Chip system capable of simultaneously detecting molecule and cell surface markers and detection method

Abstract

The invention provides a chip system and a detection method capable of simultaneously detecting molecular and cell surface markers, the chip system comprises a detection chip, a PDMS kit, a detection system and a sample introduction system, the PDMS kit is provided with a first sample introduction port and a second sample introduction port; the detection system comprises a flow cell, and the flow cell is provided with a cell detection channel, a molecule detection channel, a cell sample inlet and a molecule sample inlet; the first sample inlet, the second sample inlet, the cell sample inlet and the molecule sample inlet can be communicated with the surface of the detection chip, and the surface of the detection chip is divided into a cell detection area and a molecule detection area; the sample introduction system comprises a multi-way valve and a reciprocating delivery pump, one end of the reciprocating delivery pump is connected with the cell sample introduction port, the molecule sample introduction port, the first sample introduction port and the second sample introduction port through the multi-way valve, and the other end of the reciprocating delivery pump is connected with a sample to be detected and a modification reagent. According to the present invention, the cell and molecule detection can be simultaneously achieved, the detection efficiency is high, and the detection of molecule-molecule and molecule-cell combination kinetics can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a chip system and a detection method capable of simultaneously detecting molecules and cell surface markers. Background Art

[0002] Surface plasmon resonance (SPR) is a sensitive technology that can simultaneously detect the binding kinetics and thermodynamics between cells and molecules. It has good application prospects in the fields of chemical industry, food, materials and biomedicine. SPR technology is a surface analysis method. The detection of the effects of related substances can only be carried out on the surface of the chip. Therefore, the quality of the chip directly determines the accuracy of the test results. The development of SPR microscopy technology has provided conditions for the detection of cell-molecule interactions. However, traditional SPR technology can only perform single molecular interactions, and existing chips can only meet single detection needs. For systems that require simultaneous detection of molecule-molecule and molecule-cell interactions, separate detection is still required. In view of the high cost of biological samples and chips, this greatly increases the cost of detection. At the same time, since the chip pre-treatment during the detection process requires a lot of time, this also reduces the detection efficiency. Summary of the invention

[0003] In order to solve the problems existing in the background technology, the present invention proposes a chip system that can simultaneously detect molecules and cell surface markers, including a detection chip, a PDMS kit, a detection system and an injection system, wherein a diaphragm is provided in the middle of the top of the PDMS kit, and a first injection port and a second injection port are provided on the PDMS kit; The detection system comprises a circulation pool, on which a cell detection channel, a molecule detection channel, and a cell injection port and a molecule injection port respectively connected to the cell detection channel and the molecule detection channel are provided; The surface of the detection chip is divided into a cell detection area and a molecule detection area. When the detection chip is modified, the detection chip is arranged in a PDMS box, the molecule detection area and the cell detection area are separated by a diaphragm, and the first injection port and the second injection port are connected to the molecule detection area and the cell detection area respectively; When fixing molecules and cells, the detection chip is connected to the circulation pool, the cell injection port and the cell detection channel are connected to the cell detection area of ​​the detection chip, and the molecule injection port and the molecule detection channel are connected to the molecule detection area of ​​the detection chip; The injection system includes a multi-way valve and a reciprocating pump. One end of the reciprocating pump is connected to the cell injection port, the molecule injection port, the first injection port and the second injection port respectively through the multi-way valve, and the other end of the reciprocating pump is connected to the sample to be detected and the modification reagent.

[0004] Preferably, there are at least two cell detection channels, two adjacent cell detection channels are connected through a cell injection port, and there are at least two molecule detection channels, two adjacent molecule detection channels are connected through a molecule injection port, and the cell detection channels and the molecule detection channels are arranged in a wave shape.

[0005] Preferably, the inner diameter of the molecular detection channel is smaller than the inner diameter of the cell detection channel.

