Electrochemical multi-target joint detection electrode and preparation method thereof

Abstract

The application is suitable for the technical field of electrochemical sensing and detection, and provides an electrochemical multi-target joint detection electrode and a preparation method thereof.The joint detection electrode comprises a substrate, the substrate comprises a glass sheet, an electrode layer and an insulating film which are sequentially stacked, four working electrodes, one reference electrode and one counter electrode which are arranged on the electrode layer, and the surface of the working electrode is covalently modified with phosphorylcholine groups and carboxyl groups.The working electrode is prepared into a multi-target joint detection electrode, the detection rate of electrochemical sensing is greatly improved, and the application range is expanded.The working electrode of the joint detection electrode has a large specific surface area, compared with the electrode with a smooth surface, the oxidation-reduction current is increased, and the accessibility of the analyte is improved.The working electrodes have the same surface area, and the consistency of the working voltage applied during electrochemical joint detection is effectively ensured.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensing and detection technology, and particularly relates to an electrochemical multi-target joint detection electrode and its preparation method. Background Technology

[0002] Electrochemical detection is an important sensing technology that converts chemical energy into electrical signals, providing a sensitive and effective method for the quantitative detection of target molecules. The principle of electrochemical detection is based on electrochemical reactions, using changes in current or potential to quantitatively or qualitatively analyze chemical substances. Its core is the electrode system, typically composed of a working electrode, a reference electrode, and a counter electrode. The working electrode is the primary site of electrochemical reactions; the current is proportional to the concentration of ionized species in the solution, while the potential is related to the redox reaction. The electrochemical sensor reacts with the analyte, generating an electrical signal proportional to its concentration, thereby achieving detection. The electrochemical measurement methods used in this technology are cyclic voltammetry and chronoamperometry.

[0003] Currently, single-target electrochemical detection can only detect samples using a single label. If multiple labels need to be detected in the same sample, multiple electrodes and repeated operations are required, which not only wastes consumables but also increases time costs. Therefore, one of the challenges facing target electrochemical detection is the difficulty in achieving rapid detection of multiple labels in the same sample. How to rapidly improve the rate of electrochemical multi-target detection is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] This invention provides an electrochemical multi-target joint detection electrode and its preparation method, aiming to solve the above-mentioned problems.

[0005] This invention is implemented as follows: an electrochemical multi-target joint detection electrode, used to connect with a joint detection chip to achieve time-division multiplexing detection, includes:

[0006] The substrate comprises a glass sheet, an electrode layer, and an insulating film stacked sequentially.

[0007] Four working electrodes, one reference electrode, and one counter electrode are disposed on the electrode layer. The contacts of the four working electrodes are configured to be connected to multiple channel input terminals of the combined detection chip, respectively. The contacts of the reference electrode and the counter electrode are configured to be directly connected to an electrochemical instrument, such as the reference terminal and the counter electrode terminal of the electrochemical instrument.

[0008] By fabricating the working electrode into a multi-target electrode capable of joint detection, the detection rate of electrochemical sensing is significantly improved, and its application range is expanded. The working electrode of the joint detection electrode has a large specific surface area, which increases the redox current and improves the accessibility of analytes compared with a smooth-surfaced electrode. The working electrodes all have the same surface area, which effectively ensures the consistency of the applied working voltage during electrochemical joint detection.

[0009] Preferably, the two ends of the combined detection electrode are a contact end and a working end. The contact end consists of six electrode contacts that are in contact with the electrode adapter, and the working end consists of six electrode surfaces that are immersed in the sample solution and undergo an electrochemical reaction.

[0010] Preferably, the six electrode contacts at the contact end are in full contact with the electrode adapter, providing good contact performance. When voltage is applied or current is detected, current can flow between the electrode contacts and the electrode adapter, exhibiting good conductivity.

[0011] Preferably, the counter electrode and the working electrode form a circuit, allowing current to flow through the circuit to ensure that the required electrochemical reaction occurs on the working electrode. Since the absolute potential value of a single electrode cannot be measured, a reference electrode with a known potential value is needed to form another circuit with the working electrode to control or measure the potential of the working electrode.

[0012] Preferably, the current of the four working electrodes is detected sequentially by time-division multiplexing, and the four working electrodes share a reference electrode and a counter electrode at the same time, so that the joint detection electrode has the function of time-division multiplexing to detect the current of the working electrodes sequentially.

[0013] Preferably, during a single electrochemical detection process, the working electrode, the reference electrode, and the counter electrode operate as follows: a fixed voltage level is applied to the reference electrode as the reference potential of the electrochemical three-electrode system; a specified voltage waveform is applied to the counter electrode (a forward-biased sawtooth wave is applied for electrochemical CV detection, and a single-step waveform is applied for electrochemical CA detection), with the counter electrode serving as the voltage input channel for the three-electrode system; an electrochemical reaction occurs on the surface of the working electrode, which serves as the receiving channel for the reaction current; the counter electrode and the working electrode form a loop, allowing current to flow through the loop to ensure that the required electrochemical reaction occurs on the working electrode.

