A gold-silver alloy nanoflower SERS substrate, a preparation method and application thereof

Abstract

The application provides a gold-silver alloy nanoflower SERS substrate and a preparation method and application thereof. 3+ and Ag + The preparation method is as follows: an aluminum foil paper substrate is placed in a growth solution containing Au 3+ and Ag + , a gold-silver alloy nanoflower array pattern layer with a multi-level branch structure is formed on the surface of the aluminum foil paper substrate by a displacement reaction of aluminum and noble metal ions in situ. The gold-silver alloy nanoflower array pattern layer has rich nanotips and a three-dimensional rough structure, can form a large number of electromagnetic 'hot spots', and can significantly enhance the Raman signal intensity. Relying on the excellent SERS enhancement performance, the gold-silver alloy nanoflower SERS substrate is applied to an immune recognition mediated sandwich detection mode, and high-sensitivity and accurate detection of a hidden blood marker in a fecal sample is realized. The preparation process is simple, the cost is low, and complex equipment is not needed; the obtained SERS substrate has strong stability and excellent repeatability, and can meet the needs of early screening and on-site rapid detection of digestive diseases.

Description

Technical Field

[0001] This invention relates to the field of surface-enhanced Raman spectroscopy (SERS) technology, and particularly to a gold-silver alloy nanoflower SERS substrate, its preparation method, and its application, relating to nanomaterial preparation, biosensing, and in vitro diagnostic technologies. Background Technology

[0002] Fecal occult blood testing is an important means of screening for digestive tract diseases, especially early colorectal cancer. Traditional testing methods have obvious limitations: chemical colorimetric methods have low sensitivity and are easily affected by factors such as diet and drugs, affecting the accuracy of the test; although immunological methods have high specificity, they still have problems such as insufficient detection sensitivity and strong dependence on detection instruments, making it difficult to meet the needs of early clinical screening.

[0003] Surface-enhanced Raman scattering (SERS) technology, with its unique advantages such as ultra-high sensitivity, strong fingerprint specificity, and outstanding resistance to background interference, has shown broad application prospects in the field of biomedical detection. However, the performance of SERS detection largely depends on the structural characteristics and stability of the substrate material, and a high-performance SERS substrate is a key prerequisite for achieving accurate detection.

[0004] Currently, existing SERS substrates are mostly prepared by self-assembly of gold and silver nanoparticles or vacuum deposition, which generally suffer from problems such as complex preparation processes, high production costs, uneven distribution of electromagnetic "hot spots" and poor detection repeatability, thus limiting their large-scale application and clinical translation.

[0005] Therefore, developing a high-performance SERS substrate that is simple to prepare, low in cost, and has a high hot spot density is of great theoretical significance and practical application value. Summary of the Invention

[0006] The purpose of this invention is to provide a gold-silver alloy nanoflower SERS substrate, its preparation method, and its application, so as to solve the technical problems of complex preparation process, high cost, uneven hot spot distribution, and poor repeatability of existing SERS substrates. At the same time, it makes up for the defects of insufficient sensitivity and weak anti-interference ability of traditional fecal occult blood detection methods, so as to achieve highly sensitive and accurate detection of occult blood markers in fecal samples, and meet the actual needs of early screening and rapid on-site detection of digestive tract diseases.

[0007] To address the aforementioned technical problems, this invention first provides a method for preparing a gold-silver alloy nanoflower SERS substrate, comprising the following steps:

[0008] S10, Immerse the aluminum foil paper substrate in the etching solution for etching treatment to remove the aluminum oxide film on the surface of the aluminum foil paper substrate;

[0009] S20, the etched aluminum foil substrate is immersed in a growth solution for in-situ growth reaction, forming a gold-silver alloy nanoflower array pattern layer rich in hot spots on the surface of the aluminum foil substrate; the growth solution includes at least Au. 3+ and Ag + ;

[0010] S30, take out the aluminum foil paper substrate containing the gold and silver alloy nanoflower array pattern layer, and obtain the gold and silver alloy nanoflower SERS substrate after cleaning and drying.

[0011] Specifically, the preparation method of the present invention removes the oxide film on the surface of the aluminum foil substrate by etching in step S10 to ensure a clean interface and strong bonding for subsequent nanostructure growth; step S20 uses Au-containing materials. 3+ and Ag + In-situ growth of the growth solution can directly form a gold-silver alloy nanoflower array rich in high-density hot spots on the surface of the aluminum foil substrate, significantly improving the SERS enhancement effect; the target substrate can be obtained by simple cleaning and drying in step S30. The overall process is simple, low-cost, and has excellent controllability and repeatability.

[0012] Furthermore, the gold-silver alloy nanoflower SERS substrate obtained by the above preparation method has a sunflower-shaped gold-silver alloy nanoflower structure with multi-level branching and radial morphology. It has the structural characteristics of multi-level branching, abundant tips and dense nano gaps. The surface is distributed with a large number of nano tips and rough structures, which can form a large number of stable electromagnetic hot spots on the surface of aluminum foil paper substrate, thereby providing high-density surface-enhanced Raman scattering hot spots.

