阅读说明：本技术 数字微流控芯片、数字微流控芯片系统、酶联免疫检测系统及方法 (Digital microfluidic chip, digital microfluidic chip system, enzyme-linked immunoassay system and method ) 是由 胡思怡 马汉彬 苏阳 于 2020-05-12 设计创作，主要内容包括：本申请涉及检测设备技术领域,尤其涉及一种数字微流控芯片、包括该数字微流控芯片的数字微流控芯片系统、包括该数字微流控芯片系统的酶联免疫检测系统及采用酶联免疫检测系统的检测方法。上述酶联免疫检测系统,包括数字微流控芯片系统、驱动控制单元和光学检测系统。驱动控制单元用于驱动数字微流控芯片系统内的液体流动,光学检测系统用于检测检测点的光学性质。上述酶联免疫检测系统及检测方法,可以实现ELISA检测反应。酶联免疫检测系统具有较高的集成度且与现有技术相比具有制备方法简单、自动化程度高、成本低且灵活度高等优势。可以大大的减少在进行ELISA流程操作时的人为误差、人工时间成本和试剂的消耗,同时提高了检测的准确率和效率。(The application relates to the technical field of detection equipment, in particular to a digital microfluidic chip, a digital microfluidic chip system comprising the digital microfluidic chip, an enzyme-linked immunoassay system comprising the digital microfluidic chip system and a detection method adopting the enzyme-linked immunoassay system. The enzyme-linked immunoassay system comprises a digital microfluidic chip system, a drive control unit and an optical detection system. The driving control unit is used for driving liquid in the digital microfluidic chip system to flow, and the optical detection system is used for detecting the optical property of the detection point. The ELISA detection system and the detection method can realize ELISA detection reaction. The enzyme-linked immunoassay system has higher integration level and has the advantages of simple preparation method, high automation degree, low cost, high flexibility and the like compared with the prior art. Human errors, labor time cost and reagent consumption during ELISA flow operation can be greatly reduced, and meanwhile, the accuracy and efficiency of detection are improved.)

1. A digital microfluidic chip is characterized by comprising a sample liquid storage area group to be detected, a negative control liquid storage area, a positive control liquid storage area, a substrate reagent liquid storage area, a termination reagent liquid storage area, a washing liquid storage area, a buffer liquid storage area, an antibody liquid storage area, a waste liquid area and a reaction area;

the reaction area comprises at least three detection points which are respectively a detection point group of a sample to be detected, a negative control detection point and a positive control detection point;

the liquid storage group of the sample to be detected is communicated with the detection point group of the sample to be detected;

the negative control liquid storage area is communicated with the negative control detection point;

the positive control liquid storage area is communicated with the positive control detection point;

the substrate reagent liquid storage area, the termination reagent liquid storage area, the washing liquid storage area, the buffer liquid storage area, the antibody liquid storage area and the waste liquid area are respectively communicated with all the detection points.

2. The digital microfluidic chip according to claim 1 wherein said reaction zone comprises first and second oppositely disposed sides and third and fourth oppositely disposed sides;

the sample liquid storage group to be detected, the negative control liquid storage area, the positive control liquid storage area and the waste liquid area are all positioned on the first side of the reaction area;

the washing liquid storage area is positioned on the second side of the reaction area;

the buffer solution storage area and the antibody storage area are positioned on the third side of the reaction area;

the base reagent reservoir region and the termination reagent reservoir region are located on a fourth side of the reaction region.

3. The digital microfluidic chip according to claim 1, wherein said test sample reservoir group comprises a first test sample reservoir region, a second test sample reservoir region, and a third test sample reservoir region;

the to-be-detected sample detection point group comprises a first to-be-detected sample detection point, a second to-be-detected sample detection point and a third to-be-detected sample detection point;

the first sample storage area to be detected is communicated with the first sample detection point to be detected;

the second sample storage area to be detected is communicated with the second sample detection point to be detected;

and the third sample storage area to be detected is communicated with the third sample detection point to be detected.

4. The digital microfluidic chip according to claim 3, wherein said first point to be detected, said second point to be detected, said third point to be detected, said negative control point and said positive control point are arranged in a matrix.

