A green reduced gold nanoparticle composite nanomaterial and its preparation method and application

Abstract

The present invention discloses a green reduced gold nanoparticle composite nanomaterial, its preparation method, and application. The nanomaterial comprises amino-ion graphene and amino acid-reduced and amino acid-coated gold nanoparticles. This nanocomposite material significantly increases the effective electrode area and has promising application prospects in the field of nanosensors. It can also serve as a universal antibody immobilization interface, potentially enabling the detection of a range of immune response markers.

Description

Technical Field

[0001] The present invention relates to the technical field of composite nanomaterials, in particular to a green reduced gold nanoparticle composite nanomaterial and a preparation method and application thereof. Background Art

[0002] In recent years, graphene (rGO) has been widely used in biosensing, biomedical diagnosis, energy technology and medical fields due to its unique physical properties, high specific surface area ratio and excellent electrical conductivity. However, rGO prepared by existing methods lacks stability and dispersibility in solution. The surface properties of gold nanoparticles determine the loading efficiency and anti-interference ability of antibodies, and how to avoid nanoparticle agglomeration is crucial. The self-assembly of functionalized rGO and AA-reduced nanoparticles has not been fully explored. In general, previous studies have not addressed the compatibility of electrodes with advanced chemical technologies. Since immunosensors have significant requirements on the electrochemical properties of substrates, it is not clear how the combination of functionalized rGO and AA-coated nanoparticles can exert the advantages of immunosensors. Summary of the Invention

[0003] The purpose of the present invention is to provide a green reduced gold nanoparticle composite nanomaterial to address the problems of poor stability and dispersibility of rGO, poor stability of gold nanoparticles, and poor performance of immunosensors in the prior art.

[0004] Another object of the present invention is to provide a method for preparing the nanocomposite material.

[0005] Another object of the present invention is to provide an application of the nanocomposite material in an immunosensor.

[0006] The technical solution adopted to achieve the purpose of the present invention is:

[0007] A green reduced gold nanoparticle composite nanomaterial comprises amino ion graphene and gold nanoparticles reduced and wrapped by amino acids carried on the graphene.

[0008] In the above technical solution, the amino-ionic graphene is prepared by the following method: dispersing GO in double-distilled water, adding amino-ionic liquid, ultrasonically treating and continuously stirring, then adding alkaline substance and ultrasonically treating, heating, stirring and refluxing the obtained mixed solution, centrifuging after the reaction is completed, and washing the obtained solid alternately with ethanol and double-distilled water to obtain amino-ionic graphene IL-rGO.

[0009] In the above technical solution, the amino acid-reduced and -wrapped gold nanoparticles are prepared by the following method: an aqueous solution of HAuCl4 is heated to boiling, and an amino acid solution is added dropwise. After the reaction is completed, the obtained reaction system is quenched in an ultrasonic ice bath. After quenching, the obtained solid particles are washed with double-distilled water to obtain amino acid-reduced and -wrapped gold nanoparticles AA-Au NPs.

[0010] In the above technical solution, the gold nanoparticles reduced and wrapped by amino acids are loaded on amino-ionic graphene by the following method: the IL-rGO and AA-Au NPs are uniformly dispersed in double-distilled water respectively, mixed and stirred, wherein the mass ratio of IL-rGO and AA-Au NPs is (5-1):1, and after the reaction is completed, centrifugation is performed to obtain AA-Au NPs@IL-rGO.

[0011] In the above technical solution, in the preparation of the IL-rGO, the amino ionic liquid is 1-aminopropyl-3-methylimidazolium chloride, the mass ratio of the amino ionic liquid to GO is (400-200):1, the ultrasonic treatment time is 30-60 min, the alkaline substance is KOH, the ultrasonic treatment time is 50-80 min, the reflux temperature is 80-90° C., and the reflux time is 18-24 hours;

[0012] In the preparation of the AA-Au NPs, the molar ratio of the HAuCl4 to the amino acid is 1:(20-30), the amino acid solution is added at a flow rate of 0.1-0.2 mL / min, the amino acid solution is a saturated solution or a solution with a concentration of 80-100 mM, the reaction time is 30-40 min, and the ultrasonic quenching time is 15-20 min;

[0013] In the preparation of gold nanoparticles reduced and wrapped by amino acid on amino ionic graphene, the concentration of the amino acid reduced and wrapped gold nanoparticles is 0.1-1 mg / mL, the concentration of the amino ionic graphene is 0.5-1 mg / mL, the mixing time is 30-50 minutes, and the mixing temperature is 18-37°C.

