of exosomes secreted by cancer cells, which has a more obvious effect compared to directly detecting the concentration of cancer cells (the initial content is extremely small and difficult to detect). At the same time, the present invention has a detachable five-layer structure, which not only facilitates the preset of drugs during device production or before experimental detection, but also facilitates the subsequent cleaning and reuse of the experiment, and is more conducive to replacement or upgrade in the subsequent development process. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a three-dimensional structural schematic diagram of an embodiment of the present invention.

[0031] Figure 2 is an exploded schematic diagram of the separation layer in an embodiment of the present invention.

[0032] Figure 3 is a three-dimensional structural schematic diagram (bottom surface structure diagram) of the separation layer in an embodiment of the present invention.

[0033] Figure 4 is a three-dimensional structural schematic diagram (bottom surface structure diagram) of the reaction layer in an embodiment of the present invention.

[0034] Figure 5 is a three-dimensional structural schematic diagram (bottom surface structure diagram) of the detection layer in an embodiment of the present invention.

[0035] Figure 6 [[ID=Z6]]is a three-dimensional structural schematic diagram (bottom surface structure diagram) of the waste liquid layer in an embodiment of the present invention.

[0036] Figure 7 is a three-dimensional structural schematic diagram (bottom surface structure diagram) of the bottom plate in an embodiment of the present invention.

[0037] Reference numerals in the figure: 1-1, separation layer; 1-2, reaction layer; 1-3, detection layer; 1-4, waste liquid layer; 1-5, bottom plate; 1-6, screw; 1-7, through hole; 2-1, pressing plate; 2-2, micro pump; 2-3, sample input port; 2-4, micro pump cavity; 2-5, screw hole; 3-1, spiral channel; 3-2, spiral channel liquid inlet; 3-3, separation layer sample liquid outlet; 3-4, separation layer waste liquid outlet; 4-1, reaction layer sample liquid outlet; 4-2, first sample reaction area; 4-3, turbulence column; 4-4, sample shunt channel; 4-5, reaction layer sample liquid inlet channel; 4-6, reaction layer waste liquid channel; 5-1, detection layer sample liquid inlet channel; 5-2, second sample reaction area; 5-3, sample detection area; 5-4, detection layer liquid outlet; 5-5, detection layer waste liquid channel; 6-1, air outlet; 6-2, waste liquid collection area; 6-3, waste liquid layer liquid inlet channel. Specific implementation mode

[0038] The following further describes the present invention in combination with the embodiments shown in the drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0039] The idea of the present invention is as follows: Aiming at the invasiveness, radiation risk, long detection cycle, high cost, etc. of imaging examination and pathological examination methods in traditional cancer detection methods, tumor marker detection can be realized through a microfluidic chip. And aiming at the low content of cancer cells in the early stage of cancer and the limited sensitivity of traditional tumor marker detection, the content of exosomes secreted by cancer cells is detected instead, and the relatively higher content greatly improves the sensitivity of marker detection. Then, aiming at the traditional tumor marker detection method, a large amount of long-term processing of samples is required step by step, the operation is cumbersome and the difficulty is great. For this reason, microfluidic channels such as a separation area, a reaction area, and a detection area are designed to realize faster and more efficient sample processing and detection, reduce the operation difficulty, reduce human interference, and at the same time use an electroactive soft actuator as a micro pump to provide power, no longer requiring other large pumping devices, greatly improving the portability of detection.

[0040] According to the above idea, the technical solution of the present invention is formed.

[0041] Figure 1 The shown integrated microfluidic cancer point-of-care testing device includes four plates respectively made with a separation layer 1-1, a reaction layer 1-2, a detection layer 1-3, and a waste liquid layer 1-4; by Figures 2 - 7It can be known that: for the convenience of manufacturing and use, the cavities required for the separation layer, reaction layer, detection layer, and waste liquid layer all open at the bottom surface of the plate. The opening of each layer is closed by the upper surface of the adjacent lower layer. The bottom plate 1-5 located at the bottommost layer is used to close the waste liquid layer. The liquid input channels of each cavity penetrate upward through the plate and are connected to the adjacent plate. The plates respectively made with the separation layer, reaction layer, detection layer, and waste liquid layer and the bottom plate are placed in sequence from top to bottom, and are connected into one body by inserting four bolts at the through holes 1-7 of the five plates.

