Tumor cell detection device with easy reading, microfluidic chip and preparation method thereof

Abstract

The application discloses a tumor cell detection device easy to read, a micro-fluidic chip and a preparation method thereof, wherein the micro-fluidic chip comprises a sample inlet unit, a detection unit, a power supply unit and a waste liquid recovery unit. The power supply unit comprises a plurality of capillary tubes connected with the detection unit and providing power for liquid flow. The tumor cell detection device easy to read provided by the application comprises a box body, a result reading window is arranged on the box body, at least one detection unit is arranged in the box body, and the detection unit is used for placing the micro-fluidic chip. A smart phone is used for color analysis by being placed on the reading window. The application does not need external pumps and control equipment to control liquid flow, the detection result is convenient to read, the portability is good, the requirements for equipment and experience are low, and the application is conducive to popularization.

Description

Easily readable tumor cell detection device, microfluidic chip and its preparation method Technical Field

[0001] This application relates to the field of tumor cell detection technology, and in particular to easily readable tumor cell detection devices, microfluidic chips, and their preparation methods. Background Technology

[0002] Current tumor diagnostic methods largely rely on expensive, large-scale equipment. Their cumbersome operation and the need for specialized technicians limit their practical applicability in point-of-care (POC) diagnostics, especially in resource-scarce regions. In recent years, the rapid development of microfluidic chip technology has greatly advanced CTC detection technology. However, current microfluidic chip operation is cumbersome, the devices are complex, and additional external pumps, connectors, and control systems are required to complete the detection. Furthermore, the interpretation of microfluidic chip test results typically requires specialized knowledge, hindering large-scale market adoption.

[0003] Lateral flow immunoassay (LFIA) is a solid-phase immunochromatographic technique that combines chromatographic analysis and immunoreaction principles. It is characterized by its ease of operation, rapid detection, and portability, and has been widely applied in clinical diagnostics, drug detection, and environmental pollution control. Previous studies have shown that in resource-limited countries and regions, LFIA devices can detect pathogens from a wide range of samples. However, LFIA analyzers typically target DNA, RNA, or proteins, making them unsuitable for detecting tumor cells. Therefore, it is necessary to develop a rapid, simple, relatively economical, and widely usable circulating cell detection device to meet the clinical requirements for timely diagnosis and treatment monitoring to improve patient quality of life.

[0004] Colorectal cancer, a common malignant gastrointestinal disease, is often difficult to detect in its early stages due to the lack of symptoms. Currently, only advanced secondary tumors can be detected clinically. Early diagnosis and treatment of the tumor are the most effective ways to reduce mortality and improve quality of life.

[0005] Circulating tumor cells (CTCs) are tumor cells that detach from the primary lesion, recurrent lesion, or metastatic lesion of a solid tumor and enter the bloodstream through the blood or lymphatic system. They are the main cause of tumor metastasis and development. CTC detection in patients can dynamically monitor the occurrence and progression of tumors, which helps to achieve the goal of early diagnosis and early treatment. Summary of the Invention

[0006] This application provides an easily readable tumor cell detection device, a microfluidic chip, and a method for preparing the same. The advantages of the provided easily readable tumor cell detection device and microfluidic chip are that they do not require external pumps and control equipment to control the flow of liquid, the detection results are easy to read, they are portable, and they have low requirements for equipment and experience, which is conducive to their widespread application.

[0007] On one hand, this application provides a microfluidic chip, comprising:

[0008] A sample introduction unit, wherein the sample introduction unit has an inlet, and the sample to be detected or a signal amplification matrix is ​​introduced into the sample introduction unit;

[0009] A detection unit is connected to the sample injection unit;

[0010] A power supply unit, the power supply unit comprising a plurality of capillaries connected to the detection unit;

[0011] And a waste liquid recovery unit, which is connected to the power supply unit and has an outlet.