[0014] In the above technical solution, the amino acid is valine, asparagine, tryptophan, aspartic acid or lysine, preferably tryptophan.

[0015] In the above technical solution, the particle size of the amino acid-coated gold nanoparticles is 5-50 nm.

[0016] Another aspect of the present invention provides an application of the green reduced gold nanoparticle composite nanomaterial in an immunosensor.

[0017] In the above technical solution, the green reduced gold nanoparticle composite nanomaterial is used to modify the glassy carbon electrode to prepare the immunosensor;

[0018] Preferably, the green reduced gold nanoparticle composite nanomaterial is dispersed in double distilled water, drop-coated on a glassy carbon electrode GCE, and air-dried at room temperature to obtain AA-AuNPs@IL-rGO / GCE. After activation by EDC / NHS, the AA-AuNPs@IL-rGO / GCE is immersed in an ethylenediamine solution to block the carboxyl groups. The antibody is fixed to the exposed carboxyl groups of AA. After blocking the nonspecific binding sites with BSA, the unbound BSA is washed with a PBS buffer solution to obtain an immunosensor for detecting immune response markers.

[0019] Another aspect of the present invention provides a method for preparing a green reduced gold nanoparticle composite nanomaterial, comprising the following steps:

[0020] Step 1: GO is dispersed in double-distilled water, an amino ionic liquid is added, ultrasonic treatment is performed and continuous stirring is continued, and then an alkaline substance is added and ultrasonic treatment is performed. The resulting mixed solution is heated, stirred and refluxed. After the reaction is completed, centrifugation is performed, and the resulting solid is alternately washed with ethanol and double-distilled water to obtain amino ionic graphene IL-rGO;

[0021] Step 2: After the HAuCl4 aqueous solution is heated to boiling, the amino acid solution is added dropwise. After the reaction is completed, the resulting reaction system is quenched in an ultrasonic ice bath. After quenching, the reaction system is centrifuged and the solid particles are washed with double-distilled water to obtain amino acid-reduced and coated gold nanoparticles AA-Au NPs.

[0022] Step 3: The IL-rGO and AA-Au NPs are uniformly dispersed in double-distilled water, mixed and stirred, wherein the mass ratio of IL-rGO to AA-Au NPs is 5:1-1:1. After the reaction is completed, centrifugation is performed to obtain AA-Au NPs@IL-rGO.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention adopts a one-pot method to prepare ionic graphene (IL-rGO). During the preparation process, the surface inertness of graphene is improved and its solubility is enhanced by taking advantage of the low interfacial tension and easy formation of oxygen bonds of ionic liquids. In addition, the single-layer rGO generated by ionic liquid exfoliation has high electrochemical activity at its edges, thereby improving its ability to transmit electrons and significantly increasing the effective area of ​​the electrode. It has good application prospects in the field of nanosensors.

[0025] 2. Natural amino acids (AA) have the advantages of being environmentally friendly and having negligible cytotoxicity. They can be used as stabilizers and end-capping agents for gold nanoparticles to prevent their aggregation in solution. The abundant amino and carboxyl groups on the surface of amino acid-coated nanoparticles facilitate further modification and functionalization. The amino acid-coated gold nanoparticles provided by the present invention enable a one-step green thermal reduction, providing new ideas for the application of amino acids in the field of nanosensors.

[0026] 3. The present invention screened five natural amino acids and found that Trp-AuNPs had excellent antigen fixation and antibody detection capabilities. The reason may be that Trp-AuNPs have a smaller particle size and provide more aminocarboxyl active groups. The composite nanomaterial prepared from them has the best performance in the detection of immune response markers.

[0027] 4. The green reduced gold nanoparticle composite nanomaterial prepared by the present invention is used to modify the sensor prepared by the glassy carbon electrode, which can be used as a universal antibody immobilization interface and has the potential to realize the detection of a series of immune response markers.

[0028] 5. This invention prepares gold nanoparticles coated with five different amino acids. The amino groups, which are unique to the amino acids, undergo amidation condensation with the carboxyl groups on the Fab shoulder and Fc base of the antibody, thereby immobilizing the antibody on the nanomaterial. Because all antibodies share this characteristic, the materials prepared in this invention are universally applicable, providing a simple and rapid platform for antibody immobilization strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Shown is the flow chart of amino acid-coated gold nanoparticles and ion-loaded graphene.