5. A digital microfluidic chip system, comprising an upper cover and the digital microfluidic chip according to any one of claims 1 to 4, wherein the upper cover and the digital microfluidic chip are packaged;

the upper cover is provided with a sample inlet hole of a sample to be detected, a negative control sample inlet hole, a positive control sample inlet hole, a substrate reagent sample inlet hole, a stop reagent sample inlet hole, a washing liquid sample inlet hole, a buffer solution sample inlet hole, an antibody sample inlet hole and an anti-coating detection area;

the sample inlet hole of the sample to be detected and the sample liquid storage area group of the sample to be detected are correspondingly arranged, the negative control sample inlet hole and the negative control liquid storage area are correspondingly arranged, the positive control sample inlet hole and the positive control liquid storage area are correspondingly arranged, the substrate reagent sample inlet hole and the substrate reagent liquid storage area are correspondingly arranged, the termination reagent sample inlet hole and the termination reagent liquid storage area are correspondingly arranged, the washing liquid sample inlet hole and the washing liquid storage area are correspondingly arranged, the buffer liquid sample inlet hole and the buffer liquid storage area are correspondingly arranged, the antibody sample inlet hole and the antibody liquid storage area are correspondingly arranged, and the anti-envelope detection area and the detection point are correspondingly arranged.

6. The digital microfluidic chip system according to claim 5, wherein said upper cover is prepared by the following steps:

shielding the primary anti-coating detection area of the conductive surface of the upper cover by using a shielding piece;

preparing a hydrophobic layer on the conductive surface of the upper cover;

removing the shielding sheet on the primary anti-coating detection area;

capture primary antibody at the primary antibody coated detection zone for modification.

7. The digital microfluidic chip system of claim 6, wherein the primary antibody is captured at the primary antibody coating detection zone and modified by the following method:

modifying the primary antibody coating detection area according to the primary antibody corresponding to each row of target antigen, standing for 12-18 hours at the temperature of 4 ℃, and then washing by using a washing buffer solution;

and adding a sealing buffer solution, standing for 2 hours at room temperature, and cleaning by using a cleaning buffer solution to obtain the upper cover with primary antibody modification.

8. An ELISA detection system, comprising the digital microfluidic chip system as claimed in any one of claims 5 to 7, a driving control unit for driving the liquid flow in the digital microfluidic chip system, and an optical detection system for detecting the optical signal at the detection point.

9. A detection method using the enzyme-linked immunoassay system according to claim 8, comprising the steps of:

respectively adding a sample to be detected, a positive control, a negative control, a substrate reagent, a termination reagent, a buffer solution, an antibody and a washing solution with required volumes to corresponding liquid storage areas of the digital microfluidic chip;

quantitatively distributing the buffer solution to generate buffer solution drops, and transferring the buffer solution drops to all the detection points;

quantitatively distributing positive control to generate a positive control liquid drop, transferring the positive control liquid drop to a positive control detection point, and mixing the positive control liquid drop and the buffer liquid drop to form a positive control reaction liquid drop;

quantitatively distributing negative control to generate negative control liquid drops, transferring the negative control liquid drops to a negative control detection point, and mixing the negative control liquid drops and the buffer liquid drops to form negative control reaction liquid drops;

quantitatively distributing a sample to be detected, generating sample liquid drops to be detected, transferring the sample liquid drops to a sample detection point group to be detected, mixing the sample liquid drops to be detected and the buffer liquid drops to form sample reaction liquid drops to be detected, and standing;

sequentially transferring the positive control reaction liquid drops, the negative control reaction liquid drops and the reaction liquid drops of the sample to be detected to a waste liquid area to form waste liquid;

quantitatively distributing the washing liquid to generate washing liquid drops, washing a path through which the positive control reaction liquid drops pass, a path through which the negative control reaction liquid drops pass, a path through which the sample reaction liquid drops to be detected pass and all detection points, and transferring the used washing liquid drops to a waste liquid area;

quantitatively distributing the antibody, generating antibody liquid drops, transferring the antibody liquid drops to all the detection points, and standing;

quantitatively distributing the washing liquid to generate washing liquid drops, washing all the detection points, and transferring the used washing liquid drops to a waste liquid area;

quantitatively dispensing a substrate reagent to generate substrate reagent droplets, transferring the substrate reagent droplets to all the detection points, and standing;

quantitatively dispensing a stopping reagent, generating stopping reagent droplets, transferring to all detection points, and mixing the stopping reagent droplets with the substrate reagent droplets to form reaction liquid droplets;

and detecting optical signals respectively sent by the detection points, and judging the result.

Technical Field

The invention relates to the technical field of detection equipment, in particular to a digital microfluidic chip, a digital microfluidic chip system, an enzyme-linked immunoassay system and an enzyme-linked immunoassay method.

