CN113390862A

CN113390862A - Electrochemical luminescence immunosensor and preparation method thereof

Info

Publication number: CN113390862A
Application number: CN202110685064.XA
Authority: CN
Inventors: 徐国宝; 杜芳昕
Original assignee: Changchun Institute of Applied Chemistry of CAS
Current assignee: Changchun Institute of Applied Chemistry of CAS
Priority date: 2021-06-21
Filing date: 2021-06-21
Publication date: 2021-09-14

Abstract

The invention relates to the technical field of electrochemical sensors, in particular to an electrochemical luminescence immunosensor and a preparation method thereof. The invention uses the carbon nano material with certain resistance and excellent adsorption capacity to prepare the electrode with only one anode and one cathode connected together, and covers a layer of porous membrane to carry out the electrochemical system of multi-sample electrochemical experiment in a plurality of cells formed by the porous membrane and the electrode, and a plurality of groups of electrodes are not needed, thus the invention has simple manufacture. Meanwhile, compared with ITO/FTO, the carbon nano material as the electrode material can load the antibody through adsorption without further modification on the surface. After binding to the antigen or antibody, an electrochemiluminescent reagent is added to conduct immunoassay of multiple samples simultaneously.

Description

Electrochemical luminescence immunosensor and preparation method thereof

Technical Field

The invention relates to the technical field of biological analysis, in particular to an electrochemiluminescence immunosensor and a preparation method thereof.

Background

Currently, the electrode arrays of electrochemiluminescence immunosensors that can simultaneously perform multiple sample analyses typically include the following types: (1) a three-electrode array comprising a plurality of working electrodes, a common auxiliary electrode, and a reference electrode; or an electrode array comprising a plurality of three-electrode systems comprising a working electrode, an auxiliary electrode, and a reference electrode; (2) a bipolar electrode array comprising two drive electrodes and a plurality of bipolar electrodes. The above electrochemical systems all require a plurality of electrodes. In addition, the electrochemical workstation of the three-electrode system is used as an output power supply, so that the operation is relatively complex and inconvenient; bipolar electrode arrays have a high driving voltage, a low bipolar current efficiency, and are susceptible to background interference from the driving electrodes (anal. chem.2013,85, 5335-5339).

A new electrochemical system has recently appeared, in which only one anode and one cathode are connected, and multiple samples can be simultaneously analyzed without using multiple electrodes. The system only explores ITO/FTO as an electrode material and is not used for construction of an immunosensor, and if the system is used for construction of the immunosensor, modification needs to be carried out on the surface of an electrode for loading an antibody, so that the operation is complex.

Disclosure of Invention

In view of this, the present invention provides an electrochemiluminescence immunosensor and a method for manufacturing the same. The electrochemical luminescence immunosensor is characterized in that an electrode is prepared from a carbon nano material, the electrode only comprising an anode and a cathode which are connected together is prepared, a porous membrane is covered on the electrode, multi-sample electrochemical analysis can be realized, the preparation process is simple, and the detection limit is low.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of an electrochemiluminescence immunosensor, which comprises the following steps:

coating a carbon nano coating on a plastic substrate, and drying to obtain a substrate electrode;

adhering a porous membrane on the surface of the substrate electrode, and dividing the substrate electrode into a plurality of independent pore chambers; and connecting two ends of the substrate electrode by using a conductive copper adhesive tape to obtain the electrochemiluminescence immunosensor.

According to the invention, the copper adhesive tape is used for replacing the traditional wire, the contact area of the adhesive tape and the electrode is large, the coating is coated on the contact surface of the adhesive tape and the electrode, the cracking phenomenon can also occur, and the preparation efficiency and the uniformity of electrode signals are improved. The standard deviation of the electrochemical luminescence intensity of the improved electrode can be reduced to below 5%, and the situation that wires crack and fall off cannot occur in the using process.