[0030] Figure 2 Shown are the structural formulas representing five different types of amino acids, where A is Tryptophan (Try), B is Lysine (Lys), C is Valine (Val), D is Asparagine (Asn), and E is Aspartic acid (Asp).

[0031] Figure 3 The graph shown is a graph of the graphene solution after standing for 24 hours, where: A is rGO solution and B is amino ion type.

[0032] Figure 4 Shown are TEM images and photos of gold nanoparticles reduced with different amino acids, where: A is Trp-AuNPs; B is Lys-AuNPs; C is Asn-AuNPs; D is Asp-AuNPs; E is Val-AuNPs; F is a picture of gold nanoparticles reduced with different amino acids.

[0033] Figure 5 Shown are TEM data diagrams, where: A is rGO; B is IL-rGO; C is Trp-AuNPs-IL-rGO, and D is AuNPs@IL-rGO.

[0034] Figure 6 Shown are HPLC and XPS graphs, where A is the HPLC graph of Trp-AuNPs and Trp, and B is the XPS graph of rGO, IL-rGO, and Trp-AuNPs@IL-rGO.

[0035] Figure 7 Shown are the XPS spectral data, where: A and B are the peak fittings of rGO and IL-rGO C1s of rGO, respectively, and C and D are the peak fittings of N1 2p and Au 4f of Trp-AuNPs@IL-rGO composite materials, respectively.

[0036] Figure 8 Figure A shows the absorbance measurement of the ability of gold nanoparticles reduced with different amino acids to immobilize antibodies, while Figure B shows the current difference measurement of the ability of gold nanoparticles reduced with different amino acids to detect antigens. The image inset in Figure B shows the DPV data of gold nanoparticles reduced with different amino acids before and after antibody detection. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] Example 1

[0039] A green reduced gold nanoparticle composite nanomaterial, comprising amino ion graphene and amino acid-reduced and wrapped gold nanoparticles. Figure 1 As shown, the nanocomposite material is prepared by the following method:

[0040] Step 1: Synthesis of amino-ionized graphene (IL-rGO)

[0041] GO was dispersed in 25 mL of double-distilled water to obtain 0.5 mg mL -1To a homogeneous solution of GO, 5 g of amino ionic viscous liquid (IL-NH2, 1-aminopropyl-3-methylimidazolium chloride (purchased from Shanghai Chengjie Chemical Co., Ltd.)) was added. After 30 minutes of continuous stirring and ultrasonic treatment, 25 mg of KOH was added to the mixture, and then the mixture was ultrasonically treated for 50 minutes. After the ultrasonic treatment, the turbid mixture was transformed into a homogeneous solution (a uniformly dispersed gray solution). The homogeneous solution was heated under reflux at 80°C and vigorously stirred for 24 hours to obtain a reaction solution. The reaction solution was centrifuged to precipitate IL-rGO. After washing the precipitate alternately with ethanol and double distilled water, it was dispersed in double distilled water (1 mg / mL). It was stored in a 4°C environment for subsequent experiments.

[0042] Step 2: Synthesis of amino acid reduced and coated gold nanoparticles (AA-AuNPs)

[0043] Amino acids (Trp, Lys, Asn, Asp, Val (structural formula such as Figure 2 ))Reduced and wrapped gold nanoparticles were obtained by direct reduction of HAuCl4.

[0044] In this system, five representative natural amino acids (non-polar amino acids: Val; polar neutral amino acids (fatty chains: Asn; aromatic chains: Trp); polar acidic amino acids: Asp; polar basic amino acids: Lys;) are used as reducing agents to fully utilize the reducing properties of amino acids. In addition, in this system, amino acids also act as capping agents and bridging agents. When used as capping agents, they can improve the stability of nanomaterials and prevent them from agglomerating. When used as bridging agents, the free amino and carboxyl groups can effectively fix antibodies, and the free carboxyl groups can also fix ionic amino acids.

[0045] The specific operation is to heat 100 mL of HAuCl4 (0.1 mM) aqueous solution to boiling, and add 3.0 mL of 100 mM amino acid (if the amino acid is not soluble, use a saturated solution of amino acid) at a constant rate of 0.2 mL min -1 After 10-15 minutes, red colloid formation was observed, and the synthesis was completed after 30 minutes. To terminate the reaction, the resulting solution was quenched in an ultrasonic ice bath and sonicated for 15 minutes to ensure good dispersion of the nanoparticles. The solution was then centrifuged at 9000 rpm, and the supernatant was discarded to obtain amino acid-reduced and coated gold nanoparticles (AA-AuNPs). The solution was washed 2-3 times with deionized water, dispersed in deionized water (1 mg / mL), and stored at 4°C until use.

