CN111812329A - An antibody chip kit for quantitative detection of multiple tumor markers - Google Patents

Abstract

The invention relates to an antibody chip kit for quantitatively detecting multiple tumor markers. Combining qualitative nano-microspheres and quantitative magnetic microspheres, qualitative and quantitative detection can be carried out at the same time. The combination of ultrasonic resonance drive and magnetic field control can distinguish qualitative detection and quantitative detection. Due to the low dosage of qualitative detection, Qualitative detection does not affect quantitative detection, with simple operation and good stability. Qualitative detection using qualitative microspheres of different particle sizes has good accuracy and strong anti-interference ability. The use of quantitative detection does not require separate treatment for each tumor marker, and has good applicability; in addition, the structures controlled by ultrasound and magnetic fields can be disassembled and used multiple times, which is economical.

Description

An antibody chip kit for quantitative detection of 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 preparation method thereof.

Background technique

Tumor markers are a class of substances synthesized and released by tumor cells themselves, or increased in response to tumor cells, and their main components are proteins and carbohydrates. Tumor markers exist in blood, body fluids, and tissues, and can be detected by biochemical, immunological, genomics and other methods, and can be used for early diagnosis of tumors, prediction of prognosis, and evaluation of therapeutic effects. Abnormal elevation of tumor markers may indicate tumorigenesis, but abnormal elevation of tumor markers may also occur in non-neoplastic diseases.

The existing tumor markers are generally detected by flow cytometry. At present, many researchers detect tumor markers based on technologies such as fluorescently encoded microspheres. However, the above methods need to be set separately for different tumor markers. Only the corresponding detection reagent can be quantitatively detected, the material processing is complicated, and the amount is large. One detection requires quantitative detection reagents for all tumor markers, but in actual detection, at most one or two markers can generally be detected. In addition, in actual detection, when rapid detection is required, other detection methods such as CT are often combined. Therefore, tumor markers generally only need to be detected and the concentration is sufficient.

SUMMARY OF THE INVENTION

In view of the above content, in order to solve the above problems, an antibody chip kit for quantitatively detecting various tumor markers is provided, including a microfluidic chip, an ultrasonic exciter, a magnetic controller, qualitative nano-microspheres of different particle sizes, quantitative magnetic Spheres, sulfhydryl luminescent group reagents, antibodies for multiple tumor markers, fluorescence detectors; the surface of each qualitative nanosphere can be modified with an antibody for one tumor marker, and the surface of qualitative nanospheres can be modified with thiol luminescent groups ;The surface of the quantitative magnetic nanospheres is modified with multiple tumor marker antibodies at the same time, and the quantitative magnetic particles themselves have fluorescence or characteristic light absorption peaks;

The microfluidic chip includes a sample addition area, a reaction area, a transport area and a detection area; the sample addition area includes a reagent addition area and a sample addition area. The sample addition area is connected to the reaction area, and the reaction area is connected to the transportation area. Long transportation channel, the detection area is equipped with qualitative detection area and quantitative detection area. The number of qualitative detection areas is multiple, and each qualitative detection area is connected to the exit of the transportation area at different distances from the reaction area. The quantitative detection area is set at the end of the transport zone;

The exits in the elongated transportation channel of the transportation area include high exits and low exits, wherein the low exit is the exit connected to the qualitative detection area, and the high exit is the exit connected to the quantitative detection area, wherein the horizontal position of the high exit is higher than the low exit and the vertical height of the transport channel in the transport zone is 20 times greater than that of the nanospheres and the magnetic microspheres; the qualitative nanospheres and the quantitative magnetic microspheres are transported from the reaction zone under the action of the ultrasonic exciter and the magnetic controller to the detection area.

The ultrasonic exciter includes an ultrasonic signal conditioner and an ultrasonic transducer. The number of ultrasonic transducers is multiple groups, with two pieces in each group; the ultrasonic transducers are divided into horizontal and vertical groups, and one group of longitudinal groups is arranged in the transport area for transportation. The front and rear ends with the same direction, multiple sets of transverse groups are arranged at the two ends perpendicular to the transportation direction of the transportation area, and the openings connecting the qualitative detection area are all arranged on the side perpendicular to the transportation direction of the transportation area. The openings of the area are set with a set of horizontal groups;

The two transducer sheets of the longitudinal group form moving standing waves when working, and the working frequency of the longitudinal group is the composite frequency, and the working mode is the sweeping working mode, which pushes the qualitative nano-microspheres and quantitative magnetic microspheres from the reaction area to the detection area. Forward; the working mode of the lateral group is single frequency, and the qualitative nano-microspheres corresponding to the resonance frequency are pushed into the corresponding openings to enter the qualitative detection area;

The magnetic controller is arranged on the upper and lower surfaces of the detection chip, and controls 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, so as to directly reach the other end of the transportation area and enter the quantitative detection area.

