CN112206839A - Exosome detection microfluidic chip based on graphene oxide quenching aptamer and application - Google Patents
Exosome detection microfluidic chip based on graphene oxide quenching aptamer and application Download PDFInfo
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- CN112206839A CN112206839A CN201910622437.1A CN201910622437A CN112206839A CN 112206839 A CN112206839 A CN 112206839A CN 201910622437 A CN201910622437 A CN 201910622437A CN 112206839 A CN112206839 A CN 112206839A
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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
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
An exosome detection microfluidic chip based on graphene oxide quenching aptamer and application thereof are disclosed, and the microfluidic chip comprises the following components: the upper layer is a sample liquid path layer, the lower layer is a nucleic acid aptamer liquid path layer quenched by graphene oxide, and the bottom surface is a blank glass bottom plate; the device is specifically provided with the following structures: the sample injection device comprises a sample injection port to be detected, a sample injection channel area to be detected, a graphene oxide quenched nucleic acid aptamer injection port, a graphene oxide quenched nucleic acid aptamer injection channel area and a reaction tank. The exosome is detected by the principle that the graphene oxide quenches the aptamer fluorescent probe, and a microfluidic array chip system is adopted to simultaneously detect a plurality of markers of a plurality of samples. The exosome extraction detection flow is optimized, the exosome detection efficiency is greatly improved, the manual operation procedure is simplified, and a good exosome detection effect is achieved.
Description
Technical Field
The invention relates to the field of exosome separation, in particular to an exosome detection microfluidic chip based on a graphene oxide quenching aptamer and an application method.
Background
The exosome is an extracellular nanoscale vesicle formed by cells through a series of regulation processes such as endocytosis, fusion and efflux. It is widely distributed in human body, and urine, sweat, blood, milk, etc. of human body all contain exosome. Exosomes play two main roles in the human body, the first is immunologically active exosomes, which play a major role in antigen presentation and co-stimulation, with information transfer functions. The second is an exosome containing a considerable amount of RNA and mediating the exchange of genetic material between cells, and has a material-transferring function. With the progress of research, it is found that exosomes play an important role in adaptive immunity, inflammation process, embryogenesis, and tumor generation and development process. In the case of tumors, over a hundred years ago, it was discovered through dissection that specific tumor cells always tend to metastasize to specific tissue organs, and thus a well-known "seed and soil" metastasis hypothesis was proposed that tumor cells could only form metastases in the appropriate tissue organ environment. With the development of the technology, the metastasis mechanism of the tumor is continuously improved, people find that the tumor can actively change the microenvironment of a metastasis focus by secreting exosomes, and the exosomes can promote tumor angiogenesis and tumor metastasis by regulating the immune function, or directly act on tumor cells to influence the tumor development. Therefore, research on exosomes is expected to provide a new idea for early diagnosis of tumors, inhibition of tumor development and the like.
The microfluidic chip technology is an important technology in the 21 st century, and the core of the technology 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 or nano microchannel network, and a complex analysis process is completed by controlling fluid, so that the technology 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. In addition, the method also becomes a potential platform for exosome research due to the characteristics of micro-size, automation, trace reagent usage, high flux, realization of multifunctional integration and the like. At present, two types of technologies for separating exosomes by using a microfluidic chip are available, one is a separation technology based on size, and the separation technology mainly comprises the step of directly acting on a sample by using device structures such as a nanopore membrane, a nano array, a microfilter and the like to separate exosomes. The other is separation technology based on immunocapture, which mainly comprises planar immunocapture and microbead immunocapture. However, it is still a difficult problem to efficiently obtain high-purity exosomes and analyze and detect the exosomes.
Disclosure of Invention
The invention aims to provide an exosome detection microfluidic chip based on a graphene oxide quenching aptamer and an application method thereof, so as to solve the limitations of complex operation steps, large reagent consumption and the like existing in the separation and detection process of exosomes.
