Electrochemical multi-target joint detection electrode and preparation method thereof

Abstract

The invention is applicable to the technical field of electrochemical sensing 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 stacked in sequence; the four working electrodes, the reference electrode and the counter electrode are arranged on the electrode layer; the surface of the working electrode is covalently modified with a phosphorylcholine group and a carboxyl group, the working electrode is prepared into a multi-target electrode capable of joint detection, the detection rate of electrochemical sensing is greatly improved, and the application range of the multi-target electrode is expanded; the working electrode of the combined detection electrode has a large specific surface area, compared with an electrode with a smooth surface, the redox current can be increased, the accessibility of an analyte can be improved, the working electrode has the same surface area, and the consistency of applied working voltage during electrochemical combined detection is effectively guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensing detection, and in particular relates to an electrochemical multi-target combined detection electrode and a preparation method thereof. Background Art

[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, and chemical substances are quantitatively or qualitatively analyzed by measuring changes in current or potential. Its core is the electrode system, which usually consists of a working electrode, a reference electrode, and a counter electrode. The working electrode is the main site of electrochemical reactions, and the current is proportional to the concentration of ionized species in the solution, while the potential is related to the progress of the redox reaction. Electrochemical sensors react with the substance being measured to generate an electrical signal proportional to its concentration, thereby achieving detection. The electrochemical measurement methods used in this technology are cyclic voltammetry and chronoamperometry.

[0003] At present, single-target electrochemical detection can only use a single marker to detect samples. If multiple markers need to be detected in the same test sample, multiple electrodes and repeated operations are required, which not only wastes consumables but also increases time costs. Therefore, one challenge facing target electrochemical detection is the difficulty in achieving rapid detection of multiple markers in the same test sample. How to quickly increase the rate of electrochemical multi-target detection is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] The present invention provides an electrochemical multi-target combined detection electrode and a preparation method thereof, aiming to solve the above-mentioned problems.

[0005] The present invention is implemented as follows: an electrochemical multi-target joint detection electrode is used to connect with a joint detection chip to achieve time-sharing multiplexing detection, comprising: A substrate, comprising a glass sheet, an electrode layer, and an insulating film stacked in sequence; Four working electrodes, one reference electrode, and one counter electrode are provided on the electrode layer, wherein the contacts of the four working electrodes are configured to be respectively connected to multiple channel input terminals of the combined detection chip, and the contacts of the reference electrode and the counter electrode are configured to be directly connected to an electrochemical instrument, such as a reference terminal and a counter electrode terminal of the electrochemical instrument; The working electrode is prepared as a multi-target electrode capable of combined detection, which greatly improves the detection rate of electrochemical sensing and expands its application range; the working electrode of the combined detection electrode has a larger specific surface area, which will increase the redox current and improve the accessibility of the analyte compared with the electrode with a smooth surface. The working electrodes all have the same surface area, which effectively ensures the consistency of the applied working voltage during electrochemical combined detection.

[0006] Preferably, the two ends of the joint detection electrode are a contact end and a working end, the contact end is six electrode contacts in contact with the electrode adapter, and the working end is six electrode surfaces immersed in the sample solution and subjected to electrochemical reactions.

[0007] Preferably, the six electrode contacts of the contact end are in full contact with the electrode adapter, and the contact performance is good, and when a voltage is applied or a current is detected, the current can flow between the electrode contacts and the electrode adapter, and the conductivity is good.

[0008] Preferably, the counter electrode and the working electrode form a loop, so that current flows in the loop, ensuring that the required electrochemical reaction occurs on the working electrode; the absolute potential value of a single electrode cannot be measured, so a reference electrode with a known potential value is needed to form another loop with the working electrode to control or measure the potential of the working electrode.

[0009] Preferably, the current of the four working electrodes is detected in sequence by time division multiplexing, and the four working electrodes share one reference electrode and one counter electrode at the same time, so that the joint detection electrode has the function of detecting the current of the working electrodes in sequence by time division multiplexing.

[0010] Preferably, in a single electrochemical detection process, the working mode of the counter electrode, working electrode, and reference electrode is: a fixed 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), and the counter electrode serves as the voltage input channel of the three-electrode system; the electrode surface of the working electrode undergoes an electrochemical reaction, and the working electrode serves as the receiving channel of the reaction current; the counter electrode and the working electrode form a loop, so that current flows in the loop, ensuring that the required electrochemical reaction occurs on the working electrode.