[0014] Preferably, when the joint detection electrode is switched to other working electrodes, the reference electrode is always connected to an external fixed voltage, and the voltage applied to the counter electrode is stopped, thus ensuring the potential stability of the reference electrode and the counter electrode.

[0015] Preferably, the insulating layer is laid flat on the electrode layer, which effectively restricts the flow of liquid sample and confines the liquid sample to the working electrode area of ​​the combined detection electrode, thereby increasing the stability of electrochemical detection and effectively improving the efficiency of electrochemical reaction.

[0016] Preferably, due to the larger surface area and higher volume ratio, the combined detection electrode can enhance the interaction between the metal surface and biomolecules, thereby significantly improving sensitivity and expanding the application range of electrochemical sensors.

[0017] Preferably, in order to obtain a sensing interface resistant to non-specific interference, the present invention covalently grafts the diazonium salts of 4-aminophenylphosphonic choline (PPC) and 4-aminophenylbutyric acid (PBA) onto the working electrode surface simultaneously by electrochemical reduction. The phosphorylcholine group can effectively reduce the adsorption of non-specific proteins on the electrode surface, while the carboxyl group on butyric acid can fix the capture antibody to the electrode surface through amide bonds for target recognition.

[0018] Preferably, to enable the electrodes to achieve multi-target joint detection, this invention employs a joint detection chip, specifically an 8-channel analog multiplexer chip 74HC4051, to perform multi-channel joint control of the joint detection electrodes, thereby achieving the function of multi-target joint detection. Specifically:

[0019] The 74HCT4051 chip is suitable for analog or digital 8:1 multiplexing / demultiplexing applications. The chip has three digital selection input interfaces (S0, S1 and S2), eight independent input / output interfaces (Yn), one common input / output interface (Z) and one digital enable input interface (E). When E is HIGH, the switch is off.

[0020] The four output interfaces (Y0, Y1, Y2, Y3) of the 8-channel analog multiplexer chip 74HC4051 are connected to the four working electrodes of the multi-target joint detection electrode. When an external potential is used to control the level of the three digital selection input interfaces (S0, S1, and S2) of the 74HC4051 chip, the level of the digital selection input interface of the 74HC4051 chip presents the required logic binary value. According to the function truth table of the 74HC4051 chip, at the same time, the common input / output interface (Z) of the 74HC4051 chip can be connected to one of the four output interfaces (Y0, Y1, Y2, Y3) through this binary value. By changing this binary value, the common input / output interface (Z) of the 74HC4051 chip can be switched to another interface of the four output interfaces (Y0, Y1, Y2, Y3), thereby realizing the switching process of joint detection.

[0021] Preferably, the time-division multiplexing process of the joint detection chip is as follows: by changing the level of the three control pins (S2, S1, S0) of the joint detection chip, the channel switching and control from the main channel (Z) to the four sub-channels (Y0, Y1, Y2, Y3) can be realized according to the logic truth table of the joint detection chip; when the level of the control pins (S2, S1, S0) is 000, the main channel (Z) is connected to the sub-channel Y0; when the level of the control pins (S2, S1, S0) is 001, the main channel (Z) is connected to the sub-channel Y1; when the level of the control pins (S2, S1, S0) is 010, the main channel (Z) is connected to the sub-channel Y2; when the level of the control pins (S2, S1, S0) is 011, the main channel (Z) is connected to the sub-channel Y3; by changing the above pin levels, the switching of the working electrode of the joint detection electrode can be realized by using the joint detection chip.

[0022] Preferably, in order to enable the detection of the electrochemical reaction current of the joint detection electrode using a joint detection chip, the present invention provides an electrode adapter for a multi-target joint detection electrode, which significantly improves the accuracy and stability of electrochemical reaction current detection. The electrode adapter is used to clamp the multi-target joint detection electrode to lead out wires. During operation, the interface of the electrode adapter is connected to the interface of the joint detection electrode. The interface of the electrode adapter is designed as a clamping and fixing type to ensure the stability of mechanical contact and ensure stable current transmission.

[0023] Specifically, the electrode adapter includes:

[0024] Base;

[0025] The top seat is mounted on the base, and the top seat and the base form an elastic clamping structure. Specifically, a support lug is fixed in the middle of the upper surface of the base, and the top seat is rotatably connected to the support lug in the middle. A torsion spring is installed between the top seat and the support lug.

[0026] Multiple conductor rods installed at one end of the top base are used for electrical connection with the joint detection electrode. PIN pins are installed on one side of each conductor rod. Specifically, six groups of conductor rods can be arranged side by side to connect to four working electrodes, a reference electrode, and a counter electrode, with two rods in each group.

