An immunoassay method based on direct adsorption of nanomaterial surfaces and its application

Abstract

The present invention discloses an immunoassay method based on direct adsorption on the surface of nanomaterials and its application, wherein the immunoassay method comprises: using streptavidin as a recognition protein to directly adsorb on the surface of noble metal nanoparticles, using bovine serum albumin as a blocking protein, paving and immunoassay of noble metal nanoparticles simulating nanoscale microplates, constructing a nano-enzyme-linked immunosorbent assay model, and realizing specific recognition and detection of target molecules. The present invention also discloses an NLISA kit constructed based on the immunoassay method. The method of the present invention is simple, highly universal and highly repeatable, provides new possibilities for the application of nanoenzymes in ELISA, and promotes research progress in this field.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterials and biosensor technology, and in particular to an immunoassay method based on direct adsorption of nanomaterial surfaces and an application thereof. Background Art

[0002] Precious metal nanomaterials have potential applications in many fields such as optical metamaterials, sensing, catalysis and biomedicine due to their unique localized plasmon resonance characteristics. Enzyme-linked immunosorbent assay (ELISA) is a technology widely used in biomarker detection. Its sensitivity and specificity make it of great value in clinical diagnosis and biomedical research. In recent years, precious metal nanoparticles have gradually become an emerging material in ELISA technology due to their unique catalytic properties and excellent optical properties. Precious metal nanoparticles can not only serve as carriers of natural enzymes to enhance the detection signal, but also as color developers, which can further realize the detection of specific biomarkers by causing significant color changes through their aggregation or assembly. In addition, precious metal nanoparticles, as nanozymes, exhibit catalytic activity similar to that of enzymes, which has promoted the application exploration in nanozyme-ELISA (NLISA).

[0003] In the traditional ELISA model, due to the binding affinity of streptavidin (SA) and biotin (10 15 M -1 ) is much larger than the specific binding force between antibody and antigen (10 8 ~10 12 M -1 ), SA-biotin affinity system (ABC) is widely used in immunoassay signal amplification system. Bovine serum albumin (BSA) is usually used as a blocking agent for horseradish peroxidase (HRP) enzyme-labeled system. In this process, SA is first plated and BSA is blocked on a solid phase carrier, and then biotinylated antibodies are connected as the basis for capturing target molecules. In sandwich ELISA, the complex of SA and HRP is constructed as an enzyme-labeled secondary antibody, which is further combined with biotinylated antibodies to achieve the detection of target molecules.

[0004] The above mature strategy was borrowed into NLISA to establish ABC-NLISA, which represents an important application of nanotechnology in the field of immunoassay. Although the application of nanozymes in ELISA to replace traditional horseradish peroxidase (HRP) shows broad prospects, it still faces multiple challenges. For example, the catalytic activity of nanozymes may be affected by surface modification. In addition, the covalent binding of recognition antibodies to the surface of nanozymes usually requires complex chemical modification and cross-linking steps, such as first modifying its surface with thiol-polyethylene glycol with a carboxyl terminal, then covalently linking SA using the carbodiimide method, and finally combining with biotinylated antibodies to establish ABC-NLISA. Although this method has high sensitivity, the complex chemical modification process, the screening of covalent linking agents, and the difficulty of characterizing the nanostructure with ordinary UV-vis instruments still limit the widespread application of precious metal nanozymes as a general model for immunoassays to a certain extent. It is undoubtedly a simple strategy to directly adsorb proteins with recognition functions such as antibodies to participate in NLISA, but the high background signal limits the application of precious metal nanozymes in systems with lower detection limits.

