AuPdNPs (at) GO-MWCNTs composite material modified immunosensor with wide detection range and application thereof

Abstract

The invention discloses an AuPdNPs (at) GO-MWCNTs composite material modified immunosensor with a wide detection range and application of the AuPdNPs (at) GO-MWCNTs composite material modified immunosensor, the composite material is prepared by dispersing graphene oxide and carboxylated MWCNTs in a solution, performing an ultrasonic reaction in an ice-water bath to obtain a GO-MWCNTs compound, then dropwise adding an AuPdNPs solution onto a GO-MWCNTs film, and after the AuPdNPs solution is air-dried, forming the AuPdNPs (at) GO-MWCNTs composite material. The detection limit of the immunosensor modified by the AuPdNPs (at) GO-MWCNTs compound on lipoprotein phospholipase A2 is 0.3 ng / ml, and the detection range is 1 ng / ml to 2 [mu] g / ml; and the method has good specificity, the repeatability, the stability and the clinical property all meet the requirements, and the content of the lipoprotein phospholipase A2 in a clinical sample can be quantitatively detected.

Description

Technical Field

[0001] The invention relates to the field of detection, in particular to an AuPdNPs@GO-MWCNTs composite material with a wide detection range, and also to applications of the AuPdNPs@GO-MWCNTs composite material. Background Art

[0002] According to the latest statistics released by WHO, cardiovascular disease is the number one cause of death in the world - more people die from cardiovascular disease each year than from any other disease, so cardiovascular disease research is an important problem that needs to be overcome in the world's public health. Atherosclerosis (AS) is a disease characterized by hardening of the arterial wall and formation of intimal atherosclerotic plaques, and is the main cause of cardiovascular and cerebrovascular events. In recent years, the mortality rate of cardiovascular and cerebrovascular diseases in developed countries has gradually declined, while the prevalence of the disease in developing countries has been on the rise. It is reported that developing countries bear 80% of cardiovascular and cerebrovascular diseases worldwide. AS has a complex pathophysiological mechanism and is easily overlooked due to its hidden early clinical manifestations. Before AS causes complications, its progression period can be as long as ten years. Therefore, early diagnosis and dynamic monitoring of the development of AS are the key to reducing potential clinical complications and the total burden of cardiovascular and cerebrovascular events.

[0003] At present, the methods for early diagnosis and evaluation of AS mainly include serological index detection, imaging technology and other technologies. Although the previous early diagnosis methods of AS have a high accuracy rate, they also have certain limitations. For example, imaging technology detection is expensive, and it often needs to be combined with other methods for diagnosis. Traditional serological tests often collect human serum for ELISA experiments, which are time-consuming, require a large amount of samples, and require labeling (fluorescent labeling methods are often used in the early days). Different methods have their own advantages and disadvantages. Clinically, a variety of means should be combined to achieve early diagnosis and monitoring of AS.

[0004] For disease research, a biomarker is a biochemical indicator of a characteristic pathological or physiological process, which has sufficient accuracy and reproducibility. Finding and discovering valuable biomarkers has become an important hotspot in current research. Atherosclerosis (AS) is an inflammatory reaction, and many studies have shown that lipoprotein-associated phospholipase A2 (LP-PLA2) is an emerging biomarker with a strong pro-inflammatory effect and is an independent risk factor for coronary heart disease. Traditional inflammatory indicators of atherosclerosis include CRP, IL-6, TNF-α, etc. These substances, as acute reaction synthetic products of AS, can increase in the early stage and promote the occurrence and development of AS. However, these indicators have many interfering factors and low vascular specificity. Compared with other inflammatory markers, A2 has three major advantages in diagnosing AS. 1) LP-PLA2 is not easily affected by systemic factors such as rheumatic diseases, hypertension, blood pressure, body mass index, diabetes, and infection. 2) Plasma LP-PLA2 is relatively stable in the body, and luminescence immunoassay and ELISA are currently the main measurement methods. 3) Different from traditional risk factors, LP-PLA2 is a new independent risk factor for predicting cardiovascular events.

