CN111804351A - Integrated exosome separation and detection microfluidic chip and preparation method thereof - Google Patents
Integrated exosome separation and detection microfluidic chip and preparation method thereof Download PDFInfo
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- CN111804351A CN111804351A CN201910291437.8A CN201910291437A CN111804351A CN 111804351 A CN111804351 A CN 111804351A CN 201910291437 A CN201910291437 A CN 201910291437A CN 111804351 A CN111804351 A CN 111804351A
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L2300/12—Specific details about materials
Abstract
An integrated exosome separation and detection microfluidic chip and a preparation method thereof are disclosed, the microfluidic chip comprises the following components: the upper layer is a reagent pool layer, the middle layer is a liquid path control layer, and the bottom layer is a reaction pool layer; the following structure is specifically designed: the device comprises an exosome sample pool, an exosome sample introduction channel, an exosome dye primary antibody pool, an exosome dye primary antibody sample introduction channel, an exosome dye secondary antibody pool, an exosome dye secondary antibody sample introduction channel, a chromogenic liquid pool, a chromogenic liquid sample introduction channel, a cleaning buffer liquid pool, a buffer liquid sample introduction channel, a waste liquid pool, a waste liquid outflow channel and a reaction pool. Preparing a photoresist template with a raised channel part; developing and hardening; treating the template with a silylating agent; obtaining a polydimethylsiloxane chip with a structure; irreversible sealing, and the like. The invention has the advantages of simple structure, convenient preparation and operation, high speed, high efficiency and wide application range.
Description
Technical Field
The invention relates to the technical field of design, processing, manufacturing and application of a microfluidic technology and a polymer chip, and particularly provides an integrated exosome separation and detection microfluidic chip and a preparation method thereof.
Background
In the prior art, a microfluidic chip is a system for controlling the flow of a micro-volume fluid in a micro-channelSystem in which the channels have dimensions of tens to hundreds of microns and the quantity of carrier fluid is 10-9-10-18And L. The operation units of the microfluidic chip are mutually communicated through the flow of fluid in the microchannel network. The microfluid control is the core of the operation of the microfluidic chip laboratory, and the related processes of sample introduction, mixing, reaction, separation and the like are all completed in the motion of the controllable fluid. Valves are the core component of fluid control, whether macroscopic or microscopic. Due to its importance, the development of micro valves has attracted much attention as early as the birth of microfluidic chips. In theory, all parts capable of controlling the closed and opened states of the micro-channel can be used as micro-valves in the micro-fluidic chip. An ideal microvalve should have the following characteristics: low leakage, low power consumption, fast response speed, linear operation capability and wide application range. The microfluid and the micro valve form a complete set of microfluidic chip system.
The core of the microfluidic analysis is that a microfluidic chip is utilized to integrate basic operation units such as sample pretreatment, biological and chemical reactions, separation and detection and the like on a chip with a micro-channel network or a nano-channel network, and a complex analysis process is completed by controlling a fluid, so that the microfluidic analysis chip has the advantages of less consumption of samples and reagents, short analysis time, high flux, easiness in realization of large-scale parallel determination and the like. The micro-fluidic analysis technology can be used for conveniently realizing the miniaturization, integration and portability of the analysis system. At present, the system is widely applied to the fields of life science, disease diagnosis and treatment, drug synthesis and screening and the like.
Exosome is a nano-scale vesicle which is secreted by cells and contains a plurality of substances such as lipid, protein, mRNAs and the like, the exosome is widely distributed in a human body, and most of body fluids of the human body such as urine, blood, milk, saliva and the like contain exosome. Exosomes mainly have two major functions of substance transmission and information transmission in human bodies. Research shows that exosome plays an important role in the inflammation process, adaptive immunity, embryogenesis, and the generation and development process of tumor. However, how to effectively separate exosomes from body fluid and detect exosomes still remains a great challenge, and the separation methods such as ultracentrifugation and ultrafiltration which are commonly used at present are time-consuming and labor-consuming, and the purity of products is difficult to guarantee.
The microfluidic chip technology has the advantages of greatly reducing sample consumption, saving labor and time cost, realizing automation and high-flux experiments in a centimeter square space and the like, is widely concerned, and also occupies a place in the field of exosome research.
