A method and apparatus for perioperative fibrinogen detection

Abstract

This invention provides a method and apparatus for perioperative fibrinogen detection, comprising the following steps: S1, modifying the working electrode surface of a screen-printed electrode with antibody-functionalized self-supporting graphene aerogel material; S2, adding the serum sample to be tested to the modified working electrode surface; S3, adding horseradish peroxidase-labeled fibrinogen detection antibody to the modified working electrode surface; S4, adding a matrix-type TMB solution containing H2O2 to the modified working electrode surface; S5, collecting the reduction current of electrochemically active substances on the electrode surface using a chronoamperometry method; S6, calculating the fibrinogen concentration based on the reduction current value and a standard curve. This invention solves the technical problems of low electron transport efficiency and detection sensitivity, structural instability, poor biocompatibility, and difficulty in enriching electroactive substances when using graphene-modified electrodes.

Description

Technical Field

[0001] This invention relates to the technical field of fibrinogen detection, and more particularly to a method and apparatus for perioperative fibrinogen detection. Background Technology

[0002] Fibrinogen is a key marker of coagulation function, and its perioperative concentration monitoring is of great clinical significance. When the fibrinogen concentration in bleeding patients is below 1.5 g / L, timely supplementation is necessary; excessive supplementation can increase the risk of thrombosis.

[0003] Currently, the clinical gold standard Clauss method relies on large coagulation analyzers and specialized operation, and sample transport suffers from significant time delays, failing to meet the needs of real-time perioperative monitoring. Optical methods offer high sensitivity but require expensive equipment; acoustic methods, while facilitating equipment miniaturization, suffer from poor interference resistance; and electrochemical methods, though fast and portable, face technical bottlenecks such as low electron transfer efficiency, poor biocompatibility of electrode materials, and difficulty in accumulating electroactive substances. Therefore, innovative detection methods are urgently needed to achieve on-site, real-time monitoring.

[0004] Meanwhile, graphene, with its high specific surface area, conductivity, and biocompatibility, is a potential material for improving the sensitivity of electrochemical detection. However, direct electrode modification suffers from problems such as a limited number of active sites and low interfacial mass transfer efficiency, affecting detection sensitivity and making it difficult to meet high requirements. Assembling two-dimensional graphene nanosheets into a three-dimensional structure can improve performance, but due to the easy aggregation caused by the interactions between nanosheets, it is difficult to form a stable three-dimensional framework. How to regulate the interactions between two-dimensional graphene nanosheets and use them for electrode modification to achieve efficient electrochemical sensing has become an urgent problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a method and apparatus for perioperative fibrinogen detection, which solves the technical problems of low electron transmission efficiency and detection sensitivity, unstable structure, poor biocompatibility, and difficulty in enriching electroactive substances when using graphene-modified electrodes.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for perioperative fibrinogen detection, comprising the following steps:

[0008] S1. Electrode modification: Antibody-functionalized self-supporting graphene aerogel material is modified onto the working electrode surface of a screen-printed electrode. The self-supporting graphene aerogel is prepared by a hydrothermal method assisted by polyethylene glycol and dopamine, and its interlayer and three-dimensional structure are effectively controlled by adjusting the content of polyethylene glycol and multi-walled carbon nanotube solution during preparation.

[0009] S2. Incubation of the test sample: The serum sample to be tested is dropped onto the surface of the modified working electrode and then washed after incubation.

[0010] S3. Detect antibody binding: Add horseradish peroxidase-labeled fibrinogen detection antibody to the modified working electrode surface and wash after incubation.

[0011] S4. Add an electrochemical probe by dropping a matrix-type TMB solution containing H2O2 onto the surface of the modified working electrode and then cleaning it after incubation.

[0012] S5. Electrochemical signal acquisition: The reduction current of the electrochemically active material on the electrode surface is acquired by chronoamperometry.

[0013] S6. Concentration Calculation: Calculate the fibrinogen concentration based on the reduction current value and the standard curve.

[0014] Furthermore, the working electrode and counter electrode of the screen-printed electrode are obtained by printing carbon paste onto a PET substrate, and the reference electrode is obtained by printing silver or silver chloride paste onto a PET substrate.

[0015] Furthermore, the self-supporting graphene aerogel is prepared by a hydrothermal method assisted by polyethylene glycol and dopamine, and the specific steps include:

[0016] S1. Mix the graphene oxide solution with the multi-walled carbon nanotube solution and disperse them by ultrasonication to form a composite dispersion.

[0017] S2. Add polyethylene glycol to the composite dispersion, stir to dissolve and form a PEG-GO / MWCNTs precursor solution, and adjust the pH of the precursor solution.

[0018] S3. Add dopamine to the PEG-GO / MWCNTs precursor solution, stir to dissolve, perform water bath heating for polymerization and crosslinking, and freeze-dry to obtain self-supporting graphene aerogel.

[0019] Furthermore, the bonding mode between layers of self-supporting graphene aerogel sheets can be controlled and effective separation can be achieved by adjusting the mass fraction of polyethylene glycol.

[0020] Furthermore, it also includes controlling the self-supporting graphene aerogel structure by adjusting the concentration of multi-walled carbon nanotube solution, so that it exhibits a uniform three-dimensional structure.

[0021] Furthermore, the antibody-functionalized self-supporting graphene aerogel-modified screen-printed electrode was prepared using a bio-coupling method, the preparation steps of which included:

[0022] S1. Interface activation: EDC solution and NHS solution are sequentially added to the surface of the screen-printed electrode modified with self-supporting graphene aerogel, and the electrode surface activation treatment is completed by incubation.

[0023] S2. Antibody conjugation: After rinsing the electrode surface obtained in step S1, fibrinogen antibody solution is added and incubated to achieve bioconjugation between the antibody and the electrode surface.