The invention provides an exosome detection microfluidic chip based on a graphene oxide quenching nucleic acid aptamer, which is characterized in that:
the micro-fluidic chip is formed by sequentially connecting an upper layer, a lower layer and a bottom surface in series and laminating, wherein: the upper layer is a sample liquid path layer, the lower layer is a nucleic acid aptamer liquid path layer quenched by graphene oxide, and the bottom surface is a blank glass bottom plate;
the sample liquid path layer is provided with the following structures:
-a sample inlet: located furthest upstream of the entire sample fluid path layer;
-a sample injection channel region: the sample injection port and the reaction cell are arranged between the two for communicating;
the liquid path layer of the graphene oxide quenched aptamer is provided with the following structure:
-a graphene oxide quenched aptamer sample inlet: the aptamer liquid path layer is positioned at the most upstream of the whole graphene oxide quenched aptamer liquid path layer;
-a graphene oxide quenched aptamer sample injection channel region: the sample inlet is arranged between the graphene oxide quenched nucleic acid aptamer sample inlet and the reaction pool and is used for communicating the graphene oxide quenched nucleic acid aptamer sample inlet and the reaction pool;
the reaction tank is arranged between the oxidized graphene quenched nucleic acid aptamer sample injection channel area and the sample injection channel area, vertically penetrates through the whole chip and is used for communicating the two.
The exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
the reaction tank is provided with 4nThe sample injection port at the upstream of the corresponding sample liquid path layer is provided with 2nEach sample injection port corresponds to a sample injection channel region, and each sample injection channel region has 2nThe outlets are respectively connected with the reaction tank; the sample inlet of the aptamer at the upstream of the liquid path layer of the graphene oxide quenched aptamer has 2nEach nucleic acid adapter sample inlet corresponds to a nucleic acid adapter sample inlet channel region, and each nucleic acid adapter sample inlet channel region has 2nThe outlets are respectively connected with the reaction tank, wherein n is a natural number.
The exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets one or a combination of the following requirements:
one, the sample injection channel regions are respectively corresponding to one injection port and 2nA reaction tank;
secondly, the sample introduction channel regions of the graphene oxide quenched nucleic acid aptamer correspond to a sample introduction port and 2nA reaction tank.
The exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets one or a combination of the following requirements:
firstly, the sample liquid path layer and the graphene oxide quenched nucleic acid aptamer liquid path layer are both made of polydimethylsiloxane polymers, and the thickness of the sample liquid path layer is the same as that of the graphene oxide quenched nucleic acid aptamer liquid path layer and is 1-3 mm;
secondly, the reaction tank is a cylindrical chamber with the bottom surface diameter of 2mm and penetrates through the upper layer chip and the lower layer chip;
thirdly, the sample liquid path layer and the graphene oxide quenched aptamer liquid path layer have the same height and are both 80-200 μm;
and fourthly, the widths of the sample liquid path layer and the aptamer liquid path layer quenched by the graphene oxide are the same and are both 30-100 microns.
The exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
the sample liquid path layer is obtained by pouring a layer of polydimethylsiloxane 1-3mm higher than the template on the successfully manufactured template of the sample liquid path layer, and removing the polydimethylsiloxane after curing; the graphene oxide quenched aptamer liquid path layer is obtained by pouring a layer of polydimethylsiloxane 1-3mm higher than the template on the successfully prepared graphene oxide quenched aptamer liquid path layer template, and removing the polydimethylsiloxane after curing; one side of the sample liquid path layer with the structure is sealed and connected to the side of the graphene oxide quenched nucleic acid aptamer liquid path layer without the structure, and one side of the graphene oxide quenched nucleic acid aptamer liquid path layer with the structure is bonded to a clean glass sheet through plasma.
The application method of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer comprises the following steps:
(1) adding 5-20nM aptamer of related protein to be detected and 0.02-0.2mg/ml graphene oxide aqueous solution into buffer solution (Tris-HCl buffer solution or phosphate buffered saline solution), and reacting at room temperature for 5-20min to completely quench the fluorescence of the aptamer for later use;
(2) adding the quenched aptamer probes into reaction tanks by using a syringe pump, wherein the volume of each reaction tank is 10-30 mu L;
(3) adding a sample to be detected into reaction tanks by using a syringe pump, wherein the volume of each reaction tank is 0.1-0.3 mu L;
(4) reacting at room temperature in dark for 20-40 min;
(5) and detecting the fluorescence value of the system.