People hope to obtain an integrated exosome separation and detection microfluidic chip with excellent technical effect urgently.
Disclosure of Invention
The invention aims to provide an integrated exosome separation and detection microfluidic chip with excellent technical effect. The method solves the problems of complicated operation steps, large reagent consumption and the like in the separation and detection process of the exosome.
The invention provides an integrated exosome separation and detection microfluidic chip, which is formed by sequentially connecting an upper layer, a middle layer and a lower layer in series and laminating, wherein: the upper layer is a reagent pool layer, the middle layer is a liquid path control layer, and the bottom layer is a reaction pool layer;
the liquid path control layer is specifically provided with the following structures:
-exosome injection channel a: the front end of the sample injection channel A is connected with the exosome sample pool 1, the rear end of the sample injection channel A vertically converges into the main channel D and is used for communicating the two channels, and a pneumatic micro-valve structure is designed on the exosome sample injection channel A and is used for controlling the on-off of the channel;
-exosome staining of an anti-injection channel B: the front end of the first-antibody exosome-dyeing pool is connected with the first-antibody exosome-dyeing pool 2, the rear end of the first-antibody exosome-dyeing pool is vertically converged into the main channel D and used for communicating the two pools, and a pneumatic micro-valve structure is designed on the first-antibody exosome-dyeing sample feeding channel B and used for controlling the on-off of the channel;
-exosome-stained secondary antibody sample injection channel C: the front end of the secondary exosome-dyed antibody sample injection channel C is connected with the secondary exosome-dyed antibody pool 3, the rear end of the secondary exosome-dyed antibody sample injection channel C is vertically converged into the main channel D and is used for communicating the two, and a pneumatic micro-valve structure is designed on the secondary exosome-dyed antibody sample injection channel C and is used for controlling the on-off of the channel;
-a main channel D: the device is arranged between the reagent pool and the reaction pool 5 and is used for communicating the reagent pool and the reaction pool;
-a color development liquid sample injection channel E: the color developing liquid sampling channel E is provided with a pneumatic micro valve structure for controlling the on-off of the channel;
-a waste outflow channel F: the front end of the waste liquid outflow channel F is connected with the reaction tank 5, the rear end of the waste liquid outflow channel F is connected with the waste liquid tank 6 and used for communicating the two, and a pneumatic micro valve structure is designed on the waste liquid outflow channel F and used for controlling the on-off of the channel;
-buffer sample injection channel G: the buffer solution sampling channel G is provided with a pneumatic micro valve structure for controlling the on-off of the channel.
The reagent pool layer is provided with six reagent pools which are an exosome sample pool 1, an exosome staining primary antibody pool 2, an exosome staining secondary antibody pool 3, a chromogenic liquid pool 4, a waste liquid pool 6 and a cleaning buffer liquid pool 7 respectively; the reaction tank layer is designed with a reaction tank which is a reaction tank 5.
The exosome sample cell 1 is connected with an exosome sample introduction channel A through an exosome sample introduction hole h1, the exosome dye primary antibody cell 2 is connected with an exosome dye primary antibody sample introduction channel B through a primary antibody sample introduction hole h2, the exosome dye secondary antibody cell 3 is connected with an exosome dye secondary antibody sample introduction channel C through a secondary antibody sample introduction hole h3, the chromogenic solution cell 4 is connected with a chromogenic solution sample introduction channel E through a chromogenic solution sample introduction hole h4, the waste solution cell 6 is connected with a waste solution outflow channel F through a waste solution outlet hole h5, the cleaning buffer solution cell 7 is connected with a buffer solution sample introduction channel G through a cleaning buffer solution sample introduction hole h6, the reaction cell is connected with a main channel D through a reaction cell sample introduction hole h7, and is connected with the waste solution outflow channel F through a reaction cell outlet hole h 8; each channel is controlled by a separate micro valve, and all valves for controlling the micro control chip are normally closed valves.
The integrated exosome separation and detection microfluidic chip further meets one or a combination of the following requirements:
firstly, the upper chip is made of polycarbonate plastic and has the thickness of 0.5-2.5 cm; the upper reagent pool layer is a customized module; the upper reagent well layer is a polycarbonate plastic module customized by the company.