[0005] In summary, the application of nanozyme technology in NLISA is still in the early exploratory stage, and the commercialization process is relatively slow. Therefore, there is an urgent need to develop universal strategies in this field to simplify the surface modification process of nanozymes and ensure their catalytic activity, so as to promote their widespread application in NLISA detection. This will provide new opportunities for the popularization and development of immunoassay technology. Summary of the invention

[0006] The purpose of the present invention is to provide an immunoassay method based on direct adsorption of nanomaterials and its application, so as to solve the problems of complex chemical modification process of precious metal nanozymes, cumbersome screening process of covalent linking reagents and difficulty in structural characterization using ordinary UV-vis instruments in the prior art.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] According to a first aspect of the present invention, there is provided an immunoassay method based on direct adsorption on the surface of nanomaterials, the immunoassay method comprising: using streptavidin as a recognition protein to directly adsorb on the surface of noble metal nanoparticles, using bovine serum albumin as a blocking protein, paving and immunoassaying noble metal nanoparticles on a microplate simulating nanoscale, constructing a nano-enzyme-linked immunosorbent assay model, and realizing specific recognition and detection of target molecules.

[0009] According to the present invention, the method comprises the following steps: S1, plating-nanoparticle surface modification: synthesizing precious metal nanoparticles by a seed growth method, adsorbing streptavidin (SA) and bovine serum albumin (BSA) to the surface of precious metal nanoparticles in a certain ratio, incubating for a period of time, centrifuging the SA / BSA-precious metal nanoparticle complex, and finally dispersing it in a high concentration blocking agent for standby use; S2, detection-typical application of ELISA model: using the sandwich ELISA model to detect the target antigen sample, adding the SA / BSA-precious metal nanoparticle complex blocked in step S1 to enhance specificity and reduce background interference, and drawing a standard curve of a given antigen sample concentration to verify the sensitivity.

[0010] Preferably, in step S1, the noble metal nanoparticles are core-shell nanostructures, with gold as the core and the metal shell layer comprising palladium, platinum, etc. The morphology is not limited, and can be nanorods or nanospheres.

[0011] In the present invention, in order to increase the affinity between the enzyme and the acidic substrate, we select a negatively charged surface coating layer, such as sodium polystyrene sulfonate (PSS) and mesoporous silica (SiO2). Preferably, the nanoparticles include but are not limited to mesoporous silica-coated gold core palladium shell nanorods (Au@Pd@SiO2) and gold core platinum shell nanorods (Au@Pt@SiO2).

[0012] Preferably, in step S1, the protein incubation temperature and incubation time can be set as needed. Preferably, the incubation temperature is 4 ° C to 37 ° C, and the incubation is overnight or longer. The preferred temperature is 4 ° C and the time is 16 h.

[0013] In the present invention, the SA and BSA can remain stable in a specific buffer environment. Preferably, Tris-HCl (10 mM, pH = 9.0) or PBS (10 mM, pH = 7.4) buffer is selected.

[0014] Preferably, in step S1, the nanoparticles are dispersed in tris-HCl buffer (Tris-HCl, 10 mM, pH = 9.0) as an incubation solution.

[0015] Preferably, the blocking agent is a mixed solution containing BSA and PBST, preferably, BSA ranges from 0.1% to 2%, preferably, 0.5% (w / v, i.e., 5 mg / mL) dispersed in phosphate-buffered saline (PBST, 10 mM, pH = 7.4) buffer as the blocking concentration.

[0016] Preferably, in step S1, by adjusting the co-incubation ratio of streptavidin and bovine serum albumin, effective regulation of detection sensitivity can be achieved.

[0017] Preferably, in step S1, streptavidin as a recognition protein can capture biotinylated antibodies, and the concentration of the streptavidin should not be too high, and can be 0.5-2.0 µg / mL, preferably 0.5, 1, 1.5 and 2 µg / mL, etc., and more preferably, the concentration of streptavidin is 1 µg / mL.

[0018] Preferably, in step S1, bovine serum albumin as a blocking protein can inhibit the nonspecific adsorption of streptavidin and stabilize its spatial structure, and the concentration of the bovine serum albumin is 5 to 50 µg / mL. Preferably, 5, 10, 20, 30, 40 and 50 µg / mL, etc., and more preferably, the concentration of the bovine serum albumin is 10 µg / mL.

