CN107446797B

CN107446797B - Exosome processing chip and processing method

Info

Publication number: CN107446797B
Application number: CN201610373684.9A
Authority: CN
Inventors: 陈欲超
Original assignee: Shenzhen Huixin Bio Medical Technology Co ltd
Current assignee: Shenzhen Hui core Bio Medical Technology Co., Ltd.
Priority date: 2016-05-31
Filing date: 2016-05-31
Publication date: 2020-07-31
Anticipated expiration: 2036-05-31
Also published as: CN107446797A

Abstract

The invention provides an exosome processing chip which is used for separating and purifying exosomes. The exosome processing chip includes a substrate; two electrodes arranged at two ends of the substrate for providing positive and negative voltages; and a gel layer disposed on the upper surface of the substrate for separating impurities from the exosome mixture. The gel layer is provided with at least one sample adding groove, and the sample adding groove is used for storing the exosome after the exosome mixed solution is separated and purified. The invention also relates to an exosome processing method using the exosome processing chip. Therefore, the exosome processing chip and the exosome processing method can be used for quickly separating and purifying the exosome in a high-throughput manner.

Description

Exosome processing chip and processing method

Technical Field

The invention relates to an exosome processing chip and a processing method.

Background

Exosomes (Exosomes) are circular single-layer membrane structures with diameters of 40-100 nm, can be released by various types of cells of an organism, and are widely distributed in body fluids such as saliva, plasma, milk, urine and the like. Exosomes can carry various proteins, mRNA and miRNA and participate in processes such as cell communication, cell migration, angiogenesis, tumor cell growth and the like. Therefore, the separation and purification of exosome is more and more receiving attention.

At present, the treatment methods based on exosome include differential centrifugation, density gradient centrifugation, magnetic bead immunization and PEG-base precipitation.

Although the differential centrifugation method is simple to operate and can obtain a large amount of exosomes, the treatment process is time-consuming, the recovery rate is unstable, and the purity is not high. Furthermore, repeated centrifugation may also cause damage to the exosomes, thereby reducing their quality.

Although exosomes obtained by density gradient centrifugation are high in purity, the processing steps are cumbersome, time-consuming, and sensitive to the time of centrifugation.

The magnetic bead immunization method has the advantages of high specificity, simple and convenient operation, no influence on the complete form of the exosome and the like, but has low efficiency, the bioactivity of the exosome is easily influenced by the hydrogen ion concentration index and the salt concentration, is not beneficial to downstream experiments, and is difficult to widely popularize.

The principle of PEG-base precipitation is related to competitive binding of free water molecules. The PEG precipitated exosome has the problems of low purity and recovery rate, more impure proteins, uneven particle size, generation of polymer which is difficult to remove, damage to exosome by mechanical force or chemical additives such as Tween-20 and the like.

Disclosure of Invention

In view of the above, there is a need to provide an exosome processing chip and processing method for separating exosomes quickly and efficiently.

An exosome-processing chip for separation and purification of exosomes, comprising:

a substrate;

two electrodes arranged at two ends of the substrate for providing positive and negative voltages; and

a gel layer arranged on the upper surface of the substrate and used for separating impurities in the exosome mixed solution;

the gel layer is provided with at least one sample adding groove, and the sample adding groove is used for storing the exosome after the exosome mixed solution is separated and purified.

The middle part of the gel layer is provided with a plurality of sample adding grooves which are arranged at intervals.

The sample adding grooves are arranged linearly or in a staggered way, and after being projected towards the ends where the two electrodes are positioned, the sample adding grooves are not overlapped with each other.

The spacing distance between the central lines of the adjacent sample adding grooves is more than or equal to 6 mm.

The exosome processing chip also comprises a sealing film covering the surface of the at least one sample adding slot, and the sealing film is a preservative film.

The gel layer is a conductive porous material, and the pore diameter of the porous material is 200-1000 nanometers.

The gel layer comprises agarose gel, silica porous gel or calcium alginate porous gel.

An exosome processing method using the exosome processing chip comprises the following steps:

pretreating a biological sample to obtain an exosome mixed solution;

adding the exosome mixed solution into a sample adding groove;

connecting the two electrodes with positive and negative voltages respectively to form continuous current on the gel layer;

under the action of the current, impurities in the exosome mixed solution pass through the gel layer and move towards the two electrodes, and the exosome is retained in the sample adding groove.

The voltage value is 3-25V.

The pre-treating the biological sample comprises subjecting the biological sample to filtration membrane with a syringe or high speed centrifugation to remove cells.

