CN210071844U

CN210071844U - Cancer detection device based on PD-L1 receptor

Info

Publication number: CN210071844U
Application number: CN201821829523.7U
Authority: CN
Inventors: 孙佳姝
Original assignee: Beijing Institute of Nanoenergy and Nanosystems
Current assignee: National Center for Nanosccience and Technology China; Beijing Institute of Nanoenergy and Nanosystems
Priority date: 2018-11-07
Filing date: 2018-11-07
Publication date: 2020-02-14
Anticipated expiration: 2028-11-07

Abstract

A PD-L1 receptor-based cancer detection device comprising: a heating unit for heating the extracellular vesicles in the blood of the subject; a sample chamber unit disposed at one side of the heating unit; the signal processing unit is arranged on one side of the sample chamber unit and is used for acquiring at least one optical signal parameter, and the single-species protein expression intensity is acquired by quantifying the optical parameter and carrying out statistical calculation on the optical parameter. The utility model discloses an use aptamer or antibody to carry out the light mark to the extracellular vesicle in the patient's blood cell to use detecting element to detect and handle the light parameter in the light mark, count the calculation through quantifying the light parameter and to it, the expression protein intensity that reachs the extracellular vesicle that can be quick accurate, the detection precision is high.

Description

Cancer detection device based on PD-L1 receptor

Technical Field

The utility model relates to a cancer diagnosis technical field especially relates to a cancer detection device based on PD-L1 acceptor.

Background

PD-1 is known as programmed death receptor 1, and is called programmed death 1 in English, is an important immunosuppressive molecule and is a member of CD28 superfamily. The immunoregulation taking PD-1 as a target point has important significance in the aspects of tumor resistance, infection resistance, autoimmune disease resistance, organ transplantation survival and the like. The ligand PD-L1 can also be used as a target, and the corresponding antibody can also play the same role. PD-L1 is known as programmed cell death-Ligand 1, English name programmed cell death-Ligand 1, and is the first type of transmembrane protein with a size of 40 kDa. The immune system normally responds to foreign antigens that accumulate in the lymph nodes or spleen, promoting proliferation of antigen-specific T cells. The combination of programmed death receptor 1(PD-1) and programmed death-ligand 1(PD-L1) can transmit inhibitory signals and reduce the proliferation of T cells. One way for tumor cells to escape T cell destruction is by producing PD-L1 on its surface, which can transmit inhibitory signals when PD-1 on the T cell surface of immune cells recognizes PD-L1, and the T cells cannot find and send out attacking signals to the tumor cells.

Programmed death receptor-1 (PD-1) is the major immune checkpoint receptor that helps maintain tolerance to tumor cells by down-regulating T cell effector function by binding to its ligand programmed death ligand-1 (PD-L1). Blocking these pathways by anti-PD-1 and anti-PD-L1 antibodies may help prevent this down-regulation so that T cells maintain their anti-tumor properties and the ability to mediate tumor cell death. At present, three inhibitors aiming at PD-1 and PD-L1 are mainly available on the market, namely pembrolizumab, Nibolumab and Atezolizumab, and can be used for treating various cancers such as melanoma, non-small cell lung cancer, bladder cancer and the like. However, not all patients benefit from the treatment with PD-1/PD-L1 inhibitors, and PD-1/PD-L1 inhibitors currently produce a long lasting tumor control in only a small fraction of cancer patients. Therefore, testing a patient for positive expression of PD-L1 can be effective in helping the patient select the appropriate drug for treatment. Currently, the detection method of PD-1/PD-L1 is mainly based on the detection of cell protein level, and in clinic, an immunohistochemical method is mainly adopted, and tumor tissues obtained after operation or puncture are used for section staining. The outcome of immunohistochemistry is closely related to the experience of the pathologist. In addition to staining techniques, the specificity of the antibody is also of particular importance. At present, the detection of PD-L1 in China is relatively disordered, and firstly, the dyeing technology and conditions are not unified; secondly, the variety of the staining antibodies; thirdly, the pathological evaluation criteria are not unified, which all cause the value of the domestic patients for using immunohistochemical evaluation on the PD-L1 level to be reduced. In addition, since immunohistochemistry requires tissue acquisition and examination, it is an invasive procedure, either through surgery or puncture, and may cause injury to the patient. Meanwhile, clinical experimental research shows that the blocking of PD-L1 can effectively treat or inhibit tumor growth. Therefore, clinically, a simple and easy detection means is needed to screen out tumor patients with tumor tissues or cells highly expressing PD-L1, and guidance and basis are provided for personalized treatment of blocking PD-L1. The existing kit for detecting PD-L1 has the defects of low sensitivity, low specificity and the like. Therefore, a new noninvasive evaluation method and a PD-L1 detection method with stable standard are urgently needed.

