An Electrochemical Immunosensing Device and Method for Simultaneously Detecting PLGF and sFlt-1

Abstract

The present invention discloses an electrochemical immunosensing device and method for simultaneously detecting PLGF and sFlt-1, which relates to the technical field of electrochemical biosensors. The device includes a microprocessor module, a power supply module, an analog-to-digital conversion module, a digital-to-analog conversion module, a communication module and a reaction cavity integrated on a circuit board; the analog-to-digital conversion module and the communication module are respectively connected to the microprocessor module; the connection contact of the circuit board excitation electrode unit on the circuit board is connected to the excitation electrode unit, and the excitation electrode unit includes a first working electrode, a second working electrode, a first counter electrode, a second counter electrode and a reference electrode; the first working electrode and the second working electrode, as carriers for binding biomarkers, are respectively located in two independent reaction cavities. The present invention can accurately output the detection result on-site and can visually output the sFlt-1 / PLGF ratio in the sample, and determine the dynamic balance of the two biomarkers according to the concentration ratio. The result of the dynamic balance can assist and improve the prediction ability of preeclampsia in clinical practice.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical biosensors, and particularly to an electrochemical immunosensing device and method for simultaneously detecting PLGF and sFlt-1. Background Art

[0002] Preeclampsia is a disease that presents with symptoms of proteinuria and hypertension after 20 weeks of pregnancy, seriously endangering the lives of pregnant women. Therefore, how to accurately predict preeclampsia has become an urgent problem to be solved.

[0003] Abnormal signal pathways of placental growth factor (PLGF) and soluble FMS-like tyrosine kinase-1 (sFlt-1) can affect angiogenesis, reduce the invasion of trophoblasts into the endometrium, and thus induce preeclampsia, threatening the lives of pregnant women.

[0004] Due to the large concentration difference between PLGF and sFlt-1 in the human body, traditional detection methods need to use ELISA kits to detect them separately, which have limitations such as long detection time and inability to output detection results synchronously. Summary of the Invention

[0005] To solve the above problems, the present invention provides an electrochemical immunosensing device and method for simultaneously detecting PLGF and sFlt-1. A dual-channel electrochemical immunosensor based on a smartphone simultaneously detects PLGF and sFlt-1 in a blood sample, studies their signal pathways, and evaluates the risk of preeclampsia.

[0006] To achieve the above object, the present invention provides an electrochemical immunosensing device for simultaneously detecting PLGF and sFlt-1, including a microprocessor module, a power supply module, an analog-to-digital conversion module, a digital-to-analog conversion module, and a communication module integrated on the same circuit board; the analog-to-digital conversion module and the communication module are respectively connected to the microprocessor module.

[0007] The circuit board is provided with connection contacts for a circuit board excitation electrode unit, and the connection contacts for the circuit board excitation electrode unit are connected to the excitation electrode unit. The excitation electrode unit includes a first working electrode, a second working electrode, a first counter electrode, a second counter electrode, and a reference electrode; the first working electrode and the second working electrode, as carriers for binding markers, are respectively located in two independent reaction chambers.

[0008] Preferably, an amplifier is further included, which is electrically connected to the input end of the analog-to-digital conversion module. The microprocessor module collects and transmits the signal output by the analog-to-digital conversion module. The amplifier, the analog-to-digital conversion module, and the digital-to-analog conversion module are used to control and output the voltage required for the sensing device to perform substance detection.

[0009] Preferably, the input ends of the first working electrode, the second working electrode, the first pair of electrodes, and the second pair of electrodes are respectively electrically connected to the output end of the digital-to-analog conversion module; the input end of the reference electrode is electrically connected to the output end of the digital-to-analog conversion module.

[0010] Preferably, a power supply module and an LED lamp are further provided on the circuit board. The output end of the power supply module is electrically connected to the input end of the LED lamp. The microprocessor module sets the light-emitting mode of the LED lamp to observe whether the circuit is normal.

[0011] Preferably, the communication module is a Bluetooth module, and the Bluetooth module establishes communication with the mobile terminal to realize the real-time transmission of the detection result.

