CN107656083B - Self-injection immunoassay paper chip and preparation method thereof - Google Patents

Abstract

Self-injection immunoassay paper chip and preparation method thereof, wherein the self-injection immunoassay paper chip is used to detect antigen concentration, and from top to bottom along the stacking direction includes: a microfluidic layer having microfluidics connected through communication channels Control channel and sampling hole, the microfluidic channel is used for self-sampling, and the sampling hole is used to introduce the tissue to be tested obtained by sampling into the detection area; the auxiliary electrode layer; the detection layer, and the working electrode layer, whose surface is modified There are electroactive composite substances, and the surface of the electroactive composite substances is used to immobilize antibodies; the auxiliary electrode layer, the detection layer and the working electrode layer constitute the detection area, including the electrochemical detection circuit, which is electrochemically based on the specific binding principle of antigen-antibody and the properties of the electroactive complex substance, the concentration of the antigen in the liquid to be tested can be obtained by detection. Due to the combination of microfluidic technology and the advantages of paper, automatic sample injection is realized through the set microfluidic channel and injection hole, which is easy to operate and does not require professional operators.

Description

Self-injection immunoassay paper chip and preparation method thereof

technical field

The invention belongs to the field of biological detection, and more particularly relates to a self-injection immune detection paper chip and a preparation method thereof.

Background technique

Antigens are substances that can induce an immune response. Most antigens are specific and can specifically bind to antibodies. There are many types of antigens, including proteins and polypeptides. The content of certain antigens, such as disease markers, is of great significance to human health. The detection of these substances can be used for mechanism research, early diagnosis, early intervention and treatment of some major diseases.

Currently, a variety of immunoassays are available for highly sensitive detection of antigens, including radioimmunoassay, enzyme-linked immunoassay (ELISA), electrochemiluminescence immunoassay, etc. However, the above methods all require pretreatment of the detected antigens or antibodies, so they have certain limitations in point-of-care detection.

SUMMARY OF THE INVENTION

Based on the above problems, the main purpose of the present invention is to provide a trace electrochemical immunoassay paper chip and a preparation method thereof, which are used to solve at least one of the above technical problems.

In order to achieve the above object, as an aspect of the present invention, a self-injection immunoassay paper chip is proposed for detecting the concentration of antigens, comprising from top to bottom along the stacking direction: a microfluidic layer, having Microfluidic channel and sampling hole, the microfluidic channel is used for self-sampling, and the sampling hole is used to introduce the tissue fluid to be tested obtained by sampling into the detection area; the auxiliary electrode layer; the detection layer, and the working electrode layer, the surface of which is Modified with electroactive composite substances, the surface of the electroactive composite substances is used to immobilize antibodies; the auxiliary electrode layer, the detection layer and the working electrode layer constitute the detection area, including the electrochemical detection circuit, which is electrochemically based on the specific binding of antigen-antibody Based on the principle and the properties of the electroactive composite substance, the concentration of the antigen in the liquid to be tested can be obtained by detection.

In some embodiments of the present invention, the above-mentioned electroactive composite materials include carbon nanomaterials, electroactive materials and nano-gold composite materials; carbon nanomaterials include multi-wall carbon nanotubes or aminated graphene; electroactive materials include thionine , Prussian blue or potassium ferricyanide; preferably, in the composite material, the ratio between the carbon nanomaterial, the electroactive substance and the nanogold is 1:1:5 to 1:2:10.

In some embodiments of the present invention, the above-mentioned working electrode layer includes at least two circular working electrodes arranged in the circumferential direction; the auxiliary electrode layer includes a first through hole arranged opposite to the sample injection hole, and a first through hole arranged opposite to the circular working electrode. At least two second through holes arranged in pairs, and at least two pairs of counter electrodes and reference electrodes arranged in combination; the combinations of the counter electrodes and the reference electrodes are respectively matched with part of the edges of the at least two second through holes; The working electrode, the counter electrode and the reference electrode are all connected with electrode leads to connect external devices.

In some embodiments of the present invention, the detection layer includes a third through hole, the third through hole is facing the first through hole, and a symmetrical communication hole extends along the length direction of the detection layer, and the communication hole is directly opposite to the first through hole. For the circular working electrode, a detection area is formed.

In some embodiments of the present invention, the microfluidic layer and the detection layer both have at least two groups of edge through holes arranged in the circumferential direction, and the at least two groups of edge through holes are facing the working electrode, the counter electrode and the reference electrode. electrode lead.

In some embodiments of the present invention, the upper surface of the microfluidic layer is provided with a first filter layer; the first filter layer has first filter holes disposed opposite to the microfluidic channel for When the channel is self-sampling, the tissue fluid to be tested is filtered; preferably, the porosity of the first filter hole is less than 3 μm.