Compared with the prior art, the exosome processing chip is provided with the electrodes for providing positive and negative voltages so as to form continuous current on the gel layer. Under the action of the current, impurities in the exosome mixed solution pass through the gel layer and move towards the two electrodes, and the exosome is retained in the sample adding groove. Therefore, the exosome processing chip and the exosome processing method can be used for quickly separating and purifying the exosome in a high-throughput manner.

Drawings

FIG. 1 is a diagram of an exosome processing chip according to a preferred embodiment of the present invention.

FIG. 2 is a schematic diagram of an exosome processing chip according to another preferred embodiment of the present invention.

FIG. 3 is a flow chart of an exosome-processing method of the present invention.

Description of the main elements

Exosome processing chip	100，200
		Substrate	10
Electrode for electrochemical cell	21，22
		Gel layer	30
Sample adding groove	31
		Sealing film	40

The following detailed description will further illustrate the invention in conjunction with the above-described figures.

Detailed Description

Referring to fig. 1, a structure diagram of a first preferred embodiment of an exosome processing chip 100 according to the present invention, the exosome processing chip 100 is used for separating and purifying exosomes. The exosome processing chip 100 comprises a substrate 10, two

opposing electrodes

21, 22 clamped from two ends of the substrate 10, and a gel layer 30 disposed on the upper surface of the substrate 10. The gel layer 30 is used to separate impurities in the exosome mixture.

It will be appreciated that the exosomes are present in a bodily fluid, such as, but not limited to, blood, saliva, urine or breast milk. The exosomes have a special structure, e.g., comprising encapsulated DNA fragments, small RNAs and polypeptides, etc., so that the exosomes are electrically neutral.

The substrate 10 is, for example, but not limited to, one or a combination of glass, quartz plate, silicon plate, or polymer plate.

The gel layer 30 is an electrically conductive porous material. The gel layer 30 is, for example, but not limited to, a lipogel, a silica porous gel, a calcium alginate porous gel. It will be appreciated that the gel layer 30 may also be other similar functional materials. The pore diameter range of the conductive porous material is 200-1000 nm.

Preferably, the pore size of the conductive porous material is in the range of 1000 nm.

It will be appreciated that the gel layer 30 serves to separate impurities in the exosome mixture, which may pass through the pores of the gel layer 30. The impurities are, for example, proteins, nucleic acids, lipids or carbohydrates.

The upper surface of the gel layer 30 is provided with at least one sample adding slot 31.

In this embodiment, the upper surface of the gel layer 30 is provided with a plurality of sample adding grooves 31.

The sample addition grooves 31 are provided at intervals on the upper surface of the gel layer 30.

The sample addition grooves 31 can be arranged linearly, and after the projection of each sample addition groove 31 to the ends of the two

electrodes

21 and 22, no overlap exists.

It is understood that the position of the sample addition grooves 31 is not limited to the middle of the gel layer 30.

It can be understood that the plurality of sample application grooves 31 can simultaneously perform separation and purification of exosomes on different biological samples. The sample adding slot 31 is used for storing the exosome after the exosome mixed solution is separated and purified.

It can be understood that the sample adding slots 31 can be used for storing the exosome mixture, and can also be used for storing the exosomes separated and purified by the exosome mixture.

In order to facilitate the addition and extraction of the exosomes to be separated and purified, the spacing distance between the central lines of the adjacent sample adding grooves 31 is more than or equal to 0.6 cm.

In order to prevent the exosome from being contaminated during the separation and purification, a sealing film 40 is covered on the upper surface of the gel layer 30 to seal the sample loading slots 31.

It is understood that in other embodiments, the packaging film 40 can cover each sample loading slot 31 individually. The sealing film 40 seals the sample adding grooves 31 and also comprises other sealing modes for achieving the sealing effect.

The sealing film 40 is, for example, a preservative film, and the sealing film 40 may be made of polyethylene, polyvinyl chloride, polyvinylidene chloride, and other preservative materials.

It will be appreciated that the sealing film 40 may also be other materials that function like a cling film.

Referring to fig. 2, a structure diagram of a second preferred embodiment of the exosome processing chip 100 of the present invention, the exosome processing chip 200 has a similar structure to the exosome processing chip 100, except that: the sample addition slots 31 can be arranged in a staggered manner, and after the projection of each sample addition slot 31 to the ends of the two

electrodes

21 and 22, no overlap exists.