[0012] Preferably, both the first working electrode and the second working electrode include a substrate, a sensitive modification layer, and a functional modification layer;

[0013] The substrate is a low-resistance conductive ink, the sensitive modification layer is a composite nanomaterial of carboxylated graphene and gold nanoparticles. The carboxylated graphene and the composite nanomaterial of gold nanoparticles are carboxyl-activated. After the carboxyl functional groups of the first working electrode and the second working electrode are activated, they are covalently bonded to the PLGF antibody and the sFlt-1 antibody respectively to form a functional modification layer.

[0014] Preferably, the power supply module includes a power management unit and a battery, and the input end of the power management unit is electrically connected to the output end of the battery.

[0015] Preferably, a DC-DC converter is further included. The output end of the power supply module is electrically connected to the input end of the DC-DC converter. The power supply module outputs a voltage of 3.7V. The output end of the DC-DC converter is electrically connected to the circuit board. The input end of the power supply module is electrically connected to the circuit board to adjust the voltage to meet the different voltage requirements of each chip inside the circuit board;

[0016] The output ends of the DC-DC converter, the power supply module, the digital-to-analog conversion module, the analog-to-digital conversion module, and the power supply module together with the DC-DC converter form a power management module to stably adjust the output voltage within the range of -5V to 3.3V.

[0017] A method for using an electrochemical immunosensing device for simultaneously detecting PLGF and sFlt-1 as described above includes the following steps:

[0018] S1. Establish a standard curve

[0019] The standard solution is dripped onto the first working electrode and the second working electrode; the power supply module is turned on, and the main control chip controls the power management unit to output a scanning potential, which is output to the first working electrode and the second working electrode through the digital-to-analog conversion module and the power management unit to apply an excitation to the solution to be measured;

[0020] The voltage signal output by the main control chip is received through the analog-to-digital conversion module, the change of the output voltage is controlled, the voltage-current relationship is obtained, and the current change-marker concentration curve is established according to the voltage-current relationship and the standard solution concentration; the current change refers to the change of the peak current of the first working electrode and the second working electrode;

[0021] S2. The solution to be measured is dripped onto the first working electrode and the second working electrode for detecting the marker concentration, the marker concentration is obtained according to the output voltage-current relationship and the standard curve established in step S1, and the information is transmitted to the connected mobile terminal through the communication module.

[0022] The above-mentioned electrochemical immunosensing device and method for simultaneously detecting PLGF and sFlt-1 of the present invention have the following technical effects:

[0023] (1) The device of the present invention is a portable and simplified electrochemical workstation, and the whole device can perform biological detection, recording and analysis of the detection results of biological markers with a small amount of samples, analyze the change of the concentration ratio of the two markers over time, and further evaluate the dynamic balance of the two markers in the solution system;

[0024] (2) Through result analysis, the probability of the detected person developing preeclampsia in the future can be predicted, and the limitation that the traditional detection result analysis completely depends on doctors can be reduced.

[0025] Next, through the drawings and embodiments, the technical solutions of the present invention will be further described in detail. Description of the Drawings

[0026] Figure 1 is the system schematic diagram of the electrochemical immunosensing device of the present invention;

[0027] Figure 2 is the schematic diagram of the outer shell of the electrochemical immunosensing device of the present invention;

[0028] Figure 3 is the schematic diagram of the detection layer and the circuit board after the electrochemical immunosensing device of the present invention is disassembled;

[0029] Figure 4 is the structural diagram of the detection layer and the circuit board after the electrochemical immunosensing device of the present invention is disassembled;

[0030] Figure 5It is the schematic diagram of the working electrode modification and working process of the sensing device of the present invention.