In some embodiments of the present invention, the lower surface of the above-mentioned microfluidic layer is provided with a second filter layer, which has a second filter hole disposed opposite to the injection hole, and is used to further filter the tissue fluid to be tested obtained by sampling .

In some embodiments of the present invention, the width of the above-mentioned communication channel connecting the microfluidic channel and the sampling hole is inversely proportional to the sampling rate.

In some embodiments of the present invention, the size of the self-injecting immunoassay paper chip is 10.5 mm×35 mm.

In some embodiments of the present invention, the areas where the microfluidic channel, the communication channel and the injection hole of the microfluidic layer are located are hydrophobic, and other areas are hydrophilic.

In order to achieve the above purpose, the present invention also proposes a method for preparing a self-injection immunoassay paper chip, which is used to prepare the above-mentioned self-injection immunoassay paper chip, comprising the following steps: adding a pre-set microfluidic channel After printing on the surface of the microfluidic layer by a wax spray printer, the microfluidic layer is heated to make the area where the microfluidic channel of the microfluidic layer is located has hydrophobicity; Print the working electrode, the counter electrode and the reference electrode; modify the electroactive composite material on the surface of the working electrode; integrate the microfluidic layer, the auxiliary electrode layer, the detection layer and the working electrode layer; When the antibody is added dropwise to the surface of the working electrode modified with the electroactive composite substance.

In some embodiments of the present invention, the above-mentioned electroactive composite material is prepared by the following steps: Step 1. Dissolving the multi-wall carbon nanotubes/aminated graphene into an aqueous solution of polyethyleneimine to make the multi-wall carbon nanotubes / Non-covalent functionalization of aminated graphene; Step 2, adding an electroactive substance to the above solution, stirring vigorously for a certain period of time, so as to make the non-covalent functionalized multi-walled carbon nanotubes/aminated graphene and the electroactive substance Combining; step 3, mixing the solution obtained in step 2 with the gold nanoparticle solution, adding the chitosan solution after vigorous stirring, and ultrasonically mixing to obtain an electroactive composite substance.

The self-injection immune detection paper chip and the preparation method thereof proposed by the present invention have the following beneficial effects:

1. Due to the combination of microfluidic technology and the advantages of paper, the capillary action of the liquid on the paper is used to achieve automatic sample injection through the set microfluidic channel and injection hole, which is easy to operate and does not require professional operators. ; And the surface of the working electrode layer is modified with an electroactive composite substance, which can fix the antibody, so that when the antigen is combined with the antibody immobilized on the surface of the electroactive composite substance, the activity of the electroactive composite substance is affected, thereby affecting the electroactive composite substance and work. The current generated by the interaction of the electrode surface can indirectly measure the concentration of the antigen, so there is no need to pre-process the sample to be detected. Short, enabling rapid trace detection;

2. A first filter layer with first filter holes is provided. Therefore, by selecting filter holes with different pores, the detection of serum samples and whole blood samples can be realized; and the sample amount required for the detection process is small, about 10 μm ;

3. The microfluidic channel is printed on the surface of the microfluidic layer by a wax spray printer and then heated, so that the area where the microfluidic channel is located is hydrophobic. Combined with the capillary effect of the paper chip and the liquid, it can quickly sample and be very efficient. Good guidance of the flow direction of the liquid to achieve fast and efficient detection;

4. There are at least two combinations of working electrodes, reference electrodes, and counter electrodes. Therefore, different antibodies are dripped on the surface of at least two combinations of working electrodes, so the concentration of at least two antigens can be detected by one sampling. , thereby further reducing the amount of sample required in the detection process;

5. The production cost is low, and it can be used for one-time portable use, which is convenient for detection.

Description of drawings

1 is a schematic diagram of a layered structure of a self-injecting immunoassay paper chip proposed in an embodiment of the present invention;

Fig. 2 is the preparation method of the electroactive composite substance in the preparation method of the self-injection immunoassay paper chip proposed by an embodiment of the present invention;

FIG. 3 is a schematic diagram of the use and detection of the self-injection immunodetection paper chip according to an embodiment of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

The label-free electrochemical rapid detection technology can perform rapid and trace detection of the analyzed antigens (proteins, peptides) by directly measuring the current changes formed when the antigen-antibody binds. It has important scientific significance and application prospects.

In recent years, nanomaterials have gradually entered the forefront of analytical chemistry and played a key role in a variety of analytical methods. There are more and more researches on the application of nanomaterials in electroanalytical chemistry and electrochemical sensors. Trends in the conversion of chemical electrodes to nanomaterial-modified electrodes. This is due to the fact that nanomaterials have many unique properties that are different from traditional materials. Compared with conventional electrodes, nanomaterial-modified electrodes have larger effective surface area, faster material transport, and higher catalytic activity. The ability of the electrode surface to modulate the local environment. Among them, carbon nanotubes and graphene are widely used in the modification of biosensors due to their excellent electrical conductivity and certain electrocatalytic functions.

