CN111812329B - Antibody chip kit for quantitatively detecting multiple tumor markers - Google Patents

Antibody chip kit for quantitatively detecting multiple tumor markers Download PDF

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CN111812329B
CN111812329B CN202010739081.2A CN202010739081A CN111812329B CN 111812329 B CN111812329 B CN 111812329B CN 202010739081 A CN202010739081 A CN 202010739081A CN 111812329 B CN111812329 B CN 111812329B
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microspheres
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CN111812329A (en
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赵曙华
陈婷珺
李慧新
盖戈
王佳佳
王芬
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Shandong Shengjian Medical Research Co ltd
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Abstract

The invention relates to an antibody chip kit for quantitatively detecting multiple tumor markers. The qualitative nano microspheres and the quantitative magnetic microspheres are combined together, qualitative and quantitative detection can be simultaneously carried out at one time, the ultrasonic resonance driving and the magnetic field control are combined, the qualitative detection and the quantitative detection can be distinguished, and the qualitative detection does not influence the quantitative detection due to low use amount of the qualitative detection, so that the operation is simple and the stability is good. Qualitative detection is carried out by using qualitative microspheres with different particle sizes, so that the accuracy is good, and the anti-interference capability is strong. The quantitative detection is used, and the independent treatment is not needed for each tumor marker, so that the applicability is good; in addition, the ultrasonic and magnetic field control structure can be disassembled for multiple use, and the economy is good.

Description

Antibody chip kit for quantitatively detecting multiple tumor markers
Technical Field
The invention relates to the field of medical detection, in particular to an antibody chip kit for quantitatively detecting multiple tumor markers and a manufacturing method thereof.
Background
Tumor markers (Tumor markers) are substances which are synthesized and released by Tumor cells or are increased by the body reacting to the Tumor cells, and the main components of the Tumor markers are proteins, saccharides and the like. The tumor marker exists in blood, body fluid and tissues, can be detected by methods such as biochemistry, immunology, genomics and the like, and can be used for early diagnosis, prognosis prediction, curative effect evaluation and the like of tumors. An abnormally elevated tumor marker may indicate the occurrence of a tumor, but an abnormally elevated tumor marker may also occur in non-neoplastic diseases.
The existing tumor marker detection generally adopts a flow cytometer method, and at present, researchers detect the tumor marker based on technologies such as fluorescent coding microspheres, but the method can carry out quantitative detection only by independently setting corresponding detection reagents for different tumor markers, and has the disadvantages of complex material treatment and large usage amount. One detection needs to be provided with a reagent for quantitative detection for all tumor markers, but in actual detection, at most one or two markers can be detected. In addition, in actual detection, when rapid detection is required, other detection means such as CT are often combined, so that the concentration of the tumor marker can be determined only by detecting which type of the tumor marker is present.
Disclosure of Invention
Aiming at the content, the antibody chip kit for quantitatively detecting multiple tumor markers is provided for solving the problems, and comprises a microfluidic chip, an ultrasonic exciter, a magnetic controller, qualitative nano microspheres with different particle sizes, quantitative magnetic microspheres, a sulfhydryl luminescent group reagent, antibodies of multiple tumor markers and a fluorescence detector; the surface of each qualitative nano microsphere can be modified with an antibody of a tumor marker, and the surface of each qualitative nano microsphere can be modified with a sulfhydryl luminescent group; the surface of the quantitative magnetic nano microsphere is simultaneously decorated with a plurality of tumor marker antibodies, and the quantitative magnetic particles have fluorescence or characteristic light absorption peaks;
the micro-fluidic chip comprises a sample adding area, a reaction area, a transportation area and a detection area; the sample adding area comprises a reagent sample adding area and a sample adding area, the sample adding area is connected with the reaction area, the reaction area is connected with the transportation area, the transportation area is provided with a slender transportation channel, the detection area is provided with a qualitative detection area and a quantitative detection area, the number of the qualitative detection areas is multiple, each qualitative detection area is connected to outlets, which are different from the reaction area, of the transportation area, and the quantitative detection area is arranged at the tail of the transportation area;
outlets in the elongated transport channel of the transport zone comprise a high outlet and a low outlet, wherein the low outlet is an outlet connected to the qualitative detection zone, the high outlet is an outlet connected to the quantitative detection zone, wherein the horizontal position of the high outlet is higher than that of the low outlet, and the vertical height of the transport channel of the transport zone is more than 20 times that of the nano-microspheres and the magnetic microspheres; the qualitative nanometer microspheres and the quantitative magnetic microspheres are transported from the reaction area to the detection area under the action of the ultrasonic exciter and the magnetic controller.