[0005] Electrochemical detection method is a useful tool. Due to its strong selectivity, easy operation and rapid response, it is widely used in food safety control, environmental testing and pharmaceutical analysis. Among them, glassy carbon electrode (GCE) is widely used in the development of biosensors. The advantages of glassy carbon electrode are good conductivity, high chemical stability, small thermal expansion coefficient, hard texture, good air tightness, wide potential application range (about -1~1V, relative to saturated calomel electrode), and can be made into cylindrical, disc and other electrode shapes. It can also be used as a substrate to make mercury film glassy carbon electrode and chemically modified electrode. It is increasingly widely used in electrochemical experiments or electroanalytical chemistry. So far, there has been no report on the electrochemical method for detecting lipoprotein phospholipase A2 based on GCE electrode. Therefore, it is very necessary to develop a method for detecting lipoprotein phospholipase A2 based on GCE biosensor. Summary of the invention

[0006] In view of this, the present invention develops an AuPdNPs@GO-MWCNTs enhanced GCE-based lipoprotein phospholipase A2 immunocomposite material to meet the needs of early clinical diagnosis of AS; lipoprotein phospholipase A2 is detected by specific binding of antigen and antibody; research results show that the developed immunosensor platform can effectively and accurately detect the lipoprotein phospholipase A2 content in clinical samples, and has certain application potential; the second purpose of the present invention is to provide the application of the AuPdNPs@GO-MWCNTs composite material in the preparation of an electrochemical immunosensor; the third purpose of the present invention is to provide an electrochemical immunosensor containing the AuPdNPs@GO-MWCNTs composite material; the fourth purpose of the present invention is to provide a method for detecting lipoprotein phospholipase A2 using the electrochemical immunosensor.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The AuPdNPs@GO-MWCNTs composite material with a wide detection range is prepared by dispersing graphene oxide and carboxylated MWCNTs in a solution, subjecting the composite material to continuous ultrasonic reaction at 100 kHz for 0.5 h in an ice-water bath, and then centrifuging at 20°C and 12,000 rpm for 0.5 h to obtain a GO-MWCNTs composite. The AuPdNPs solution is then dripped onto a GO-MWCNTs film, which is then air-dried to form an AuPdNPs@GO-MWCNTs composite material.

[0009] Preferably, the GO-MWCNTs composite is prepared by adding carboxylated multi-arm carbon nanotubes to graphene oxide at a concentration of 2.5 mg / mL to a final concentration of 2.5 mg / mL, subjecting the mixture to ultrasonic reaction in an ice-water bath for 1 hour, centrifuging to remove the supernatant, placing the precipitate at -70°C for 12 hours, and vacuum drying to obtain the GO-MWCNTs composite.

[0010] Preferably, the preparation method of AuPdNPs is as follows: a 20 mM K2PdCl4 solution, H2O, a 6.0 M HCl and pluronic F127 are mixed in a volume mass ratio of 0.6 ml: 3 ml: 60 ul: 60.0 mg, and after pluronic F127 is completely dissolved, a 0.1 M ascorbic acid solution is added to the above solution in an equal volume to the H2O, the mixed solution is ultrasonically treated in a 45°C water bath for 3 h, a 10 mM HAuCl4.4H2O solution is added to the reaction system, ultrasonicated for 1 h, the product is washed three times with ethanol and water, and collected by centrifugation to obtain AuPdNPs.

[0011] 2. Application of the AuPdNPs@GO-MWCNTs composite material in the preparation of electrochemical immunosensors.

[0012] 3. An electrochemical immunosensor containing the AuPdNPs@GO-MWCNTs composite material, wherein the working electrode of the electrochemical immunosensor is first formed into a film on a glassy carbon electrode with the GO-MWCNTs composite, and then an AuPdNPs solution is added and air-dried to obtain AuPdNPs@GO-MWCNTs / GCE, and then the composite material is activated and modified with an activation solution prepared with N,N′-carbonyldiimidazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The electrode is immersed in 1 mL of a solution containing 2 μg / mL detection antibody overnight, and finally sealed with 5% skimmed milk powder to obtain an immunosensor modified with AuPdNPs@GO-MWCNTs.

[0013] Preferably, the detection antibody is a lipoprotein phospholipase A2 monoclonal antibody.

[0014] Preferably, the lipoprotein phospholipase A2 monoclonal antibody is fixed on the AuPdNPs@GO-MWCNTs composite material of the working electrode, and then incubated in 1 mL of the sample to be tested for 30 min, and then 5 mM [Fe (CN) 6] 3- / 4- +0.1M KCl solution at 50mV.S -1 The CV was scanned in the voltage range of -0.5 to +0.8 V at a scan rate of .

