A high-throughput optoelectrochemical biological detection device and method

Abstract

The application discloses a high-flux photoelectrochemical biological detection device and method, wherein the device comprises an embedded thin film counter electrode, a thin film working electrode, a reference electrode, an electrolytic cell, a light source, an electrochemical workstation and a computer, and the detection method comprises separate incubation and centralized detection; the embedded thin film counter electrode is uniformly deposited or coated on the inner wall of a groove at the top of the electrolytic cell, so that errors caused by changes in electrode distance during the test are avoided; the light source provides stable light for the thin film working electrode through a through hole at the bottom of the electrolytic cell; during detection, the antigen-antibody is first separately incubated in the through hole of the electrolytic cell, and then high-flux detection is centrally carried out by synchronously linking the electrochemical workstation and the light source under the control of the computer program. The high-flux photoelectrochemical biological detection device and method provided in the application have the advantages of simple structure, low cost, convenient maintenance, strong anti-interference ability, fast detection speed and the like, and will help to promote large-scale practical application of photoelectrochemical biological detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of photoelectrochemical biological detection, in particular to a high-throughput photoelectrochemical biological detection device and method. BACKGROUND

[0002] With the development of society and the improvement of living standards, biological detection technology is increasingly applied in medical care and disease diagnosis. Among them, photoelectrochemical biological detection technology has attracted widespread attention due to its low background signal, fast response speed, high sensitivity and other advantages. In photoelectrochemical biological detection, long-term incubation between antigens and antibodies is often required. Therefore, in the application scenario of a large number of samples to be detected, high-throughput biological detection can greatly reduce time and labor costs compared to single-channel biological detection, realize rapid and efficient detection, and ensure the timeliness of the detection results. Currently, high-throughput photoelectrochemical biological detection methods mainly include optical addressing, electrical addressing and double addressing. In these detection methods, the detection device often requires precise moving machinery to meet the requirements of precise positioning and addressing, so the equipment cost and maintenance cost are relatively high (CN202211399485.7). In the optical addressing device, the position of the counter electrode is fixed, while the position of the working electrode changes with the optical addressing, which can cause changes in the distance between the working electrode and the counter electrode, affecting the accuracy and stability of the detection. In addition, the working electrode, as a key component in the photoelectrochemical biological detection system, its stability and repeatability of the preparation process have a great influence on the detection signal of the biological sensor, and usually complex correction is required for different batches of working electrodes. SUMMARY

[0003] The purpose of the present application is to provide a high-throughput photoelectrochemical biological detection device and method, wherein the detection device does not involve any moving mechanical components, has a simple structure, low cost and convenient maintenance; the scheme of uniformly depositing a thin film counter electrode inside the electrolytic cell avoids errors caused by changes in electrode distance during testing; the single uniform large-area working electrode prepared by the magnetron sputtering method reduces the interference caused by different batches of working electrode materials; the strategy of separate incubation of antigens and antibodies and photoelectrochemical centralized testing avoids contamination between adjacent samples, and at the same time realizes rapid and efficient high-throughput detection.

[0004] The present application is realized at least by one of the following technical solutions.

[0005] A high-throughput photoelectrochemical biological detection device comprises an embedded thin film counter electrode, a thin film working electrode, a reference electrode, an electrolytic cell, a light source, an electrochemical workstation and a computer; the embedded thin film counter electrode and the reference electrode are integrated in the inner wall of the groove at the top of the electrolytic cell; the light source is located below the electrolytic cell and provides stable light for the thin film working electrode fixed in the middle of the electrolytic cell through the through hole at the bottom of the electrolytic cell; the electrochemical workstation is connected with the working electrode, the counter electrode and the reference electrode respectively, and the detected electrical signal is transmitted to the computer for processing, display and storage.

[0006] Further, the embedded thin film counter electrode is a metal thin film or a carbon thin film uniformly deposited or coated on the inner wall of the groove at the top of the electrolytic cell, and the distance between the working electrode and the counter electrode in each through hole is consistent, eliminating the error caused by different electrode spacings.

[0007] Further, the working electrode is prepared by magnetron sputtering technology.

[0008] Further, the electrolytic cell is divided into upper and lower parts, and there are corresponding through holes in the center position, the through holes in the upper part are used to carry the buffer solution, and the through holes in the lower part are used for light irradiation.

