阅读说明：本技术 一种基于磁阻生物传感器的微流控磁敏免疫装置及其使用方法 (Micro-fluidic magnetic-sensitive immunization device based on magnetic-resistance biosensor and use method thereof ) 是由 彭年才 张航 胡飞 李治鹏 郭晓牛 于 2019-09-17 设计创作，主要内容包括：本发明一种基于磁阻生物传感器的微流控磁敏免疫装置及其使用方法,微流控磁敏免疫装置中,上盖上设置超顺磁珠存储区和抗体存储区,微流控芯片上设置第一生化反应腔和第一微流控通道,使用时,生物素化的超顺磁珠和生物素化的抗体依次在第一生化反应腔和第一微流控通道中进行混合反应,反应后的标记抗体的超顺磁珠再与待测样品反应,生成标记超顺磁珠的免疫蛋白,然后与磁传感器阵列中的捕获抗体反应结合,从而将超顺磁珠固定在磁传感器阵列中的传感器上进行后续的检测过程。本发明将磁敏免疫分析中超顺磁珠与生物素化的抗体的结合整合到芯片中完成,提高了集成度,简化了生化反应的操作步骤,提高了生化反应效率。(The invention relates to a micro-fluidic magnetic-sensing immunization device based on a magnetoresistive biosensor and a using method thereof. The invention integrates the combination of the superparamagnetic beads and the biotinylated antibodies in the magnetic-sensitive immunoassay into a chip to complete, improves the integration level, simplifies the operation steps of biochemical reaction and improves the biochemical reaction efficiency.)

1. A micro-fluidic magnetic-sensing immune device based on a magnetic-resistance biosensor is characterized by comprising a bottom plate (4) and an upper cover (1) which are fixedly connected, wherein a micro-fluidic chip (2) is arranged between the bottom plate (4) and the upper cover (1);

a superparamagnetic bead storage region (12), an antibody storage region (13), a sample storage region to be detected (14) and a washing liquid storage region (15) are arranged on the upper cover (1); the micro-fluidic chip (2) is provided with a first biochemical reaction cavity (213), a second biochemical reaction cavity (205), a detection area (208) and a waste liquid cavity (207); a sample inlet of the first biochemical reaction cavity (213) is communicated with the superparamagnetic bead storage area (12) and the antibody storage area (13), a sample outlet is communicated with a sample inlet of the second biochemical reaction cavity (205) through the first microfluidic channel (203), the sample inlet of the second biochemical reaction cavity (205) is also communicated with a sample storage area (14) to be detected, the sample outlet of the second biochemical reaction cavity (205) is communicated with a sample inlet of the detection area (208) through the second microfluidic channel (206), and the sample inlet of the detection area (208) is also communicated with the washing solution storage area (15); a sample outlet of the detection area (208) is communicated with the waste liquid cavity (207);

the micro-fluidic chip (2) is also provided with a groove (215), the groove (215) corresponds to the detection area (208) in the upper and lower positions and is communicated with the detection area, a magnetic sensor array (3) capable of combining immune protein is embedded in the groove (215), and the opening of the groove (215) is sealed.

2. The microfluidic magnetosensitive immune device based on a magnetoresistive biosensor as claimed in claim 1, wherein the upper cover (1) is provided with an exhaust hole (16), and the exhaust hole (16) is communicated with the waste liquid cavity (207).

3. The microfluidic magnetosensitive immune device based on a magnetoresistive biosensor as claimed in claim 2, wherein the microfluidic chip (2) is provided with a waste liquid hole (209), the waste liquid hole (209) corresponds to the exhaust hole (16) up and down, and the waste liquid cavity (207) is communicated with the exhaust hole (16) through the waste liquid hole (209).

4. A magnetoresistive biosensor-based microfluidic magnetosensitive immune device according to claim 1, characterized in that the microfluidic chip (2) comprises a substrate (201) and a thin film (202) fixed on the substrate (201), and a first biochemical reaction chamber (213), a second biochemical reaction chamber (205), a detection zone (208) and a waste liquid chamber (207) are enclosed between the thin film (202) and the substrate (201).

5. A magnetoresistive biosensor-based microfluidic magnetosensitive immunization device as claimed in claim 4, wherein the membrane (202) is a PDMS membrane and the substrate is a PMMA substrate.