Chinese patent publication No.: cn201810312287.x discloses a method for detecting circulating tumor cell surface marker molecule PD-L1, comprising the following steps: (1) treating whole blood with erythrocyte lysate, separating out nucleated cells, and fixing with formaldehyde; (2) positive screening is carried out through a tumor immune light marker cytokeratin antibody anti-CK, all cells are incubated by a PD-L1 primary antibody, then a PD-L1 secondary antibody marked with FITC light groups is used for incubation, and all cells are marked by a cell nucleus light dye DAPI; (3) and (3) adopting high-throughput multicolor imaging analysis, selecting filters of CY5, FITC and DAPI, observing the light color of the channel surface, and finally realizing the detection of the circulating tumor cell surface marker molecule PD-L1. It can be seen that the above detection method has the following problems.

First, in the detection method, a blood sample is lysed using a red blood cell lysate during sampling, and when nucleated cells are obtained, residues after lysis remain in the liquid, thereby affecting subsequent detection.

Secondly, after the PD-L1 receptor is optically labeled, light is distributed in multiple colors, accurate measurement cannot be carried out on single color during observation, and the measurement precision is low.

Third, the optical signature distribution is too diffuse and there is no way to accumulate it, which can result in too weak an optical signal to obtain a high accuracy result when detecting the optical signature.

SUMMERY OF THE UTILITY MODEL

Therefore, the utility model provides a cancer detection device based on PD-L1 acceptor for overcome among the prior art because the fluorescence colour kind is many leads to the problem that the detection precision is low.

The invention provides a PD-L1 receptor detection device, which comprises:

a heating unit for heating extracellular vesicles in blood of a subject;

the sample chamber unit is arranged on one side of the heating unit and used for loading extracellular vesicles, PD-L1 receptors in the extracellular vesicles in the sample chamber unit can be specifically combined with aptamers or antibodies to label optical signals, and after the sample chamber unit is heated by the heating unit, thermophoresis effect and convection are generated in the sample chamber unit, so that the extracellular vesicles are gathered on the side, with lower temperature, of the sample chamber unit;

the signal processing unit is arranged on one side of the sample chamber unit and used for acquiring and calculating the amplified optical signals, and the signal processing unit acquires at least one optical signal parameter and judges the corresponding optical signal parameter by quantifying the optical parameter to acquire the expression intensity of the PD-L1 receptor in the single extracellular vesicle.

Further, the sample chamber unit is used for catalyzing the luminescent substrate by the luminescent catalytic substance in the incubated extracellular vesicles to enable the substrate to reach an excited state, and releasing light energy in the process of converting the substrate to a ground state so as to enable the extracellular vesicles to carry light signals on the surfaces.

Further, the detection physical quantity of the signal processing unit includes at least one of a wavelength λ, a brightness L, an intensity C, and an absorbance a of the optical signal.

Further, the heating unit is a laser heater provided with a focusing device, and the specific heating position is adjusted by changing the focus.

Further, the sample chamber unit is disposed on one side of the heating unit, and the signal processing unit is disposed on one side of the sample chamber unit away from the heating unit, and includes:

the first heat conducting surface is arranged on one side of the heating unit and used for absorbing heat of the heating unit;

a second heat-conducting surface arranged below the first heat-conducting surface and used for absorbing the heat of the heating unit;

and the gasket is arranged between the first heat-conducting surface and the second heat-conducting surface, is provided with a through hole in the center and is used for loading the sample liquid.

Furthermore, the first heat conduction surface and the second heat conduction surface are both made of transparent materials, and the heat conductivity of the second heat conduction surface is higher than that of the first heat conduction surface.

Furthermore, a through hole is formed in the gasket and is used for being matched with the first heat-conducting surface and the second heat-conducting surface to form a space for loading extracellular vesicles.