[0031] Figure 6 It is the scanning electron microscope image of the first working electrode of the sensing device of the present invention;

[0032] Figure 7 It is the scanning electron microscope image of the second working electrode of the sensing device of the present invention;

[0033] Figure 8 It is the goodness-of-fit graph of the working electrode of the sensing device of the present invention, which is the goodness-of-fit curve of the first working electrode;

[0034] Figure 9 It is the goodness-of-fit graph of the working electrode of the sensing device of the present invention, which is the goodness-of-fit curve of the second working electrode;

[0035] Figure 10 It is the bio-detection specificity graph of the working electrode of the sensing device of the present invention, which is the specificity graph of the first working electrode;

[0036] Figure 11 It is the bio-detection specificity graph of the working electrode of the sensing device of the present invention, which is the specificity graph of the second working electrode.

[0037] Reference numerals

[0038] 100 - Device housing;

[0039] 200 - Detection layer; 300 - Circuit board;

[0040] 201 - Detection cavity; 202 - Reference electrode; 203 - First pair of electrodes; 204 - First working electrode; 205 - Detection cavity electrical connection port; 206 - Second working electrode; 207 - Second pair of electrodes; 208 - Collection tank;

[0041] 301 - Circuit board excitation electrode unit connection contact; 302 - Analog-to-digital conversion module; 303 - Power supply; 304 - DC-DC converter; 305 - Digital-to-analog conversion module; 306 - Main control chip; 307 - Bluetooth module; 308 - LED lamp; 309 - Amplifier. Detailed implementation manners

[0042] In order to make the objectives, technical solutions, and advantages of the embodiments disclosed in the present invention clearer and more understandable, the following further elaborates on the embodiments of the present invention in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely used to explain the embodiments of the present invention and are not used to limit the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts belong to the scope of protection of this application. Examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions throughout.

[0043] It should be noted that the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or server that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products, or devices.

[0044] Similar reference numerals and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0045] Embodiment 1

[0046] An electrochemical immunosensing device for simultaneously detecting PLGF and sFlt-1 includes a device housing 100, a detection layer 200, and a circuit board 300. The device housing includes a housing and an internal fixing component for protecting and fixing the detection layer 200 and the circuit board 300.

[0047] As shown in the device housing 100 Figure 2 The schematic diagrams of the detection layer 200 and the circuit board 300 after the device housing 100 is disassembled are as shown in Figure 3 As shown in Figure 4 As shown.

[0048] The system schematic diagram of the circuit board 300 is as shown in Figure 1 As shown, and includes: the connection contact 301 of the circuit board excitation electrode unit, the analog-to-digital conversion module 302, the power module 303, the DC-DC converter 304, the digital-to-analog conversion module 305, the main control chip 306, the Bluetooth module 307, the LED lamp 308, and the amplifier 309.

[0049] The power supply 303 includes a power management unit and a battery. The input end of the power module management unit is electrically connected to the output end of the battery. The output end of the power module 303 is electrically connected to the input end of the LED lamp 308, and the power consumption of the LED lamp 308 can be ignored. The main control chip 306 is used to set the lighting mode of the LED lamp 308 to flashing, and the time interval is 1 second. When the power module 303 starts to output voltage and it is observed that the LED lamp 308 starts to flash with an interval of 1 second, it indicates that the circuit board can work normally.

[0050] The digital-to-analog conversion module 305, the analog-to-digital conversion module 302, the Bluetooth module 307 and the main control chip 306 are integrated on the same circuit board to achieve the miniaturized design of the device.

[0051] The output end of the power module 303 is electrically connected to the input end of the DC-DC converter 304. The power module 303 can output a voltage of 3.7V. The output end of the DC-DC converter 304 is electrically connected to the circuit board 300, and the input end of the power module 303 is electrically connected to the circuit board 300, which is used to adjust the voltage to meet the different voltage requirements of each chip inside the circuit board 300.

[0052] Specifically, the DC-DC converter 304, the power module 303, the digital-to-analog conversion module 305, the analog-to-digital conversion module 302 and the output end of the power module 303 and the DC-DC converter 304 together form a power management module, which can stably adjust the output voltage in the range of -5V to 3.3V.

[0053] The power module 303 converts 3.7V of direct current into 3.3V to supply power to each chip on the circuit board 300, and the DC-DC converter 304 converts 3.3V of current into 5V to supply power to the digital-to-analog conversion module 305.