Secondly, the materials of the middle layer chip and the lower layer chip are polydimethylsiloxane polymers, the thickness of the middle layer chip is 100-500 mu m, and the thickness of the lower layer chip is 1-2 mm;
thirdly, the reagent pool is of a cylindrical structure with the bottom surface diameter of 0.2-1cm and the height of 0.4-2.4 cm;
fourthly, a round hole with the diameter of 0.2-0.5mm is formed at the bottom of the reagent pool, and the upper chip is punched through to be communicated with the middle chip;
fifthly, the height and the width of a liquid path of a middle layer liquid path layer of the chip are the same and are both 80-200 mu m;
sixthly, the height of the channel of the lower reaction pool layer of the chip is 0.8-1.6mm, the diameter of the reaction pool is 2-8mm, and the width of the connecting channel is 80-200 mu m.
The middle liquid path control layer is formed by throwing a polydimethylsiloxane membrane with the thickness of 500 mu m higher than that of the template 100-fold on the successfully manufactured liquid path template;
the reaction tank layer is formed by throwing a polydimethylsiloxane membrane 1-2mm higher than a template on a successfully manufactured reaction tank template;
the unstructured side of the polydimethylsiloxane film of the middle liquid path control layer is bonded to the bottom of the reaction tank layer through plasma;
and one side of the polydimethylsiloxane structure of the middle layer liquid path control layer is bonded with one side of the reaction pool layer with the structure through plasma.
A preparation method of an integrated exosome separation and detection microfluidic chip sequentially comprises the following steps:
(1) preparing a middle-layer liquid path control layer and a lower-layer reaction tank layer photoresist SU-8 template with raised channel parts by adopting a photoetching and corrosion method;
(2) developing the photoresist SU-8 template by ethyl lactate, and hardening the photoresist for 1-3 h at 165-180 ℃;
(3) treating the chip photoresist SU-8 template with a silanization reagent for 5-10 min to enable PDMS to be easily stripped from the bottom surface of the template;
(4) mixing polydimethylsiloxane PDMS and an initiator according to a volume ratio of (5-20): 1, uniformly mixing, respectively pouring the mixture into SU-8 templates of the photoresist of the middle and lower layers of the chip, curing the mixture in an oven at 80 ℃ for 20-40 min, and stripping the SU-8 templates of the polydimethylsiloxane PDMS photoresist to obtain a polydimethylsiloxane PDMS chip with a structure;
(5) punching holes one by one at corresponding positions of the middle-layer liquid path control layer and the upper-layer reagent pool layer at the bottoms of the reagent pools by using a puncher;
(6) carrying out oxygen plasma treatment on one side with the structure on the middle layer of the chip and one side with the structure on the lower layer of the chip for 1-3 min, and carrying out irreversible sealing at 70-90 ℃ for 30-60 min;
(7) and (3) carrying out oxygen plasma treatment on the sealed polydimethylsiloxane PDMS chip and the upper polycarbonate plastic for 1-3 min, and carrying out irreversible sealing at 70-90 ℃ for 30-60 min to obtain the integrated exosome separation and detection microfluidic chip.
The preparation method of the integrated exosome separation and detection microfluidic chip further meets the following requirements: after the exosome sample is introduced into the microfluidic chip, buffer solution cleaning, primary antibody staining, cleaning, secondary antibody staining and color development liquid developing are sequentially carried out, and finally, an enzyme-linked immunosorbent assay (ELISA) instrument is used for detection.
The invention utilizes microfluid and microvalve technology to prepare a microfluidic chip which integrates exosome enrichment, primary antibody incubation, horseradish peroxidase labeled secondary antibody incubation, color development and cleaning, and all the steps are carried out in sequence. The whole chip platform is simple in structure, convenient to operate, high in integration level, high in analysis speed and high in efficiency, and does not need any complex and expensive equipment and a large number of samples and reagents. In summary, the invention of the integrated exosome separation and detection microfluidic chip is convenient and rapid, high in integration level and wide in application range, and has very important significance.
