WO2019010787A1

WO2019010787A1 - Blood cell capture chip and method

Info

Publication number: WO2019010787A1
Application number: PCT/CN2017/100740
Authority: WO
Inventors: 韩琳; 丁庆; 刘荣跃; 李辰
Original assignee: 华讯方舟科技有限公司; 深圳市太赫兹科技创新研究院有限公司
Priority date: 2017-07-12
Filing date: 2017-09-06
Publication date: 2019-01-17
Also published as: CN107290534A

Abstract

Provided are a blood cell capture chip and method. The method comprises steps of: arranging specific antibody-fluorescent molecule-magnetic nanoparticles in a micro-channel, and fixing a magnetic structure at the bottom of a glass substrate, so that the specific antibody-fluorescent molecule-magnetic nanoparticles are attracted and fixed by the magnetic structure; capturing target cell antigen in blood by the specific antibody-fluorescent molecule-magnetic nanoparticles, so that fluorescent molecules in the specific antibody-fluorescent molecule-magnetic nanoparticles emit fluorescence due to the fact that the fluorescent molecules gather to be in a stimulated state, realizing effective enrichment and distinguish of the target cell antigen, and the blood cell capture chip and method can be widely applied to detection operation of pathological cells such as circulating tumor cells, cancer cells and the like.

Description

Title: A blood cell capture chip and method

Technical field

[0001] Embodiments of the present invention belong to the field of biomedical technology, and in particular, to a blood cell capturing chip and method.

[0002] In the field of biomedical technology, it is generally involved in performing detection operations on some diseased cells in the blood.

Taking circulating tumor cells (CTC) as an example, it is usually necessary to separate tumor cells before detecting and diagnosing tumor cells. Commonly used tumor cell separation techniques include Isolation by size of epithelial tumor cells (ISET), density gradient centrifugation (density gradient centrifuge), and immunomagnetic bead (1MB).

[0003] However, membrane filtration separation of tumor cells does not effectively collect small-sized tumor cells. Density gradient centrifugation leads to the mixing of leukocytes and monocytes in tumor cells and blood samples after separation, reducing detection. Efficiency, immunomagnetic beads technology results in a small number of non-specifically bound cells that cannot be distinguished in the isolated tumor cells.

technical problem

Embodiments of the present invention provide a blood cell capture chip and method, which can achieve effective enrichment of target cell antigens in blood, and effectively distinguish between specific binding cells and non-specific binding cells by fluorescent markers.

Problem solution

Technical solution

[0005] An embodiment of the present invention provides a blood cell capture chip including a micro flow channel chip and a glass substrate bonded to each other, wherein the micro flow channel chip is provided with a micro flow channel of a preset length, A specific antibody-fluorescent molecule-nanomagnetic particle is disposed in the microchannel, and the specific antibody-fluorescent molecule-nanomagnetic particle is a specific antibody coated on the nano magnetic particle and labeled with a fluorescent molecule, the glass substrate The bottom portion is fixedly fixed with a plurality of magnetic structures, and the specific antibody-fluorescent molecule-nanomagnetic particles are subjected to the magnetic junction a structure is fixed in the micro flow channel;

Blood flows from the inlet of the microchannel, and the target cell antigen in the blood is captured by the specific antibody-fluorescent molecule-nanomagnetic particle, the specific antibody-fluorescent molecule-nanomagnetic particle Fluorescent molecules in the emission emit fluorescence due to aggregation in an excited state.

[0007] The target cell antigen is an antigen of circulating tumor cells, and the specific antibody is a tumor cell-specific antibody.

In one embodiment, the micro flow channel includes at least one micro flow channel unit connected end to end.

[0009] In one embodiment, the micro flow channel unit is a planar spiral structure, and the adjacent micro flow channel units are connected by micro pipes, and the micro pipes are not in the same plane as the micro flow channel unit. Inside.

[0010] In one embodiment, the planar spiral structure is a circular spiral structure or a polygonal spiral structure.

[0011] In one embodiment, the microchannel unit is an S-shaped structure.

[0012] In one embodiment, the magnetic structure is detachably fixed to the bottom of the glass substrate.

[0013] In one embodiment, the magnetic structure is a ferromagnetic structure or an electromagnet structure.

[0014] Another aspect of the embodiments of the present invention further provides a blood cell capturing method, which is implemented based on the blood cell capturing chip described above, and the method includes:

[0015] arranging a specific antibody-fluorescent molecule-nanomagnetic particle in the microchannel;

[0016] fixing a magnetic structure at a predetermined position at the bottom of the glass substrate, wherein the specific antibody-fluorescent molecule-nanomagnetic particles are attracted and fixed by the magnetic structure;

[0017] injecting blood from the inlet of the microchannel, and capturing the target cell antigen in the blood by the specific antibody-fluorescent molecule-nanomagnetic particle, so that the specific antibody-fluorescent molecule-nanomagnetic particle Fluorescent molecules emit fluorescence due to aggregation in an excited state.