6. A magnetorsistive sensing immune set based on a magnetoresistive biosensor as claimed in claim 1, wherein the microfluidic chip (2) is provided with a first sample well (212), a second sample well (211), a third sample well (204) and a fourth sample well (214); the first biochemical reaction cavity (213) is communicated with the superparamagnetic bead storage region (12) through a first sample adding hole (212), the first biochemical reaction cavity (213) is communicated with the antibody storage region (13) through a second sample adding hole (211), the second biochemical reaction cavity (205) is communicated with the sample storage region (14) to be detected through a third sample adding hole (204), and the detection region (208) is communicated with the washing liquid storage region (15) through a fourth sample adding hole (214); the superparamagnetic bead storage region (12), the antibody storage region (13), the sample storage region to be detected (14) and the washing liquid storage region (15) respectively correspond to the positions of the first sample adding hole (212), the second sample adding hole (211), the third sample adding hole (204) and the fourth sample adding hole (214) up and down.

7. A magnetoresistive biosensor-based microfluidic magnetosensitive immunization device as claimed in claim 1, wherein the first microfluidic channel (203) is a serpentine channel and the second microfluidic channel (206) is an S-shaped channel.

8. A magnetoresistive biosensor-based microfluidic magnetosensitive immune device according to claim 1, characterized in that the magnetic sensor array (3) comprises a substrate (31), a plurality of sensors (33) and a plurality of electrodes (32); the sensor (33) and the electrode (32) are arranged on the substrate (31), a power supply interface of the sensor (33) is connected with an external power supply through the electrode (32), a signal output interface of the sensor (33) is connected with an external PCB through the electrode (32), the electrode (32) is connected with the external PCB through a lead (34), and the electrode (32) is connected with the external PCB through the lead; a layer of SiO is arranged on the sensor (33)₂Protective film, SiO₂The protective film is provided with a layer of gold film, and the gold film is marked with a capture antibody.

9. A micro-fluidic magnetic-sensing immunization device based on a magnetoresistive biosensor as claimed in claim 1, wherein the upper surface of the bottom plate (4) is provided with a coil (44) and a heating plate (43), the coil (44) is electrically connected with the outside through a first lead (45), and the heating plate (43) is electrically connected with the outside through a second lead (42); the center of the coil (44) is positioned right below the detection area (208); the heating plate (43) is positioned below the second biochemical reaction chamber (205) and the first biochemical reaction chamber (213).

10. Use of a microfluidic magnetosensitive immunization device based on a magnetoresistive biosensor as claimed in any of claims 1 to 9, characterized in that it comprises the following steps:

(1) adding superparamagnetic beads into a superparamagnetic bead storage area (12), adding biotinylated antibodies into an antibody storage area (13), adding samples to be detected into a sample storage area (14) to be detected, and adding washing liquid into a washing liquid storage area (15);

(2) applying pressure, pressing the superparamagnetic beads and the biotinylated antibodies into a first biochemical reaction cavity (213) and then into a first microfluidic channel (206) to react to form superparamagnetic beads for labeling the antibodies; continuously applying pressure, pressing the superparamagnetic beads marked with the antibodies into a second biochemical reaction cavity (205), pressing the sample to be detected into the second biochemical reaction cavity (205), continuously applying pressure, and allowing the superparamagnetic beads marked with the antibodies and the sample to be detected to enter a second microfluidic channel (206) to react to form the immunoprotein marked with the superparamagnetic beads;

(3) continuously applying pressure to press the immune protein labeled with the superparamagnetic beads into the detection area (208) and fixing the immune protein by the magnetic sensor array (3); applying pressure to inject the washing solution into the detection zone (208) for washing;

(4) and applying a magnetic field perpendicular to the magnetic sensor array (3), magnetizing the superparamagnetic beads, collecting resistance signals of the magnetic sensor array (3) through an external detection system, and calculating the concentration of the immune protein in the sample to be detected according to a standard curve of the concentration of the immune protein and the change of the resistivity.

Technical Field

The invention relates to magnetic-sensitive immunoassay, in particular to a micro-fluidic magnetic-sensitive immunoassay device based on a magnetic-resistance biosensor and a using method thereof.