The invention solves the technical limitations of complex operation steps, large reagent consumption and the like in the previous exosome separation and detection process. The preparation method has the advantages of stable preparation process, simple operation and high integration level.
The integrated exosome separation and detection microfluidic chip provided by the invention has the application specification that: the chip can be used for capturing and enriching exosomes expressing specific proteins, and identifying the captured exosomes by enzyme-linked immunosorbent assay. The integrated microfluidic chip can be used for enriching exosomes of different samples, capturing exosomes expressed by different specific proteins and dyeing different types of antibodies.
The invention has the advantages that: 1. the operation is simple, convenient and quick; 2. the dosage of the sample and the reagent is small, and the experiment cost is low; 3. does not contact toxic and harmful reagents, and is environment-friendly; 4. high integration and wide application range.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of a structure of an integrated exosome separation and detection microfluidic chip;
FIG. 2 is a schematic diagram of the structure of an upper reagent pool layer of the integrated exosome separation and detection microfluidic chip;
FIG. 3 is a schematic diagram of a layer liquid control layer in an integrated exosome separation and detection microfluidic chip;
fig. 4 is a schematic diagram of a lower reaction cell layer structure of the integrated exosome separation and detection microfluidic chip.
The device comprises an exosome sample pool 1, an exosome staining primary antibody pool 2, an exosome staining secondary antibody pool 3, a chromogenic solution pool 4, a reaction pool 5, a waste solution pool 6, a cleaning buffer solution pool 7, an exosome sample introduction channel A, an exosome staining primary antibody sample introduction channel B, an exosome staining secondary antibody sample introduction channel C, a main channel D, a chromogenic solution sample introduction channel E, a waste solution outflow channel F and a cleaning buffer solution sample introduction channel G, wherein the exosome staining primary antibody pool 2 is connected with the cleaning buffer solution pool 3; except the main channel D, the other channels are provided with a pneumatic micro valve for controlling the on-off of the liquid path;
h1-h6 are through holes at the bottom of the reagent pool for communicating the reagent pool with a middle layer liquid channel, an exosome sample inlet hole h1, a primary antibody inlet hole h2, a secondary antibody inlet hole h3, a chromogenic solution inlet hole h4, a waste liquid outlet hole h5 and a cleaning buffer solution inlet hole h 6; a reaction cell sample inlet h7 and a reaction cell outlet h 8.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The integrated exosome separation and detection microfluidic chip and the preparation method thereof according to the embodiment of the invention are described in detail below.
The invention provides an integrated exosome separation and detection microfluidic chip, as shown in figure 1, the microfluidic chip is formed by sequentially connecting an upper layer, a middle layer and a lower layer in series and laminating, wherein: the upper layer is a reagent pool layer (figure 2), the middle layer is a liquid path control layer (figure 3), and the bottom layer is a reaction pool layer (figure 4);
the liquid path control layer is specifically provided with the following structures:
-exosome injection channel a: the front end of the sample injection channel A is connected with the exosome sample pool 1, the rear end of the sample injection channel A vertically converges into the main channel D and is used for communicating the two channels, and a pneumatic micro-valve structure is designed on the exosome sample injection channel A and is used for controlling the on-off of the channel;
-exosome staining of an anti-injection channel B: the front end of the first-antibody exosome-dyeing pool is connected with the first-antibody exosome-dyeing pool 2, the rear end of the first-antibody exosome-dyeing pool is vertically converged into the main channel D and used for communicating the two pools, and a pneumatic micro-valve structure is designed on the first-antibody exosome-dyeing sample feeding channel B and used for controlling the on-off of the channel;
-exosome-stained secondary antibody sample injection channel C: the front end of the secondary exosome-dyed antibody sample injection channel C is connected with the secondary exosome-dyed antibody pool 3, the rear end of the secondary exosome-dyed antibody sample injection channel C is vertically converged into the main channel D and is used for communicating the two, and a pneumatic micro-valve structure is designed on the secondary exosome-dyed antibody sample injection channel C and is used for controlling the on-off of the channel;
-a main channel D: the device is arranged between the reagent pool and the reaction pool 5 and is used for communicating the reagent pool and the reaction pool;
-a color development liquid sample injection channel E: the color developing liquid sampling channel E is provided with a pneumatic micro valve structure for controlling the on-off of the channel;
-a waste outflow channel F: the front end of the waste liquid outflow channel F is connected with the reaction tank 5, the rear end of the waste liquid outflow channel F is connected with the waste liquid tank 6 and used for communicating the two, and a pneumatic micro valve structure is designed on the waste liquid outflow channel F and used for controlling the on-off of the channel;
-buffer sample injection channel G: the buffer solution sampling channel G is provided with a pneumatic micro valve structure for controlling the on-off of the channel.