Further, the signal amplification unit is disposed on a side of the sample chamber unit away from the heating unit, and is used for amplifying and reflecting the light signal on the surface of the extracellular vesicle, and the signal amplification unit includes:

the objective lens is arranged on one side of the second heat-conducting surface, which is far away from the heating unit, and is used for observing optical signals;

the collecting reflector is arranged on one side of the objective lens, which is far away from the heating unit, and is used for reflecting the light mark;

the magnifying reflector is arranged on one side of the objective lens, which is far away from the heating unit, and is used for reflecting a light source;

and the observation light source is arranged on one side of the magnifying reflector and used for providing a magnifying light source for the optical mark.

Furthermore, the collecting reflector and the amplifying reflector form a certain included angle with the objective lens so as to respectively reflect the optical signal and the amplifying light source to the designated positions.

Furthermore, the signal acquisition unit is one or more of a CCD camera, a luminometer, a spectrometer, a monochromator, an sCMOS, an EMCCD and a PMT according to the optical parameters to be detected by the detection device.

Compared with the prior art, the method has the advantages that the PD-L1 receptor of the extracellular vesicle of the blood of the patient is optically labeled by using the aptamer or the antibody, the physical parameter of the light in the optical label is detected and processed by using the detection unit, the weighted expression intensity of the PD-L1 receptor of the extracellular vesicle can be rapidly and accurately obtained by analyzing the optical parameter, and the detection precision is high. Particularly, the invention adopts the accumulation detection of various physical parameters instead of the direct biological reaction detection, the detection process is simpler and easier to operate, the sample dosage is small, and the expression of the detection dosage is easier to quantify compared with the biological reaction detection through the accumulation detection of the physical parameters, so that the expression of the PD-L1 receptor can be clearly judged. In particular, the invention detects and parametrizes the luminosity of the extracellular vesicle PD-L1 receptor, and then determines the expression intensity of the PD-L1 receptor according to the standard function relationship of the standard PD-L1 receptor concentration and a certain parameter in an optical signal. In the detection process based on the same antibody or aptamer, through detection and calculation of various physical parameters and mutual comparison, the expression of the optimal physical parameters is selected to determine the final canceration result.

In particular, the invention adopts a light accumulation mode combining chemiluminescence, thermophoresis effect and convection, achieves an excited state through catalysis of a luminous catalytic substance in an aptamer or antibody combined with the extracellular vesicles, releases light energy in the process of converting the luminous catalytic substance into a ground state so as to mark optical signals on the surfaces of the extracellular vesicles, and the luminous catalytic substance can keep emitting light for a long time during detection.

Furthermore, the sample chamber unit is provided with the first heat-conducting surface and the second heat-conducting surface which are made of transparent materials, the sample liquid in the sample chamber unit is heated to enable the extracellular vesicles to generate thermophoresis effect and move to a low temperature position, meanwhile, the sample liquid can generate thermal convection after the temperature is increased and enable the extracellular vesicles to be accumulated at a designated position, so that optical signals of the extracellular vesicles are amplified, specific values of optical parameters of the extracellular vesicles can be observed more accurately when the optical parameters are detected, and the detection precision of the detection system is further improved.

Further, the detection system of the present invention accumulates extracellular vesicles using a thermophoresis effect and a thermal convection effect, and thus, the detection system has no particular limitation on the size of the sample chamber unit. The sample liquid in the sample chamber unit is used for loading extracellular vesicles and aptamers or antibodies and can enable the extracellular vesicles and the aptamers or the antibodies to generate thermophoretic effect and convection, so that the detection system is not particularly limited in selection of the sample liquid as long as the sample liquid can drive the extracellular vesicles to move and accumulate under the effect of thermal convection. Furthermore, the extracellular vesicles and the aptamer or the antibody are connected together in a specific combination mode, so that the optical marker can be stably connected to the extracellular vesicles, the optical parameters of the extracellular vesicles can be observed more accurately when the extracellular vesicles accumulate, and the detection precision of the detection system is further improved. The force of the extracellular vesicles under the thermophoresis effect is in direct proportion to the square of the diameters of the extracellular vesicles and is independent of the number of the extracellular vesicles, so that the detection can be completed only by a small amount of blood samples, the sample dosage of the extracellular vesicles is only 0.1 microliter, and the samples do not need to be subjected to pretreatment.