[0009] Further, there are corresponding bolt holes at the edge position of the electrolytic cell, which are used for installing and fixing bolts.

[0010] Further, the rubber gasket has a through hole corresponding to the electrolytic cell, which ensures the sealing when the buffer solution contacts the working electrolyte and prevents liquid leakage.

[0011] Further, the light source is a programmable array of lamp beads, which can control the on-off state, on-off duration and brightness of any lamp bead, and can realize automatic detection by cooperating with the electrochemical workstation.

[0012] Further, the counter electrode is a thin film uniformly deposited inside the groove at the top of the electrolytic cell or a net uniformly laid inside the groove at the top of the electrolytic cell, and the material of the counter electrode is platinum or carbon.

[0013] Further, the length and width of the groove at the top of the electrolytic cell are determined according to the through hole area, and the depth of the groove is 10mm-20mm.

[0014] The detection method of the high-throughput photoelectrochemical biological detection device comprises the steps of separate incubation and centralized detection: 1) separate incubation: incubating antigen-antibody in the through hole of the separate electrolytic cell;

[0015] 2) centralized detection: adding buffer solution in the groove of the electrolytic cell, and using the computer to control the electrochemical workstation and the light source to detect the sensing signal in the through hole of the electrolytic cell one by one.

[0016] Further, the specific operation is: a small amount of buffer solution containing the antibody is added in the through hole, and incubation is performed for 30min-120min, one of the through holes is added with the buffer solution without the antibody and used as an electrode correction; after the antibody incubation is completed, the through hole is washed with the buffer solution, a large amount of the buffer solution is added to cover the through hole, and then the photoelectrochemical signals are detected one by one in batches, and recorded as a reference signal; a small amount of the buffer solution of the actual sample to be detected (which may contain the antigen) is added in the through hole, and incubation is performed for 30min-120min, one of the through holes is added with the buffer solution without the antigen and used as a blank control group; after the antigen incubation is completed, the through hole is washed with the buffer solution, and then a large amount of the buffer solution is added to cover the through hole, and then the photoelectrochemical signals are detected one by one in batches, and recorded as a sample signal.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application provides a high-throughput photoelectrochemical biological detection device and method, which adopts the scheme of uniformly depositing a thin film counter electrode in an electrolytic cell, thereby avoiding errors caused by changes in electrode distance during the test; adopts a magnetron sputtering method to prepare a single uniform working electrode with a large area, thereby reducing interference caused by different preparation batches of the working electrode material; adopts an antigen-antibody separate incubation and photoelectrochemical batch testing strategy, thereby avoiding pollution between adjacent samples, and simultaneously realizing rapid and efficient high-throughput detection; the detection device does not involve any moving mechanical parts, and has a simple structure, low cost and convenient maintenance. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Fig. 1 is a schematic diagram of a general high-throughput photoelectrochemical analysis test device according to an embodiment of the present application;

[0020] Figure 2 Fig. 4 is a schematic diagram of the coding of the lamp beads and the through holes according to an embodiment of the present application;

[0021] Figure 3 Fig. 5 is a schematic diagram of a photoelectric linkage batch detection procedure according to an embodiment of the present application. DETAILED DESCRIPTION

[0022] In order to enable personnel in the technical field to better understand the present application scheme, the present application will be further described in detail below in combination with the drawings and specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application. The high-throughput photoelectrochemical biological detection method provided in the present application will be described in detail below in combination with the detection device provided in the present application.

[0023] As Figure 1The high-flux photoelectrochemical biological detection device shown includes an embedded thin film counter electrode 1, a uniform large-area working electrode 2, a reference electrode 3, an electrolytic cell 4, a light source 5, a rubber gasket 6, a fastening bolt 7, an electrochemical workstation 8, and a computer 9, etc.; the electrolytic cell 4 has a groove opened at the top, and the counter electrode 1 and the reference electrode 3 are integrated on the inner wall of the groove; the embedded thin film counter electrode 1 is uniformly deposited or coated on the inner wall of the groove at the top of the electrolytic cell 4; the light source 5 is arranged below the electrolytic cell 4, and a plurality of through holes are arranged at the bottom of the electrolytic cell 4 to provide stable light for testing; the electrochemical workstation 8 is connected with the working electrode 2, the counter electrode, and the reference electrode 3 respectively, and transmits the detected electrical signal to the computer for processing, display, and storage.