The electrochemical luminescence immunosensor (the structure schematic diagram is shown in figure 1), figure 1 is a schematic diagram of 5 pore chambers, a gray electrode is a whole piece of thin film electrode, and after voltage is applied, voltage difference is generated between two ends of the thin film electrode which is in contact with each sample cell due to the resistance of the thin film (namely, one end is an anode (+), and the other end is a cathode (-), so that the electrode surface of each pore generates electrochemical luminescence. The structure of the conventional three-electrode system (fig. 2-a) and the bipolar electrode system (fig. 2-B) is schematically shown in fig. 2, wherein the blue electrode horizontally arranged in the middle of fig. 2-B is a bipolar electrode, and the gray electrode vertically arranged on two sides is a driving electrode connected with a power supply.

Both conventional three-electrode systems and bipolar electrode systems require multiple electrodes and require a multiple of the number of electrodes when performing multiple sample analyses. Compared with the traditional three-electrode system and the two-electrode system, the single-electrode system prepared by the invention only has one electrode with the anode and the cathode connected together, and can be used for carrying out multi-sample electrochemical experiments in a plurality of cells formed by the porous membrane and the electrode by covering a layer of porous membrane. And the system size and the number of independent reaction tanks can be adjusted by adjusting the size of the holes of the porous plastic sheet and the number of the holes according to actual requirements, the solution added into the independent reaction tanks is an alkaline solution containing luminol and hydrogen peroxide, the stability of the electrodes is high, the point chemiluminescence signals of different reaction tanks are basically the same, and the accuracy of subsequent experiments is ensured.

In some embodiments, the carbon nanomaterials employed in the present invention comprise a volume fraction of 0.05% to 0.5% of a thickener. The addition of the thickener significantly increases the viscosity of the coating, improves the retraction phenomenon, and significantly reduces the standard deviation compared to a control without the addition of the thickener. The volume concentration of the thickening agent is specifically 0.05%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%; the thickener is an associated alkali swelling thickener. Associative alkali swelling thickeners, i.e., hydrophobically modified polyacrylate alkali swellable emulsions, such as SN636 by Nopco, TT-935 by Rohm & Haas, and the like, include but are not limited to these.

In some embodiments, the method of preparing the base electrode comprises:

a1, sticking a sticker with the same size as the electrode of the needed substrate on the plastic substrate;

a 2: uniformly coating the carbon nano-coating on the paster by using a glass slide, and drying to obtain a substrate electrode; the carbon nanomaterial completely covers the decal area.

In some embodiments, the plurality is two or more, the individual chambers have a diameter of 6mm and the two orifices are spaced 3mm apart. In some embodiments, the plurality of cells is 24 cells having a diameter of 6mm and a 3mm separation between two cells.

The invention also provides the electrochemiluminescence immunosensor prepared by the preparation method.

The invention also provides the application of the electrochemiluminescence immunosensor prepared by the preparation method in detecting antigens or antibodies.

The antigen is one of cardiac troponin I, cardiac fatty acid binding protein and peptide.

In some embodiments, the antibodies are novel coronavirus antibodies lgG and lgM.

Specifically, the method for detecting the antigen or the antibody by adopting the electrochemical luminescence immunosensor comprises the following steps:

adding an antibody of an antigen to be detected or an antigen of the antibody to be detected into a pore chamber of the electrochemiluminescence immunosensor prepared by the method disclosed by the invention, incubating overnight at 4 ℃, washing with PBS buffer solution, then sequentially adding bovine serum albumin liquid, antigen solution or antibody solution, incubating for 50 minutes at 37 ℃, washing with PBS buffer solution, adding electrochemiluminescence solution, connecting a power supply, and obtaining the concentration of the antigen or the antibody according to the change of electrochemiluminescence signals. In some embodiments, after the addition of bovine serum albumin solution, incubation is performed for 50 minutes at 37 ℃; then, the antigen or antibody solution was added thereto, and the mixture was incubated at 37 ℃ for 50 minutes.