The ultrasonic exciter comprises an ultrasonic signal regulator and ultrasonic transduction pieces, the number of the ultrasonic transduction pieces is multiple, and each group of the ultrasonic signal regulator comprises two pieces; the ultrasonic energy conversion sheet is divided into transverse combined longitudinal groups, one group of longitudinal groups are arranged at the front end and the rear end in the same transport direction with the transport area, a plurality of groups of transverse groups are arranged at the two ends perpendicular to the transport direction of the transport area, openings connected with the qualitative detection area are arranged on the side face perpendicular to the transport direction of the transport area, and each opening connected with the qualitative detection area is provided with one group of transverse groups;
two energy conversion sheets of the longitudinal group form moving standing waves when working, the working frequency of the longitudinal group is a composite frequency, the working mode is a frequency sweeping working mode, and qualitative nano microspheres and quantitative magnetic microspheres are pushed to advance from a reaction area to a detection area; the working mode of the transverse group is single frequency, and qualitative nano microspheres corresponding to the resonance frequency are pushed into corresponding openings so as to enter a qualitative detection area;
the magnetic controller is arranged on the upper surface and the lower surface of the detection chip, and controls the quantitative magnetic microspheres to be positioned on the upper surface of the transportation channel during transportation, so that the quantitative detection area is prevented from entering, and the other end of the transportation area directly enters the quantitative detection area.
The fluorescence detector detects the fluorescence intensity in each qualitative detection area, and the qualitative analysis of the tumor marker is carried out on the sample according to the fluorescence intensity of different qualitative detection areas; the photodetector detects the fluorescence intensity change or the characteristic light absorption change in the quantitative detection area, and the tumor marker is quantitatively analyzed on the sample by combining the frequency sweeping frequency of the longitudinal group.
The manufacturing method of the antibody chip kit for quantitatively detecting a plurality of tumor markers is characterized by comprising the following steps:
manufacturing the micro-fluidic chip:
processing a substrate, an intermediate layer and a top plate by using femtosecond laser, wherein the substrate, the intermediate layer and the top plate are made of PMMA (polymethyl methacrylate), the number of the intermediate layer is two, a sample adding area, a reaction area, a transportation area and a detection through hole are processed in the intermediate layer by using the femtosecond laser, mounting grooves for mounting a transducer plate and a magnetic controller are processed on the substrate and the top plate, a sample adding hole of the sample adding area is processed on the top plate, a channel connected to a qualitative detection area is processed in the lower intermediate layer, a channel connected to a quantitative detection area is processed in the upper intermediate layer, and the substrate, the intermediate layer and the top plate are sealed and bonded in sequence;
installation of an ultrasonic exciter and an ultrasonic controller:
mounting an ultrasonic energy conversion sheet on the microfluidic chip, fixing the ultrasonic energy conversion sheet by using an elastic clamp, and connecting a lead of the energy conversion sheet to an ultrasonic generator; mounting a magnetic controller on the microfluidic chip and connecting the magnetic controller to a power supply;
preparation of reagents:
using ready-made Fe 3 O 4 Nanoparticles, antibodies of a plurality of different tumor markers including AFP, CEA, CYFRA21-1, CA19-9, CA125, PSA, NSE, beta-HCG, etc.; the corresponding antibody is connected to Fe by using DNA or sulfhydrylation treatment 3 O 4 Obtaining quantitative magnetic microspheres from the nano particles;
using SiO of different particle sizes 2 The microsphere or polyethylene microsphere has particle sizes of 150nm, 200nm, 500nm, 700nm, 1100nm, etc., and has a particle size floating range of less than 5% as qualitative nanometer microsphere; and the surface of the qualitative nano microsphere is activated so that the qualitative nano microsphere can be connected with a sulfhydrylation fluorescent group.