[0013] Preferably, before step S10, the process further includes: ultrasonically cleaning the aluminum foil substrate sequentially with acetone, ethanol, and deionized water.

[0014] Specifically, before step S10, the aluminum foil substrate is ultrasonically cleaned sequentially with acetone, ethanol, and deionized water. This effectively removes oil, impurities, and particles from the substrate surface, resulting in a highly clean growth interface. This lays the foundation for subsequent uniform etching and the stable and uniform growth of gold and silver alloy nanoflowers, which is beneficial for improving the uniformity of the substrate structure and its reliability.

[0015] Preferably, in step S10, the concentration of the etching solution is 0.05–0.15 mol / L, the etching solution is NaOH solution or KOH solution, and the etching time is 1–5 min.

[0016] Specifically, the above etching process can accurately and efficiently remove the aluminum oxide film on the surface of the aluminum foil substrate without damaging it. The etching degree is moderate and highly controllable, which not only ensures the cleanliness and regularity of the interface for subsequent nanostructure growth, but also significantly improves the process stability and substrate preparation repeatability.

[0017] Preferably, in step S20, the in-situ growth reaction time is 5 to 15 minutes, and the in-situ growth temperature is 20 to 30°C.

[0018] Specifically, the above-mentioned in-situ growth reaction conditions are mild and easy to control, and do not require complex temperature control equipment. This can ensure that gold and silver alloy nanoflowers grow uniformly and densely on the substrate surface, forming sufficient and stable SERS hot spots, and can effectively improve the repeatability of the preparation process and the consistency of the substrate product.

[0019] Preferably, Au in the growth solution 3+ and Ag + The molar concentration ratio is 1:(1~2.8).

[0020] Specifically, the above molar concentration range ensures that gold-silver alloy nanoflowers grow smoothly and uniformly on aluminum foil substrates, forming a nanoflower array with a regular structure and sufficient SERS hotspots.

[0021] Furthermore, when Ag + molar concentration reaches Au 3+ At three times the molar concentration, Ag elemental is difficult to grow on aluminum foil substrates. The reasons may be as follows: an excessively high concentration of silver ions will lead to an excessively fast reduction rate of silver, which will easily cause homogeneous nucleation in the growth solution, making it difficult to achieve stable heterogeneous nucleation and in-situ growth on the surface of the aluminum foil substrate; at the same time, an excessively high proportion of silver ions will destroy the interfacial compatibility of gold-silver alloy co-deposition, reduce the bonding force between the metal and the aluminum foil substrate, and ultimately prevent the formation of continuous and dense alloy nanostructures on the surface of the aluminum foil substrate.

[0022] Preferably, the growth solution is a mixture of 0.2–2 mmol / L HAuCl4 solution and 0.2–5.6 mmol / L AgNO3 solution.

[0023] Specifically, the growth solution containing the above-mentioned suitable molar concentration of metal cations can achieve controllable co-reduction and synergistic deposition of gold and silver ions, ensuring that gold and silver alloy nanoflowers grow uniformly, densely, and stably on the surface of aluminum foil paper substrate, effectively forming high-density SERS hotspots, and significantly improving the reinforcement performance of the substrate and the stability and repeatability of the preparation process.

[0024] Preferably, when Ag in the growth solution + When the molar concentration remains constant, both the size and surface roughness of the gold-silver alloy nanoflower array pattern layer increase with increasing molar concentration of HAuCl4 solution.

[0025] Preferably, when Au in the growth solution 3+ When the molar concentration remains constant, the size and surface roughness of the gold-silver alloy nanoflower array pattern layer both increase with increasing molar concentration of AgNO3 solution.

[0026] Specifically, by adjusting the molar concentrations of HAuCl4 and AgNO3 in the growth solution, the size and surface roughness of the gold-silver alloy nanoflower array pattern layer can be flexibly controlled. Increasing the concentration of both can effectively increase the array structure and surface roughness, making it easier to accurately construct high-density SERS hotspots and significantly improving the adjustability and application adaptability of the substrate's SERS performance.

[0027] Accordingly, the present invention also provides a gold-silver alloy nanoflower SERS substrate, which is prepared by any of the above methods for preparing gold-silver alloy nanoflower SERS substrate; the gold-silver alloy nanoflower SERS substrate includes an aluminum foil substrate and a gold-silver alloy nanoflower array pattern layer grown in situ on the surface of the aluminum foil substrate.

[0028] Specifically, the gold-silver alloy nanoflower SERS substrate is prepared by the aforementioned method. It uses aluminum foil as a substrate and grows a gold-silver alloy nanoflower array pattern layer on the surface in situ. The structure is uniform and it is firmly bonded to the substrate. It can stably form high-density SERS hot spots and has both excellent enhancement performance and ease of preparation.

[0029] Accordingly, the present invention also provides an application of a gold-silver alloy nanoflower SERS substrate, which is used for fecal occult blood detection based on SERS sandwich immunoassay.

