SERS (Surface Enhanced Raman Scattering) immunodetection substrate based on gold and silver nanoflowers as well as preparation method and application thereof

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) immunodetection substrate based on gold and silver nanoflowers as well as a preparation method and application thereof. The gold-silver nanoflower has excellent uniformity on the microscopic scale, the interior of the gold-silver nanoflower is of a silver core-gold shell structure, the surface of the gold-silver nanoflower is in a flower shape, and the gold-silver nanoflower can provide a large number of SERS hot spots and carries Raman signal molecules intrinsically. Through chemical modification, the gold and silver nanoflowers and magnetic beads with carboxyl groups on the surfaces are chemically activated and coupled with a specific antibody to form an immune probe, and then an immune sandwich structure is constructed when target protein is detected. Finally, high-sensitivity SERS detection of ultralow-concentration target protein can be realized by means of magnetic enrichment. The constructed SERS immunodetection substrate can be used for detecting t-PSA, and the lowest limit of detection (LOD) can reach 100 fg / mL. Besides, the prepared SERS substrate has good signal uniformity, shows excellent anti-interference capability and has wide application prospects in the fields of biological analysis, clinical detection and the like.

Description

Technical Field

[0001] The present invention relates to a preparation method of a novel gold and silver nanoflower, a preparation method of a SERS immunosensing substrate formed by compounding the novel gold and silver nanoflower with magnetic beads, and an application thereof in protein detection, belonging to the technical field of nanomaterial manufacturing and SERS biosensing detection. Background Art

[0002] Surface-Enhanced Raman Scattering (SERS) is a super-high-sensitivity spectroscopic detection technology, which can greatly enhance the Raman scattering signal and realize the rapid detection of trace target molecules. Through the synergistic effect of plasmonic nanomaterials and special nanostructures, SERS can even achieve biomolecular recognition at the single-molecule level, and thus has wide application value in the fields of early disease diagnosis, tumor marker detection, metabolite monitoring, etc. In recent years, there have been many research reports on SERS detection using nanoparticles conjugated with target proteins, and this method has been proven to have the potential as an early screening method for diseases such as cancer.

[0003] Free total prostate-specific antigen (t-PSA) is an important serum marker for prostate cancer and prostate-related diseases (such as prostate hyperplasia, inflammation), and its concentration change is directly related to the early diagnosis, staging and treatment monitoring of the diseases. Clinically, it is generally considered that a t-PSA concentration exceeding 4 ng / mL indicates prostate-related diseases, and when it exceeds 10 ng / mL, the risk of prostate cancer increases significantly, and 4 ng / mL to 10 ng / mL is considered the "detection gray zone". Therefore, constructing a t-PSA detection substrate with a detection limit (LOD) of at least as low as 10 ng / mL and being able to accurately analyze the "detection gray zone" is crucial for the early screening and accurate diagnosis of early prostate cancer. However, the current mainstream detection methods (such as ELISA, chemiluminescence method) are limited by sensitivity and it is difficult to achieve efficient, stable and low-background-interference t-PSA detection in a complex biological environment.

[0004] Therefore, based on the SERS detection technology, constructing a nanoscale immune probe that intrinsically carries Raman signals, has excellent SERS enhancement performance and can be efficiently separated is expected to develop a highly sensitive, highly specific, stable and reliable SERS immunosensing technology. Ultimately, this technology can be used for t-PSA detection and make great contributions to the early screening and accurate diagnosis of prostate cancer. Summary of the Invention

[0005] Objective of the Invention: The first objective of the present invention is to provide a preparation method of novel intrinsic Raman signal-carrying molecules and gold-silver nanoflowers that can provide strong SERS amplification.

[0006] The second objective of the present invention is to provide gold-silver nanoflowers prepared by the above method.

[0007] The third objective of the present invention is to provide a SERS immunoprobe.

[0008] The fourth objective of the present invention is to provide a preparation method of a SERS immunoprobe.

[0009] The fifth objective of the present invention is to provide a preparation method of a SERS immunosensing substrate based on gold-silver nanoflowers.

[0010] The sixth objective of the present invention is to provide a SERS immunosensing substrate based on gold-silver nanoflowers.

[0011] The seventh objective of the present invention is to provide an application of a SERS immunosensing substrate based on gold-silver nanoflowers.

[0012] Technical Solution: A preparation method of gold-silver nanoflowers of the present invention, the preparation method comprising the following steps: adding surface amino-functionalized silver nanoseeds to a growth solution, and after the reaction is completed, collecting the product, which is the gold-silver nanoflower; wherein, the growth solution is composed of ethanol, chloroauric acid, ascorbic acid, and 4-mercaptobenzoic acid, and wherein the concentration of 4-mercaptobenzoic acid in the growth solution is at least 2.13 mM.