[0024] The embedded thin film counter electrode 1 is a metal thin film or a carbon thin film uniformly deposited or coated on the bottom surface of the groove of the electrolytic cell 4, which serves to ensure that the distance between the working electrode 2 and the counter electrode 1 in each through hole at the bottom of the electrolytic cell 4 is consistent relative to any detection hole, thereby eliminating errors caused by different electrode spacings.

[0025] The uniform large-area working electrode 2 is prepared by using a magnetron sputtering technology to ensure the uniformity of materials at different positions on the working electrode and the consistency of material properties. The preparation method of the uniform large-area working electrode 2 in this embodiment adopts a preparation method of a CuV2O6-based photoelectric sensor and its application in arginine detection (CN113295744A).

[0026] As an embodiment, the electrolytic cell 4 is divided into two parts, and there are corresponding through holes at the center position, the through hole diameter is 1mm-5mm, the spacing between the through holes is 1mm-3mm, and the through hole depth is 10mm-40mm; the upper part of the through hole is used to bear the buffer solution, and the lower part of the through hole is used for light; there are corresponding bolt holes at the edge position of the electrolytic cell for installing and fixing the fastening bolt 7; the light source 5 is located below the electrolytic cell, and provides stable light for testing through the bottom through hole; the rubber gasket 6 has a through hole corresponding to the electrolytic cell, which ensures the sealing when the buffer solution 4 and the working electrolyte contact, and prevents liquid leakage; the electrochemical workstation 8 is connected with the working electrode 2, the counter electrode, and the reference electrode 3 respectively, and transmits the detected electrical signal to the computer for processing, display, and storage.

[0027] As an embodiment, the groove length and width at the top of the electrolytic cell are determined according to the through hole area, and the groove depth is 10mm-20mm.

[0028] The counter electrode is a thin film uniformly deposited inside the groove or a net uniformly laid inside the groove, and the material is platinum or carbon.

[0029] The light source 5 is a programmable control array lamp bead plate, which can control any lamp bead switch state, switch duration, lamp bead brightness, and can realize full-automatic detection through cooperation with the programming control of the electrochemical workstation; the lamp bead plate is easy to disassemble, and different wavelength light sources can be conveniently replaced. Figure 2 The lamp beads and their corresponding through holes are uniformly coded in the shown manner. Figure 3 The program shown is used for testing samples one by one. In the program, M is the maximum column number of the electrolytic cell through hole and the lamp bead array, N is the maximum row number of the electrolytic cell through hole and the lamp bead, the position of the electrolytic cell through hole and the lamp bead is numbered as (i, j), indicating that the position of the electrolytic cell through hole and the lamp bead is in the i-th column and the j-th row. The number of the currently tested through hole is determined in the form of a loop statement nested with a judgment statement, and the lamp bead with the same number is activated, the electrochemical workstation is called to detect the signal, and the final test result is named as the through hole number. The specific execution logic of the high-throughput photoelectric chemical biological detection program is as follows:

[0030] 1. The initial value of i is assigned as i = 1, and then the next operation is performed.

[0031] 2. The size relationship between i and M is judged, if i is greater than M, the program is ended, if i is less than or equal to M, the next operation is performed.

[0032] 3. The value of j is set as 1.

[0033] 4. The size relationship between j and N is judged, if j is greater than N, the value of the current i is added by 1 and is re-assigned to i, jumps to the second step, and the program is executed from the second step.

[0034] 5. The electrochemical workstation is called to start detection, the (i, j) lamp bead is called, is turned off for 1 second, is turned on for 1 second, is turned off for 1 second, the electrochemical workstation is called to stop detection, and the test result is saved as sample (i, j), and then the next operation is performed.

[0035] 6. The value of the current j is added by 1 and is re-assigned to j, jumps to the fourth step, and the program is executed from the fourth step.

[0036] The high-throughput photoelectric chemical biological detection device and method provided by the application can also be used for electrochemical biological detection, and the difference from photoelectric chemical biological detection is that only the electrochemical workstation needs to be called in the detection process, and the light source does not need to be called. The specific execution logic of the high-throughput electrochemical biological detection program is as follows:

[0037] 1. The initial value of i is assigned as i = 1, and then the next operation is performed.