Background

The magnetic-sensitive immunoassay is a new immunoassay method which appears after fluorescence immunoassay, chemiluminescence, electrochemiluminescence, electrochemistry and surface Raman enhancement, is a method for indirectly quantifying a marker to be detected by detecting and analyzing a magnetic signal of an antigen marked by magnetic beads, and compared with other immunoassay methods, the magnetic-sensitive immunoassay has the advantages of simple operation, lower cost and extremely high sensitivity. The magnetic sensor is an important device for magnetic-sensing immunoassay, and commonly used sensors include hall sensors, magnetoresistive sensors (AMR), giant magnetoresistive sensors (GMR), tunneling magnetoresistive sensors (TMR), giant magnetoresistive sensor (GMI), and the like. Because of simple process steps, stable performance, high sensitivity, low background noise and good thermal stability, Giant Magnetoresistance Sensors (GMRs) are more widely used in biomedical detection including immunoassays.

The microfluidic chip is also called a lab-on-a-chip, can integrate a series of complex and tedious biochemical reaction operations, biomedical detection and other functions into a chip with the size of a few square centimeters, and has the advantages of less required samples, simpler and more convenient operation, sample pollution prevention, system miniaturization and the like. However, few examples of combining microfluidic chips with magnetic-sensitive immunoassay techniques exist.

Chinese patent CN 109917139 a proposes a magnetic sensitive immunodetection card based on GMR sensor array and microfluidic chip. The invention fixes the sensing array and the bottom plate and utilizes the PDMS film to prepare the micro-fluidic chip. The designed microfluidic device injects a sample to be detected, immunomagnetic beads and washing liquid into a reaction area step by step, and the used immunomagnetic beads can be used only by being pre-labeled, so that the steps are complicated and the operation is complex. Therefore, it is of great significance to design a microfluidic system capable of completing magnetic bead biomarker in the chip.

Disclosure of Invention

The invention aims to provide a micro-fluidic magnetic-sensing immune device based on a magnetic-resistance biosensor, which improves the integration level of a chip, simplifies the operation steps of biochemical reaction and shortens the analysis time.

The invention is realized by the following technical scheme:

a micro-fluidic magnetic-sensing immune device based on a magnetic-resistance biosensor comprises a bottom plate and an upper cover which are fixedly connected, wherein a micro-fluidic chip is arranged between the bottom plate and the upper cover;

the upper cover is provided with a superparamagnetic bead storage area, an antibody storage area, a sample storage area to be detected and a washing liquid storage area; the micro-fluidic chip is provided with a first biochemical reaction cavity, a second biochemical reaction cavity, a detection area and a waste liquid cavity; the sample inlet of the first biochemical reaction cavity is communicated with the superparamagnetic bead storage area and the antibody storage area, the sample outlet is communicated with the sample inlet of the second biochemical reaction cavity through a first microfluidic channel, the sample inlet of the second biochemical reaction cavity is also communicated with the sample storage area to be detected, the sample outlet of the second biochemical reaction cavity is communicated with the sample inlet of the detection area through a second microfluidic channel, and the sample inlet of the detection area is also communicated with the washing liquid storage area; the sample outlet of the detection area is communicated with the waste liquid cavity;

the micro-fluidic chip is also provided with a groove, the groove corresponds to the detection area in the upper and lower positions and is communicated with the detection area, a magnetic sensor array capable of combining immune protein is embedded in the groove, and the opening of the groove is sealed.

Preferably, the upper cover is provided with an exhaust hole which is communicated with the waste liquid cavity.

Furthermore, a waste liquid hole is formed in the micro-fluidic chip, the waste liquid hole vertically corresponds to the exhaust hole in position, and the waste liquid cavity is communicated with the exhaust hole through the waste liquid hole.

Preferably, the microfluidic chip comprises a substrate and a film fixed on the substrate, and a first biochemical reaction chamber, a second biochemical reaction chamber, a detection zone and a waste liquid chamber are enclosed between the film and the substrate.

Furthermore, the film adopts a PDMS film, and the substrate adopts a PMMA substrate.

Preferably, the microfluidic chip is provided with a first sample adding hole, a second sample adding hole, a third sample adding hole and a fourth sample adding hole; the first biochemical reaction cavity is communicated with the superparamagnetic bead storage region through a first sample adding hole, the first biochemical reaction cavity is communicated with the antibody storage region through a second sample adding hole, the second biochemical reaction cavity is communicated with a sample storage region to be detected through a third sample adding hole, and the detection region is communicated with the washing liquid storage region through a fourth sample adding hole; the superparamagnetic bead storage area, the antibody storage area, the to-be-detected sample storage area and the washing liquid storage area respectively correspond to the first sample adding hole, the second sample adding hole, the third sample adding hole and the fourth sample adding hole in position from top to bottom.