[0042] Figure 2 , Figure 3 In the structure of the separation layer plate (the plate made with the separation layer) shown in Figure 3 : a micro-pump cavity 2-4 for installing the micro-pump 2-2 is made on the upper surface; the IPMC driver (electroactive soft driver) serving as the micro-pump is connected and fixed to the separation layer plate through the pressure plate 2-1 and the screw 1-6 (the screw hole 2-5 matches the screw 1-6), and seals the micro-pump cavity. The micro-pump adopts a microfluidic pump panel based on the IPMC driver (see CN202211172398.8); the required power supply for the IPMC driver is introduced from the outside.

[0043] Among them, the contour of the pressure plate 2-1 fits the contour of the IPMC driver to ensure the installation reliability of the IPMC driver; the micro-pump cavity is in a conical shape with a larger upper part and a smaller lower part, so that the cavity can hold a sufficient amount of liquid, and at the same time relatively increases the pumping pressure of the micro-pump during operation. A sample input port 2-3 (blind hole) is opened beside the micro-pump cavity. The sample input port is in a conical structure with a larger upper part and a smaller lower part, so as to provide a certain space as a temporary storage area for the sample, and increase the pressure of the fluid sample entering the channel to achieve an accelerated flow rate of the sample; the bottom end of the sample input port communicates with the side surface of the micro-pump cavity, and the bottom end of the micro-pump cavity communicates with the spiral channel on the bottom surface; check valves are respectively installed at the two communicating places to prevent liquid backflow.

[0044] Figure 3 The spiral channel shown in Figure 3 utilizes the principle of inertial focusing to achieve different sample separation effects by providing centrifugal forces with different flow rates, and can effectively achieve the plasma separation of blood samples, removing large-volume cells such as blood cells and retaining small-volume vesicles such as exosomes in the sample. Two shunt channels are led out from the outside of the spiral channel; the separated plasma sample flows to the separation layer sample outlet 3-3 on the inner side of the spiral channel and then flows to the reaction layer; the waste liquid of large cells such as separated blood cells is transported to the separation layer waste liquid outlet 3-4 at the shunt channel on the outside of the spiral channel, and then sequentially passes through the reaction layer waste liquid channel 4-6 and the detection layer waste liquid channel 5-5 and converges to the waste liquid layer.

[0045] The sample can be a whole blood sample, a serum sample, or a plasma sample.

[0046] Figure 4 In the structure of the reaction layer plate (the plate made with the reaction layer) shown: A reaction layer sample inlet channel 4-5 connecting to the separation layer sample outlet is provided at the starting section of the reaction layer. This channel extends towards the other end of the microfluidic panel and communicates with four microchannels for splitting (i.e., a one-to-four channel), and each microchannel communicates with a sample reaction area; such that the passing sample fluid has the same flow rate in different channels and respectively leads to their respective sample reaction areas; the said sample reaction area is called the first sample reaction area (shown as a hexagonal cavity in the figure; the cavity structure shape can also be selected according to needs). Specific antigens or specific antibodies are coated inside the cavity, and a number of flow disturbance columns are arranged in a certain rule, such that the sample can achieve sufficient reaction in this area. The end of the reaction area refocuses on the microchannel and leads to the detection layer.

[0047] The flow disturbance columns are preferably micro-sized vertical cylinders. A number of flow disturbance columns are regularly arranged in the cavity of the sample reaction area, which can appropriately hinder the forward flow velocity of the sample fluid, thereby disturbing the laminar flow phenomenon of the fluid, increasing the degree of fluid chaos in this area, realizing a function similar to stirring, and thus promoting the specific reaction between the sample and the preset marker antibody.

[0048] The sample fluid flows forward after flowing from the separation layer to the reaction layer sample inlet channel 4-5. After passing through the four microchannels, the liquid is evenly divided into four parts, and the flow velocity of each channel is the same (the one-to-multiple channel design enables the present invention to achieve the function of measuring multiple indicators at one time, greatly improving the detection efficiency); subsequently, the sample fluids in each channel respectively enter the first sample reaction area 4-2; the first sample reaction area is coated with an antigen or an antibody, and the antigen or antibody can specifically react with the specific cancer cell exosome surface marker protein in the sample, thereby forming an antigen-antibody complex (primary antibody); at the same time, a number of regularly arranged flow disturbance columns are provided inside the first sample reaction area to disturb the fluid, such that the target exosomes in the sample can achieve sufficient reaction in this area, and then respectively converge to the reaction layer sample outlet 4-1, and flow to the detection layer through the detection layer sample inlet channel 5-1.