[0011] Preferably, when the joint detection electrode switches to other working electrodes, the reference electrode is always externally connected to an external fixed voltage, and the voltage applied to the counter electrode is stopped, ensuring the potential stability of the reference electrode and the counter electrode.

[0012] Preferably, the insulating layer is laid on the electrode layer, effectively limiting the flow of the liquid sample, confining the liquid sample to the working electrode area of the joint detection electrode, increasing the stability of the electrochemical detection, and effectively improving the efficiency of the electrochemical reaction.

[0013] Preferably, due to the larger surface area and higher volume ratio, the joint detection electrode can enhance the interaction between the metal surface and the biomolecules, thereby significantly improving the sensitivity and expanding the application range of the electrochemical sensor.

[0014] Preferably, in order to obtain a sensing interface with resistance to nonspecific interference, the present invention simultaneously covalently grafts 4-aminophenylphosphorylcholine (PPC) and diazonium salt of 4-aminophenylbutyric acid (PBA) to the surface of the working electrode by an electrochemical reduction method. The phosphorylcholine group can effectively reduce the adsorption of nonspecific proteins on the electrode surface, while the carboxyl group on the butyric acid can fix the capture antibody to the electrode surface through an amide bond for target recognition.

[0015] Preferably, in order to enable the electrodes to realize multi-target joint detection, the present invention adopts a joint detection chip, specifically an 8-channel analog multiplexer chip 74HC4051, to perform multi-channel joint control on the joint detection electrodes to realize the function of multi-target joint detection, specifically: 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 a digital enable input interface (E). When E is HIGH, the switch is closed.

[0016] The four output interfaces (Y0, Y1, Y2, and Y3) of an 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 levels of the three digital selection input interfaces (S0, S1, and S2) of the 74HC4051 chip, the levels of the digital selection input interfaces of the 74HC4051 chip present a required logical binary value. According to the functional 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, and Y3) by using 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, and Y3) by changing this binary value, thereby realizing the switching process of joint detection.

[0017] Preferably, the process for the joint detection chip to achieve time-sharing multiplexing is as follows: by changing the levels of the three control pins (S2, S1, S0) of the joint detection chip, according to the logical truth table of the joint detection chip, channel switching and control from the main channel (Z) to four sub-channels (Y0, Y1, Y2, Y3) can be achieved; when the levels of the control pins (S2, S1, S0) are 000, the main channel (Z) is connected to the sub-channel Y0; when the levels of the control pins (S2, S1, S0) are 001, the main channel (Z) is connected to the sub-channel Y1; when the levels of the control pins (S2, S1, S0) are 010, the main channel (Z) is connected to the sub-channel Y2; when the levels of the control pins (S2, S1, S0) are 011, the main channel (Z) is connected to the sub-channel Y3; by changing the levels of the above pins, the joint detection chip can be used to control the switching of the working electrodes of the joint detection electrode.

[0018] Preferably, in order to be able to use the combined detection chip to detect the electrochemical reaction current of the combined detection electrode, the present invention provides an electrode adapter for a multi-target combined detection electrode, which greatly improves the accuracy and stability of the electrochemical reaction current detection. The electrode adapter is used to clamp the multi-target combined detection electrode to lead out the wire. During operation, the interface of the electrode adapter is connected to the interface of the combined detection electrode. The interface of the electrode adapter is designed to be clamped and fixed to ensure the stability of the mechanical contact and ensure stable current transmission. Specifically, the electrode adapter includes: base; A top seat mounted on the base, the top seat and the base forming an elastic clamping structure, specifically, a lug is fixed to the middle of the upper surface of the base, the middle of the top seat is rotatably connected to the lug, and a torsion spring is installed between the top seat and the lug; Multiple conductor rods installed at one end of the top seat are used to electrically connect to the joint detection electrode. A PIN pin column is installed on one side of the conductor rod. Specifically, 6 groups of conductor rods can be arranged side by side to respectively connect 4 working electrodes, a reference electrode and a counter electrode, with 2 rods in each group.

