CN118477706A - A microfluidic sensor chip and a method for detecting protein tumor markers - Google Patents

Abstract

The invention provides a microfluidic sensing chip for detecting protein tumor markers and a protein tumor marker detection method, wherein the microfluidic sensing chip comprises: a reaction layer substrate comprising a base and a PARYLENE C polymeric material film, wherein a tumor marker capture antibody is modified on the PARYLENE C polymeric material film; the micro-channel modification layer and the micro-channel sample adding layer are respectively provided with a plurality of micro-flow channels at the bottom, each micro-flow channel is arranged in parallel, two ends of each micro-flow channel are respectively provided with a round sample adding hole and a flow hole which are vertically communicated, and the round sample adding holes and the flow holes are communicated with two ends of the micro-flow channels; the micro-flow channel between the micro-flow channel modification layer and the micro-flow channel sample adding layer is vertically arranged; the reaction layer substrate is sequentially combined with the micro-channel modification layer and the micro-channel sample adding layer to form a micro-fluidic chip for modification and a micro-fluidic chip for detection; the microfluidic channels in the microfluidic chip for detection are vertically intersected to form a detection point array, and the detection result of a single detection sample is presented in a bar code form to form a tumor marker bar code detection band. The invention can detect a plurality of different protein tumor markers simultaneously, and the detection process is more convenient, simple and rapid.

Description

A microfluidic sensor chip and a method for detecting protein tumor markers

Technical Field

The present invention relates to the technical field of biochemical sensor chips, and in particular to a microfluidic sensor chip for detecting protein tumor markers and a method for detecting protein tumor markers.

Background Art

Tissue biopsy is considered the gold standard for molecular analysis of tumors and can be used to confirm, diagnose and classify tumor types, as well as guide treatment. However, biopsy of patients is an invasive procedure with inherent health hazards, possible surgical complications and economic considerations that make this measure limited.

Currently, researchers are keen on detecting tumor markers. Traditional methods for detecting tumor markers mainly include enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence analysis, mass spectrometry, etc.

However, the above methods cannot meet the requirements of instant detection. Enzyme-linked immunosorbent assay kits need to consume a large amount of samples to complete the detection of multiple biomarkers one by one; the electrochemical method has high sensitivity, but its high-throughput detection capability is limited.

Summary of the invention

The present invention provides a microfluidic sensor chip for detecting protein tumor markers and a method for detecting protein tumor markers, which are used to solve the defects of the prior art such as the inability to meet the requirements of real-time detection, large sample consumption and limited high-throughput detection capability, and achieve the purpose of rapid, stable and on-site high-throughput detection.

The present invention provides a microfluidic sensor chip for detecting protein tumor markers, comprising: a reaction layer substrate, the reaction layer substrate comprising a substrate and a Parylene C polymer material film deposited on the substrate, the Parylene C polymer material film being modified with a tumor marker capture antibody;

A microfluidic channel modification layer and a microfluidic channel sample loading layer, wherein a plurality of microfluidic channels are respectively arranged at the bottom of the microfluidic channel modification layer and the microfluidic channel sample loading layer, wherein the microfluidic channels in the microfluidic channel modification layer and the microfluidic channel sample loading layer are arranged in parallel, and circular sample loading holes and outflow holes are arranged at both ends of each microfluidic channel, which are connected with both ends of the microfluidic channel; and the microfluidic channels between the microfluidic channel modification layer and the microfluidic channel sample loading layer are arranged vertically;

The reaction layer substrate is sequentially pressed together with the microfluidic modification layer and the microfluidic sample loading layer to form microfluidic chips with different functions; the reaction layer substrate and the microfluidic modification layer are pressed together for the first time to form a modified microfluidic chip; the reaction layer substrate modified by the modified microfluidic chip and the microfluidic sample loading layer are pressed together for the second time to form a detection microfluidic chip; the microfluidic channels in the detection microfluidic chip intersect vertically to form a detection point array, and the detection result of a single detection sample is presented in the form of a barcode to form a tumor marker barcode detection band.

According to the microfluidic sensor chip provided by the present invention, the base material of the reaction layer substrate is a transparent hard material, and the length and width dimensions are 20mm×20mm;

The height of each microfluidic channel of the microfluidic channel modification layer and the microfluidic channel sample loading layer is 50-100 μm, the width is 100-300 μm, and the aperture size of each sample loading hole and each outflow hole is 400-600 μm.

According to the microfluidic sensor chip provided by the present invention, the reaction layer substrate is obtained by depositing the Parylene C polymer material film on the base by a chemical vapor deposition method, and the Parylene C polymer material film has a thickness of 8-15 μm.

According to the microfluidic sensor chip provided by the present invention, the microfluidic modification layer and the microfluidic sample loading layer are both made by casting from a silicon mold; the materials of the microfluidic modification layer and the microfluidic sample loading layer are both PDMS; the microfluidic modification layer and the microfluidic sample loading layer are combined with the substrate by clamp pressing.

According to the microfluidic sensor chip provided by the present invention, the tumor marker capture antibody is fixed by physical adsorption between the tumor marker protein antibody and the substrate on which the Parylene C polymer material film is deposited.

According to the microfluidic sensor chip provided by the present invention, the microfluidic channel modification layer contains 20 microfluidic channels, and 20 different types of tumor marker barcode detection bands and control bands can be printed simultaneously, wherein the tumor marker barcode detection bands and control bands are arranged in parallel in the form of barcodes and are evenly spaced; the tumor marker barcode detection bands and control bands are printed on the Parylene C polymer material film to form an antigen capture area;

The microfluidic sample loading layer contains 20 microfluidic channels, and each microfluidic channel is arranged vertically with each tumor marker barcode detection band and control band to form a two-dimensional array of 400 detection points.