The reagent pool layer is provided with six reagent pools which are an exosome sample pool 1, an exosome staining primary antibody pool 2, an exosome staining secondary antibody pool 3, a chromogenic liquid pool 4, a waste liquid pool 6 and a cleaning buffer liquid pool 7 respectively; the reaction tank layer is designed with a reaction tank which is a reaction tank 5.
The exosome sample cell 1 is connected with an exosome sample introduction channel A through an exosome sample introduction hole h1, the exosome dye primary antibody cell 2 is connected with an exosome dye primary antibody sample introduction channel B through a primary antibody sample introduction hole h2, the exosome dye secondary antibody cell 3 is connected with an exosome dye secondary antibody sample introduction channel C through a secondary antibody sample introduction hole h3, the chromogenic solution cell 4 is connected with a chromogenic solution sample introduction channel E through a chromogenic solution sample introduction hole h4, the waste solution cell 6 is connected with a waste solution outflow channel F through a waste solution outlet hole h5, the cleaning buffer solution cell 7 is connected with a buffer solution sample introduction channel G through a cleaning buffer solution sample introduction hole h6, the reaction cell is connected with a main channel D through a reaction cell sample introduction hole h7, and is connected with the waste solution outflow channel F through a reaction cell outlet hole h 8; each channel is controlled by a separate micro valve, and all valves for controlling the micro control chip are normally closed valves.
The integrated exosome separation and detection microfluidic chip further meets one or a combination of the following requirements:
firstly, the upper chip is made of polycarbonate plastic and has the thickness of 0.5-2.5 cm; the upper reagent pool layer is a customized module; the upper reagent well layer is a polycarbonate plastic module customized by the company.
Secondly, the materials of the middle layer chip and the lower layer chip are polydimethylsiloxane polymers, the thickness of the middle layer chip is 100-500 mu m, and the thickness of the lower layer chip is 1-2 mm;
thirdly, the reagent pool is of a cylindrical structure with the bottom surface diameter of 0.2-1cm and the height of 0.4-2.4 cm;
fourthly, a round hole with the diameter of 0.2-0.5mm is formed at the bottom of the reagent pool, and the upper chip is punched through to be communicated with the middle chip;
fifthly, the height and the width of a liquid path of a middle layer liquid path layer of the chip are the same and are both 80-200 mu m;
sixthly, the height of the channel of the lower reaction pool layer of the chip is 0.8-1.6mm, the diameter of the reaction pool is 2-8mm, and the width of the connecting channel is 80-200 mu m.
The middle liquid path control layer is formed by throwing a polydimethylsiloxane membrane with the thickness of 500 mu m higher than that of the template 100-fold on the successfully manufactured liquid path template;
the reaction tank layer is formed by throwing a polydimethylsiloxane membrane 1-2mm higher than a template on a successfully manufactured reaction tank template;
the unstructured side of the polydimethylsiloxane film of the middle liquid path control layer is bonded to the bottom of the reaction tank layer through plasma;
and one side of the polydimethylsiloxane structure of the middle layer liquid path control layer is bonded with one side of the reaction pool layer with the structure through plasma.