During detection, each qualitative nano microsphere with different particle sizes is connected with an antibody of a tumor marker in a DNA (deoxyribonucleic acid) connection mode or a sulfhydrylation connection mode, and then the qualitative nano microspheres and the antibody are mixed to be modified by a fluorescent luminescent group; mixing the modified nano microspheres with quantitative magnetic microspheres in a mixing ratio of qualitative nano microspheres: the quantitative magnetic microsphere 1 is characterized in that the ratio of the magnetic microsphere 1 to the magnetic microsphere 1 is 100 to 1 10000, so that the influence of qualitative detection on quantitative detection is reduced;
injecting the mixed microspheres into the sample adding hole, adding a sample to be detected into the sample adding hole, so that the mixed microspheres react with the sample to be detected, and combining the tumor marker with the qualitative nano microspheres and the quantitative magnetic microspheres, wherein the qualitative nano microspheres are completely reacted during reaction because the quantity of the qualitative nano microspheres is far lower than that of the quantitative magnetic microspheres;
starting the ultrasonic energy conversion sheets of the longitudinal group, and sweeping frequency to enable all the microspheres to move to a detection area along the transportation area; simultaneously starting the transverse group, wherein the vibration frequency of the transverse group is preset, so that the qualitative nano microspheres which are not connected with the tumor marker under the driving of the transverse group can enter the qualitative detection area from the corresponding openings, and the qualitative nano microspheres connected with the tumor marker cannot enter the qualitative detection area because the tumor marker changes the resonance frequency and cannot be driven by the corresponding transverse group; detecting the fluorescence intensity of different qualitative detection areas, wherein the qualitative detection area which can not detect the fluorescence intensity all the time indicates that the corresponding qualitative nano-microsphere detects the tumor marker;
the quantitative magnetic microspheres move along the transportation area under the driving of the longitudinal group, and the magnetic controller is started at the same time, so that the magnetic microspheres move along the top of the transportation area and can enter the quantitative detection area;
because the longitudinal group is in a frequency sweeping mode, the quantitative magnetic microspheres can be driven to move only by scanning the corresponding resonance frequency, so that the magnetic microspheres entering the quantitative detection area are increased in a pulse mode, the fluorescence or characteristic light absorption intensity of the magnetic microspheres in the quantitative detection area is caused to change intermittently, the resonance frequency for driving the quantitative magnetic microspheres can be obtained according to the time point of the intermittent change, the type of the tumor marker is obtained by qualitative detection, the concentration of the tumor marker combined on the surface of the quantitative magnetic microspheres can be reversely deduced according to the resonance frequency, and the influence of each tumor marker with different concentrations on the resonance frequency of the quantitative magnetic microspheres can be obtained in advance through experiments, and a standard curve or a table is drawn.
The beneficial effects of the invention are as follows: the invention combines the qualitative nano-microspheres and the quantitative magnetic microspheres, can simultaneously carry out qualitative and quantitative detection at one time, uses the combination of ultrasonic resonance driving and magnetic field control to distinguish the qualitative detection from the quantitative detection, has low dosage of the qualitative detection, does not influence the quantitative detection, and has simple operation and good stability.
Qualitative detection is carried out by using qualitative microspheres with different particle diameters, so that the accuracy is good, and the anti-interference capability is strong. The quantitative detection is used, and the independent treatment is not needed for each tumor marker, so that the applicability is good; in addition, the ultrasonic and magnetic field control structure can be disassembled for multiple use, and the economy is good.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosed subject matter, are incorporated in and constitute a part of this specification. The drawings illustrate the implementations of the disclosed subject matter and, together with the detailed description, serve to explain the principles of implementations of the disclosed subject matter. No attempt is made to show structural details of the disclosed subject matter in more detail than is necessary for a fundamental understanding of the disclosed subject matter and various modes of practicing the same.
Fig. 1 is a schematic structural diagram of a microfluidic chip according to the present invention.
Detailed Description
The advantages, features and methods of accomplishing the same will become apparent from the drawings and the detailed description that follows.