The application method of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
(1) the aptamers are all synthesized by a reagent company, and the 5' ports of the aptamers are all modified by fluorescent groups;
(2) the exosome detection microfluidic chip based on the graphene oxide quenching aptamer can simultaneously realize 2 at most at one timenA sample 2nDetection of individual markers.
The application of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer provided by the invention is as follows: the chip can be applied to the detection of a plurality of protein markers in different exosome samples (blood, urine, saliva and the like).
The invention has the advantages that:
1. the invention selects the protein specific aptamer to detect the exosome, thereby improving the specificity and the reliability of the detection.
2. The invention introduces the sample and detects the aptamer to be integrated on one chip, thereby optimizing the detection process of the exosome.
3. The invention introduces the reagent by the injection pump, simplifies the manual operation, reduces the error and realizes the space-time resolution which is difficult to realize by the traditional method.
4. The invention can detect a plurality of proteins of a plurality of samples at one time through the array design on the chip, thereby greatly improving the detection efficiency.
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 microfluidic chip for detecting exosomes based on graphene oxide quenched nucleic acid aptamers;
FIG. 2 is a schematic diagram of an upper layer structure of a microfluidic chip for detecting exosomes based on graphene oxide quenching aptamers;
FIG. 3 is a schematic diagram of the lower layer structure of a microfluidic chip for detecting exosomes based on graphene oxide quenching aptamers;
FIG. 4 is a graph showing the relationship between quenching effect of graphene oxide quenched aptamer and concentration of graphene oxide;
FIG. 5 is a graph showing the relationship between quenching effect of graphene oxide quenched aptamer and quenching time;
FIG. 6 is a graph showing the relationship between the fluorescence recovery effect of a graphene oxide quenching aptamer and the concentration of graphene oxide;
FIG. 7 is a graph showing the relationship between the fluorescence recovery effect and the recovery time of a graphene oxide quenched aptamer.
Wherein: 1 is a sample injection port; 2 is a sample injection channel area; 3 is a reaction tank; 4, a sample inlet of a graphene oxide quenched nucleic acid aptamer; 5 is a sample introduction channel area of the graphene oxide quenched nucleic acid aptamer.
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 microfluidic chip for detecting exosome based on the graphene oxide quenching aptamer and the application method thereof are specifically described below.
The invention provides an exosome detection microfluidic chip based on a graphene oxide quenching nucleic acid aptamer, as shown in figure 1, the microfluidic chip is formed by sequentially connecting an upper layer, a lower layer and a bottom surface in series, wherein: the upper layer is a sample liquid path layer (figure 2), the lower layer is a nucleic acid aptamer liquid path layer quenched by graphene oxide (figure 3), the bottom surface is a blank glass bottom plate, and the sample liquid path layer is provided with the following structures:
-sample inlet 1: located furthest upstream of the entire sample fluid path layer;
-the sample injection channel region 2: the sample injection port is arranged between the sample injection port to be detected and the reaction tank and is used for communicating the sample injection port to be detected and the reaction tank;
the liquid path layer of the graphene oxide quenched aptamer is provided with the following structure:
-graphene oxide quenched aptamer sample inlet 4: the aptamer liquid path layer is positioned at the most upstream of the whole graphene oxide quenched aptamer liquid path layer;
-graphene oxide quenched aptamer sample injection channel region 5: the sample inlet is arranged between the graphene oxide quenched nucleic acid aptamer sample inlet and the reaction pool and is used for communicating the graphene oxide quenched nucleic acid aptamer sample inlet and the reaction pool;
the reaction tank 3 is arranged between the sample introduction channel area of the graphene oxide quenched nucleic acid aptamer and the sample introduction channel area, vertically penetrates through the whole chip and is used for communicating the two.
The exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
the reaction pool is provided with 64 reaction pools, the upstream sample injection ports of the sample liquid path layer corresponding to the reaction pools are 8, each sample injection port corresponds to one sample injection channel area, and each sample injection channel area is provided with 8 outlets which are respectively connected with the reaction pool; the number of sample inlets of the graphene oxide quenched nucleic acid aptamer liquid path layer upstream nucleic acid aptamer is 8, each sample inlet of the graphene oxide quenched nucleic acid aptamer corresponds to one sample inlet channel region of the graphene oxide quenched nucleic acid aptamer, and 8 outlets of each sample inlet channel region of the graphene oxide quenched nucleic acid aptamer are respectively connected with the reaction tank.
The exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets one or a combination of the following requirements:
firstly, each sample injection channel area corresponds to one injection port and 8 reaction cells;
secondly, the sample injection channel regions of the graphene oxide quenched nucleic acid aptamers correspond to a sample injection port and 8 reaction cells respectively.
The exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets one or a combination of the following requirements:
firstly, the sample liquid path layer and the graphene oxide quenched nucleic acid aptamer liquid path layer are both made of polydimethylsiloxane polymers, and the thickness of the two layers of chips is the same and is 2 mm;
secondly, the reaction tank is a cylindrical chamber with the bottom surface diameter of 2mm and penetrates through the upper layer chip and the lower layer chip;
thirdly, the sample liquid path layer and the graphene oxide quenched nucleic acid aptamer liquid path layer have the same height and are both 100 micrometers;
and fourthly, the widths of the sample liquid path layer and the aptamer liquid path layer quenched by the graphene oxide are the same and are both 100 micrometers.
The exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
the sample liquid path layer is obtained by pouring a layer of polydimethylsiloxane 2mm higher than the template on the successfully manufactured sample liquid path layer template, and removing the polydimethylsiloxane after curing; the graphene oxide quenched aptamer liquid path layer is obtained by pouring a layer of polydimethylsiloxane 2mm higher than the template on the successfully prepared graphene oxide quenched aptamer liquid path template, and removing the polydimethylsiloxane after curing; one side of the sample liquid path layer with the structure is sealed and connected to the side of the graphene oxide quenched nucleic acid aptamer liquid path layer without the structure, and one side of the graphene oxide quenched nucleic acid aptamer liquid path layer with the structure is bonded to a clean glass sheet through plasma.
The application method of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer comprises the following steps:
(1) adding 10nM aptamer of related protein to be detected and 0.1mg/ml graphene oxide aqueous solution into buffer solution (Tris-HCl buffer solution or phosphate buffered saline solution), and reacting at room temperature for 5min to completely quench the aptamer fluorescence for later use;
(2) adding the aptamer probes quenched by the graphene oxide into reaction tanks by using a syringe pump, wherein the volume of each reaction tank is 20 mu L;
(3) adding a sample to be detected into reaction tanks by using a syringe pump, wherein the volume of each reaction tank is 0.2 mu L;
(4) reacting for 30min at room temperature in a dark place;
(5) and detecting the fluorescence value of the system.
The application method of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
(1) the aptamers are all synthesized by a reagent company, and the 5' ports of the aptamers are all modified by fluorescent groups;
(2) the exosome detection microfluidic chip based on the graphene oxide quenching aptamer can simultaneously detect 8 markers in 8 samples at most at one time.
The application of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer provided by the invention is as follows: the chip can be applied to the detection of a plurality of protein markers in different exosome samples (blood, urine, saliva and the like).
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 protruding channel part by adopting a photoetching and corrosion method on the basis of the exosome detection microfluidic chip of the graphene oxide quenching aptamer, wherein the lower layer structure in the chip is respectively composed of two SU-8 template reverse-mode PDMS;
the sample liquid path layer chip template 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 for 20min at 95 ℃, naturally cooling, placing a mask of a chip sample liquid path layer structure on an SU-8 glue flat plate, performing ultraviolet exposure for 30s, post-baking for 20min at 95 ℃, 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 preparation method of the graphene oxide quenched nucleic acid aptamer liquid circuit layer chip template comprises the following steps: 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 of a chip graphene oxide quenched nucleic acid aptamer liquid path layer structure on an SU-8 glue flat plate, performing ultraviolet exposure for 30s, post-baking at 95 ℃ for 20min, and naturally cooling; and finally, developing the SU-8 photoresist for 5min by using ethyl lactate, hardening the film for 2h at 180 ℃, and naturally cooling to obtain the chip template.