[0015] The beneficial effects of the present invention are as follows: the present invention successfully synthesizes AuPdNPs@GO-MWCNTs composite materials, and a certain amount of lipoprotein phospholipase A2 monoclonal antibody can be bound by activating the carboxyl group of the composite material. By this method, the immune response can be amplified to achieve highly sensitive detection of lipoprotein phospholipase A2. Under optimized conditions, the prepared immunosensor has good performance in detecting lipoprotein phospholipase A2, a wide linear range (covering clinical needs), and a low detection limit. In addition, the immunosensor also has good stability and selectivity. Moreover, the content of lipoprotein phospholipase A2 in the sample can be preliminarily judged by the value of ΔI detected by the immunosensor. In general, this method has great application potential and constructs a rapid and sensitive biosensor platform for the application of biomarker analysis methods and early disease diagnosis of atherosclerosis. Our future research will focus on further evaluating and optimizing the performance of the immunosensor by testing the content of lipoprotein phospholipase A2 in more serum specimens from patients with atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0017] Figure 1 SEM results (a: AuPdNPs@GO-MWCNTs SEM results (10μm); b: GO-MWCNTs SEM results (10μm); c: AuPdNPs@GO-MWCNTs SEM results (3μm); d: GO-MWCNTs SEM results (2μm);

[0018] Figure 2 This is the EDS (line scan) scanning result of AuPdNPs@GO-MWCNTs composite material;

[0019] Figure 3 The construction process of the sensor;

[0020] Figure 4 It is the CV curve diagram of electrode modification;

[0021] Figure 5 To detect different concentrations of A2 sensitivity curve (DPV);

[0022] Figure 6 Linear regression equation for detecting the sensitivity of A2 at different concentrations;

[0023] Figure 7 It is the repeatability test of the sensor;

[0024] Figure 8 It is a sensor stability test;

[0025] Fig. 9 It is a sensor specificity test;

[0026] Fig.10 For clinical trials of sensors. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0028] The materials used in the present invention are as follows: phosphate buffer (0.01M PBS, pH: 7.4), deionized water, blocking solution (prepared by skim milk powder and PBST in a certain proportion, SMPSF), lipoprotein phospholipase A2 monoclonal mouse antibody (self-made by hybridoma system) and carbon nanotubes (Aladdin).

[0029] Glassy carbon electrode (Poten), HAuCl4.4H2O (Aladdin), K2PdCl4 (Aladdin), N,N′-carbonyldiimidazole (Macklin), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (Macklin), potassium ferrocyanide (Aladdin) / potassium ferrocyanide (Macklin), (K4[Fe(CN)6·3H2O) / (C6FeK4N6) and potassium chloride (KCl) (Aladdin).

[0030] Example 1. Preparation of GO-MWCNTs composite material

[0031] Weigh 4g of graphite, 16.875g of NaNO3, and 180 mL of concentrated H2SO4 in a three-necked flask. Stir mechanically in an ice-water bath for 1h, then add 11.25g of KMnO4 and continue the reaction for 2h. Next, raise the water temperature to 35°C, react for 12h, then slowly add 200mL of deionized water, and add 250mL of water and 25mL of H2O2 after 1h of reaction. Ultrasonic peel the reaction suspension for 30min and let it stand for stratification, and centrifuge to remove the supernatant. Prepare a 20% HCl solution for washing the above mixture, centrifuge the mixture and wash it with HCl solution until the supernatant is added to a 0.1mol / L BaCl2 solution without precipitation. Then wash it with deionized water until it is nearly neutral to prepare a graphene oxide (GO) suspension.

[0032] Prepare the prepared GO solution (2.5 mg / mL) and add a certain amount of carboxylated multi-arm carbon nanotubes (COOH-MWCNTS) to the suspension to make the final concentration 2.5 mg / mL. Ultrasonic reaction in an ice water bath for 0.5 h and centrifuge to remove the supernatant. Wash the centrifuged precipitate mixture three times with deionized water and place it in an ultra-low temperature refrigerator at -70 ° C for 12 h, and then place it in a vacuum drying oven for 12 h to make a GO-MWCNTs composite powder material.

[0033] Example 2. Preparation of AuPdNPs

[0034] Mix 0.6 mL of K2PdCl4 solution (20 mM), 3.0 mL of H2O, 6.0 M HCl, and 60.0 mg of pluronic F127. After pluronic F127 is completely dissolved, add 3.0 mL (0.1 M) of ascorbic acid solution to the above solution. Ultrasonicate the mixed solution in a 45°C water bath for 3 h. Then, add 1.2 mL (10 mM) of HAuCl4.4H2O solution to the reaction system, ultrasonicate continuously for 1 h, and collect the final product through three washing / centrifugation cycles of ethanol and water.