The preparation method of the electrochemical luminescence immunosensor provided by the invention comprises the following steps: coating a carbon nano coating on a plastic substrate, and drying to obtain a substrate electrode; adhering a porous membrane on the surface of the substrate electrode, and dividing the substrate electrode into a plurality of independent pore chambers; connecting two ends of the substrate electrode by using a conductive copper adhesive tape; and sequentially adding the antibody of the antigen to be detected or the antigen of the antibody to be detected and bovine serum albumin into the independent pore chamber for incubation to obtain the electrochemical luminescence immunosensor. The invention uses the carbon nano material with certain resistance and excellent adsorption capacity to prepare the electrode with only one anode and one cathode connected together, and covers a layer of porous membrane to carry out the electrochemical system of multi-sample electrochemical experiment in a plurality of cells formed by the porous membrane and the electrode, and a plurality of groups of electrodes are not needed, thus the invention has simple manufacture. Meanwhile, compared with ITO/FTO, the carbon nano material as the electrode material can load the antibody through adsorption without further modification on the surface. After binding to the antigen or antibody, an electrochemiluminescent reagent is added to conduct immunoassay of multiple samples simultaneously.

Drawings

FIG. 1 shows a schematic structural diagram of an electrochemiluminescence immunosensor of the present invention;

FIG. 2 is a schematic diagram showing the structure of a conventional three-electrode and two-electrode system, wherein 2-A is a three-electrode system and 2-B is a two-electrode system;

FIG. 3 shows a flow chart of a process for preparing an electrochemiluminescence immunosensor;

FIG. 4 shows a process flow diagram for electrochemical detection;

FIG. 5 is a linear equation showing the results of detection in example 1;

FIG. 6 shows the results of the detection of the carbon nanomaterial electrode in the reaction cell of example 1;

FIG. 7 is a linear equation showing the results of detection in example 2;

fig. 8 shows a schematic of a 3 x 8 pore electrode, porous cell (left) and box (right) in example 3.

Detailed Description

The invention provides an electrochemiluminescence immunosensor and a preparation method thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides a preparation method of an electrochemiluminescence immunosensor, a process flow chart is shown in figure 3, and the method specifically comprises the following steps:

(1) a piece of plastic paster is taken, and a square frame and a porous pattern with the required electrode size are carved on the paster by using a carving machine. And (3) attaching the square sticker to a clean plastic sheet, adding a proper amount of carbon nano material coating, coating a glass slide in a scraping mode to cover the area of the square sticker and spread the square sticker uniformly, and naturally drying the square sticker in a dust-free environment to obtain the carbon nano material electrode.

(2) The porous pattern sticker is pasted on the carbon nano material electrode, and the whole electrode is divided into a plurality of independent pore chambers. And (3) attaching conductive copper adhesive tapes at two ends of the electrode, and uniformly coating carbon nano material coating at the joint of the adhesive tapes and the electrode, so that two ends of the electrode are conveniently connected to a power supply to be electrified.

(3) And adding an antibody of the antigen to be detected or an antigen of the antibody to be detected into the pore chamber, adding bovine serum albumin, and incubating to obtain the electrochemical luminescence immunosensor.

Wherein, the excellent adsorption capacity of the carbon material electrode is utilized to load an antibody or an antigen, and when the object to be detected is the antigen, the antibody of the antigen to be detected is added into the pore chamber; when the analyte is an antibody, an antigen of the antibody to be detected is added to the well.

When the electrochemical luminescence immunosensor is used for detecting antigens or antibodies, the antigen detection is taken as an example for description, the schematic diagram of the detection process is shown in figure 4, and the specific steps are as follows:

adding the antigen to be detected into a well chamber of the antibody combined with the antigen to be detected, incubating, and washing three times by using PBS (pH 7.4) buffer solution;

b: adding an electrochemiluminescence reagent, connecting two ends of an electrode of the sensor to a power supply, electrifying the sensor to be connected with the power supply, and obtaining the concentration of the antigen or the antibody according to the change of an electrochemiluminescence signal.