A preparation method of an integrated exosome separation and detection microfluidic chip sequentially comprises the following steps:
(1) preparing a middle-layer liquid path control layer and a lower-layer reaction tank layer photoresist SU-8 template with raised channel parts by adopting a photoetching and corrosion method;
(2) developing the photoresist SU-8 template by ethyl lactate, and hardening the photoresist for 1-3 h at 165-180 ℃;
(3) treating the chip photoresist SU-8 template with a silanization reagent for 5-10 min to enable PDMS to be easily stripped from the bottom surface of the template;
(4) mixing polydimethylsiloxane PDMS and an initiator according to a volume ratio of (5-20): 1, uniformly mixing, respectively pouring the mixture into SU-8 templates of the photoresist of the middle and lower layers of the chip, curing the mixture in an oven at 80 ℃ for 20-40 min, and stripping the SU-8 templates of the polydimethylsiloxane PDMS photoresist to obtain a polydimethylsiloxane PDMS chip with a structure;
(5) punching holes one by one at corresponding positions of the middle-layer liquid path control layer and the upper-layer reagent pool layer at the bottoms of the reagent pools by using a puncher;
(6) carrying out oxygen plasma treatment on one side with the structure on the middle layer of the chip and one side with the structure on the lower layer of the chip for 1-3 min, and carrying out irreversible sealing at 70-90 ℃ for 30-60 min;
(7) and (3) carrying out oxygen plasma treatment on the sealed polydimethylsiloxane PDMS chip and the upper polycarbonate plastic for 1-3 min, and carrying out irreversible sealing at 70-90 ℃ for 30-60 min to obtain the integrated exosome separation and detection microfluidic chip.
The preparation method of the integrated exosome separation and detection microfluidic chip further meets the following requirements: after the exosome sample is introduced into the microfluidic chip, buffer solution cleaning, primary antibody staining, cleaning, secondary antibody staining and color development liquid developing are sequentially carried out, and finally, an enzyme-linked immunosorbent assay (ELISA) instrument is used for detection.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Preparing an SU-8 template with a raised channel part by adopting a photoetching and corrosion method for the integrated exosome separation and detection microfluidic chip, wherein the lower layer structure in the chip is respectively composed of two SU-8 template reverse-mode PDMS;
the template of the middle-layer liquid path control chip is manufactured as follows: taking a clean glass sheet, throwing SU-8 glue on a glue throwing machine to a thickness of 100 mu m, pre-baking at 95 ℃ for 20min, naturally cooling, placing a mask with a middle layer structure on a SU-8 glue flat plate, performing ultraviolet exposure for 30s, post-baking at 95 ℃ for 20min, and naturally cooling; finally, developing the SU-8 photoresist for 5min by using ethyl lactate, hardening the film for 2h at 180 ℃, and naturally cooling to obtain a chip template;
the lower reaction tank layer chip template is manufactured as follows: taking a clean glass sheet, pouring SU-8 glue with the thickness of 1mm on the glass sheet, baking for 12h at 95 ℃, naturally cooling, placing a mask of a chip lower layer structure on an SU-8 glue flat plate, performing ultraviolet exposure for 100s, baking for 20min at 95 ℃, and naturally cooling; and finally, developing the SU-8 photoresist for 30min by using ethyl lactate, hardening the film for 2h at 180 ℃, and naturally cooling to obtain a chip template.
Example 2
Treating SU-8 templates of the middle and lower layer structures of the chip with a silylation reagent for 10min to make PDMS easily peel off the bottom surface of the template; PDMS to initiator in a volume ratio of 10: 1, uniformly mixing, respectively pouring into SU-8 templates of the middle and lower structures of the chip, curing in an oven at 80 ℃ for 40min, and stripping PDMS from the SU-8 templates of the chip to obtain a PDMS chip with a structure; punching holes one by one at corresponding positions of the middle-layer liquid path control layer and the upper-layer reagent pool layer at the bottoms of the reagent pools by using a puncher; performing oxygen plasma treatment on the side with the structure on the middle layer and the side with the structure on the lower layer of the chip for 2min, baking for 45min at 80 ℃, and performing irreversible sealing; and (3) carrying out oxygen plasma treatment on the sealed polydimethylsiloxane PDMS chip and the upper polycarbonate plastic for 2min, and carrying out irreversible sealing by baking at 80 ℃ for 45min to obtain the integrated exosome separation and detection microfluidic chip.