Background

Enzyme-linked immunosorbent assay (ELISA) is the most widely used Immunoassay method, and at present, 96-well plates are generally used in clinic and laboratories as carriers of the detection reaction, and the primary antibody is embedded by performing operations such as adsorption, sealing, cleaning and the like on the primary antibody. Then adding a sample to be detected or a standard substance containing a specific antigen, incubating for a period of time, washing redundant antigen, adding a second antibody with a specific enzyme label, incubating for a period of time, fully combining the second antibody with the antigen, and washing the redundant second antibody. And finally, adding a substrate to perform a color reaction, and realizing the detection of signals. Therefore, the manual intensive operation required by the conventional ELISA detection is very complicated, the reagent consumption is high, and the high misoperation risk exists.

Therefore, many researchers have introduced microfluidic chips as reaction carriers for ELISA instead of 96-well plates. At present, continuous microfluidic chips with various microchannel structures are used to integrate the whole flow of ELISA, such as the control of target reagents, antibody-antigen binding reaction, and detection of reaction results, on the microfluidic chips.

The traditional ELISA microfluidic chip based on the microfluidic channel structure is mainly manufactured by methods such as soft lithography or injection molding. If the ELISA microfluidic chip for multi-index joint inspection is developed, the chip has a complex space microfluidic channel structure, the processing difficulty is high by applying a soft lithography or injection molding process, and the detection cost of ELISA is greatly improved.

Disclosure of Invention

In view of this, it is necessary to provide a digital microfluidic chip, a digital microfluidic chip system, an enzyme-linked immunoassay system and a method with simple manufacturing method, which can perform multi-index joint detection.

A digital microfluidic chip comprises a sample liquid storage area group to be detected, a negative control liquid storage area, a positive control liquid storage area, a substrate reagent liquid storage area, a termination reagent liquid storage area, a washing liquid storage area, a buffer liquid storage area, an antibody liquid storage area, a waste liquid area and a reaction area;

the reaction area comprises at least three detection points which are respectively a detection point group of a sample to be detected, a negative control detection point and a positive control detection point;

the liquid storage group of the sample to be detected is communicated with the detection point group of the sample to be detected;

the negative control liquid storage area is communicated with the negative control detection point;

the positive control liquid storage area is communicated with the positive control detection point;

the substrate reagent liquid storage area, the termination reagent liquid storage area, the washing liquid storage area, the buffer liquid storage area, the antibody liquid storage area and the waste liquid area are respectively communicated with all the detection points.

In one embodiment, the reaction zone comprises first and second oppositely disposed sides, and third and fourth oppositely disposed sides;

the sample liquid storage group to be detected, the negative control liquid storage area, the positive control liquid storage area and the waste liquid area are all positioned on the first side of the reaction area;

the washing liquid storage area is positioned on the second side of the reaction area;

the buffer solution storage area and the antibody storage area are positioned on the third side of the reaction area;

the base reagent reservoir region and the termination reagent reservoir region are located on a fourth side of the reaction region.

In one embodiment, the test sample reservoir group comprises a first test sample reservoir region, a second test sample reservoir region, and a third test sample reservoir region;

the to-be-detected sample detection point group comprises a first to-be-detected sample detection point, a second to-be-detected sample detection point and a third to-be-detected sample detection point;

the first sample storage area to be detected is communicated with the first sample detection point to be detected;

the second sample storage area to be detected is communicated with the second sample detection point to be detected;

and the third sample storage area to be detected is communicated with the third sample detection point to be detected.

In one embodiment, the first test sample detection point, the second test sample detection point, the third test sample detection point, the negative control detection point and the positive control detection point are arranged in a matrix.

A digital micro-fluidic chip system comprises an upper cover and the digital micro-fluidic chip, wherein the upper cover and the digital micro-fluidic chip are packaged;

the upper cover is provided with a sample inlet hole of a sample to be detected, a negative control sample inlet hole, a positive control sample inlet hole, a substrate reagent sample inlet hole, a stop reagent sample inlet hole, a washing liquid sample inlet hole, a buffer solution sample inlet hole, an antibody sample inlet hole and an anti-coating detection area;

the sample inlet hole of the sample to be detected and the sample liquid storage area group of the sample to be detected are correspondingly arranged, the negative control sample inlet hole and the negative control liquid storage area are correspondingly arranged, the positive control sample inlet hole and the positive control liquid storage area are correspondingly arranged, the substrate reagent sample inlet hole and the substrate reagent liquid storage area are correspondingly arranged, the termination reagent sample inlet hole and the termination reagent liquid storage area are correspondingly arranged, the washing liquid sample inlet hole and the washing liquid storage area are correspondingly arranged, the buffer liquid sample inlet hole and the buffer liquid storage area are correspondingly arranged, the antibody sample inlet hole and the antibody liquid storage area are correspondingly arranged, and the anti-envelope detection area and the detection point are correspondingly arranged.