The electrode material has a certain resistance, and the voltage drop generated at the two ends of the hole enables one end of the carbon nano material electrode in contact with the solution to generate electrochemical oxidation reaction to generate electrochemical luminescence. The detection is carried out by using a smart phone or a CCD, and an obvious electrochemiluminescence phenomenon with the anode end of each hole can be observed. The combination of the antigen on the surface of the electrode can change the impedance of the electrode and generate steric hindrance, so that the electrochemical luminescence signal is changed, and the concentration of the antigen to be detected is obtained according to the relation between the change of the electrochemical luminescence signal and the concentration of the antigen.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1

The squares and porous patterns required for the 3 x 3 pore electrodes were scribed on the decal using a scribing machine. Each hole was 6mm in diameter, the spacing between two holes was 3mm, the desired box size was 48 x 48mm, and the box width was 4 mm. The paster that this step used is thickness for 0.2 mm.

The white plastic sheet (54 x 86mm) was washed clean with water and ethanol in sequence, and the square sticker was attached to the clean plastic sheet. 0.3mL of carbon nanomaterial paint with 0% of thickening agent content is absorbed by a plastic dropper and dripped at one end of the square frame, a glass slide is used for blade coating to cover the area of the square frame-shaped sticker and spread uniformly, and the square frame-shaped sticker is placed in a moisture-proof box or a dust-free environment for room temperature drying to obtain a 40 x 40mm carbon nanomaterial electrode.

And sticking the porous pattern sticker on the carbon nano material electrode, and dividing the whole electrode into 3 × 3 independent pore chambers. And (3) pasting conductive copper adhesive at two ends of the electrode, uniformly coating the carbon nano material coating at the joint of the adhesive tape and the electrode by using a lamb brush, and connecting two ends of the electrode to a power supply for electrifying to obtain the electrochemical luminescence immunosensor.

The method for detecting the myocardial infarction marker cardiac troponin I (cTnI) by using the electrochemical luminescence immunosensor comprises the following specific steps:

30. mu.L of 0.1mg/mL cTnI antibody was added to each individual well of the carbon nanoelectrodes prepared above, and incubated overnight at 4 ℃. Then, 30. mu.L of a 1% Bovine Serum Albumin (BSA) solution was added thereto, and the mixture was incubated at 37 ℃ for 50 minutes. Finally, 30. mu.L of cTnI with a concentration of 0, 0.1, 1, 5, 10, 50, 100, 200, 1000ng/mL was added and incubated at 37 ℃ for 50 minutes. After the end of each step, the cells were washed three times with PBS (pH 7.4) buffer. When performing the electrochemiluminescence test, 10 μ L of electrochemiluminescence solution was added to each well, connected to a power supply (applied voltage of 6.5V), and photographed and analyzed with a smartphone, and the results are shown in fig. 5 and 6. Wherein the electrochemiluminescence solution comprises 70% of 0.1M CBS buffer (pH 10), 10% of 1mM luminol, 10% of 10mM hydrogen peroxide and 10% of triton X-100 with a volume fraction of 10%. As can be seen from FIG. 6, the detection limit was 0.89 ng/mL.

Example 2

The white plastic sheet (54 x 86mm) was washed clean with water and ethanol in sequence, and the square sticker was attached to the clean plastic sheet. 0.3mL of carbon nanomaterial paint with 0.2% of thickening agent content is absorbed by a plastic dropper and dripped at one end of the square frame, a glass slide is used for blade coating to cover the area of the square frame-shaped sticker and spread uniformly, and the square frame-shaped sticker is placed in a moisture-proof box or a dust-free environment for room temperature drying to obtain a 40 x 40mm carbon nanomaterial electrode.

And sticking the porous pattern sticker on the carbon nano material electrode, and dividing the whole electrode into 3 × 3 independent pore chambers. And (3) attaching conductive copper adhesive tapes at two ends of the electrode, and uniformly coating the joint of the adhesive tapes and the electrode with carbon nano material coating, so that the two ends of the electrode are conveniently connected to a power supply to be electrified, and the chemiluminescence immunosensor is obtained.

cTnI was detected by the immunosensor prepared in this example according to the method of example 1, and the detection result is shown in FIG. 7, with a detection limit of 9.5 ng/mL.