[0046] Step 3: Load the amino acid-coated gold nanoparticles onto amino-ion graphene to obtain AA-AuNPs@IL-rGO

[0047] Equal volumes of the IL-rGO aqueous solution obtained in step 1 and the AA-AuNPs aqueous solution obtained in step 2 were stirred and mixed at 37°C for 30 minutes. The two substances were bound to each other through the amino groups on the surface of IL-rGO and the carboxyl groups exposed on the surface of AA-Au NPs. At the same time, the AA-Au NPs were loaded on the surface of IL-rGO through physical adsorption.

[0048] Comparative Example 1

[0049] 1.1. Preparation of graphene by PDDA reduction

[0050] rGO was prepared by PDDA reduction of graphene oxide.

[0051] 1.2. Preparation of Uncoated Gold Nanoparticles

[0052] Uncoated gold nanoparticles (AuNPs) were synthesized according to conventional methods. Specifically, 50 mL of double-distilled water was added to a 150 mL round-bottom flask, followed by 1.16 mL of a 10 mM HAuCl4 aqueous solution. Under continuous magnetic stirring, the reaction system was boiled in an oil bath below 120°C and then maintained for 3 minutes. Afterwards, 417 μL of a 1% sodium citrate aqueous solution was quickly added under vigorous magnetic stirring, and its color changed from colorless to purple-red after boiling for 6 minutes. Afterwards, the resulting colloidal AuNPs were gradually cooled to room temperature and stored at 4°C until use.

[0053] Preparation of IL-rGO-loaded AuNPs

[0054] The preparation method of IL-rGO is consistent with step 1 of Example 1, the preparation method of AuNPs is consistent with the method in 1.2 of Comparative Example 1, and the preparation method of IL-rGO-loaded AuNPs (AuNPs@IL-rGO) is consistent with step 3 of Example 1.

[0055] Example 2

[0056] The IL-rGO, rGO, AA-AuNPs (including Trp-AuNPs, Lys-AuNPs, Asn-AuNPs, Asp-AuNPs, and Val-AuNPs) and AA-AuNPs@IL-rGO (including Trp-AuNPs@IL-rGO, Lys-AuNPs@IL-rGO, Asn-AuNPs@IL-rGO, Asp-AuNPs@IL-rGO, and Val-AuNPs@IL-rGO) described in Example 1 or Comparative Example 1 above were characterized.

[0057] 2.1. Photos of rGO and IL-rGO after 24 hours of storage

[0058] like Figure 3 As shown in the figure, rGO has obvious precipitation after storage for 24 hours, while IL-rGO is still well dispersed, which indicates that compared with the graphene prepared by PDDA reduction of graphene oxide, IL-rGO can be better dispersed in water and can remain without precipitation for a longer time.

[0059] TEM characterization and images of gold nanoparticles reduced with different amino acids

[0060] like Figure 4 As shown, the color of the amino acid-reduced gold nanoparticles varies from colorless to pink to brown-purple depending on the type of amino acid. As the color deepens, the particle size of the gold nanoparticles gradually increases. Specifically, the average particle sizes of Trp-AuNPs, Lys-AuNPs, Asn-AuNPs, Asp-AuNPs, and Val-AuNPs are 5.186±0.629nm, 19.92±0.45nm, 20.22±1.07nm, 29.78±0.61nm, and 49.09±1.44nm, respectively. This is due to the different reducing properties of different amino acids.

[0061] TEM Characterization of rGO, IL-rGO, Trp-AuNPs@IL-rGO, and AuNPs@IL-rGO

[0062] like Figure 5 As shown in Figures A and B, it can be observed from the TEM images of IL-rGO that IL-rGO has a smoother layered structure than rGO, which may be due to its better solubility and stability. In addition, IL-rGO can provide more active sites, making it difficult for gold nanoparticles to fall off. Figure 5 As can be seen from Figures C and D, the gold nanoparticles on AA-AuNPs and AuNPs loaded with IL-rGO are more evenly attached than those on AuNPs@IL-rGO. This may be because Trp-AuNPs have rich carboxyl groups, which can interact with a considerable number of amino groups (-NH2) on IL-rGO to establish specific and stable connections.