The present invention also provides a method for detecting protein tumor markers, which is implemented by using any of the microfluidic sensor chips for detecting protein tumor markers as described above, and the method comprises:

Inject the same or different samples to be tested from each sample addition well, wherein the samples to be tested contain antigens to be tested;

Suction is performed from each outflow hole to make each of the samples to be tested evenly distributed in the microfluidic channel, and a first incubation is performed to allow the antigen to be tested in each of the samples to be tested to fully combine with the tumor marker capture antibody on the reaction layer substrate;

After the first incubation is completed, the antigen to be tested in each of the samples to be tested specifically binds to the tumor marker capture antibody on the reaction layer substrate and is then fixed on the reaction layer substrate;

Discharging excess sample from each of the outflow holes;

Injecting 1% bovine serum albumin by mass from each of the sample loading holes, aspirating from each of the outflow holes to make the bovine serum albumin uniformly distributed in the microfluidic channel, performing a second incubation, and sealing the unoccupied base surface of the reaction layer substrate;

After the second incubation is completed, PBS buffer solution is injected from each of the sample addition wells for cleaning, and each of the outflow wells is continuously aspirated to remove the unabsorbed antigens to be tested and bovine serum albumin;

Injecting antibodies coupled with fluorescent dyes from each of the sample loading holes, aspirating from each of the outflow holes to make the antibodies coupled with fluorescent dyes evenly distributed in the microfluidic channel, and performing a third incubation;

After the third incubation is completed, the antibody coupled with the fluorescent dye specifically binds to the tumor marker capture antibody and is fixed on the reaction layer substrate;

The tumor markers are fluorescently detected and the test results are stored in the tumor marker barcode detection band.

According to the protein tumor marker detection method provided by the present invention, the first incubation time is 10-20 minutes, the second incubation time is 10-15 minutes, and the third incubation time is 15-30 minutes.

According to the method for detecting protein tumor markers provided by the present invention, the fluorescent detection of tumor markers comprises:

A fluorescent image is obtained by photographing with a photographing tool, and a fluorescent imaging sensor detection system is used to perform fluorescence intensity analysis on the fluorescent image.

According to the method for detecting protein tumor markers provided by the present invention, the method further comprises:

When injecting the sample to be tested, the microfluidic chip for detection is clamped by a sample adding fixture, and the sample to be tested is injected into the sample adding hole through a sample injector, and the amount of a single sample to be tested is less than 1 μL each time;

When suction is performed on each of the outflow holes, the sample to be tested is sucked by a vacuum pump so that the sample to be tested is evenly distributed in the microfluidic channel, thereby completing the sample loading;

When performing fluorescence detection on tumor markers, the detection microfluidic chip is clamped by a detection fixture, the bottom of the detection fixture is connected to a light source, and observation and shooting are performed at a preset observation window by a shooting tool.

The present invention also provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the program, the method for detecting protein tumor markers as described above is implemented.

The present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, and when the computer program is executed by a processor, the method for detecting a protein tumor marker as described above is implemented.

The present invention also provides a computer program product, comprising a computer program, wherein when the computer program is executed by a processor, the computer program implements any of the above-mentioned methods for detecting protein tumor markers.

The present invention provides a microfluidic sensor chip for protein tumor marker detection and a protein tumor marker detection method, which are integrated with a microfluidic detection chip through a Parylene C polymer film substrate; the microfluidic sample loading layer comprises a plurality of microfluidic channels arranged evenly and perpendicular to a plurality of barcode detection bands and a control band, and can perform parallel detection of a plurality of biological or chemical indicators on a plurality of samples at the same time; when the chip is in use, only the outflow hole and the sample loading hole are connected to the outside world, and the reaction process is completed in a closed microfluidic system, effectively reducing the interference of the external environment on the reaction and detection process; the detection result is stored in the form of a barcode, so it can be quickly obtained by a mobile device by scanning the barcode strip, which is user-friendly and convenient, and suitable for timely on-site care occasions; the microfluidic sensor chip provided by the present invention can simultaneously detect a variety of different protein tumor markers in a plurality of samples to be tested, and is suitable for large-scale rapid detection; and the chip has a simple structure, is easy to integrate and combine with matching automated equipment to realize automated detection, has the functions of rapid, stable, and on-site high-throughput detection, and makes the detection of on-site samples more convenient, simple, and rapid.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a schematic diagram of the modification and detection process of a microfluidic sensor chip for detecting protein tumor markers provided by an embodiment of the present invention;

FIG2 is a schematic diagram of a substrate layer deposited with a Parylene C film provided in an embodiment of the present invention;

FIG3 is a schematic diagram of the structure of a microchannel modification layer provided in an embodiment of the present invention;

FIG4 is a schematic diagram of the structure of a microchannel sample loading layer provided in an embodiment of the present invention;

FIG5 is a cross-sectional schematic diagram of a modified detection chip provided by an embodiment of the present invention;

FIG6 is a top view of a detection chip provided in an embodiment of the present invention;

FIG7 is a silicon mold for forming a microchannel sample loading layer and a modification layer provided by an embodiment of the present invention;

FIG8 is a schematic diagram of a sample adding fixture provided in an embodiment of the present invention;

FIG. 9 is a schematic diagram of a detection fixture provided in an embodiment of the present invention.