[0035] The morphology of the synthesized AuPdNPs@GO-MWCNTs nanocomposites was characterized by scanning electron microscopy (SEM). Figure 1 As shown. The SEM results show that the microscopic characterization results of GO are black flakes and evenly distributed, which is consistent with the characterization results reported in the literature. The microscopic characterization structure of MWCNTs is multi-arm tubular, which is consistent with the characterization results reported in the literature. As can be seen from Figure c, AuPdNPs are evenly wrapped around MWCNTs. The EDS element content analysis results show that the composite material contains four elements, C, O, Pd, and Au, and the content ratio is roughly the same as that of the materials added during the synthesis of the composite material. In summary, the AuPdNPs@GO-MWCNTs composite material was successfully synthesized. Therefore, AuPdNPs were successfully compounded on the surface of the GO-MWCNTs composite nanomaterial.

[0036] The element types and contents of the composite materials were analyzed by energy dispersive spectrometer (EDS). Figure 2 And as shown in Table 1.

[0037] Table 1. Element content of AuPdNPs@GO-MWCNTs composite materials

[0038]

[0039] Example 3: Construction of A2 Immunosensor

[0040] The A2 immunosensor (GCE) consists of a working electrode, a carbon counter electrode, and a silver reference electrode. The construction process of the sensor is shown in Figure 3 . The specific method is to drop 6μL of GO-MWCNTs ethylene glycol solution onto the working electrode once, air-dry it naturally, and obtain GO-MWCNTs / GCE. Then, the AuPdNPs solution is dropped onto the GO-MWCNTs film, and it is air-dried to form AuPdNPs@GO-MWCNTs / GCE. Before the lipoprotein phospholipase A2 antibody is fixed, the composite modified electrode is activated with an activation solution prepared with N,N′-carbonyldiimidazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide at 25°C for 1h. Next, the activated electrode is immersed in the lipoprotein phospholipase A2 antibody dilution solution at 4°C overnight. Finally, the electrode (AuPdNPs@GO-MWCNTs / GCE) is modified with a monoclonal antibody, washed with PBS, and blocked with 5% skim milk powder for 1h to prevent false positive signals.

[0041] The differently modified sensors were exposed to 5 mM [Fe(CN)6] 3- / 4- +0.1M KCl solution at 50mV.S -1The CV was scanned in the voltage range of -0.5 to +0.8 V at a scan rate of Figure 4 As shown. The results show that the bare GCE electrode exhibits a pair of redox peaks. Due to the poor conductivity of GO-MWCNTs, the redox peak current increased significantly after AuPdNPs were modified on GO-MWCNTs. This shows that AuPdNPs can effectively enhance electron transfer. After the skim milk powder was blocked on the surface of AuPdNPs@GO-MWCNTs, the peak current decreased. Moreover, after 1mL of antibody (2μg / mL) was immobilized on the surface of AuPdNPs@GO-MWCNTs, the current was further reduced. After the antibody specifically recognized lipoprotein phospholipase A2, an immune complex was formed on the surface of AuPdNPs@GO-MWCNTs, further inhibiting electron transfer and thus further reducing the current. These characterizations reveal the successful preparation of the electrode.

[0042] Example 4: Electrochemical Detection of Lipoprotein Phospholipase A2 Immunosensor

[0043] Under the optimal reaction conditions, differential pulse voltammetry (DPV) scanning analysis was performed from -0.5 to +0.8 v, and the current signal of the immunosensor (AuPdNPs@GO-MWCNTs / GCE) was recorded as I0. After the introduction of the target analyte, the DPV signal generated from the immunosensor (AuPdNPs@GO-MWCNTs / GCE) was recorded as I1. At different concentrations of lipoprotein phospholipase A2 protein (1-2000 ng / mL) in the test reactor, the DPV signal was recorded as ΔI (ΔI = I0-I1). All experimental data were analyzed using Origin Lab Software v.6, and the test results are shown in Figure 2. Figure 5 The results showed that the peak current increased with the increase of lipoprotein phospholipase A2 concentration from 1 to 2000 ng.mL -1 The linear regression equation of A2 sensitivity at different concentrations is as follows: Figure 6 The results show that the current signal (I) has a good correspondence with the logarithmic value of the actual lipoprotein phospholipase A2 concentration. The corresponding relationship is: I (μA) = -6.9556log C A2 (ng mL -1 )+70.319(R 2 =0.9966), and the lower limit of detection (LOD) was 0.30 ng mL -1(S / N=3). Obviously, the change of current signal can be used to indicate immune response, and the sensitivity can be effectively amplified. This method has the characteristics of wide linear range and low detection limit. However, ultra-sensitive sensors are still the goal of our efforts. The sensitivity of its sensor can be improved by selecting ideal metals or metal oxides as signal amplification materials, adjusting the morphology and increasing the specific surface area of ​​the substrate material.