[0006] Preferably, the reciprocating delivery pump is a multi-channel peristaltic pump, the multi-way valve comprises a first multi-way valve and a five-way valve, the five-way valve comprises a first injection end, a second injection end, a discharge end, a first addition end and a second addition end, the first injection end is connected to the first injection port, and the second injection end is connected to the second injection port; The first multi-way valve comprises a third sample addition end, a fourth sample addition end, a cell injection end and a molecule injection end, the cell injection port is connected to the cell injection end, and the molecule injection port is connected to the molecule injection end; The multi-channel peristaltic pump comprises a first delivery channel, a second delivery channel, a third delivery channel, a fourth delivery channel and a fifth delivery channel. One end of the first delivery channel, the second delivery channel, the third delivery channel, the fourth delivery channel and the fifth delivery channel are respectively connected to the first loading end, the second loading end, the discharge end, the third loading end and the fourth loading end. The other end of the first delivery channel and the other end of the second delivery channel are respectively connected to the modification reagent, the other end of the fourth delivery channel is connected to the molecular sample to be detected, the other end of the fifth delivery channel is connected to the cell sample to be detected, and the other end of the third delivery channel is used to discharge waste.

[0007] Preferably, the modification reagent includes compounds and a cleaning solution, the compounds used in the molecule detection zone include thiol PEG-OH, thiol PED-COOH, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxysuccinimide and components for fixing the molecules to be detected, the compounds used in the cell detection zone include thiol PEG-OH, thiol PED-COOH, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and components for fixing the cells to be detected, and the cleaning solution includes ethanol and deionized water.

[0008] Preferably, the component for fixing the cells to be detected includes an adhesion protein, and the adhesion protein includes fibronectin; and the component for fixing the molecules to be detected includes a biomolecule detection antibody.

[0009] Preferably, it also includes a first sample pool, a second sample pool, a first reagent pool and a second reagent pool, the molecular sample to be detected is placed in the first sample pool, the cell sample to be detected is placed in the second sample pool, the modification reagent for the cell detection area is placed in the first reagent pool, and the modification reagent for the molecular detection area is placed in the second reagent pool, and the first delivery channel, the second delivery channel, the fourth delivery channel and the fifth delivery channel are respectively connected to the first reagent pool, the second reagent pool, the first sample pool and the second sample pool.

[0010] A detection method for simultaneously detecting molecular and cell surface markers comprises the following steps: S1. Place the detection chip in the PDMS box, and use the sample injection system to add modification reagents to the cell detection area and the molecule detection area to modify the detection chips of the two detection areas; S2. After the modification is completed, the detection chip is placed in the detection system, and the reciprocating delivery pump is started to introduce the sample to be detected into the cell detection channel and the molecular detection channel through the cell injection port and the molecular injection port, respectively, so that the molecular sample to be detected and the cell sample to be detected are in contact with the surface of the molecular detection area and the surface of the cell detection area of ​​the detection chip, respectively; S3, the reciprocating delivery pump reciprocates to drive the sample to be detected, so that the sample to be detected repeatedly flows through the surface of the detection chip, the molecular sample to be detected flowing through the surface of the detection chip is fixed in the molecular detection area, and the cell sample to be detected flowing through the surface of the detection chip is fixed in the cell detection area; S4. After the cells and the surface of the molecule detection chip are fixed, take out the detection chip for detection.

[0011] Preferably, the step S1 specifically comprises: S11, 10 μg / mL of thiol PEG-OH and thiol PED-COOH were mixed evenly in a ratio of 1:1, added to the detection chip in the molecular detection area through the injection system, and the detection chip was placed at room temperature overnight; 10 μg / mL of thiol PEG-OH and thiol PED-COOH were mixed evenly at a ratio of 1:1, added to the detection chip in the cell detection area using the injection system, and the detection chip was placed at room temperature overnight; S12, using the injection system to remove the thiol PEG-OH and thiol PED-COOH in the cell detection area and the thiol PEG-OH and thiol PED-COOH in the molecule detection area, and then using the injection system to deliver ethanol to the surface of the detection chip, and using ethanol to clean the cell detection area and the molecule detection area respectively; S13, after the cleaning is completed, the ethanol is removed, and then a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide is added to the cell detection area and the molecular detection area respectively by using the injection system, and left at room temperature for 1 hour; S14, using the injection system to remove the mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide, and again washing the cell detection area and the molecular detection area with ethanol in the manner of step S12; S15. After the washing is completed, remove the ethanol, add 0.1 μg / mL of fibronectin to the cell detection area and add a biomolecule detection antibody to the molecule detection area using the injection system, and react for half an hour; S16. After the reaction is completed, the detection chip is taken out from the PDMS box, cleaned with deionized water, and then blown dry with nitrogen.