[0024] S3. Electrode sealing: Rinse the electrode surface obtained in step S2, add bovine serum albumin solution for incubation, and seal the non-specific binding sites on the electrode surface.

[0025] S4. Electrode cleaning: Rinse the electrode surface obtained in step S3 to finally obtain an antibody-functionalized self-supporting graphene aerogel-modified screen-printed electrode.

[0026] Furthermore, the fibrinogen concentration was calculated based on the standard curve I = 5.38 + 1.34 ln [Cfib], where I is the current value (μA) and Cfib is the fibrinogen concentration (g / L).

[0027] In a second aspect, the present invention also provides an apparatus for perioperative fibrinogen detection, comprising the method described in any of the preceding claims, including:

[0028] Electrode interface circuit for connecting screen-printed electrodes modified with antibody-functionalized self-supporting graphene aerogel material;

[0029] An electrochemical detection circuit, connected to an electrode interface circuit, includes a constant potential output unit and a current detection unit, used to provide electrochemical reaction voltage and detect current signals;

[0030] Wireless communication circuitry is used to interact with the user interface and transmit detection commands and results.

[0031] The microprocessor circuit is connected to the electrochemical detection circuit and the wireless communication circuit respectively, and is used to regulate the output voltage parameters and the amplification parameters of the small electrochemical current signal of the electrochemical detection circuit.

[0032] Furthermore, the electrochemical detection circuit includes:

[0033] The digital-to-analog converter module is used to convert the digital voltage signal given by the microprocessor circuit into an analog signal;

[0034] The transimpedance amplifier module is used to amplify the weak electrochemical current signal generated by the screen-printed electrode and convert it into a measurable voltage signal.

[0035] The analog-to-digital converter module is used to sample the amplified voltage signal and convert it into a digital signal, and then transmit the digitized detection results to the microprocessor circuit.

[0036] Furthermore, the electrochemical detection circuit, wireless communication circuit, and microprocessor circuit are integrated on the first circuit board; the electrode interface circuit is integrated on the second circuit board; the first circuit board and the second circuit board are connected by flexible printed cables.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] The porous three-dimensional network structure of the self-supporting graphene aerogel of this invention provides a high specific surface area, offering ample space for antibody modification and immobilization, effectively inhibiting antibody separation and aggregation, and improving the stability of the biorecognition interface. At the same time, the porous structure and high specific surface area of ​​the self-supporting graphene aerogel provide more adsorption sites for electroactive substances, increasing the loading of electroactive substances on the electrode surface, effectively solving the problem of difficult enrichment of electroactive substances, and improving detection sensitivity.

[0039] This invention prepares self-supporting graphene aerogels via a hydrothermal method assisted by polyethylene glycol and dopamine. By adjusting the mass fraction of polyethylene glycol, the bonding mode between the layers can be effectively controlled, avoiding the formation of blocky structures through stacking. At the same time, by adjusting the concentration of multi-walled carbon nanotubes, a uniform three-dimensional structure can be obtained, solving the problem of structural instability caused by the easy aggregation of graphene nanosheets.

[0040] This invention uses polyethylene glycol and dopamine to synergistically prepare self-supporting graphene aerogels. Dopamine can form a polydopamine coating on the surface of reduced graphene oxide. In addition, the structure is controlled by adjusting parameters during the preparation process, which helps to improve the biocompatibility of the electrode.

[0041] The excellent conductivity of reduced graphene oxide and multi-walled carbon nanotubes in the self-supported graphene aerogel of this invention can significantly improve electron transport efficiency and enhance detection sensitivity. Attached Figure Description

[0042] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0043] Figure 1 A three-dimensional schematic diagram of a perioperative fibrinogen detection device according to an embodiment of the present invention;

[0044] Figure 2 A schematic diagram of a circuit board for a perioperative fibrinogen detection device according to an embodiment of the present invention;

[0045] Figure 3 A system structural block diagram of a perioperative fibrinogen detection device according to an embodiment of the present invention;

[0046] Figure 4 A schematic diagram of a user interface for a perioperative fibrinogen detection device according to an embodiment of the present invention;

[0047] Figure 5 A flowchart of the operating procedure for a perioperative fibrinogen detection device according to an embodiment of the present invention;

[0048] Figure 6 A flowchart for the preparation of self-supporting graphene aerogels provided in this embodiment of the invention;

[0049] Figure 7 A schematic diagram of a screen-printed electrode modified with antibody-functionalized graphene aerogel prepared by bioconjugation method provided in this embodiment of the invention.

[0050] Figure 8 A schematic diagram illustrating the detection principle of the perioperative fibrinogen detection method provided in this embodiment of the invention;

[0051] Figure 9 Scanning electron microscope (SEM) images of self-supporting graphene aerogels prepared at different PEG mass fractions in a perioperative fibrinogen detection method provided in this embodiment of the invention;

[0052] Figure 10 Scanning electron microscope (SEM) images of self-supporting graphene aerogels prepared at different MWCNT concentrations in a perioperative fibrinogen detection method provided in this embodiment of the invention;

[0053] Figure 11 XPS characterization of a self-supporting graphene aerogel-modified electrode in a perioperative fibrinogen detection method provided in this embodiment of the invention;

[0054] Figure 12 XRD characterization of a self-supporting graphene aerogel-modified electrode in a perioperative fibrinogen detection method provided in this embodiment of the invention;

[0055] Figure 13 Electrochemical characterization of a self-supporting graphene aerogel-modified electrode in a perioperative fibrinogen detection method provided in this embodiment of the invention;

[0056] Figure 14 The electrochemical response characteristics of a self-supporting graphene aerogel-modified electrode to TMB in a perioperative fibrinogen detection method provided in this embodiment of the invention;

[0057] Figure 15 The cyclic voltammograms of the perioperative fibrinogen detection method provided in this embodiment of the invention for detecting fibrinogen standard solutions of different concentrations;

[0058] Figure 16 The chronoamperometry current response diagram of the perioperative fibrinogen detection method provided in this embodiment of the invention when detecting fibrinogen standard solutions of different concentrations;

[0059] Figure 17 The present invention provides a standard fitting curve of the chronoamperometry current response value and the concentration and logarithm of the fibrinogen standard solution when detecting fibrinogen standard solutions of different concentrations in a perioperative fibrinogen detection method.