FIG10 is a schematic diagram of a process for detecting protein tumor markers according to an embodiment of the present invention;

FIG11 is a schematic flow chart of a method for modifying and detecting a barcode sensor chip based on a Parylene C thin film flexible substrate according to an embodiment of the present invention;

FIG. 12 is a schematic diagram of the structure of an electronic device provided by the present invention.

Reference numerals:

1: Reaction layer substrate; 2: Microfluidic modification layer; 3: Microfluidic loading layer; 4: Microfluidic chip for detection; 5: Microfluidic chip for modification; 6: Parylene C polymer film; 7: Chip substrate; 8: PDMS microfluidic layer; 9: Loading hole; 10: Outflow hole; 11: Microfluidic channel; 12: Tumor marker capture antibody; 13: Barcode detection signal; 14: Silicon mold for loading layer and modification layer; 15: Loading fixture; 16: Throttle valve; 17: Pipette; 18: Detection fixture; 19: Shooting window.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

In recent years, various types of cancer, as diseases with extremely high morbidity and mortality, have seriously endangered human health. The incidence of various malignant tumors has been increasing and has a trend of younger patients. If tumor lesions can be detected in time, early treatment and intervention can greatly improve the patient's survival period and survival rate. Tissue biopsy is considered the gold standard for tumor molecular analysis, which can be used to confirm, diagnose and classify tumor types, as well as guide treatment. However, biopsy of patients is an invasive operation, and its inherent health hazards, possible surgical complications and economic considerations make this measure limited. Therefore, in order to achieve early diagnosis and treatment of malignant tumors, researchers are more keen to detect tumor markers. Traditional tumor marker detection methods mainly include enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence analysis, mass spectrometry, etc. However, these methods cannot meet the requirements of instant detection. Enzyme-linked immunosorbent assay kits need to detect multiple biomarkers one by one and consume a large amount of samples. Electrochemical methods have high sensitivity, but their high-throughput detection capabilities are limited. Based on this, biochemical sensor chips have shown broad application prospects with their high throughput and low sample usage. In addition, barcodes, as a representation of object information, can read a large amount of information in a short time through a defined decoding device. In recent years, they have flourished in assisting multiple biological detection to meet the increasingly urgent needs of rapid diagnosis and disease treatment. The combination of biochemical sensor chips and barcodes has shown great potential in further improving detection throughput.

In view of this, the present invention provides a microfluidic sensor chip for detecting protein tumor markers, which can simultaneously detect multiple different protein tumor markers to achieve the purpose of rapid, stable and high-throughput detection.

The microfluidic sensor chip for protein tumor marker detection provided in an embodiment of the present invention comprises: a reaction layer substrate, wherein the reaction layer substrate comprises a substrate and a Parylene C polymer material film deposited on the substrate, wherein the Parylene C polymer material film is modified with a tumor marker capture antibody;

A microfluidic channel modification layer and a microfluidic channel sample loading layer, wherein a plurality of microfluidic channels are respectively arranged at the bottom of the microfluidic channel modification layer and the microfluidic channel sample loading layer, wherein the microfluidic channels in the microfluidic channel modification layer and the microfluidic channel sample loading layer are arranged in parallel, and circular sample loading holes and outflow holes are arranged at both ends of each microfluidic channel, which are connected with both ends of the microfluidic channel; and the microfluidic channels between the microfluidic channel modification layer and the microfluidic channel sample loading layer are arranged vertically;

The reaction layer substrate is sequentially pressed together with the microfluidic modification layer and the microfluidic sample loading layer to form microfluidic chips with different functions; the reaction layer substrate and the microfluidic modification layer are pressed together for the first time to form a modified microfluidic chip; the reaction layer substrate modified by the modified microfluidic chip and the microfluidic sample loading layer are pressed together for the second time to form a detection microfluidic chip; the microfluidic channels in the detection microfluidic chip intersect vertically to form a detection point array, and the detection result of a single detection sample is presented in the form of a barcode to form a tumor marker barcode detection band.