[0044] The stability of a biosensor refers to its ability to maintain detection performance over a certain period of time. Stability includes indicators such as repeatability, reproducibility, and sustainability, which can be improved through long-term use and quality control. Biosensor repeatability and stability have always been considered the most important aspects of biosensor performance for true biomarker detection.

[0045] In order to evaluate the repeatability of this biosensor in detecting lipoprotein phospholipase A2, a certain concentration of lipoprotein phospholipase A2 antibody was incubated on the modified electrode, and the same concentration of lipoprotein phospholipase A2 protein solution was repeatedly detected 5 times. The results showed that ( Figure 7 ), under the same test conditions, the same concentration of A2 antigen was detected and the current generated was basically the same, indicating that the sensor has good repeatability.

[0046] In addition, the stability of the biosensor was evaluated. To study the stability, five biosensors from the same batch of biosensors were prepared for the detection of lipoprotein phospholipase A2 under the same assay conditions. The same concentration of lipoprotein phospholipase A2 protein solution was detected under the same conditions on days 1, 3, 5, 7, and 9. All these sensors performed the same DPV detection parameters. The results showed that the current change was smaller as the detection time increased. Figure 8 ). This indicates that the biosensor prepared by this method has good stability.

[0047] In order to verify whether the sensor has high affinity detection performance for the target antigen, a specificity test was performed. Different proteins were used as interfering molecules, including heart-type fatty acid binding protein (hFABP), placental growth factor (PLGF), anti-angiogenic factor (CYFRA21-1) and cytokeratin 19. Fig. 9 As shown, when the biosensor was evaluated under the same conditions, only the detection of lipoprotein phospholipase A2 protein solution caused a strong current signal change. The current response of other interfering proteins was smaller. The results showed that excessive interfering molecules had no effect on the detection system, indicating that the biosensor has good specificity.

[0048] In order to verify the ability of the immunosensor to be applied to real clinical samples, the immunosensor was used to test clinical serum. Samples SN1, SN2, SN3, and SN4 were positive samples (C A2 >175ng / mL), SP1, SP 6 and SP 7 were negative samples. In the specificity test, peak current inhibition was used to determine whether the antigen and antibody were bound. The analysis results of A2 clinical samples showed that the electrical signal inhibition of samples SN1-4 was greater than 17μA, while the electrical signal inhibition of samples SP5-7 was basically the same, not exceeding 5μA, proving that the sensor can successfully distinguish positive and negative samples ( Fig.10 The results confirmed that the electrochemical biosensor has certain application potential in detecting the content of lipoprotein phospholipase A2 in real serum samples.

[0049] In order to further study whether the sensor can accurately detect the A2 content in positive samples, the concentration of samples that have been judged to be A2 positive was calculated. The values ​​detected by the electrochemical method were compared with the actual sample concentrations. It was found that the A2 concentrations in the four samples measured by the immunosensor were highly consistent with the ELISA results, and the recovery rate was between 97.54% and 100.59% (Table 2). These results show that the developed immunosensor has good reliability and can be used for quantitative detection of A2 concentrations in clinical samples.

[0050] Table 2. Clinical sample concentration measurement data

[0051]

[0052] In order to further evaluate the clinical use value of the sensor, we compared the present invention with the biosensors reported in the literature for the detection of lipoprotein phospholipase A2, as shown in Table 3. The detection limit of the existing lipoprotein phospholipase A2 biosensors is ng.mL -1 This may be related to the structure of the A2 protein molecule itself. The modified antibody in the sensor is easily saturated with the A2 antigen in the sample, and the content of lipoprotein phospholipase A2 is relatively high in human serum (the average content of A2 in the serum of normal Han people is 175ng.mL -1 , when it exceeds 175ng.mL -1The detection limit of the molecule is relatively low, but the detection range is relatively high. The detection range and detection limit of the sensor of the present invention are close to the results of foreign top journal papers, but the electrochemical sensor method of the present invention has a simpler operation process and higher stability. The clinical sample detection results are highly consistent with the results of the commercially available ELISA kit, and the relative standard deviation is within 2% (as shown in Table 2). Compared with the ELISA method, no enzyme labeling is required and the detection time is shorter, indicating that the sensor detection method of the present invention can meet the clinical detection requirements and has potential clinical application value.