All reagent pools are controlled by independent valves, different types of samples flow into a main channel D through an exosome sample injection channel A of an exosome sample pool 1 and enter a reaction pool with the reference numeral 5, after the exosomes are enriched in the reaction pool, primary antibodies, secondary antibodies, chromogenic solutions and PBS are respectively added to structures with the reference numerals of B, C, E, F, G through control, and then the enzyme-labeling instrument is used for detection.
Claims (7)
1. An integrated exosome separation and detection microfluidic chip is characterized in that:
the micro-fluidic chip is formed by sequentially connecting an upper layer, a middle layer and a lower layer in series and laminating, wherein: the upper layer is a reagent pool layer, the middle layer is a liquid path control layer, and the bottom layer is a reaction pool layer;
the liquid path control layer is specifically provided with the following structures:
-exosome injection channel (a): the front end of the sample injection channel is connected with the exosome sample pool (1), the rear end of the sample injection channel vertically converges into the main channel (D) and is used for communicating the two, and a pneumatic micro-valve structure is designed on the exosome sample injection channel (A) and is used for controlling the on-off of the channel;
-exosome staining-anti-injection channel (B): the front end of the first-antibody sample injection channel (B) is connected with the first-antibody exosome staining pool (2), the rear end of the first-antibody exosome staining pool is vertically converged into the main channel (D) and is used for communicating the first-antibody exosome staining pool and the main channel, and a pneumatic micro-valve structure is designed on the first-antibody exosome staining sample injection channel (B) and is used for controlling the on-off of the channel;
-exosome-stained secondary antibody sampling channel (C): the front end of the secondary exosome-dyed antibody sample injection channel is connected with the secondary exosome-dyed antibody pool (3), the rear end of the secondary exosome-dyed antibody sample injection channel vertically converges into the main channel (D) and is used for communicating the secondary exosome-dyed antibody pool and the main channel, and a pneumatic micro-valve structure is designed on the secondary exosome-dyed antibody sample injection channel (C) and is used for controlling the on-off of the channel;
-a main channel (D): the device is arranged between the reagent pool and the reaction pool (5) and is used for communicating the reagent pool and the reaction pool;
-a color development liquid sample injection channel (E): the color developing liquid sampling channel (E) is provided with a pneumatic micro valve structure for controlling the on-off of the channel;
-a waste outflow channel (F): the front end of the waste liquid outflow channel (F) is connected with the reaction tank (5), the rear end of the waste liquid outflow channel is connected with the waste liquid tank (6) and used for communicating the two, and a pneumatic micro-valve structure is designed on the waste liquid outflow channel (F) and used for controlling the on-off of the channel;
-buffer sample injection channel (G): the buffer solution sampling channel (G) is provided with a pneumatic micro valve structure for controlling the on-off of the channel.
2. The integrated exosome separation and detection microfluidic chip according to claim 1, characterized in that: the reagent pool layer is provided with six reagent pools which are an exosome sample pool (1), an exosome staining primary antibody pool (2), an exosome staining secondary antibody pool (3), a chromogenic solution pool (4), a waste solution pool (6) and a cleaning buffer solution pool (7) respectively; the reaction tank layer is designed with a reaction tank which is a reaction tank (5).
3. The integrated exosome separation and detection microfluidic chip according to claim 2, characterized in that: the exosome sample cell (1) is connected with an exosome sample introduction channel (A) through an exosome sample introduction hole (h1), the exosome-staining primary antibody cell (2) is connected with an exosome-staining primary antibody sample feeding channel (B) through a primary antibody sample feeding hole (h2), the second exosome-dyed antibody pool (3) is connected with a second exosome-dyed antibody sample feeding channel (C) through a second exosome-dyed antibody sample feeding hole (h3), the color developing liquid pool (4) is connected with a color developing liquid sample feeding channel (E) through a color developing liquid sample feeding hole (h4), the waste liquid tank (6) is connected with a waste liquid outflow channel (F) through a waste liquid outlet hole (h5), the washing buffer solution pool (7) is connected with a buffer solution sample feeding channel (G) through a washing buffer solution sample feeding hole (h6), the reaction tank is connected with the main channel (D) through a reaction tank sample inlet (h7), connected with the waste liquid outflow channel (F) through the reaction cell outlet hole (h 8).
Each channel is controlled by a separate micro valve, and all valves for controlling the micro control chip are normally closed valves.