[0027] The present invention also provides a method for preparing the above-mentioned electrochemical multi-target joint detection electrode, comprising the following steps:

[0028] Using 0.1 mol / L H2SO4 as the electrolyte, the CV curve was scanned in the range of -0.2 to +1.3 V until the curve stabilized. The surface of the joint detection electrode was then washed with deionized water.

[0029] PPC and PBA were dissolved in 0.1 mol / L HCl solution, and NaNO2 aqueous solution was added dropwise under ice bath conditions. The reaction was carried out under stirring to prepare a diazonium salt solution. Preferably, the concentration of PPC was 900 μmol / L, the concentration of PBA was 100 μmol / L, the final concentration of NaNO2 was 1 mmol / L, and the reaction time was 10 minutes.

[0030] The surface of the joint detection electrode is covered with a diazonium salt solution as an electrolyte. The CV curve is scanned once in the range of -0.8 to +0.1V (the joint detection chip controls the four working electrodes to perform one scan each). The surface of the joint detection electrode is then washed with deionized water. Preferably, the scan rate is controlled at 50mV / s.

[0031] PBS buffer containing carbodiimide (EDC) and N-hydroxysuccinimide (NHS) was dripped onto the surface of four working electrodes and incubated at room temperature to activate the carboxyl groups; preferably, the concentration of EDC was 25 mmol / L, the concentration of NHS was 40 mmol / L, and the incubation time was set to 1 hour.

[0032] Wash away EDC / NHS with PBS buffer, drop the capture antibody solutions of the four myocardial injury markers onto the surface of the four working electrodes, and refrigerate overnight to fix the capture antibodies on the electrode surface.

[0033] After washing with PBS buffer, the combined detection electrode is ready for subsequent detection.

[0034] This invention also provides an electrochemical multi-target joint detection system, comprising:

[0035] The aforementioned electrochemical multi-target joint detection electrode;

[0036] The aforementioned electrode adapter is used to hold the electrode and lead out the wire;

[0037] The joint detection chip is used to perform multi-channel joint control of the joint detection electrodes to achieve joint detection of multiple targets.

[0038] The present invention also provides a detection method based on the above-mentioned multi-target joint detection electrode, comprising the following steps:

[0039] The blood sample was mixed evenly with a horseradish peroxidase (HRP)-labeled antibody mixture (containing equal amounts of four myocardial injury marker antibodies), coated onto the surface of four working electrodes, and incubated at room temperature for a period of time; preferably, the volume of the blood sample and the antibody mixture was 100 μL each, and the incubation time was 5 minutes.

[0040] After washing the surfaces of the four working electrodes with PBS buffer, the surfaces of the combined detection electrode were covered with PBS buffer containing 2 mmol / L hydrogen peroxide (H2O2) and 0.1 mmol / L hydroxymethyl ferrocene (Fc).

[0041] Square wave voltammetry was scanned at a frequency of 20Hz in the range of 0 to +0.3V (the four working electrodes were controlled by the joint detection chip to perform one scan each), and the peak current intensity was recorded. The concentration of each myocardial marker was obtained by comparing with the standard curve.

[0042] Compared with the prior art, the embodiments of this application have the following main advantages:

[0043] This invention prepares the working electrode into a multi-target electrode that can be used for joint detection, which greatly improves the detection rate of electrochemical sensing and expands its application range.

[0044] An electrochemical sensor was fabricated using a multi-target co-detection electrode as the working electrode. The working electrode of the co-detection electrode has a large specific surface area, which increases the redox current and improves the accessibility of analytes compared to electrodes with smooth surfaces. The working electrodes all have the same surface area, which effectively ensures the consistency of the applied working voltage during electrochemical co-detection. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the structure of an electrochemical multi-target joint detection electrode provided by the present invention.

[0046] Figure 2 This is a flowchart of a method for preparing an electrochemical multi-target joint detection electrode provided by the present invention.

[0047] Figure 3 This is a schematic diagram of the structure of an electrode adapter provided by the present invention.

[0048] Figure 4 This is a schematic diagram of the structure of a combined detection chip provided by the present invention.

[0049] Figure 5 This is a connection diagram for the joint detection control of an electrochemical multi-target joint detection electrode provided by the present invention.

[0050] Figure 6 This is a standard curve diagram of the four myocardial markers CK-MB obtained in this invention.

[0051] Figure 7 This is a standard curve of the myocardial marker CTnI obtained in this invention.

[0052] Figure 8 This is a standard curve of the myocardial marker H-FBAP obtained in this invention.

[0053] Figure 9 This is a standard curve of the myocardial marker NT-proBNP obtained in this invention.

[0054] Figure 10 This is a waveform diagram of the applied electrode voltage of an electrochemical multi-target joint detection electrode provided by the present invention.