Microfluidic paper chips are referred to as paper chips, which combine the advantages of microfluidic technology and paper. Compared with traditional electrodes, the advantages of paper chips are: 1. The paper is rich in sources and can be mass-produced; 2. No external pump is required, the main component of the paper is cellulose, and the fluid flows on the paper through capillary action, which can realize automatic feeding. 3. The sample consumption is lower, the biocompatibility is good, and the properties of the paper can be changed by chemical modification; 4. One-time portable analysis, easy to operate, and does not even require professional operators. Paper chips provide a broad platform for portable detection and on-site real-time monitoring required in clinical diagnosis, environmental monitoring, and food safety analysis. For underdeveloped areas where medical staff and medical equipment are in short supply, paper chips are low-cost, rapid detection point-of-care diagnosis. powerful tool.

The invention intends to meet the needs of rapid portable detection of antigens (proteins, polypeptides), based on the principle of label-free electrochemical rapid detection technology and antigen-antibody specific binding, by synthesizing nanocomposite materials, to prepare a new type of automatic feeding type trace amount Electrochemical immunoassay paper chip, and use tumor markers (carcinoembryonic antigen CEA, neuron-specific enolase NSE, etc.), neurotrophic factors (brain-derived neurotrophic factor BDNF, etc.) and hormones as entry points to develop traces Quantitative, rapid detection test.

Specifically, the present invention proposes a self-injection immunodetection paper chip for detecting antigen concentration, comprising from top to bottom along the stacking direction: a microfluidic layer having a microfluidic channel connected by a communication channel and an inlet The sample hole, the microfluidic channel is used for self-sampling, the sample injection hole is used to introduce the tissue fluid to be tested obtained by sampling into the detection area; the auxiliary electrode layer; The surface of the electroactive composite material is used to immobilize the antibody; the auxiliary electrode layer, the detection layer and the working electrode layer constitute the detection area, including the electrochemical detection circuit. characteristic, and the concentration of the antigen in the liquid to be tested can be obtained by detection.

Therefore, because the present invention combines the advantages of microfluidic technology and paper, utilizes the capillary action of liquid on paper, and realizes automatic sample injection through the set microfluidic channel and injection hole, which is easy to operate and does not require professional Operators; and the surface of the working electrode layer is modified with electroactive composite substances, which can immobilize antibodies, so that when the antigen binds to the antibodies immobilized on the surface of the electroactive composite substances, the activity of the electroactive composite substances is affected, thereby affecting the electroactive composite substances. The current generated by the interaction with the surface of the working electrode can indirectly measure the concentration of the antigen, so there is no need to pre-process the sample to be detected. It takes less time and can realize rapid trace detection.

In some embodiments of the present invention, the above-mentioned electroactive composite materials include composite materials of carbon nanomaterials, electroactive materials and nano-gold; the carbon nanomaterials can be multi-walled carbon nanotubes or aminated graphene, but not It is limited, any carbon nanomaterials that can form the composite material required by the present invention can be used; electroactive substances include thionine, Prussian blue or potassium ferricyanide, but are not limited to this, and when the antigen is combined with the antibody, Any electroactive species that can affect its activity and thus the magnitude of the current generated by its interaction with the surface of the working electrode can be used.

In some embodiments of the present invention, in the above-mentioned composite material, the ratio between the carbon nanomaterial, the electroactive substance and the nano-gold is 1:1:5-1:2:10.

In some embodiments of the present invention, the above-mentioned working electrode layer includes at least two circular working electrodes arranged in the circumferential direction; the auxiliary electrode layer includes a first through hole arranged opposite to the sample injection hole, and a first through hole arranged opposite to the circular working electrode. At least two second through holes arranged in pairs, and at least two pairs of counter electrodes and reference electrodes arranged in combination; the combinations of the counter electrodes and the reference electrodes are respectively matched with part of the edges of the at least two second through holes; The working electrode, the counter electrode and the reference electrode are all connected with electrode leads to connect external devices. The working electrode, the counter electrode and the reference electrode form a three-electrode system. The three electrodes are all connected to the electrochemical workstation through the electrode leads to form an electrochemical detection circuit. The properties of the electroactive complex substance are detected to obtain the concentration of the antigen in the liquid to be tested. And because there are at least two groups of working electrodes, reference electrodes, and counter electrodes, different antibodies are dropped on the surfaces of at least two groups of working electrodes, so the concentration of at least two antigens can be detected by one sampling. , thereby further reducing the amount of sample required during the detection process.