[0012] Furthermore, the detection unit includes a first chamber and a second chamber, which are connected by a connecting channel. One end of the first chamber is connected to the sample injection unit, and both the first chamber and the second chamber are connected to the power supply unit.

[0013] Furthermore, the power supply unit includes a first capillary group and a second capillary group, which are respectively connected to the first chamber and the second chamber in the detection unit.

[0014] Furthermore, the waste liquid recovery unit includes a first recovery chamber and a second recovery chamber, both of which have outlets. The first and second recovery chambers are respectively connected to the first capillary group and the second capillary group in the power supply unit.

[0015] Furthermore, the first chamber and the second chamber are provided with a number of triangular micropillars. The triangular micropillars in the first chamber are modified with antibodies that can specifically bind to the target tumor cells to be tested, and the triangular micropillars in the second chamber are modified with strong cationic polyelectrolytes. In the sample to be tested, tumor cells are labeled using CoPt3 probes modified with specific recognition antibodies.

[0016] Furthermore, the second capillary assembly is used to provide power so that the sample to be tested placed in the sample introduction unit can flow sequentially through the first chamber and the second chamber to the second capillary assembly.

[0017] The first capillary assembly is used to provide power so that the signal amplification matrix placed in the injection unit can flow from the injection unit through the first chamber to the first capillary assembly.

[0018] On the other hand, this application provides a method for detecting tumor cells, which utilizes a microfluidic chip as described in the above scheme for detection, including the following steps:

[0019] Sample pretreatment: Add peripheral circulating blood / fecal samples, CoPt3 probe and buffer to a centrifuge tube, incubate for 30 minutes, then separate CoPt3 probe and CoPt3 probe-circulating tumor cell complex with a magnet, wash two to three times with buffer, and finally add 200 μL of buffer.

[0020] Sample introduction and detection: Keep the outlet of the second recovery chamber unobstructed, keep the second capillary group open, and block the outlet of the first recovery chamber with sealing film to close the first capillary group; add the pretreated sample to the inlet of the sample introduction unit, and the sample automatically flows through the detection unit under the action of capillary force, and flows through the first chamber and the second chamber in sequence; the first chamber serves as the detection line and the second chamber serves as the control line. During this process, the antibody on the triangular micropillar of the detection line binds to the surface protein of circulating tumor cells in the sample, realizing the recognition and capture of circulating tumor cells, and the strong cationic polyelectrolyte on the control line physically adsorbs the CoPt3 probe;

[0021] Signal amplification: After the sample flows out of the control line, close the second capillary group and open the first capillary group to add the signal amplification matrix to the injection port;

[0022] Detection: The matrix to be amplified is filled with the detection line and control line of the detection unit. After five minutes, the color depth of the detection line is used for intuitive and qualitative detection analysis. The color of the control line is used to determine whether the detection is effective, or color information can be obtained and analyzed using an image acquisition device.

[0023] On the other hand, this application provides a method for fabricating a microfluidic chip, which is used to fabricate a microfluidic chip as described above, comprising the following steps:

[0024] The channels of the microfluidic chip were drawn and printed on two black films. One film included the sample injection channel and the upper pump-free capillary channel, while the other film included the first chamber, the second chamber, and the lower pump-free capillary channel.

[0025] The master mold was fabricated on a silicon wafer with a radius of 10 cm using black film, SU-8 photoresist, and an ultraviolet exposure machine via soft lithography.

[0026] The PDMS prepolymer and curing agent were mixed at a mass ratio of 10:1. The PDMS mixture was poured onto the master mold and then cured at 80°C for 120 minutes to form a PDMS substrate.

[0027] The prepared PDMS substrate was peeled off from the master mold and holes were punched at the injection inlet and the outlets of the two pump-free capillary pumps. The surface of the capture micropillars was modified with polyethylene glycol to increase the hydrophilicity of the internal channels of the chip. The micropillars were washed three times with isopropanol to remove the residual polyethylene glycol inside the channels and cooled at 4°C for 1 hour.