Further, when the sample chamber unit is heated, as long as the temperature difference exists between the first heat-conducting surface and the second heat-conducting surface, a thermophoresis effect and a thermal convection can be generated in the sample chamber unit, and the extracellular vesicles with the optical markers can be accumulated to a specified position. The detection system only needs to detect the optical parameters of the accumulated extracellular vesicles without using other special instruments, and the cost of the detection device is saved under the condition that the detection precision of the detection system is not influenced.

Furthermore, the detection system is provided with a data acquisition unit which can extract specified optical parameters from the acquired protein expression map and bring the optical parameters into a non-weighted sum model to calculate the non-weighted expression intensity of the extracellular vesicle expressed protein, and the detection precision of the detection system is further improved by converting visual images into specific numbers.

Furthermore, the detection system is provided with a data acquisition unit which can extract specified optical parameters from the acquired protein expression map, and the detection precision of the detection system is further improved by converting visual images into specific numbers.

Further, the detection device can select corresponding aptamers or antibodies for labeling high-expression proteins in different cancers, so as to measure abundance maps of the corresponding expression proteins and calculate expression intensity, and thus, the detection device can not only detect the PD-L1 receptor, but also rapidly and accurately detect other cancers, such as: lung cancer, pancreatic cancer, colorectal cancer, stomach cancer, prostate cancer, head and neck cancer, skin cancer, kidney cancer, testicular cancer, thyroid cancer, bladder cancer, uterine cancer, vaginal cancer, endometrial cancer, ovarian cancer, esophageal cancer, oral cancer, salivary gland cancer, laryngeal cancer, peritoneal cancer, nasal cancer, laryngeal cancer, fallopian tube cancer, nephrocyte cancer, lymphatic cancer, biliary duct cancer, and also sarcoma of swing, synovial sarcoma, medulloblastoma, trophoblastoma, glioma, glioblastoma, cholesteatoma, chondrosarcoma, ependymoma, schwannoma, neuroma, rhabdomyosarcoma.

Drawings

FIG. 1 is a schematic diagram of the PD-L1 receptor-based cancer detection device of the present invention;

FIG. 2 is a schematic diagram of a detection device based on the use of chemiluminescence for the PD-L1 receptor;

FIG. 3 is a schematic diagram of the present invention using a monochromator to detect the absorbance of extracellular vesicle samples.

Detailed Description

In order to make the objects and advantages of the present invention more apparent, the present invention will be further described with reference to the following embodiments; it should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The above and further features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

Preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the present invention, and do not limit the scope of the present invention.

It should be noted that in the description of the present invention, the terms of direction or positional relationship indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.

Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

Embodiment of the System

The embodiment of the present invention is a PD-L1 receptor detection apparatus based on thermophoretic extracellular vesicles, please refer to fig. 1, which is a schematic structural diagram of a PD-L1 receptor detection apparatus based on thermophoretic extracellular vesicles detection according to an embodiment of the present invention, the system of the present embodiment includes a heating unit 1, a sample chamber unit 2, a signal amplification unit 3 and a signal processing unit 4, wherein the heating unit 1 is disposed above the sample chamber unit 2 for heating a sample in the sample chamber unit 2; the sample chamber unit 2 is filled with sample liquid for loading extracellular vesicles and aptamers with fluorescent labels; the signal amplification unit 3 is arranged below the sample chamber unit 2 and used for amplifying the fluorescence signal of the fluorescence mark, the signal processing unit 4 is arranged on the side surface of the signal amplification unit 3 and used for collecting and recording the amplified fluorescence signal, and carrying out or acquiring one or more of the brightness, the light intensity and the light wavelength parameter of the fluorescence signal, and meanwhile, a non-weighted model is used for carrying out cancer lesion degree detection on the extracellular vesicle of the sample to be detected.