Preferably, the first microfluidic channel is a serpentine channel and the second microfluidic channel is an S-shaped channel.

Preferably, the magnetic sensor array comprises a substrate, a plurality of sensors and a plurality of electrodes; the sensor and the electrode are arranged on the substrate, a power supply interface of the sensor is connected with an external power supply through an electrode, a signal output interface of the sensor is connected with an external PCB through an electrode and is connected with the electrode through a lead, and the electrode is connected with the external PCB through the lead; a layer of SiO is arranged on the sensor₂Protective film, SiO₂The protective film is provided with a layer of gold film, and the gold film is marked with a capture antibody.

Preferably, the upper surface of the bottom plate is provided with a coil and a heating sheet, the coil is electrically connected with the outside through a first lead, and the heating sheet is electrically connected with the outside through a second lead; the center of the coil is positioned right below the detection area; the heating plate is positioned below the second biochemical reaction cavity and the first biochemical reaction cavity.

The use method of the microfluidic magnetic-sensing immune device based on the magnetoresistive biosensor comprises the following steps:

(1) adding superparamagnetic beads into a superparamagnetic bead storage region, adding biotinylated antibodies into an antibody storage region, adding a sample to be detected into a sample storage region to be detected, and adding a cleaning solution into a cleaning solution storage region;

(2) applying pressure, pressing the superparamagnetic beads and the biotinylated antibodies into a first biochemical reaction cavity, pressing the superparamagnetic beads and the biotinylated antibodies into a first microfluidic channel, and reacting to form superparamagnetic beads for marking the antibodies; continuously applying pressure, pressing the superparamagnetic beads marked with the antibodies into a second biochemical reaction cavity, pressing the sample to be detected into the second biochemical reaction cavity, continuously applying pressure, and allowing the superparamagnetic beads marked with the antibodies and the sample to be detected to enter a second microfluidic channel to react to form the immunoprotein marked with the superparamagnetic beads;

(3) continuously applying pressure, pressing the immune protein marked with the superparamagnetic beads into the detection area, and combining and fixing the immune protein by the magnetic sensor array; applying pressure to inject the washing liquid into the detection area for washing;

(4) and applying a magnetic field perpendicular to the magnetic sensor array, magnetizing the superparamagnetic beads, collecting resistance signals of the magnetic sensor array through an external detection system, and calculating the concentration of the immune protein in the sample to be detected according to a standard curve of the concentration of the immune protein and the change of the resistivity.

Compared with the prior art, the invention has the following beneficial technical effects:

in the microfluidic magnetic-sensing immune device, a superparamagnetic bead storage area and an antibody storage area are arranged on an upper cover, a first biochemical reaction chamber and a first microfluidic channel are arranged on a microfluidic chip, when the microfluidic magnetic-sensing immune device is used, biotinylated superparamagnetic beads and biotinylated antibodies are sequentially subjected to mixed reaction in the first biochemical reaction chamber and the first microfluidic channel, superparamagnetic beads of the labeled antibodies after reaction are reacted with a sample to be detected to generate immune protein labeled with the superparamagnetic beads, and then the immune protein is reacted and combined with capture antibodies in a magnetic sensor array, so that the superparamagnetic beads are fixed on a sensor in the magnetic sensor array to perform a subsequent detection process. The invention integrates the combination of the superparamagnetic beads and the biotinylated antibodies in the magnetic-sensitive immunoassay into the chip to complete, improves the integration level, carries out the biological markers of the superparamagnetic beads in the chip, simplifies the operation steps of biochemical reaction and improves the biochemical reaction efficiency.

Furthermore, the exhaust hole is arranged, so that the sample adding is smoothly carried out by exhausting gas when the micro-fluidic chip samples, and after the detection is finished, waste liquid after the reaction is extracted from the exhaust hole, magnetic beads are recycled, and reagents are saved.

Furthermore, the film adopts a PDMS film, and because PDMS has extremely strong hydrophobicity, a flow channel in the film structure has a capillary effect, so that the reagents in the reagent storage areas can be effectively prevented from flowing into the microfluidic chip in advance.

Furthermore, most of the flow channels of the micro-fluidic device designed in the prior art are straight flow channels, and uneven mixing is easily caused by a mode of directly injecting a sample into a reaction area.