[0019] Preferably, in step S1, the centrifugation condition is 12000 rpm, 5 min.

[0020] According to the present invention, a universal ABC-NLISA model based on direct adsorption of SA and BSA is provided. The method has simple operation steps, mild reaction conditions, and the reagents used are cheap and non-toxic.

[0021] According to a preferred embodiment of the present invention, human IL-6 is selected as an embodiment of target antigen detection, and a sensitivity and LOD value similar to that of the HRP commercial kit are achieved, which proves the practicality and sensitivity of the NLISA model. It should be understood that based on the versatility of biotin in modifying different antibodies, the ELISA model is also universal. It only needs to change the target antigen and the corresponding antibody, and the nanoparticles still participate in the final color development reaction as a quasi-enzyme, which can be used universally in different antigen detection models. Because the concentration of the commercial kit plating process is unknown, the operation details of self-plating are provided here. The method comprises the following steps:

[0022] 1) Prepare IL-6 capture antibody at 10 µg / mL in sodium carbonate buffer (50 mM, pH = 9.6), add 100 µL per well to the plate, and place in a 4 °C refrigerator for at least 12 h. Wash three times with PBST (10 mM, pH = 7.4, the same below);

[0023] 2) Add 300 µL of 1% BSA-PBST (w / v) solution to each well, place at 37 °C for 2 h, and wash three times with PBST;

[0024] 3) Dilute 300 pg / mL human IL-6 antigen to the corresponding concentration with PBST containing 0.1% BSA, add 100 µL of antigen to each well, react at 37°C for 1.5 h, and then wash three times with PBST;

[0025] 4) Add 5 µg / mL biotin-labeled antibody at 100 µL per well, react at 37 °C for 1 h, and then wash three times with PBST;

[0026] 5) Add 0.2 nM SA / BSA-noble metal nanoparticle complex to each well at 100 µL, react at 37 °C for 0.5 h, then wash five times with PBST (10 mM, pH = 7.4) and three times with PBS (10 mM, pH = 7.4);

[0027] 6) The NLISA colorimetric solution is a mixture of 1 mM TMB, 1M H2O2, and 50 mM HAc. 50 µL of the colorimetric reagent is added to each well, and after reacting at 37°C for 20 min, 100 µL of 2 M H2SO4 is added to each well to terminate the reaction, and the OD absorbance at 450 nm is read.

[0028] In the present invention, the inventors have found that the co-incubation process of SA and BSA has a significant effect on the final detection sensitivity. It has been reported that the protein apparent binding constant (Ka) can be calculated based on the change in the relative displacement of the gold nanorod LSPR. In the present invention, based on the similar molecular weight of the proteins and the same adsorption surface, the ratio of SA and BSA can be adjusted according to the size of Ka to achieve improved sensitivity.

[0029] In the present invention, specifically, when studying direct adsorption, we selected antigen concentrations of 0 and 300 pg / mL for each sample system and read OD 450 (respectively denoted as OD 0 , O.D. 300 ), used to explore nonspecific adsorption and sensitivity. The steps include the following parts: 1) synthesizing noble metal nanoparticles by seed growth method, adsorbing SA and BSA on the surface of nanoparticles in a certain ratio, and incubating for a period of time; 2) centrifuging the complex and finally dispersing it in a high concentration blocking agent for use.

[0030] Taking the Au@Pt@SiO2 system as an example, preferably, when a mixture of 10 µg / mL BSA and 1 µg / mL SA is added, that is, when the BSA:SA ratio is 10:1, the OD 0 (< 0.2) with low blank background and OD 300 It reaches a relative maximum (~1.5). However, it should be understood that the optimal ratio of BSA and SA for co-incubation is determined by the binding constant of the protein on the surface ligand layer and the signal-to-noise ratio. It is not the optimal ratio for nanoparticle systems of any size. Those skilled in the art can adjust it according to actual conditions.