In one embodiment, the method of making the cover is as follows:

shielding the primary anti-coating detection area of the conductive surface of the upper cover by using a shielding piece;

preparing a hydrophobic layer on the conductive surface of the upper cover;

removing the shielding sheet on the primary anti-coating detection area;

capture primary antibody at the primary antibody coated detection zone for modification.

In one embodiment, the method of capturing primary antibodies at the primary antibody coated detection zone for modification is as follows:

modifying the primary antibody coating detection area according to the primary antibody corresponding to each row of target antigen, standing for 12-18 hours at the temperature of 4 ℃, and then washing by using a washing buffer solution;

and adding a sealing buffer solution, standing for 2 hours at room temperature, and cleaning by using a cleaning buffer solution to obtain the upper cover with primary antibody modification.

An enzyme-linked immunoassay system comprises the digital microfluidic chip system, a drive control unit and an optical detection system, wherein the drive control unit is used for driving liquid in the digital microfluidic chip system to flow, and the optical detection system is used for detecting optical signals of detection points.

A detection method adopting the enzyme-linked immunoassay system comprises the following steps:

respectively adding a sample to be detected, a positive control, a negative control, a substrate reagent, a termination reagent, a buffer solution, an antibody and a washing solution with required volumes to corresponding liquid storage areas of the digital microfluidic chip;

quantitatively distributing the buffer solution to generate buffer solution drops, and transferring the buffer solution drops to all the detection points;

quantitatively distributing positive control to generate a positive control liquid drop, transferring the positive control liquid drop to a positive control detection point, and mixing the positive control liquid drop and the buffer liquid drop to form a positive control reaction liquid drop;

quantitatively distributing negative control to generate negative control liquid drops, transferring the negative control liquid drops to a negative control detection point, and mixing the negative control liquid drops and the buffer liquid drops to form negative control reaction liquid drops;

quantitatively distributing a sample to be detected, generating sample liquid drops to be detected, transferring the sample liquid drops to a sample detection point group to be detected, mixing the sample liquid drops to be detected and the buffer liquid drops to form sample reaction liquid drops to be detected, and standing;

sequentially transferring the positive control reaction liquid drops, the negative control reaction liquid drops and the reaction liquid drops of the sample to be detected to a waste liquid area to form waste liquid;

quantitatively distributing the washing liquid to generate washing liquid drops, washing a path through which the positive control reaction liquid drops pass, a path through which the negative control reaction liquid drops pass, a path through which the sample reaction liquid drops to be detected pass and all detection points, and transferring the used washing liquid drops to a waste liquid area;

quantitatively distributing the antibody, generating antibody liquid drops, transferring the antibody liquid drops to all the detection points, and standing;

quantitatively distributing the washing liquid to generate washing liquid drops, washing all the detection points, and transferring the used washing liquid drops to a waste liquid area;

quantitatively dispensing a substrate reagent to generate substrate reagent droplets, transferring the substrate reagent droplets to all the detection points, and standing;

quantitatively dispensing a stopping reagent, generating stopping reagent droplets, transferring to all detection points, and mixing the stopping reagent droplets with the substrate reagent droplets to form reaction liquid droplets;

and detecting optical signals respectively sent by the detection points, and judging the result.

The digital microfluidic chip, the digital microfluidic chip system, the enzyme-linked immunoassay system and the enzyme-linked immunoassay method can realize ELISA detection reaction. The enzyme-linked immunoassay system comprises a digital microfluidic chip utilizing an electrowetting principle, a drive control system and a photoelectric detection system, has higher integration level, and has the advantages of simple preparation method, high automation degree, low cost, high flexibility and the like compared with the prior art.

Drawings

Fig. 1 is a schematic structural diagram of a digital microfluidic chip according to an embodiment;

FIG. 2 is a schematic flow chart illustrating the preparation of the upper lid according to one embodiment;

FIG. 3 is a schematic diagram of an embodiment of a cover capture primary antibody;

FIG. 4 is a schematic diagram of an embodiment of a cover and a digital microfluidic chip package;

FIG. 5 is a schematic cross-sectional view of a digital microfluidic chip according to an embodiment;

FIG. 6 is a schematic diagram of a digital microfluidic chip incorporating various reagents according to an embodiment;

FIG. 7 is a schematic diagram of a digital microfluidic chip for quantitatively distributing buffer according to an embodiment;