In some embodiments of the present invention, the detection layer includes a third through hole, the third through hole is facing the first through hole, and a symmetrical communication hole extends along the length direction of the detection layer, and the communication hole is directly opposite to the first through hole. For the circular working electrode, a detection area is formed.

In some embodiments of the present invention, the microfluidic layer and the detection layer both have at least two groups of edge through holes arranged in the circumferential direction, and the at least two groups of edge through holes are facing the working electrode, the counter electrode and the reference electrode. Electrode leads to connect to the electrochemical workstation.

In some embodiments of the present invention, the upper surface of the microfluidic layer is provided with a first filter layer; the first filter layer has first filter holes disposed opposite to the microfluidic channel for When the channel is self-sampling, the liquid to be tested is filtered; preferably, the porosity of the first filter hole is less than 3 μm. Specifically, the pore diameter of the first filter hole can be specifically set. Therefore, the use of small filter pores can effectively filter cells and molecules with large diameters such as white blood cells and red blood cells in the whole blood, and can realize the detection of whole blood samples. .

In some embodiments of the present invention, the lower surface of the above-mentioned microfluidic layer is provided with a second filter layer, which has a second filter hole disposed opposite to the injection hole, and is used to further filter the tissue fluid to be tested obtained by sampling .

In some embodiments of the present invention, the width of the above-mentioned communication channel connecting the microfluidic channel and the sampling hole is inversely proportional to the sampling rate. Therefore, the communication channel can be set to a larger width according to the actual sampling rate, so as to achieve the purpose of rapid detection.

In some embodiments of the present invention, the size of the above-mentioned self-injection immunoassay paper chip is 10.5 mm×35 mm, and this size is adopted to fully consider the convenience of the connection between the self-injection immunoassay paper chip and the back-end instrument. .

In some embodiments of the present invention, the areas where the microfluidic channel, the communication channel and the injection hole of the above-mentioned microfluidic layer are located are hydrophobic, and other areas are hydrophilic, so that the microfluidic channel can pass through the microfluidic channel well. It guides the flow direction of the liquid to achieve fast and efficient detection.

In order to achieve the above purpose, the present invention also proposes a method for preparing a self-injection immunoassay paper chip, which is used to prepare the above-mentioned self-injection immunoassay paper chip, comprising the following steps: adding a pre-set microfluidic channel After printing on the surface of the microfluidic layer by a wax spray printer, the microfluidic layer is heated to make the area where the microfluidic channel of the microfluidic layer is located has hydrophobicity; Print the working electrode, the counter electrode and the reference electrode; modify the electroactive composite material on the surface of the working electrode; integrate the microfluidic layer, the auxiliary electrode layer, the detection layer and the working electrode layer; When the antibody is added dropwise to the surface of the working electrode modified with the electroactive composite substance.

Wherein, the above-mentioned electroactive composite material is prepared by the following steps: Step 1. Dissolving the multi-wall carbon nanotubes/aminated graphene into the polyethyleneimine aqueous solution, so that the multi-wall carbon nanotubes/aminated graphene are non-co-coated valence functionalization; step 2, adding an electroactive substance to the above solution, stirring vigorously for a certain period of time, so as to combine the non-covalent functionalized multi-wall carbon nanotubes/aminated graphene with the electroactive substance; step 3, combining the step The solution obtained in 2 is mixed with the nano-gold particle solution, the chitosan solution is added after vigorous stirring, and the electroactive composite substance is obtained by ultrasonic mixing.

To sum up, the self-injecting immune whole blood detection paper chip designed in the present invention is based on the combined application of microfluidic paper chip, carbon nanocomposite preparation and modification technology, label-free electrochemical detection technology, etc. (disease markers, neurotrophic factors) and polypeptide molecules for label-free, high-sensitivity, trace, immediate and rapid detection, low detection requirements, and the ability to detect serum and whole blood samples, breaking through the traditional detection technology, providing protein And polypeptide molecule detection provides a new type of detection device and method.