[0019] The present invention also provides a method for preparing the electrochemical multi-target combined detection electrode, comprising the following steps: Use 0.1 mol / L H2SO4 as the electrolyte and scan the CV curve in the range of -0.2~+1.3V until the curve is stable. Wash the surface of the combined detection electrode with deionized water. Dissolve PPC and PBA in a 0.1 mol / L HCl solution, add a NaNO2 aqueous solution dropwise in an ice bath, and react under stirring to prepare a diazonium salt solution; preferably, the concentration of PPC is 900 μmol / L, the concentration of PBA is 100 μmol / L, the final concentration of NaNO2 is 1 mmol / L, and the reaction time is 10 minutes; A diazonium salt solution was dripped onto the surface of the combined detection electrode as an electrolyte, and the CV curve was scanned for one cycle in the range of -0.8 to +0.1 V (the combined detection chip controls each of the four working electrodes to perform one scan), and the surface of the combined detection electrode was washed with deionized water; preferably, the scan rate was controlled to be 50 mV / s; PBS buffer containing EDC and N-hydroxysuccinimide (NHS) is dripped onto the surface of four working electrodes and incubated at room temperature to activate the carboxyl groups; preferably, the concentration of EDC is 25 mmol / L, the concentration of NHS is 40 mmol / L, and the incubation time is set to 1 hour; EDC / NHS was washed off with PBS buffer, and capture antibody solutions of four myocardial injury markers were dropped onto the surfaces of four working electrodes respectively. The solution was refrigerated overnight to allow the capture antibodies to be fixed on the electrode surfaces. After washing with PBS buffer, the combined detection electrode is prepared and used for subsequent detection.

[0020] The present invention also provides an electrochemical multi-target joint detection system, comprising: The above-mentioned electrochemical multi-target combined detection electrode; The aforementioned electrode adapter is used to clamp the electrode and lead out the wire; The combined detection chip is used to perform multi-channel combined control of combined detection electrodes to achieve multi-target combined detection.

[0021] The present invention also provides a detection method based on the above-mentioned multi-target joint detection electrode, comprising the following steps: Mix the blood sample and a horseradish peroxidase (HRP)-labeled antibody mixture (containing equal amounts of four myocardial injury marker antibodies) evenly, apply it to the surface of four working electrodes, and incubate at room temperature for a period of time; preferably, the volume of the blood sample and the antibody mixture is 100 μL each, and the incubation time is 5 minutes; After washing the surfaces of the four working electrodes with PBS buffer, PBS buffer containing 2 mmol / L hydrogen peroxide (H2O2) and 0.1 mmol / L hydroxymethylferrocene (Fc) was dripped onto the surface of the combined detection electrode; The square wave voltammogram was scanned at a frequency of 20 Hz in the range of 0~+0.3V (the combined detection chip controlled the four working electrodes to perform one scan each), the peak current intensity was recorded, and the concentration of each myocardial marker was obtained by comparing it with the standard curve.

[0022] Compared with the prior art, the embodiments of the present application have the following beneficial effects: The present invention prepares the working electrode into a multi-target electrode capable of joint detection, greatly improving the detection rate of electrochemical sensing and expanding its application range.

[0023] The multi-target combined detection electrode is used as the working electrode to prepare an electrochemical sensor. The working electrode of the combined detection electrode has a larger specific surface area, which will increase the redox current and improve the accessibility of the analyte compared with the electrode with a smooth surface. The working electrodes all have the same surface area, which effectively ensures the consistency of the applied working voltage during electrochemical combined detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a structural schematic diagram of an electrochemical multi-target combined detection electrode provided by the present invention.

[0025] Figure 2 This is a flow chart of a method for preparing an electrochemical multi-target combined detection electrode provided by the present invention.

[0026] Figure 3 It is a structural schematic diagram of an electrode adapter provided by the present invention.

[0027] Figure 4 It is a structural schematic diagram of a combined detection chip provided by the present invention.

[0028] Figure 5 This is a joint detection control connection diagram of an electrochemical multi-target joint detection electrode provided by the present invention.

[0029] Figure 6 This is a standard curve diagram of the four myocardial markers CK-MB tested in the present invention.

[0030] Figure 7 It is a standard curve diagram of the myocardial marker CTnI tested in the present invention.