Specifically, before the PD-L1 receptor detection device based on thermophoretic extracellular vesicle detection works, extracellular vesicles and aptamers with fluorescent labels are placed in the sample chamber unit 2, and the aptamers are specifically combined with PD-L1 receptors in the extracellular vesicles, so that the fluorescent labels are marked on the extracellular vesicles. After the labeling is finished, the heating unit 1 starts to heat the sample chamber unit 2, and after the sample chamber unit 2 is heated, the sample liquid in the sample chamber unit starts to generate thermophoresis effect and generate convection, and the labeled extracellular vesicles are gathered at the designated position; after the aggregation is finished, the signal amplification unit 3 emits a contrast light source to the position where the extracellular vesicles aggregate, the signal processing unit 4 collects relevant information of the aggregated extracellular vesicles, corresponding analysis is carried out on the relevant information, one or more of light brightness, light intensity and wavelength parameters are obtained, and the cancer lesion degree is judged in a non-weighted summation mode. It can be understood by those skilled in the art that the PD-L1 receptor detection apparatus based on thermophoretic extracellular vesicle detection in the present embodiment can be used for not only aggregating and detecting extracellular vesicles, but also detecting extracellular vesicles or other types of micro-nano biological particles, as long as the PD-L1 receptor detection apparatus based on thermophoretic extracellular vesicle detection can reach its designated working state.

Referring to fig. 1, the heating unit 1 according to the embodiment of the present invention is a laser heater disposed above the sample chamber unit 2 for heating the sample liquid inside the sample chamber unit 2 to generate a circular heating region therein. When the labeling of the extracellular vesicles is completed, the heating unit 1 heats the sample liquid inside the sample chamber unit 2 to aggregate the extracellular vesicles. It is understood that the heating manner of the heating unit 1 is not limited to laser irradiation, and the selection of the direction and power of laser irradiation is not particularly limited in this embodiment as long as the heating unit 1 can generate a temperature difference inside the sample chamber unit 2 to converge extracellular vesicles.

As shown in fig. 1, the sample chamber unit 2 of the embodiment of the present invention is disposed below the heating unit 1, and is used for containing a sample liquid containing extracellular vesicles and aptamers, and includes a first heat-conducting surface 21, a second heat-conducting surface 22, and a gasket 23; the gasket 23 is disposed under and in contact with the first heat-conducting surface 21 for containing a sample liquid, and the second heat-conducting surface 22 is disposed under the gasket 23 for sealing the sample liquid inside the gasket 23 together with the first heat-conducting surface 21. When the heating unit 1 heats the sample chamber unit 2, the laser sequentially passes through the first heat conduction surface 21 and the second heat conduction surface 22 to heat the sample chamber unit 2, and the sample liquid is heated, so that the temperature of the extracellular vesicles also rises after heating, at this time, the extracellular vesicles generate thermophoresis effect and move towards the first heat conduction surface 21 and the second heat conduction surface 22 with lower temperature, and because the first heat conduction surface 21 and the second heat conduction surface 22 have different heat conductivities, the temperature at the heating point of the first heat conduction surface 21 is higher than that at the heating point of the second heat conduction surface 22 after heating, so that the sample liquid generates temperature difference, and the sample liquid generates heat convection towards a high temperature formula in the sample chamber unit 2 along a low temperature, so that the extracellular vesicles in the sample migrate and are accumulated to the second heat conduction surface 22. It is understood that the sample chamber unit 2 of the present embodiment may be disposed below, above, to the left, or to the right of the heating unit 1, as long as the heating unit 1 can heat the sample chamber unit 2 to raise the temperature of the sample liquid therein.

Specifically, the first heat-conducting surface 21 is a glass plate, which is disposed above the gasket 23, and is used for sealing the sample liquid inside the gasket 23 and heating the sample liquid together with the heating unit 1. When the laser of the heating unit 1 passes through the first heat conducting surface 21, it heats the center of the first heat conducting surface 21 and increases the temperature of the heated portion, and after the temperature is increased, the first heat conducting surface 21 transfers heat to the sample liquid in the gasket 23 and generates convection to the sample liquid to accumulate extracellular vesicles. It is understood that the material of the first thermal conductive surface 21 may be glass, polymethyl methacrylate (PMMA), Polydimethylsiloxane (PDMS), sapphire or other transparent materials, as long as the first thermal conductive surface 21 can be heated.

