Immune biosensor for human tissue plasminogen activator antigen
By dispersing nanomaterials on the electrode surface and introducing aldehyde groups and combining t-PA antibodies, the existing t-PA detection methods are solved, and fast and economical t-PA detection is achieved.
Patent Information
- Application Number
- CN202411802324.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-07
AI Technical Summary
The existing t-PA detection methods are complex, costly and long detection time, making it difficult to achieve rapid detection.
The composite of porous nanocopper, multi-walled carbon nanotubes and nanogold particles dispersed in chitosan solution was used to modify the electrode surface, and the aldehyde group was introduced through glutaraldehyde and the detection was carried out in combination with t-PA antibodies.
The electroactivity and sensitivity of the electrode interface are improved, the detection time is shortened, the cost is reduced, and t-PA detection in the range of 1μg/L to 12μg/L is achieved.
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Figure CN119574668A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of human tissue plasminogen activator antigen detection, in particular to an immune biosensor for human tissue plasminogen activator antigen. Background Art
[0002] Tissue plasminogen activator (t-PA) is a serine protease that is widely distributed in vascular endothelial cells, mature oocytes, pancreas and other tissues in the body. t-PA is a key substance in the fibrinolytic system. It activates plasminogen to convert into plasmin, thereby degrading fibrin (the main component of thrombus), and is widely used in thrombolytic therapy for acute myocardial infarction.
[0003] When t-PA levels are elevated, it indicates increased fibrinolytic activity, which may be seen in primary or secondary fibrinolysis, such as disseminated intravascular coagulation (DIC); when t-PA levels are decreased, it indicates decreased fibrinolytic activity, which may be seen in hypercoagulable states and thrombotic diseases.
[0004] Thrombotic disease is one of the common diseases in clinical practice, including myocardial infarction, cerebral infarction, etc. t-PA detection can be used as one of the indicators for auxiliary detection of thrombotic diseases. During the thrombosis process, the activity of t-PA may decrease, making the thrombus difficult to dissolve. Therefore, by detecting the level of t-PA, the presence and severity of thrombotic diseases can be indirectly judged.
[0005] Existing t-PA detection methods include bubble rise method, fibrin plate method, real-time fluorescence quantitative RT-PCR method, solid-phase liquid phase two-step method, chromatography and mass spectrometry, etc. On the one hand, these methods require more complicated pre-processing of samples, the detection cost is relatively high, and more sophisticated instruments and technical support are also required; on the other hand, the detection time is long, and it is difficult to achieve rapid detection. Summary of the invention
[0006] In view of the technical defects of the existing human tissue plasminogen activator antigen detection, the present invention aims to provide a complex for human tissue plasminogen activator antigen detection, a modified layer based on the complex, and an immunobiosensor for human tissue plasminogen activator antigen based on the modified layer.
[0007] The present invention protects a complex for detecting human tissue plasminogen activator antigen, wherein porous nano copper NPC, multi-walled carbon nanotubes MWCNTs, and nano gold particles AuNP are dispersed in a chitosan solution to obtain a solution I, wherein the mass ratio of NPC, MWCNTs, AuNP to the chitosan solution is 1-5:1-5:0.1-0.5:500;
[0008] Dissolving 1-hexyl-3-methylimidazolium hexafluorophosphate HMIM[PF6] in N,N-dimethylformamide DMF solution to obtain solution II, wherein the mass ratio of HMIM[PF6] to DMF solution is 1 to 3:10;
[0009] The MWCNTs-AuNP-NPC-CTS-HMIM[PF6] composite was prepared by uniformly dispersing solution I and solution II by ultrasonic mixing, wherein the volume ratio of solution I to solution II was 1:1-3.
[0010] Preferably, the preparation process of solution I comprises:
[0011] (1) Preparation of nanogold-chitosan solution: Mix nanogold solution with a volume ratio of 0.5:1 to 3:1 and chitosan solution with a concentration of 1.5%, and disperse by ultrasonication at room temperature;
[0012] (2) Dispersing MWCNTs and NPC in the nanogold-chitosan solution to obtain solution I, wherein the mass ratio of MWCNTs, NPC to the nanogold-chitosan solution is 1-5:1-5:500.
[0013] Preferably, the concentration of the nanogold solution is 0.5-2 mg / mL, and the concentration of the chitosan solution is 0.5%-10%.