[0054] The analog-to-digital conversion module 302 and the Bluetooth module 307 send the detected voltage change and current call to the mobile terminal to achieve real-time monitoring.

[0055] The connection contact 301 of the circuit board excitation electrode unit is connected to the excitation electrode unit of the detection layer 200, including the first working electrode 204, the second working electrode 206, the first pair of electrodes 203, the second pair of electrodes 207, and the reference electrode 202. The detection cavity electrical connection port 205 is electrically connected to the connection contact 301 of the circuit board excitation electrode unit.

[0056] The first working electrode 204 and the second working electrode 206 are respectively located in two detection cavities 201 for detecting two target substances.

[0057] The first pair of electrodes 203 and the second pair of electrodes 207 are used to form a current loop with the first working electrode 204 and the second working electrode 206.

[0058] The reference electrode 202 compares the electrode potentials of the first working electrode 204 and the second working electrode 206, and measures the electrode potentials of the first working electrode 204 and the second working electrode 206.

[0059] The first working electrode 204, the second working electrode 206, the first pair of electrodes 203, and the second pair of electrodes 207 adopt low-resistance conductive ink electrodes, and the reference electrode 202 adopts a silver / silver chloride electrode to stably complete biosensing detection.

[0060] The input ends of the first working electrode 204 and the second working electrode 206 are electrically connected to the output end of the digital-to-analog conversion module 305. The first working electrode 204 and the second working electrode 206 are independent of each other and can be used to detect different markers respectively.

[0061] The input end of the reference electrode 202 is electrically connected to the output end of the digital-to-analog conversion module 305.

[0062] The input ends of the first pair of electrodes 203 and the second pair of electrodes 207 are electrically connected to the output end of the digital-to-analog conversion module 305 through the contact 301 of the circuit board excitation electrode unit, and are used to generate excitation for the solution to be measured. The excitation is applied to the solution to be measured in the form of a scanning potential, so that the solution to be measured undergoes an oxidation-reduction reaction, thereby realizing biosensing applications.

[0063] For the first working electrode 204 and the second working electrode 206, a collection tank 208 is provided to prevent the samples from intersecting and to ensure that the two electrodes do not affect each other.

[0064] The first working electrode 204 and the second working electrode 206 include a substrate, a functional modification layer, and a sensitive modification layer, and the substrate is a low-resistance conductive ink.

[0065] The preparation of the sensitive modification layer is as Figure 5 shown, including:

[0066] (1) A 1 mg / mL carboxylated graphene (GO-COOH) solution and a 0.5% gold nanoparticle (HAuCl4) solution are mixed in a ratio of 20:1 to prepare a GO-COOH / HAuCl4 composite material mixture.

[0067] (2) 100 μL of the GO-COOH / HAuCl4 composite material mixture is extracted and dropped onto the surfaces of the first working electrode 204 and the second working electrode 206, and the composite material is deposited on the surfaces of the first working electrode 204 and the second working electrode 206 by an electrochemical method.

[0068] The scanning electron microscope images of the working electrode modified with the composite material are as Figure 6 and Figure 7 shown, Figure 6is the first working electrode 204, Figure 7 is the second working electrode 206; It can be seen that the gold nanoparticles on the surface of the working electrode after being modified with the composite material are evenly dispersed, which is beneficial to binding more detection substances and improving the sensitivity of the electrode.

[0069] The preparation of the functional modification layer includes:

[0070] (1) Select 15 μL of HS-PEG8-COOH solution with a concentration of 1 mg / mL, and evenly drop it on the surfaces of the first working electrode 204 and the second working electrode 206 modified with the composite material, and leave it overnight in a refrigerator at 4 °C to introduce a large number of -COOH groups on the surface of the composite material.

[0071] (2) Mix 5 mM of EDC and 2 mM of NHS in a volume ratio of 1:1, and drop 10 μL of the EDC and NHS mixed solution on the surfaces of the first working electrode 204 and the second working electrode 206, and incubate for 30 min to activate the -COOH functional groups on the electrode surface to bind with PLGF and sFlt-1 antibodies. Use a pipette to aspirate the excess solution and place it at room temperature to dry for 5 min.