[0031] In the present invention, the detection sensitivity can be regulated according to the ratio of SA and BSA in the direct adsorption process. SA has a strong binding force and the nonspecific adsorption it brings is also obvious. In order to better promote the binding of SA and biochemical antibodies, BSA is introduced into the incubation process. BSA protein is used as a blocking agent and incubated with the recognition protein to promote its spatial structure and adsorption state stability. BSA not only blocks the contact between nanoparticles and the ELISA plate, but also inhibits the nonspecific adsorption of SA.

[0032] In the present invention, the covalent modification strategy of the nanozyme surface needs to be screened appropriately, and the surface blocking strategy often uses PEG to prevent the nonspecific adsorption of proteins on the surface, but the activity of the nanozyme is inhibited due to the blocking of the surface sites. However, in the present invention, through the physical adsorption process, the adsorption of BSA and SA will not block the active sites of the nanozyme, and will not affect the activity of the nanozyme. In addition, the activity of the nanozyme can be enhanced by regulating the buffer environment and the metal components of the nanozyme.

[0033] In the present invention, the guarantee of catalytic activity is closely related to the stability of the synthesis batch, the synthesis of precious metal nanozymes is controllable and can be stable for a long time, and the repeatability of NLISA detection has certain technical support.

[0034] In the present invention, the pH of the environment is regulated near the isoelectric point (pI = 4.7) of the blocking agent BSA. As the ionic strength of the buffer increases, the color development becomes more obvious and the sensitivity increases accordingly. The protein structure becomes loose and the diffusion barrier of the substrate is reduced. Preferably, the reaction is carried out in an acetate buffer (pH = 4.5), and the buffer concentration is 10 mM to 200 mM, such as 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, etc., and 50 mM is further preferred.

[0035] In the present invention, the concentration of the complex particles of nanoparticles and protein adsorption should be appropriate, preferably, 0.1 nM~0.5 nM, such as 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, etc.; more preferably, the particle concentration is 0.2 nM.

[0036] In the present invention, human IL-6 capture antibody and SA were coated on the surface of polystyrene microplate and noble metal nanoparticles at 4°C overnight, respectively, and then both were subjected to BSA blocking step and immune reaction process.

[0037] According to the second aspect of the present invention, a NLISA kit constructed based on the above-mentioned immunoassay method is also provided.

[0038] In NLISA, the precious metal nanozyme is designed to simulate the peroxidase activity of HRP. Through the surface modification strategy, the present invention proposes the concept of coating and blocking "nanoscale microwells (nanowell)" for the first time. Specifically, the surface of the precious metal nanozyme simulates the solid phase carrier (such as polystyrene microwell plate) of the enzyme labeling plate in the traditional ELISA. Through the formation of SA and nanozyme complex and the blocking of BSA, the early nanowell blocking and plating steps are established, and then the biotinylated labeled antibody is connected to achieve specific recognition of the target molecule. One of the inventive points of the present invention is mainly to synthesize a precious metal nanoparticle complex, which acts like HRP as a catalyst in the color development reaction step, effectively improves the electron transfer efficiency, enhances the sensitivity and signal intensity of the chemical reaction, and the catalytic activity is more flexible and adjustable than HRP. The complex replaces the avidin-coated HRP and the streptavidin-coated nanozyme in the system. After the surface is blocked by BSA, it is fixed on the plate surface through the bridge effect of biotin, which reduces nonspecific adsorption, and the properties of the nanozyme are reflected in the sensitivity.

[0039] In NLISA, SA and BSA are physically adsorbed on the surface of nanozymes. The method and process are relatively simple, easy to operate and mass produce. However, compared with covalent modification, its adsorption state is often uncertain. The present invention uses the particle size change of gold nanospheres (AuNS) and the longitudinal surface plasmon resonance (LSPR) characteristics of gold nanorods (AuNR) to characterize the adsorption characteristics after protein coating. Specifically, the binding constant is an important parameter to describe the interaction strength between proteins and their ligands. Through dynamic light scattering (DLS) and ultraviolet-visible spectroscopy (UV-vis) tests, the changes in particle size and LSPR peak position before and after protein and nanoparticle adsorption were measured, and the apparent binding constant of the protein was calculated. This method can be used to effectively evaluate the adsorption binding force of proteins. This lays a theoretical basis for the state measurement during nanowell coating and closure.