Example 2
Treating SU-8 templates of upper and lower layer structures of the chip with a silylation agent 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 the mixture on SU-8 templates of the upper layer structure and the lower layer structure of the chip, curing the mixture 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 the corresponding positions of the sample injection ports of the upper layer sample liquid path layer by using a puncher; performing oxygen plasma treatment on the side with the structure on the upper layer of the chip and the side without the structure on the lower layer of the chip for 2min, baking for 45min at 80 ℃, and performing irreversible sealing; punching holes one by one at corresponding positions of a sample inlet of the aptamer quenched by the lower graphene oxide and the reaction tank by using a puncher; and (3) carrying out oxygen plasma treatment on the sealed polydimethylsiloxane PDMS chip and a piece of glass for 2min, and carrying out irreversible sealing by baking at 80 ℃ for 45min to obtain the exosome detection microfluidic chip based on the graphene oxide quenching nucleic acid aptamer.
In FIG. 2, 1 is a sample inlet, 2 is a sample inlet channel region, and 3 is a reaction cell.
In fig. 3, 3 is a reaction cell, 4 is a sample inlet of graphene oxide quenched aptamer, and 5 is a sample channel region of graphene oxide quenched aptamer.
Example 3
Quenching effect of graphene oxide quenching aptamer and exploration of relationship between graphene oxide concentration and quenching time
The micro-fluidic chip prepared in example 2 was selected for exploration and detection, and the specific steps were as follows:
(1) 8 1mL centrifuge tubes were filled with the oxidized graphene Tris-HCl buffer solutions at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.175, and 1.5875. mu.g/mL, respectively, and the aptamer was added thereto to give a final concentration of 10 nM/L.
(2) Adding a nucleic acid aptamer graphene oxide reaction system into reaction tanks by using a syringe pump, wherein the volume of each reaction tank is 20 mu L;
(3) the fluorescence intensity values were read every 5 minutes.
The relationship between the fluorescence quenching effect and the concentration of graphene oxide is shown in fig. 4, and it can be seen from the graph that the higher the concentration of graphene oxide is, the better the fluorescence quenching effect is; when the concentration is higher than 50. mu.g/mL, the effect of the increase in concentration on the fluorescence quenching effect is insignificant. The relationship between the fluorescence quenching effect and the quenching time is shown in FIG. 5, and it can be seen from the graph that the quenching is complete when the quenching time is 5 min.
Example 4
Exploration of relationship between fluorescence recovery effect of graphene oxide quenching aptamer and concentration and recovery time of graphene oxide
The method comprises the following steps of selecting a graphene oxide microfluidic chip in which the aptamer is added into a reaction cell in the embodiment 3 for exploration and detection:
(1) adding a serum sample into reaction tanks by using a syringe pump, wherein the volume of each reaction tank is 0.2 mu L;
(2) the fluorescence intensity values were read every 5 minutes.
The relationship between the fluorescence recovery effect and the graphene oxide concentration is shown in fig. 6, and it can be seen from the graph that the fluorescence recovery effect of the aptamer probe is better when the graphene oxide concentration is lower, and the fluorescence of the fluorescence probe is hardly recovered when the graphene oxide concentration is higher. The relationship between the fluorescence recovery effect and the fluorescence recovery time is shown in FIG. 7, and it can be seen from the graph that the longer the recovery time is, the better the fluorescence recovery effect is, and when the recovery time is greater than 30min, the increase of the recovery time has no significant effect on the fluorescence intensity recovery effect.