[0014] The present invention also protects a modified layer of an immune biosensor. The above-mentioned complex solution is dripped onto the surface of a working electrode and dried to obtain a complex film. A phosphate buffer solution is used to rinse off the nanoparticles not fixed on the electrode surface, and an aldehyde group introduction solution is then dripped onto the modified layer. The reaction is carried out at room temperature in the dark for 1 hour, and then the electrode surface is rinsed with a phosphate buffer solution to remove excess aldehyde group introduction solution to obtain the desired modified layer. The aldehyde group introduction solution is preferably a glutaraldehyde solution with a concentration of 0.1%-2%.
[0015] The introduction of MWCNTs can improve the electrical activity of the electrode interface, increase the effective area of the electrochemical reaction, and improve the electrochemical response signal. On the one hand, NPC, as a nanometal material, improves conductivity. On the other hand, it has a large specific surface area. A large number of probe molecules will attach to the tube wall and channel surface of NPC, providing more sites for antigen binding, and further improving the sensitivity of the electrode. The introduction of AuNP improves the biocompatibility of the material. By combining with chitosan, its biocompatibility is further improved, and the stability and service life of the sensor are improved. In addition, the introduction of AuNP also further improves the conductivity of the sensor and increases the sensitivity of the sensor.
[0016] The composite film obtained after the complex solution is dripped onto the surface of the working electrode and dried solves the dispersion problem of MWCNTs, AuNPs, and NPCs. Since chitosan itself has a large number of amino groups, the aldehyde groups in glutaraldehyde and the amino groups on the surface of chitosan are covalently bonded by dripping glutaraldehyde solution, and finally aldehyde groups are introduced on the surface of chitosan. During the detection process, by dripping the probe molecule solution (t-PA antibody) on the electrode surface, the probe molecules in the solution are combined with the aldehyde groups on the surface of chitosan to capture the analyte.
[0017] The present invention also protects an immune biosensor for human tissue plasminogen activator antigen, which is characterized by comprising a PET substrate, an electrode layer, an insulating layer, and a modification layer; the electrode layer is printed on the PET substrate, including a working electrode, a counter electrode, a reference electrode, and a pin, the reaction area is placed on the working electrode, and the modification layer is loaded on the reaction area and is the above-mentioned modification layer; the insulating layer is located on the electrode layer, covering other areas except the working electrode, the counter electrode, the reference electrode, and the pin.
[0018] The working electrode and the counter electrode are used to measure the characteristic current. The electrode material can be a metal material, such as platinum, gold or their alloys, or a carbon material or other inert conductive material; preferably a metal material. The insulating layer can be a photocurable insulating material such as polyimide and polyamide to prevent the electrode from short-circuiting during use.
[0019] The present invention proposes a complex for modifying an immunosensor, wherein the complex solution is dripped onto the electrode surface by a drop coating method to form a complex film, and then the film surface is modified by introducing an aldehyde group into the solution, and the aldehyde group is introduced for binding with the antibody to be detected. NPC, MWCNTs, and AuNP are dispersed in a chitosan solution to improve the electrical activity of the electrode interface, increase the effective area of the electrochemical reaction, and enhance the sensitivity of the electrode. While increasing the electrochemical signal, a wide range of active sites is provided; the linear range of the immunosensor is large (1 μg / L to 12 μg / L), and the detection of human tissue plasminogen activator antigen within the normal concentration range can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 Schematic diagram of the immune biosensor structure;
[0021] Figure 2 Electrochemical detection CV graphs of the immunobiosensor for human tissue plasminogen activator antigen obtained in groups A, B, C, and D;
[0022] Figure 3a It current test graphs of the immune biosensor of human tissue plasminogen activator antigen obtained by four groups A, B, C, and D;
[0023] Figure 3b This is a linear relationship diagram between current and concentration during the detection of human tissue plasminogen activator antigen of various concentrations by the immune biosensor of human tissue plasminogen activator antigen obtained by the four groups A, B, C, and D. DETAILED DESCRIPTION
[0024] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below in conjunction with the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.
[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The technical scheme of the present invention is described more clearly and completely below in conjunction with specific embodiments and comparative examples.
[0026] Example 1
[0027] Preparation of complex for human tissue plasminogen activator antigen detection:
[0028] 1. Preparation of nanogold-chitosan solution: Mix 5 mL of 1 mg / mL nanogold solution and 10 mL of 1.5% chitosan solution, ultrasonicate for 2 h at room temperature, and store at 4°C for later use.
[0029] The chitosan solution with a concentration of 1.5% is prepared by using 1% glacial acetic acid, specifically: first weigh 15g chitosan solid, add 980mL water to disperse evenly, then add 20mL 1% glacial acetic acid, stir until the solid is completely dissolved, and obtain a chitosan solution with a concentration of 1.5%. The preparation method of the chitosan solution in the following examples is the same as that of this example, except that the gram weight of the chitosan solid is adjusted.