[0055] Figure 11 This invention provides a logic control truth table for a joint detection chip.

[0056] Figure 12 This is a schematic diagram of time-division multiplexing timing control of a joint detection chip provided by the present invention.

[0057] Figure 13 This is a schematic diagram of the structure of an electrochemical multi-target joint detection system provided by the present invention.

[0058] Figure label annotations: 100, Joint detection electrode; 200, Electrode adapter; 300, Joint detection chip; 1, Working electrode; 2, Reference electrode; 3, Counter electrode; 4, Base; 5, Top mount; 6, PIN pin post; 7, Conductor rod. Detailed Implementation

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0060] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0061] Example 1

[0062] This invention provides an electrochemical multi-target joint detection electrode, such as... Figure 1 As shown, it includes:

[0063] The substrate comprises a glass sheet, an electrode layer, and an insulating film stacked sequentially; the glass sheet has dimensions of 50 mm in length × 12.5 mm in width × 1.0 mm in thickness; the metal layer has a thickness of 18-22 nm, preferably 20 nm in this embodiment; the insulating film has a thickness of 148-152 nm, preferably 150 nm in this embodiment.

[0064] The electrode layer comprises four working electrodes 1, one reference electrode 2, and one counter electrode 3. Specifically, the electrode layer area of ​​each working electrode 1 is 19.98-20.02 mm². 2 In this embodiment, 20.00 mm is preferred. 2 The electrode layer area of ​​counter electrode 3 is 23.86-23.89 mm². 2 In this embodiment, the preferred diameter is 23.875 mm. 2 The electrode layer area of ​​reference electrode 2 is 3.11-3.14 mm². 2 In this embodiment, 3.125mm is preferred. 2 .

[0065] Through time-division multiplexing (time-division multiplexing timing control such as...) Figure 7 As shown, the current of the four working electrodes 1 is detected sequentially. The four working electrodes 1 share a reference electrode 2 and a counter electrode 3 at the same time, so that the joint detection electrode has the function of time-division multiplexing to detect the current of the working electrodes sequentially.

[0066] In this embodiment, the preparation method of the electrochemical multi-target joint detection electrode 100 is as follows: Figure 2 As shown, it includes the following steps:

[0067] Using 0.1 mol / L H2SO4 as the electrolyte, the CV curve was scanned in the range of -0.2 to +1.3 V until the curve stabilized, and the electrode surface was washed with deionized water.

[0068] PPC and PBA are dissolved in 0.1 mol / L HCl solution, and NaNO2 aqueous solution is added dropwise under ice bath conditions. The reaction is carried out under stirring to obtain a diazonium salt solution. The concentration of the PPC solution is 700-900 μmol / L, preferably 900 μmol / L in this embodiment; the concentration of the PBA solution is 100-300 μmol / L, preferably 100 μmol / L in this embodiment; the final concentration of the NaNO2 solution is 0.8-1.2 mmol / L, preferably 1 mmol / L in this embodiment; the reaction time is 10-15 minutes, preferably 10 minutes in this embodiment.

[0069] A diazonium salt solution was used as the electrolyte to cover the surface of the combined detection electrode (including WE, RE, CE). The CV curve was scanned once in the range of -0.8 to +0.1V (the combined detection chip controlled the four working electrodes to perform one scan each). The surface of the combined detection electrode was then washed with deionized water. The scanning rate was 50-100mV / s, and in this embodiment, it was preferably 50mV / s.

[0070] Four working electrodes were covered with PBS buffer containing carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and incubated at room temperature to activate the carboxyl groups. The concentration of the EDC solution was 20-30 mmol / L, preferably 25 mmol / L in this embodiment; the concentration of the NHS solution was 40-50 mmol / L, preferably 40 mmol / L in this embodiment; and the incubation time was 40-60 minutes, preferably 60 minutes in this embodiment.

[0071] Wash away EDC / NHS with PBS buffer, drop the capture antibody solutions of the four myocardial injury markers onto the surface of the four working electrodes, and refrigerate overnight to fix the capture antibodies onto the surface of the four working electrodes.

[0072] After washing with PBS buffer, the combined detection electrode 100 is prepared and used for subsequent detection.

[0073] The preparation and detection process of the combined detection electrode in this invention are as follows:

[0074] Based on the design dimension drawing of the combined detection electrode 100, the designed multi-target combined detection electrode was modeled in three dimensions using three-dimensional software to obtain the three-dimensional model of the multi-target combined detection electrode.

[0075] Based on the structure of the three-dimensional model of the multi-target joint detection electrode, the joint detection electrode is fabricated and modified to obtain the multi-target joint detection electrode.

[0076] Based on the interface size of the multi-target joint detection electrode, an electrode adapter 200 interface adapted to the multi-size is designed, and the overall structure of the electrode adapter 200 is fabricated to obtain the joint detection electrode adapter.