[0013] In the above technical solution, the synthesis of gold-silver nanoflowers is simple and rapid, and uniformly shaped, structurally stable gold-silver nanoflowers with a silver core and a gold shell (Ag@Au) can be prepared in large quantities within several hours. The above synthesis method is derived from the synthesis idea of growing gold wires at the solid-phase interface, but it is not a simple modification of the synthesis environment. The formula of the growth solution needs to be optimized to obtain uniformly shaped gold-silver nanoflowers suitable for SERS detection.

[0014] Preferably, the preparation method of the growth solution is: mixing 600 μL of ethanol, 50 μL of 4-mercaptobenzoic acid (4-MBA, 60 mM), 100 μL of L(+)-ascorbic acid (AA, 0.4 M), and 660 μL of (HAuCl4, 25 mM) to obtain the growth solution.

[0015] On the other hand, the present invention provides gold-silver nanoflowers obtained by the above preparation method, the gold-silver nanoflowers having a silver core-gold shell structure inside and a nanoflower shape on the surface.

[0016] On the other hand, the present invention provides a SERS immunoprobe, which includes a signal probe and a capture probe. Among them, the above-mentioned gold-silver nanoflowers are used as the signal probe after amino-functionalization, and magnetic beads with carboxyl groups on the surface are used as the capture probe.

[0017] Further, the method for amino-functionalizing the gold-silver nanoflowers is as follows: the surface of the silver nanoseeds is amino-functionalized by silanization to obtain amino-functionalized silver nanoseeds; the amino-functionalized silver nanoseeds are added to the growth solution, and after the reaction is completed, the amino-functionalized gold-silver nanoflowers are obtained.

[0018] Preferably, the method for amino-functionalizing the gold-silver nanoflowers is as follows: First, take silver spheres and add them to the APTES solution to amino-functionalize the surface of the silver nanoparticles by silanization to obtain a mixed solution; take the growth solution and add it to the mixed solution; after reacting for 30-40 minutes, collect the synthesized nanoflowers.

[0019] Further, magnetic beads with carboxyl groups on the surface are synthesized by a solvothermal method.

[0020] Preferably, the method for synthesizing magnetic beads with carboxyl groups on the surface by the solvothermal method is as follows: First, dissolve ferric chloride hexahydrate in a well-mixed solution of diethylene glycol ((CH2OH)2) and ethylene glycol (C4H 10 O3). Then, add sodium acetate (CH3COONa) and sodium acrylate (C3H3O2Na) to the mixed solution, and the mixture is completely dissolved. Transfer the obtained homogeneous solution into a reaction kettle and heat it at 200-220 °C for 10-12 h. After the reaction is completed, magnetically separate and collect the black precipitate, which is the magnetic beads with carboxyl groups on the surface.

[0021] The present invention constructs a SERS immunodetection substrate for t-PSA detection based on gold-silver nanoflowers and nanomagnetic beads. Among them, the gold-silver nanoflowers are used as nanoprobes that intrinsically carry Raman signal molecules and have excellent SERS enhancement performance, while the nanomagnetic beads provide magnetic separation characteristics. The gold-silver nanoflowers and nanomagnetic beads are respectively conjugated with antibodies after chemical modification to form two different immunoprobes. When detecting the target protein (t-PSA), a "sandwich" immune structure is formed through antigen-antibody specific recognition to construct a SERS immunodetection substrate, and the concentration of t-PSA is accurately detected by analyzing the intensity of the detected Raman signal.

[0022] In the above technical solution, the gold-silver nanoflowers have a silver core-gold shell structure. The nanoflower structure on the surface can provide a large number of stable "SERS hot spots", ensuring the high sensitivity and stability of the SERS signal. At the same time, the magnetic beads are modified by carboxyl groups, enabling them to covalently connect antibodies and providing a magnetic separation function during the immunoassay, improving the sensitivity and anti-interference ability of the detection substrate. The SERS immunoassay substrate of the present invention can achieve the detection of t-PSA concentration as low as 100 fg / mL, enabling the sensitive detection of the target protein. And the concentration of t-PSA can be quantitatively analyzed and evaluated by analyzing the intensity of the Raman signal molecule 4-MBA, showing excellent detection performance.

[0023] Among them, the preparation method process of the nanoparticles for constructing the immune probe includes any of the following methods

[0024] A. 4nm silver nanospheres: Mix 75 mL of deionized water with 20 mL of 1% sodium citrate solution and heat to 70 °C. Add 1.7 mL of 1% AgNO3 solution under vigorous stirring, and then quickly add 2 mL of freshly prepared 0.1% NaBH4 solution all at once. Continue stirring for 1 h, then let the mixed solution cool naturally to room temperature and make up the volume to 100 mL.