[0060] Figure 18 The anti-interference evaluation results of the perioperative fibrinogen detection method provided in the embodiments of the present invention;

[0061] Figure 19 Example 3: Schematic diagram of serum sample collection from patients at different perioperative stages in a clinical trial.

[0062] Figure 20 Comparison of fibrinogen test results at different stages of the perioperative period;

[0063] Figure 21 Correlation analysis of fibrinogen detection results at different perioperative stages using the method described in this invention and the Clauss method.

[0064] icon:

[0065] 10-First circuit board; 11-Electrochemical detection circuit; 12-Microprocessor circuit; 13-Wireless communication circuit; 14-Power management circuit; 15-Flexible printed wiring;

[0066] 20 - Second circuit board; 21 - Electrode interface circuit;

[0067] 30 - Screen-printed electrode;

[0068] 40 - Base; 50 - Circuit fixing slot; 60 - Top cover. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] Example 1

[0071] like Figure 1 As shown, this embodiment of the invention provides a device for perioperative fibrinogen detection, such as... Figure 1 As shown, it is characterized by including the following functional modules:

[0072] The electrochemical detection circuit 11 includes a constant potential output unit for providing the voltage required for the electrochemical reaction of the three-electrode system and a current detection unit for detecting the small electrochemical current signal generated during the three-electrode electrochemical reaction.

[0073] Electrode interface circuit 21 is used to connect a screen-printed three-electrode system. Electrode interface circuit 21 is designed with a dedicated electrode connector, whose three metal contacts can be connected to the inserted screen-printed three-electrode, thereby forming a connection path with electrochemical detection circuit 11.

[0074] The wireless communication circuit 13 is used for interaction between the fibrinogen detection device and the user interface. The user can input detection commands via a mobile phone or other platform, and the wireless communication circuit will transmit these commands to the fibrinogen detection device. After the detection is completed, the wireless communication circuit will wirelessly transmit the detection results back to the user interface.

[0075] The microprocessor circuit 12, as the control core of the device, is connected to the electrochemical detection circuit 11 and the wireless communication circuit 13 respectively, and is used to regulate the output voltage parameters of the electrochemical detection circuit 11 and the amplification parameters of the small electrochemical current signal.

[0076] According to the above technical solution, the device described in this embodiment of the invention can adjust the output voltage parameters and current detection parameters of the electrochemical detection circuit 11 through the microprocessor circuit 12 according to the detection needs, and send the detection results to the user in real time through the wireless communication circuit.

[0077] The electrochemical detection circuit 11, wireless communication circuit 13, and microprocessor circuit 12 involved in the embodiments of the present invention are small in size and simple in structure, which can realize the miniaturization and portability of the perioperative coagulation function detection device.

[0078] In this embodiment, as Figure 2As shown, the electrochemical detection circuit 11, wireless communication circuit 13, and microprocessor circuit 12 are all packaged on the first circuit board 10 to achieve device integration. The first circuit board 10 uses an FR-4 substrate with a thickness of 1.6 mm and a size no greater than 28 mm in length and 16 mm in width. All components are integrated using surface mount technology. The electrode interface circuit 21 is packaged on the second circuit board 20. The first circuit board 10 also uses an FR-4 substrate with a thickness of 1.6 mm and a size no greater than 12 mm in length and 10 mm in width. All components are also integrated using surface mount technology. The first circuit board 10 and the second circuit board 20 are connected by a flexible printed wiring 15, facilitating the adjustment of the electrode interface circuit 21 according to detection needs.

[0079] The first circuit board 10 and the second circuit board 20 are both fixed inside the housing. The outer dimensions of the housing are no greater than 60 mm in length × 38 mm in width × 35 mm in height, which enables the detection device to be portable.

[0080] The outer casing comprises a top cover 60, a base 40, and a circuit fixing slot 50. The top cover 60 and base 40 can be fully assembled to form a shell structure with a hollow inner cavity. The circuit fixing slot 50 has positioning holes for fixing the first circuit board 10. The outer casing is designed with an electrode interface slot, and the second circuit board 20 is fixed to the electrode interface slot of the outer casing. The electrode connector on the second circuit board 20 corresponds to the electrode interface slot of the outer casing, allowing the electrode connector to be stably fixed to the outer casing for easy insertion and removal of electrodes. The outer casing also has a battery fixing slot, which can store a lithium battery with a volume not exceeding 30 mm (length) × 20 mm (width) × 12 mm (height) to provide power for the first circuit board 10 and the second circuit board 20, enabling portable applications without an external power source.

[0081] In this embodiment, the outer shell has splicing and fixing holes for stable splicing connection between the top cover 60 and the base 40.

[0082] In one possible implementation, a screen-printed three-electrode is inserted into the electrode connector. The screen-printed three-electrode includes a working electrode, a counter electrode, a reference electrode, and an electrode substrate. The connection port of the screen-printed three-electrode is connected to three metal contacts in the electrode connector, respectively, to form a connection path between the screen-printed three-electrode and the electrochemical detection circuit 11 and the electrode interface circuit 21, thereby realizing electrochemical detection. The screen-printed three-electrode uses screen printing technology to print carbon ink onto the electrode substrate to form a specific shape, serving as the working electrode and counter electrode, and to print silver / silver chloride paste onto the electrode substrate to form a specific shape, serving as the reference electrode.