[0030] 2. Take 40 mg of MWCNTs and 40 mg of NPC and disperse them in 15 mL of nanogold-chitosan solution;
[0031] 3. Take 2g of HMIM[PF6] and dissolve it in 20g of DMF solution;
[0032] 4. Mix the two solutions of step 2 and step 3 in a volume ratio of 1:2 and disperse them ultrasonically to obtain the desired composite solution.
[0033] Example 2
[0034] Preparation of complex for human tissue plasminogen activator antigen detection:
[0035] 1. Prepare nanogold-chitosan solution by mixing 5 mL of 2 mg / mL nanogold solution and 10 mL of 2% chitosan solution, ultrasonicate for 2 h at room temperature, and store at 4 °C for later use;
[0036] 2. Take 100 mg of MWCNTs and 100 mg of NPC and disperse them in 15 mL of nanogold-chitosan solution;
[0037] 3. Take 5g of HMIM[PF6] and dissolve it in 20g of DMF solution;
[0038] 4. Mix the two solutions of step 2 and step 3 in a volume ratio of 1:1 and disperse them ultrasonically to obtain the desired composite solution.
[0039] Example 3
[0040] Preparation of complex for human tissue plasminogen activator antigen detection:
[0041] 1. Prepare nanogold-chitosan solution by mixing 10 mL of 0.5 mg / mL nanogold solution and 5 mL of 4% chitosan solution, ultrasonicating for 2 h at room temperature, and storing at 4 °C for later use;
[0042] 2. Take 80 mg of MWCNTs and 80 mg of NPC and disperse them in 15 mL of nanogold-chitosan solution;
[0043] 3. Take 4g of HMIM[PF6] and dissolve it in 20g of DMF solution;
[0044] 4. Mix the two solutions of step 2 and step 3 in a volume ratio of 1:3 and disperse them ultrasonically to obtain the desired composite solution.
[0045] Example 4
[0046] Preparation of complex for human tissue plasminogen activator antigen detection:
[0047] 1. Disperse 40 mg of MWCNTs, 40 mg of NPC, and 5 mg of gold nanoparticles in 15 mL of gold nanoparticle-chitosan solution;
[0048] 2. Take 2g of HMIM[PF6] and dissolve it in 20g of DMF solution;
[0049] 3. Mix the two solutions of step 1 and step 2 in a volume ratio of 1:2 and disperse them ultrasonically to obtain the desired composite solution.
[0050] The complex solutions prepared in Examples 1-4 were respectively added dropwise to the reaction areas of the working electrodes of three groups of immunobiosensors (respectively designated as Groups A, B, C, and D), and a complex film was obtained after drying; a 2% phosphate buffer solution was then used to rinse off the unfixed nanoparticles on the electrode surface, and then 2 μL of an aldehyde introduction solution (1% glutaraldehyde solution) was added dropwise to the modified layer, and the reaction was carried out at room temperature in the dark for 1 h, and then the electrode surface was rinsed with a 2% phosphate buffer solution to remove excess aldehyde introduction solution to obtain the desired modified layer.
[0051] The immune biosensor includes a PET substrate 1, an electrode layer, an insulating layer, and a modified layer; the electrode layer is printed on the PET substrate 1, including a working electrode 2, a counter electrode 3, a reference electrode 4, and a pin 5, and the reaction area is placed on the working electrode; the insulating layer is located on the electrode layer, covering other areas except the working electrode, the counter electrode, the reference electrode, and the pin.
[0052] The following electrochemical detection was performed on the immunobiosensors of human tissue plasminogen activator antigen obtained in groups A, B, C, and D. The immunobiosensors of human tissue plasminogen activator antigen in each group were taken and measured at 5 mM [Fe(CN6)] 3- / 4- The test was carried out in 0.1 mol KCl solution. The modification of the electrode layer was verified by CV. The scanning range of CV was -0.6 V to 1 V, and the scanning rate was 100 mV / S. The results are shown in Figure 2 shown.
[0053] from Figure 2 It can be seen that the redox peak current of the unmodified electrode is low. When the MWCNTs-AuNP-NPC-CTS-HMIM[PF6] complex solution is added, the electrode is modified and the redox peak current increases significantly. This indicates that the addition of the MWCNTs-AuNP-NPC-CTS-HMIM[PF6] complex solution significantly increases the peak current and improves the sensitivity of the electrode reaction interface.