[0028] After the prepared PDMS substrate is placed in a plasma cleaner for 1 minute, it is aligned and bonded according to the interface. After plasma treatment, it is bonded to obtain a microfluidic chip.

[0029] The antibody and strong cationic polyelectrolyte were injected into the detection and control line chambers, respectively, and incubated in a refrigerator for 12 hours. After incubation, the cells were washed with phosphate buffer and dried for later use.

[0030] On the other hand, this application provides an easily readable tumor cell detection device, including a housing with a result reading window; at least one detection unit is provided inside the housing, the detection unit being used to place a microfluidic chip as described in the above scheme, and after the microfluidic chip is placed inside the housing, the detection module is located directly opposite the reading window.

[0031] Furthermore, the enclosure is equipped with a light shield, and the reading window is used for visual observation or for placing image acquisition equipment.

[0032] In summary, the beneficial effects of this application are as follows:

[0033] 1. The microfluidic chip provided in this application does not require an external pump or control equipment to control the liquid flow, nor does it require large instruments. Its structure is simpler, reducing detection costs and lowering the requirements for equipment and personnel operation, which is conducive to promotion. Moreover, the detection results are easy to read.

[0034] 2. The tumor detection process described in this application is simple and easy to master, which facilitates rapid screening of tumor cells to determine whether further accurate pathological examination is needed, and greatly improves the diagnostic efficiency of POC.

[0035] 3. The easily readable tumor cell detection device provided in this application is compact in size, easy to carry, and can simplify the operation process, save time, and reduce costs. Attached Figure Description

[0036] Figure 1 is a schematic diagram illustrating the principle of tumor cell detection in this application;

[0037] Figure 2 is a schematic diagram of the microfluidic chip of this application;

[0038] Figure 3 is a schematic diagram of the detection unit in the microfluidic chip of this application;

[0039] Figure 4 is a schematic diagram of the power supply unit in the microfluidic chip of this application;

[0040] Figure 5 is a schematic diagram of the easily readable tumor cell detection device of this application, wherein the microfluidic chip is located inside the box;

[0041] Figure 6 is a schematic diagram of the easily readable tumor cell detection device of this application, in which the microfluidic chip is pulled out of the box.

[0042] In the figure, 1-placement unit; 2-light shield; 3-reading window; 4-sample injection unit; 5-detection unit; 6-power supply unit; 7-waste liquid recovery unit; 8-sample capillary pump outlet; 9-signal capillary pump outlet; 10-connection channel; 11-sample inlet. Detailed Implementation Methods

[0043] The specific embodiments of this application are described in detail below with reference to the accompanying drawings.

[0044] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this utility model according to the specific circumstances.

[0045] Example: This application provides a microfluidic chip, referring to 1-5. The microfluidic chip is composed of three layers of polydimethylsiloxane (PDMS) bonded together, including a sample injection unit 4, a detection unit 5, a power supply unit 6, and a waste liquid recovery unit 7.

[0046] The sample introduction unit has an inlet, and the sample to be tested or a signal amplification matrix is ​​added inside the sample introduction unit.

[0047] The detection unit is connected to the sample injection unit; the detection unit includes a first chamber and a second chamber, connected by a connecting channel 10. One end of the first chamber is connected to the sample injection unit, and both the first and second chambers are connected to a power supply unit. The first chamber serves as a detection line, and the second chamber serves as a control line.

[0048] The power supply unit includes a plurality of capillaries connected to the detection unit. The capillaries include a first capillary group and a second capillary group, which respectively form a first capillary pump and a second capillary pump. The first capillary group and the second capillary group are respectively connected to a first chamber and a second chamber in the detection unit.

[0049] The waste liquid recovery unit is connected to the power supply unit, and the waste liquid recovery unit has an outlet. The waste liquid recovery unit includes a first recovery chamber and a second recovery chamber, each of which has an outlet. The first recovery chamber and the second recovery chamber are respectively connected to a first capillary assembly and a second capillary assembly in the power supply unit.