In some embodiments of the present invention, as shown in FIG. 1, a new type of self-injecting immunoassay paper chip is proposed, which includes 6 layers in total, namely a first filter layer 1, a microfluidic layer 2, and a second filter layer. layer 3 , auxiliary electrode layer 4 , detection layer 5 and working electrode layer 6 , specifically, the first filter layer 1 is provided with a first filter hole 11 , and the microfluidic layer 2 is provided with a microfluidic channel connected by a communication channel 21 The channel 22 and the injection hole 23, wherein, the microfluidic channel 22 is arranged in a position directly opposite to the first filter hole 11; the second filter layer 3 has a second filter hole 31 directly opposite to the injection hole 23; auxiliary The electrode layer 4 has a first through hole 41 facing the second filter hole 31 , and two second through holes 42 symmetrically arranged on both sides of the first through hole 41 ; the edges of the two second through holes 42 are The counter electrode 43 and the reference electrode 44 that are in agreement with the partial edges are provided, and the counter electrode 43 and the reference electrode 44 are separately arranged, and are respectively connected with the external equipment through the counter electrode lead 45 and the reference electrode lead 46; the detection layer 5 has a The first through hole 41 is opposite to the third through hole 51 . The third through hole 51 has a symmetrical communication hole 52 extending in the length direction of the detection layer 5 , and the end 520 of the communication hole 52 is the detection layer of the detection layer 5 . The position of the detection area is directly opposite to the position of the second through hole 42; the working electrode layer 6 is provided with a circular working electrode 61 that is directly opposite to the detection area, which is connected to the external device through the working electrode lead 62, and its size is the same as The dimensions of the second through holes are matched.

The paper chip designed in this embodiment is a 3D structure paper chip, which is a 6-layer structure composed of an integrated package. Since the fluid flows on the paper through capillary action, the paper chip uses the microfluidic channel 22 to realize the self-feeding of the tissue fluid to be tested. Sample, the self-injected tissue fluid to be tested is simultaneously filtered by the first filter hole 11 to filter out cells and molecules with large diameters such as leukocytes and red blood cells in the whole blood; the tissue fluid to be tested reaches the injection hole 23 through the connecting channel 21, and then After infiltration through the second filter hole 31, the remaining large-diameter cells and molecules are further filtered; after further filtration, the liquid to be tested reaches the detection area 520 at the end of the communication hole 52 through the first through hole 41 and the third through hole 51, The counter electrode 43, the reference electrode 44 and the working electrode 61 are made to be in the same liquid environment.

Among them, the first layer 1, the third layer 3 and the fifth layer 5 of the above-mentioned paper chip use filter paper with a porosity of less than 3 μm, and the particle retention degree is less than 3 μm, which can effectively filter cells and molecules with large diameters such as leukocytes and red blood cells; the second The porosity of the filter paper used in the layer 2, the fourth layer 4 and the sixth layer 6 is smaller than that of the first, third and fifth layers. By setting the porosity of the first, third and fifth layers, the detection of serum and whole blood samples can be realized.

The surface of the working electrode is modified with an electroactive composite substance, and the electroactive composite substance is specifically obtained by the following preparation method:

As shown in Figure 2, first, take 1 mg of multi-walled carbon nanotubes, dissolve them into 10 mL of polyethyleneimine (PEI) aqueous solution, and take 2 mL of the dispersion liquid of multi-walled carbon nanotubes with a concentration of 1 mg/mL ^-1 and Mix 2 mL of thionine solution or Prussian blue solution with a concentration of 2 mg/mL ^-1 , stir vigorously for 24 hours, remove unbound thionine or Prussian blue molecules by centrifugation, and take 100 μL of thionine/Prussian blue-multi-walled carbon nanoparticle The tube mixture was mixed with 250 μL of gold nanoparticles solution, and after vigorous stirring for 12 hours, 100 μL of chitosan solution was added, and ultrasonically mixed to make a multi-walled carbon nanotube-thionine/Prussian blue-nano-gold-chitosan mixture. liquid, that is, the electroactive composite substance.

Since the non-covalent function of polyethyleneimine is used to modify multi-walled carbon nanotubes in the preparation of electroactive composite materials, it has a stronger catalytic effect than ordinary graphene, so it can make the final signal current larger. , so that the detection range of the self-injection immunoassay paper chip of this embodiment is wider.

In some embodiments of the present invention, a method for preparing a self-injection immunodetection paper chip is proposed, and the specific steps are as follows:

1. Use computer software to design the structure of each layer of the self-injection immunodetection paper chip. The main structure includes the microfluidic channel structure of the paper chip and the structure of the electrode. The paper chip adopts a three-electrode system, the working electrode is circular with a diameter of 3mm, and the counter electrode and the reference electrode are both arc-shaped. The size of the electrode area is consistent;

2. Due to the limited printing size of the wax spray printer, the filter paper of A1 size is cut into A4 size with a paper cutter, which is suitable for printing. The first, third and fifth layers of the paper chip are made of porosity of 1μ, The filter paper has a particle retention of 1 μm, which can effectively filter cells and molecules with large diameters such as white blood cells and red blood cells; the second, fourth and sixth layers use filter paper with a porosity of 11 and a particle retention of 11 μm. ;

3. Using a digital wax spray printer, print the designed paper chip channel pattern on the surface of the second layer of filter paper, and at the same time print the corresponding graphics on the filter paper of other layers;