[0018] In an embodiment, the method further includes:

[0019] repeatedly flushing the microchannels a predetermined number of times to reduce non-specifically bound cells within the microchannels.

Advantageous effects of the invention

Beneficial effect

[0020] In the embodiment of the present invention, a specific antibody-fluorescent molecule-nanomagnetic particle is disposed in a microchannel, and a magnetic structure is fixed at a bottom of the glass substrate, so that the specific antibody-fluorescent molecule-nanomagnetic particle is attracted and fixed by the magnetic structure. Capturing target cell antigens in blood by specific antibody-fluorescent molecule-nanomagnetic particles The specific antibody-fluorescent molecule-fluorescent molecule in the nano-magnetic particle emits fluorescence due to aggregation in an excited state, thereby realizing efficient enrichment and differentiation of the target cell antigen, and can be widely applied to diseases such as circulating tumor cells and cancer cells. Cell detection operation.

Brief description of the drawing

DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly described below. Obviously, the drawings in the following description are some implementations of the present invention. For example, other drawings may be obtained from those of ordinary skill in the art in light of the inventive work.

1 is a schematic diagram showing the basic structure of a blood cell capture chip according to an embodiment of the present invention;

2 is a schematic diagram of a basic structure of a blood cell capture chip according to another embodiment of the present invention; [0024] FIG. 3 is a schematic diagram of a basic structure of a blood cell capture chip according to still another embodiment of the present invention; [0025] 4 is a basic flow chart of a blood cell capturing method provided by an embodiment of the present invention.

Embodiments of the invention

The technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings in the embodiments of the present invention. It is an embodiment of the invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without departing from the inventive scope are intended to fall within the scope of the invention.

[0027] The term "comprising" and variations of the invention in the specification and claims of the invention and the above description are intended to cover a non-exclusive inclusion. For example, a process, method or system, product or device comprising a series of steps or units is not limited to the listed steps or units, but optionally also includes steps or units not listed, or alternatively also includes Other steps or units inherent to these processes, methods, products or equipment. Further, the terms "first", "second", "third", etc. are used to distinguish different objects, and are not intended to describe a particular order.

[0028] As shown in FIG. 1, an embodiment of the present invention provides a blood cell capture chip 100 including a micro flow channel chip 10 and a glass substrate 20 bonded to each other, and a preset is provided on the micro flow channel chip 10. Length of micro flow channel 1 1. The micro-channel 11 is provided with a specific antibody-fluorescent molecule-nanomagnetic particle (not shown), and the specific antibody-fluorescent molecule-nanomagnetic particle is coated on the nano-magnetic particle and labeled by the fluorescent molecule. The specific antibody, a plurality of magnetic structures (not shown) are distributed and fixed at the bottom of the glass substrate 20, and the specific antibody-fluorescent molecule-nanomagnetic particles are fixed in the microchannel 11 by the magnetic structure.

[0029] In a specific application, bonding refers to the surface cleaning and activation treatment of two clean or atomic level homogenous or heterogeneous semiconductor materials, which are directly combined under certain conditions, through van der Waals forces, molecular forces and even The technique of atomic force synthesizing wafer bonds into one body, that is, in the present embodiment, the micro flow channel chip and the glass substrate are bonded together by this bonding technique.

[0030] FIG. 1 exemplarily shows that the micro flow channel chip 10 and the glass substrate 20 are two-layer structure stacked on top of each other. In practical applications, the micro flow channel chip and the glass substrate may also be an integrated structure. China does not specifically limit the relationship between the two.

[0031] In a specific application, the shape and length of the microchannel can be selected according to actual needs, as long as the length of the microchannel is long enough, the target cell antigen can be fully combined with the specific antibody, that is, Set the length to be set according to actual needs. The micro flow path 11 is exemplarily shown in Fig. 1 as a wave-shaped curved structure.

[0032] In a specific application, the magnetic structure may be fixed to the bottom of the glass substrate by any means.

[0033] In one embodiment, the magnetic structure is detachably fixed to the bottom of the glass substrate, and the magnetic structure may be removed or mounted according to actual needs to increase or decrease the number of magnetic structures or change the fixed position of the magnetic structure.

[0034] In a particular application, the magnetic structure can be any magnet having an electromagnetic attraction function.