Specifically, the second heat-conducting surface 22 is a glass plate having a higher heat conductivity than the first heat-conducting surface 21, and is disposed below the gasket 23 to seal the sample liquid inside the gasket 23 and heat the sample liquid together with the heating unit 1. When the laser of the heating unit 1 passes through the second heat conduction surface 22, it will heat the center of the second heat conduction surface 22 and raise the temperature of the heated place, after the temperature is raised, the second heat conduction surface 22 will transfer the heat to the sample liquid in the pad 23, because the heat conductivity of the second heat conduction surface 22 is higher than that of the first heat conduction surface 21, after the heating unit 1 finishes heating, the temperature of the second heat conduction surface 22 will be lower than that of the first heat conduction surface 21, a temperature difference is generated in the pad 23, and the sample liquid generates convection, so as to accumulate the extracellular vesicles. It is understood that the material of the second thermal conductive surface 22 can be glass, polymethyl methacrylate (PMMA), Polydimethylsiloxane (PDMS), sapphire or other transparent materials, as long as the second thermal conductive surface 22 can be heated to a temperature lower than the core temperature of the sample liquid.

Specifically, the gasket 23 is a circular plate with through holes, which is disposed between the first heat-conducting surface 21 and the second heat-conducting surface 22, and is used for loading sample liquid and accumulating extracellular vesicles. When the heating unit 1 heats the sample chamber unit 2, the focal point of the heating laser is located at the sample liquid inside the pad, the sample liquid is heated to generate a thermophoretic effect on the extracellular vesicles inside the sample liquid and the extracellular vesicles are gathered to the second heat conduction surface 22, and meanwhile, due to the temperature difference between the first heat conduction surface 21 and the second heat conduction surface 22, the sample liquid starts to generate convection and gather the extracellular vesicles at the laser irradiation position of the second heat conduction surface 22. It will be appreciated that the sample fluid in the pad 23 may be plasma, serum or any form of blood or processed derivative thereof, provided that the sample fluid is capable of carrying extracellular vesicles and aptamers and of causing thermophoretic effects and convection.

As shown in fig. 1, the signal amplifying unit 3 according to the embodiment of the present invention is disposed below the sample chamber unit 2, and is configured to irradiate the extracellular vesicles collected in the second heat-conducting surface 22 and amplify the fluorescence signals in the extracellular vesicles, and includes an objective lens 31, a collecting mirror 32, a magnifying mirror 33, and an observation light source 34. The objective lens 31 is disposed below the second heat-conducting surface 22 for collecting the fluorescence signal of the fluorescently-labeled extracellular vesicle, the collecting mirror 32 is disposed below the ocular lens 31 for reflecting the amplified fluorescence signal to the signal processing unit 4, the magnifying mirror 33 is disposed below the collecting mirror 32 for reflecting the light from the observation light source 34 to the objective lens 31, and the observation light source 34 is disposed at the right side of the magnifying mirror 33 for providing the light of the amplified fluorescence signal. When the signal amplification unit starts to work, the observation light source 34 emits light, the light is reflected to the objective lens 31 through the amplification reflector 33, the objective lens 31 irradiates the light to the gathering position of the extracellular vesicles in the second heat conduction surface 22, the fluorescence signals of the extracellular vesicles are amplified according to the light, and after the amplification is completed, the signal processing unit 4 collects the amplified fluorescence signals through the collection reflector 32, so that the collection and processing of the fluorescence signals are completed. It is understood that the signal amplification unit 3 may be disposed above, below, to the left, or to the right of the sample chamber unit 2, as long as it is capable of collecting a fluorescence signal in the sample chamber.

Specifically, the objective lens 31 is disposed below the location where the extracellular vesicles of the second heat-conducting surface 22 are gathered, and is used for collecting the fluorescence signals in the extracellular vesicles, and when the objective lens 31 is irradiated by the light from the observation light source 34, the objective lens 31 irradiates the light onto the second heat-conducting surface 22, so as to amplify the fluorescence signals on the extracellular vesicles. It is to be understood that the type of the objective lens 31 is not particularly limited in this embodiment, as long as the objective lens 31 can achieve its specified operation state.

Specifically, the collecting mirror 32 according to the embodiment of the present invention is a plane mirror, and is disposed below the objective lens 31 and forms an included angle of 45 ° with the objective lens 31, so as to reflect the amplified fluorescent signal. After the fluorescent signal of the extracellular vesicle is amplified, the collecting mirror 32 reflects the fluorescent label to the signal processing unit 4 to complete the collection of the fluorescent signal. It is understood that the size of the collecting reflector 32 is not limited in this embodiment, as long as the collecting reflector 32 can completely reflect the fluorescence signal to the signal collecting unit.