[0012] The present invention has the following beneficial effects: the present invention can simultaneously realize the fixation and detection of cells and molecules, which can meet the needs of cell and molecule fixation on the one hand, and can meet the detection of molecule-molecule and molecule-cell binding dynamics on the other hand. At the same time, the chip can also realize online detection of cell secretions, metabolites, etc. In addition, the present invention saves the time of pre-processing of the detection chip and improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the overall structure of the chip system of the present invention; Figure 2 A cross-sectional view of a PDMS box when the detection chip of the present invention is arranged in the PDMS box; Figure 3 This is a schematic diagram of the positional relationship between the detection chip and the flow cell of the present invention; Figure 4 It is a schematic diagram of the structure of the circulation pool of the present invention close to the detection chip.

[0014] Numbers in the figure: 1. detection chip; 2. PDMS box; 3. box cover; 4. diaphragm; 5. first injection port; 6. second injection port; 7. detection system; 8. five-way valve; 81. first sample addition end; 82. first injection end; 83. second sample addition end; 84. discharge end; 85. second injection end; 9. first multi-way valve; 91. cell injection end I; 92. molecular injection end I; 93. molecular injection end II; 94. cell injection end II; 95. third sample addition end; 96. fourth sample addition end; 10. circulation pool; 101. molecular injection port; 102. molecular detection channel; 103. cell injection port; 104. cell detection channel; 11. multi-channel peristaltic pump; 12. first reagent cell; 13. second reagent cell; 14. first sample cell; 15. second sample cell. DETAILED DESCRIPTION

[0015] In order to make the present invention clearer and more understandable, the technical solution of the present invention is further described in detail below in combination with the accompanying drawings and embodiments. It should be understood that the given embodiment is only one implementation method and does not represent all embodiments.

[0016] In this article, the terms "left, right, up, down" and the like are established based on the positional relationships shown in the drawings. Depending on the different drawings, the corresponding positional relationships may also change accordingly. Therefore, they cannot be understood as absolute limitations on the scope of protection.

[0017] Combined with Figure 1 -Attached Figure 4 , a chip system capable of simultaneously detecting molecules and cell surface markers, comprising a detection chip 1, a PDMS box 2, a detection system 7 and an injection system, wherein a diaphragm 4 is provided at the middle position of the top of the PDMS box 2, and a first injection port 5 and a second injection port 6 are provided on the PDMS box 2; The detection system 7 includes a circulation pool 10, on which a cell detection channel 104, a molecule detection channel 102, and a cell injection port 103 and a molecule injection port 101 respectively connected to the cell detection channel 104 and the molecule detection channel 102 are provided; The surface of the detection chip 1 is divided into a cell detection area and a molecule detection area. When the detection chip 1 is modified, the detection chip 1 is set in a PDMS box 2, the molecule detection area and the cell detection area are separated by a diaphragm 4, and the first injection port 5 and the second injection port 6 are connected to the molecule detection area and the cell detection area respectively; When fixing molecules and cells, the detection chip 1 is connected to the circulation pool 10, the cell injection port 103 and the cell detection channel 104 are connected to the cell detection area of ​​the detection chip 1, and the molecule injection port 101 and the molecule detection channel 102 are connected to the molecule detection area of ​​the detection chip 1; The injection system includes a multi-way valve and a reciprocating pump. One end of the reciprocating pump is connected to the cell injection port 103, the molecule injection port 101, the first injection port 5 and the second injection port 6 through the multi-way valve, and the other end of the reciprocating pump is connected to the sample to be detected and the modification reagent.