Furthermore, the conventional magnetosensitive microfluidic device also needs complex external driving devices, such as temperature control, external magnetic field generation, fluid driving and the like, so that the whole system is too complex and large. According to the invention, the coil is arranged on the bottom plate to provide the magnetic field required by detection, so that the integration level is improved, and a complex and huge external magnetic field generating device is avoided. Because the required reaction temperature is usually higher than room temperature, the heating sheet is arranged on the bottom plate to provide the temperature required by the reaction, so that the reaction is fully performed, the speed is increased, the integration level is improved, and a complex and huge external heating device is avoided.

According to the method, the superparamagnetic beads and the biotinylated antibody are directly fed into the chip for reaction, so that the reaction automation is realized, the complicated steps of immunoassay are simplified, and the problems of complicated immunoassay steps, complicated operation, long analysis period and the like are solved.

Drawings

Fig. 1 is a schematic structural view of the present invention.

Fig. 2 is a schematic view of the structure of the upper cover.

Fig. 3 is a schematic structural diagram of the upper surface of the microfluidic chip.

Fig. 4 is a schematic structural diagram of the lower surface of the microfluidic chip.

Fig. 5 is a schematic structural diagram of a magnetic sensor array.

Fig. 6 is a schematic view of the structure of the base plate.

Wherein: 1-an upper cover, 11-a shell, 12-a superparamagnetic bead storage region, 13-an antibody storage region, 14-a sample storage region to be detected, 15-a washing solution storage region and 16-an exhaust hole; 2-microfluidic chip, 201-substrate, 202-film, 203-first microfluidic channel, 204-third sample adding hole, 205-second biochemical reaction cavity, 206-second microfluidic channel, 207-waste liquid cavity, 208-detection zone, 209-waste liquid hole, 210-straight channel, 211-second sample adding hole, 212-first sample adding hole, 213-first biochemical reaction cavity 213, 214-fourth sample adding hole, 215-groove; 3-magnetic sensor array, 31-substrate, 32-electrode, 33-sensor, 34-lead; 4-bottom plate, 41-bottom shell, 42-wire, 43-heating plate, 44-coil, 45-wire.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

Referring to fig. 1, the invention provides a microfluidic magnetic-sensing immune device based on a magnetoresistive biosensor, which comprises an upper cover 1, a microfluidic chip 2, a magnetic sensor array 3 and a bottom plate 4. The upper cover 1 is buckled on the microfluidic chip 2, and the two are connected in a gluing or mechanical fixing mode.

Referring to fig. 2, the upper cover 1 includes a housing 11, and the housing 11 is provided with 4 reagent storage areas, which are a superparamagnetic bead storage area 12, an antibody storage area 13, a sample storage area to be detected 14, and a washing solution storage area 15. The superparamagnetic bead storage region 12, the antibody storage region 13, the to-be-detected sample storage region 14 and the washing liquid storage region 15 are all stepped holes, the small-aperture hole section is located on the lower surface of the shell 11, and the large-aperture hole section is located on the upper surface of the shell 11. The superparamagnetic bead storage area 12 and the antibody storage area 13 are used for storing superparamagnetic beads and biotinylated antibodies respectively, the sample storage area 14 to be detected is used for storing samples to be detected, and the washing liquid storage area 15 is used for storing washing liquid. The volume of each reagent storage region is 10-40 microlitres. The storage area can be sealed by PDMS film or plastic film. The housing 11 is provided with an air outlet 16. When the micro-fluidic chip 2 is used for sample application, the gas is discharged to ensure that the sample application is carried out smoothly, and after the detection is finished, the waste liquid after the reaction is extracted from the vent hole 16.

The upper cover 1 is made of hard plastics or ceramics and is manufactured in a 3D printing mode.

Referring to fig. 3, in an embodiment of the present invention, the microfluidic chip 2 includes a substrate 201 and a thin film 202 fixed on the substrate 201, and a microstructure including a first well 212, a second well 211, a third well 204, a fourth well 214, a first biochemical reaction chamber 213, a second biochemical reaction chamber 205, a detection region 208, a waste liquid chamber 207, and a waste liquid well 209 is disposed on the thin film 202. The sample inlet of the first sample adding hole 212 is communicated with the small-aperture hole section of the superparamagnetic bead storage area 12, the sample inlet of the second sample adding hole 211 is communicated with the small-aperture hole section of the antibody storage area 13, the sample inlet of the third sample adding hole 204 is communicated with the small-aperture hole section of the sample storage area 14 to be detected, and the sample inlet of the fourth sample adding hole 214 is communicated with the small-aperture hole section of the washing liquid storage area 15, so that the first sample adding hole 212, the second sample adding hole 211, the third sample adding hole 204 and the fourth sample adding hole 214 are respectively used for injecting a sample to be detected, a superparamagnetic bead, a biotinylated antibody and a washing liquid into the microfluidic chip 2.