The fluorescence detector detects the fluorescence intensity in each qualitative detection area, and performs qualitative analysis of tumor markers on the sample according to the fluorescence intensities of different qualitative detection areas; the photodetector detects the fluorescence intensity change or characteristic light absorption change in the quantitative detection area, Combined with the sweep frequency of the longitudinal group, the samples were subjected to quantitative analysis of tumor markers.

A manufacturing method of the antibody chip kit for quantitatively detecting multiple tumor markers, which is characterized by comprising the following steps:

Fabrication of microfluidic chip:

The substrate, the middle layer and the top plate are processed by femtosecond laser. The materials of the substrate, the middle layer and the top plate are all PMMA. The number of layers in the middle layer is two layers. The sample addition area and the reaction area are processed in the middle layer by the femtosecond laser. , transport area, through holes for detection, process the installation grooves for the transducer and magnetic controller on the base and top plate, process the sample injection holes in the sample adding area on the top plate, and process the connection to the bottom of the middle layer. For the channel of the qualitative detection area, the channel connected to the quantitative detection area is processed on the upper middle layer, and the substrate, the middle layer and the top plate are sealed and bonded in sequence;

Installation of ultrasonic exciter and ultrasonic controller:

Install the ultrasonic transducer on the microfluidic chip and fix it with elastic clips, and connect the wires of the transducer to the ultrasonic generator; install the magnetic controller on the microfluidic chip, and connect the magnetic controller to the power supply;

Preparation of reagents:

Using ready-made Fe ₃ O ₄ nanoparticles, a variety of different tumor marker antibodies, including AFP, CEA, CYFRA21-1, CA19-9, CA125, PSA, NSE, β-HCG, etc.; The antibody is linked to Fe ₃ O ₄ nanoparticles by means of DNA or thiolation treatment to obtain quantitative magnetic microspheres;

Use SiO ₂ microspheres or polyethylene microspheres with different particle sizes, the particle sizes are 150nm, 200nm, 500nm, 700nm, 1100nm, etc., and the particle size floating range is required to be less than 5%, as qualitative nano-microspheres; and qualitative nano-microspheres are used. The surface of the sphere is activated to allow the attachment of thiolated fluorophores.

During detection, each qualitative nanosphere with different particle sizes is connected with an antibody of a tumor marker. The connection method can be DNA connection method or thiolated connection method, and then they are mixed and subjected to fluorescent luminescence. group modification; after modification, it is mixed with quantitative magnetic microspheres, and the mixing ratio of qualitative nano-microspheres: quantitative magnetic microspheres is 1:100 to 1:10000, so that the influence of qualitative detection on quantitative detection is reduced;

The mixed microspheres are injected into the sample well, and the sample to be tested is added to the sample well, so that the mixed microspheres and the sample to be tested react, and the tumor markers are combined with the qualitative nano-microspheres and quantitative magnetic microspheres. The amount of microspheres is much lower than the quantitative magnetic microspheres, so the qualitative nano-microspheres are fully reacted during the reaction;

Activate the ultrasonic transducers in the longitudinal group to sweep the frequency, so that all the microspheres move along the transport area to the detection area; at the same time, activate the transverse group. The vibration frequency of the transverse group is preset, so that there is no The qualitative nanospheres connected with tumor markers can enter the qualitative detection area from the corresponding openings, while the qualitative nanospheres connected with tumor markers cannot be driven by the corresponding lateral group because the tumor markers change the resonance frequency, so they cannot enter Qualitative detection area; at this time, the fluorescence intensity of different qualitative detection areas is detected, and the qualitative detection area where fluorescence intensity cannot be detected from beginning to end indicates that the corresponding qualitative nanospheres have detected tumor markers;

The quantitative magnetic microspheres move along the transport area under the driving of the longitudinal group, and at the same time, the magnetic controller is activated, so that the magnetic microspheres move along the top of the transport area and can enter the quantitative detection area;