[0040] In addition, the present invention also found that in the process of forming the complex, the co-coating of SA and BSA has a favorable effect on the detection sensitivity. We can optimize the binding constant of the two proteins adsorbed on the ligand surface to achieve a nanozyme "plating" design with both low nonspecific adsorption and high specific recognition. The main point of the present invention is to optimize the synthesis of a nanoparticle complex with enzyme-like activity, and the nanoparticles and streptavidin can also construct a high-sensitivity model of ABC-NLISA through direct adsorption. This innovative design idea provides new possibilities for the application of nanozymes in ELISA and promotes research progress in this field.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1) Simplicity and practicality: The present invention provides a method for improving the sensitivity of immunoassay, characterized in that BSA protein is used as a blocking agent and co-incubated with a recognition protein. The synergistic effect of the two is beneficial to the detection sensitivity. For example, during co-incubation, BSA has a positive effect on inhibiting the nonspecific adsorption of SA on the adsorption behavior of SA; regulating the ionic strength at the isoelectric point of BSA can promote the improvement of the sensitivity of the color development reaction; and without the need for high-concentration nanoparticles, the cost is reduced. All of these indicate that based on this simple modification method and characterization means, the construction of enzyme-labeled secondary antibodies with noble metal nanozymes and SA has simple operation and practicality;

[0043] 2) Universality of affinity system: The streptavidin-biotin system is easy to modify and suitable for the detection of different target molecules. Combined with the LSPR characteristics of noble metal nanoparticles, simple spectral monitoring can be achieved, which improves sensitivity, applicability and ease of operation;

[0044] 3) Research and development of NLISA commercial test kits: Proteins in the immune system are all physically adsorbed. For the first time, the concept of nanoscale microplate design based on the NLISA system was proposed. Nano-sized coating and blocking strategies were used to simplify sample preparation steps, significantly reduce nonspecific adsorption, and provide commercial application potential for precious metal nanoparticles in immune detection kits.

[0045] In summary, the present invention discloses a method for improving the sensitivity of immunoassays that is simple, practical, and universal, and develops the commercial application potential of noble metal nanoparticles in immunoassay kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The present invention shows a method of co-incubating two proteins on the surface of noble metal nanoparticles in a direct adsorption manner to simulate a nanoscale microplate for plating;

[0047] Figure 2 A schematic diagram of an ABC-NLISA model according to the present invention is shown;

[0048] Figure 3 TEM image (A) and corresponding UV-vis spectrum (B) of Au@Pd@SiO2 nanorods are shown;

[0049] Figure 4 The calculation of the apparent binding constant of the protein based on the change of LSPR peak position is shown;

[0050] Figure 5 The effect of SA and BSA adsorption on the surface of noble metal nanozymes on the detection sensitivity is shown;

[0051] Figure 6The ELISA and NLISA standard curves for detecting human IL-6 antigen based on Au@Pd@SiO2 and Au@Pt@SiO2 nanozymes are shown. DETAILED DESCRIPTION

[0052] In order to better illustrate the present invention and facilitate understanding of the technical solution of the present invention, the present invention is further described in detail below. However, the following embodiments are only illustrative of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0053] The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used are all purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0054] In NLISA, the noble metal nanozyme is designed to mimic the peroxidase activity of HRP. Through the surface modification strategy, the present invention first proposes the concept of coating and blocking "nanoscale microwells". Specifically, the surface of the noble metal nanozyme simulates the solid phase carrier (such as polystyrene microwell plate) of the ELISA plate in traditional ELISA. Through the formation of SA and nanozyme complexes and the blocking of BSA, the early nanowell blocking and plating steps (such as Figure 1 ), followed by linking a biotinylated labeled antibody to achieve specific recognition of the target molecule (as shown in Figure 2 As shown). One of the inventive points of the present invention is to synthesize a noble metal nanoparticle complex, which has the same function as HRP, as a catalyst in the color development step, effectively improving the electron transfer efficiency, enhancing the sensitivity and signal intensity of the chemical reaction, and the catalytic activity is more flexible and adjustable than HRP. The complex replaces the avidin-coated HRP in the system, such as Figure 2 The model shown shows that the streptavidin-coated nanozyme has a surface blocked with BSA, which is fixed on the plate surface through the bridge effect of biotin, reducing nonspecific adsorption. At the same time, the properties of the nanozyme are reflected in sensitivity.