The first biochemical reaction chamber 213 is communicated with the second biochemical reaction chamber 205 through the first microfluidic channel 203, the second biochemical reaction chamber 205 is communicated with the detection zone 208 through the second microfluidic channel 206, and the detection zone 208 is communicated with the waste liquid chamber 207. The sample outlets of the first and second wells 212 and 211 communicate with the biochemical reaction chamber 213, the third well 204 communicates with the second biochemical reaction chamber 205, and the fourth well 214 communicates with the detection region 208 via the flow channel 210. The first biochemical reaction chamber 213 provides a reaction space for the magnetic bead biomarker, and the first microfluidic channel 203 allows the reagents to be mixed more uniformly and the reaction to be more complete. The second biochemical reaction chamber 205 provides a reaction space for the combination of the immunoprotein and the superparamagnetic beads in the sample to be detected, and the second microfluidic channel 206 connected at the rear is used for the reaction of the immunoprotein and the superparamagnetic beads. The lower part of the detection area 208 corresponds to the magnetic sensor array 3, and is used for detecting the immune protein marked by the superparamagnetic beads. Waste liquid hole 209 upper portion and exhaust hole intercommunication, waste liquid hole 209 and waste liquid chamber 207 intercommunication, waste liquid chamber 207 deposits by washing liquid back exhaust impurity, unreacted super paramagnetic bead etc to discharge waste liquid through waste liquid hole 209 and exhaust hole. The waste liquid hole 209 has both functions of exhausting gas and recovering waste liquid.

In this embodiment, the superparamagnetic bead storage region 12, the antibody storage region 13, the to-be-detected sample storage region 14, and the washing solution storage region 15 correspond to the first sample adding hole 212, the second sample adding hole 211, the third sample adding hole 204, and the fourth sample adding hole 214 up and down, respectively. The exhaust hole 16 is positioned to correspond to the waste liquid hole 209 up and down.

The film 202 is made of a PDMS film, the substrate is a PMMA substrate, and the microfluidic chip 2 is specifically manufactured by bonding the PDMS film 202 to the PMMA substrate 201 by adhesive bonding. Because PDMS has extremely strong hydrophobicity, the flow channel in the film structure has a capillary effect, and reagents in each reagent storage area can be effectively prevented from flowing into the microfluidic chip 2 in advance. The volume of the biochemical reaction chamber 213 is 10-20 microliter. The volume of the detection zone 208 is 30-40 microliters.

Referring to fig. 4, a groove 215 is formed in the lower surface of the microfluidic chip 2, that is, the lower surface of the substrate 201, a magnetic sensor array 3 is embedded in the groove 215, and the magnetic sensor array 3 corresponds to the detection region 208 up and down.

The preparation method of the microfluidic chip 2 comprises the following steps: (1) by a photolithographic process. Etching a required pattern on a silicon wafer to be used as a mold; (2) uniformly coating a layer of PDMS on a silicon wafer, and manufacturing a required microstructure in a mode of reverse molding; (3) cleaning the PMMA substrate, cutting the PMMA substrate into a required shape by using a laser cutting machine, and cutting a groove 215 required by embedding a sensor array on the lower surface of the PMMA substrate; (4) and fixing the PDMS film containing the microstructures on the upper surface of the PMMA substrate by means of adhesive bonding.

Referring to fig. 5, the magnetic sensor array 3 is located directly below the detection zone 208, and includes a substrate 31, a plurality of sensors 33, and a plurality of electrodes 32. The sensors 33 and the electrodes 32 are arranged on the substrate 31, a power interface and a signal output interface of each sensor 33 are respectively connected with one electrode 32 through leads 34, one electrode 32 is connected with a power interface in the PCB through an external lead, and the other electrode 32 is connected with a sampling and amplifying circuit interface in the PCB through an external lead, so that the acquisition of signals is realized. Sensor 33 is sputtered with a layer of SiO₂Protective film, SiO₂A gold film is sputtered on the protective film and used for biomarkers (labeled capture antibodies), and the capture antibodies modified on the gold film are combined with the immune protein in the sample to be detected in the detection area 208, so that the superparamagnetic beads are fixed on the surface of the sensor 33, and the magnetic field generated by the superparamagnetic beads is detected to generate corresponding electric signals. Different types of capture antibodies are modified on the surface of the sensor 33, so that the same magnetic sensor array 3 can be realizedVarious immunity proteins were detected. Each sensor 33 is 100-120 microns wide and 120-200 microns long, and the array may include 4-20 sensors 33 arranged in a linear or checkerboard pattern. The magnetic sensor array 3 is embedded in the groove 215 on the back of the PMMA substrate 201 by means of adhesive bonding. The substrate 31 is made of Si substrate or SiO₂A substrate. The sensor 33 employs GMR.