[0031] Figure 8 It is a standard curve diagram of the myocardial marker H-FBAP tested in the present invention.

[0032] Figure 9 It is a standard curve diagram of the myocardial marker NT-proBNP tested in the present invention.

[0033] Figure 10 This is a waveform diagram of the voltage applied to the electrode of an electrochemical multi-target combined detection electrode provided by the present invention.

[0034] Figure 11 This is a logic control truth table of a joint detection chip provided by the present invention.

[0035] Figure 12 This is a schematic diagram of time-division multiplexing timing control of a joint detection chip provided by the present invention.

[0036] Figure 13This is a structural schematic diagram of an electrochemical multi-target combined detection system provided by the present invention.

[0037] Notes on the accompanying drawings: 100, combined detection electrode; 200, electrode adapter; 300, combined detection chip; 1, working electrode; 2, reference electrode; 3, counter electrode; 4, base; 5, top seat; 6, PIN pin column; 7, conductor rod. DETAILED DESCRIPTION

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The terms used in the specification of the application are only for the purpose of describing specific embodiments and are not intended to limit this application. The terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0039] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0040] Example 1 The embodiment of the present invention provides an electrochemical multi-target joint detection electrode, such as Figure 1 Shown, including: A substrate comprising a glass sheet, an electrode layer, and an insulating film stacked in sequence; the glass sheet has dimensions of 50 mm in length, 12.5 mm in width, and 1.0 mm in thickness; the metal layer has a thickness of 18-22 nm, preferably 20 nm in this embodiment; and the insulating film has a thickness of 148-152 nm, preferably 150 nm in this embodiment. Four working electrodes 1, a reference electrode 2 and a counter electrode 3 are provided on the electrode layer. Specifically, the electrode layer area of ​​each working electrode 1 is 19.98-20.02 mm 2 , in this embodiment, preferably 20.00mm 2 ; The electrode layer area of ​​electrode 3 is 23.86-23.89mm 2 , in this embodiment, preferably 23.875mm 2 ; The electrode layer area of ​​reference electrode 2 is 3.11-3.14mm 2, in this embodiment, preferably 3.125mm 2 .

[0041] Through time division multiplexing (time division multiplexing timing control such as Figure 7 As shown in FIG, the current of the four working electrodes 1 is detected in sequence. 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-sharing multiplexing to detect the current of the working electrodes in sequence.

[0042] In this embodiment, the preparation method of the electrochemical multi-target combined detection electrode 100 is as follows: Figure 2 As shown, the following steps are included: Use 0.1 mol / L H2SO4 as the electrolyte, scan the CV curve in the range of -0.2~+1.3V until the graph is stable, and wash the electrode surface with deionized water; PPC and PBA are dissolved in a 0.1 mol / L HCl solution, and a NaNO2 aqueous solution is added dropwise in an ice bath, and reacted under stirring to prepare 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; A diazonium salt solution was dripped onto the surface of the combined detection electrode (including WE, RE, and CE) as an electrolyte, and the CV curve was scanned for one cycle in the range of -0.8 to +0.1 V (the combined detection chip controls each of the four working electrodes to perform one scan), and the surface of the combined detection electrode was rinsed with deionized water; the scan rate was 50-100 mV / s, preferably 50 mV / s in this embodiment; A PBS buffer solution containing EDC and N-hydroxysuccinimide (NHS) was dripped onto the surfaces of four working electrodes 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. The incubation time was 40-60 minutes, preferably 60 minutes in this embodiment. EDC / NHS was washed off with PBS buffer, and capture antibody solutions of four myocardial injury markers were dropped onto the surfaces of four working electrodes respectively. The solution was refrigerated overnight to allow the capture antibodies to be fixed on the surfaces of the four working electrodes. After washing with PBS buffer, the combined detection electrode 100 is completed and used for subsequent detection.