[0050] The first chamber and the second chamber are equipped with a number of triangular micropillars. The triangular micropillars in the first chamber are modified with antibodies that can specifically bind to the target tumor cells to be tested, and the triangular micropillars in the second chamber are modified with strong cationic polyelectrolytes. In the sample to be tested, tumor cells are labeled using CoPt3 probes modified with specific recognition antibodies.

[0051] The second capillary assembly provides power, allowing the sample to be tested placed in the injection unit to flow sequentially through the first and second chambers to the second capillary assembly. The second capillary pump is a sample capillary pump; the sample capillary pump outlet 8 is connected to the control line. The first capillary assembly provides power, allowing the signal amplification matrix placed in the injection unit to flow from the injection unit through the first chamber to the first capillary assembly. The first capillary pump is a signal capillary pump; the signal capillary pump outlet 9 is connected to the detection line, and the connection point is located on the opposite side of the injection unit connection point.

[0052] In a sufficiently thin capillary channel, the solution can spontaneously flow from the inlet to the outlet under the action of surface tension, thus providing the driving force for the liquid flow.

[0053] Another embodiment of this application provides a method for detecting tumor cells, which utilizes a microfluidic chip as described in the above scheme. Referring to Figure 1, the method includes the following steps:

[0054] Sample pretreatment: Add peripheral circulating blood / fecal samples, CoPt3 probe and buffer to a centrifuge tube, incubate for 30 minutes, then separate the CoPt3 probe and CoPt3 probe-circulating tumor cell complex with a magnet, wash two to three times with buffer, and finally add 200 μL of buffer; use CoPt3 probe to label hyaluronic acid antibody, and cells in the sample to be tested bind to hyaluronic acid to form CoPt3-cell complex.

[0055] Sample introduction and detection: Keep the outlet of the second recovery chamber unobstructed, keep the second capillary group open, and block the outlet of the first recovery chamber with sealing film to close the first capillary group; add the pretreated sample to the inlet of the sample introduction unit. The sample automatically flows through the detection unit under the force of the capillary, and then flows through the first and second chambers in sequence; the first chamber serves as the detection line and the second chamber serves as the control line. During this process, the antibody on the triangular micropillar of the detection line binds to the surface protein of circulating tumor cells in the sample, realizing the recognition and capture of circulating tumor cells. The strong cationic polyelectrolyte on the control line physically adsorbs the CoPt3 probe; the triangular micropillar of the detection line is modified with antibody, and the interaction between the antibody and the cell surface protein is used to realize the recognition of tumor cells. The triangular micropillar of the control line is modified with strong cationic polyelectrolyte, and the negatively charged CoPt3 probe is physically adsorbed to it to realize the capture of the CoPt3 probe.

[0056] Signal amplification: After the sample flows out of the control line, close the second capillary group and open the first capillary group, adding the signal amplification matrix (H2O2 and dopamine) to the injection port. Signal amplification principle: CoPt3's peroxidase activity catalyzes the formation of polydopamine (PDA) from dopamine, achieving signal amplification. PDA has good cell adhesion and biocompatibility, and its black color provides a strong visual signal, improving the reliability of the detection results. In negative samples, due to the lack of target tumor cells, no cells are captured within the detection line; all CoPt3 probes are captured at the control line, resulting in only a brown to black signal. In positive samples, target tumor cells form a CoPt3-cell complex with the CoPt3 probe and are captured by the detection line microcolumns. Excess CoPt3 probes are captured by the control line microcolumns, so both the detection and control lines show signals. Therefore, if the control line does not show a signal, the test is invalid regardless of whether the detection line shows a signal.