4. Put the wax-sprayed filter paper into the oven. Due to heating, the wax on the surface of the paper will melt and penetrate into the inside of the paper, so that the wax-sprayed area becomes a hydrophobic area, and the unsprayed area becomes a hydrophilic area. As the wax penetrates into the interior, it will diffuse to the surroundings. Therefore, by precisely controlling the heating temperature and time, the area of the hydrophilic and hydrophobic regions can be precisely controlled;

5. Use a screen printing machine to print the three-electrode system (including the working electrode, the counter electrode, and the reference electrode) on the surface of the paper heated by spraying wax. The materials of working electrode and counter electrode are conductive carbon paste, and the reference electrode is conductive Ag/AgCl paste.

6. Design engraving graphics, and use the engraving machine to make double-sided tapes with corresponding structures;

7. Based on carbon nanomaterials and electroactive substances, carbon nanomaterials-electroactive substances composite nanomaterials are prepared;

8. Add nano-gold to prepare carbon nano-material-electroactive material-nano-gold composite nano-material, and modify the working electrode of the paper chip; take 10 μL of multi-walled carbon nanotube-thionine/Prussian blue-nano-gold mixed solution, It was drop-coated on the surface of the working electrode, placed in an oven, and baked at 50 °C for 20 min.

9. Use the engraved double-sided tape to encapsulate the modified working electrode with the structure of other paper chips for detection;

10. Use the specific adsorption principle of antigen-antibody to conduct actual test on the sample.

The working principle and use method of the self-injection immunoassay paper chip prepared in this example are shown in Figure 3. Take 10 μL of the tissue fluid to be tested with a concentration of 200 mg/mL ^-1 and drop the antibody corresponding to the antigen to be tested on the surface of the electrode. surface, placed in a refrigerator, and placed at 4°C for 6 hours for antigen binding. The remaining active sites on the electrode surface were then blocked with 10 μL of 1% bovine serum albumin at room temperature to reduce specific repair.

Then, after the self-injection immunodetection paper chip self-sampling the tissue fluid to be detected, the tissue fluid flows to the detection layer, and the antigen in it is combined with the antibody immobilized on the surface of the electroactive composite material. Although the specific binding of the antigen and the antibody does not produce electron transfer, Since the electroactive substance is immobilized on the electrode surface, the detected current is the current generated by the interaction between the electroactive substance and the electrode surface. After the antigen binds to the antibody immobilized on the electrode surface, it will affect the activity of the immobilized electroactive substance, thereby It affects the magnitude of the detected current. The more antigens are bound, the smaller the detected current. The antigen concentration is inversely proportional to the current. Therefore, by detecting the current, the concentration of the antigen in the tissue fluid to be tested can be indirectly measured.

The self-injection immunodetection paper chip proposed by the present invention, its preparation method, and its use method will be described in detail below through specific examples.

Example 1

This embodiment proposes a paper chip for rapid electrochemical immunoassay detection of whole blood with automatic feeding type and a preparation method thereof, which are used for the rapid electrochemical detection of trace quantities with automatic sample feeding of CEA and NSE two parameters at the same time. 1 shows the structure.

The preparation and use process of the automatic feeding type trace rapid electrochemical immunoimmunity whole blood detection paper chip to detect tumor markers are as follows:

(1) The two parameters of carcinoembryonic antigen (CEA) and neuron-specific ene purifying enzyme (NSE) were selected for combined determination;

(2) Preparation of automatic feed-type trace rapid electrochemical immunoassay whole blood detection paper chip: On the surface of the corresponding filter paper, the designed channel pattern is printed on the surface of the paper by a wax spray printer; Heating will melt the wax on the surface of the paper and penetrate into the inside of the paper, so that the wax sprayed area becomes a hydrophobic area, and the unsprayed area becomes a hydrophilic area. Therefore, by precisely controlling the heating temperature and time, the hydrophilic area can be precisely controlled. , the area of the hydrophobic area; use a screen printing machine to print the counter electrode and the reference electrode on the surface of the fourth layer of paper, and print the working electrode on the surface of the sixth layer of paper;

(3) Preparation of multi-walled carbon nanotubes-electroactive material-nano-gold composite nanomaterials: take 1 mg of multi-walled carbon nanotubes, dissolve them in 10 mL of PEI aqueous solution, and take 2 mL of multi-walled carbon nanotubes with a concentration of 1 mg/mL ^-1 The nanotube dispersion was mixed with 2 mL of thionine solution or Prussian blue solution with a concentration of 2 mg/mL ^-1 , stirred vigorously for 24 hours, and centrifuged to remove unbound thionine or Prussian blue molecules, and take 100 μL of thionine/Prussian blue. -Mix the multi-walled carbon nanotube mixture with 250 μL of gold nanoparticles solution, stir vigorously for 12 hours, add 100 μL of chitosan solution, and mix by ultrasonic to prepare multi-walled carbon nanotubes-thionine/Prussian blue-nano-gold - Chitosan mixture.