[0025] B. 20nm silver nanospheres: Mix 75 mL of deionized water with 2 mL of 1% sodium citrate solution and heat to boiling. Under vigorous stirring, add 10 mL of the above 4nm silver nanosphere solution and 1.7 mL of 1% AgNO3 solution successively, and continue stirring for 1 h under the condition of condensation reflux. Then add 2 mL of 1% sodium citrate solution and 1.7 mL of AgNO3 solution, and continue stirring for 1 h under the condition of condensation reflux. This step is repeated twice. Finally, let the mixed solution cool naturally to room temperature and make up the volume to 100 mL.

[0026] C. Gold and silver nanoflowers: First, take 100 μL of the prepared 20-nm silver spheres and centrifuge them at 8000 rpm for 10 min to remove the supernatant. Subsequently, add 800 μL of 0.005 M APTES solution to functionalize the surface of the silver nanoparticles with amino groups through silanization. The mixed solution is oscillated on a shaker at 300 rpm for 1 h. Subsequently, prepare a growth solution containing 600 μL of ethanol (EtOH), 50 μL of 4-mercaptobenzoic acid (4-MBA, 60 mM), 100 μL of L(+)-ascorbic acid (AA, 0.4 M), and 660 μL of (HAuCl4, 25 mM). Take 300 μL of the growth solution and add it to the mixed solution. After reacting for 30 min, centrifuge at 6000 rpm for 10 min to remove the supernatant, redisperse it in 1 mL of phosphate buffer solution (PBS), and repeat this step twice. Finally, collect the synthesized nanoflowers, redisperse them in 1 mL of PBS, and store them in a refrigerator at 4 °C for later use.

[0027] D. Nanomagnetic beads: First, dissolve 0.54 g of ferric chloride hexahydrate (FeCl3·6H2O) in a well-mixed solution of 10 mL of diethylene glycol ((CH2OH)2) and 10 mL of ethylene glycol (C4H 10 O3). Then, add 1.50 g of sodium acetate (CH3COONa) and 1.50 g of sodium acrylate (C3H3O2Na) to the solution, and completely dissolve the mixture by alternately using magnetic stirring and ultrasound. Transfer the resulting homogeneous solution into a stainless-steel autoclave lined with polytetrafluoroethylene and heat it at 200 °C for 10 h. After the reaction is completed, collect the black precipitate by magnetic separation, wash it 3 times with deionized water (DI) and 3 times with ethanol, and finally dry it in vacuo at 60 °C for 12 h.

[0028] On the other hand, the present invention provides a method for preparing the above-mentioned SERS immunoprobe. By using the chemical activation method, the signal probe and the capture probe are respectively conjugated with specific antibodies to form an SERS immunoprobe.

[0029] On the other hand, the present invention provides a method for preparing an SERS immunodetection substrate based on the above-mentioned gold and silver nanoflowers, comprising the following steps:

[0030] Step 1): First, synthesize initial silver nanoseeds and regulate their size by thermal growth method. After the silver nanoseeds are amino-functionalized, add them to the growth solution to synthesize gold and silver nanoflowers;

[0031] Step 2): Synthesize magnetic beads with carboxyl groups on the surface by solvothermal method;

[0032] Step 3): By using the chemical activation method, conjugate the gold and silver nanoflowers and the nanomagnetic beads with specific antibodies respectively to form an SERS immunoprobe;

[0033] Step 4): Bind the SERS immunoprobe to the protein to be detected to form a "sandwich" immune structure;

[0034] Step 5): Drop the mixed solution containing the "sandwich" structure onto the substrate material to form a SERS immunodetection substrate.

[0035] On the other hand, the present invention provides a preparation method of an immunoprobe based on gold-silver nanoflowers and magnetic beads, and jointly constructing a SERS immunodetection substrate, including the following steps:

[0036] Step 1): First, synthesize initial silver nanoseeds and regulate their size by thermal growth method. After the silver nanoseeds are amino-functionalized, add them to the growth solution to synthesize gold-silver nanoflowers;

[0037] Step 2) Gold-silver nanoflower immunoprobe: Take the prepared gold-silver nanoflowers, activate the carboxyl group through EDC / NHS reaction, and incubate with t-PSA antibody to form a gold-silver nanoflower probe.

[0038] Step 3) Magnetic immunoprobe: Take the prepared magnetic beads, activate the carboxyl group through EDC / NHS reaction, and incubate with t-PSA antibody to form a magnetic bead probe.

[0039] Step 4) Preparation of the "sandwich" structure: Take a specific concentration of t-PSA solution and bind it to the magnetic immunoprobe. After incubation, use magnetic separation and wash the precipitate. After redispersion, add the prepared gold-silver nanoflower immunoprobe to bind it to the conjugate of the magnetic immunoprobe and t-PSA. After incubation, use magnetic separation and wash the precipitate, and redisperse it in phosphate buffered saline (PBS).

[0040] Step 5) SERS detection of the immunodetection substrate: Take a certain amount of the mixed solution prepared in Step 4) and drop it on a clean silicon wafer, and then perform SERS detection. Determine the t-PSA concentration by detecting the intensity of the 4-MBA Raman signal molecule.