The magnetic sensor array 3 is manufactured by adopting a photoetching and magnetron sputtering method, and the manufacturing method comprises the following steps: (1) depositing a film with GMR effect on a silicon substrate by using a multi-target magnetron sputtering machine; (2) making the film into a required pattern by using a photoetching machine and a dry etching process to serve as a sensor 33; (3) manufacturing the electrode 32 by using a photoetching machine and a magnetron sputtering method; (4) sputtering a layer of SiO with the thickness of 200-300nm on the sensor 33 by using a magnetron sputtering machine₂A protective film; (5) using magnetron sputtering machine on SiO₂Sputtering a layer of gold film with the thickness of 100-250nm on the protective film; (6) the magnetic sensor array 33 is fixed in the groove 215 on the lower surface of the microfluidic chip 2 by means of adhesive bonding.

Referring to fig. 6, the bottom plate 4 is made of plastic or ceramic, the bottom plate 4 includes a bottom case 41, a coil 44 and a heating plate 43 are disposed on an upper surface of the bottom case 41 through a mechanical fixing or gluing method, the coil 44 is electrically connected to the outside through a first wire 45, and the heating plate 43 is electrically connected to the outside through a second wire 42. The coil 44 is located right below the detection area 208 of the microfluidic chip 2, and is formed by winding a copper wire or an aluminum wire, and the area of the coil is much larger than that of the sensing array 3. The first wire 45 is electrified to generate a magnetic field of 200-350 oe, which is used for magnetizing the superparamagnetic beads adsorbed on the surface of the sensor 33 and generating the magnetic field for the sensor 33 to detect. When the GMR sensor is adopted, because the GMR sensor has demagnetization to the vertical magnetic field, the resistance of the sensor 33 can be changed only temporarily by the magnetic field generated by the coil 44, and the signal measured after demagnetization is finished is the stray magnetic field signal generated by the superparamagnetic beads. The heating plate 43 is located under the second biochemical reaction chamber 205 and the first biochemical reaction chamber 213, and the second lead 42 supplies power to heat the heating plate 43 and provide a temperature of 37-63 ℃, so that the biochemical reaction can be smoothly performed. The heating plate 43 is a silica gel heating plate. The base plate 4 is connected to the upper cover 1 by adhesive bonding or mechanical fastening. The soleplate 4 can be reused.

The following describes the working procedure of the microfluidic magnetic-sensing immune device by taking the detection of prostate specific antigen PSA as an example:

(1) the magnetic sensor array 3 is subjected to bioprocessing: firstly, respectively using NaOH, hydrochloric acid, ethanol and deionized water to carry out ultrasonic cleaning on the magnetic sensor array 3, so that a gold film on the surface of the sensor 33 forms a self-assembly layer; then incubating in MPA solution for 3 hours, dripping mercaptan onto the surface of the Au membrane, and washing the Au membrane with ethanol and deionized water; the gold film was washed with PBS (saline) after 40 minutes by treating with certain concentrations of EDC and NHS solution at 25 degrees Celsius, and left to dry in air.

(2) Modification of the capture antibody on the sensor 33 surface: the dropping concentration of the solution on the surface of the sensor 33 is 0.8-1.5mg/mL^-1Standing the PSA antibody solution at 37 ℃ for 45-65 minutes; then repeatedly washing with Phosphate Buffered Saline (PBS) containing 0.5% -1.2% Bovine Serum Albumin (BSA); next, in order to avoid non-specific adsorption of the immune protein by the gold film, the gold film is treated by BSA with the concentration of 1 percent and is kept stand for 2 to 3 hours at the low temperature of 4 ℃; finally, the sensor 33 was washed with PBS. Among them, the sensors 33 to be modified with the capture antibody are only a part, and are referred to as experimental group sensors, and the remaining sensors 33 are only subjected to biological treatment as a control group, and are referred to as control group sensors. The processed magnetic sensor array 3 is embedded in a groove 215 on the back side of the microfluidic chip 2.