(4) Modification of working electrode: 10 μL of the above-mentioned multi-walled carbon nanotube-electroactive substance-gold nanocomposite nanomaterial was dropped onto the surface of the working electrode with a pipette, and baked at 50°C for 20min. Subsequently, 10 μL of CEA and NSE antibodies at a concentration of 200 mg/mL ^-1 were dropped onto the surface of the electrode, placed in a refrigerator, and placed at 4°C for 6 h for binding antibodies. Finally, at room temperature, 10 μL of 1% BSA solution was used to cover the remaining active sites.

(5) Using a plotter to make double-sided adhesive tape, the above-mentioned paper chips are integrated and packaged.

The paper chip prepared in this example for rapid electrochemical immunoassay whole blood detection with automatic injection type can automatically inject the sample, and the required sample volume is about 10 μL, and the sample is automatically injected through the microfluidic channel and the injection hole; , let stand for 10 minutes at room temperature to allow the antigen and antibody to combine, connect the above paper chip directly to the electrochemical workstation through the back-end interface, and use differential pulse voltammetry to detect the current response of the working electrode. Change the value, inversely calculate the concentration of CEA and NSE in the sample to be tested.

Example 2

The present embodiment proposes an automatic-type trace-level rapid electrochemical immune whole blood detection paper chip and a preparation method thereof. The automatic-type trace-level rapid electrochemical immune whole blood detection paper chip is used to detect neurotrophic factor (BDNF), Its preparation and use process are as follows:

(1) Select BDNF parameters for measurement;

(2) Preparation of automatic feed-type trace rapid electrochemical immunoassay whole blood detection paper chip: On the surface of the corresponding filter paper, the designed channel pattern is printed on the surface of the paper by a wax spray printer; Heating will melt the wax on the surface of the paper and penetrate into the inside of the paper, so that the wax sprayed area becomes a hydrophobic area, and the unsprayed area becomes a hydrophilic area. Therefore, by precisely controlling the heating temperature and time, the hydrophilic area can be precisely controlled. , the area of the hydrophobic area; use a screen printing machine to print the counter electrode and the reference electrode on the surface of the fourth layer of paper, and print the working electrode on the surface of the sixth layer of paper;

(3) Preparation of Aminated Graphene-Electroactive Substance-Nano Gold Composite Nanomaterials: Take 2mL of dispersion liquid of graphene aminated with a concentration of 1mg/mL ^-1 and 2mL of a thionine solution with a concentration of 2mg/mL ^-1 or Prussian blue solution, stir vigorously for 24h, remove unbound thionine or Prussian blue molecules by centrifugation, take 100 μL of thionine/Prussian blue-multi-walled carbon nanotube mixture and 250 μL of gold nanoparticles solution, mix vigorously After stirring for 12 hours, 100 μL of chitosan solution was added and mixed by ultrasonic, thereby preparing the mixed solution of amidated graphene-thionine/Prussian blue-nano-gold-chitosan.

(4) Modification of working electrode 8: Using a pipette, 10 μL of the above-mentioned aminated graphene-electroactive substance-gold nanocomposite nanomaterial was dropped onto the surface of the working electrode, and baked at 50° C. for 20 min. Subsequently, 10 μL of BDNF antibody at a concentration of 200 mg/mL ^-1 was dripped onto the surface of the electrode, placed in a refrigerator, and placed at 4°C for 6 h for binding to the antibody. Finally, at room temperature, 10 μL of 1% BSA solution was used to cover the remaining active sites.

(5) Using a plotter to make double-sided adhesive tape, the above-mentioned paper chips are integrated and packaged.

The paper chip prepared in this example for rapid electrochemical immunoassay whole blood detection with automatic injection type can automatically inject the sample. The required sample volume is about 10 μL, and the sample is automatically injected through the microfluidic channel and the injection hole. After sample injection, let stand for 10 minutes at room temperature to allow the antigen and antibody to combine. The above paper chip is directly connected to the electrochemical workstation through the back-end interface, and the current response of the working electrode is detected by differential pulse voltammetry. The change value of the current signal, inversely calculate the concentration of BDNF in the sample to be tested.

The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in further detail. It should be understood that the above are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principle of the present invention, any modifications, equivalent replacements, improvements, etc. made should be included within the protection scope of the present invention.