FIG. 8 is a schematic diagram of a digital microfluidic chip for quantitatively distributing a positive control and a negative control according to an embodiment;

FIG. 9 is a schematic diagram of a digital microfluidic chip for quantitatively distributing a sample to be detected according to an embodiment;

FIG. 10 is a schematic diagram of a digital microfluidic chip for quantitatively dispensing a washing solution according to an embodiment;

FIG. 11 is a diagram illustrating a digital microfluidic chip for quantitatively distributing antibodies according to an embodiment;

FIG. 12 is a schematic diagram of a digital microfluidic chip for quantitatively dispensing a washing solution according to an embodiment;

FIG. 13 is a schematic diagram of a digital microfluidic chip for quantitatively dispensing a substrate reagent according to an embodiment;

FIG. 14 is a schematic diagram of a digital microfluidic chip for quantitatively dispensing a stop reagent according to an embodiment;

fig. 15 is a schematic illustration of the detection of an optical signal emitted by a detection spot using optical detection techniques.

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 with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

In the description of the present invention, it should be understood that the relation indicating the orientation or position such as "above" is based on the orientation or position relation shown in the drawings, or the orientation or position relation which the product of the present invention is usually put into use, or the orientation or position relation which is usually understood by those skilled in the art, and is only for convenience of describing the present invention and simplifying the description, but does not indicate or imply that the device or element to be referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, cannot be construed as limiting the present invention.

The following detailed description of embodiments of the invention refers to the accompanying drawings.

As shown in fig. 1, the digital microfluidic chip of an embodiment includes a sample reservoir group 21 to be tested, a negative control reservoir region 22A, a positive control reservoir region 22B, a substrate reagent reservoir region 23A, a stop reagent reservoir region 23B, a wash solution reservoir region 24, a buffer solution reservoir region 23C, an antibody reservoir region 23D, a waste solution region 25, and a reaction region 26.

The reaction area 26 comprises at least three detection points 27, and the detection points 27 are a detection point group of a sample to be detected, a negative control detection point 26F and a positive control detection point 26D respectively.

The liquid storage group 21 of the sample to be detected is communicated with the detection point group of the sample to be detected.

The negative control reservoir zone 22A is in communication with the negative control test spot 26F.

The positive control reservoir zone 22B is in communication with the positive control checkpoint 26D.

The substrate reagent storage area 23A, the termination reagent storage area 23B, the washing solution storage area 24, the buffer solution storage area 23C, the antibody storage area 23D, and the waste liquid area 25 are respectively communicated with all the detection points 27.

The digital microfluidic chip can quantitatively distribute the sample to be detected, the negative control, the positive control, the substrate reagent, the termination reagent, the buffer solution and the antibody, and realize the simultaneous detection of a plurality of samples. Through setting up washing liquid storage area, can quantitative distribution washing liquid carry out many times self-cleaning to digital micro-fluidic chip to can realize automatic many indexes joint inspection.

The digital microfluidic chip is suitable for detecting 5 indexes of 3 samples.

The antibody liquid storage area 23D of the digital microfluidic chip is used for storing the secondary antibody.

In one embodiment, the reaction zone 26 is rectangular in shape. The reaction zone 26 includes first and second oppositely disposed sides and third and fourth oppositely disposed sides.

The test sample reservoir block 21, the negative control reservoir block 22A, the positive control reservoir block 22B and the waste block 25 are all located on a first side of the reaction zone 26.

A wash liquid reservoir 24 is located on a second side of the reaction zone 26.

The buffer reservoir region 23C and the antibody reservoir region 23D are located on the third side of the reaction region 26.

The base reagent reservoir region 23A and the termination reagent reservoir region 23B are located on the fourth side of the reaction region 26.

The arrangement mode of the positions of the sample liquid storage area group 21 to be detected, the negative control liquid storage area 22A, the positive control liquid storage area 22B, the substrate reagent liquid storage area 23A, the termination reagent liquid storage area 23B, the washing solution liquid storage area 24, the buffer liquid storage area 23C, the antibody liquid storage area 23D and the waste liquid area 25 can enable all areas of the digital microfluidic chip to be compactly arranged and reduce the volume. It is to be understood that the shape of the reaction region 26 is not limited to being rectangular, and may be any electrode shape or combination of electrode shapes.

It can be understood that the positions of the sample reservoir group 21, the negative control reservoir region 22A, the positive control reservoir region 22B, the substrate reagent reservoir region 23A, the stop reagent reservoir region 23B, the washing solution reservoir region 24, the buffer solution reservoir region 23C, the antibody reservoir region 23D, and the waste liquid region 25 can also be adjusted according to actual needs.