[0077] In this embodiment, as Figure 3 As shown, the electrode adapter includes:

[0078] Base 4;

[0079] The top seat 5 is installed on the base 4. The top seat 5 and the base 4 form an elastic clamping structure. Specifically, a support lug is fixed in the middle of the upper surface of the base 4. The top seat 5 is rotatably connected to the support lug in the middle. A torsion spring is installed between the top seat 5 and the support lug.

[0080] Multiple conductor rods 7 installed at one end of the top base are used for electrical connection with the joint detection electrode. PIN pin posts 6 are installed on one side of each conductor rod 7. Specifically, 6 groups of conductor rods can be arranged side by side to connect to 4 working electrodes 1, reference electrodes 2 and counter electrodes 3 respectively, with 2 rods in each group.

[0081] According to the electrode adapter interface, use wires to connect the 74HCT4051 to the detection chip 300 (e.g., Figure 4 The working interface of the multi-target joint detection electrode 100 is connected to the working interface of the electrode adapter, the joint detection electrode 100 is connected to the electrode adapter 200, and the reference electrode and counter electrode are connected to the external electrochemical instrument to obtain the hardware system of the multi-target joint detection electrode (as shown). Figure 13 As shown), the truth table of 74HC4051 is as follows: Figure 11 As shown, the timing control logic diagram of 74HC4051 is as follows: Figure 12 As shown, the connection method between 2, 74HC4051 and STM32, 6-electrode detection, and electrochemical instrument is as follows: Figure 5 As shown;

[0082] Based on the hardware system of the multi-target joint detection electrode, a control method for the joint detection electrode is designed. Based on this control method, C language program code for joint detection control based on STM32 is written to obtain the software system of the multi-target joint detection electrode.

[0083] After cleaning the electrodes with dilute sulfuric acid, using a diazonium salt solution of PPC and PBA as the electrolyte, phosphorylcholine groups and carboxyl groups were covalently modified onto the surface of the working electrodes by cyclic voltammetry. The carboxyl groups on the four working electrodes were activated by EDC / NHS and used to immobilize antibodies against four myocardial injury markers.

[0084] Securely connect the electrode adapter to the joint detection electrode interface. The joint detection chip connects to the joint detection electrode adapter and performs the adapter's on / off state.

[0085] Standards for four myocardial injury markers were tested, and standard curves were plotted. Figure 6 , Figure 7 , Figure 8 , Figure 9 The figure shows the standard curves of the four myocardial markers (CK-MB, CTnI, H-FBAP and NT-proBNP) obtained from the test.

[0086] Example 2

[0087] The difference between this embodiment and Embodiment 1 is that this embodiment provides an electrochemical multi-target joint detection electrode, such as... Figure 1 As shown, it includes:

[0088] The substrate comprises a glass sheet, an electrode layer, and an insulating film stacked sequentially; the glass sheet has dimensions of 50 mm in length × 12.5 mm in width × 1.0 mm in thickness; the metal layer has a thickness of 18 nm; and the insulating film has a thickness of 148 nm.

[0089] The electrode layer comprises four working electrodes 1, one reference electrode 2, and one counter electrode 3. Specifically, the electrode layer area of ​​each working electrode 1 is 19.98 mm². 2 The electrode layer area of ​​counter electrode 3 is 23.86 mm². 2 The electrode layer area of ​​reference electrode 2 is 3.11 mm². 2 .

[0090] Through time-division multiplexing (time-division multiplexing timing control such as...) Figure 7 As shown, the current of the four working electrodes 1 is detected sequentially. The four working electrodes 1 share a reference electrode 2 and a counter electrode 3 at the same time, so that the joint detection electrode has the function of time-division multiplexing to detect the current of the working electrodes sequentially.

[0091] In this embodiment, the preparation method of the electrochemical multi-target joint detection electrode 100 is as follows: Figure 2 As shown, it includes the following steps:

[0092] Using 0.1 mol / L H2SO4 as the electrolyte, the CV curve was scanned in the range of -0.2 to +1.3 V until the curve stabilized, and the electrode surface was washed with deionized water.

[0093] PPC and PBA were dissolved in 0.1 mol / L HCl solution, and NaNO2 aqueous solution was added dropwise under ice bath conditions. The reaction was carried out under stirring to obtain a diazonium salt solution. The concentration of the PPC solution was 850 μmol / L; the concentration of the PBA solution was 150 μmol / L; the final concentration of the NaNO2 solution was 0.8 mmol / L; and the reaction time was 10-15 minutes, preferably 10 minutes in this embodiment.