[0041] On the other hand, the present invention provides a SERS immunodetection substrate prepared by the above preparation method.

[0042] On the other hand, the present invention provides an application of the above SERS immunodetection substrate in protein detection.

[0043] On the other hand, the present invention provides an application of the above SERS immunodetection substrate in the preparation of a SERS detection platform.

[0044] The SERS immunosensing substrate based on gold-silver nanoflowers and magnetic beads provided by the present invention has the characteristics of simple preparation, high sensitivity, high specificity and good reproducibility. The silver core-gold shell structure and the surface nanoflower-like morphology of the gold-silver nanoflowers provide abundant "SERS hot spots", ensuring the stable enhancement of SERS signals. At the same time, the magnetic separation function of the magnetic beads significantly improves the sensitivity and anti-interference ability of the detection substrate, enabling it to maintain good detection performance in the detection of complex biological samples. The SERS immunosensing substrate of the present invention can be widely applied to the detection of biomarkers, and the detection limit of t-PSA can be as low as 100 fg / mL. In addition, this immunosensing substrate also has the potential to be extended to the detection of other proteins, biomolecules and pathogenic microorganisms, showing application prospects in many other fields such as early disease diagnosis, cancer screening, environmental monitoring, food safety and forensic analysis.

[0045] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. The gold-silver nanoflowers prepared by the present invention adopt a novel synthesis technology that has not been disclosed. The preparation method is simple, efficient and has good repeatability.

[0046] 2. The obtained gold-silver nanoflowers have uniform sizes, with a silver core-gold shell structure inside and a nanoflower-like shape on the surface, which can provide a large number of highly efficient and stable "SERS hot spots", thus significantly enhancing the enhancement effect of Raman signals.

[0047] 3. During the synthesis process of the gold-silver nanoflowers, the Raman signal molecule 4-MBA is naturally adsorbed on the surface. Subsequently, the change in the Raman intensity of 4-MBA can be directly used as the quantitative detection signal for the target protein concentration, improving the convenience and accuracy of detection.

[0048] 4. By combining magnetic beads with carboxyl groups on the surface, the present invention effectively solves the problems of incomplete separation and poor signal enhancement effect existing when traditional nanoparticles are used for protein detection, thereby improving the detection sensitivity and signal stability.

[0049] 5. The SERS immunosensing substrate constructed by combining gold-silver nanoflowers and magnetic beads has excellent signal uniformity, and the lowest detectable t-PSA concentration can be as low as 100 fg / mL. In addition, this detection substrate has good uniformity and anti-interference ability, and has the potential to be applied in actual clinical serum detection. Description of the Drawings

[0050] Figure 1 : Schematic diagram of the synthesis steps of gold-silver nanoflowers and magnetic beads and their construction and application in the SERS immunosensing substrate.

[0051] Figure 2: Characterization and analysis of the gold-silver nanoflowers of Example 1. Among them, (a) is the transmission electron microscope (TEM) image and the energy dispersive spectroscopy (EDS) elemental analysis image, (b) is the particle size analysis image, and (c-e) are the X-ray photoelectron spectroscopy (XPS) images.

[0052] Figure 3 : TEM images of the gold-silver nanoflowers of Comparative Examples 5-6. Among them, (a-b) are the TEM images of Comparative Example 5, and (c) is the TEM image of Comparative Example 6.

[0053] Figure 4 : Performance evaluation of the SERS immunosensing substrate, including (a) detection sensitivity, (b) linear regression analysis between the Raman intensity at the characteristic peak (1587 cm-1) of 4-MBA and different t-PSA concentrations, and (c-d) verification experiments and characterization results of signal uniformity and anti-interference ability. Detailed implementation manners

[0054] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0055] The embodiment of the present invention provides a preparation method of a SERS immunosensing substrate based on gold-silver nanoflowers and magnetic beads, which specifically includes the following steps:

[0056] Step 1) Preparation of gold-silver nanoflowers: First, uniformly sized silver nanoseeds are synthesized, and their size is further grown by the thermal growth method. Subsequently, the silver nanoparticles are aminated, and a growth solution is added to form gold-silver nanoflowers with a silver core-gold shell structure. Finally, after centrifugal purification, the obtained gold-silver nanoflowers are resuspended in PBS buffer and stored at 4 °C for later use.

[0057] Step 2) Preparation of magnetic beads: Solvothermal method is used to synthesize magnetic beads with good magnetic response performance and carboxyl groups on the surface. After magnetic separation, the residual reactants are repeatedly washed away, and finally the obtained magnetic beads are resuspended in PBS buffer and stored at 4 °C for later use.

[0058] Step 3) Preparation of immune probes: Using the EDC / NHS chemical activation method, the gold-silver nanoflowers and carboxyl-modified magnetic beads are respectively surface-functionalized to covalently couple with specific antibodies to form two types of nano-immune probes.