In a specific embodiment, FIG. 1 is a schematic diagram of the modification and detection process of a microfluidic sensor chip for detecting protein tumor markers provided in an embodiment of the present invention. As shown in FIG. 1 , the microfluidic sensor chip includes a reaction layer substrate 1, a microfluidic sample loading layer 3 and a modification layer 2 located thereon. The reaction layer substrate 1 and the microfluidic modification layer 2 are pressed together for the first time to form a modified microfluidic chip 5. The reaction layer substrate 1 and the microfluidic sample loading layer 3 are pressed together for the second time to form a detection microfluidic chip 4. FIG. 2 is a schematic diagram of a substrate layer deposited with a Parylene C film provided in an embodiment of the present invention. As shown in FIG. 2 , the reaction layer substrate 1 includes a substrate 7 and a Parylene C film deposited on the substrate. C polymer material film 6, the material of the substrate 7 is transparent hard material such as glass, quartz or silicon wafer; Figure 3 is a schematic diagram of the structure of the microfluidic modification layer provided in an embodiment of the present invention, and Figure 4 is a schematic diagram of the structure of the microfluidic sample loading layer provided in an embodiment of the present invention. As shown in Figures 3 and 4, a plurality of microfluidic channels 11 are arranged at the bottom of the microfluidic sample loading layer 3 and the modification layer 2, and the plurality of microfluidic channels 11 are arranged in parallel. The two ends of the microfluidic channels 11 in the microfluidic sample loading layer 3 and the modification layer 2 are provided with circular sample loading holes 9 and outflow holes 10 that are connected from top to bottom; Figure 5 is a schematic diagram of the cross-section of the modified detection chip provided in an embodiment of the present invention. As shown in Figure 5, the circular sample loading holes 9 and the outflow holes 10 are connected to the two ends of the microfluidic channel 11, and Parylene is deposited on the bottom substrate of the microfluidic channel 11. C polymer material film 6, on which tumor marker capture antibody 12 is modified; FIG. 6 is a top view of the detection chip provided in an embodiment of the present invention. As shown in FIG. 6, the microfluidic channel 11 between the microfluidic sample loading layer 3 and the modification layer 2 is arranged vertically to form a detection point array, and the detection results between single detection samples are presented in the form of barcode 13; FIG. 7 is a silicon mold for mold-making the microfluidic sample loading layer and the modification layer provided in an embodiment of the present invention. As shown in FIG. 7, the microfluidic sample loading layer 3 and the modification layer 2 are molded by a silicon mold 14; FIG. 8 is a sample loading fixture provided in an embodiment of the present invention Schematic diagram; as shown in Figure 8, when adding the sample to be tested, the microfluidic chip 4 for detection is clamped by the sample adding fixture 15, and added to the sample adding hole 9 by the pipette gun 17, and then the liquid sample is sucked by the vacuum pump connected to the throttle valve 16 to make it evenly distributed in the microfluidic channel to complete the sample adding; Figure 9 is a schematic diagram of the detection fixture provided by an embodiment of the present invention. As shown in Figure 9, when detecting the fluorescent signal, the microfluidic chip 4 for detection is clamped by the detection fixture 18, and the light source is connected to the bottom of the detection fixture 18, and the CMOS camera or mobile phone is used to observe and shoot at the observation window 19.

The Parylene C film provided by the embodiment of the present invention is chemically inert, non-biodegradable, and is widely used as a coating for implantable biomedical devices. Parylene C film can be deposited onto a substrate by vapor deposition, and the resulting film is uniform and non-porous. The all-carbon structural backbone, high molecular weight, and non-polar entities make Parylene C highly resistant to most chemicals as well as fungal and bacterial growth. Studies have shown that Parylene C is extremely hydrophobic and has good adsorption capacity for proteins. It has a stronger adsorption capacity than quartz and polystyrene (commonly used matrix materials in ELISA biosensors), and is very suitable as an immunoprotein adsorption substrate.

Optionally, the base material of the reaction layer substrate is a transparent hard material such as glass, quartz or silicon wafer, a Parylene C polymer material film is deposited on the base, and the base has a length and width of 20 mm×20 mm.

Optionally, the reaction layer substrate is obtained by depositing the Parylene C polymer material film on the substrate by a chemical vapor deposition method, and the Parylene C polymer material film has a thickness of 8-15 μm.

Optionally, the microfluidic modification layer and the microfluidic sample loading layer are both made of silicon molds; the materials of the microfluidic modification layer and the microfluidic sample loading layer are both PDMS; the microfluidic modification layer and the microfluidic sample loading layer are combined with the substrate for a short time by clamping.

Optionally, the tumor marker capture antibody is fixed on the substrate by physical adsorption between the tumor marker protein antibody and the substrate on which the Parylene C polymer material film is deposited.

Optionally, the height of each microfluidic channel of the microfluidic channel modification layer and the microfluidic channel loading layer is 50-100 μm, the width is 100-300 μm, and the pore size of each loading hole and outflow hole is 400-600 μm.

Optionally, the microfluidic channel modification layer contains 20 microfluidic channels, and 20 different types of tumor marker barcode detection bands and control bands can be printed simultaneously, wherein the tumor marker barcode detection bands and control bands are arranged in parallel in the form of barcodes and are evenly spaced; the tumor marker barcode detection bands and control bands are printed on the Parylene C polymer material film to form an antigen capture area.

Optionally, the microfluidic sample loading layer contains 20 microfluidic channels, and each microfluidic channel is arranged vertically to each tumor marker barcode detection band and control band to form a two-dimensional array of 400 detection points.

On the basis of the above embodiments, the present invention further provides a method for detecting protein tumor markers, which is implemented using the microfluidic chip for detecting protein tumor markers as described in any of the above items. FIG. 10 is a flow chart of the method for detecting protein tumor markers provided by an embodiment of the present invention. As shown in FIG. 10 , the method includes:

Step 1010, injecting the same or different samples to be tested from each sample injection hole, wherein the samples to be tested contain antigens to be tested;

Step 1020, aspirating from each outflow hole to make each of the samples to be tested evenly distributed in the microfluidic channel, and performing a first incubation to allow the antigen to be tested in each of the samples to be tested to fully combine with the tumor marker capture antibody on the reaction layer substrate;

Step 1030, after the first incubation is completed, the antigen to be tested in each of the samples to be tested specifically binds to the tumor marker capture antibody on the reaction layer substrate and is then fixed on the reaction layer substrate;

Step 1040, discharging excess sample from each of the outflow holes;

Step 1050, injecting 1% bovine serum albumin from each of the sample injection holes, aspirating from each of the outflow holes to make the bovine serum albumin uniformly distributed in the microfluidic channel, performing a second incubation, and sealing the unoccupied base surface of the reaction layer substrate;

Step 1060, after the second incubation is completed, PBS buffer solution is injected from each of the sample addition wells for washing, and each of the outflow wells is continuously aspirated to remove the unadsorbed antigens and bovine serum albumin;

Step 1070, injecting antibodies coupled with fluorescent dyes from each of the sample injection holes, aspirating from each of the outflow holes to make the antibodies coupled with fluorescent dyes evenly distributed in the microfluidic channel, and performing a third incubation;

Step 1080, after the third incubation is completed, the antibody coupled with the fluorescent dye specifically binds to the tumor marker capture antibody and is fixed on the reaction layer substrate;

Step 1090: Perform fluorescence detection on the tumor marker and store the detection result in the tumor marker barcode detection band.