[0063] HPLC Characterization of Trp-AuNPs and Trp and XPS Characterization of rGO, IL-rGO, and Trp-AuNPs@IL-rGO

[0064] like Figure 6As shown in Figure A, Trp-AuNPs and Trp were characterized by HPLC, and the retention time of the two peaks was consistent, which was 6.215min. This shows that Trp not only acts as a reducing agent, but also acts as a capping agent attached to Trp-AuNPs. Figure 6 As shown in Figure B, compared with rGO, IL-rGO has N1s signal. rGO and IL-rGO show two main binding energies of graphite sp2 carbon atoms and oxygen-containing groups, which are about 285eV and 530eV respectively. Compared with rGO, IL-rGO has a new peak of 285.55eV in the peak fitting, and a decrease in CO. According to the literature, it is the CN peak of the newly appeared C after the reaction and the N of the imidazole ring of the ionic liquid ( Figure 7 A and B). Figure 7 As shown in Figure C, 401.7eV is the center and 399.9eV is the low binding energy shoulder, indicating that IL-NH2 is successfully attached to the graphene sheet. In addition, Figure 6 In the XPS spectrum of Figure B, Au 4f signals appeared in Trp-AuNPs@IL-rGO. Figure 7 Figure D shows that the peaks of Au4f (83.7eV and 87.45eV) on Trp-AuNPs@IL-rGO are consistent with the Au0 state, confirming the presence of Au(0) on the IL-rGO surface, namely Trp-AuNPs. The above results further prove the successful preparation of Trp-AuNPs@IL-rGO.

[0065] Example 3

[0066] The antibody fixation and antigen detection capabilities of gold nanoparticles reduced with five different amino acids were investigated. First, different materials such as AA-AuNPs@IL-rGO were adsorbed into a 96-well plate, 20 μL of material was added to each well, incubated at 37°C for two hours, the unbound portion was discarded, and the plate was washed three times with PBS. Then 10 μL of EDC / NHS (concentration of 100 mM; volume ratio of 1:1) was added to activate the material, activated at room temperature for 30 minutes, the supernatant was discarded, and the plate was washed three times with PBS. Then 3.75 μL mL -1 Incubate the material with the antibody at 4°C for 90 minutes, and wash as above. Add 10 μL of 1:500 HRP-labeled secondary antibody to each well and incubate for 1 hour, and wash as above. Then use 150 μL of TMB to develop at 37°C for 30 minutes, and finally use 50 μL of 2mM sulfuric acid to terminate the reaction. Test the absorbance at 650 nm. Figure 8 As shown in middle A, the results show that Trp-AuNPs@IL-rGO exhibits the best performance for antibody immobilization.

[0067] When performing antigen detection, the following method was used: AA-AuNPs@IL-rGO / GCE was dispersed in double-distilled water, drop-coated on a glassy carbon electrode, and air-dried at room temperature to obtain AA-AuNPs@IL-rGO / GCE. It was activated by EDC and NHS, and then the AA-AuNPs@IL-rGO / GCE was immersed in ethylenediamine solution to block the carboxyl groups. The carboxyl groups at the bottom of the antibody Fc and the shoulders of the Fab can be fixed to the exposed carboxyl groups of AA. BSA was used to remove nonspecific binding sites and block the electrode surface. The unbound BSA was washed with PBS buffer solution, and the assembled sensor was immersed in the antigen solution for detection.

[0068] Since proteins are non-conductors and will hinder the transfer of electrons, DPV is used to examine the detection performance of antigens. Figure 8 As shown in Figure B, the changes in DPV peak current before and after antigen detection were used to evaluate the antigen detection capabilities of different amino acid-reduced gold. The results showed that Trp-AuNPs@IL-rGO had the best detection performance.

[0069] By adjusting the process parameters according to the present invention, the Trp-AuNPs@IL-rGO of the present invention can be prepared, and the performance is basically the same as that of Example 1.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A green reduced gold nanoparticle composite nanomaterial, characterized in that: The nanomaterial includes amino-ion graphene and gold nanoparticles reduced and wrapped with amino acids on the graphene; The amino-ionic graphene is prepared by the following method: dispersing GO in double-distilled water, adding amino-ionic liquid, ultrasonically treating and continuously stirring, then adding alkaline substance and ultrasonically treating, heating, stirring and refluxing the obtained mixed solution, centrifuging after the reaction is completed, and washing the obtained solid with ethanol and double-distilled water alternately to obtain the amino-ionic graphene IL-rGO.