[0049] Figure 5In the structure of the detection layer plate (the plate with the detection layer): Samples enter the second sample reaction area 5-2 and the sample detection area 5-3 one by one from the four detection layer sample inlet channels 5-1; The structure of the second sample reaction area is the same as that of the first sample reaction area. The second sample reaction area is coated with a specific antigen or a specific antibody (secondary antibody) labeled with signal particles. The labeled antigen or antibody can continue to specifically react with the primary antibody in the sample, so as to form an antigen-antibody-secondary antibody-fluorescent microsphere complex and then continue to flow forward to the sample detection area 5-3; Each detection channel in the sample detection area is preset with a corresponding capture antigen or capture antibody. The capture antigen or capture antibody can capture and fix the antigen-antibody-secondary antibody-fluorescent microsphere complex formed in the second sample reaction area, while the unlabeled small vesicles will flow to the detection layer outlet 5-4 and flow to the waste liquid layer; The large cell waste liquid in the reaction layer also flows to the waste liquid layer after passing through the waste liquid outlet 5-4 of the detection layer.

[0050] Figure 5 In the structure of the waste liquid layer plate (the plate with the waste liquid layer): All the waste liquid flowing out from the detection layer will flow to the waste liquid collection area 6-2 for storage through the respective waste liquid layer inlet channels 6-3. The waste liquid collection area has a relatively large space and can retain a large amount of liquid, thus avoiding the pollution of the reaction waste liquid to the environment. An air outlet 6-1 is provided in the waste liquid layer to form a closed loop of external environment-internal channel-external environment to ensure the smooth flow of liquid in the channel.

[0051] The lower bottom plate covers the opening of the waste liquid layer. The bottom plate is a fully solid and non-channel pressing plate, which is only used for the fixation and sealing of the bottom layer.

[0052] The point-of-care testing device provided by the present invention, wherein the horizontal microchannel areas of each layer are all open semi-channel structures, and are sealed by overlapping and pressing with the upper plane of the lower layer, which is more convenient for the preset work and subsequent cleaning of the device, can be recycled, and is more environmentally friendly. Through holes are respectively opened at the four ends of the five-layer structure, and fixation and sealing can be achieved through bolt connection.

[0053] In the present invention, check valves are respectively arranged in the channels communicating between the separation layer, the reaction layer, the detection layer and the waste liquid layer to prevent the liquid from flowing back during the flow process.

[0054] The separation layer, the reaction layer, the detection layer and the waste liquid layer are preferably made of insulating transparent materials and can be directly prepared by 3D printing technology; They can be freely selected according to user needs, such as using transparent photosensitive resin for 3D printing to facilitate observing the flow of liquid in the microfluidic chip channel.

[0055] The using method of the integrated microfluidic cancer point-of-care testing device includes the following steps:

[0056] Step 1: Drop the sample to be detected from the sample input port;

[0057] Step 2: After dropping the sample in Step 1, apply alternating current with a set voltage and frequency to the IPMC driver, so that the deformation generated by the driver causes a change in the volume inside the cavity of the micro-pump, and then the sample is pushed to flow through the pressure difference and enters the separation zone, where plasma samples without large-volume cells such as blood cells are centrifugally separated. The waste liquid containing large cells such as blood cells directly flows to the waste liquid layer cavity of the detection layer;

[0058] Step 3: As the plasma sample continues to flow, it undergoes one-to-many channel shunting in the reaction layer and reaches the first sample reaction zone connected to each microchannel respectively; the first sample processing area is coated with an antigen or a first antibody (a specific antibody of a specific cancer protein marker), which can specifically bind to the exosome protein molecule detection marker secreted by specific cancer cells in the sample to form an antigen-antibody complex;