[0055] In the following examples, the sources of the reagents used are as follows: streptavidin (Sigma), bovine serum albumin (Alfa). The human IL-6 antibody and the corresponding antibody were purchased from Biolegend. The components of the color development solution, tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), were purchased from Aladdin.

[0056] In the following examples, sensitive detection was calculated by the change in absorbance at 450 nm after the color development reaction was terminated, using OD 450 Taking the detection of human IL-6 antigen as an example, when the concentration is 0 pg / mL, OD 0 When the concentration is 300 pg / mL, OD300 express.

[0057] Example 1 Difference in adsorption capacity between SA and BSA

[0058] The present invention uses different bimetallic core-shell nanorods such as Au@Pd@PSS and Au@Pd@SiO2 systems. The TEM images and corresponding UV-vis spectra of the Au@Pd@SiO2 nanorods are shown in Figure 2. Figure 3 As shown in A and B in the figure, the nanoparticles show typical rod-like morphology and LSPR characteristics (TEM scale is 50 nm). Before using them in NLISA, it is necessary to explore the adsorption strength of SA and BSA on the PSS and SiO2 surfaces to establish a theoretical basis for the construction of enzyme-labeled secondary antibodies.

[0059] 0.1nM gold nanoparticles were dispersed in 10mM Tris-HCl (pH = 9), and the specified concentration of BSA or SA was added to make the final volume 1.5 mL, and incubated at 37 °C for 2h. Taking the adsorption of 1 µg / mL SA by silica-coated gold nanoparticles (AuNS@SiO2) as an example: 150 µL AuNS@SiO2 (1 nM) was dispersed in 150 µL SA (10 µg / mL, prepared with Tris-HCl) and 1.2 mL Tris-HCl (10 mM, pH = 9), mixed and placed at 37 °C, incubated for 2h, and then taken out to measure its DLS particle size. K was calculated by measuring the DLS changes of adsorption systems of different concentrations such as 0.5 / 1 / 2 / 5 / 10 µg / mL. a .

[0060] AuNR@SiO2 and AuNR@PSS nanorods adsorbed SA and BSA systems, and the corresponding Ka was also obtained through the changes in LSPR peak position shift. Figure 4 The calculation results of the AuNR@SiO2 system before and after LSPR shift show SA binding ability (K a-SA ) than BSA (K a-BSA ) is one order of magnitude higher. And on similar SiO2 surfaces, both DLS and LSPR results show that the K a It is about 50 times higher than BSA (Table 1).

[0061] Table 1 Apparent binding constants of BSA and SA on SiO2 ligand layer in Example 1

[0062] BSA SA <![CDATA[AuNR@SiO2]]> <![CDATA[6.1*10 5 M -1 ]]> <![CDATA[4*10 7 M -1 ]]> AuNR@PSS <![CDATA[1.7*10 6 M -1 ]]> <![CDATA[2.6*10 8 M -1 ]]> <![CDATA[AuNS@SiO2]]> <![CDATA[4.5*10 5 M -1 ]]> <![CDATA[1.8*10 7 M -1 ]]> .