[0059] Step 4) Preparation of the "sandwich" immune structure: The magnetic immune probe prepared in Step 3) is added to the sample to be tested to specifically bind with t-PSA. After magnetic separation to remove the unbound protein, the gold-silver nanoflower immune probe prepared in Step 3) is further added to finally form a stable "sandwich" immune structure.

[0060] Step 5) SERS immunoassay: Enrich the "sandwich" immune structure by applying an external magnetic field, and remove unbound interferents by thorough washing. Subsequently, take a certain amount and drop it on a clean silicon wafer, and collect the characteristic Raman signal of 4-MBA by Raman detection. Analyze the concentration of t-PSA by measuring the Raman intensity at the Raman characteristic peak of the signal molecule, so as to achieve highly sensitive and specific SERS immunoassay.

[0061] The following takes specific examples for detailed description:

[0062] Example 1

[0063] As Figure 1 shown, the preparation process of gold-silver nanoflowers, magnetic beads and the SERS immunoassay substrate constructed by them is as follows: First, prepare gold-silver nanoflowers and magnetic beads; then, couple them with specific antibodies through chemical activation to form immune probes; finally, add the protein to be detected to react with the immune probes to form a sandwich immune structure. After the reaction is completed, perform Raman detection on the solution to analyze the concentration of the protein to be detected. Figure 1 shown is the schematic diagram of the preparation of the SERS immune substrate constructed by gold-silver nanoflowers and magnetic beads. The detailed preparation process is as follows:

[0064] (1) Preparation of gold-silver nanoflowers

[0065] 4 nm silver nanospheres: Mix 75 mL of deionized water with 20 mL of 1% sodium citrate solution and heat to 70 °C. Under vigorous stirring, add 1.7 mL of 1% AgNO3 solution, and then quickly add 2 mL of freshly prepared 0.1% NaBH4 solution at one time. Continue stirring for 1 h, then naturally cool the mixed solution to room temperature and make up the volume to 100 mL.

[0066] 20 nm silver nanospheres: Mix 75 mL of deionized water with 2 mL of 1% sodium citrate solution and heat to boiling. Under vigorous stirring, add 10 mL of the above 4 nm silver nanosphere solution and 1.7 mL of 1% AgNO3 solution successively, and continue stirring for 1 h under the condition of condensation reflux. Subsequently, add 2 mL of 1% sodium citrate solution and 1.7 mL of AgNO3 solution, and continue stirring for 1 h under the condition of condensation reflux. This step is repeated twice. Finally, naturally cool the mixed solution to room temperature and make up the volume to 100 mL.

[0067] Gold and silver nanoflowers: First, take 100 μL of the prepared 20-nm silver spheres, centrifuge them at 8000 rpm for 10 min, and remove the supernatant. Subsequently, add 800 μL of 0.005 M APTES solution to functionalize the surface of the silver nanoparticles with amino groups through silanization. The mixed solution is oscillated on a shaker at 300 rpm for 1 h. Subsequently, prepare a growth solution containing 600 μL of ethanol (EtOH), 50 μL of 4-mercaptobenzoic acid (4-MBA, 60 mM), 100 μL of L(+)-ascorbic acid (AA, 0.4 M), and 660 μL of (HAuCl4, 25 mM). Take 300 μL of the growth solution and add it to the mixed solution. After reacting for 30 min, centrifuge at 6000 rpm for 10 min, remove the supernatant, redisperse it in 1 mL of phosphate buffer solution (PBS), and repeat this step twice. Finally, collect the synthesized nanoflowers, redisperse them in 1 mL of PBS, and store them in a refrigerator at 4 °C for later use.

[0068] (2) Preparation of magnetic beads

[0069] Magnetic beads: First, dissolve 0.54 g of ferric chloride hexahydrate (FeCl3·6H2O) in a well-mixed solution of 10 mL of diethylene glycol ((CH2OH)2) and 10 mL of ethylene glycol (C4H 10 O3). Then, add 1.50 g of sodium acetate (CH3COONa) and 1.50 g of sodium acrylate (C3H3O2Na) to the solution, and completely dissolve the mixture by alternately using magnetic stirring and ultrasound. Transfer the obtained homogeneous solution into a stainless-steel autoclave lined with polytetrafluoroethylene, and heat it at 200 °C for 10 h. After the reaction is completed, magnetically separate and collect the black precipitate, wash it 3 times with deionized water (DI), wash it 3 times with ethanol, and finally dry it in vacuo at 60 °C for 12 h.