[0083] In one possible implementation, the first circuit board 10 further includes a power management circuit 14, which converts a 3.7V DC voltage to a 3.3V DC voltage for powering the various circuit modules in the first circuit board 10. More specifically, the power management circuit 14 includes a power management chip TPS7333 and corresponding peripheral circuitry to convert the 3.7V DC voltage of the lithium battery to a 3.3V DC voltage for powering the various circuit modules in the circuit board.

[0084] In one possible implementation, the electrochemical detection circuit 11 includes a digital-to-analog converter (DAC), an analog-to-digital converter (ADC), and a transimpedance amplifier module. The DAC converts the digital voltage signal from the microprocessor circuit 12 into an analog signal and outputs it to the screen-printed three electrodes. Its output voltage range is -1.2 to +1.2 V, and the voltage control accuracy is less than 1 mV. The transimpedance amplifier module amplifies the weak electrochemical current signal generated in the screen-printed three-electrode system and converts it into a measurable voltage signal. The ADC samples the analog voltage signal amplified by the transimpedance amplifier, converts it into a digital signal, and transmits the digitized detection result to the microprocessor circuit 12. Its sampling accuracy is 12 bits, and the current detection resolution is less than 0.1 μA.

[0085] More specifically, the digital-to-analog conversion module is implemented using the integrated circuit chip DAC1220, the analog-to-digital conversion module is implemented using the integrated circuit chip ADS1115, and the transimpedance amplification module is implemented using the integrated circuit chip AD8605.

[0086] In one possible implementation, the core chip of the microprocessor circuit 12 on the first circuit board 10 can be programmed via an SWD interface to control the operating logic of the entire device. More specifically, the core chip of the microprocessor circuit 12 can be an STM32L432KBU6 microprocessor from STMicroelectronics.

[0087] In one possible implementation, the wireless communication circuit in the first circuit board 10 is connected to the microprocessor circuit 12 via a serial interface to enable wireless communication between the device and the user interface. More specifically, the wireless communication circuit can receive detection commands given by the user through the user interface of a mobile phone or tablet via the Bluetooth protocol, parse the detection commands, and send them to the microprocessor circuit 12 via the serial interface. After the detection is completed, the wireless communication circuit receives the detection result sent by the microprocessor circuit 12 through the serial interface and sends it to the user's mobile phone or tablet user interface via the Bluetooth protocol.

[0088] In one possible implementation, the device for perioperative fibrinogen detection provided by the present invention can, through Figure 3 The block diagram shown is used for illustration.

[0089] More specifically, the microprocessor circuit 12 is the control core of the entire device. While controlling the electrochemical detection circuit 11 to apply the voltage required for the electrochemical reaction, it also measures the weak current signal generated by the electrochemical reaction. Simultaneously, the microprocessor circuit 12 is also connected to a wireless communication circuit via a serial interface to enable wireless communication with the user interface of a mobile phone or tablet computer.

[0090] In one possible implementation, the user interface of the mobile phone or tablet computer is as follows: Figure 4 As shown, it can be developed based on the Android Studio platform.

[0091] In one possible implementation, the program flow in the microprocessor circuit 12 can be... Figure 5 To elaborate further, the procedure can be summarized in the following steps:

[0092] Step S1, Parameter Reception and Configuration. The system receives setting parameters sent from the user interface of a mobile phone or tablet via a wireless communication circuit. These parameters include those for applying the voltage required for the electrochemical reaction and those for detecting the electrochemical current signal. Upon receiving the parameters, the relevant parameters are set in the digital-to-analog conversion module and the analog-to-digital conversion module, respectively.

[0093] Step S2: Perform detection and collect data. After receiving the start detection command sent by the mobile phone or tablet user interface, control the digital-to-analog converter module to apply voltage according to the specified voltage parameters, and simultaneously control the analog-to-digital converter module to sample according to the specified sampling parameters, and transmit the sampling results to the wireless communication circuit, thereby sending them to the mobile phone or tablet user interface.

[0094] Step S3, Data Transmission and Processing. During the detection process, the detected current value is fed back to the user interface in real time via a wireless communication circuit. At the end of the detection, the analog-to-digital conversion module and the analog-to-digital conversion module are stopped. The results obtained from the analog-to-digital conversion are processed to obtain the final detection result, which is then transmitted to the user interface of a mobile phone or tablet computer via wireless communication circuit 13.

[0095] Example 2

[0096] The present invention also provides a method for perioperative fibrinogen detection, specifically comprising the following steps:

[0097] Step S1, Electrode Modification and Connection. Antibody-functionalized self-supporting graphene aerogel material is modified onto the surface of the working electrode of the screen-printed electrode 30. The modified electrode is then inserted into the electrode connector of the detection device of Example 1. The three-electrode system is connected to the electrochemical detection circuit 11 via metal contacts in the electrode connector. The screen-printed three-electrode system includes a working electrode, a counter electrode, and a reference electrode. The working electrode and counter electrode are obtained by screen printing carbon paste onto a polyethylene terephthalate (PET) substrate, and the reference electrode is obtained by screen printing silver / silver chloride paste onto a PET substrate.

[0098] Step S2, sample incubation. Add 20 μL of the serum sample to be tested onto the working electrode surface and incubate at room temperature for 10 minutes. Wash with PBS to remove unbound material.

[0099] Step S3, detect antibody binding. Apply 10 μL of horseradish peroxidase (HRP)-labeled fibrinogen detection antibody to the working electrode surface, incubate at room temperature for 10 minutes, and then wash with PBS.