[0043] The preparation and detection process of the combined detection electrode in the present invention are as follows: According to the design dimension drawing of the combined detection electrode 100, a three-dimensional model of the designed multi-target combined detection electrode is performed using three-dimensional software to obtain a three-dimensional model of the multi-target combined detection electrode; According to the structure of the three-dimensional model of the multi-target joint detection electrode, the joint detection electrode is processed and prepared, and the multi-target joint detection electrode is modified to obtain the multi-target joint detection electrode; According to the interface size of the multi-target joint detection electrode, an electrode adapter 200 interface adapted to the multiple sizes is designed, and the overall structure of the electrode adapter 200 is processed and prepared to obtain a joint detection electrode adapter; In this embodiment, Figure 3 As shown, the electrode adapter includes: Base 4; A top seat 5 is mounted on the base 4. The top seat 5 and the base 4 form an elastic clamping structure. Specifically, a lug is fixed to the middle of the upper surface of the base 4. The middle of the top seat 5 is rotatably connected to the lug, and a torsion spring is installed between the top seat 5 and the lug. A plurality of conductor rods 7 are installed at one end of the top seat for electrically connecting to the joint detection electrode. A PIN pin column 6 is installed on one side of the conductor rod 7. Specifically, 6 groups of conductor rods can be arranged side by side to respectively connect to 4 working electrodes 1, reference electrode 2 and counter electrode 3, with 2 rods in each group.

[0044] According to the interface of the electrode adapter, use the wire to connect the 74HCT4051 joint detection chip 300 (such as Figure 4 The working interface of the electrode adapter is connected to the working interface of the electrode adapter, the combined detection electrode 100 is connected to the electrode adapter 200, and the reference electrode and the counter electrode are connected to the external electrochemical instrument to obtain the hardware system of the multi-target combined detection electrode (as shown in FIG. 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 in Figure 2, the connection method between 74HC4051, STM32, 6 joint detection electrodes and electrochemical instrument is as follows: Figure 5 As shown; According to the hardware system of the multi-target joint detection electrode, a control method of the joint detection electrode is designed, and based on the control method, a 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; Using dilute sulfuric acid-cleaned electrodes and diazonium salt solutions of PPC and PBA as electrolytes, cyclic voltammetry was used to covalently modify phosphorylcholine and carboxyl groups onto the working electrode surface. Four carboxyl groups on the working electrodes were activated with EDC / NHS and then used to immobilize antibodies against four myocardial injury markers. Put the electrode adapter firmly in contact with the joint detection electrode interface, and the joint detection chip connects to the joint detection electrode adapter and performs the conduction and cutoff of the adapter; The standards of four myocardial injury markers were tested and standard curves were drawn, such as Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 As shown in the figure, the standard curves of the four myocardial markers (CK-MB, CTnI, H-FBAP and NT-proBNP) obtained by testing.

[0045] Example 2 The difference between this embodiment and embodiment 1 is that the embodiment of the present invention provides an electrochemical multi-target joint detection electrode, such as Figure 1 Shown, including:

[0046] A substrate comprising a glass sheet, an electrode layer, and an insulating film stacked in sequence; the glass sheet has dimensions of 50 mm in length, 12.5 mm in width, and 1.0 mm in thickness; the metal layer has a thickness of 18 nm; and the insulating film has a thickness of 148 nm. Four working electrodes 1, a reference electrode 2 and a counter electrode 3 are provided on the electrode layer. Specifically, the electrode layer area of ​​each working electrode 1 is 19.98 mm 2 ; The electrode layer area of ​​electrode 3 is 23.86mm 2 The electrode layer area of ​​reference electrode 2 is 3.11mm 2 .

[0047] Through time division multiplexing (time division multiplexing timing control such as Figure 7 As shown in FIG, the current of the four working electrodes 1 is detected in sequence. 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-sharing multiplexing to detect the current of the working electrodes in sequence.