[0054] Next, the performance of the four groups of immunosensors A, B, C, and D was evaluated using the it current test method. Human serum human tissue plasminogen activator antibody buffer was added to the working electrode with the modified layer. After incubation for 12 hours, a 0.1% bovine serum albumin BSA solution was added to block the nonspecific binding sites; then human tissue plasminogen activator antigen with a concentration range of 1μg / L to 12μg / L was introduced, incubated for 30 minutes to wait for the antigen and antibody to bind, and then 5mM [Fe(CN6)] was added to the working electrode area. 3- / 4- and 0.1mol KCl solution, the it current test voltage is 0.3V, the running time is 10s, and the results are as follows Figure 3a , Figure 3b shown.
[0055] Figure 3a The results showed that after the introduction of human tissue plasminogen activator antigen, the current signal of the immunosensor decreased. This may be because the antigen-antibody binding continuously hindered the transfer of electrons, causing the resistance of the sensor to further increase, indicating the successful progress of the antigen-antibody reaction. When the antigen-antibody binding time was 30 minutes, the current signal on the immunosensor further decreased with the increase of antigen concentration.
[0056] Figure 3b It shows that when the concentration of human tissue plasminogen activator antigen is 1μg / L~12μg / L, the magnitude of the current signal shows a good linear relationship with the concentration of human tissue plasminogen activator antigen, and the linear regression equation is y=-0.702C t-PA +8.834×10 -6 , the linear correlation coefficient is R 2 =0.97096.
[0057] In summary, the present invention is used to design a complex for modifying an immunosensor, the complex solution is dripped onto the electrode surface by a drop coating method to form a corresponding complex film, the film surface is modified by glutaraldehyde, an aldehyde group is introduced for binding with the antibody of the object to be detected, and porous nano-copper materials, multi-walled carbon nanotubes and colloidal gold materials are introduced at the same time, while increasing the electrochemical signal, a wide range of active sites is provided. The linear range of the sensor is large (1μg / L to 12μg / L), and it can realize the detection of human tissue plasminogen activator antigen within the normal concentration range.
[0058] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A complex for detecting human tissue plasminogen activator antigen, characterized in that: Solution I is obtained by dispersing porous nano-copper NPC, multi-walled carbon nanotubes MWCNTs, and nano-gold particles AuNP in a chitosan solution, wherein the mass ratio of NPC, MWCNTs, AuNP, and chitosan solution is 1-5:1-5:0.1-0.5:500; Dissolving 1-hexyl-3-methylimidazolium hexafluorophosphate HMIM[PF6] in N,N-dimethylformamide DMF solution to obtain solution II, wherein the mass ratio of HMIM[PF6] to DMF solution is 1 to 3:10; The MWCNTs-AuNP-NPC-CTS-HMIM[PF6] composite was prepared by uniformly dispersing solution I and solution II by ultrasonic mixing, wherein the volume ratio of solution I to solution II was 1:1-3.
2. The composite according to claim 1, characterized in that The preparation process of solution I includes: (1) Preparation of nanogold-chitosan solution: Mix nanogold solution and chitosan solution in a volume ratio of 0.5:1 to 3:1, and disperse by ultrasonic at room temperature; (2) Dispersing MWCNTs and NPC in the nanogold-chitosan solution to obtain solution I, wherein the mass ratio of MWCNTs, NPC to the nanogold-chitosan solution is 1-5:1-5:
500.
3. The composite according to claim 1, characterized in that The concentration of the nano-gold solution is 0.5-2 mg / mL, and the concentration of the chitosan solution is 0.5%-10%.
4. A modified layer of an immune biosensor, characterized in that: Adding the composite solution of claim 1 dropwise onto the surface of the working electrode, and drying to obtain a composite film; The unfixed nanoparticles on the electrode surface were washed away with phosphate buffer, and then the aldehyde introduction solution was added dropwise on the modified layer. The reaction was allowed to proceed at room temperature in the dark for 1 hour, and then the electrode surface was rinsed with phosphate buffer to remove the excess aldehyde introduction solution to obtain the desired modified layer.
5. The modification layer according to claim 4, characterized in that The aldehyde group introduction solution is a glutaraldehyde solution with a concentration of 0.1%-2%.
6. An immunobiosensor for human tissue plasminogen activator antigen, characterized in that: It comprises a PET substrate (1), an electrode layer, an insulating layer, and a modification layer; The electrode layer is printed on the PET substrate (1), comprising a working electrode (2), a counter electrode (3), a reference electrode (4), and a lead (5); the reaction zone is placed on the working electrode; the modification layer is loaded on the reaction zone and is the modification layer according to claim 3 or 4; The insulating layer is located on the electrode layer, and covers other areas except the working electrode, the counter electrode, the reference electrode, and the pins.