In one embodiment, the test sample reservoir group 21 includes a first test sample reservoir region 21A, a second test sample reservoir region 21B, and a third test sample reservoir region 21C.

The set of point samples includes a first point sample 26A, a second point sample 26B and a third point sample 26C.

The first sample detection point 26A is connected to the first sample reservoir area 21A.

The second sample storage area 21B to be tested is communicated with the second sample detection point 26B to be tested.

The third sample storage area 21C to be tested is communicated with the third sample detection point 26C to be tested.

It can be understood that the number of the sample reservoir groups 21 to be tested can be set according to actual needs, and is not limited to three sample reservoir groups to be tested. When the number of the liquid storage areas of the sample to be detected is increased or decreased, the number of the detection points of the sample to be detected is correspondingly increased or decreased.

In one embodiment, the first test sample detection spot 26A, the second test sample detection spot 26B, the third test sample detection spot 26C, the negative control detection spot 26F, and the positive control detection spot 26D are arranged in a 5 × 5 matrix. Namely, the first to-be-detected sample detection point 26A, the second to-be-detected sample detection point 26B, the third to-be-detected sample detection point 26C, the negative control detection point 26F and the positive control detection point 26D each have 5 detection points, and the 5 detection points are arranged in a row.

Specifically, the first to-be-detected sample detection point 26A in the first row is communicated with the first to-be-detected sample storage area 21A, the second to-be-detected sample detection point 26B in the second row is communicated with the second to-be-detected sample storage area 21B, the third to-be-detected sample detection point 26C in the third row is communicated with the third to-be-detected sample storage area 21C, the negative control detection point 26F in the fourth row is communicated with the negative control storage area 22A, and the positive control detection point 26D in the fifth row is communicated with the positive control storage area 22B.

According to the digital microfluidic chip, cross contamination can be completely avoided through path design and automatic cleaning between the sample and the reaction.

It is understood that the first test sample detection point 26A, the second test sample detection point 26B, the third test sample detection point 26C, the negative control detection point 26F and the positive control detection point 26D may also be arranged in a matrix of 5 × n, where n is not limited to 5, and n may be other positive integers. That is, the number of the detection points of the first to-be-detected sample detection point 26A, the second to-be-detected sample detection point 26B, the third to-be-detected sample detection point 26C, the negative control detection point 26F, and the positive control detection point 26D is not limited to 5.

The digital microfluidic chip is mainly used for accurately distributing and controlling ELISA reaction reagents and completing on-chip experiments of ELISA full-reaction processes.

In addition, referring to fig. 4, a digital microfluidic chip system according to an embodiment is further provided, which includes the digital microfluidic chip 16 with the upper cover 17 as above. The upper cover 17 and the digital microfluidic chip 16 are packaged and arranged.

Referring to fig. 2, the upper cover is provided with a sample inlet, a negative control sample inlet, a positive control sample inlet, a substrate reagent sample inlet, a stop reagent sample inlet, a washing solution sample inlet, a buffer solution sample inlet, an antibody sample inlet, and an anti-coating detection zone 6. The wells are not individually numbered and reference may be made to reference numeral 10 in fig. 2.

The sample inlet hole of the sample to be detected is arranged corresponding to the sample liquid storage group 21 to be detected, the negative control sample inlet hole is arranged corresponding to the negative control liquid storage region 22A, the positive control sample inlet hole is arranged corresponding to the positive control liquid storage region 22B, the substrate reagent sample inlet hole is arranged corresponding to the substrate reagent liquid storage region 23A, the termination reagent sample inlet hole is arranged corresponding to the termination reagent liquid storage region 23B, the washing liquid sample inlet hole is arranged corresponding to the washing liquid storage region 24, the buffer liquid sample inlet hole is arranged corresponding to the buffer liquid storage region 23C, the antibody sample inlet hole is arranged corresponding to the antibody liquid storage region 23D, and the anti-coating detection region 6 is arranged corresponding to the detection point 27. The upper cover 17 and the digital microfluidic chip 16 are packaged to realize the pre-embedding of primary impedance.

The digital microfluidic chip system can expand the detection flux by adjusting the design of the patterned upper cover and the digital microfluidic chip.

Referring to fig. 2, in one embodiment, the upper cover is prepared as follows:

s1, the primary anti-coating detection area 6 of the conductive surface of the upper cover is shielded by the shielding sheet 11.

And S2, preparing a hydrophobic layer 5 on the conductive surface of the upper cover.