Since the longitudinal group is in a frequency sweep mode, only the corresponding resonance frequency can be scanned to drive the quantitative magnetic microspheres to move, which leads to a pulsed increase of the magnetic microspheres entering the quantitative detection area, resulting in the magnetic microspheres in the quantitative detection area. The fluorescence or characteristic light absorption intensity changes intermittently, and the resonance frequency that drives the quantitative magnetic microspheres can be obtained according to the time point of the intermittent change. Since the type of tumor marker has been obtained by qualitative detection, it can be inversely bound to the quantitative magnetic microspheres according to the resonance frequency. The concentration of tumor markers on the surface and the effect of different concentrations of each tumor marker on the resonance frequency of quantitative magnetic microspheres can be obtained through experiments in advance, and a standard curve or table can be drawn.

The beneficial effects of the present invention are as follows: the present invention combines qualitative nano-microspheres and quantitative magnetic microspheres, and can simultaneously perform qualitative and quantitative detection at one time, and uses the combination of ultrasonic resonance drive and magnetic field control to distinguish qualitative detection and quantitative detection. , and because the amount of qualitative detection is low, the qualitative detection does not affect the quantitative detection, the operation is simple, and the stability is good.

Qualitative detection using qualitative microspheres of different particle sizes has good accuracy and strong anti-interference ability. The use of quantitative detection does not require separate treatment for each tumor marker, and has good applicability; in addition, the structures controlled by ultrasound and magnetic fields can be disassembled and used multiple times, which is economical.

Description of drawings

The accompanying drawings, which are included to provide a further understanding of the disclosed subject matter, are incorporated into and constitute a part of this specification. The drawings also illustrate implementations of the disclosed subject matter, and together with the detailed description serve to explain principles of implementation of the disclosed subject matter. No attempt has been made to show more structural detail than is required for a basic understanding of the disclosed subject matter and its various ways of practicing.

FIG. 1 is a schematic structural diagram of a microfluidic chip of the present invention.

Detailed ways

The advantages and features of the present invention and the method for achieving the stated objects will be apparent from the accompanying drawings and the following detailed description.

Example:

An antibody chip kit for quantitative detection of multiple tumor markers, including a microfluidic chip, an ultrasonic exciter, a magnetic controller, qualitative nano-microspheres of different particle sizes, quantitative magnetic microspheres, thiol luminophore reagents, multi- Antibodies and fluorescence detectors for various tumor markers; the surface of each qualitative nanosphere can be modified with an antibody of one tumor marker, and the surface of qualitative nanospheres can be modified with sulfhydryl luminescent groups; the surface of quantitative magnetic nanospheres can be modified at the same time Various tumor marker antibodies, and the quantitative magnetic particles themselves have fluorescence or characteristic light absorption peaks;

The microfluidic chip includes a sample addition area 11, a reaction area 12, a transport area 13 and a detection area; the sample addition area 11 includes a reagent sample addition area 11 and a sample addition area 11, and the sample addition area 11 is connected to the reaction area 12 and the reaction area 12. Connect 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 qualitative detection areas 14 is multiple, and each qualitative detection area 14 is connected to the transportation area On the exits at different distances from the reaction zone 12 of 13, the quantitative detection zone 15 is arranged at the end of the transport zone 13;

The outlets in the elongated transport channel of the transport zone 13 include a high outlet and a low outlet, wherein the low outlet is the outlet connected to the qualitative detection zone 14, and the high outlet is the outlet connected to the quantitative detection zone 15, wherein the horizontal position of the high outlet is high In the horizontal position of the low outlet, and the vertical height of the transport channel of the transport zone 13 is 20 times greater than that of the nano-microspheres and the magnetic microspheres; the qualitative nano-microspheres and the quantitative magnetic Transported from the reaction zone 12 to the detection zone.

The ultrasonic exciter includes an ultrasonic signal conditioner and an ultrasonic transducer sheet 16. The number of ultrasonic transducer sheets 16 is multiple groups, with two sheets in each group; The front and rear ends of the transportation area 13 have the same transportation direction, and multiple sets of transverse groups are arranged at the two ends perpendicular to the transportation direction of the transportation area 13. At the same time, the openings connecting the qualitative detection area 14 are arranged on the side perpendicular to the transportation direction of the transportation area 13. On the top, each opening connected to the qualitative detection area 14 is provided with a set of transverse groups;

The two transducers of the longitudinal group form moving standing waves when working, and the working frequency of the longitudinal group is the composite frequency, and the working mode is the sweeping working mode, which pushes the qualitative nano-microspheres and quantitative magnetic microspheres from the reaction area 12 to the detection. The working mode of the lateral group is a single frequency, and the qualitative nano-microspheres corresponding to the resonance frequency are pushed into the corresponding opening 18 to enter the qualitative detection area 14;

The magnetic controller 17 is arranged on the upper and lower surfaces of the detection chip, and controls 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, so as to directly reach the other end of the transportation area 13 and enter the quantitative detection area 15.