Claims (8)
1. An exosome detection microfluidic chip based on graphene oxide quenching aptamer is characterized in that:
the micro-fluidic chip is formed by sequentially connecting an upper layer, a lower layer and a bottom surface in series and laminating, wherein: the upper layer is a sample liquid path layer, the lower layer is a nucleic acid aptamer liquid path layer quenched by graphene oxide, and the bottom surface is a blank glass bottom plate;
the sample liquid path layer is provided with the following structures:
-a sample inlet: located furthest upstream of the entire sample fluid path layer;
-a sample injection channel region: the sample injection port and the reaction cell are arranged between the two for communicating;
the liquid path layer of the graphene oxide quenched aptamer is provided with the following structure:
-a graphene oxide quenched aptamer sample inlet: the aptamer liquid path layer is positioned at the most upstream of the whole graphene oxide quenched aptamer liquid path layer;
-a graphene oxide quenched aptamer sample injection channel region: the sample inlet is arranged between the graphene oxide quenched nucleic acid aptamer sample inlet and the reaction pool and is used for communicating the graphene oxide quenched nucleic acid aptamer sample inlet and the reaction pool;
the reaction tank is arranged between the sample injection channel area of the graphene oxide quenched nucleic acid aptamer and the sample injection channel area of a sample to be detected, vertically penetrates through the whole chip and is used for communicating the two.
2. The microfluidic chip for detecting exosome based on graphene oxide quenching aptamer according to claim 1, characterized in that: the exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
the reaction tank is provided with 4n(n can be any natural number such as 0,1,2,3,4, etc.) and 2 sample injection ports at the upstream of the corresponding sample liquid path layernEach sample injection port corresponds to a sample injection channel region, and each sample injection channel region has 2nThe outlets are respectively connected with the reaction tank; the sample inlet of the aptamer at the upstream of the liquid path layer of the graphene oxide quenched aptamer has 2nEach nucleic acid adapter sample inlet corresponds to a nucleic acid adapter sample inlet channel region, and each nucleic acid adapter sample inlet channel region has 2nThe outlets are respectively connected with the reaction tanks.
3. The microfluidic chip for detecting exosome based on graphene oxide quenching aptamer according to claim 1, characterized in that: the exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets one or a combination of the following requirements:
one, the sample injection channel regions are respectively corresponding to one injection port and 2nA reaction tank;
II, theThe sample introduction channel regions of the graphene oxide quenched nucleic acid aptamer correspond to one sample introduction port and 2nA reaction tank.
4. The microfluidic chip for detecting exosome based on graphene oxide quenching aptamer according to claim 1, characterized in that: the exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets one or a combination of the following requirements:
firstly, the sample liquid path layer and the graphene oxide quenched nucleic acid aptamer liquid path layer are both made of polydimethylsiloxane polymers, and the thickness of the two layers of chips is the same and is 1-3 mm;
secondly, the reaction tank is a cylindrical chamber with the bottom surface diameter of 2mm and penetrates through the upper layer chip and the lower layer chip;
thirdly, the sample liquid path layer and the graphene oxide quenched aptamer liquid path layer have the same height and are both 80-200 μm;
and fourthly, the widths of the sample liquid path layer and the aptamer liquid path layer quenched by the graphene oxide are the same and are both 30-100 microns.
5. The microfluidic chip for detecting exosome based on graphene oxide quenching aptamer according to claim 1, characterized in that: the exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
the sample liquid path layer is obtained by pouring a layer of polydimethylsiloxane 1-3mm higher than the template on the successfully manufactured template of the sample liquid path layer, and removing the polydimethylsiloxane after curing; the graphene oxide quenched aptamer liquid path layer is obtained by pouring a layer of polydimethylsiloxane 1-3mm higher than the template on the successfully prepared graphene oxide quenched aptamer liquid path layer template, and removing the polydimethylsiloxane after curing; one side of the sample liquid path layer with the structure is sealed and connected to the side of the graphene oxide quenched nucleic acid aptamer liquid path layer without the structure, and one side of the graphene oxide quenched nucleic acid aptamer liquid path layer with the structure is bonded to a clean glass sheet through plasma.