Specifically, the hydrophobic layer can be prepared by a suspension coating method. It will be appreciated that the hydrophobic layer may also be prepared by other methods.

S3, removing the shielding sheet 11 on the primary anti-coating detection zone 6.

S4, capturing the primary antibody at the primary antibody coating detection zone 6 for modification.

Through carrying out patterning to the upper cover, carry out the cover of hydrophobic layer again after sheltering from one anti parcel detection zone 6, then remove to shelter from, can realize carrying out one anti embedding to the detection area and handle.

In one embodiment, the shielding sheet 11 may be a single-sided adhesive.

In one embodiment, the method of capturing primary antibodies at the primary antibody coated detection zone 6 for modification is as follows:

s42, modifying the primary antibody coating detection zone 6 according to the primary antibody corresponding to each target antigen, standing for 12-18 hours in the environment of 4 ℃, and then washing by using a washing buffer solution.

And S44, adding a sealing buffer solution, standing for 2 hours at room temperature, and cleaning by using a cleaning buffer solution to obtain the upper cover with the primary antibody modification.

Specifically, referring to FIG. 3, according to the sequence from left to right, primary antibodies 12 corresponding to each row of a target antigen are listed₁、12₂、12₃、12₄And 12₅Modifying, standing for 12-18 hours at 4 ℃, then washing twice by using a washing buffer solution, then adding a sealing buffer solution, standing for 2 hours at room temperature, and finally washing for 2 times by using the washing buffer solution to obtain the digital microfluidic chip upper cover with primary antibody modification. The detection area corresponds to one sample to be detected 13 in each of the first three rows from top to bottom₁、13₂And 13₃The fourth row is a positive control group 14, and the fifth row is a negative control group 15.

The washing buffer may be Phosphate Buffered Saline (PBS) containing 0.05% Tween-20.

The blocking buffer may be a PBS solution containing 5% sucrose and 2% bovine serum albumin.

The cross-sectional view of the digital microfluidic chip system is shown in fig. 5. The digital microfluidic chip system comprises a substrate 1, an electrode array 2, a dielectric layer 3, a hydrophobic layer 4, an upper cover hydrophobic layer 5, an anti-coating detection area 6, an upper cover 7, a gap control structure 8 and a liquid moving space 9. The digital microfluidic chip comprises a substrate 1, an electrode array 2, a dielectric layer 3 and a hydrophobic layer 4.

As shown in fig. 4, the digital microfluidic chip 16 is encapsulated with a patterned upper cover 17 in the manner shown. The projection position of the primary anti-coating detection area 6 of the upper cover 7 in the vertical direction on the digital microfluidic chip 16 is the detection point (as shown in fig. 1).

In addition, an enzyme-linked immunoassay system of an embodiment is also provided, which comprises a digital microfluidic chip system, a drive control unit and an optical detection system. The driving control unit is used for driving liquid in the digital microfluidic chip system to flow, and the optical detection system is used for detecting an optical signal of a detection point.

According to the ELISA detection system, the relevant paths are loaded in the on-chip ELISA detection process, the relevant reagents and samples are loaded in the digital microfluidic chip system, the path program is operated, the automatic ELISA detection can be completed, the optical detection system can be used for collecting optical signals of detection points, and accordingly an ELISA detection result is obtained.

In addition, the detection method adopting the enzyme-linked immunoassay system in the embodiment comprises the following steps:

s10, please refer to fig. 6, the sample 28 to be tested, the positive control 29, the negative control 30, the substrate reagent 31, the stop reagent 32, the buffer 33, the antibody 34 and the washing solution 35 are added to the corresponding liquid storage areas of the digital microfluidic chip shown in fig. 1.

S20, buffer 33 is dispensed quantitatively to generate buffer droplets 36, and the resulting droplets are transferred to all detection spots 27.

Specifically, referring to FIG. 7, 25 drops 36 of 4 microliters are generated by dispensing buffer 33 and transferred to 25 detection spots 27.

And S30, quantitatively distributing the positive control 29 to generate a positive control droplet, transferring the positive control droplet to a positive control detection point, and mixing the positive control droplet with the buffer droplet 36 to form a positive control reaction liquid droplet 37B.

And S40, quantitatively distributing the negative control 30 to generate a negative control droplet, transferring the negative control droplet to a negative control detection point, and mixing the negative control droplet with the buffer droplet 36 to form a negative control reaction liquid droplet 37A.

Specifically, referring to FIG. 8, positive control 29 and negative control 30 were dosed, each resulting in 5 drops of 4. mu.l in volume and transferred to the detection site to be mixed with buffer drop 36 to form reaction solution drops 37B and 37A of 8. mu.l in volume.