Preferably, the first incubation time is 10-20 minutes, the second incubation time is 10-15 minutes, and the third incubation time is 15-30 minutes.

Optionally, the fluorescent detection of tumor markers includes:

A fluorescent image is obtained by photographing with a photographing tool, and a fluorescent imaging sensor detection system is used to perform fluorescence intensity analysis on the fluorescent image.

Specifically, a fluorescence imaging sensor detection system is used to perform fluorescence intensity analysis, and images can be taken by a CMOS camera or a mobile phone camera. Since the detection band is arranged in the form of a barcode, the qualitative detection results can be quickly obtained by the barcode encoding pre-packaged in the customized software, and the quantitative results can also be calculated by comparing with the standard curve pre-measured and packaged in the customized software. The entire detection time does not exceed 10 minutes.

Based on the above embodiment, the method further includes:

When injecting the sample to be tested, the microfluidic chip for detection is clamped by a sample adding fixture, and the sample to be tested is injected into the sample adding hole through a sample injector, and the amount of a single sample to be tested is less than 1 μL each time;

When suction is performed on each of the outflow holes, the sample to be tested is sucked by a vacuum pump so that the sample to be tested is evenly distributed in the microfluidic channel, thereby completing the sample loading;

When performing fluorescence detection on tumor markers, the detection microfluidic chip is clamped by a detection fixture, the bottom of the detection fixture is connected to a light source, and observation and shooting are performed at a preset observation window by a shooting tool.

Optionally, the sample injector is a pipette or a syringe.

Based on any of the above embodiments, the present invention further provides a specific embodiment of a method for modifying and detecting a barcode sensor chip based on a Parylene C film flexible substrate. FIG. 11 is a flow chart of a method for modifying and detecting a barcode sensor chip based on a Parylene C film flexible substrate according to an embodiment of the present invention. As shown in FIG. 11 , the method includes:

Step 1110, generating a Parylene C film substrate:

In this step, the Parylene C polymer material film 6 is prepared by placing a clean substrate 7 into a CVD system for growing Parylene film, setting CVD growth parameters, growing the Parylene C film, calculating and weighing the raw materials according to the growth data, controlling the thickness of the grown Parylene C film, and completing the film preparation after ultrasonic cleaning and drying.

Preferably, in this embodiment, the substrate 7 is made of quartz or glass, with a length and width of 20 mm and a thickness of 1.5 mm; the thickness of the Parylene C film is 8-15 μm.

Step 1120, printing barcode detection tape:

In this step, a plurality of detection band and control band patterns are printed using a microfluidic modification layer 2. Specifically, a silicon mold 14 is used as a template, a PDMS material is used to mold, and holes are punched at the circular sample loading hole 9 and the outflow hole 10 to obtain a microfluidic modification layer 2; the PDMS microfluidic modification layer 2 is covered on the reaction layer substrate 1, and is clamped in a sample loading fixture 15. By utilizing the elasticity of the PDMS material, the microfluidic modification layer 2 and the reaction layer substrate 1 can be tightly fitted through non-bonding, and the through holes and flow channels are sealed to prevent solution leakage here. The tumor marker capture antibody 12 can be added from the sample loading hole 9 using a flat-head needle or a pipette. Preferably, a pipette 17 with a range of 10 μL is used for loading. Different tumor marker capture antibodies 12 and blank controls of 0.5 μL are added to each flow channel, respectively. The number of repetitions of certain antibodies or blank controls can be increased depending on the specific application scenario to increase the detection accuracy and reduce errors.

Preferably, in this embodiment, the tumor marker antibodies used include alpha-fetoprotein (AFP) antibody, carcinoembryonic antigen (CEA) antibody, cancer antigen 125 (CA125) antibody, cancer antigen 15-3 (CA15-3) antibody, prostate specific antigen (PSA) antibody, cancer antigen 19-9 (CA19-9) antibody, cancer antigen 72-4 (CA72-4) antibody, neuron-specific enolization (NSE) antibody, cytokeratin fragment 19 (CYFRA21-1) antibody, squamous cell carcinoma antigen (SCC) antibody and progastrin-releasing peptide (ProGRP) antibody.

After the sample is added, the vacuum pump is connected to the throttle valve 16 to adjust the air intake. After the liquid is evenly distributed in the microfluidic channel 11, the vacuum pump is turned off. Incubate at 37°C for 1 hour, then wash three times with phosphate buffered saline (PBS) solution, and peel off the microfluidic modification layer 2 after drying to complete the capture antibody printing.

Step 1130, adding the sample to be tested:

In this step, the silicon mold 14 is used as a template in the same manner as in the above step, and the PDMS material is used to mold the microfluidic sample layer 3; the PDMS microfluidic sample layer 3 is attached to the reaction layer substrate 1, and the microfluidic channel 11 is kept vertically distributed with the previously printed capture antibody detection band and control band. Similarly, the detection microfluidic chip 4 covering the PDMS microfluidic layer 8 is placed in the sample clamp 15 for clamping, and 0.5 μL of the sample to be tested from different sources is injected from multiple sample holes 9 with a pipette 17. After the vacuum pump is sucked to evenly distribute the liquid in the microfluidic channel 11, the vacuum pump is turned off and incubated for 20 minutes to allow the antigen to be tested in the sample to be tested to fully bind to the protein tumor marker capture antibody printed on the reaction layer substrate 1. After the incubation is completed, the excess sample to be tested is drained from the outflow hole 10. At this time, the antigen to be tested is specifically bound to the protein tumor marker capture antibody and fixed on the reaction layer substrate 1.