[0018] Specifically, the cell injection port 103 and the molecule injection port 101 pass through the circulation pool 10, and the cell detection channel 104 and the molecule detection channel 102 are opened on the bottom surface of the circulation pool 10; the detection system 7 also includes a shell, the circulation pool 10 is fixed in the shell, and the top of the shell is provided with interfaces respectively connected to the cell injection port 103 and the molecule injection port 101, and a receiving groove for placing the detection chip 1 is provided at a position below the circulation pool 10 in the shell. When fixing molecules and cells, the modified detection chip 1 is placed in the receiving groove, and the modified side of the detection chip 1 contacts the bottom surface of the circulation pool 10. At this time, the molecule detection channel 102 and the cell detection channel 104 are connected to the surface of the detection chip 1.

[0019] Specifically, a box cover 3 is provided on the top of the PDMS box 2. The box cover 3 is made of transparent optical glass, which is convenient for observing the status of the detection chip 1 during the modification process.

[0020] Specifically, there are at least two cell detection channels 104, and two adjacent cell detection channels 104 are connected through a cell injection port 103. There are at least two molecule detection channels 102, and two adjacent molecule detection channels 102 are connected through a molecule injection port 101. The cell detection channels 104 and the molecule detection channels 102 are arranged in a wave shape. The wave-like arrangement can extend the path of molecules and cells flowing through the surface of the detection chip 1, thereby improving the fixation effect of molecules and cells. More specifically, there are two cell injection ports 103 and two molecule injection ports 101, one cell injection port 103 and one molecule injection port 101 are arranged at one end of the circulation pool 10 near the middle, and the other cell injection port 103 and the other molecule injection port 101 are arranged at the other end of the circulation pool 10 near the two sides; there are two molecular detection channels 102 and two cell detection channels 104, one molecule injection port 101 is located at one end of a molecular detection channel 102, and the other molecule injection port 101 is located at the connection of the two molecular detection channels 102, connecting the two molecular detection channels 102; one cell injection port 103 is located at one end of a cell detection channel 104, and the other cell injection port 103 is located at the connection of the two cell detection channels 104, connecting the two cell detection channels 104. Correspondingly, the first multi-way valve 9 is a five-way valve 8, the cell injection end of the five-way valve 8 includes a cell injection end I 91 and a cell injection end II 94, each of which is connected to a cell injection port 103, and the molecule injection end of the five-way valve 8 includes a molecule injection end I 92 and a molecule injection end II 93, each of which is connected to a molecule injection port 101. The simultaneous delivery of samples by multiple injection ports can improve the efficiency of sample inflow or outflow in the detection channel.

[0021] Specifically, considering that the volume of cells / bacteria is larger than the volume of molecules, the inner diameter of the molecular detection channel 102 is smaller than the inner diameter of the cell detection channel 104 .

[0022] Specifically, the reciprocating pump is a multi-channel peristaltic pump 11, which can realize the individual and reciprocating delivery of each substance. The multi-way valve includes a first multi-way valve 9 and a five-way valve 8. The five-way valve 8 includes a first injection end 82, a second injection end 85, a discharge end, a first addition end 81 and a second addition end 83. The first injection end 82 is connected to the first injection port 5, and the second injection end 85 is connected to the second injection port 6. The first multi-way valve 9 comprises a third sample addition end 95, a fourth sample addition end 96, a cell injection end and a molecule injection end, the cell injection port 103 is connected to the cell injection end, and the molecule injection port 101 is connected to the molecule injection end; The multi-channel peristaltic pump 11 includes a first delivery channel, a second delivery channel, a third delivery channel, a fourth delivery channel, and a fifth delivery channel. One end of the first delivery channel, the second delivery channel, the third delivery channel, the fourth delivery channel, and the fifth delivery channel are respectively connected to the first loading end 81, the second loading end 83, the discharge end, the third loading end 95, and the fourth loading end 96. The other end of the first delivery channel and the other end of the second delivery channel are respectively connected to the modification reagent, the other end of the fourth delivery channel is connected to the molecular sample to be detected, the other end of the fifth delivery channel is connected to the cell sample to be detected, and the other end of the third delivery channel is used to discharge waste.