[0057] Detection: After the signal amplification matrix fills the detection line and control line of the detection unit (the signal amplification matrix flows into the detection line and control line chambers from the sample inlet 11 at the bottom of the detection unit, and after the two chambers are filled with solution, it flows out from the channel located at the top of the chamber to the signal capillary pump outlet 9), wait for five minutes, and perform intuitive visual qualitative detection analysis based on the color depth of the detection line. The color of the control line is used to determine whether the detection is effective, or color information can be obtained and analyzed using image acquisition equipment.

[0058] Another embodiment of this application provides a method for fabricating a microfluidic chip, which is used to fabricate a microfluidic chip as described above, and includes the following steps:

[0059] The channels of the microfluidic chip were drawn using AutoCAD software and printed on two black films. One film included the sample injection channel and the upper pump-free capillary channel, while the other film included the first chamber, the second chamber, and the lower pump-free capillary channel.

[0060] The master mold was fabricated on a silicon wafer with a radius of 10 cm using black film, SU-8 photoresist, and an ultraviolet exposure machine via soft lithography.

[0061] The PDMS prepolymer and curing agent were mixed at a mass ratio of 10:1. The PDMS mixture was poured onto the master mold and then cured at 80°C for 120 minutes to form a PDMS substrate.

[0062] The prepared PDMS substrate was peeled off from the master mold and holes were punched at the injection inlet and the outlets of the two pump-free capillary pumps. The surface of the capture micropillars was modified with polyethylene glycol to increase the hydrophilicity of the internal channels of the chip. The micropillars were washed three times with isopropanol to remove the residual polyethylene glycol inside the channels and cooled at 4°C for 1 hour.

[0063] After the prepared PDMS substrate is placed in a plasma cleaner for 1 minute, it is aligned and bonded according to the interface. After plasma treatment, it is bonded to obtain a microfluidic chip.

[0064] The antibody and strong cationic polyelectrolyte were injected into the detection and control line chambers, respectively. After incubation in a refrigerator for 12 hours, the mixture was washed with phosphate buffer and dried for further processing.

[0065] Another embodiment of this application provides an easily readable tumor cell detection device. Referring to Figures 5-6, it includes a housing with a result reading window 3 on the housing. The housing contains three detection units 1, which are used to place microfluidic chips as described in the above scheme. The microfluidic chips are retractable. After the microfluidic chips are placed in the housing, the detection module is located directly opposite the reading window.

[0066] The chamber is a dark chamber, and a light-shielding plate 2 is installed on the chamber to maintain consistent brightness inside the dark chamber during the detection process, thereby reducing errors. The reading window is used for visual observation or to place an image acquisition device. The image acquisition device is a smartphone, and the color analysis software on the smartphone is used to analyze the color of the detection line to obtain more accurate data.

[0067] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of this application, and these all fall within the protection scope of this application.

Claims

1. A microfluidic chip, characterized in that, include: A sample introduction unit, wherein the sample introduction unit has an inlet, and the sample to be detected or a signal amplification matrix is ​​introduced into the sample introduction unit; The detection unit is connected to the sample injection unit. The detection unit includes a first chamber and a second chamber, connected by a connecting channel. One end of the first chamber is connected to the sample injection unit, and both the first and second chambers are connected to a power supply unit. Each of the first and second chambers contains several triangular micropillars. The triangular micropillars in the first chamber are modified with antibodies that specifically bind to the target tumor cells, while the triangular micropillars in the second chamber are modified with a strong cationic polyelectrolyte. Tumor cells in the sample are labeled using a CoPt3 probe modified with a specific recognition antibody. The power supply unit... The power supply unit includes a plurality of capillaries connected to the detection unit; the power supply unit includes a first capillary group and a second capillary group, which are respectively connected to a first chamber and a second chamber in the detection unit, and a waste liquid recovery unit, which is connected to the power supply unit and has an outlet; the waste liquid recovery unit includes a first recovery chamber and a second recovery chamber, each of which has an outlet, and the first recovery chamber and the second recovery chamber are respectively connected to the first capillary group and the second capillary group in the power supply unit.