[0063] Example 2 Blocking effect of BSA in NLISA

[0064] 0.1 nM Au@Pd@SiO2 was dispersed in a mixture of 10 / 50 / 300 μg / mL BSA and 10 mM Tris-HCl (pH = 9), incubated at 4 °C for 16 h, and then dispersed in 0.5% BSA-PBST at a particle concentration of 0.2 nM after centrifugation and placed on a shaker for 2 h. NLISA was performed and OD was recorded. 0 and OD 300 In Table 2, OD 0 and OD 300 The values ​​were all less than 0.1, indicating that appropriate BSA protein concentration coated on the particle surface could inhibit the nonspecific adsorption of nanoparticles to the polystyrene microplate.

[0065] Table 2 OD values ​​after BSA blocking on the surface of nanozymes in Example 2

[0066] .

[0067] Example 3 Effect of co-adsorption of SA and BSA on detection sensitivity

[0068] In the present invention, both sensitivity and background signal can be regulated by simple physical adsorption, and the adsorption behavior of SA and BSA is specifically investigated. Figure 5 As shown in Figure 2, the higher the SA concentration, the higher the specificity of recognition in OD 300 (antigen concentration was 300pg / mL) did not increase significantly, but OD 0 (antigen concentration is 0 pg / mL), which indicates that SA will cause a strong background signal through direct adsorption. Taking the mixed system of 10 µg / mL BSA and 10 µg / mL SA and the single system of 25 µg / mL SA as examples, it can be best shown that the presence of SA will produce obvious nonspecific adsorption. In addition, Example 1 is the basic basis of Example 3 and the data support for the co-incubation design. SA has a strong binding ability, so a large amount of SA is not required, and an effective recognition effect can be achieved by controlling the low concentration adsorption. This can be clearly observed in the mixed system of 10~25 µg / mL BSA and 0.5~1 µg / mL SA. The coexistence of extremely low background signals and high sensitive responses. This not only simplifies the system model, but also reduces costs and improves the practical application value of nanoparticles.

[0069] Example 4 NLISA standard curve based on Au@Pd@SiO2

[0070] In the Au@Pd@SiO2 system, a mixture of 10 µg / mL BSA and 1 µg / mL SA was added, and the mixture was allowed to stand at 4 °C for 16 h. After centrifugation, the particles were dispersed in 0.5% BSA-PBST at a concentration of 0.2 nM and placed on a shaker for 2 h. NLISA was performed and the OD was recorded. 0 and OD 300 The best system presented in Example 3 was selected, that is, when BSA:SA was 10, OD 0 (< 0.2) with low blank background and OD 300 Reaching the highest relative value in the system (~0.45). NLISA standard curve y = 0.0012x + 0.13, R 2 =0.9907.

[0071] Example 5 NLISA standard curve based on Au@Pt@SiO2

[0072] Unlike Example 4, Example 5 uses a Pt metal shell with higher catalytic activity. A mixture of 10 µg / mL BSA and 1 µg / mL SA was added to the Au@Pt@SiO2 system. The other conditions and steps were exactly the same as those in Example 4. The best system presented in Example 3 was selected, that is, when the BSA:SA ratio was 10, the OD 0 (< 0.2) with low blank background and OD 300 Reached a relative maximum (~1.5). The results are as follows Figure 6 As shown, the NLISA standard curve of Au@Pt@SiO2 system is y = 0.0042x +0.32, R 2 = 0.9866, with a detection limit as low as 2.4 pg / mL, which is 3.5 times more sensitive than Au@Pd@SiO2 and is also better than HRP-based ELISA.

[0073] Through Example 4 and Example 5, it is proved that the strategy of co-incubating the two proteins on the particle surface by direct adsorption in the present invention is universal, and the two-protein adsorption ratio adjusted by the Au@Pd@SiO2 system is also applicable to other surfaces of the same size and the same SiO2. The metal component is controlled as the only variable, and the detection sensitivity of Au@Pt@SiO2 is higher. This shows that under the same ELISA model and incubation conditions, the regulation of the adsorption state of BSA on SA is beneficial when enzyme-like active nanomaterials are applied to ELISA.