[0023] Specifically, the modification reagent includes compounds and a cleaning solution. The compounds used in the molecule detection area include thiol PEG-OH (HS-PEG-COOH), thiol PED-COOH (HS-PEG-OH), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxysuccinimide, and components for fixing the molecules to be detected. The compounds used in the cell detection area include thiol PEG-OH, thiol PED-COOH, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), and components for fixing the cells to be detected. The cleaning solution includes ethanol and deionized water.

[0024] Specifically, the components used to fix the cells to be detected all include adhesion proteins, and the adhesion proteins include fibronectin; the components used to fix the molecules to be detected include biological molecule detection antibodies.

[0025] Specifically, it also includes a first sample pool 14, a second sample pool 15, a first reagent pool 12 and a second reagent pool 13. The molecular sample to be detected is placed in the first sample pool 14, the cell sample to be detected is placed in the second sample pool 15, the modification reagent for the cell detection area is placed in the first reagent pool 12, and the modification reagent for the molecular detection area is placed in the second reagent pool 13. The first delivery channel, the second delivery channel, the fourth delivery channel and the fifth delivery channel are respectively connected to the first reagent pool 12, the second reagent pool 13, the first sample pool 14 and the second sample pool 15. More specifically, the first reagent pool 12 includes a sulfhydryl PEG-OH and sulfhydryl PED-COOH mixed solution placement chamber, an ethanol placement chamber, an EDC and NHS mixed solution placement chamber, and a biomolecule detection antibody placement chamber, and the first delivery channel is connected to the sulfhydryl PEG-OH and sulfhydryl PED-COOH mixed solution placement chamber, the ethanol placement chamber, the EDC and NHS mixed solution placement chamber, and the fibronectin placement chamber through a multi-way valve, and the second reagent pool 13 includes a sulfhydryl PEG-OH and sulfhydryl PED-COOH mixed solution placement chamber, an ethanol placement chamber, an EDC and NHS mixed solution placement chamber, a fibronectin placement chamber, and an antibody protein placement chamber, and the second delivery channel is connected to the sulfhydryl PEG-OH and sulfhydryl PED-COOH mixed solution placement chamber, the ethanol placement chamber, the EDC and NHS mixed solution placement chamber, the antibody protein placement chamber, and the fibronectin placement chamber through a multi-way valve. The delivery of the required reagents is achieved by selectively opening the multi-way valve.

[0026] A detection method for simultaneously detecting molecular and cell surface markers comprises the following steps: S1, placing the detection chip 1 in the PDMS box 2, and using the sample injection system to add modification reagents to the cell detection area and the molecule detection area respectively, to modify the detection chips 1 of the two detection areas; S2. After the modification is completed, the detection chip 1 is placed in the detection system 7, and the reciprocating delivery pump is started to pass the sample to be detected into the cell detection channel 104 and the molecular detection channel 102 through the cell injection port 103 and the molecular injection port 101, respectively, so that the molecular sample to be detected and the cell sample to be detected are in contact with the surface of the molecular detection area and the surface of the cell detection area of ​​the detection chip 1, respectively; S3, the reciprocating conveying pump reciprocates to drive the sample to be detected, so that the sample to be detected repeatedly flows through the surface of the detection chip 1, and the molecular sample to be detected flowing through the surface of the detection chip 1 is fixed in the molecular detection area, but the molecules flowing through this place do not produce non-specific adsorption in this area, or even if non-specific adsorption is produced, the amount does not exceed 10% of the specific binding; the cell sample to be detected flowing through the surface of the detection chip 1 is fixed in the cell detection area, but the molecules flowing through this place do not produce non-specific adsorption in this area, or even if non-specific adsorption is produced, the amount does not exceed 10% of the specific binding; specifically, when conveying the cell sample to be detected, the cell loading end and the third injection end of the first multi-way valve 9 are opened, and the other ports are closed, and the fourth conveying channel of the multi-channel peristaltic pump 11 is opened, and forward conveying and reverse conveying are performed alternately to realize the reciprocating flow of the cell sample to be detected; when conveying the molecular sample to be detected, the molecular loading end and the fourth injection end of the first multi-way valve 9 are opened, and the other ports are closed, and the fifth conveying channel of the multi-channel peristaltic pump 11 is opened, and forward conveying and reverse conveying are performed alternately to realize the reciprocating flow of the molecular sample to be detected. The sample repeatedly flows through the surface of the detection chip 1 , which can ensure the mass transfer of the sample and increase the fixed amount of the sample on the surface of the detection chip 1 .