[0059] Step 4: The antigen-antibody complex obtained in Step 3 will continue to flow to the second sample reaction zone. The sample reaction zone is coated with an antigen or a second antibody (secondary antibody) labeled with signal particles, and specifically binds to the antigen-antibody complex generated by the reaction in the first sample reaction zone of each channel again to form an antigen-antibody-secondary antibody-fluorescent microsphere complex;

[0060] Step 5: The fluorescently labeled complex sample obtained in Step 4 will continue to be transported to the sample detection area and is specifically captured by the corresponding preset capture antigen or capture antibody during the flow in the detection area cavity;

[0061] Step 6: The remaining sample continues to flow until it is collected by the waste liquid layer;

[0062] Step 7: After all reactions are completed, remove the fixing bolts to separate each layer of the detection device, take out the detection layer plate and insert it into the fluorescence analysis immunoassay instrument. The antigen-antibody-secondary antibody-fluorescent microsphere complex fixed in the sample detection area will generate a fluorescence signal after being excited by a light source with a specific wavelength from the outside, and provide the fluorescence immunoassay analyzer for detecting the fluorescence signal intensity. From this, the concentration of the corresponding cancer cell exosomes in the sample is automatically converted with a preset calibration curve and compared with the normal value, so as to judge whether the sample provider has cancer;

[0063] The above embodiments are only used to describe the technical solutions of the present invention. It should be noted that those of ordinary skill in the art can make certain improvements and deformations according to their own understandings without departing from the principles of the technical solutions of the present invention. The purpose of the above-provided embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

Claims

1. Integrated microfluidic cancer point-of-care testing device, characterized in that: The instant detection device includes a separation layer (1-1), a reaction layer (1-2), a detection layer (1-3), and a waste liquid layer (1-4) that are connected in sequence from top to bottom; an IPMC driver serving as a micro pump (2-2) is provided on the separation layer, and a spiral channel (3-1) for plasma separation of a blood sample is provided; antigens or first antibodies capable of reacting with the blood sample are coated in the sample reaction area of the reaction layer; antigens or second antibodies labeled with signal particles are coated in the sample reaction area of the detection layer, and corresponding capture antigens or capture antibodies are preset in the sample detection area of the detection layer; the waste liquid collection area of the waste liquid layer is used for temporarily storing all waste liquid to be processed. The blood sample can be a whole blood sample, a serum sample, or a plasma sample.

2. The integrated microfluidic cancer point-of-care testing device according to claim 1, wherein: The separation layer, the reaction layer, the detection layer, the waste liquid layer, and the bottom plate (1-5) are detachably connected as a whole from top to bottom; the spiral channel, the sample reaction area of the reaction layer, the sample reaction area and the sample detection area of the detection layer, and the waste liquid collection area of the waste liquid layer are all cavities provided on the bottom surface of the plate and the openings penetrate the bottom surface, and the bottom plate located at the bottommost layer is used to close the cavity opening of the waste liquid layer.

3. The integrated microfluidic cancer point-of-care testing device according to claim 2, wherein: The separation layer is provided with a sample input port (2-3), a micro pump cavity (2-4) installed with a micro pump, and the spiral channel that are connected in sequence. The sample input port and the micro pump cavity penetrate the upper surface of the separation layer plate; the spiral channel is a spiral structure, the spiral center of the spiral channel communicates with the lower end of the micro pump cavity, and the separation layer waste liquid outlet (3-4) at the outermost end of the spiral channel communicates with the waste liquid collection area through a downward waste liquid channel; the separation layer sample outlet (3-3) at the second outermost end of the spiral channel communicates with the reaction layer.

4. The integrated microfluidic cancer point-of-care testing device according to claim 3, characterized in that: The reaction layer sample inlet channel (4-5) of the reaction layer communicates with the separation layer sample outlet at the end of the spiral channel, and is connected to a plurality of micro channels with the same flow rate through a one-to-many channel. Each micro channel is respectively connected to its own first sample reaction area (4-2) and then respectively communicates with the detection layer through the reaction layer sample outlet (4-1); a number of turbulence columns (4-3) arranged regularly are provided inside the cavity so that the sample can react sufficiently.