(3) Adding reagent into the upper cover 1, specifically adding about 10 microliters of streptavidin protein with the concentration of 75-100 mug.mL into the super paramagnetic bead storage region 12 for functionalization^-1Superparamagnetic beads with the diameter of 100-200 nm; about 10 microliters of the antibody storage region 13 is added with the concentration of about 0.8-1.2 mg/mL^-1A biotinylated PSA antibody; adding tens of microliters of samples to be detected into the sample storage area 14; tens of microliters of phosphate buffered saline containing bovine serum albumin was added as a wash solution to the wash solution storage region 15. The reagents are sealed by PDMS film, and if the detection is not started immediately, the device needs to be stored in a low-temperature environment of 4 ℃.

(4) The superparamagnetic bead and the biotinylated PSA antibody are first pressed into the biochemical reaction chamber 213 through the well 212 and the well 211 by an external pumping means, such as a syringe pump, to perform a preliminary mixing reaction of the superparamagnetic bead and the biotinylated PSA antibody. Meanwhile, the heating sheet 43 on the bottom plate 4 is electrified, so that the temperature in the microfluidic chip 2 reaches and is maintained at 37 ℃, and the reaction requirement is met. Further pressure is applied to force the mixed reagents into the first microfluidic flow channel 203 for thorough mixing and reaction. And continuously applying pressure, adding the sample to be detected through the sample adding hole 204, enabling the sample to enter a second biochemical reaction cavity 205 for primary mixing with the superparamagnetic beads with the surface fixed with the biotinylated PSA antibody, then entering a second microfluidic flow channel 206, and fully reacting to enable the biotinylated PSA antibody fixed on the superparamagnetic beads to be combined with the immune protein PSA in the sample to be detected, so as to form the immune protein marked with the superparamagnetic beads.

(5) The sensor 33 with the capture antibody modified on the surface can be combined with the immune protein marked with the superparamagnetic beads and adsorb the superparamagnetic beads at the same time. The washing solution is injected into the detection region 208 through the fourth sample application hole 214 by applying pressure, so that impurities and residual superparamagnetic beads which do not participate in the reaction are washed away, and interference of other components and background noise of the magnetic field are reduced. After the detection is completed, the waste liquid is sucked through the waste liquid hole 209 by the pumping device, and the magnetic beads are recovered.

(6) The coil 44 on the bottom plate 4 is energized by an external circuit to generate a magnetic field perpendicular to the sensor 33, and the current is controlled to keep the magnetic field strength at a certain value between 200 DEG and 300 DEG for magnetizing the superparamagnetic beads adsorbed on the surface of the sensor 33. While an external circuit supplies an excitation current of 0.01-0.1mA to the sensor 33 through the electrode 32. When the GMR sensor is adopted, considering the demagnetization of the GMR sensor, namely the characteristic that the resistance of the sensor 33 is changed and quickly recovered under the action of a vertical magnetic field, the signal is acquired through an external PCB (printed circuit board) after the sensor 33 is demagnetized, namely the signal generated by the vertical magnetic field is eliminated. And comparing signals of the experimental group sensor modified with the capture antibody with signals of the control group sensor, analyzing the influence of the magnetic field generated by the superparamagnetic beads on the resistance of the sensor 33, and calculating the concentration of the immune protein to be detected according to the resistance change rate.

Before this, a sample with a certain concentration gradient and known concentration is required to test the system, the relation between the antigen concentration change and the GMR biosensor resistivity change is measured and researched, a standard curve of the antigen concentration and the resistivity change is drawn, an antigen sample with unknown concentration is detected, and the antigen concentration of the sample to be detected is obtained by comparing the detected signal with the standard curve and analyzing.

The invention designs the micro-fluidic magnetic-sensing immunoassay device which can complete magnetic bead biological labeling, magnetic bead and antibody combination and integrate a heating function and an external magnetic field generating device in a chip, can well solve the problems, can realize the rapidness, integration, automation and miniaturization of magnetic-sensing immunoassay, greatly improves the performance of a magnetic-sensing immunoassay system, and has important significance.

The above description is only for the purpose of illustrating embodiments of the present application and is not intended to limit the scope of the present application, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the specification and the drawings of the present application or are directly or indirectly applied to other related technical fields, are also included in the scope of the present application.