[0100] Step S4: Add electrochemical probe. Take 30 μL of matrix-type TMB solution containing H2O2, drop it onto the surface of the working electrode, incubate at room temperature for 10 minutes, and then wash with PBS.

[0101] Step S5: Configure the device to detect the required parameters through the user interface of a mobile phone or tablet, and start the detection program.

[0102] Step S6: Electrochemical signal acquisition and transmission. The microprocessor circuit 12 controls the device to execute specified logic, acquiring the reduction current of the electrochemically active material deposited on the electrode surface in real time. After detection, the microprocessor circuit 12 transmits the data to the user interface of a mobile phone or tablet via Bluetooth.

[0103] Step S7: Concentration Calculation and Result Display. The user reads the test data through the user interface of a mobile phone or tablet. The current data is processed according to the standard curve I = 5.38 + 1.34ln[Cfib], and the test results are automatically calculated and displayed as the concentration of fibrinogen in the serum to be tested.

[0104] In one possible implementation, the screen-printed electrode 30 is fabricated using screen printing technology. The three-electrode system includes a working electrode, a counter electrode, and a reference electrode, wherein the working electrode and the counter electrode are formed using carbon paste printing, and the reference electrode is formed using silver / silver chloride paste printing.

[0105] In one possible implementation, the self-supporting graphene aerogel is prepared by a hydrothermal method assisted by polyethylene glycol and dopamine, and then obtained by freeze-drying.

[0106] More specifically, the steps for preparing self-supporting graphene aerogels are as follows: Figure 6 As shown, the details are as follows:

[0107] Step S1: Mix 1 mL of graphene oxide (GO) solution with a concentration of 4 mg / mL with 1 mL of multi-walled carbon nanotube (MWCNT) solution with a concentration of 0.5 mg / mL, and sonicate for 30 minutes to obtain GO / MWCNT composite dispersion solution.

[0108] Step S2: Add 20 mg of polyethylene glycol (PEG) with a mass fraction of 2% to the dispersion solution and stir continuously to promote its dissolution to obtain a PEG-GO / MWCNTs precursor solution.

[0109] Step S3: Adjust the pH of the PEG-GO / MWCNTs precursor solution to 7.0 (±0.2) using dilute hydrochloric acid or sodium hydroxide solution.

[0110] Step S4: Add 4 mg of dopamine (DA) to the PEG-GO / MWCNTs precursor solution, stir continuously to promote its dissolution, and place at 80°C. o In-situ polymerization and crosslinking were carried out by heating at a constant temperature in a water bath for 6 hours, followed by freeze-drying for 24 hours to obtain self-supporting graphene aerogel. Figure 9 , 10 As shown.

[0111] In one possible implementation, the self-supporting graphene aerogel is modified onto the working electrode surface of the screen-printed electrode 30 by drop coating.

[0112] In one possible implementation, the antibody-functionalized self-supporting graphene aerogel-modified screen-printed electrode is prepared via a bio-coupling method, the preparation steps of which are as follows: Figure 7 As shown, the details are as follows:

[0113] Step S1, Interface Activation. 30 μL of a 10 mg / mL solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 30 μL of a 10 mg / mL solution of N-hydroxysuccinimide (NHS) were sequentially added to the surface of the self-supporting graphene aerogel-modified screen-printed electrode 30. The electrode surface was incubated at room temperature for 40 minutes to complete the electrode surface activation treatment.

[0114] Step S2, antibody conjugation. After rinsing the electrode surface obtained in step S1 with PBS buffer, add 10 μL of a 10 mg / mL fibrinogen antibody solution and incubate at 4°C. o Incubation at C for 24 hours achieves bioconjugation between the antibody and the electrode surface.

[0115] Step S3, electrode sealing. Rinse the electrode surface obtained in step S2 with PBS buffer solution, then add 30 μL of 1% bovine serum albumin (BSA) solution and incubate at 37°C. o Incubate at C for 15 minutes to seal non-specific binding sites on the electrode surface.

[0116] Step S4, electrode cleaning. The electrode surface obtained in step S3 is rinsed with PBS buffer to finally obtain the antibody-functionalized self-supporting graphene aerogel modified screen-printed electrode 30.

[0117] The principle of fibrinogen detection described in this embodiment is as follows: Figure 8 As shown, the specific process is as follows:

[0118] First, fibrinogen-capturing antibodies are immobilized on the surface of a self-supporting graphene aerogel-modified electrode using bioconjugation technology, constructing a specific biorecognition interface. When the target analyte fibrinogen is present in the sample solution, it specifically binds to the capture antibody on the electrode surface, forming an antigen-antibody immune complex.

[0119] Subsequently, an HRP-labeled detection antibody is added, which binds to the epitope at the other end of fibrinogen in the immune complex, forming a "sandwich-type" immune complex.

[0120] Next, a matrix-type TMB solution containing H2O2 is added to the electrode surface. The HRP immobilized on the electrode surface catalyzes the oxidation of TMB molecules, forming electrochemically active and electrochemically detectable TMB-TMB oxide precipitates (TMB-TMBox).

[0121] Finally, the first circuit board 10 described in Example 1 performs a chronoamperometry method. After the redox reaction reaches a stable state (approximately 120 seconds), the electrochemical reduction current is measured. Since the oxidation current value of the TMB-TMBox deposited on the electrode surface is positively correlated with the amount of HRP bound to the electrode surface, and the amount of HRP is linearly related to the fibrinogen concentration, the concentration of fibrinogen in the sample solution can be calculated using a standard curve by measuring the reduction current of the TMB-TMBox.