[0070] (3) Preparation of nano-immunoprobes

[0071] Gold-silver nanoflower immune probe: Add 50 μL of EDC (100 mM) and 50 μL of NHS (100 mM) to 1 mL of the prepared gold-silver nanoflower PBS solution. The mixed solution is incubated on a shaker at 400 rpm for 30 min at room temperature. Subsequently, the solution is centrifuged at 6000 rpm and washed with PBS twice. The precipitate is redispersed in 1 mL of PBS, and then 20 μL of 1 mg / mL t-PSA antibody is added to the dispersion and incubated at room temperature for 3 h. After incubation, the solution is centrifuged at 6000 rpm and washed with PBS twice. Then, 100 μL of bovine serum albumin solution (BSA, 3%) is added as a capping agent, and the mixed solution is incubated at room temperature for 2 h. After the reaction, the solution is centrifuged at 6000 rpm and washed with PBS twice. Finally, the precipitate is redispersed in 1 mL of PBS and stored in a refrigerator at 4 °C.

[0072] Preparation of magnetic immune probe: First, prepare 1 mL of PBS solution containing 0.5 mg of carboxyl magnetic beads (Fe3O4-COOH). Subsequently, 50 μL of EDC (100 mM) and 50 μL of NHS (100 mM) are added, and the mixed solution is incubated at 400 rpm for 30 min at room temperature. After the reaction is completed, the supernatant is removed by magnetic separation, and then washed with PBS twice. The precipitate is redispersed in 1 mL of PBS. Then, 20 μL of 1 mg / mL t-PSA antibody is added to the dispersion and incubated at room temperature for 3 h. After incubation, the supernatant is removed by magnetic separation, and then washed with PBS twice. The precipitate is redispersed in 1 mL of PBS. Then, 100 μL of bovine serum albumin solution (BSA, 3%) is added as a capping agent, and the mixed solution is incubated at room temperature for 2 h. After the reaction, the obtained magnetic immune probe is washed twice by magnetic separation with PBS. Finally, the precipitate is redispersed in 1 mL of PBS and stored in a refrigerator at 4 °C.

[0073] (4) Construction and detection of SERS immune detection substrate

[0074] Formation of sandwich immune structure: Take 20 μL of t-PSA solution with a specific concentration and bind it to 50 μL of magnetic immune probe. After the reaction is completed, the precipitate is separated by magnetic separation and thoroughly washed with PBS, and then the precipitate is redispersed in PBS. Then, 50 μL of the prepared gold-silver nanoflower immune probe is added to make it bind to the conjugate of magnetic immune probe and t-PSA. After the reaction, the precipitate is separated by magnetic separation and thoroughly washed with PBS, and then the precipitate is redispersed in PBS and stored in a refrigerator at 4 °C for subsequent SERS detection.

[0075] SERS immunosensing substrate detection: Take 10 μL of the solution containing the sandwich immune structure prepared, drop it on a clean silicon wafer to form a detection substrate, and then perform SERS detection. The concentration of t-PSA is judged by the intensity of the Raman signal molecule of 4-MBA obtained by detection.

[0076] Comparative example

[0077] Select the gold-silver nanoflowers prepared in the prior art as the comparative example.

[0078] Comparative example 1 was prepared by the method described in "Cheng, H., Chen, R., Zhan, Y., Dong, W., Chen, Q., Wang, Y., Zhou, P., Gao, S., Huang, W., Li, L., Feng, J., 2024. Novel Ratiometric Surface-Enhanced RamanScattering (SERS) Biosensor for Ultrasensitive Quantitative Monitoring of HumanCarboxylesterase-1in Hepatocellular Carcinoma Cells Using Ag–Au Nanoflowersas SERS Substrate. Analytical chemistry(Washington) 96(46), 18555-18563."

[0079] Comparative example 2 was prepared by the method described in "Xu, Y., Kutsanedzie, F.Y.H., Hassan, M.M., Zhu, J., Li, H., Chen, Q., 2020. Functionalized hollow Au@Ag nanoflower SERS matrix for pesticide sensingin food. Sensors and actuators. B, Chemical 324, 128718."

[0080] Comparative Example 3 was prepared by the method described in "Zhang, Z., Luan, Y., Ru, S., Teng, H., Li, Y., Liu, M., Wang, J., 2023. A novel electrochemical aptasensor for ultrasensitive detection of herbicide prometryn based on its highly specific aptamer and Ag@Au nanoflowers. Talanta (Oxford) 265, 124838 - 124838."

[0081] Comparative Example 4 was prepared by the method described in "Zhu, A., Ali, S., Xu, Y., Ouyang, Q., Chen, Q., 2021. A SERS aptasensor based on AuNPs functionalized PDMS film for selective and sensitive detection of Staphylococcus aureus. Biosens Bioelectron 172, 112806."