Claims (8)

1. A self-injection immunoassay paper chip for detecting antigen concentration, comprising from top to bottom along the stacking direction: The microfluidic layer has a microfluidic channel connected by a communication channel and a sample injection hole, the microfluidic channel is used for self-sampling, and the sample injection hole is used to introduce the sampled tissue fluid to be tested into the detection area ; an auxiliary electrode layer, the auxiliary electrode layer includes a first through hole arranged directly opposite to the injection hole, and at least two pairs of counter electrodes and reference electrodes arranged in combination; The detection layer, wherein the detection layer includes a third through hole, the third through hole is facing the first through hole, and the third through hole has symmetrical communication holes extending along the length direction of the detection layer, and a working electrode layer, the surface of which is modified with an electroactive composite substance, and the surface of the electroactive composite substance is used to immobilize the antibody; wherein, the working electrode layer comprises at least two circular working electrodes arranged along the circumferential direction, The communication hole is facing the circular working electrode; the circular working electrode, the counter electrode and the reference electrode are all connected with electrode leads to connect external devices; And the auxiliary electrode layer also includes at least two second through holes arranged opposite to the circular working electrode, and the combination of the counter electrode and the reference electrode is respectively connected with the part of the at least two second through holes. the edges match; The auxiliary electrode layer, the detection layer and the working electrode layer constitute the detection area, including an electrochemical detection circuit. Through electrochemical means, based on the specific binding principle of antigen-antibody and the characteristics of the electroactive composite substance, the detection to be detected is obtained. The concentration of the antigen in the tissue fluid. 2. The self-injection immunodetection paper chip according to claim 1, wherein: The electroactive composite material includes a composite material containing carbon nanomaterials, electroactive materials and nano-gold; The carbon nanomaterials include multi-walled carbon nanotubes or aminated graphene; The electroactive substances include thionine, Prussian blue or potassium ferricyanide; In the composite material, the ratio between the carbon nanomaterial, the electroactive material and the nano gold is 1:1:5-1:2:10. 3. The self-injection immunodetection paper chip according to claim 1, wherein, The microfluidic layer and the detection layer have at least two sets of edge through holes arranged in the circumferential direction, and the at least two sets of edge through holes face the electrode leads of the working electrode, the counter electrode and the reference electrode. 4. The self-injection immunodetection paper chip according to claim 1, wherein, The upper surface of the microfluidic layer is provided with a first filter layer; The first filter layer has a first filter hole that is directly opposite to the microfluidic channel, and is used to filter the tissue fluid to be tested when the microfluidic channel is self-sampling; The porosity of the filter pores is less than 3 μm; The lower surface of the microfluidic layer is provided with a second filter layer, which has a second filter hole directly opposite to the sample injection hole, which is used to further filter the tissue fluid to be tested obtained by sampling. 5. The self-injection immunodetection paper chip according to claim 1, wherein, The width of the communication channel connecting the microfluidic channel and the injection hole is inversely proportional to the sampling rate; The size of the self-injection immunoassay paper chip is 10.5 mm×35 mm. 6. The self-injection immunodetection paper chip according to claim 1, wherein the microfluidic channel, the communication channel and the injection hole of the microfluidic layer have hydrophobicity in the region, and other regions have hydrophilicity . 7. A method for preparing a self-injection immunodetection paper chip, for preparing the self-injection immunodetection paper chip according to any one of claims 1 to 6, comprising the following steps: After the pre-set microfluidic channel is printed on the surface of the microfluidic layer by a wax spray printer, the microfluidic layer is heated to make the microfluidic channel, communication channel and the microfluidic layer of the microfluidic layer. The area where the injection hole is located is hydrophobic; The screen printer prints the working electrode, the counter electrode and the reference electrode on the working electrode layer and the auxiliary electrode layer respectively; modifying the electroactive composite material on the surface of the working electrode; The microfluidic layer, the auxiliary electrode layer, the detection layer and the working electrode layer are integrated and packaged; When the self-injection immunodetection paper chip is used, an antibody is dropped on the surface of the working electrode modified with the electroactive composite substance. 8. The method for preparing a self-injection immunodetection paper chip according to claim 7, wherein the electroactive composite substance is prepared by the following steps: Step 1, dissolving multi-wall carbon nanotubes or aminated graphene into an aqueous solution of polyethyleneimine, so that the multi-wall carbon nanotubes or aminated graphene are non-covalently functionalized; Step 2, adding an electroactive substance to the above solution, stirring vigorously for a certain period of time, so as to combine the non-covalently functionalized multi-walled carbon nanotubes or aminated graphene with the electroactive substance; Step 3. Mix the solution obtained in step 2 with the gold nanoparticle solution, add the chitosan solution after vigorous stirring, and ultrasonically mix to obtain the electroactive composite substance.

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