[0122] The morphology of the prepared self-supporting graphene aerogel modified electrode was characterized. The results showed that the mass fraction of polyethylene glycol (PEG) in the preparation method of the self-supporting graphene aerogel described in this invention has a significant impact on the surface roughness of the self-supporting graphene aerogel. Figure 9The images shown are scanning electron microscope (SEM) images of self-supporting graphene aerogels prepared with different PEG mass fractions. Reduced graphene oxide (rGO) nanosheets readily self-aggregate after hydrothermal treatment, forming smooth, multilayered stacks or bulk structures. As the PEG mass fraction increases from 0% to 3.0%, the surface roughness of the rGO sheets significantly increases, achieving effective separation between the sheets. However, when the PEG mass fraction exceeds 5%, the high concentration of PEG inhibits "side-to-surface" bonding between rGO sheets, promoting "surface-to-surface" stacking and ultimately forming an undesirable bulk structure. Therefore, adjusting the PEG mass fraction can effectively control the bonding mode between the self-supporting graphene aerogel sheets; in this embodiment, 2% was determined to be the optimal PEG mass fraction.

[0123] Since the preparation process of the self-supporting graphene aerogel described in this invention involves high temperature and freeze-drying, it is necessary to introduce MWCNTs during the synthesis process. The bridging effect exhibited by MWCNTs can increase the interlayer spacing between rGO nanosheets in the self-supporting graphene aerogel, thereby obtaining uniformly distributed monolayer graphene sheets. Figure 10 As shown, when the MWCNTs concentration is 0.25 mg / mL, the aerogel exhibits a uniform three-dimensional network structure. However, when the MWCNTs concentration is increased to 0.75-100 mg / mL, their random distribution leads to a decrease in the stability of the aerogel structure. During heating and freeze-drying, the formed three-dimensional network will be damaged, ultimately resulting in an undesirable blocky structure. Based on this, the interlayer spacing of rGO sheets and the microstructure of self-supporting graphene aerogel can be effectively controlled by adjusting the MWCNTs concentration. In this embodiment, 0.25 mg / mL is selected as the optimal MWCNTs concentration.

[0124] The chemical structure of the prepared self-supporting graphene aerogel modified electrode was characterized by XPS and XRD, and the characterization results are as follows:

[0125] The characterization results of XPS are as follows Figure 11 As shown, characteristic C 1s and O 1s peaks were observed in both the graphene oxide / multi-walled carbon nanotube (GO / MWCNTs) composite and the self-supporting graphene aerogel. The significantly reduced intensity of the O 1s peak indicates that graphene oxide (GO) has been successfully reduced to reduced graphene oxide (rGO). The appearance of the N 1s peak in the self-supporting graphene aerogel confirms the formation of a polydopamine (PDA) coating on the surface of the rGO nanosheets. Further deconvolution of the C 1s peak in GO / MWCNTs corresponds to C=C / CC. Characteristic peaks for C (~283.2 eV), CO (epoxide and hydroxyl, ~285.4 eV), C=O (carbonyl, ~286.9 eV), and OC=O. The corresponding O 1s spectrum unconvolved yields C=O (~531.4 eV), O... C=O (carbonyl group, ~532.1 eV) and C The O (epoxide and hydroxyl, ~534.5 eV) peak, compared with the C 1s spectrum Figure 1 After reduction with dopamine, the C=C / CC peak at 283.2 eV in the C 1s spectrum becomes the main peak, indicating that sp... 2 The carbon structure predominates, further confirming the reduction reaction of GO. Furthermore, the O 1s peak of the self-supported graphene aerogel shows characteristic peaks of C=O (~531.4 eV), OC=O, and CO (~532.3 eV), consistent with its C1s spectrum. Figure 1 The N 1s peak corresponds to various nitrogen-containing groups, including... N = (~399.6 eV) NH (~401.0 eV) and NH2 (~401.5 eV).

[0126] The characterization results of XRD are as follows Figure 12 As shown, 10 can be observed in the XRD pattern of GO. o The sharp diffraction peak at 24 disappeared after dopamine reduction. Meanwhile, in the XRD pattern of rGO, 24... o A broad diffraction peak appears at 26, indicating that GO has been successfully reduced to rGO. The XRD pattern of MWCNTs at 26... o and 43 o Two characteristic peaks are observed at the 10° position, corresponding to the (002) and (100) crystal planes of carbon atoms in graphene, respectively. The XRD pattern of the self-supported graphene aerogel shows characteristic peaks for rGO and MWCNTs, indicating that rGO and MWCNTs achieve effective conformation and retain their respective crystal structures in the self-supported graphene aerogel. Notably, in the XRD pattern of the self-supported graphene aerogel, 10°... o No obvious diffraction peaks were observed, further confirming that GO was completely reduced by dopamine during the preparation process.

[0127] The electrochemical performance of the prepared self-supporting graphene aerogel-modified electrode was characterized, and the results are as follows: Figure 13 As shown: The modified electrode is located at the positively charged probe hexaammineruthenium (Ru(NH3)6C) l3Both the cyclic voltammetry tests of the graphene oxide aerogel and the negatively charged probe potassium ferricyanide (K3[Fe(CN)6]) showed obvious redox peaks. However, the oxidation current amplitude of K3[Fe(CN)6] was significantly lower, indicating that the prepared self-supported graphene aerogel was negatively charged. This may be because the abundant oxygen-containing functional groups (such as carboxyl and hydroxyl groups) in the reduced graphene oxide (rGO) in the self-supported graphene aerogel endow the material surface with negative charge, generating an electrostatic repulsion effect on the negatively charged probe, thereby inhibiting its electron transfer process on the electrode surface and resulting in a weakened response current.