[0072] (3) Dilute the PLGF antibody with a concentration of 1 mg / mL with 0.1 M PBS (pH 7.2 - 7.4) to obtain a PLGF antibody solution with a concentration of 20 μg / mL. When in use, first take out the first working electrode 204 after being activated by EDC / NHS, and drop 10 μL of the PLGF antibody solution onto the first working electrode 204, and incubate in a refrigerator at 4 °C for 7 hours.

[0073] (4) Dilute the sFlt-1 antibody with a concentration of 1 mg / mL with 0.1 M PBS (pH 7.2 - 7.4) to obtain a sFlt-1 antibody solution with a concentration of 20 μg / mL. When in use, first take out the second working electrode 206 after being activated by EDC / NHS, and drop 10 μL of the sFlt-1 antibody solution onto the second working electrode 206, and incubate in a refrigerator at 4 °C for 7 hours.

[0074] (5) The BSA solution is used to block the free sites on the electrode surface that have not bound antibodies, prevent the oxidation of the electrode surface, and improve the stability of the electrode surface. Drop 10 μL of BSA with a mass-volume percentage of 0.05% onto the surfaces of the first working electrode 204 and the second working electrode 206 that have already bound antibodies, and incubate at room temperature for 30 min.

[0075] Example Two

[0076] The detection method of an electrochemical immunosensing device for simultaneously detecting PLGF and sFlt-1 in Example One includes the following steps:

[0077] S1. Establish a standard curve

[0078] Drop the standard solution onto the first working electrode 204 and the second working electrode 206; turn on the power module, and the system starts to operate. The main control chip 306 controls the power management unit to output a scanning potential, which is output to the first working electrode 204 and the second working electrode 206 through the digital-to-analog conversion module 305 and the power management unit to apply an excitation to the solution to be measured;

[0079] Receive the voltage signal output by the main control chip 306 through the analog-to-digital conversion module 302, control the change of the output voltage, obtain the voltage-current relationship, and establish a current change-marker concentration curve according to the voltage-current relationship and the standard solution concentration; the current change refers to the change in the peak current of the first working electrode and the second working electrode;

[0080] S2. Drop the solution to be measured onto the first working electrode 204 and the second working electrode 206 to detect the marker concentration, obtain the marker concentration according to the output voltage-current relationship and the standard curve established in step S1, and transmit the information to the connected mobile terminal through the Bluetooth module 307.

[0081] Experimental test

[0082] (1) Goodness-of-fit evaluation

[0083] Prepare a PLGF solution with 0.1M PBS (pH 7.2 - 7.4). The selected concentration gradients are 1 pg / mL, 5 pg / ml, 10 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, and 250 pg / mL. The above concentration gradients are selected according to the normal content in human serum and the concentration range detected by traditional detection methods.

[0084] Start measuring from 1 pg / mL and progress in sequence until 250 pg / mL as a set of concentration gradients. Wash between each concentration with PBS solution of 0.1M PBS (pH 7.2 - 7.4), and the total interval does not exceed 0.5 minutes. Repeat this 3 times. After processing and fitting analysis of the obtained results, a goodness-of-fit curve is obtained, as shown in Figure 8 shown. It can be seen from the goodness-of-fit curve that the R 2 of PLGF is 0.989.

[0085] The sFlt-1 solution is made with 0.1M PBS (pH 7.2 - 7.4). The selected concentration gradients are 10 pg / ml, 100 pg / mL, 1000 pg / mL, 2000 pg / mL, 4000 pg / mL, 6000 pg / mL, and 8000 pg / mL. These concentration gradients are selected based on the normal content in human serum and the concentration range detected by traditional detection methods.