[0035] In one embodiment, the magnetic structure is a ferromagnetic structure or an electromagnet structure.

[0036] In one embodiment, when the magnetic structure is an electromagnet structure, the blood cell capture chip further includes an electromagnetic control circuit or device connected to the electromagnet structure, and the power is turned on even if the electromagnet structure generates an electromagnetic field due to energization.幵The power supply loses its magnetic properties, and the magnetic size of the electromagnet structure can be adjusted by adjusting the magnitude of the power supply current. Correspondingly, in one embodiment, the blood cell chip further comprises a control device connected to the electromagnetic control circuit or the device, wherein the control device is used for controlling the on and off of the power source, and the control device can be through a general-purpose integrated circuit, such as a single chip microcomputer. The CPU (Central Processing Unit) is implemented by an ASIC (Application Specific Integrated Circuit). [0037] The fluorescent molecules in the present embodiment have a property of being excited to emit fluorescence in an aggregated state and freely rotating in a free state without fluorescence.

[0038] The working principle of the blood cell capture chip provided by this embodiment is:

[0039] Blood flows in from the inlet of the microchannel, and the target cell antigen in the blood is captured by the specific antibody-fluorescent molecule-nanomagnetic particle, and the fluorescent molecule in the specific antibody-fluorescent molecule-nanomagnetic particle is subjected to aggregation The state is excited to emit fluorescence.

[0040] In a specific application, the target cell antigen may be an antigen of a circulating tumor cell, an antigen of various types of cancer cells, a lymphocyte antigen, or the like, or a normal cell antigen that can enter the peripheral blood, and the corresponding specific antibody may be Is a tumor cell-specific antibody, various cancer cell antibodies, lymphocyte antibodies, etc.

[0041] In one embodiment, the target cell antigen specifically refers to an antigen of a circulating tumor cell, and the specific antibody specifically refers to a tumor cell-specific antibody.

[0042] In this embodiment, a specific antibody-fluorescent molecule-nanomagnetic particle is disposed in a microchannel, and a magnetic structure is fixed at a bottom of the glass substrate, so that the specific antibody-fluorescent molecule-nanomagnetic particle is attracted and fixed by the magnetic structure. Capturing the target cell antigen in the blood by the specific antibody-fluorescent molecule-nanomagnetic particle, and binding the specific antibody to the specific antibody in the specific antibody-fluorescent molecule-nanomagnetic particle, the specific antibody-fluorescent molecule-nano Fluorescent molecules in magnetic particles emit fluorescence due to aggregation in an excited state, thereby achieving efficient enrichment and differentiation of target cell antigens, and can be widely applied to diseased or non-lesional cells such as circulating tumor cells, cancer cells, and lymphocytes. Detection operation.

[0043] In one embodiment of the invention, the microchannel comprises at least one microchannel unit connected end to end.

[0044] In a particular application, the microchannel unit can be a planar spiral structure, an S-structure, or any other structure capable of increasing the microchannel length.

As shown in FIG. 2, the micro flow channel 11 is exemplarily shown to include three micro flow channel units 111, the micro flow channel unit 111 is a square planar spiral structure, and the adjacent micro flow channel units 111 pass through micro Pipe 112 connection, micro pipe 1

12 is not in the same plane as the micro runner unit 111.

[0046] In a specific application, the planar spiral structure may be any one of a circular spiral structure, an elliptical spiral structure, a heterogeneous spiral structure, or a polygonal spiral structure, for example, a square spiral structure, a regular hexagonal spiral structure, or the like. As shown in FIG. 3, the micro flow path 11 is exemplarily shown to include a plurality of micro flow path units 111, and the micro flow path unit 111 has an S-shaped structure. In a specific application, the turning point of the S-shaped structure may be a smooth curved micro-pipe or a right-angled bent micro-pipe. Illustratively shown in FIG. 3, the turning point of the S-shaped structure is a right-angled bent micro-pipe

[0048] As shown in FIG. 4, an embodiment of the present invention provides a blood cell capturing method, which is implemented based on the blood cell capturing chip 100 described above, and the method includes:

[0049] Step S101: Deploying a specific antibody-fluorescent molecule-nanomagnetic particle in the microchannel.

[0050] In a specific application, step S101 includes pre-treating the nano magnetic particles in advance, coating the specific antibody on the nano magnetic particles, and labeling the specific antibody with the fluorescent molecule to obtain a specific antibody-fluorescent molecule- Nano magnetic particles.

[0051] Step S102: fixing a magnetic structure at a predetermined position at the bottom of the glass substrate, wherein the specific antibody-fluorescent molecule-nanomagnetic particles are attracted and fixed by the magnetic structure.