[0074] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the contents of the specification of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. An immunoassay method based on direct adsorption of nanomaterial surfaces, characterized in that: The immunoassay method comprises: using streptavidin as a recognition protein to directly adsorb on the surface of noble metal nanoparticles, using bovine serum albumin as a blocking protein, simulating the noble metal nanoparticles on a nanoscale microplate for plating and immunoassay, and constructing a nano-enzyme-linked immunosorbent assay model to achieve specific recognition and detection of target molecules; The method comprises the following steps: S1, plating-nanoparticle surface modification: synthesizing noble metal nanoparticles by seed growth method, adsorbing streptavidin and bovine serum albumin at a certain ratio to the surface of noble metal nanoparticles to prepare SA / BSA-noble metal nanoparticle complexes, wherein the concentration of streptavidin is 0.5-2.0 µg / mL, the concentration of bovine serum albumin is 10-50 µg / mL, the noble metal nanoparticles are core-shell nanostructures, with gold as the core, the metal shell layer is selected from palladium or platinum, and the surface ligand layer of the noble metal nanoparticles is mesoporous silica. After incubation for a period of time, the SA / BSA-noble metal nanoparticle complexes are centrifuged and finally dispersed in a high concentration blocking agent for standby use; S2, detection - Typical application of ELISA model: Use the sandwich ELISA model to detect the target antigen sample, add the SA / BSA-precious metal nanoparticle complex blocked in step S1 to enhance specificity and reduce background interference, and draw a standard curve for a given antigen sample concentration to verify sensitivity.

2. The immunoassay method according to claim 1, characterized in that: In step S1, the incubation temperature is 4°C to 37°C, and the incubation time is 2 to 24 h.

3. The immunoassay method according to claim 1, characterized in that: In step S1, the incubation solution is 10 mM Tris-HCl buffer at pH 9.

0.

4. The immunoassay method according to claim 1, characterized in that: In step S1, by adjusting the co-incubation ratio of streptavidin and bovine serum albumin, effective regulation of the detection sensitivity can be achieved.

5. The immunoassay method according to claim 1, characterized in that: The method comprises the following steps: 1) Disperse 0.1 nM Au@Pd@SiO2 nanorods in 10 mM Tris-HCl at pH 9, add 1 µg / mL SA and 10 µg / mL BSA, mix well and place in a 4 °C refrigerator for 16 h, then disperse into 0.5% BSA-PBST by centrifugation and place on a shaker for 2 h. The particle concentration is 0.2 nM for later use; 2) Prepare IL-6 capture antibody at 10 µg / mL in 50 mM sodium carbonate buffer at pH 9.6, add 100 µL per well to a polystyrene microplate, place in a 4 °C refrigerator for 16 h, and wash three times with 10 mM PBS at pH 7.4; 3) Add 300 µL of 1% BSA-PBST solution to each well, incubate at 37 °C for 2 h, and wash three times with 10 mM PBST (pH 7.4); 4) Dilute 300 pg / mL human IL-6 antigen to gradient concentrations with PBST containing 0.1% BSA, add 100 µL of antigen to each well, react at 37 °C for 1.5 h, and then wash three times with 10 mM PBST at pH 7.4; 5) Add 5 µg / mL biotin-labeled antibody at 100 µL per well, react at 37 °C for 1 h, and then wash three times with 10 mM PbST at pH 7.4; 6) Add 0.2 nM of the reserved noble metal nanoparticle complex to each well at 100 µL, react at 37 °C for 0.5 h, then wash five times with 10 mM PBS at pH 7.4, and wash three times with 10 mM PBS at pH 7.4; 7) The NLISA colorimetric solution is a mixture of 1 mM TMB, 1M H2O2 and 50 mM HAc. 50 µL of the colorimetric solution is added to each well. After reacting at 37°C for 20 min, 100 µL of 2 M H2SO4 is added to each well to terminate the reaction and the OD absorbance at 450 nm is read.

6. A NLISA kit constructed based on the immunoassay method according to any one of claims 1 to 5.

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