Example 3

The box and porous pattern required for the 3 x 8 pore electrode was scribed on the decal using a scribing machine. Each hole was 6mm in diameter, the spacing between two holes was 3mm, the desired box size was 48 x 85mm, the box width was 4mm (as shown in figure 8) and the decal used in this step was 0.2mm thick.

The white plastic sheet (54 x 86mm) was washed clean with water and ethanol in sequence, and the square sticker was attached to the clean plastic sheet. 0.5mL of carbon nanomaterial paint with 0% of thickening agent content is absorbed by a plastic dropper and dripped at one end of the square frame, a glass slide is used for blade coating to cover the area of the square frame-shaped sticker and spread uniformly, and the square frame-shaped sticker is placed in a moisture-proof box or a dust-free environment for room temperature drying to obtain a 40 × 77mm carbon nanomaterial electrode.

And sticking the porous pattern sticker on the carbon nano-material electrode, and dividing the whole electrode into 3-8 independent pore chambers. And (3) attaching conductive copper adhesive tapes at two ends of the electrode, and uniformly coating carbon nano material coating at the joint of the adhesive tapes and the electrode, so that two ends of the electrode are conveniently connected to a power supply to be electrified.

The electrode of this example detected cTnI, and the obtained results were in accordance with example 1.

Example 4

An immunosensor was prepared according to the method of example 1, and the carbon nanomaterial paint was added with thickeners in the amounts of 0, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% by volume, respectively. The electrochemiluminescence intensity and standard deviation of each individual well on the corresponding electrode are shown in table 1, and the two concentrations of 0% and 0.2% are determined by combining the two parameters of intensity and standard deviation.

TABLE 1 Effect of different thickener concentrations on the electrochemiluminescence of the electrode

Concentration/%)	Strength/RLU	Standard deviation/%
			0	3079.535	3.32
0.05	2931.051	3.36
			0.1	2782.726	4.53
0.2	2640.6	1.57
			0.3	2543.887	4.01
0.4	2503.682	2.83
			0.5	2392.245	3.31

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

Claims

1. A method for preparing an electrochemiluminescence immunosensor, comprising:

2. The electrochemiluminescence immunosensor of claim 1, wherein the carbon nanopaint comprises a volume fraction of 0.05% to 0.5% of a thickener; the thickener is an associated alkali swelling thickener.

3. The electrochemiluminescence immunosensor according to claim 1, wherein the base electrode is prepared by a method comprising:

4. The electrochemiluminescence immunosensor of claim 1, wherein the plurality is two or more, the independent well chamber has a diameter of 6mm, and the two wells are spaced 3mm apart.

5. An electrochemiluminescence immunosensor prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the electrochemiluminescence immunosensor prepared by the preparation method according to any one of claims 1 to 4 or the electrochemiluminescence immunosensor according to claim 5 in detection of an antigen or an antibody.

7. The use according to claim 6, wherein the antigen is one of cardiac troponin I, cardiac fatty acid binding protein and peptin.

8. The use according to claim 6, wherein the antibodies are the novel coronavirus antibodies lgG and lgM.

9. The use according to claim 6, wherein said detecting comprises the steps of:

adding an antibody of an antigen to be detected or an antigen of the antibody to be detected into a well chamber of the electrochemiluminescence immunosensor prepared by the preparation method of any one of claims 1-4, incubating overnight at 4 ℃, washing with a PBS buffer solution, then sequentially adding a bovine serum albumin solution, an antigen solution or an antibody solution, incubating for 50 minutes at 37 ℃, washing with the PBS buffer solution, adding an electrochemiluminescence solution, connecting a power supply, and obtaining the concentration of the antigen or the antibody according to the change of an electrochemiluminescence signal.