5. The integrated microfluidic cancer point-of-care testing device according to claim 4, wherein: The detection layer includes a number of detection layer sample inlet channels (5-1) that are respectively connected to the sample outlets of a number of the first sample reaction areas. Each detection layer sample inlet is then sequentially connected to a second sample reaction area (5-2) and a sample detection area, and then is connected to the waste liquid layer inlet channel (6-3) through the detection layer outlet (5-4); among them, the structure of the second sample reaction area is the same as that of the first sample reaction area; the sample detection area receives samples with different labeling effects from the second sample reaction area to complete fluorescence detection.

6. The integrated microfluidic cancer point-of-care testing device according to claim 5, wherein: The waste liquid layer is provided with a waste liquid collection area (6-2) and a number of waste liquid layer inlet channels (6-3) that connect the waste liquid collection area and a number of detection layer outlets, and an air outlet (6-1) is also provided.

7. The integrated microfluidic cancer point-of-care testing device according to claim 6, characterized in that: The sample input port has a tapered structure with a larger upper part and a smaller lower part to accelerate the flow rate of the sample. The cavity of the micro pump is in a conical shape with a larger upper part and a smaller lower part, and the side is connected to the sample input port, and the lower end is connected to the spiral channel; check valves are respectively arranged at the connection parts of the micro pump cavity with the sample input area and the spiral channel to prevent the reverse flow of the fluid sample.

8. The integrated microfluidic cancer point-of-care testing device according to claim 7, wherein: The antigen or the first antibody can specifically react with the surface marker protein of cancer cell exosomes in the sample, thereby forming an antigen-antibody complex; The antibody is an antibody for cancer cell exosome protein markers, including EDIL3 antibody (breast cancer), Survivin antibody (prostate cancer), MAGE3 / 6 antibody (ovarian cancer), CD63 antibody (melanoma), CD151 antibody (non-small cell lung cancer), GPC1 antibody (pancreatic cancer), CD147 antibody (colorectal cancer).

9. The integrated microfluidic cancer point-of-care testing device according to claim 8, wherein: The antigen or the second antibody can specifically react with the antigen-antibody complex, thereby forming an antigen-antibody-secondary antibody-fluorescent microsphere complex; The signal particles include at least one of organic fluorescent dyes, synthetic fluorescent dyes, inorganic nanomaterials, time-resolved fluorescent markers, near-infrared fluorescent dyes, metal nanoparticles, and enzyme-linked fluorescent amplification systems.

10. A method for using the integrated microfluidic cancer point-of-care testing device according to claim 1, comprising the following steps: Step 1: Drop the sample to be tested from the sample input port; Step 2: Apply an alternating current with a suitable voltage and frequency to the IPMC driver, thereby driving the sample to flow into the separation area through the driver, centrifugally separating the plasma sample without large-volume cells, and directly discharging the waste liquid containing large cells separated to the waste liquid layer cavity of the detection layer; Step 3: The plasma sample continues to flow into the reaction layer and after being shunted, reaches the first sample reaction area communicating with each microchannel respectively. The exosome protein molecule detection marker secreted by specific cancer cells in the plasma sample specifically reacts and binds with the antigen or the first antibody coated in the first sample treatment area, forming an antigen-antibody complex; Step 4: The antigen-antibody complex obtained through step 3 continues to be transported to the second sample reaction area of the detection layer, and specifically binds again with the antigen or the second antibody labeled with signal particles coated in the second sample reaction area, forming an antigen-antibody-secondary antibody-fluorescent microsphere complex; Step 5: The complex sample labeled with signal particles obtained in step 4 continues to be transported to the sample detection area and is specifically captured by the corresponding preset capture antigen or capture antibody during the flow in the detection area cavity; Step 6: The remaining sample continues to flow until it is collected by the waste liquid layer; Step 7: After all reactions are completed, remove the fixing bolts to separate each layer of the detection device, take out the detection layer plate and insert it into the fluorescence analysis immunoassay instrument for detection; the antigen-antibody-secondary antibody-fluorescent microsphere complex in the sample detection area will generate a fluorescence signal after being excited by a light source with a specific wavelength from the outside, and provide the fluorescence immunoassay analyzer for detecting the fluorescence signal intensity. From this, the concentration of the corresponding cancer cell exosomes in the sample is automatically converted with a preset calibration curve and compared with the normal value, thereby determining whether the sample provider has cancer; Detection completed.

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