[0038] 2, judge the size relationship between i and M, if i is greater than M, end the program, if i is less than or equal to M, execute the next operation.

[0039] 3, set the value of j to 1.

[0040] 4, judge the size relationship between j and N, if j is greater than N, add 1 to the current value of i and reassign i, jump to step 2, execute the program from step 2.

[0041] 5, call the electrochemical workstation to start detection, the detection time lasts for 5s, call the electrochemical workstation to stop detection, save the test results as sample (i, j), and then execute the next operation.

[0042] 6, add 1 to the current value of j and reassign j, jump to step 4, execute the program from step 4.

[0043] The antigen-antibody incubation step in the detection method is carried out in the through holes of the electrolytic cell, which aims to avoid contamination between adjacent samples; the photoelectric linkage centralized detection step is realized by programming control of the light source array and electrochemical test synchronous linkage, and the photoelectrochemical signal is centralized detected after incubation in the electrolytic cell groove to cover all the through holes, which can avoid frequent replacement of electrolyte, improve the detection speed, and realize high-throughput detection.

[0044] The detection method of the high-throughput photoelectrochemical biological detection device is realized, which includes the following steps:

[0045] 1, assemble the detection device according to Figure 1 .

[0046] 2, add a small amount of buffer solution containing antibody in the through hole of the electrolytic cell 4 to ensure sufficient contact with the working electrode 2. One hole is added with buffer solution without antibody for electrode correction.

[0047] 3, after incubation for 1h in a suitable environment, the antibody is attached to the working electrode 2. At this time, the through hole is washed with buffer solution to remove the antibody not attached to the working electrode 2.

[0048] 4, add buffer solution in the groove of the electrolytic cell 4 to ensure contact of the buffer solution with the working electrode 2, the counter electrode 1 and the reference electrode 3. Connect the working electrode 2, the counter electrode and the reference electrode 3 with the electrochemical workstation 8, start the pre-written control program, let the electrochemical workstation 8 and the light source 5 detect the photoelectric current or photoelectric voltage of each hole under the preset program, and record the baseline photoelectric current or photoelectric voltage.

[0049] 5、Remove the buffer solution, and add the actual sample to be tested into the through hole, ensuring that it is in full contact with the working electrode 2. One of the holes is added with a buffer solution without antigen, serving as a blank control group.

[0050] 6、After incubation for 1 hour in a suitable environment, the antigen and antibody are fully coupled, forming an antigen-antibody structure on the surface of the working electrode 2. At this time, the through hole is flushed with a buffer solution, with the purpose of removing the residual actual sample in the through hole.

[0051] 7、Add a buffer solution to the groove of the electrolytic cell 4, ensuring that the buffer solution is in contact with the working electrode 2, the counter electrode, and the reference electrode 3. Connect the working electrode 2, the counter electrode, the reference electrode 3, and the electrochemical workstation 8, and start the pre-written control program. Let the electrochemical workstation 8 and the light source 5 detect the photocurrent and photovoltage of each hole under the preset program, and record them as the sample photocurrent or photovoltage.

[0052] 8、After the photoelectrochemical detection is completed, the computer automatically calculates the change value of the photocurrent or photovoltage, and outputs the quantitative detection results of the sample in each through hole according to the standard curve.

[0053] The high-throughput photoelectrochemical biological detection device and method provided by the application can also be used for electrochemical biological detection. Unlike photoelectrochemical biological detection, only an electrochemical workstation needs to be called during the detection process, without the need to call a light source. The detection method of the high-throughput electrochemical biological detection device comprises the following steps:

[0054] 1、Assemble the detection device in the manner shown in Figure 1 .

[0055] 2、Add a small amount of buffer solution containing antibodies to the through hole of the electrolytic cell 4, ensuring that it is in full contact with the working electrode 2. One of the holes is added with a buffer solution without antibodies, serving as an electrode correction.

[0056] 3、After incubation for 1 hour in a suitable environment, the antibodies are attached to the working electrode 2. At this time, the through hole is flushed with a buffer solution, with the purpose of removing the antibodies that are not attached to the working electrode 2.

[0057] 4、Add a buffer solution to the groove of the electrolytic cell 4, ensuring that the buffer solution is in contact with the working electrode 2, the counter electrode 1, and the reference electrode 3. Connect the working electrode 2, the counter electrode, the reference electrode 3, and the electrochemical workstation 8, and start the pre-written control program. Let the electrochemical workstation 8 detect the current or voltage of each hole under the preset program, and record them as the reference current or voltage.