[0094] A diazonium salt solution was used as the electrolyte to cover the surface of the combined detection electrode (including WE, RE, CE). The CV curve was scanned once in the range of -0.8 to +0.1V (the combined detection chip controlled the four working electrodes to perform one scan each). The surface of the combined detection electrode was then washed with deionized water. The scanning rate was 50-100mV / s, and in this embodiment, it was preferably 50mV / s.

[0095] Four working electrodes were covered with PBS buffer containing carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and incubated at room temperature to activate the carboxyl groups; the concentration of the EDC solution was 20 mmol / L; the concentration of the NHS solution was 42 mmol / L; and the incubation time was 55 minutes.

[0096] Wash away EDC / NHS with PBS buffer, drop the capture antibody solutions of the four myocardial injury markers onto the surface of the four working electrodes, and refrigerate overnight to fix the capture antibodies onto the surface of the four working electrodes.

[0097] After washing with PBS buffer, the combined detection electrode 100 is prepared and used for subsequent detection.

[0098] Example 3

[0099] The difference between this embodiment and Embodiment 1 is that this embodiment provides an electrochemical multi-target joint detection electrode, such as... Figure 1 As shown, it includes:

[0100] The substrate comprises a glass sheet, an electrode layer, and an insulating film stacked sequentially; the glass sheet has dimensions of 50 mm in length × 12.5 mm in width × 1.0 mm in thickness; the metal layer has a thickness of 19 nm; and the insulating film has a thickness of 149 nm.

[0101] The electrode layer comprises four working electrodes 1, one reference electrode 2, and one counter electrode 3. Specifically, the electrode layer area of ​​each working electrode 1 is 19.99 mm². 2 The electrode layer area of ​​counter electrode 3 is 23.87 mm². 2 The electrode layer area of ​​reference electrode 2 is 3.12 mm². 2 .

[0102] Through time-division multiplexing (time-division multiplexing timing control such as...) Figure 7 As shown, the current of the four working electrodes 1 is detected sequentially. The four working electrodes 1 share a reference electrode 2 and a counter electrode 3 at the same time, so that the joint detection electrode has the function of time-division multiplexing to detect the current of the working electrodes sequentially.

[0103] In this embodiment, the preparation method of the electrochemical multi-target joint detection electrode 100 is as follows: Figure 2 As shown, it includes the following steps:

[0104] Using 0.1 mol / L H2SO4 as the electrolyte, the CV curve was scanned in the range of -0.2 to +1.3 V until the curve stabilized, and the electrode surface was washed with deionized water.

[0105] PPC and PBA were dissolved in 0.1 mol / L HCl solution, and NaNO2 aqueous solution was added dropwise under ice bath conditions. The reaction was carried out under stirring to obtain a diazonium salt solution. The concentration of the PPC solution was 800 μmol / L; the concentration of the PBA solution was 200 μmol / L; the final concentration of the NaNO2 solution was 0.9 mmol / L; and the reaction time was 10-15 minutes, preferably 10 minutes in this embodiment.

[0106] A diazonium salt solution was used as the electrolyte to cover the surface of the combined detection electrode (including WE, RE, CE). The CV curve was scanned once in the range of -0.8 to +0.1V (the combined detection chip controlled the four working electrodes to perform one scan each). The surface of the combined detection electrode was then washed with deionized water. The scanning rate was 50-100mV / s, and in this embodiment, it was preferably 50mV / s.

[0107] Four working electrodes were covered with PBS buffer containing carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and incubated at room temperature to activate the carboxyl groups; the concentration of the EDC solution was 22 mmol / L; the concentration of the NHS solution was 45 mmol / L; and the incubation time was 50 minutes.

[0108] Wash away EDC / NHS with PBS buffer, drop the capture antibody solutions of the four myocardial injury markers onto the surface of the four working electrodes, and refrigerate overnight to fix the capture antibodies onto the surface of the four working electrodes.

[0109] After washing with PBS buffer, the combined detection electrode 100 is prepared and used for subsequent detection.

[0110] Example 4

[0111] The difference between this embodiment and Embodiment 1 is that this embodiment provides an electrochemical multi-target joint detection electrode, such as... Figure 1 As shown, it includes:

[0112] The substrate comprises a glass sheet, an electrode layer, and an insulating film stacked sequentially; the glass sheet has dimensions of 50 mm in length × 12.5 mm in width × 1.0 mm in thickness; the metal layer has a thickness of 21 nm; and the insulating film has a thickness of 151 nm.

[0113] The electrode layer comprises four working electrodes 1, one reference electrode 2, and one counter electrode 3. Specifically, the electrode layer area of ​​each working electrode 1 is 20.01 mm². 2 The electrode layer area of ​​counter electrode 3 is 23.88 mm². 2 The electrode layer area of ​​reference electrode 2 is 3.13 mm². 2 .

[0114] Through time-division multiplexing (time-division multiplexing timing control such as...) Figure 7 As shown, the current of the four working electrodes 1 is detected sequentially. The four working electrodes 1 share a reference electrode 2 and a counter electrode 3 at the same time, so that the joint detection electrode has the function of time-division multiplexing to detect the current of the working electrodes sequentially.