[0048] In this embodiment, the preparation method of the electrochemical multi-target combined detection electrode 100 is as follows: Figure 2 As shown, the following steps are included: Use 0.1 mol / L H2SO4 as the electrolyte, scan the CV curve in the range of -0.2~+1.3V until the graph is stable, and wash the electrode surface with deionized water; PPC and PBA were dissolved in a 0.1 mol / L HCl solution, and a NaNO2 aqueous solution was added dropwise in an ice bath, and reacted under stirring to prepare 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; the reaction time was 10 to 15 minutes, preferably 10 minutes in this embodiment; A diazonium salt solution was dripped onto the surface of the combined detection electrode (including WE, RE, and CE) as an electrolyte, and the CV curve was scanned for one cycle in the range of -0.8 to +0.1 V (the combined detection chip controls each of the four working electrodes to perform one scan), and the surface of the combined detection electrode was rinsed with deionized water; the scan rate was 50-100 mV / s, preferably 50 mV / s in this embodiment; PBS buffer containing EDC and N-hydroxysuccinimide (NHS) was dripped onto the surfaces of four working electrodes 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; EDC / NHS was washed off with PBS buffer, and capture antibody solutions of four myocardial injury markers were dropped onto the surfaces of four working electrodes respectively. The solution was refrigerated overnight to allow the capture antibodies to be fixed on the surfaces of the four working electrodes. After washing with PBS buffer, the combined detection electrode 100 is completed and used for subsequent detection.

[0049] Example 3 The difference between this embodiment and embodiment 1 is that the embodiment of the present invention provides an electrochemical multi-target joint detection electrode, such as Figure 1 Shown, including: A substrate comprising a glass sheet, an electrode layer, and an insulating film stacked in sequence; the glass sheet has dimensions of 50 mm in length, 12.5 mm in width, and 1.0 mm in thickness; the metal layer has a thickness of 19 nm; and the insulating film has a thickness of 149 nm; Four working electrodes 1, a reference electrode 2 and a counter electrode 3 are provided on the electrode layer. Specifically, the electrode layer area of ​​each working electrode 1 is 19.99 mm 2 ; The electrode layer area of ​​electrode 3 is 23.87mm 2 The electrode layer area of ​​reference electrode 2 is 3.12 mm 2 .

[0050] Through time division multiplexing (time division multiplexing timing control such as Figure 7As shown in FIG, the current of the four working electrodes 1 is detected in sequence. 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-sharing multiplexing to detect the current of the working electrodes in sequence.

[0051] In this embodiment, the preparation method of the electrochemical multi-target combined detection electrode 100 is as follows: Figure 2 As shown, the following steps are included: Use 0.1 mol / L H2SO4 as the electrolyte, scan the CV curve in the range of -0.2~+1.3V until the graph is stable, and wash the electrode surface with deionized water; PPC and PBA were dissolved in a 0.1 mol / L HCl solution, and a NaNO2 aqueous solution was added dropwise under ice bath conditions, and reacted under stirring to prepare 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; the reaction time was 10 to 15 minutes, preferably 10 minutes in this embodiment; A diazonium salt solution was dripped onto the surface of the combined detection electrode (including WE, RE, and CE) as an electrolyte, and the CV curve was scanned for one cycle in the range of -0.8 to +0.1 V (the combined detection chip controls each of the four working electrodes to perform one scan), and the surface of the combined detection electrode was rinsed with deionized water; the scan rate was 50-100 mV / s, preferably 50 mV / s in this embodiment; PBS buffer containing EDC and N-hydroxysuccinimide (NHS) was dripped onto the surfaces of four working electrodes 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; EDC / NHS was washed off with PBS buffer, and capture antibody solutions of four myocardial injury markers were dropped onto the surfaces of four working electrodes respectively. The solution was refrigerated overnight to allow the capture antibodies to be fixed on the surfaces of the four working electrodes. After washing with PBS buffer, the combined detection electrode 100 is completed and used for subsequent detection.

[0052] Example 4 The difference between this embodiment and embodiment 1 is that the embodiment of the present invention provides an electrochemical multi-target joint detection electrode, such as Figure 1 Shown, including: A substrate comprising a glass sheet, an electrode layer, and an insulating film stacked in sequence; the glass sheet has dimensions of 50 mm in length, 12.5 mm in width, and 1.0 mm in thickness; the metal layer has a thickness of 21 nm; and the insulating film has a thickness of 151 nm; Four working electrodes 1, one reference electrode 2 and one counter electrode 3 are arranged on the electrode layer, and specifically, the electrode layer area of each working electrode 1 is 20.01mm 2 ; the electrode layer area of the counter electrode 3 is 23.88mm 2 ; and the electrode layer area of the reference electrode 2 is 3.13mm 2 .

[0053] The current of the four working electrodes 1 is detected in sequence through time division multiplexing (the time sequence control of time division multiplexing is shown in Figure 7 ), and the four working electrodes 1 share one reference electrode 2 and one counter electrode 3 at the same time, so that the combined detection electrode has the function of sequentially detecting the current of the working electrode through time division multiplexing.