Example (b):
an antibody chip kit for quantitatively detecting multiple tumor markers comprises a microfluidic chip, an ultrasonic exciter, a magnetic controller, qualitative nano microspheres with different particle sizes, quantitative magnetic microspheres, a sulfhydryl luminescent group reagent, antibodies of multiple tumor markers and a fluorescence detector; the surface of each qualitative nano microsphere can be modified with an antibody of a tumor marker, and the surface of each qualitative nano microsphere can be modified with a sulfhydryl luminescent group; the surface of the quantitative magnetic nano microsphere is simultaneously decorated with a plurality of tumor marker antibodies, and the quantitative magnetic particles have fluorescence or characteristic light absorption peaks;
the micro-fluidic chip comprises a sample adding area 11, a reaction area 12, a transportation area 13 and a detection area; the sample adding area 11 comprises a reagent sample adding area 11 and a sample adding area 11, the sample adding area 11 is connected with a reaction area 12, the reaction area 12 is connected with a transportation area 13, the transportation area 13 is provided with a slender transportation channel, the detection area is provided with a qualitative detection area 14 and a quantitative detection area 15, the number of the qualitative detection areas 14 is multiple, each qualitative detection area 14 is connected with an outlet of the transportation area 13, which is different from the reaction area 12 in distance, and the quantitative detection area 15 is arranged at the tail end of the transportation area 13;
the outlets in the elongated transport channel of the transport zone 13 comprise a high outlet and a low outlet, wherein the low outlet is an outlet connected to the qualitative detection zone 14 and the high outlet is an outlet connected to the quantitative detection zone 15, wherein the horizontal position of the high outlet is higher than the horizontal position of the low outlet, and the vertical height of the transport channel of the transport zone 13 is more than 20 times that of the nano-microspheres and the magnetic microspheres; the qualitative nano-microspheres and the quantitative magnetic microspheres are transported from the reaction zone 12 to the detection zone under the action of the ultrasonic exciter and the magnetic controller.
The ultrasonic exciter comprises an ultrasonic signal adjuster and ultrasonic transducer plates 16, the number of the ultrasonic transducer plates 16 is multiple, and each group of the ultrasonic transducer plates 16 comprises two plates; the ultrasonic transducer 16 is divided into transverse combined longitudinal groups, one group of longitudinal groups is arranged at the front end and the rear end in the same transport direction as the transport area 13, a plurality of groups of transverse groups are arranged at the two ends perpendicular to the transport direction of the transport area 13, openings connected with the qualitative detection areas 14 are arranged on the side faces perpendicular to the transport direction of the transport area 13, and each opening connected with the qualitative detection area 14 is provided with one group of transverse groups;
the two energy conversion sheets of the longitudinal group form moving standing waves when working, the working frequency of the longitudinal group is composite frequency, the working mode is a frequency sweeping working mode, and qualitative nano microspheres and quantitative magnetic microspheres are pushed to move forward from the reaction area 12 to the detection area; the transverse group operates at a single frequency to push the qualitative nanospheres corresponding to the resonance frequency into the corresponding openings 18 and thus into the qualitative detection zone 14;
the magnetic controller 17 is arranged on the upper surface and the lower surface of the detection chip, controls the quantitative magnetic microspheres to be positioned on the upper surface of the transportation channel during transportation, and avoids entering the qualitative detection area 14, so that the other end of the quantitative magnetic microspheres directly reaching the transportation area 13 enters the quantitative detection area 15.
The fluorescence detector detects the fluorescence intensity in each qualitative detection area 14, and performs qualitative analysis on the tumor marker of the sample according to the fluorescence intensity of different qualitative detection areas 14; the photodetector detects the change of fluorescence intensity or the change of characteristic light absorption in the quantitative detection area 15, and the sample is subjected to quantitative analysis of the tumor marker by combining the frequency sweeping frequency of the longitudinal group.