[0082] Compared with the silver - gold nanoflowers prepared in the above - mentioned comparative examples, the advantages of the silver - gold nanoflowers prepared in Example 1 are as follows:

[0083] 1. There is no reported literature on the same synthesis strategy. The existing synthesis strategies for silver - gold nanoflowers are limited, and the synthesis methods are not simple. The silver - gold nanoflowers in Example 1 can be synthesized simply and rapidly, and a large number of silver - gold nanoflowers with uniform morphology, stable structure, and silver - core and gold - shell (Ag@Au) can be prepared within several hours. The synthesis method in Example 1 is derived from the synthesis idea of growing gold wires at the solid - phase interface, but it is not just a simple modification of the synthesis environment. The formula of the growth solution needs to be optimized to obtain the synthesis strategy of the silver - gold nanoflowers in Example 1.

[0084] 2. The structure and morphology of the honeysuckle in Example 1 are very suitable for application in SERS detection. First of all, the enhancement of SERS by gold and silver materials is particularly obvious among many materials and is widely used. Secondly, the nanoflower in Example 1 has a silver core and gold shell structure, which can couple the SERS effects of the two materials to achieve SERS enhancement that cannot be obtained by a single material. Moreover, the dense flower-like structure on the surface of the nanoflower structure in Example 1 can provide a large number of SERS hot spots for SERS detection. Finally, the gold shell structure can well protect the silver core from being oxidized by the solution or oxygen in the air, thus protecting the stability of the gold-silver nanoflower structure and ensuring the stability of its SERS enhancement.

[0085] 3. In other studies, signal molecules for Raman detection, such as 4-mercaptobenzoic acid (4-MBA) or other Raman signal probes, are added with additional Raman signal molecules by ligand binding or surface modification methods after the synthesis of nanoparticles for detection. However, in the synthesis process of the present patent, the growth solution contains an excessive amount of 4-mercaptobenzoic acid (4-MBA), which makes the surface of the synthesized gold-silver nanoflowers densely covered with 4-MBA after the synthesis. This result ensures the stability of subsequent SERS detection and provides SERS marker detection molecules for subsequent detection.

[0086] Comparative Example 5

[0087] The difference between this comparative example and Example 1 lies in that the formulation of the growth solution in the preparation process of the gold-silver nanoflowers is different. The preparation method of the growth solution in this comparative example is: prepare a growth solution containing 600 μL of ethanol (EtOH), 50 μL of 4-mercaptobenzoic acid (4-MBA, 6 mM), 100 μL of L(+)-ascorbic acid (AA, 0.4 M), and 660 μL of (HAuCl4, 25 mM). The remaining steps are the same as those in Example 1. The finally prepared gold-silver nanoflowers are as shown in Figure 3 a and b in. When the content of 4-MBA in the growth solution is too low, the nanoflower structure cannot be formed.

[0088] Comparative Example 6

[0089] The difference between this comparative example and Example 1 lies in that silver nanoparticles without amino functionalization are used for the preparation of gold-silver nanoflowers, and the remaining steps are the same as those in Example 1.

[0090] The specific preparation process of the gold-silver nanoflowers is as follows: First, take 100 μL of the prepared 20-nm silver spheres, centrifuge them at 8000 rpm for 10 min, and remove the supernatant. The mixed solution is oscillated on a shaker at 300 rpm for 1 h. Subsequently, a growth solution containing 600 μL of ethanol (EtOH), 50 μL of 4-mercaptobenzoic acid (4-MBA, 60 mM), 100 μL of L(+)-ascorbic acid (AA, 0.4 M), and 660 μL of (HAuCl4, 25 mM) is prepared, and 300 μL of the growth solution is added to the mixed solution. After reacting for 30 min, centrifuge at 6000 rpm for 10 min, remove the supernatant, redisperse it in 1 mL of phosphate buffer solution (PBS), and repeat this step twice. Finally, collect the synthesized nanoflowers, redisperse them in 1 mL of PBS, and store them in a refrigerator at 4 °C for later use.

[0091] The finally prepared gold-silver nanoflowers are as shown in Figure 3 c in the figure. It can be seen from the figure that when the silver seeds are added to the growth solution without amino-functionalization, the formation of the nanoflower structure is prevented.

[0092] Comparative Example 7

[0093] The difference between this comparative example and Example 1 lies in the different addition amounts of the 20-nm silver spheres in the preparation process of the gold-silver nanoflowers, which are 500 μL and 300 μL respectively. The remaining steps are the same as those in Example 1.

[0094] The finally synthesized gold-silver nanoflowers show poor dispersibility in the PBS buffer solution, are prone to severe aggregation, forming large particle agglomerates, and the aggregation phenomenon will seriously affect their usability and performance as immune probes in subsequent detections, restricting their application in actual biological systems.

[0095] Example 2: Performance characterization of the SERS immunosensing substrate.