[0054] In the embodiment, the preparation method of the electrochemical multi-target combined detection electrode 100, as shown in Figure 2 , includes the following steps: 0.1mol / L H2SO4 as electrolyte, CV curve scanning in-0.2~+1.3V range to the graph stable, electrode surface with deionized water clean; Dissolve PPC and PBA in 0.1mol / L HCl solution, drop NaNO2 aqueous solution under ice bath condition, react under stirring condition to prepare diazonium salt solution; the concentration of PPC solution is 750μmol / L; the concentration of PBA solution is 250μmol / L; the final concentration of NaNO2 solution is 1.1mmol / L; the reaction time is 10-15 minutes, and 10 minutes is preferred in the embodiment; Diazonium salt solution as electrolyte drop full combined detection electrode (including WE, RE, CE) surface, CV curve scanning in-0.8~+0.1V range for one circle (combined detection chip control four working electrodes to scan respectively once), combined detection electrode surface with deionized water clean; the scanning rate is 50-100mV / s, and 50mV / s is preferred in the embodiment; Drop PBS buffer solution containing carbodiimide (EDC) and N-hydroxysuccinimide (NHS) on the surface of the four working electrodes, and incubate at room temperature to activate the carboxyl group; the concentration of EDC solution is 28mmol / L; the concentration of NHS solution is 48mmol / L; and the incubation time is 45 minutes; Wash away EDC / NHS with PBS buffer solution, and drop the capture antibody solution of four kinds of myocardial injury markers on the surface of the four working electrodes respectively, and store overnight in cold storage to fix the capture antibody on the surface of the four working electrodes; After washing with PBS buffer solution, the combined detection electrode 100 is prepared and used for subsequent detection.

[0055] Example 5 The difference between this embodiment and embodiment 1 is that the embodiment of the present invention provides an electrochemical multi-target joint detection electrode, such as Figure 1 Shown, including: A substrate comprising a glass sheet, an electrode layer, and an insulating film stacked in sequence; the glass sheet has dimensions of 50 mm in length, 12.5 mm in width, and 1.0 mm in thickness; the metal layer has a thickness of 22 nm; and the insulating film has a thickness of 152 nm; Four working electrodes 1, a reference electrode 2 and a counter electrode 3 are provided on the electrode layer. Specifically, the electrode layer area of ​​each working electrode 1 is 20.02 mm 2 The electrode layer area of ​​electrode 3 is 23.89mm 2 ; The electrode layer area of ​​reference electrode 2 is 3.14mm 2 .

[0056] Through time division multiplexing (time division multiplexing timing control such as Figure 7 As shown in FIG, the current of the four working electrodes 1 is detected in sequence. 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-sharing multiplexing to detect the current of the working electrodes in sequence.

[0057] In this embodiment, the preparation method of the electrochemical multi-target combined detection electrode 100 is as follows: Figure 2 As shown, the following steps are included: Use 0.1 mol / L H2SO4 as the electrolyte, scan the CV curve in the range of -0.2~+1.3V until the graph is stable, and wash the electrode surface with deionized water; PPC and PBA were dissolved in a 0.1 mol / L HCl solution, and a NaNO2 aqueous solution was added dropwise in an ice bath, and reacted under stirring to prepare a diazonium salt solution; the concentration of the PPC solution was 700 μmol / L; the concentration of the PBA solution was 300 μmol / L; and the final concentration of the NaNO2 solution was 1.2 mmol / L; the reaction time was 10 to 15 minutes, preferably 10 minutes in this embodiment; A diazonium salt solution was dripped onto the surface of the combined detection electrode (including WE, RE, and CE) as an electrolyte, and the CV curve was scanned for one cycle in the range of -0.8 to +0.1 V (the combined detection chip controls each of the four working electrodes to perform one scan), and the surface of the combined detection electrode was rinsed with deionized water; the scan rate was 50-100 mV / s, preferably 50 mV / s in this embodiment; PBS buffer containing EDC and N-hydroxysuccinimide (NHS) was dripped onto the surfaces of four working electrodes 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; EDC / NHS was washed off with PBS buffer, and capture antibody solutions of four myocardial injury markers were dropped onto the surfaces of four working electrodes respectively. The solution was refrigerated overnight to allow the capture antibodies to be fixed on the surfaces of the four working electrodes. After washing with PBS buffer, the combined detection electrode 100 is completed and used for subsequent detection.