[0115] In this embodiment, the preparation method of the electrochemical multi-target joint detection electrode 100 is as follows: Figure 2 As shown, it includes the following steps:

[0116] Using 0.1 mol / L H2SO4 as the electrolyte, the CV curve was scanned in the range of -0.2 to +1.3 V until the curve stabilized, and the electrode surface was washed with deionized water.

[0117] PPC and PBA were dissolved in 0.1 mol / L HCl solution, and NaNO2 aqueous solution was added dropwise under ice bath conditions. The reaction was carried out under stirring to prepare a diazonium salt solution. The concentration of the PPC solution was 750 μmol / L; the concentration of the PBA solution was 250 μmol / L; the final concentration of the NaNO2 solution was 1.1 mmol / L; and the reaction time was 10-15 minutes, preferably 10 minutes in this embodiment.

[0118] A diazonium salt solution was used as the electrolyte to cover the surface of the combined detection electrode (including WE, RE, CE). The CV curve was scanned once in the range of -0.8 to +0.1V (the combined detection chip controlled the four working electrodes to perform one scan each). The surface of the combined detection electrode was then washed with deionized water. The scanning rate was 50-100mV / s, and in this embodiment, it was preferably 50mV / s.

[0119] Four working electrodes were covered with PBS buffer containing carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and incubated at room temperature to activate the carboxyl groups; the concentration of the EDC solution was 28 mmol / L; the concentration of the NHS solution was 48 mmol / L; and the incubation time was 45 minutes.

[0120] Wash away EDC / NHS with PBS buffer, drop the capture antibody solutions of the four myocardial injury markers onto the surface of the four working electrodes, and refrigerate overnight to fix the capture antibodies onto the surface of the four working electrodes.

[0121] After washing with PBS buffer, the combined detection electrode 100 is prepared and used for subsequent detection.

[0122] Example 5

[0123] The difference between this embodiment and Embodiment 1 is that this embodiment provides an electrochemical multi-target joint detection electrode, such as... Figure 1 As shown, it includes:

[0124] The substrate comprises a glass sheet, an electrode layer, and an insulating film stacked sequentially; the glass sheet has dimensions of 50 mm in length × 12.5 mm in width × 1.0 mm in thickness; the metal layer has a thickness of 22 nm; and the insulating film has a thickness of 152 nm.

[0125] The electrode layer comprises four working electrodes 1, one reference electrode 2, and one counter electrode 3. Specifically, the electrode layer area of ​​each working electrode 1 is 20.02 mm². 2 The electrode layer area of ​​counter electrode 3 is 23.89 mm². 2 The electrode layer area of ​​reference electrode 2 is 3.14 mm². 2 .

[0126] Through time-division multiplexing (time-division multiplexing timing control such as...) Figure 7 As shown, the current of the four working electrodes 1 is detected sequentially. The four working electrodes 1 share a reference electrode 2 and a counter electrode 3 at the same time, so that the joint detection electrode has the function of time-division multiplexing to detect the current of the working electrodes sequentially.

[0127] In this embodiment, the preparation method of the electrochemical multi-target joint detection electrode 100 is as follows: Figure 2 As shown, it includes the following steps:

[0128] Using 0.1 mol / L H2SO4 as the electrolyte, the CV curve was scanned in the range of -0.2 to +1.3 V until the curve stabilized, and the electrode surface was washed with deionized water.

[0129] PPC and PBA were dissolved in 0.1 mol / L HCl solution, and NaNO2 aqueous solution was added dropwise under ice bath conditions. The reaction was carried out under stirring to obtain a diazonium salt solution. The concentration of the PPC solution was 700 μmol / L; the concentration of the PBA solution was 300 μmol / L; the final concentration of the NaNO2 solution was 1.2 mmol / L; the reaction time was 10-15 minutes, preferably 10 minutes in this embodiment.

[0130] A diazonium salt solution was used as the electrolyte to cover the surface of the combined detection electrode (including WE, RE, CE). The CV curve was scanned once in the range of -0.8 to +0.1V (the combined detection chip controlled the four working electrodes to perform one scan each). The surface of the combined detection electrode was then washed with deionized water. The scanning rate was 50-100mV / s, and in this embodiment, it was preferably 50mV / s.

[0131] Four working electrodes were covered with PBS buffer containing carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and incubated at room temperature to activate the carboxyl groups; the concentration of the EDC solution was 30 mmol / L; the concentration of the NHS solution was 50 mmol / L; and the incubation time was 40 minutes.

[0132] Wash away EDC / NHS with PBS buffer, drop the capture antibody solutions of the four myocardial injury markers onto the surface of the four working electrodes, and refrigerate overnight to fix the capture antibodies onto the surface of the four working electrodes.