[0027] S4, after the cell and molecule detection chip 1 are fixed on the surface, the detection chip 1 is taken out for detection. Before cell detection, antibody protein is added to the cell detection area of ​​the detection chip 1, and the activity of the cell surface molecule is determined by the antibody protein before cell detection. The protein detection antibody includes one of a glycoprotein recognition molecule and a cell membrane surface protein.

[0028] Specifically, the step S1 is as follows: S11, 10 μg / mL of thiol PEG-OH and thiol PED-COOH are mixed evenly in a ratio of 1:1, added to the detection chip 1 in the molecular detection area through the injection system, and the detection chip 1 is placed at room temperature overnight; specifically, the first injection end 82 and the first addition end 81 of the six-way valve are opened, and the other ports are closed, and the first delivery channel of the multi-channel peristaltic pump 11 starts forward delivery, and the mixed solution of thiol PEG-OH and thiol PED-COOH is input into the PDMS kit 2 through the first injection port 5, so that the mixed solution is located in the molecular detection area of ​​the detection chip 1; 10 μg / mL of thiol PEG-OH and thiol PED-COOH were mixed evenly in a ratio of 1:1, added to the detection chip 1 in the cell detection area using an injection system, and the detection chip 1 was placed at room temperature overnight; specifically, the second injection port 85 and the second addition port 83 of the six-way valve were opened, and the other ports were closed, and the second delivery channel of the multi-channel peristaltic pump 11 started forward delivery, and the mixed solution of thiol PEG-OH and thiol PED-COOH was input into the PDMS kit 2 through the first injection port 5, so that the mixed solution was located in the cell detection area of ​​the detection chip 1.

[0029] S12. Use the injection system to remove the thiol PEG-OH and thiol PED-COOH in the cell detection area and the thiol PEG-OH and thiol PED-COOH in the molecule detection area, and then use the injection system to deliver ethanol to the surface of the detection chip 1, and use ethanol to clean the cell detection area and the molecule detection area respectively; specifically, the first injection end 82, the second injection end 85 and the discharge end 84 of the six-way valve are opened, and the other ports are closed, and the third delivery channel of the multi-channel peristaltic pump 11 is started, and the third delivery channel delivers in reverse to extract and discharge the thiol PEG-OH and thiol PED-COOH in the cell detection area and the thiol PEG-OH and thiol PED-COOH in the molecule detection area, and a waste liquid tank can be connected to the end of the third delivery channel away from the six-way valve, so that the discharged solution enters the waste liquid tank for temporary storage; after the solution is discharged.

[0030] S13. After the cleaning is completed, the ethanol is removed, and then a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide is added to the cell detection area and the molecular detection area respectively by using the injection system, and left at room temperature for 1 hour; specifically, the concentration of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is 0.2M, the concentration of N-hydroxysuccinimide (NHS) is 0.05M, and the solvent for the mixture of EDC and NHS is water; S14, using the injection system to remove the mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide, and again washing the cell detection area and the molecular detection area with ethanol in the manner of step S12; S15. After cleaning, remove the ethanol, add 0.1 μg / mL of fibronectin to the cell detection area using the injection system, add a biological molecule detection antibody to the molecular detection area, and react for half an hour; specifically, the biological molecule detection antibody can be an antibody for the recognition site of glycoprotein on the surface of the cell membrane (such as wheat germ agglutinin antibody, etc.), and the type of the biological molecule detection antibody can be selected according to the specific molecule to be detected.

[0031] Specifically, when ethanol is introduced and discharged, and the mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide is introduced and discharged, the six-way valve opens and closes the port according to the introduction and discharge of the mixed solution of thiol PEG-OH and thiol PED-COOH and the thiol PEG-OH solution, and the multi-channel peristaltic pump 11 also starts the corresponding delivery channel according to the introduction and discharge of the mixed solution of thiol PEG-OH and thiol PED-COOH.