The fluorescence detector detects the fluorescence intensity in each qualitative detection area 14, and performs qualitative analysis of tumor markers on the sample according to the fluorescence intensities of different qualitative detection areas 14; the light detector detects the fluorescence intensity change or characteristic light in the quantitative detection area 15 Changes in uptake and quantification of tumor markers were performed on the samples in combination with the sweep frequency of the longitudinal groups.

A manufacturing method of the antibody chip kit for quantitatively detecting multiple tumor markers, which is characterized by comprising the following steps:

Fabrication of microfluidic chip:

The substrate, the middle layer and the top plate are processed by femtosecond laser. The materials of the substrate, the middle layer and the top plate are all PMMA. Area 12, transportation area 13, through holes for detection, the mounting grooves for installing the transducer and the magnetic controller 17 are machined on the base and the top plate, and the sample adding holes for the sample adding area 11 are machined on the top plate. A channel connected to the qualitative detection area 14 is processed in the layers, a channel connected to the 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 ultrasonic exciter and ultrasonic controller:

Install the ultrasonic transducer 16 on the microfluidic chip and fix it with elastic clips, and connect the lead of the transducer to the ultrasonic generator; install the magnetic controller 17 on the microfluidic chip, and connect the magnetic controller 17 Connect to the power supply;

Preparation of reagents:

Using ready-made Fe ₃ O ₄ nanoparticles, a variety of different tumor marker antibodies, including AFP, CEA, CYFRA21-1, CA19-9, CA125, PSA, NSE, β-HCG, etc.; The antibody is linked to Fe ₃ O ₄ nanoparticles by means of DNA or thiolation treatment to obtain quantitative magnetic microspheres;

Use SiO ₂ microspheres or polyethylene microspheres with different particle sizes, the particle sizes are 150nm, 200nm, 500nm, 700nm, 1100nm, etc., and the particle size floating range is required to be less than 5%, as qualitative nano-microspheres; and qualitative nano-microspheres are used. The surface of the sphere is activated to allow the attachment of thiolated fluorophores.

During detection, each qualitative nanosphere with different particle sizes is connected with an antibody of a tumor marker. The connection method can be DNA connection method or thiolated connection method, and then they are mixed and subjected to fluorescent luminescence. group modification; after modification, it is mixed with quantitative magnetic microspheres, and the mixing ratio of qualitative nano-microspheres: quantitative magnetic microspheres is 1:100 to 1:10000, so that the influence of qualitative detection on quantitative detection is reduced;

The mixed microspheres are injected into the sample well, and the sample to be tested is added to the sample well, so that the mixed microspheres and the sample to be tested react, and the tumor markers are combined with the qualitative nano-microspheres and quantitative magnetic microspheres. The amount of microspheres is much lower than the quantitative magnetic microspheres, so the qualitative nano-microspheres are fully reacted during the reaction;

The ultrasonic transducers 16 of the longitudinal group are activated, and the frequency is swept, so that all the microspheres move along the transport area 13 to the detection area; the transverse group is activated at the same time, and the vibration frequency of the transverse group is preset, so that the drive in the transverse group is The qualitative nanospheres that are not connected with tumor markers can enter the qualitative detection area 14 from the corresponding opening, while the qualitative nanospheres connected with tumor markers cannot be driven by the corresponding lateral group because the tumor markers change the resonance frequency. Therefore, the qualitative detection area 14 cannot be entered; at this time, the fluorescence intensities of different qualitative detection areas 14 are detected, and the qualitative detection area 14 whose fluorescence intensity cannot be detected from beginning to end indicates that the corresponding qualitative nanospheres have detected tumor markers;

The quantitative magnetic microspheres are moved along the transport area 13 under the driving of the longitudinal group, and the magnetic controller 17 is activated at the same time, so that the magnetic microspheres move along the top of the transport area 13 and can enter the quantitative detection area 15;