[0096] (1) Sensitivity evaluation

[0097] To evaluate the sensitivity of the signal uniformity of the SERS immunosensing substrate, an SERS immunosensing substrate with a t-PSA concentration range covering 10 ng / mL to 100 fg / mL was constructed. The characteristic Raman signal intensity of 4-MBA was measured by Raman spectroscopy and used to evaluate the concentration of t-PSA. As shown in Figure 4 a, the Raman intensity of 4-MBA decreases with the decrease in the t-PSA concentration, and the lowest detection limit (LOD) can reach 100 fg / mL. In addition, by analyzing the data of the Raman peak intensity of 4-MBA at 1587 cm -1 and the corresponding t-PSA concentration, a linear regression equation was obtained by fitting (as shown in Figure 4as shown in b):

[0098] Y = 1043.77*X - 1970.19

[0099] As Figure 4 shown in b. Among them, the calculated correlation coefficient R 2 = 0.996, indicating a good linear correlation between the signal intensity and the t-PSA concentration, and proving the high sensitivity and reliability of this detection substrate.

[0100] (2) Homogeneity detection

[0101] To evaluate the signal homogeneity of the SERS immunosensing substrate, a sample with a t-PSA concentration of 10 ng / mL was selected, and 15 different measurement points were randomly selected on its surface for Raman detection. As Figure 4 shown in c, the Raman peak intensity of 4-MBA at 1587 cm -1 showed a good distribution pattern, and the signal homogeneity was relatively high. Further calculation showed that the relative standard deviation (RSD) was 7.93%, indicating that the immunosensing substrate had high signal stability and homogeneity.

[0102] (3) Anti-interference detection

[0103] To evaluate the anti-interference performance of the SERS immunosensing substrate, carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) were selected as potential interferents in this study, and different experimental groups were designed for comparative analysis. As Figure 4 shown in d, the CEA group, AFP group, t-PSA group (the antigen concentration in all three groups was 10 ng / mL), and the CEA + t-PSA group and AFP + t-PSA group (the different antigen concentrations in the two groups were both 10 ng / mL) were tested respectively. The experimental results showed that when t-PSA was absent, the Raman signal intensity of 4-MBA at 1587 cm -1 was close to 0, and regardless of whether t-PSA existed alone or coexisted with the interferent, its Raman signal intensity remained consistent and was not significantly affected. This result indicates that this SERS immunosensing substrate has good specificity and anti-interference ability and can accurately detect the target protein.

Claims

1. A preparation method of gold and silver nano-flowers, characterized in that, The preparation method includes the following steps: adding silver nanoseeds functionalized with surface amino groups into a growth solution, and after the reaction ends, collecting the product, which is the gold-silver nanoflower; wherein, the growth solution is composed of ethanol, chloroauric acid, ascorbic acid, and 4-mercaptobenzoic acid; wherein, the concentration of 4-mercaptobenzoic acid in the growth solution is at least 2.13 mM.

2. A gold-silver nanoflower obtained by the preparation method according to claim 1, characterized in that, The interior of the gold-silver nanoflower has a silver core-gold shell structure, and the surface is in the shape of nanoflowers.

3. A SERS immunosensor, characterized in that, The SERS immunoprobe includes a signal probe and a capture probe, wherein the gold-silver nanoflower functionalized with amino groups as described in claim 2 is used as the signal probe, and magnetic beads with carboxyl groups on the surface are used as the capture probe.

4. The SERS immunoprobe according to claim 3, wherein The method for functionalizing the gold-silver nanoflower with amino groups is as follows: the surface of the silver nanoseeds is functionalized with amino groups through silanization to obtain amino-functionalized silver nanoseeds; adding the amino-functionalized silver nanoseeds into the growth solution, and after the reaction ends, the amino-functionalized gold-silver nanoflower is obtained.

5. The SERS immune probe according to claim 3, characterized in that, Magnetic beads with carboxyl groups on the surface are synthesized by a solvothermal method.

6. A method for preparing the SERS immunoprobe according to claim 3, characterized in that, Using a chemical activation method, the signal probe and the capture probe are respectively conjugated with specific antibodies to form a SERS immunoprobe.

7. A method for preparing a SERS immunodetection substrate based on the gold-silver nanoflower as described in claim 2, including the following steps: Step 1): First, synthesize initial silver nanoseeds, and regulate their size by a thermal growth method. After the silver nanoseeds are functionalized with amino groups, add them into the growth solution to synthesize gold-silver nanoflowers; Step 2): Synthesize magnetic beads with carboxyl groups on the surface by a solvothermal method; Step 3): Using a chemical activation method, conjugate the gold-silver nanoflower and the magnetic beads with specific antibodies respectively to form a SERS immunoprobe; Step 4): Bind the SERS immunoprobe to the protein to be detected to form a "sandwich" immune structure; Step 5): Drop the mixed solution containing the "sandwich" structure onto the substrate material to form a SERS immunodetection substrate.

8. A SERS immunodetection substrate prepared by the preparation method as described in claim 7.

9. An application of the SERS immunodetection substrate as described in claim 8 in protein detection.

10. An application of the SERS immunodetection substrate as described in claim 8 in preparing a SERS detection platform.

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