4. The integrated exosome separation and detection microfluidic chip according to claims 1 and 2, characterized in that: the integrated exosome separation and detection microfluidic chip further meets one or a combination of the following requirements:
firstly, the upper chip is made of polycarbonate plastic and has the thickness of 0.5-2.5 cm;
secondly, the materials of the middle layer chip and the lower layer chip are polydimethylsiloxane polymers, the thickness of the middle layer chip is 100-500 mu m, and the thickness of the lower layer chip is 1-2 mm;
thirdly, the reagent pool is of a cylindrical structure with the bottom surface diameter of 0.2-1cm and the height of 0.4-2.4 cm;
fourthly, a round hole with the diameter of 0.2-0.5mm is formed at the bottom of the reagent pool, and the upper chip is punched through to be communicated with the middle chip;
fifthly, the height and the width of a liquid path of a middle layer liquid path layer of the chip are the same and are both 80-200 mu m;
sixthly, the height of the channel of the lower reaction pool layer of the chip is 0.8-1.6mm, the diameter of the reaction pool is 2-8mm, and the width of the connecting channel is 80-200 mu m.
5. The integrated exosome separation and detection microfluidic chip according to claim 4, characterized in that: the upper reagent pool layer is a customized module;
the middle liquid path control layer is formed by throwing a polydimethylsiloxane membrane with the thickness of 500 mu m higher than that of the template 100-fold on the successfully manufactured liquid path template;
the reaction tank layer is formed by throwing a polydimethylsiloxane membrane 1-2mm higher than a template on a successfully manufactured reaction tank template;
the unstructured side of the polydimethylsiloxane film of the middle liquid path control layer is bonded to the bottom of the reaction tank layer through plasma;
and one side of the polydimethylsiloxane structure of the middle layer liquid path control layer is bonded with one side of the reaction pool layer with the structure through plasma.
6. The method for preparing the integrated exosome separation and detection microfluidic chip according to claim 1, which is characterized in that: the preparation method of the integrated exosome separation and detection microfluidic chip sequentially requires the following steps:
(1) preparing a middle-layer liquid path control layer and a lower-layer reaction tank layer photoresist SU-8 template with raised channel parts by adopting a photoetching and corrosion method;
(2) developing the photoresist SU-8 template by ethyl lactate, and hardening the photoresist for 1-3 h at 165-180 ℃;
(3) treating the chip photoresist SU-8 template with a silanization reagent for 5-10 min to enable PDMS to be easily stripped from the bottom surface of the template;
(4) mixing polydimethylsiloxane PDMS and an initiator according to a volume ratio of (5-20): 1, uniformly mixing, respectively pouring the mixture into SU-8 templates of the photoresist of the middle and lower layers of the chip, curing the mixture in an oven at 80 ℃ for 20-40 min, and stripping the SU-8 templates of the polydimethylsiloxane PDMS photoresist to obtain a polydimethylsiloxane PDMS chip with a structure;
(5) punching holes one by one at corresponding positions of the middle-layer liquid path control layer and the upper-layer reagent pool layer at the bottoms of the reagent pools by using a puncher;
(6) carrying out oxygen plasma treatment on one side with the structure on the middle layer of the chip and one side with the structure on the lower layer of the chip for 1-3 min, and carrying out irreversible sealing at 70-90 ℃ for 30-60 min;
(7) and (3) carrying out oxygen plasma treatment on the sealed polydimethylsiloxane PDMS chip and the upper polycarbonate plastic for 1-3 min, and carrying out irreversible sealing at 70-90 ℃ for 30-60 min to obtain the integrated exosome separation and detection microfluidic chip.
7. The method for preparing the integrated exosome separation and detection microfluidic chip according to claim 6, which is characterized in that: the preparation method of the integrated exosome separation and detection microfluidic chip further meets the following requirements: after the exosome sample is introduced into the microfluidic chip, buffer solution cleaning, primary antibody staining, cleaning, secondary antibody staining and color development liquid developing are sequentially carried out, and finally, an enzyme-linked immunosorbent assay (ELISA) instrument is used for detection.
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Application publication date: 20201023 |