Since the vertical group is in a frequency sweep mode, the quantitative magnetic microspheres can only be driven to move when the corresponding resonance frequency is scanned, which leads to the increase of the magnetic microspheres entering the quantitative detection area 15 in a pulsed manner, thus causing the magnetic microspheres in the quantitative detection area 15 to increase in a pulsed manner. The fluorescence or characteristic light absorption intensity of the sphere changes intermittently, and the resonance frequency that drives the quantitative magnetic microspheres can be obtained according to the time point of the intermittent change. Since the type of tumor marker has been obtained by qualitative detection, it can be reversed based on the resonance frequency. The concentration of tumor markers on the surface of the microspheres, and the effect of each tumor marker at different concentrations on the resonance frequency of the quantitative magnetic microspheres can be obtained through experiments in advance, and a standard curve or table can be drawn.

The above are only the preferred embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention. should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (5)

1. an antibody chip kit for quantitative detection of multiple tumor markers, is characterized in that comprising microfluidic chip, ultrasonic exciter, magnetic controller, qualitative nano-microspheres of different particle sizes, quantitative magnetic microspheres, thiol luminescence Group reagents, antibodies for multiple tumor markers, fluorescence detectors; each qualitative nanosphere surface can be modified with a tumor marker antibody, and the surface of qualitative nanospheres can be modified with thiol luminescent groups; quantitative magnetic nanospheres The surface of the microspheres is modified with a variety of tumor marker antibodies, and the quantitative magnetic particles themselves have fluorescence or characteristic light absorption peaks; The microfluidic chip includes a sample adding area (11), a reaction area (12), a transport area (13) and a detection area; the sample adding area (11) includes a reagent adding area (11) and a sample adding area (11), The sample adding area (11) is connected to the reaction area (12), the reaction area (12) is connected to the transportation area (13), the transportation area (13) is provided with an elongated transportation channel, and the detection area is provided with a qualitative detection area (14) and a quantitative detection area (14). The number of detection areas (15) and qualitative detection areas (14) is multiple, and each qualitative detection area (14) is connected to the outlet of the transport area (13) at different distances from the reaction area (12), and the quantitative detection area ( 15) Set at the end of the transport area (13); The exits in the elongated transportation channel of the transportation area (13) include a high exit and a low exit, wherein the low exit is an exit connected to the qualitative detection area (14), and the high exit is an exit connected to the quantitative detection area (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 in the transport zone (13) is 20 times greater than that of the nano-microspheres and the magnetic microspheres; and the magnetic controller is transported from the reaction zone (12) to the detection zone. 2. a kind of antibody chip kit for quantitatively detecting multiple tumor markers according to claim 1, is characterized in that: The ultrasonic exciter includes an ultrasonic signal conditioner and an ultrasonic transducer sheet (16), and the number of the ultrasonic transducer sheets (16) is in multiple groups, with two sheets in each group; The longitudinal groups of the groups are arranged at the front and rear ends that are the same as the transportation direction of the transportation area (13), and the transverse groups of the plurality of groups are arranged at both ends perpendicular to the transportation direction of the transportation area (13). It is arranged on the side surface perpendicular to the transportation direction of the transportation area (13), and each opening connected to the qualitative detection area (14) is provided with a set of transverse groups; The two transducer sheets of the longitudinal group form moving standing waves when working, and the working frequency of the longitudinal group is the composite frequency, and the working mode is the sweeping working mode, which pushes the qualitative nano-microspheres and quantitative magnetic microspheres from the reaction zone (12) Advance to the detection area; the working mode of the lateral group is single frequency, and the qualitative nano-microspheres corresponding to the resonance frequency are pushed into the corresponding opening (18) to enter the qualitative detection area (14); The magnetic controller (17) is arranged on the upper and lower surfaces of the detection chip, and controls 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), so as to directly reach the other end of the transportation area (13). Enter the quantitative detection area (15). 3. The antibody chip kit for quantitatively detecting multiple tumor markers according to claim 2, characterized in that the fluorescence detector detects the fluorescence intensity in each qualitative detection area (14), and according to different qualitative detection areas The fluorescence intensity of (14) is used for qualitative analysis of tumor markers; the photodetector detects the changes of fluorescence intensity or characteristic light absorption in the quantitative detection area (15), and the samples are subjected to tumor markers in combination with the sweep frequency of the longitudinal group. Quantitative analysis of substances. 