[0128] Furthermore, the electrochemical response characteristics of the self-supporting graphene aerogel-modified electrode to TMB were verified. Figure 14 As shown, the two distinct oxidation peaks observed at +0.23 V and +0.52 V can be attributed to the sequential oxidation of two amino groups in the TMB molecule. This result confirms the excellent electrochemical performance of the self-supporting graphene aerogel-modified electrode and the feasibility of using TMB as an electrochemical signal probe.

[0129] Based on the above detection principle and characterization results, it can be seen that the special structure of the self-supporting graphene aerogel plays a key role in the performance of the fibrinogen detection method described in this embodiment, specifically in the following aspects:

[0130] First, the porous structure of the self-supporting graphene aerogel provides a high surface area for antibody modification and immobilization, which can effectively inhibit the separation and aggregation of modified antibodies and improve the stability of the biorecognition interface.

[0131] Secondly, the excellent conductivity of rGO and MWCNTs in self-supporting graphene aerogel can significantly improve electron transport efficiency and enhance detection sensitivity.

[0132] Furthermore, the negatively charged nature of the self-supporting graphene aerogel surface can promote the deposition of TMB-TMBox precipitates on the electrode surface, increase the loading of electroactive materials on the electrode surface, and thus further improve the detection sensitivity.

[0133] The fibrinogen detection method described in this embodiment was used to detect fibrinogen solutions of different concentrations to verify the feasibility of the method. More specifically, fibrinogen standard solutions with concentrations of 0 (control group), 1, and 10 g / L were prepared, using fibrinogen-free artificial serum as the solvent. The fibrinogen detection method described in this embodiment was used for testing via cyclic voltammetry (cyclic voltammetry test parameters: onset voltage 0 V, termination voltage 0.8 V, scan rate 0.1 V / s). The detection results are as follows: Figure 15 As shown, the electrochemical response of TMB can be observed in solutions containing different concentrations of fibrinogen, and the peak value of the cyclic voltammetric current increases with increasing concentration.

[0134] The effective detection range of the fibrinogen detection method described in this embodiment is 0.1 to 10 g / L. More specifically, fibrinogen standard solutions with concentrations of 0 (control group), 0.1, 1, 2, 5, and 10 g / L were prepared, using fibrinogen-free artificial serum as the solvent. The fibrinogen detection method described in this embodiment was used for testing via chronoamperometry (chronoamperometry test parameters: test voltage 0.38 V, test time 120 s). The detection results are as follows: Figure 16 As shown, it can be observed that the current response value obtained by the chronoamperometry method gradually increases with the increase of fibrinogen concentration.

[0135] Based on these test results, a standard curve was established between the chronoamperometry current response value and fibrinogen concentration. For example... Figure 17 As shown, a good linear relationship can be observed between the current value and the logarithm of the fibrinogen concentration. The resulting concentration fitting curve is shown below, where the unit of current I is μA, the unit of fibrinogen concentration is g / L, and the fibrinogen concentration range is 0.1-10 g / L. Within this range, the current value changes linearly with the fibrinogen concentration, with a linear correlation coefficient of 0.96, thus enabling the detection of fibrinogen concentration.

[0136] I = 5.38 + 1.34ln[Cfib]

[0137] The anti-interference ability of the fibrinogen detection method described in this embodiment was verified through an anti-interference experiment. More specifically, a solution containing 5 mM glucose, 1 mM lactate, 5 mM potassium ions (K+), 150 mM sodium ions (Na+), 1.5 mM calcium ions (Ca2+), 10 ng / mL bovine serum albumin (BSA), 10 ng / mL immunoglobulin G (IgG), 10 ng / mL tumor necrosis factor α (TNF-α), 100 μg / mL tranexamic acid (TXA), and 1600 ng / mL tissue plasminogen activator (tPA) was prepared for selective characterization, such as... Figure 18 As shown, with the addition of different concentrations of interfering substances, the response current value did not differ significantly with the constant concentration of fibrinogen precursor, indicating that the fibrinogen detection method described in this embodiment has strong anti-interference ability against common coexisting substances in serum.

[0138] Example 3

[0139] To verify the clinical applicability of the perioperative fibrinogen detection device and method proposed in this invention, a third perioperative clinical verification example is provided.

[0140] This study recruited 20 patients who underwent type A aortic dissection repair surgery. Type A aortic dissection repair surgery involves intimal tear resection and ascending aortic replacement. Serum samples were collected from patients at key stages of the surgery.

[0141] More specifically, the key phases of the procedure include three phases: anesthesia induction, before supplementation with human fibrinogen concentrate (HFC), and after supplementation with HFC.

[0142] The fibrinogen concentration of each sample was detected using the device and method of this invention. At the same time, the same sample was sent to the laboratory for testing using the Clauss method, and the results of the Clauss method test were used as a reference standard for changes in fibrinogen concentration.

[0143] During surgery, cardiopulmonary bypass is initiated after anesthesia induction. At this time, the patient's heart stops beating, and their cardiopulmonary function is maintained by the cardiopulmonary bypass machine. Cardiopulmonary bypass often leads to acquired hypofibrinogenemia due to blood dilution and consumption of clotting factors. Sufficient clinical evidence shows that this condition is significantly associated with increased bleeding risk, increased transfusion requirements, and poor prognosis. According to European clinical guidelines, HFC supplementation is a necessary clinical intervention.

[0144] like Figure 19 As shown, serum fibrinogen levels decreased significantly during cardiopulmonary bypass, reaching their lowest point before HFC supplementation. Fibrinogen levels gradually recovered after HFC supplementation.

[0145] like Figure 20 As shown, the dynamic trend of fibrinogen concentration detected by the method of the present invention throughout the entire operation is highly consistent with the results of the Clauss method.