The manufacturing method of the antibody chip kit for quantitatively detecting a plurality of tumor markers is characterized by comprising the following steps:
manufacturing the micro-fluidic chip:
processing a substrate, an intermediate layer and a top plate by using femtosecond laser, wherein the substrate, the intermediate layer and the top plate are made of PMMA (polymethyl methacrylate), the number of the intermediate layer is two, a sample adding area 11, a reaction area 12, a transportation area 13 and a detection through hole are processed in the intermediate layer by using the femtosecond laser, mounting grooves for mounting a transducer plate and a magnetic controller 17 are processed in the substrate and the top plate, a sample adding hole of the sample adding area 11 is processed in the top plate, a channel connected to a qualitative detection area 14 is processed in the lower intermediate layer, a channel connected to a quantitative detection area 15 is processed in the upper intermediate layer, and the substrate, the intermediate layer and the top plate are sealed and bonded in sequence;
installation of an ultrasonic exciter and an ultrasonic controller:
mounting the ultrasonic transducer plate 16 on the microfluidic chip and fixing the plate by using an elastic clamp, and connecting the lead of the transducer plate to an ultrasonic generator; mounting the magnetic controller 17 on the microfluidic chip and connecting the magnetic controller 17 to a power supply;
preparation of reagents:
using ready-made Fe 3 O 4 Nanoparticles, antibodies of various tumor markers including AFP, CEA, CYFRA21-1, CA19-9, CA125, PSA, NSE, beta-HCG, etc.; the corresponding antibody is connected to Fe by using DNA or sulfhydrylation treatment 3 O 4 Obtaining quantitative magnetic microspheres from the nano particles;
using SiO of different particle sizes 2 The microsphere or polyethylene microsphere has particle sizes of 150nm, 200nm, 500nm, 700nm, 1100nm, etc., and has a particle size floating range of less than 5% as qualitative nanometer microsphere; and the surface of the qualitative nano microsphere is activated to be connected with a sulfhydrylation fluorescent group.
During detection, each qualitative nano microsphere with different particle sizes is connected with an antibody of a tumor marker in a DNA (deoxyribonucleic acid) connection mode or a sulfhydrylation connection mode, and then the qualitative nano microspheres and the antibody are mixed to be modified by a fluorescent luminescent group; mixing the modified nano microspheres with quantitative magnetic microspheres in a mixing ratio of qualitative nano microspheres: the quantitative magnetic microsphere 1;
injecting the mixed microspheres into a sample adding hole, adding a sample to be detected into the sample adding hole, so that the mixed microspheres react with the sample to be detected, and combining the tumor marker with the qualitative magnetic microspheres and the quantitative magnetic microspheres, wherein the qualitative magnetic microspheres are completely reacted during reaction because the quantity of the qualitative magnetic microspheres is far lower than that of the quantitative magnetic microspheres;
starting the longitudinal groups of ultrasonic transducers 16, and sweeping the frequency so that all the microspheres move along the transport zone 13 to the detection zone; simultaneously starting the transverse group, wherein the vibration frequency of the transverse group is preset, so that the qualitative nano-microspheres which are not connected with the tumor marker can enter the qualitative detection area 14 from the corresponding openings under the driving of the transverse group, and the qualitative nano-microspheres which are connected with the tumor marker cannot be driven by the corresponding transverse group due to the fact that the tumor marker changes the resonance frequency, and therefore cannot enter the qualitative detection area 14; at this time, the fluorescence intensities of the different qualitative detection areas 14 are detected, and the qualitative detection area 14 in which the fluorescence intensities are not detected all the time indicates that the tumor marker is detected by the corresponding qualitative nano-microsphere;
the quantitative magnetic microspheres move along the transportation area 13 under the driving of the longitudinal group, and meanwhile, the magnetic controller 17 is started, so that the magnetic microspheres move along the top of the transportation area 13 and can enter the quantitative detection area 15;
because the longitudinal group is in a frequency sweeping mode, the quantitative magnetic microspheres can be driven to move only by scanning to the corresponding resonance frequency, so that the magnetic microspheres entering the quantitative detection area 15 are increased in a pulse mode, the fluorescence or characteristic light absorption intensity of the magnetic microspheres in the quantitative detection area 15 is intermittently changed, the resonance frequency for driving the quantitative magnetic microspheres can be obtained according to the time point of the intermittent change, the tumor markers can be obtained by qualitative detection, the concentration of the tumor markers combined on the surface of the quantitative magnetic microspheres can be reversely deduced according to the resonance frequency, the influence of each tumor marker with different concentrations on the resonance frequency of the quantitative magnetic microspheres can be obtained by experiments in advance, and a standard curve or a table can be drawn.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (3)