4. A method for making an antibody chip kit for quantitatively detecting multiple tumor markers according to any one of claims 1-3, characterized in that it comprises the steps of: Fabrication of microfluidic chip: The substrate, the middle layer and the top plate are processed by femtosecond laser. The materials of the substrate, the middle layer and the top plate are all PMMA. The number of layers in the middle layer is two layers. The sample application area is processed in the middle layer by femtosecond laser (11) , the reaction area (12), the transport area (13), the through hole for detection, the mounting grooves for installing the transducer and the magnetic controller (17) are machined on the base and the top plate, and the sample adding area (11) is machined on the top plate. ), a channel connected to the qualitative detection area (14) is processed in the lower intermediate layer, a channel connected to the quantitative detection area (15) is processed in the upper intermediate layer, and the substrate and the intermediate layer are processed in sequence. Seal and bond with the top plate; Installation of ultrasonic exciter and ultrasonic controller: Install the ultrasonic transducer sheet (16) on the microfluidic chip, and use elastic clips to fix it, and connect the wires of the transducer sheet to the ultrasonic generator; install the magnetic controller (17) on the microfluidic chip, and connect the magnetic controller (17) to the power supply; Preparation of reagents: Using ready-made Fe ₃ O ₄ nanoparticles, a variety of different tumor marker antibodies, including AFP, CEA, CYFRA21-1, CA19-9, CA125, PSA, NSE, β-HCG, etc.; The antibody is linked to Fe ₃ O ₄ nanoparticles by means of DNA or thiolation treatment to obtain quantitative magnetic microspheres; Use SiO ₂ microspheres or polyethylene microspheres with different particle sizes, the particle sizes are 150nm, 200nm, 500nm, 700nm, 1100nm, etc., and the particle size floating range is required to be less than 5%, as qualitative nano-microspheres; and qualitative nano-microspheres are used. The surface of the sphere is activated to allow the attachment of thiolated fluorophores. 5. preparation method according to claim 4, is characterized in that: During detection, each qualitative nanosphere with different particle sizes is connected with an antibody of a tumor marker. The connection method can be DNA connection method or thiolated connection method, and then they are mixed and subjected to fluorescent luminescence. group modification; after modification, it is mixed with quantitative magnetic microspheres, and the mixing ratio of qualitative nano-microspheres: quantitative magnetic microspheres is 1:100 to 1:10000, so that the influence of qualitative detection on quantitative detection is reduced; The mixed microspheres are injected into the sample well, and the sample to be tested is added to the sample well, so that the mixed microspheres and the sample to be tested react, and the tumor markers are combined with the qualitative nano-microspheres and quantitative magnetic microspheres. The amount of microspheres is much lower than the quantitative magnetic microspheres, so the qualitative nano-microspheres are fully reacted during the reaction; Activate the ultrasonic transducer sheet (16) of the longitudinal group to perform frequency sweep, so that all the microspheres move to the detection area along the transport area (13); at the same time start the transverse group, the vibration frequency of the transverse group is preset, so that in the Driven by the transverse group, qualitative nanospheres without tumor markers can enter the qualitative detection area from the corresponding opening (14), while qualitative nanospheres connected with tumor markers cannot be detected due to the change of the resonance frequency of tumor markers. The corresponding horizontal group is driven, so it cannot enter the qualitative detection area (14); at this time, the fluorescence intensities of different qualitative detection areas (14) are detected, and the qualitative detection area (14) whose fluorescence intensity cannot be detected from beginning to end indicates that its corresponding Qualitative nanospheres detected tumor markers; The quantitative magnetic microspheres move along the transport zone (13) under the driving of the longitudinal group, and simultaneously start the magnetic controller (17), so that the magnetic microspheres move along the top of the transport zone (13) and can enter the quantitative detection zone (15). ); Since the longitudinal group is in a frequency sweep mode, the quantitative magnetic microspheres can only be driven to move when the corresponding resonant frequency is scanned, which leads to a pulsed increase of the magnetic microspheres entering the quantitative detection area (15), which leads to the quantitative detection area (15). ), the fluorescence or characteristic light absorption intensity of the magnetic microspheres changes intermittently, and the resonance frequency that drives the quantitative magnetic microspheres can be obtained according to the time point of the intermittent change. Since the type of tumor markers has been obtained by qualitative detection, it can be reversed according to the resonance frequency. The concentration of tumor markers bound to the surface of the quantitative magnetic microspheres, the effect of each tumor marker at different concentrations on the resonance frequency of the quantitative magnetic microspheres can be obtained through experiments in advance, and a standard curve or table can be drawn.

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