[0032] S16. After the reaction is completed, the detection chip 1 is taken out from the PDMS box 2, cleaned with deionized water, and then blown dry with nitrogen. The above embodiments are only to illustrate the basic principles and characteristics of the present invention, but are not limited to the above embodiments. It should be understood that for ordinary technicians in this field, various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention, and these changes and modifications fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A chip system capable of simultaneously detecting molecular and cell surface markers, characterized in that: It comprises a detection chip (1), a PDMS box (2), a detection system (7) and an injection system, wherein a diaphragm (4) is provided at the middle position of the top of the PDMS box (2), and a first injection port (5) and a second injection port (6) are provided on the PDMS box (2); The detection system (7) comprises a circulation pool (10), wherein the circulation pool (10) is provided with a cell detection channel (104), a molecule detection channel (102), and a cell injection port (103) and a molecule injection port (101) respectively connected to the cell detection channel (104) and the molecule detection channel (102); The surface of the detection chip (1) is divided into a cell detection area and a molecule detection area. When the detection chip (1) is modified, the detection chip (1) is arranged in a PDMS box (2), the molecule detection area and the cell detection area are separated by a diaphragm (4), and the first injection port (5) and the second injection port (6) are respectively connected to the molecule detection area and the cell detection area; When fixing molecules and cells, the detection chip (1) is connected to the circulation pool (10), the cell injection port (103) and the cell detection channel (104) are connected to the cell detection area of ​​the detection chip (1), and the molecule injection port (101) and the molecule detection channel (102) are connected to the molecule detection area of ​​the detection chip (1); The injection system comprises a multi-way valve and a reciprocating pump, one end of the reciprocating pump is respectively connected to the cell injection port (103), the molecule injection port (101), the first injection port (5) and the second injection port (6) through the multi-way valve, and the other end of the reciprocating pump is connected to the sample to be detected and the modification reagent.

2. The chip system capable of simultaneously detecting molecules and cell surface markers according to claim 1, characterized in that: There are at least two cell detection channels (104), two adjacent cell detection channels (104) are connected via a cell injection port (103); there are at least two molecule detection channels (102), two adjacent molecule detection channels (102) are connected via a molecule injection port (101); the cell detection channels (104) and the molecule detection channels (102) are arranged in a wave shape.

3. The chip system capable of simultaneously detecting molecules and cell surface markers according to claim 1 or 2, characterized in that: The inner diameter of the molecular detection channel (102) is smaller than the inner diameter of the cell detection channel (104).

4. The chip system capable of simultaneously detecting molecules and cell surface markers according to claim 1, characterized in that: The reciprocating pump is a multi-channel peristaltic pump (11); the multi-way valve comprises a first multi-way valve (9) and a five-way valve (8); the five-way valve (8) comprises a first injection end (82), a second injection end (85), a discharge end, a first addition end (81) and a second addition end (83); the first injection end (82) is connected to the first injection port (5), and the second injection end (85) is connected to the second injection port (6); The first multi-way valve (9) comprises a third sample addition end (95), a fourth sample addition end (96), a cell injection end and a molecule injection end, the cell injection port (103) is connected to the cell injection end, and the molecule injection port (101) is connected to the molecule injection end; The multi-channel peristaltic pump (11) comprises a first delivery channel, a second delivery channel, a third delivery channel, a fourth delivery channel, and a fifth delivery channel; one end of the first delivery channel, the second delivery channel, the third delivery channel, the fourth delivery channel, and the fifth delivery channel are respectively connected to a first sample loading end (81), a second sample loading end (83), a discharge end, a third sample loading end (95), and a fourth sample loading end (96); the other end of the first delivery channel and the other end of the second delivery channel are respectively connected to a modification reagent; the other end of the fourth delivery channel is connected to a molecular sample to be detected; the other end of the fifth delivery channel is connected to a cell sample to be detected; and the other end of the third delivery channel is used to discharge waste.