[0086] Starting from 10 pg / mL, measure incrementally until 8000 pg / mL to form a set of concentration gradients. Between each concentration, clean with PBS solution of 0.1M PBS (pH 7.2 - 7.4). The total interval does not exceed 0.5 minutes. Repeat this 3 times. After processing and fitting analysis of the obtained results, a goodness-of-fit curve is obtained, as Figure 9 shown. It can be seen from the goodness-of-fit curve that the R 2 of sFlt-1 is 0.961.

[0087] (II) Specificity test

[0088] The selected substances are brain natriuretic peptide, bovine serum albumin, placental growth factor, and soluble tyrosine kinase. They are prepared into solutions with a concentration of 500 pg / mL using 0.1M PBS (pH 7.2 - 7.4). These substances are selected based on the substances existing in human interstitial fluid that can affect or may affect the detection of PLGF and sFlt-1.

[0089] For the PLGF specificity experimental procedure: Detect 100 pg / mL of PLGF using differential pulse voltammetry, and then clean with PBS solution. Repeat 3 groups. The same method is used for artificial serum, mixed solution containing 100 pg / mL PLGF, brain natriuretic peptide, bovine serum albumin, and soluble tyrosine kinase. The blank control group is PBS solution.

[0090] For the sFlt-1 specificity experimental procedure: Detect 100 pg / mL of sFlt-1 using differential pulse voltammetry, and then clean with PBS solution. Repeat 3 groups. The same method is used for artificial serum, mixed solution containing 100 pg / mL sFlt-1, brain natriuretic peptide, bovine serum albumin, and placental growth factor. The blank control group is PBS solution.

[0091] The results of the PLGF specificity experiment are as Figure 10 shown, and the results of the sFlt-1 specificity experiment are as Figure 11 shown. It can be seen from the specificity graph that the currents of the target substances, namely 100 pg / mL of PLGF and sFlt-1, are significantly lower than the currents of other substances. Therefore, the electrochemical immunosensing device proposed by the present invention has excellent specificity.

[0092] As shown in Table 1, the sensor was used to test real samples, and the ratio of sFlt-1 / PLGF was obtained. The detection results were compared with those of enzyme-linked immunosorbent assay (ELISA), showing high accuracy.

[0093] Table 1

[0094]

[0095] An electrochemical immunosensing device and method for simultaneously detecting PLGF and sFlt-1 according to the present invention. The device is a portable and simplified electrochemical workstation, and the whole device uses a flexible material harmless to the human body as a carrier. Therefore, it can perform real-time biological detection, recording and supervision of detection results without affecting the normal movement of the user; the device can perform biological detection, recording and analysis of biomarkers with a small amount of samples; through result analysis, the dynamic balance of the concentrations of the two biomarkers in the sample can be determined, solving the limitation that the analysis of traditional detection results completely depends on doctors.

[0096] After considering the specification and practicing the content disclosed herein, those skilled in the art will readily think of other embodiments of the present application. The present application is intended to cover any variations, uses or adaptations of itself, which follow the general principles of the present application and include the common general knowledge or conventional technical means in the technical field not disclosed in the present application. The specification and examples are only regarded as exemplary, and the true scope and spirit of the present application are pointed out by the following claims.

[0097] It should be understood that the present application is not limited to the exact structures already described and shown in the drawings, and various modifications and changes can be made without departing from its scope. The scope of the present application is only limited by the appended claims.

[0098] According to the disclosure and teachings of the above specification, those skilled in the art to which the present invention pertains can also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the above specific embodiments, and any obvious improvements, substitutions or variations made by those skilled in the art on the basis of the present invention all fall within the protection scope of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. An electrochemical immunosensor device for simultaneous detection of PLGF and sFlt-1, characterized in that: It includes a microprocessor module, a power module, an analog-to-digital conversion module, a digital-to-analog conversion module and a communication module integrated on the same circuit board; the analog-to-digital conversion module and the communication module are respectively connected to the microprocessor module; The circuit board is provided with a circuit board excitation electrode unit connection contact, which is connected to the excitation electrode unit. The excitation electrode unit includes a first working electrode, a second working electrode, a first pair of electrodes, a second pair of electrodes, and a reference electrode. The first working electrode and the second working electrode are respectively located in two independent reaction chambers as carriers for binding markers. The first working electrode and the second working electrode both include a substrate, a sensitive modification layer and a functional modification layer; The substrate is a low-resistance conductive ink, and the sensitive modification layer is a composite nanomaterial of carboxylated graphene and gold nanoparticles. The composite nanomaterial of carboxylated graphene and gold nanoparticles is carboxy-activated, and the carboxyl functional groups of the first working electrode and the second working electrode are covalently bonded to the PLGF antibody and the sFlt-1 antibody respectively after activation to form a functional modification layer; The voltage signal output by the main control chip is received through the analog-to-digital conversion module, the change of the output voltage is controlled, the voltage-current relationship is obtained, and the current change-marker concentration curve is established according to the voltage-current relationship and the concentration of the standard solution.