Step 1140, sealing the reaction layer substrate:

In this step, bovine serum albumin (BSA) with a mass concentration of 1% is added to block the reaction layer substrate 1 to occupy the unbound surface vacancies. After incubation for 3 minutes, it is rinsed with PBS buffer solution to rinse away the antigen to be tested and other impurities that are not adsorbed on the reaction layer substrate 1.

Step 1150, labeling antigen:

In this step, the corresponding second antibody coupled with fluorescent dye is added to the microfluidic channel 11, and after incubation for 20 minutes, the excess second antibody is discharged. At this time, the second antibody labeled with fluorescent dye specifically binds to the antigen that may exist in the sample to be tested on the reaction layer substrate 1, and then the protein tumor marker is fluorescently detected.

Preferably, in this embodiment, the fluorescent dye-conjugated secondary antibody used is an antibody conjugated with Alexa Fluor 488 fluorescent dye.

Step 1160, fluorescence detection:

In this step, a fluorescence imaging sensor detection system is used to analyze the fluorescence intensity. The image can be taken by a CMOS camera or a mobile phone camera. Since the detection band 4 is arranged in the form of a barcode, the qualitative detection result can be quickly obtained by the barcode encoding pre-packaged in the customized software. The quantitative result can be calculated by comparing with the standard curve pre-measured and packaged in the customized software. The entire detection time does not exceed 10 minutes.

Specifically, a fluorescent dye-coupled antibody solution diluted to different concentrations is prepared and added to a microfluidic channel without a printed capture antibody detection band. Preferably, Alexa Fluor 488 goat anti-mouse IgG antibody is used to prepare a gradient solution with a concentration of 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, 400 ng/mL, 500 ng/mL, 800 ng/mL, and 1000 ng/mL. The solution is photographed using a CMOS camera, and the resulting image is processed by ImageJ software. The central rectangular area is selected, the green channel is separated, the color intensity is averaged, a standard curve is drawn with the antibody concentration as the abscissa and the color intensity as the ordinate, and Origin is used for linear fitting to obtain the standard curve equation.

The Parylene C polymer film substrate of the present invention is integrated with a microfluidic detection chip. The substrate is extremely hydrophobic, has good adsorption capacity for proteins, has low manufacturing cost, and can accurately control thickness. The microfluidic sample loading layer comprises a plurality of microfluidic channels evenly arranged and perpendicular to a plurality of barcode detection bands and control bands, and can perform parallel detection of a plurality of biological or chemical indicators on a plurality of samples at the same time. When the chip is in use, only the outflow hole and the sample loading hole are connected to the outside world, and most of the reaction processes are completed in a closed microfluidic system, effectively reducing the interference of the external environment on the reaction and detection process. The width of the microfluidic channel is less than 40 The chip has a diameter of 0μm and a thickness of less than 70μm, and a single test can be completed with an extremely low sample volume of less than 1μL; the test results can be quickly obtained by scanning the barcode strips through mobile devices such as smart phones, which is user-friendly and convenient, and suitable for timely on-site care; the chip can integrate 400 detection points on an area of 20mm×20mm, and can detect up to 20 different protein tumor markers in 20 samples to be tested at the same time, which is suitable for large-scale rapid testing; and the above-mentioned chip has a simple structure and is easy to integrate and combine with supporting automated equipment to realize automated testing.

The chip can detect a variety of different protein tumor markers at the same time, and has the functions of rapid, stable, and on-site high-throughput detection, making on-site sample detection more convenient, simple, and rapid.

Figure 12 illustrates a schematic diagram of the physical structure of an electronic device. As shown in Figure 12, the electronic device may include: a processor (processor) 1210, a communication interface (Communications Interface) 1220, a memory (memory) 1230 and a communication bus 1240, wherein the processor 1210, the communication interface 1220, and the memory 1230 communicate with each other through the communication bus 1240. The processor 1210 can call the logic instructions in the memory 1230 to execute the protein tumor marker detection method, which includes: injecting the same or different samples to be tested from each sample addition hole, wherein the samples to be tested contain antigens to be tested; aspirating from each outflow hole to make each sample to be tested evenly distributed in the microfluidic channel, and performing a first incubation to make the antigens to be tested in each sample to be tested fully bind with the tumor marker capture antibodies on the reaction layer substrate; after the first incubation is completed, the antigens to be tested in each sample to be tested specifically bind with the tumor marker capture antibodies on the reaction layer substrate and are fixed on the reaction layer substrate; discharging excess samples to be tested from each outflow hole; injecting bovine serum albumin with a mass fraction of 1% from each sample addition hole, aspirating from each outflow hole, The bovine serum albumin is evenly distributed in the microfluidic channel, and a second incubation is performed to close the unoccupied base surface of the reaction layer substrate; after the second incubation is completed, PBS buffer solution is injected from each sample loading hole for cleaning, and each outflow hole is continuously aspirated to remove the unadsorbed antigen to be tested and bovine serum albumin; antibodies coupled with fluorescent dyes are injected from each sample loading hole, and aspiration is performed from each outflow hole to make the antibodies coupled with fluorescent dyes evenly distributed in the microfluidic channel, and a third incubation is performed; after the third incubation is completed, the antibodies coupled with fluorescent dyes are fixed on the reaction layer substrate after specific binding with the tumor marker capture antibody; fluorescence detection is performed on the tumor marker, and the detection results are stored in the tumor marker barcode detection band.