5. The chip system capable of simultaneously detecting molecules and cell surface markers according to claim 4, characterized in that: The modification reagent includes compounds and cleaning fluid. The compounds used in the molecule detection area include thiol PEG-OH, thiol PED-COOH, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxysuccinimide and components for fixing the molecules to be detected. The compounds used in the cell detection area include thiol PEG-OH, thiol PED-COOH, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxysuccinimide and components for fixing the cells to be detected. The cleaning fluid includes ethanol and deionized water.

6. The chip system capable of simultaneously detecting molecules and cell surface markers according to claim 5, characterized in that: The components used for fixing the cells to be detected include adhesion proteins, and the adhesion proteins include fibronectin; the components used for fixing the molecules to be detected include biological molecule detection antibodies.

7. The chip system capable of simultaneously detecting molecules and cell surface markers according to claim 5, characterized in that: The method further comprises a first sample pool (14), a second sample pool (15), a first reagent pool (12) and a second reagent pool (13); the molecular sample to be detected is placed in the first sample pool (14); the cell sample to be detected is placed in the second sample pool (15); a reagent for modifying the cell detection zone is placed in the first reagent pool (12); and a reagent for modifying the molecular detection zone is placed in the second reagent pool (13); and the first transport channel, the second transport channel, the fourth transport channel and the fifth transport channel are respectively connected to the first reagent pool (12), the second reagent pool (13), the first sample pool (14) and the second sample pool (15).

8. A method for simultaneously detecting molecular and cell surface markers, characterized in that: The following steps are involved: S1, placing the detection chip (1) in the PDMS box (2), and using the sample injection system to add modification reagents to the cell detection area and the molecule detection area respectively, thereby modifying the detection chips (1) in the two detection areas; S2. After the modification is completed, the detection chip (1) is placed in the detection system (7), and the reciprocating delivery pump is started to pass the sample to be detected into the cell detection channel (104) and the molecular detection channel (102) through the cell injection port (103) and the molecular injection port (101), respectively, so that the molecular sample to be detected and the cell sample to be detected are in contact with the surface of the molecular detection area and the surface of the cell detection area of ​​the detection chip (1), respectively; S3, the reciprocating delivery pump reciprocates to drive the sample to be detected, so that the sample to be detected repeatedly flows through the surface of the detection chip (1), the molecular sample to be detected flowing through the surface of the detection chip (1) is fixed in the molecular detection area, and the cell sample to be detected flowing through the surface of the detection chip (1) is fixed in the cell detection area; S4. After the cells and molecules are fixed on the surface of the detection chip (1), the detection chip (1) is taken out for detection.

9. The method for simultaneously detecting molecules and cell surface markers according to claim 8, characterized in that: The step S1 is specifically as follows: S11, 10 μg / mL of thiol PEG-OH and thiol PED-COOH are mixed evenly in a ratio of 1:1, and added to the detection chip (1) in the molecular detection area through the injection system, and the detection chip (1) is placed at room temperature overnight; 10 μg / mL of thiol PEG-OH and thiol PED-COOH were mixed evenly in a ratio of 1:1, and added to the detection chip (1) in the cell detection area using a sample injection system, and the detection chip (1) was placed at room temperature overnight; S12, using the injection system to remove the thiol PEG-OH and thiol PED-COOH in the cell detection area, and the thiol PEG-OH and thiol PED-COOH in the molecule detection area, and then using the injection system to deliver ethanol to the surface of the detection chip (1), and use ethanol to clean the cell detection area and the molecule detection area respectively; S13, after the cleaning is completed, the ethanol is removed, and then a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide is added to the cell detection area and the molecular detection area respectively by using the injection system, and left at room temperature for 1 hour; S14, using the injection system to remove the mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide, and again washing the cell detection area and the molecular detection area with ethanol in the manner of step S12; S15, after the washing is completed, remove the ethanol, add 0.1 μg / mL of fibronectin to the cell detection area and add a biomolecule detection antibody to the molecule detection area using the injection system, and react for half an hour; S16. After the reaction is completed, the detection chip (1) is taken out from the PDMS box (2), cleaned with deionized water, and then blown dry with nitrogen.

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