And S50, quantitatively distributing the sample to be detected 28, generating sample liquid drops to be detected, transferring the sample liquid drops to the sample detection point group to be detected, mixing the sample liquid drops to be detected and the buffer liquid drops 36 to form sample reaction liquid drops to be detected 37C, and standing.

Specifically, referring to fig. 9, the sample 28 to be tested is quantitatively dispensed, 5 drops each having a volume of 4 μ l are generated and transferred to the detection point to be mixed with the buffer drop 36 to form a reaction solution drop 37C having a volume of 8 μ l, and the reaction solution is allowed to stand for 1 hour.

S60, transferring the positive control reaction liquid drop 37B, the negative control reaction liquid drop 37A and the sample reaction liquid drop 37C to be detected to the waste liquid area 25 in sequence to form waste liquid 38.

S70, quantitatively distributing the washing liquid to generate washing liquid drops 39, washing the path through which the positive control reaction liquid drops 37B pass, the path through which the negative control reaction liquid drops 37A pass, the path through which the sample reaction liquid drops 37C to be detected pass, and all detection points, and transferring the used washing liquid drops 39 to the waste liquid area 25.

Specifically, referring to fig. 10, the washing solution is quantitatively dispensed to generate a plurality of washing solution droplets 39 each having a volume of 4 μ l, and the used washing solution droplets 39 are transferred to the waste solution area 25 by passing the washing detection point and the reaction solution droplets 37A, 37B, and 37C 4 times.

S80, quantitatively distributing the antibody 34 to generate antibody droplets 40, transferring the antibody droplets to all detection spots, and standing.

Specifically, referring to FIG. 11, antibody 34 was dosed to produce 25 antibody droplets 40 with a volume of 8 microliters, transferred to a test site, and allowed to stand for 1 hour.

In S80, antibody 34 is a secondary antibody.

S90, dispensing the washing liquid in a fixed amount to generate washing liquid droplets 39, washing all the detection points, and transferring the used washing liquid droplets 39 to the waste liquid area 25.

Specifically, referring to fig. 12, the washing solution is quantitatively dispensed to generate a plurality of washing solution droplets 39 with a volume of 4 μ l, the detection point is washed 4 times, and the used washing solution droplets 39 are transferred to the waste solution area 25.

S110, quantitatively distributing the substrate reagent 31 to generate substrate reagent droplets 41, transferring the droplets to all detection points, and standing.

Specifically, referring to FIG. 13, the substrate reagent 31 was dosed to produce 25 drops 41 of 4 microliter volume of substrate reagent and transferred to the test site, and left to stand for 15 minutes.

S120, quantitatively distributing the stopping reagent 32 to generate stopping reagent drops, transferring the stopping reagent drops to all detection points, and mixing the stopping reagent drops with the substrate reagent drops 41 to form reaction liquid drops 42.

Specifically, referring to FIG. 14, the stop reagent 32 is dosed to produce 25 stop reagent droplets having a volume of 4. mu.l and transferred to the detection site to be mixed with the base reagent droplet 41 to form a reaction solution droplet 42 having a volume of 8. mu.l.

S130, please refer to fig. 15, detecting the optical signals 43 respectively emitted from the detecting points, and determining the result.

In one embodiment, the buffer droplet, the positive control droplet, the negative control droplet, the sample droplet to be tested, the wash droplet, the base reagent droplet, and the stop reagent droplet are all the same volume V1. The volume of the antibody droplet was V2, where V2 was twice as large as V1.

The detection system of the enzyme-linked immunoassay system and the detection method adopting the detection system can greatly reduce human errors, labor time cost and reagent consumption during ELISA process operation, and improve the accuracy and efficiency of detection. The pre-embedding of the antigen reagent is realized through the patterned upper cover, and the patterned upper cover and the digital microfluidic chip are packaged, so that the whole process is automatically completed in the closed digital microfluidic chip. The reaction sample containing the negative control and the positive control can be used as a reference standard to ensure the accuracy of the detection result. Cross-contamination between the sample and the reaction can be completely avoided by the path design and washing solution washing. The method can be applied to various optical detection means to realize result judgment. Based on the digital microfluidic chip, more groups of one-to-many, many-to-one and many-to-many immunodetection reactions can be realized through reasonable amplification design of chip electrodes and corresponding detection points, and the method is not limited by the structure and layout design of a 96-well plate.

The detection method of the enzyme-linked immunoassay system can be compatible with the detection process of the conventional universal ELISA detection kit on the market.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.