According to the exosome processing chip, the exosome is neutral in electricity, and the exosome processing chip is provided with the electrodes for providing positive and negative voltages. Therefore, the protein with positive electricity can rapidly move to the negative electrode after gel electrophoresis, the gene with negative electricity rapidly moves to the positive electrode after gel electrophoresis, and the exosome basically keeps motionless under a specific electric field due to electroneutrality, so that the exosome is rapidly purified at high flux. In addition, the exosome processing chip is small in size and convenient to carry.

Referring to fig. 3, the exosome processing method 300 using the above exosome processing chip 100 according to the present invention includes the following steps:

step S10, pretreating a biological sample to obtain an exosome mixed solution;

the biological sample pretreatment step can be to filter the biological sample with a 200 nm syringe filter membrane; or the biological sample is centrifuged at high speed.

The high speed centrifugation condition is 2000g centrifugation for 10min to collect supernatant, and the supernatant is centrifuged again, and the centrifugation condition is 10000g centrifugation for 15 min.

In step S20, the exosome mixture is added to the sample addition slot of the exosome processing chip 100.

The gel layer 30 is first placed on the substrate 10, and then the exosome mixture is dropped into the sample loading slots 31 of the exosome processing chip 100. The sealing film 40 is then applied to the gel layer 30 to enclose each sample addition slot 31.

It can be understood that the plurality of sample adding chambers 31 can be used for adding different exosome mixtures to be separated and purified respectively.

In step S30, the exosome processing chip 100 is subjected to electrophoresis.

The exosome processing chip 100 is placed in a conducting buffer, and the two electrodes 31, 32 are connected to positive and negative voltages, respectively, to form a continuous current on the gel layer 30.

It will be appreciated that this voltage value is 3-25V.

Preferably, the voltage value is 4.5V.

It can be understood that the impurities in the exosome mixture in the loading chamber 31 pass through the gel layer 30 and move towards the two electrodes 31, 32 under the action of the current, and the exosome is retained in the loading chamber 31.

The positively charged impurities (such as protein) can rapidly move to the negative electrode after gel electrophoresis, the negatively charged impurities (such as gene) can rapidly move to the positive electrode after gel electrophoresis, and the exosome is basically immobile under a specific electric field due to the electroneutrality, so that the exosome can be rapidly purified at high flux.

It is understood that the impurities are, for example, proteins, genes, nucleic acids, and the like.

Step S40, the exosomes are removed.

After the gel layer 30 is finished, the sealing film 40 is opened, and the purified exosomes are separated by pipette.

The exosome treatment method provided by the invention is low in cost, simple to operate, capable of quickly separating and purifying different types of exosome samples with high flux, and capable of obtaining exosomes with high purity and high yield.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and the above embodiments are only used for explaining the claims. The scope of the invention is not limited by the description. Any changes or substitutions that can be easily made by those skilled in the art within the technical scope of the present disclosure are included in the scope of the present invention.

Claims

1. An exosome-processing chip for separation and purification of exosomes, comprising:

a substrate;

the gel layer is arranged on the upper surface of the substrate, is made of a conductive porous material and is used for separating impurities in the exosome mixed solution;

2. The exosome processing chip according to claim 1, characterized in that: the middle part of the gel layer is provided with a plurality of sample adding grooves which are arranged at intervals.

3. The exosome processing chip according to claim 2, characterized in that: the sample adding grooves are arranged linearly or in a staggered way, and after being projected towards the ends where the two electrodes are positioned, the sample adding grooves are not overlapped with each other.

4. The exosome processing chip according to claim 2, characterized in that: the spacing distance between the central lines of the adjacent sample adding grooves is more than or equal to 6 mm.

5. The exosome processing chip according to claim 1, characterized in that: the exosome processing chip also comprises a sealing film covering the surface of the at least one sample adding slot, and the sealing film is a preservative film.

6. The exosome processing chip according to claim 1, characterized in that: the pore diameter of the porous material is 200-1000 nm.

7. The exosome processing chip according to claim 1, characterized in that: the gel layer comprises agarose gel, silica porous gel or calcium alginate porous gel.

8. An exosome-processing method using the exosome-processing chip according to any one of claims 1 to 7, comprising the steps of:

pretreating the biological sample to remove cells and obtain an exosome mixture;

adding the exosome mixed solution into a sample adding groove;

9. Exosome processing method according to claim 8, characterized in that: the voltage value is 3-25V.

10. Exosome processing method according to claim 8, characterized in that: the pre-treating the biological sample comprises subjecting the biological sample to filtration membrane with a syringe or high speed centrifugation.