In addition, the logic instructions in the above-mentioned memory 1230 can be implemented in the form of a software functional unit and can be stored in a computer-readable storage medium when it is sold or used as an independent product. Based on such an understanding, the technical solution of the present invention can be essentially or partly embodied in the form of a software product that contributes to the prior art. The computer software product is stored in a storage medium, including several instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the method described in each embodiment of the present invention. The aforementioned storage medium includes: various media that can store program codes, such as a USB flash drive, a mobile hard disk, a read-only memory (ROM), a random access memory (RAM), a magnetic disk or an optical disk.

On the other hand, the present invention also provides a computer program product, which includes a computer program. The computer program can be stored in a non-transitory computer-readable storage medium. When the computer program is executed by a processor, the computer can execute the protein tumor marker detection method provided by the above methods, which includes: injecting the same or different samples to be tested from each sample addition hole, wherein the samples to be tested contain antigens to be tested; aspirating from each outflow hole so that each sample to be tested is evenly distributed in the microfluidic channel, and performing a first incubation so that the antigens to be tested in each sample to be tested are fully combined with the tumor marker capture antibodies on the reaction layer substrate; after the first incubation is completed, the antigens to be tested in each sample to be tested are specifically combined with the tumor marker capture antibodies on the reaction layer substrate and then fixed on the reaction layer substrate; discharging excess sample to be tested from each outflow hole; and Inject 1% bovine serum albumin by mass into the sample wells, aspirate from the outflow holes to make the bovine serum albumin uniformly distributed in the microfluidic channel, perform a second incubation, and seal the unoccupied base surface of the reaction layer substrate; after the second incubation is completed, inject PBS buffer solution from the sample wells for cleaning, and continuously aspirate from the outflow holes to remove the unadsorbed antigens and bovine serum albumin; inject antibodies coupled with fluorescent dyes from the sample wells, aspirate from the outflow holes to make the antibodies coupled with fluorescent dyes uniformly distributed in the microfluidic channel, and perform a third incubation; after the third incubation is completed, the antibodies coupled with fluorescent dyes are fixed on the reaction layer substrate after specifically binding to the tumor marker capture antibody; perform fluorescence detection on the tumor markers, and store the detection results in the tumor marker barcode detection band.

In another aspect, the present invention further provides a non-transitory computer-readable storage medium having a computer program stored thereon, and when the computer program is executed by a processor, the method for detecting protein tumor markers provided by the above methods is implemented, the method comprising: injecting the same or different samples to be tested from each sample addition hole, wherein the samples to be tested contain antigens to be tested; aspirating from each outflow hole so that each sample to be tested is evenly distributed in the microfluidic channel, and performing a first incubation so that the antigens to be tested in each sample to be tested fully bind to the tumor marker capture antibodies on the reaction layer substrate; after the first incubation is completed, the antigens to be tested in each sample to be tested specifically bind to the tumor marker capture antibodies on the reaction layer substrate and are fixed on the reaction layer substrate; discharging excess samples to be tested from each outflow hole; injecting 1% by mass fraction of 1% from each sample addition hole. bovine serum albumin, aspirating from each of the outflow holes so that each of the bovine serum albumin is evenly distributed in the microfluidic channel, performing a second incubation, and sealing the unoccupied base surface of the reaction layer substrate; after the second incubation is completed, PBS buffer solution is respectively injected from each of the sample loading holes for cleaning, and each of the outflow holes is continuously aspirated to remove the unadsorbed antigen to be tested and bovine serum albumin; antibodies coupled with fluorescent dyes are respectively injected from each of the sample loading holes, aspirating from each of the outflow holes so that the antibodies coupled with fluorescent dyes are evenly distributed in the microfluidic channel, and performing a third incubation; after the third incubation is completed, the antibodies coupled with fluorescent dyes are specifically combined with the tumor marker capture antibody and then fixed on the reaction layer substrate; fluorescent detection of tumor markers is performed, and the detection results are stored in the tumor marker barcode detection band.

The device embodiments described above are merely illustrative, wherein the units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they may be located in one place, or they may be distributed on multiple network units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the scheme of this embodiment. Those of ordinary skill in the art may understand and implement it without creative work.