[0053] Table 3. Comparison of the present invention and the published LP-PLA 2 biosensor

[0054]

[0055] In summary, AuPdNPs@GO-MWCNTs composites were successfully synthesized in this paper. After activating the carboxyl group of the composite, a certain amount of lipoprotein phospholipase A2 monoclonal antibody can be bound. In this way, the immune response can be amplified to achieve highly sensitive detection of lipoprotein phospholipase A2. Under optimized conditions, the prepared immunosensor has good performance in detecting lipoprotein phospholipase A2, a wide linear range (covering clinical needs), and a low detection limit. In addition, the immunosensor also has good stability and selectivity. Moreover, the value of ΔI detected by the immunosensor can be used to preliminarily judge the content of lipoprotein phospholipase A2 in the sample. In general, this method has great application potential and constructs a rapid and sensitive biosensor platform for the application of biomarker analysis methods and early disease diagnosis of atherosclerosis. Our future research will focus on further evaluating and optimizing the performance of the immunosensor by testing the content of lipoprotein phospholipase A2 in more serum specimens from patients with atherosclerosis.

[0056] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. AuPdNPs@GO-MWCNTs composite material with wide detection range, characterized by: The composite material is prepared by dispersing graphene oxide and carboxylated MWCNTs in a solution, and then continuously ultrasonically reacting at 100 kHz for 0.5 h in an ice water bath, and then centrifuging at 20° C. and 12,000 rpm for 0.5 h to obtain a GO-MWCNTs composite. Then, an AuPdNPs solution is dripped onto the GO-MWCNTs film, and the film is air-dried to form an AuPdNPs@GO-MWCNTs composite material.

2. The AuPdNPs@GO-MWCNTs composite material with a wide detection range according to claim 1, characterized in that: The GO-MWCNTs composite is prepared by adding carboxylated multi-arm carbon nanotubes to graphene oxide at a concentration of 2.5 mg / mL to a final concentration of 2.5 mg / mL, subjecting the mixture to ultrasonic reaction in an ice-water bath for 1 hour, centrifuging to remove the supernatant, placing the precipitate at -70°C for 12 hours, and vacuum drying to obtain the GO-MWCNTs composite.

3. The AuPdNPs@GO-MWCNTs composite material with a wide detection range according to claim 1, characterized in that: The preparation method of AuPdNPs is as follows: a 20mM K2PdCl4 solution, H2O, a 6.0M HCl and pluronic F127 are mixed in a volume mass ratio of 0.6ml:3ml:60ul:60.0mg, and after pluronic F127 is completely dissolved, a 0.1M ascorbic acid solution of equal volume to H2O is added to the above solution, the mixed solution is ultrasonically treated in a 45°C water bath for 3h, a 10mM HAuCl4.4H2O solution is added to the reaction system, ultrasonicated for 1h, and the product is washed three times with ethanol and water, and collected by centrifugation to obtain AuPdNPs.

4. Use of the AuPdNPs@GO-MWCNTs composite material according to any one of claims 1 to 3 in the preparation of an electrochemical immunosensor.

5. An electrochemical immunosensor comprising the AuPdNPs@GO-MWCNTs composite material according to any one of claims 1 to 3, characterized in that: The working electrode of the electrochemical immunosensor is first formed into a film on a glassy carbon electrode using a GO-MWCNTs composite, and then an AuPdNPs solution is added and air-dried to obtain AuPdNPs@GO-MWCNTs / GCE. The composite material is activated and modified with an activation solution prepared with N,N′-carbonyldiimidazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The electrode is immersed in 1 mL of a solution containing 2 ug / mL of a detection antibody overnight, and finally sealed with 5% skimmed milk powder to obtain an AuPdNPs@GO-MWCNTs modified immunosensor.

6. The electrochemical immunosensor according to claim 5, characterized in that: The detection antibody is a lipoprotein phospholipase A2 monoclonal antibody.

7. A method for detecting lipoprotein phospholipase A2 using the electrochemical immunosensor according to claim 5 or 6, characterized in that: The monoclonal antibody of lipoprotein phospholipase A2 was immobilized on the AuPdNPs@GO-MWCNTs composite material of the working electrode, and then incubated in 1 mL of the sample for 30 min. 3- / 4- +0.1M KCl solution at 50mV.S -1 The CV was scanned in the voltage range of -0.5 to +0.8 V at a scan rate of .

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