As shown in fig. 1, the signal processing unit 4 of the embodiment of the invention includes a CCD camera disposed at the right side of the collecting mirror 32 for collecting the fluorescence signal of the extracellular vesicle. After the fluorescence signal is amplified, the collecting mirror 32 reflects the amplified fluorescence signal to the signal processing unit 4, and the signal processing unit 4 collects and arranges the fluorescence signal to form a single-detection map, it can be understood that the signal processing unit 4 may include a CDD camera, or may be any instrument capable of detecting the fluorescence signal, as long as the signal processing unit 4 can photograph the extracellular vesicles with the fluorescence markers through the signal amplifying unit 3 to obtain information. Of course, the signal processing unit 4 may be located on the left side, right side, upper side, or lower side of the signal amplifying unit 3 as long as it is satisfied that the signal processing unit 4 can collect and process the fluorescence signal through the signal amplifying unit 3.

This system embodiment detection device links to each other fluorescence labeling and aptamer through now when detecting, incubates aptamer and the sample extracellular vesicle that awaits measuring together again in order to mark the fluorescence labeling with extracellular vesicle, easy operation, easily execution, when using this system to detect a plurality of determiners, the patient only need provide a small amount of blood samples, just can carry out quick diagnosis to patient's the state of an illness.

Second embodiment of the System

The embodiment of the utility model provides an extracellular vesicle detection device based on chemiluminescence, as the utility model discloses a preferred embodiment, please show with reference to fig. 2, it does the embodiment of the utility model provides a PD-L1 receptor detection device's structural schematic diagram based on thermophoresis extracellular vesicle detects, the system of this embodiment includes heating unit 1, sample bin unit 2 and signal processing unit 4, and this above-mentioned unit is the same with above-mentioned embodiment one.

Different from the above-mentioned embodiment, the utility model discloses a light mark adopts chemiluminescence marking method, before using PD-L1 receptor detection device based on thermophoresis extracellular vesicle detects, with the aptamer or the antibody that mark the luminous catalytic substance of extracellular vesicle await measuring sample together incubation, luminous catalytic substance can be the enzyme, express protein specific binding with extracellular vesicle mark enzyme through aptamer or antibody and extracellular vesicle, after the mark is accomplished, will incubate the sample of accomplishing and put into sample bin unit 2, and to the inside luminous substrate that adds of sample bin unit, the enzyme catalysis luminous substrate makes it reach the excited state to send light signal when it turns into the ground state.

When the PD-L1 receptor detection device based on thermophoretic extracellular vesicle detection starts to work, the heating unit 1 heats the sample chamber unit 2, so that the extracellular vesicles inside the sample chamber unit generate thermophoretic effect and start to move to the surface with low temperature, meanwhile, the temperatures of the two sides of the sample chamber unit are different, so that convection starts to be generated inside the sample chamber unit 2, the extracellular vesicles are accumulated at a specified position, and after the accumulation is finished, the signal acquisition unit 3 starts to acquire optical signals of the extracellular vesicles and obtains an abundance map of the protein expressed on the surface of the extracellular vesicles. Referring to fig. 2, the signal processing unit 4 according to the embodiment of the present invention is disposed below the sample chamber unit 2 for observing and collecting the aggregated extracellular vesicles.

Specifically, in the chemiluminescence-based extracellular vesicle detection system of the present embodiment, the labeled enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (ALP), or other kinds of labeled enzymes; the luminescent substrate is luminol (32 aminophthalic hydrazide), isoluminol (42 aminophthalic hydrazide) or other derivatives, and the luminescent substrate can react with the labeling enzyme and emit light.

Compared with the first embodiment of the detection device, the present embodiment has the same structure, principle and operation function as the heating unit 1 and the sample chamber unit 2, but because the PD-L1 receptor in the extracellular vesicle is labeled by the light generated by the chemical reaction and the surface of the extracellular vesicle keeps a high light signal for a long time after the labeling is completed, the present embodiment can accurately observe and collect the light signal of the extracellular vesicle without using the magnifying mirror 33 and the observation light source 34 to amplify the light signal.