[0058] It should be noted that, for the sake of simplicity, the aforementioned embodiments are described as a series of action combinations. However, those skilled in the art should be aware that the present invention is not limited by the order of the actions described, because according to the present invention, certain steps may be performed in other orders or simultaneously. Secondly, those skilled in the art should also be aware that the embodiments described in this specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present invention.

[0059] In the embodiments provided in this application, it should be understood that the disclosed devices can be implemented in other ways. For example, the device embodiments described above are merely schematic. For example, the division of the above-mentioned units is only a logical function division. In actual implementation, there may be other division methods, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or communication connection shown or discussed can be through some interfaces, and the indirect coupling or communication connection between devices or units can be in the form of telecommunications or other forms.

[0060] The units described above as separate components may or may not be physically separate, and 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 these units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work 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, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and 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 to a joint detection chip to achieve time-sharing multiplexing detection, characterized in that: include: A substrate, comprising a glass sheet, an electrode layer, and an insulating film stacked in sequence; Four working electrodes, one reference electrode, and one counter electrode are provided on the electrode layer, wherein the contacts of the four working electrodes are configured to be respectively connected to multiple channel input terminals of the combined detection chip, and 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 a phosphorylcholine group and a carboxyl group.

2. The electrochemical multi-target joint detection electrode according to claim 1, characterized in that: The electrode layer is laid flat on the glass sheet, and the insulating layer is laid flat on the electrode layer.

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

4. The electrochemical multi-target joint detection electrode according to claim 1, characterized in that: The glass sheet has the following dimensions: length 50 mm×width 12.5 mm×thickness 1.0 mm.

5. The electrochemical multi-target joint detection electrode according to claim 4, characterized in that: The metal thickness of the electrode layer is 18-22 nm, and the thickness of the insulating film is 148-152 nm.

6. The method for preparing the electrochemical multi-target combined detection electrode according to any one of claims 1 to 5, characterized in that: The steps include: Use 0.1 mol / L H2SO4 as the electrolyte and scan the CV curve in the range of -0.2~+1.3V until the curve is stable. Wash the surface of the combined detection electrode with deionized water. Dissolve 700-900 μmol / L PPC and 100-300 μmol / L PBA in 0.1 mol / L HCl solution. Add NaNO2 aqueous solution dropwise in an ice bath to a final concentration of 0.8-1.2 mmol / L. Stir and react for 10-15 minutes to obtain a diazonium salt solution. The diazonium salt solution was dripped onto the surface of the combined detection electrode as the electrolyte, and the CV curve was scanned for one cycle in the range of -0.8~+0.1V at a rate of 50-100mV / s. The surface of the combined detection electrode was washed with deionized water; Drop PBS buffer containing 20-30 mmol / L carbodiimide and 40-50 mmol / L N-hydroxysuccinimide onto the surface of four working electrodes and incubate at room temperature for 40-60 minutes to activate the carboxyl groups. EDC / NHS was washed off with PBS buffer, and capture antibody solutions of four myocardial injury markers were dropped onto the surfaces of four working electrodes respectively. The solution was refrigerated overnight to allow the capture antibodies to be fixed on the electrode surfaces. After washing with PBS buffer, the combined detection electrode is prepared.

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

8. An electrochemical multi-target joint detection method, characterized in that: The steps include: Mix 80-120 μL of blood sample and 80-120 μL of horseradish peroxidase-labeled antibody mixture, apply it on the surface of 4 working electrodes, and let it stand at room temperature for 5-10 minutes; After washing the surfaces of the four working electrodes with PBS buffer, PBS buffer containing 2 mmol / L hydrogen peroxide and 0.1 mmol / L hydroxymethylferrocene was dripped onto the surface of the combined detection electrode; The square wave voltammogram was scanned in the range of 0~+0.3V at a frequency of 20Hz, and the peak current intensity was recorded. The concentration of each myocardial marker was obtained by comparing it with the standard curve.

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