[0052] In a specific application, if the magnetic structure is an electromagnet structure, step S102 further includes performing the magnetic structure before the specific antibody-fluorescent molecule-nanomagnetic particle is attracted and fixed by the magnetic structure. Steps to power up.

[0053] Step S103: injecting blood from the inlet of the microchannel, and capturing the target cell antigen in the blood by the specific antibody-fluorescent molecule-nanomagnetic particle, so that the specific antibody-fluorescent molecule-nanomagnetic Fluorescent molecules in the particles emit fluorescence due to aggregation in an excited state.

In a specific application, blood specifically refers to peripheral blood including a target cell antigen, and the target cell antigen may be any antigen capable of entering a diseased or non-lesional cell of peripheral blood.

[0055] In an embodiment, the foregoing method further includes:

[0056] repeatedly flushing the microchannels a predetermined number of times to reduce non-specific binding cells in the microchannels; [0057] scanning the microchannels by a fluorescence microscope to identify target cells.

[0058] In a specific application, the preset number of times may be set according to actual needs, so that the non-specific binding cells in the microchannel can be washed out. The preset number can be counted based on historical experience. The purpose of rinsing the microchannel is to wash away non-specifically bound cells that do not bind to specific antibody-fluorescent molecule-nanomagnetic particles, reducing the false positive probability of detection. If not washed, it may cause the proportion of non-specifically bound cells in the microchannel to be significantly higher than that of specific binding cells, resulting in specific binding to cells. There is a serious error in the concentration detection.

[0059] The steps in the method of the embodiment of the present invention may be sequentially adjusted, merged, and deleted according to actual needs.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the present invention. Within the scope of protection of the invention.

Claims

Claim

[Claim 1] A blood cell trapping chip, comprising: a micro flow channel chip and a glass substrate bonded to each other, wherein the micro flow channel chip is provided with a micro flow channel of a predetermined length, the micro flow The inner membrane is provided with a specific antibody-fluorescent molecule-nanomagnetic particle, and the specific antibody-fluorescent molecule-nanomagnetic particle is a specific antibody coated on the nano magnetic particle and labeled with a fluorescent molecule, and the bottom of the glass substrate is distributed. Fixed with a plurality of magnetic structures, the specific antibody-fluorescent molecule-nanomagnetic particles being attracted by the magnetic structure in the microchannel; blood flowing from the inlet of the microchannel, the target in the blood The cell antigen is captured by the specific antibody-fluorescent molecule-nanomagnetic particle, and the fluorescent molecule in the specific antibody-fluorescent molecule-nanomagnetic particle emits fluorescence due to aggregation in an excited state.

[Claim 2] The blood cell-trapping chip according to claim 1, wherein the target cell antigen is an antigen of a circulating tumor cell, and the specific antibody is a tumor cell-specific antibody.

[Claim 3] The blood cell trapping chip according to claim 1, wherein the microchannel comprises at least one microchannel unit connected in series at the beginning and the end.

[Claim 4] The blood cell capture chip according to claim 3, wherein the micro flow channel unit is a planar spiral structure, and adjacent micro flow channel units are connected by micro pipes, The microchannels are not in the same plane as the microchannel unit.

[Claim 5] The blood cell-trapping chip according to claim 4, wherein the planar spiral structure is a circular spiral structure or a polygonal spiral structure.

[Claim 6] The blood cell trapping chip according to claim 3, wherein the microchannel unit has an S-shaped structure.

[Claim 7] The blood cell-trapping chip according to claim 1, wherein the magnetic structure is detachably fixed to a bottom of the glass substrate.

[Claim 8] The blood cell-trapping chip according to claim 1 or 7, wherein the magnetic structure is a ferromagnetic structure or an electromagnet structure.

[Claim 9] A blood cell capturing method, wherein the method is based on any one of claims 1 to 8.

Implemented by the blood cell capture chip, the method comprising: Deploying specific antibody-fluorescent molecule-nanomagnetic particles in the microchannel;

Fixing a magnetic structure at a predetermined position at the bottom of the glass substrate, wherein the specific antibody-fluorescent-nanomagnetic particles are attracted and fixed by the magnetic structure;

Injecting blood from the inlet of the microchannel, and capturing the target cell antigen in the blood by the specific antibody-fluorescent molecule-nanomagnetic particle, so that the fluorescent molecule in the specific antibody-fluorescent molecule-nanomagnetic particle Aggregation is in an excited state and emits fluorescence.

[Claim 10] The blood cell capturing method according to claim 9, wherein the method further comprises:

The microchannels are repeatedly rinsed a predetermined number of times to reduce non-specific binding cells within the microchannels.