[0058] 5、Remove the buffer solution, and add the actual sample to be tested into the through hole, ensuring that it is in full contact with the working electrode 2. One of the holes is added with a buffer solution without antigen, serving as a blank control group.

[0059] 6、After 1h incubation in suitable environment, the antigen and antibody are fully coupled, and antigen-antibody structure is formed on the surface of working electrode 2. At this time, the through hole is washed with buffer solution, and the residual sample in the through hole is removed.

[0060] 7、Buffer solution is added into the groove of electrolytic cell 4, and the buffer solution is in contact with working electrode 2, counter electrode and reference electrode 3. Working electrode 2, counter electrode and reference electrode 3 are connected with electrochemical workstation 8, and the pre-written control program is started. Electrochemical workstation 8 detects the current and voltage of each hole under the preset program, and the sample current or voltage is recorded.

[0061] 8、After the electrochemical detection is completed, the computer automatically calculates the change value of current or voltage, and outputs the quantitative detection result of the sample in each through hole according to the standard curve.

[0062] The above description of the embodiments is only used to help understand the technical solutions of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principle of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A high-throughput optoelectrochemical biological detection device, characterized in that, The device comprises an in-built thin film counter electrode (1), a thin film working electrode (2), a reference electrode (3), an electrolytic cell (4), a light source (5), an electrochemical workstation (8) and a computer (9). The in-built thin film counter electrode (1) and the reference electrode (3) are integrated in the inner wall of the groove on the top of the electrolytic cell (4). The light source (5) is located below the electrolytic cell (4) and provides stable light for the thin film working electrode (2) fixed in the middle of the electrolytic cell (4) through the through holes in the bottom of the electrolytic cell. The distance between the working electrode (2) and the counter electrode in each through hole is consistent, which eliminates the error caused by different electrode spacings. The electrochemical workstation (8) is connected with the working electrode (2), the counter electrode (1) and the reference electrode (3) respectively, and transmits the detected electrical signals to the computer for processing, display and storage. The electrolytic cell (4) is divided into two parts, and there are corresponding through holes in the center. The upper through holes are used to carry the buffer solution, and the lower through holes are used for light irradiation. The light source (5) is a programmable array of lamp beads, which can control the on-off state, on-off duration and brightness of any lamp bead. In combination with the linkage with the electrochemical workstation (8), full-automatic detection is realized.

2. The high-throughput optoelectrochemical bioassay device of claim 1, wherein, The in-built thin film counter electrode (1) is a metal thin film or a carbon thin film uniformly deposited or coated on the inner wall of the groove on the top of the electrolytic cell (4).

3. The high-throughput optoelectrochemical bioassay device of claim 1, wherein, The working electrode (2) is prepared by magnetron sputtering technology.

4. The high-throughput optoelectrochemical bioassay device of claim 1, wherein, There are corresponding bolt holes in the edge position of the electrolytic cell (4) for installing the fixing bolts (7).

5. The high-throughput optoelectrochemical bioassay device of claim 1, wherein, The rubber gasket (6) has a through hole corresponding to the electrolytic cell, which ensures the sealing when the buffer solution contacts the working electrode and prevents liquid leakage.

6. The high-throughput optoelectrochemical bioassay device of claim 1, wherein, The counter electrode (1) is a thin film uniformly deposited in the groove on the top of the electrolytic cell (4) or a net uniformly laid in the groove on the top of the electrolytic cell (4). The material of the counter electrode (1) is platinum or carbon.

7. The high-throughput optoelectrochemical bioassay device according to any one of claims 2 to 6, wherein, The length and width of the groove on the top of the electrolytic cell (4) are determined according to the through hole area, and the depth of the groove is 10-20 mm.

8. A method for detecting using the high-throughput optoelectrochemical bioassay device according to claim 1, characterized in that, The device comprises separate incubation and centralized detection steps: 1) Separate incubation: incubate the antigen-antibody in the through hole of the separate electrolytic cell (4); 2) Centralized detection: add buffer solution in the groove of the electrolytic cell (4), and use the computer to control the electrochemical workstation (8) and the light source (5) to detect the sensing signals in the through holes of the electrolytic cell one by one.

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