[0133] After washing with PBS buffer, the combined detection electrode 100 is prepared and used for subsequent detection.

[0134] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0135] It should be understood that the disclosed apparatus can be implemented in other ways, as illustrated in the embodiments provided in this application. For example, the apparatus embodiments described above are merely illustrative; the division of units described above is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or communication connections shown or discussed may be through some interfaces; the indirect coupling or communication connections between devices or units may be telecommunications or other forms.

[0136] The units described above as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0137] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. An electrochemical multi-target joint detection electrode, used to connect with a joint detection chip to achieve time-division multiplexing detection, characterized in that, include: The substrate comprises a glass sheet, an electrode layer, and an insulating film stacked sequentially. Four working electrodes, one reference electrode, and one counter electrode are disposed on the electrode layer. The contacts of the four working electrodes are configured to be connected to multiple channel input terminals of the combined detection chip, respectively. The contacts of the reference electrode and the counter electrode are configured to be directly connected to an electrochemical instrument. The surface of the working electrode is covalently modified with phosphorylcholine groups and carboxyl groups by diazonium salt electrochemical reduction of 4-aminophenylphosphonylcholine and 4-aminophenylbutyric acid.

2. The electrochemical multi-target joint detection electrode as described in claim 1, characterized in that, The electrode layer is laid flat on the glass plate, and the insulating film is laid flat on the electrode layer.

3. The electrochemical multi-target joint detection electrode as described in claim 1, characterized in that, The working electrode and counter electrode are made of Au, and the reference electrode is made of Ag / AgCl; the area of ​​each working electrode is 19.98-20.02 mm². 2 The area of ​​the counter electrode is 23.86-23.89 mm². 2 The area of ​​the reference electrode is 3.11-3.14 mm². 2 .

4. The electrochemical multi-target joint detection electrode as described in claim 1, characterized in that, The dimensions of the glass sheet are: 50mm long × 12.5mm wide × 1.0mm thick.

5. The electrochemical multi-target joint detection electrode as described in claim 4, characterized in that, The electrode layer has a metal thickness of 18-22 nm and an insulating film thickness of 148-152 nm.

6. The method for preparing the electrochemical multi-target joint detection electrode as described in any one of claims 1-5, characterized in that, Includes the following steps: Using 0.1 mol / L H2SO4 as the electrolyte, the CV curve was scanned in the range of -0.2 to +1.3 V until the curve stabilized. The surface of the joint detection electrode was then washed with deionized water. Dissolve 700-900 μmol / L 4-aminophenylphosphocholine (PPC) and 100-300 μmol / L 4-aminophenylbutyric acid (PBA) in 0.1 mol / L HCl solution, and add NaNO2 aqueous solution dropwise under ice bath conditions to make the final concentration of NaNO2 0.8-1.2 mmol / L. React under stirring for 10-15 minutes to obtain a diazonium salt solution. The surface of the combined detection electrode was covered with a diazonium salt solution as an electrolyte. The CV curve was scanned once in the range of -0.8 to +0.1V at a rate of 50-100mV / s. The surface of the combined detection electrode was then washed with deionized water. PBS buffer containing 20-30 mmol / L carbodiimide and 40-50 mmol / L N-hydroxysuccinimide was dripped onto the surface of the four working electrodes and incubated at room temperature for 40-60 minutes to activate the carboxyl groups. Wash away carbodiimide (EDC) / N-hydroxysuccinimide (NHS) with PBS buffer, drop the capture antibody solutions of the four myocardial injury markers onto the surface of the four working electrodes, and refrigerate overnight to fix the capture antibodies on the electrode surface. After washing with PBS buffer, the combined detection electrode is ready.

7. An electrochemical multi-target joint detection system, characterized in that, include: The electrochemical multi-target joint detection electrode as described in any one of claims 1-5; Electrode adapter, used to hold electrodes and lead out wires; The joint detection chip is used to perform multi-channel joint control of the joint detection electrodes to achieve joint detection of multiple targets.

8. An electrochemical multi-target joint detection method, characterized in that, Includes the following steps: Mix 80-120 μL of blood sample with 80-120 μL of horseradish peroxidase-labeled antibody solution, coat the mixture onto the surface of 4 working electrodes, and let stand at room temperature for 5-10 minutes. After washing the surfaces of the four working electrodes with PBS buffer, the surface of the electrochemical multi-target joint detection electrode as described in any one of claims 1-5 was filled with PBS buffer containing 2 mmol / L hydrogen peroxide and 0.1 mmol / L hydroxymethyl ferrocene. Square wave voltammograms were scanned at a frequency of 20 Hz within the range of 0 to +0.3 V, and the peak current intensity was recorded. The concentrations of various myocardial markers were obtained by comparing with a standard curve.

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