[0146] Furthermore, such as Figure 21 The correlation analysis results show that the detection results of the method of the present invention are highly correlated with the results of the Clauss method in the three stages of anesthesia induction, before HFC supplementation and after HFC supplementation, with correlation coefficients (r) of 0.97, 0.93 and 0.96, respectively.

[0147] The above results fully demonstrate the feasibility and reliability of the method and apparatus of the present invention for perioperative fibrinogen detection.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for perioperative fibrinogen detection, characterized by, Includes the following steps: S1. Electrode modification: Antibody-functionalized self-supporting graphene aerogel material is modified onto the working electrode surface of a screen-printed electrode (30). The self-supporting graphene aerogel is prepared by a hydrothermal method assisted by polyethylene glycol and dopamine. During preparation, its interlayer and three-dimensional structure are effectively controlled by adjusting the content of polyethylene glycol and multi-walled carbon nanotube solution, including controlling the self-supporting graphene aerogel structure by adjusting the concentration of multi-walled carbon nanotube solution, so that it presents a uniform three-dimensional structure. The self-supporting graphene aerogel is prepared by a hydrothermal method assisted by polyethylene glycol and dopamine, specifically step... The steps include: S1, mixing graphene oxide solution and multi-walled carbon nanotube solution, and ultrasonically dispersing to form a composite dispersion; S2, adding polyethylene glycol to the composite dispersion, stirring and dissolving to form a PEG-GO / MWCNTs precursor solution, and adjusting the pH of the precursor solution; S3, adding dopamine to the PEG-GO / MWCNTs precursor solution, stirring and dissolving, water bath heating for polymerization and crosslinking, and freeze-drying to obtain self-supporting graphene aerogel; wherein the mass fraction of polyethylene glycol is less than 3.0%, and the concentration of the multi-walled carbon nanotubes is 0.25 mg / mL; S2. Incubation of the test sample: The serum sample to be tested is dropped onto the surface of the modified working electrode and then washed after incubation. S3. Detect antibody binding: Add horseradish peroxidase-labeled fibrinogen detection antibody to the modified working electrode surface and wash after incubation. S4. Add an electrochemical probe by dropping a matrix-type TMB solution containing H2O2 onto the surface of the modified working electrode and then cleaning it after incubation. S5. Electrochemical signal acquisition: The reduction current of the electrochemically active material on the electrode surface is acquired by chronoamperometry. S6. Concentration Calculation: Calculate the fibrinogen concentration based on the reduction current value and the standard curve.

2. The method for perioperative fibrinogen detection according to claim 1, characterized in that, The working electrode and counter electrode of the screen-printed electrode (30) are obtained by printing carbon paste onto a PET substrate, and the reference electrode is obtained by printing silver or silver chloride paste onto a PET substrate.

3. The method for perioperative fibrinogen detection according to claim 1, characterized in that, By adjusting the mass fraction of polyethylene glycol, the bonding mode between layers of self-supporting graphene aerogel sheets can be controlled and effective separation can be achieved.

4. The method for perioperative fibrinogen detection according to claim 1, characterized in that, Antibody-functionalized self-supporting graphene aerogel-modified screen-printed electrodes were prepared using a bio-coupling method. The preparation steps included: S1. Interface activation: EDC solution and NHS solution are sequentially added to the surface of the self-supporting graphene aerogel modified screen-printed electrode (30) and incubated to complete the electrode surface activation treatment. S2. Antibody conjugation: After rinsing the electrode surface obtained in step S1, fibrinogen antibody solution is added and incubated to achieve bioconjugation between the antibody and the electrode surface. S3. Electrode sealing: Rinse the electrode surface obtained in step S2, add bovine serum albumin solution for incubation, and seal the non-specific binding sites on the electrode surface. S4. Electrode cleaning: Rinse the surface of the electrode obtained in step S3 to finally obtain the antibody-functionalized self-supporting graphene aerogel modified screen-printed electrode (30).

5. The method for perioperative fibrinogen detection according to claim 1, characterized in that, The fibrinogen concentration is calculated based on the standard curve I = 5.38 + 1.34ln [Cfib], where I is the current value in μA and Cfib is the fibrinogen concentration in g / L.

6. A device for perioperative fibrinogen detection, using the method described in any one of claims 1-5, characterized in that, include: Electrode interface circuit (21) is used to insert screen-printed electrodes (30) modified with antibody-functionalized self-supporting graphene aerogel material. An electrochemical detection circuit (11) is connected to an electrode interface circuit (21) and includes a constant potential output unit and a current detection unit, used to provide electrochemical reaction voltage and detect current signals. Wireless communication circuit (13) is used to interact with the user interface to transmit detection commands and results; The microprocessor circuit (12) is connected to the electrochemical detection circuit (11) and the wireless communication circuit (13) respectively, and is used to regulate the output voltage parameters and the amplification parameters of the small electrochemical current signal of the electrochemical detection circuit (11).

7. The device for perioperative fibrinogen detection according to claim 6, characterized in that, The electrochemical detection circuit (11) includes: The digital-to-analog converter module is used to convert the voltage digital signal given by the microprocessor circuit (12) into an analog signal; The transimpedance amplifier module is used to amplify the weak electrochemical current signal generated by the screen-printed electrode (30) and convert it into a measurable voltage signal; The analog-to-digital converter module is used to sample the amplified voltage signal and convert it into a digital signal, and transmit the digitized detection result to the microprocessor circuit (12).

8. The device for perioperative fibrinogen detection according to claim 6, characterized in that, The electrochemical detection circuit (11), wireless communication circuit (13) and microprocessor circuit (12) are integrated on the first circuit board (10); the electrode interface circuit (21) is integrated on the second circuit board (20); the first circuit board (10) and the second circuit board (20) are connected by a flexible printed cable (15).

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