1. An antibody chip kit for quantitatively detecting multiple tumor markers is characterized by comprising a microfluidic chip, an ultrasonic exciter, a magnetic controller, qualitative nano microspheres with different particle sizes, quantitative magnetic microspheres, a sulfhydryl luminescent group reagent, antibodies of the multiple tumor markers and a fluorescence detector; each qualitative nano microsphere is surface-modified with an antibody of a tumor marker, and the qualitative nano microsphere is surface-modified with a sulfhydryl luminescent group; the surface of the quantitative magnetic nano microsphere is simultaneously modified with a plurality of tumor marker antibodies, and the quantitative magnetic particles have fluorescence or characteristic light absorption peaks;
the micro-fluidic chip comprises a sample adding area (11), a reaction area (12), a transportation area (13) and a detection area; the sample adding area (11) comprises a reagent sample adding area (11) and a sample adding area (11), the sample adding area (11) is connected with the reaction area (12), the reaction area (12) is connected with the transportation area (13), the transportation area (13) is provided with a slender transportation channel, the detection area is provided with a qualitative detection area (14) and a quantitative detection area (15), the number of the qualitative detection areas (14) is multiple, each qualitative detection area (14) is connected to outlets of the transportation area (13) which are different in distance from the reaction area (12), and the quantitative detection area (15) is arranged at the tail of the transportation area (13);
the outlet in the elongated transport channel of the transport zone (13) comprises a high outlet and a low outlet, wherein the low outlet is an outlet connected to the qualitative detection zone (14) and the high outlet is an outlet connected to the quantitative detection zone (15), wherein the horizontal position of the high outlet is higher than the horizontal position of the low outlet, and the vertical height of the transport channel of the transport zone (13) is more than 20 times that of the nano-and magnetic microspheres; the qualitative nano microspheres and the quantitative magnetic microspheres are transported to a detection area from a reaction area (12) under the action of an ultrasonic exciter and a magnetic controller;
the ultrasonic exciter comprises an ultrasonic signal adjuster and ultrasonic energy conversion sheets (16), the number of the ultrasonic energy conversion sheets (16) is multiple groups, and each group comprises two sheets; the ultrasonic energy conversion sheets (16) are divided into transverse combined longitudinal groups, one group of longitudinal groups are arranged at the front end and the rear end in the same transportation direction with the transportation area (13), a plurality of groups of transverse groups are arranged at the two ends perpendicular to the transportation direction of the transportation area (13), openings connected with the qualitative detection areas (14) are arranged on the side face perpendicular to the transportation direction of the transportation area (13), and one group of transverse groups is arranged at each opening connected with the qualitative detection area (14);
the two energy conversion sheets of the longitudinal group form moving standing waves when working, the working frequency of the longitudinal group is composite frequency, the working mode is a frequency sweeping working mode, and qualitative nano microspheres and quantitative magnetic microspheres are pushed to move forward from the reaction area (12) to the detection area; the working mode of the transverse group is single frequency, and qualitative nano microspheres corresponding to the resonance frequency are pushed into the corresponding openings (18) so as to enter the qualitative detection area (14);
the magnetic controller (17) is arranged on the upper surface and the lower surface of the detection chip and is used for controlling the quantitative magnetic microspheres to be positioned on the upper surface of the transportation channel during transportation so as to avoid entering the qualitative detection area (14) and directly reach the other end of the transportation area (13) to enter the quantitative detection area (15); the fluorescence detector detects the fluorescence intensity in each qualitative detection area (14), and the qualitative analysis of the tumor marker is carried out on the sample according to the fluorescence intensity of different qualitative detection areas (14); the photodetector detects the change of fluorescence intensity or the change of characteristic light absorption in the quantitative detection area (15), and the tumor marker is quantitatively analyzed on the sample by combining the frequency sweeping frequency of the longitudinal group.