2. An electrochemical immunosensor device for simultaneous detection of PLGF and sFlt-1 according to claim 1, characterized in that: It also includes an amplifier, which is electrically connected to the input end of the analog-to-digital conversion module. The microprocessor module collects and transmits the signal output by the analog-to-digital conversion module. The amplifier, the analog-to-digital conversion module and the digital-to-analog conversion module are used to control and output the voltage required by the sensor device for material detection.

3. An electrochemical immunosensor device for simultaneous detection of PLGF and sFlt-1 according to claim 1, characterized in that: The input ends of the first working electrode, the second working electrode, the first pair of electrodes, and the second pair of electrodes are electrically connected to the output end of the digital-to-analog conversion module respectively; the input end of the reference electrode is electrically connected to the output end of the digital-to-analog conversion module.

4. The electrochemical immunosensor device for simultaneous detection of PLGF and sFlt-1 according to claim 1, characterized in that: A power module and an LED lamp are also provided on the circuit board. The output end of the power module is electrically connected to the input end of the LED lamp. The microprocessor module sets the light-emitting mode of the LED lamp to observe whether the circuit is normal.

5. The electrochemical immunosensor device for simultaneous detection of PLGF and sFlt-1 according to claim 1, characterized in that: The communication module is a Bluetooth module, which establishes communication with the mobile terminal to achieve real-time transmission of detection results.

6. The electrochemical immunosensor device for simultaneous detection of PLGF and sFlt-1 according to claim 4, characterized in that: The power module comprises a power management unit and a battery, wherein an input end of the power management unit is electrically connected to an output end of the battery.

7. An electrochemical immunosensor device for simultaneous detection of PLGF and sFlt-1 according to claim 4, characterized in that: It also includes a DC-DC converter, the output end of the power module is electrically connected to the input end of the DC-DC converter, the power module outputs a voltage of 3.7V, the output end of the DC-DC converter is electrically connected to the circuit board, and the input end of the power module is electrically connected to the circuit board to adjust the voltage to meet the different voltage requirements of various chips inside the circuit board; The DC-DC converter, power module, digital-to-analog conversion module, analog-to-digital conversion module and output end of the power module together with the DC-DC converter form a power management module to achieve stable adjustment of the output voltage within the range of -5V to 3.3V.

8. A method for using the electrochemical immunosensor device for simultaneously detecting PLGF and sFlt-1 according to any one of claims 1 to 7, characterized in that: The steps include: S1. Establish standard curve The standard solution is dripped onto the first working electrode and the second working electrode; the power module is turned on, and the main control chip controls the power management unit to output the scanning potential, which is output to the first working electrode and the second working electrode through the digital-to-analog conversion module and the power management unit to apply excitation to the solution to be tested; The voltage signal output by the main control chip is received through the analog-to-digital conversion module, the change of the output voltage is controlled, the voltage-current relationship is obtained, and a current change-marker concentration curve is established according to the voltage-current relationship and the concentration of the standard solution; The current change refers to the change in the peak current of the first working electrode and the second working electrode; S2. Add the solution to be tested to the first working electrode and the second working electrode to detect the marker concentration, and obtain the marker concentration based on the output voltage-current relationship and the standard curve established in step S1; transmit the information to the connected mobile terminal through the communication module.

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