The detection device of the embodiment incubates the antibody and the extracellular vesicles together and connects the antibodies and the extracellular vesicles with each other, the extracellular vesicles and the luminescent substrate are placed into the sample chamber unit 2 after incubation is completed, the luminescent substrate is catalyzed by the luminescent catalytic substance in the antibody to reach an excited state, and light energy is released when the luminescent substrate is converted into a ground state, so that light signals are marked by a PD-L1 receptor in the extracellular vesicles, meanwhile, the chemical reaction is a catalytic reaction, and the luminescent catalytic substance is used as a catalyst and can catalyze the luminescent substrate to react and continuously generate light, so that the extracellular vesicles can keep emitting light for a long time during detection.

Furthermore, because the optical signal can be maintained for a long time, the optical signal does not need to be amplified for the second time, and when the optical signal is collected, compared with the first system embodiment, the first system embodiment can clearly and accurately collect the optical signal only once, so that the detection time of the system is saved, and the detection efficiency is improved.

Furthermore, during detection, the marked extracellular vesicles are transferred from the incubation container to the sample chamber unit 2 filled with sample liquid, so that the phenomenon that redundant antibody bodies react and emit light together is eliminated, and the cancer detection device based on the chemical detection of the thermophoretic extracellular vesicles has high accuracy on the basis of the advantages of the cancer detection device based on the thermophoretic extracellular vesicles when the cancer detection device based on the chemical detection of the thermophoretic extracellular vesicles detects the extracellular vesicles.

Based on the two embodiments, the photometric detection and parameter of the extracellular vesicles are obtained based on a non-weighted calculation method, and then the canceration degree is determined according to a standard functional relationship between the standard protein marker concentration and a certain parameter of light. For example, the physical quantity is determined by detecting optical characteristics such as light intensity, light brightness, light frequency, and sample concentration at a specific wavelength absorbance.

So far, the technical solution of the present invention has been described with reference to the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, a person skilled in the art can make equivalent changes or substitutions to the related technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention; various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A PD-L1 receptor-based cancer detection device, comprising:

a heating unit for heating extracellular vesicles in blood of a subject;

2. The PD-L1 receptor-based cancer detection apparatus according to claim 1, wherein the sample compartment unit is configured to catalyze the luminescent substrate to an excited state by the luminescent catalytic substance in the incubated extracellular vesicles, and release light energy during the transition to the ground state, so as to make the extracellular vesicles have light signals on their surfaces.

3. The PD-L1 receptor-based cancer detection device according to claim 1, wherein the detected physical quantity of the signal processing unit includes at least one of wavelength λ, brightness L, intensity C, and absorbance a of the optical signal.

4. The PD-L1 receptor-based cancer detection device according to claim 2, wherein the heating unit is a laser heater provided with focusing means to adjust specific heating positions by changing focus.

5. The PD-L1 receptor-based cancer detection device according to claim 2, wherein the sample compartment unit is disposed on a side of the heating unit and the signal processing unit is disposed on a side of the sample compartment unit remote from the heating unit, including:

6. The PD-L1 receptor-based cancer detection device according to claim 5, wherein the first and second heat conduction surfaces are transparent, and the second heat conduction surface has a higher thermal conductivity than the first heat conduction surface.

7. The PD-L1 receptor-based cancer detection device according to claim 5, wherein the gasket has a through hole formed therein for cooperating with the first and second heat conduction surfaces to form an extracellular vesicle-containing space.

8. The PD-L1 receptor-based cancer detection device according to claim 5, wherein the signal amplification unit is disposed at a side of the sample chamber unit away from the heating unit for amplifying and reflecting the light signal from the surface of the extracellular vesicle, and comprises:

9. The PD-L1 receptor-based cancer detection device according to claim 8, wherein the collection mirror and the magnifying mirror are both angled with respect to the objective lens to reflect the optical signal and the magnifying light source, respectively, to a designated location.

10. The PD-L1 receptor-based cancer detection device according to claim 2, wherein the signal collection unit is one or more of a CCD camera, illuminometer, spectrometer, monochromator, sCMOS, EMCCD, PMT, depending on the optical parameters to be detected by the detection device.