2. The method for preparing the antibody chip kit for quantitatively detecting multiple tumor markers as claimed in claim 1, which comprises the following steps:
manufacturing the micro-fluidic chip:
processing a substrate, an intermediate layer and a top plate by using femtosecond laser, wherein the substrate, the intermediate layer and the top plate are all made of PMMA, the number of the intermediate layer is two, processing a sample adding area (11), a reaction area (12), a transportation area (13) and a through hole for detection on the intermediate layer by using the femtosecond laser, processing mounting grooves for mounting a transducer chip and a magnetic controller (17) on the substrate and the top plate, processing a sample adding hole of the sample adding area (11) on the top plate, processing a channel connected to a qualitative detection area (14) on the intermediate layer below, processing a channel connected to a quantitative detection area (15) on the intermediate layer above, and sealing and bonding the substrate, the intermediate layer and the top plate in sequence;
installation of an ultrasonic exciter and an ultrasonic controller:
mounting an ultrasonic transducer plate (16) on the microfluidic chip, fixing the ultrasonic transducer plate by using an elastic clamp, and connecting a lead of the transducer plate to an ultrasonic generator; mounting a magnetic controller (17) on the microfluidic chip and connecting the magnetic controller (17) to a power supply;
preparation of reagents:
using ready-made Fe 3 O 4 Nanoparticles incorporating a plurality of different tumor marker antibodies, the tumor markers including AFP, CEA, CYFRA21-1, CA19-9, CA125, PSA, NSE, β -HCG; the corresponding antibody is connected to Fe by using DNA or sulfhydrylation treatment 3 O 4 Obtaining quantitative magnetic microspheres from the nano particles;
using SiO of different particle sizes 2 The microsphere or polyethylene microsphere has particle sizes of 150nm, 200nm, 500nm, 700nm and 1100nm, and the floating range of the particle size is required to be less than 5%, and is used as qualitative nanometer microsphere; and the surface of the qualitative nano microsphere is activated to be connected with a sulfhydrylation fluorescent group.
3. The method of manufacturing according to claim 2, wherein:
during detection, each qualitative nano microsphere with different particle sizes is connected with an antibody of a tumor marker in a DNA (deoxyribonucleic acid) connection mode or a sulfhydrylation connection mode, and then the qualitative nano microspheres and the antibody are mixed to be modified by a fluorescent luminescent group; mixing the modified nano microspheres with quantitative magnetic microspheres in a mixing ratio of qualitative nano microspheres: the quantitative magnetic microsphere 1 is characterized in that the ratio of the magnetic microsphere 1 to the magnetic microsphere 1 is 100 to 1 10000, so that the influence of qualitative detection on quantitative detection is reduced;
injecting the mixed microspheres into the sample adding hole, adding a sample to be detected into the sample adding hole, so that the mixed microspheres react with the sample to be detected, and combining the tumor marker with the qualitative nano microspheres and the quantitative magnetic microspheres, wherein the qualitative nano microspheres are completely reacted during reaction because the quantity of the qualitative nano microspheres is far lower than that of the quantitative magnetic microspheres;
starting the longitudinal group of ultrasonic transducer plates (16) to sweep frequency, so that all microspheres move to a detection area along the transportation area (13); simultaneously starting the transverse group, wherein the vibration frequency of the transverse group is preset, so that the qualitative nano microspheres which are not connected with the tumor marker enter the qualitative detection area (14) from the corresponding openings under the driving of the transverse group, and the qualitative nano microspheres which are connected with the tumor marker cannot enter the qualitative detection area (14) because the tumor marker changes the resonance frequency and cannot be driven by the corresponding transverse group; detecting the fluorescence intensity of different qualitative detection areas (14), wherein the qualitative detection area (14) which cannot detect the fluorescence intensity indicates that the corresponding qualitative nano-microsphere detects the tumor marker;
the quantitative magnetic microspheres move along the transportation area (13) under the driving of the longitudinal group, and meanwhile, a magnetic controller (17) is started, so that the magnetic microspheres move along the top of the transportation area (13) and enter a quantitative detection area (15);
because the longitudinal group is in a frequency sweeping mode, the quantitative magnetic microspheres can be driven to move only by scanning to the corresponding resonance frequency, so that the magnetic microspheres entering the quantitative detection area (15) are increased in a pulse mode, the intermittent change of the fluorescence or characteristic light absorption intensity of the magnetic microspheres in the quantitative detection area (15) is caused, the resonance frequency of the quantitative magnetic microspheres is driven to be obtained according to the time point of the intermittent change, the type of the tumor marker is obtained by qualitative detection, the concentration of the tumor marker combined on the surface of the quantitative magnetic microspheres is reversely deduced according to the resonance frequency, the influence of each tumor marker with different concentrations on the resonance frequency of the quantitative magnetic microspheres is obtained in advance through experiments, and a standard curve or a table is drawn.
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