2. The microfluidic chip according to claim 1, characterized in that, The second capillary assembly provides power so that the sample to be tested placed in the sample injection unit can flow sequentially through the first chamber and the second chamber to the second capillary assembly; the first capillary assembly provides power so that the signal amplification matrix placed in the sample injection unit can flow from the sample injection unit through the first chamber to the first capillary assembly.

3. A method for detecting tumor cells, characterized in that, Detection using the microfluidic chip as described in claim 1 or 2 includes the following steps: Sample pretreatment: Peripheral circulating blood / fecal sample, CoPt3 probe, and buffer are added to a centrifuge tube. After incubation for 30 minutes, the CoPt3 probe and the CoPt3 probe-circulating tumor cell complex are separated using a magnet. The sample is then washed two to three times with buffer, and finally 200 μL of buffer is added. Sample injection and detection: The outlet of the second recovery chamber is kept open, and the second capillary assembly is in the open state. The outlet of the first recovery chamber is blocked with a sealing film, thus closing the first capillary assembly. The pretreated sample is then... A good sample is added to the inlet of the injection unit. Under the action of capillary force, the sample automatically flows through the detection unit and sequentially through the first and second chambers. The first chamber serves as the detection line, and the second chamber serves as the control line. During this process, the antibody on the triangular micropillar of the detection line binds to the surface protein of circulating tumor cells in the sample, realizing the recognition and capture of circulating tumor cells. The strong cationic polyelectrolyte on the control line undergoes physical adsorption with the CoPt3 probe. Signal amplification: After the sample flows out of the control line, the second capillary group is closed and the first capillary group is opened, and the signal amplification matrix is ​​added to the injection port. Detection: The matrix to be amplified is filled with the detection line and control line of the detection unit. After five minutes, the color depth of the detection line is used for intuitive and qualitative detection analysis. The color of the control line is used to determine whether the detection is effective, or color information can be obtained and analyzed using an image acquisition device.

4. A method for fabricating a microfluidic chip, characterized in that, The method for fabricating a microfluidic chip as described in any one of claims 1-2 includes the following steps: drawing the channels of the microfluidic chip and printing them on two black films, one of which includes an injection channel and an upper pump-free capillary channel, and the other film includes a first chamber, a second chamber, and a lower pump-free capillary channel; fabricating a master mold on a silicon wafer with a radius of 10 cm using black films, SU-8 photoresist, and an ultraviolet exposure machine via soft lithography; mixing polydimethylsiloxane (PDMS) prepolymer and a curing agent at a mass ratio of 10:1, pouring the PDMS mixture onto the master mold, and then curing it at 80°C for 120 minutes to form a PDMS substrate; The prepared PDMS substrate was peeled off from the master mold, and holes were punched at the injection inlet and the outlets of the two pump-free capillary pumps. The surface of the micropillars was modified with polyethylene glycol to increase the hydrophilicity of the channels inside the chip. The micropillars were washed three times with isopropanol to remove residual polyethylene glycol inside the channels and cooled at 4°C for 1 hour. The prepared PDMS substrate was placed in a plasma cleaner for 1 minute and then aligned and bonded according to the interface. After plasma treatment, the microfluidic chip was obtained. Antibodies and strong cationic polyelectrolytes were injected into the first and second chambers, respectively. After incubation in a refrigerator for 12 hours, the chips were washed with phosphate buffer and dried for further processing.

5. An easily readable tumor cell detection device, characterized in that, The device includes a housing with a result reading window; at least one detection unit is provided inside the housing, the detection unit being used to place the microfluidic chip as described in any one of claims 1-2, wherein after the microfluidic chip is placed inside the housing, the detection module is located directly opposite the reading window.

6. The easily readable tumor cell detection device according to claim 5, characterized in that, The enclosure is equipped with a light shield, and the reading window is used for visual observation or for placing image acquisition equipment.

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