Through the description of the above implementation methods, those skilled in the art can clearly understand that each implementation method can be implemented by means of software plus a necessary general hardware platform, and of course, it can also be implemented by hardware. Based on this understanding, the above technical solution is essentially or the part that contributes to the prior art can be embodied in the form of a software product, and the computer software product can be stored in a computer-readable storage medium, such as ROM/RAM, a disk, an optical disk, etc., including a number of instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in each embodiment or some parts of the embodiments.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A microfluidic sensor chip for detecting protein tumor markers, comprising: A reaction layer substrate, the reaction layer substrate comprising a substrate and a Parylene C polymer material film deposited on the substrate, wherein the Parylene C polymer material film is modified with a tumor marker capture antibody; A microfluidic channel modification layer and a microfluidic channel sample loading layer, wherein a plurality of microfluidic channels are respectively arranged at the bottom of the microfluidic channel modification layer and the microfluidic channel sample loading layer, wherein the microfluidic channels in the microfluidic channel modification layer and the microfluidic channel sample loading layer are arranged in parallel, and circular sample loading holes and outflow holes are arranged at both ends of each microfluidic channel, which are connected with both ends of the microfluidic channel; and the microfluidic channels between the microfluidic channel modification layer and the microfluidic channel sample loading layer are arranged vertically; The reaction layer substrate is sequentially pressed together with the microfluidic modification layer and the microfluidic sample loading layer to form microfluidic chips with different functions; the reaction layer substrate and the microfluidic modification layer are pressed together for the first time to form a modified microfluidic chip; the reaction layer substrate modified by the modified microfluidic chip and the microfluidic sample loading layer are pressed together for the second time to form a detection microfluidic chip; the microfluidic channels in the detection microfluidic chip intersect vertically to form a detection point array, and the detection result of a single detection sample is presented in the form of a barcode to form a tumor marker barcode detection band. 2. The microfluidic sensor chip according to claim 1, characterized in that: The base material of the reaction layer substrate is a transparent hard material, and the length and width dimensions are 20mm×20mm; The height of each microfluidic channel of the microfluidic channel modification layer and the microfluidic channel sample loading layer is 50-100 μm, the width is 100-300 μm, and the aperture size of each sample loading hole and each outflow hole is 400-600 μm. 3. The microfluidic sensor chip according to claim 1, characterized in that: The reaction layer substrate is obtained by depositing the Parylene C polymer material film on the base by a chemical vapor deposition method, and the Parylene C polymer material film has a thickness of 8-15 μm. 4. The microfluidic sensor chip according to claim 1, characterized in that: The microfluidic channel modification layer and the microfluidic channel loading layer are both made of silicon molds; the materials of the microfluidic channel modification layer and the microfluidic channel loading layer are both PDMS; the microfluidic channel modification layer and the microfluidic channel loading layer are combined with the substrate by clamping. 5. The microfluidic sensor chip according to claim 1, characterized in that: The tumor marker capture antibody is fixed by physical adsorption between the tumor marker protein antibody and the substrate on which the Parylene C polymer material film is deposited. 6. The microfluidic sensor chip according to any one of claims 1 to 5, characterized in that the microfluidic channel modification layer contains 20 microfluidic channels, and 20 different types of tumor marker barcode detection bands and control bands can be printed simultaneously, wherein the tumor marker barcode detection bands and control bands are arranged in parallel in the form of barcodes and are evenly spaced; the tumor marker barcode detection bands and control bands are printed on the Parylene C polymer material film to form an antigen capture area; The microfluidic sample loading layer contains 20 microfluidic channels, and each microfluidic channel is arranged vertically with each tumor marker barcode detection band and control band to form a two-dimensional array of 400 detection points. 7. A method for detecting protein tumor markers, characterized in that it is implemented by using the microfluidic chip for detecting protein tumor markers according to any one of claims 1 to 6, and the method comprises: Inject the same or different samples to be tested from each sample addition well, wherein the samples to be tested contain antigens to be tested; Suction is performed from each outflow hole to make each of the samples to be tested evenly distributed in the microfluidic channel, and a first incubation is performed to allow the antigen to be tested in each of the samples to be tested to fully combine with the tumor marker capture antibody on the reaction layer substrate; After the first incubation is completed, the antigen to be tested in each of the samples to be tested specifically binds to the tumor marker capture antibody on the reaction layer substrate and is then fixed on the reaction layer substrate; Discharging excess sample from each of the outflow holes; Injecting 1% bovine serum albumin by mass from each of the sample loading holes, aspirating from each of the outflow holes to make the bovine serum albumin uniformly distributed in the microfluidic channel, performing a second incubation, and sealing the unoccupied base surface of the reaction layer substrate; After the second incubation is completed, PBS buffer solution is injected from each of the sample addition wells for cleaning, and each of the outflow wells is continuously aspirated to remove the unabsorbed antigens to be tested and bovine serum albumin; Injecting antibodies coupled with fluorescent dyes from each of the sample loading holes, aspirating from each of the outflow holes to make the antibodies coupled with fluorescent dyes evenly distributed in the microfluidic channel, and performing a third incubation; After the third incubation is completed, the antibody coupled with the fluorescent dye specifically binds to the tumor marker capture antibody and is fixed on the reaction layer substrate; The tumor markers are fluorescently detected and the test results are stored in the tumor marker barcode detection band. 8. The method for detecting protein tumor markers according to claim 7, characterized in that the first incubation time is 10-20 minutes, the second incubation time is 10-15 minutes, and the third incubation time is 15-30 minutes. 9. The method for detecting protein tumor markers according to claim 7, wherein the fluorescence detection of tumor markers comprises: A fluorescent image is obtained by photographing with a photographing tool, and a fluorescent imaging sensor detection system is used to perform fluorescence intensity analysis on the fluorescent image. 10. The method for detecting protein tumor markers according to claim 7, characterized in that the method further comprises: When injecting the sample to be tested, the microfluidic chip for detection is clamped by a sample adding fixture, and the sample to be tested is injected into the sample adding hole through a sample injector, and the amount of a single sample to be tested is less than 1 μL each time; When suction is performed on each of the outflow holes, the sample to be tested is sucked by a vacuum pump so that the sample to be tested is evenly distributed in the microfluidic channel, thereby completing the sample loading; When performing fluorescence detection on tumor markers, the detection microfluidic chip is clamped by a detection fixture, the bottom of the detection fixture is connected to a light source, and observation and shooting are performed at a preset observation window by a shooting tool.