6. Use of the graphene oxide quenched aptamer based exosome detection microfluidic chip according to claims 1-5, wherein: the exosome detection microfluidic chip based on the graphene oxide quenching aptamer can be used for detecting exosomes in body fluid (blood, urine, saliva and the like) and a culture medium.
7. The application of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer according to claim 6, wherein the microfluidic chip comprises: the application method of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer comprises the following steps:
(1) adding 5-20nM aptamer of related protein to be detected and 0.02-0.2mg/ml graphene oxide aqueous solution into buffer solution (Tris-HCl buffer solution or phosphate buffered saline solution), and reacting at room temperature for 5-20min to completely quench the fluorescence of the aptamer for later use;
(2) adding the graphene oxide quenched aptamer probes into reaction tanks by using a syringe pump, wherein the added volume of each reaction tank is 10-30 mu L;
(3) adding the sample into reaction cells by using a syringe pump, wherein the added volume of each reaction cell is 0.1-0.3 mu L;
(4) reacting at room temperature in dark for 20-40 min;
(5) and detecting the fluorescence value of the system.
8. The application of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer according to claim 7, wherein the microfluidic chip comprises: the application method of the exosome detection microfluidic chip based on the graphene oxide quenching aptamer further meets the following requirements:
(1) the 5' ports of the aptamers are all modified by fluorescent groups;
(2) the exosome detection microfluidic chip based on the graphene oxide quenching aptamer simultaneously realizes 2nA sample 2nDetection of individual markers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910622437.1A CN112206839A (en) | 2019-07-11 | 2019-07-11 | Exosome detection microfluidic chip based on graphene oxide quenching aptamer and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022252004A1 (en) * | 2021-05-31 | 2022-12-08 | 京东方科技集团股份有限公司 | Detection chip and manufacturing method therefor, and sampling method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103801412A (en) * | 2012-11-08 | 2014-05-21 | 中山大学 | Integrated micro-fluidic chip used for fluorescence detection of enzyme catalysis product and application thereof |
| CN106497771A (en) * | 2016-10-21 | 2017-03-15 | 辽宁中医药大学 | A kind of multifunctional microflow control chip for being screened for multi-medicament and cell simultaneously |
| CN108148751A (en) * | 2016-12-06 | 2018-06-12 | 中国科学院大连化学物理研究所 | A kind of integrated drug screening and dyeing micro-fluidic chip and preparation method thereof |
| CN108795732A (en) * | 2017-04-27 | 2018-11-13 | 京东方科技集团股份有限公司 | A kind of gene detecting chip, its detection method and micro-fluidic chip system |
| CN112763708A (en) * | 2020-12-24 | 2021-05-07 | 生物岛实验室 | Exosome detection method |
-
2019
- 2019-07-11 CN CN201910622437.1A patent/CN112206839A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103801412A (en) * | 2012-11-08 | 2014-05-21 | 中山大学 | Integrated micro-fluidic chip used for fluorescence detection of enzyme catalysis product and application thereof |
| CN106497771A (en) * | 2016-10-21 | 2017-03-15 | 辽宁中医药大学 | A kind of multifunctional microflow control chip for being screened for multi-medicament and cell simultaneously |
| CN108148751A (en) * | 2016-12-06 | 2018-06-12 | 中国科学院大连化学物理研究所 | A kind of integrated drug screening and dyeing micro-fluidic chip and preparation method thereof |
| CN108795732A (en) * | 2017-04-27 | 2018-11-13 | 京东方科技集团股份有限公司 | A kind of gene detecting chip, its detection method and micro-fluidic chip system |
| US20200164366A1 (en) * | 2017-04-27 | 2020-05-28 | Boe Technology Group Co., Ltd. | Gene detection chip, detection method thereof, and microfluidic control chip system |
| CN112763708A (en) * | 2020-12-24 | 2021-05-07 | 生物岛实验室 | Exosome detection method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022252004A1 (en) * | 2021-05-31 | 2022-12-08 | 京东方科技集团股份有限公司 | Detection chip and manufacturing method therefor, and sampling method |
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