2. A green reduced gold nanoparticle composite nanomaterial according to claim 1, characterized in that: The amino acid-reduced and -encapsulated gold nanoparticles are prepared by the following method: heating an aqueous solution of HAuCl4 to boiling, adding an amino acid solution dropwise, quenching the resulting reaction system in an ultrasonic ice bath after the reaction is completed, centrifuging after the quenching, and washing the resulting solid particles with double-distilled water to obtain amino acid-reduced and -encapsulated gold nanoparticles AA-Au NPs.

3. A green reduced gold nanoparticle composite nanomaterial according to claim 2, characterized in that: The amino acid-reduced and -wrapped gold nanoparticles were loaded onto amino-ion graphene by the following method: the IL-rGO and AAAuNPs were uniformly dispersed in double-distilled water, respectively, and then mixed and stirred, wherein the mass ratio of IL-rGO to AA-Au NPs was (5-1):

1. After the reaction was completed, the mixture was centrifuged to obtain AA-Au NPs@IL-rGO.

4. A green reduced gold nanoparticle composite nanomaterial according to claim 3, characterized in that: In the preparation of the IL-rGO, the amino ionic liquid is 1-aminopropyl-3-methylimidazolium chloride, the mass ratio of the amino ionic liquid to GO is (400-200):1, the ultrasonic treatment time is 30-60 minutes, the alkaline substance is KOH, the ultrasonic treatment time is 50-80 minutes, the reflux temperature is 80-90° C., and the reflux time is 18-24 hours; In the preparation of the AA-Au NPs, the molar ratio of the HAuCl4 to the amino acid is 1:(20-30), the flow rate of the amino acid solution is 0.1-0.2 mL / min, the amino acid solution is a saturated solution or a solution with a concentration of 80-100 mM, the reaction time is 30-40 min, and the ultrasonic quenching time is 15-20 min; In the preparation of gold nanoparticles reduced and wrapped by amino acid on amino ionic graphene, the concentration of the amino acid reduced and wrapped gold nanoparticles is 0.1-1 mg / mL, the concentration of the amino ionic graphene is 0.5-1 mg / mL, the mixing time is 30-50 minutes, and the mixing temperature is 18-37°C.

5. The green reduced gold nanoparticle composite nanomaterial according to claim 1, characterized in that: The amino acid is valine, asparagine, tryptophan, aspartic acid or lysine.

6. The green reduced gold nanoparticle composite nanomaterial according to claim 1, characterized in that: The particle size of the amino acid-wrapped gold nanoparticles is 5-50 nm.

7. Use of the green reduced gold nanoparticle composite nanomaterial according to any one of claims 1 to 6 in an immunosensor, characterized in that: The green reduced gold nanoparticle composite nanomaterial is used as a matrix to fix the antibody.

8. The use according to claim 7, characterized in that The immunosensor is prepared by modifying the glassy carbon electrode with the green reduced gold nanoparticle composite nanomaterial; The green reduced gold nanoparticle composite nanomaterial was dispersed in double-distilled water, drop-coated on a glassy carbon electrode (GCE), and air-dried at room temperature to obtain AA-AuNPs@IL-rGO / GCE. After activation by EDC / NHS, the AA-AuNPs@ILrGO / GCE was immersed in an ethylenediamine solution to block the carboxyl groups. Antibodies were immobilized on the exposed carboxyl groups of AA. After nonspecific binding sites were blocked with BSA, unbound BSA was washed with PBS buffer solution to obtain an immunosensor for detecting immune response markers.

9. A method for preparing green reduced gold nanoparticle composite nanomaterials, characterized in that: The following steps are involved: Step 1: GO is dispersed in double-distilled water, an amino ionic liquid is added, ultrasonic treatment is performed and continuous stirring is continued, and then an alkaline substance is added and ultrasonic treatment is performed. The resulting mixed solution is heated, stirred and refluxed. After the reaction is completed, centrifugation is performed, and the resulting solid is alternately washed with ethanol and double-distilled water to obtain amino ionic graphene IL-rGO; Step 2: After the HAuCl4 aqueous solution is heated to boiling, the amino acid solution is added dropwise. After the reaction is completed, the resulting reaction system is quenched in an ultrasonic ice bath. After quenching, the reaction system is centrifuged and the solid particles are washed with double-distilled water to obtain amino acid-reduced and coated gold nanoparticles AA-Au NPs. Step 3: The IL-rGO and AA-Au NPs are uniformly dispersed in double-distilled water, mixed and stirred, wherein the mass ratio of IL-rGO to AA-Au NPs is 5:1-1:

1. After the reaction is completed, centrifugation is performed to obtain AA-Au NPs@IL-rGO.

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