[0030] Specifically, the gold-silver alloy nanoflower SERS substrate is applied to the detection of fecal occult blood based on SERS sandwich immunoassay. By relying on the strong enhancement characteristics of the high-density SERS hotspots on the substrate, the detection sensitivity and signal stability can be greatly improved, achieving efficient, accurate and highly sensitive detection of fecal occult blood.

[0031] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention provides a gold-silver alloy nanoflower SERS substrate, its preparation method, and its application. The preparation method involves removing the aluminum oxide layer from the surface of an aluminum foil substrate through alkali treatment, and then placing it in an Au-containing environment. 3+ and Ag +In the growth solution, a gold-silver alloy nanoflower array pattern layer with a multi-level branched structure is grown in situ on the surface of an aluminum foil substrate using a displacement reaction between aluminum and noble metal ions. This gold-silver alloy nanoflower array pattern layer possesses abundant nano-tip structures and a three-dimensional rough structure, which can form numerous electromagnetic "hot spots" on its surface, significantly enhancing the Raman signal intensity. Based on these excellent SERS enhancement properties, this invention further applies this gold-silver alloy nanoflower SERS substrate to an immune recognition-mediated sandwich detection mode, ultimately achieving highly sensitive and accurate detection of occult blood markers in fecal samples. Simultaneously, the preparation process of this invention is simple and low-cost, requiring no complex equipment, and the resulting substrate exhibits strong stability and excellent repeatability, effectively meeting the practical application needs of early screening and rapid on-site detection of digestive tract diseases. Attached Figure Description

[0032] Figure 1 This is a schematic diagram illustrating the preparation process of the gold-silver alloy nanoflower SERS substrate provided in Example 1 and the process of conducting SERS detection on fecal occult blood.

[0033] Figure 2 The image shows the SEM (Scanning Electron Microscope) image at 2.0K magnification of the gold-silver alloy nanoflower SERS substrate prepared using a growth solution containing 0.125 mmol / L HAuCl4 solution and 0.25 mmol / L AgNO3 solution in Example 1.

[0034] Figure 3 The image shows the SEM pattern of the gold-silver alloy nanoflower SERS substrate prepared using a growth solution containing 0.125 mmol / L HAuCl4 solution and 0.25 mmol / L AgNO3 solution in Example 1, at a magnification of 5.0 K.

[0035] Figure 4 The SERS spectrum obtained by detecting fecal occult blood after functionalizing the gold-silver alloy nanoflower SERS substrate prepared in this Example 1 with a growth solution containing 0.125 mmol / L HAuCl4 solution and 0.25 mmol / L AgNO3 solution with hemoglobin antibody solution (with comparison results of blank PBS solution control group) is shown in the image.

[0036] Figure 5The SERS spectrum obtained by detecting fecal occult blood after functionalizing the gold-silver alloy nanoflower SERS substrate prepared in this Example 1 with a growth solution containing 0.125 mmol / L HAuCl4 solution and 0.25 mmol / L AgNO3 solution with transferrin antibody solution (with comparison results of blank PBS solution control group) is shown in the image.

[0037] Figure 6 The SERS spectrum obtained by detecting fecal occult blood after functionalizing the gold-silver alloy nanoflower SERS substrate prepared in this Example 1 with a growth solution containing 0.125 mmol / L HAuCl4 solution and 0.25 mmol / L AgNO3 solution with a mixed antibody solution of hemoglobin antibody and transferrin antibody in a 1:1 ratio (with comparison results of blank PBS solution control group) is shown in the image. Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] To address the technical problems existing in current SERS substrates, aluminum, as a reactive metal, can achieve gold deposition through noble metal ion substitution, which is a cheap and efficient preparation approach. However, there is currently a lack of a systematic method for the controllable preparation of gold / silver alloy nanoflowers using this substitution reaction, and no published literature applies this method to the SERS detection of fecal occult blood. Therefore, this invention provides a novel and efficient solution. Through a technical route of "etching away Al2O3 + HAuCl4 + AgNO3 spontaneous substitution reaction," the substitution reaction between aluminum and noble metal ions enables the rapid and uniform growth of gold / silver alloy nanoflowers (Au / Agnanoflowers) on the surface of an aluminum foil substrate, successfully preparing a gold / silver alloy nanoflower SERS substrate with high SERS hotspot density.

[0040] The technical solution of the present invention will now be further described with reference to specific embodiments.

[0041] Example 1:

[0042] Please see Figure 1 Example 1 provides a gold-silver alloy nanoflower SERS substrate and its preparation method. The preparation process of the gold-silver alloy nanoflower SERS substrate specifically includes the following steps:

[0043] Step (1): Cut commercial aluminum foil to the required size, ultrasonically clean it in acetone, ethanol and deionized water in sequence to thoroughly remove surface contaminants, and then place it in a 40°C oven to dry completely.

[0044] Step (2): Immerse the cleaned and dried aluminum foil in 5 mL of 0.1 mol / L sodium hydroxide aqueous solution and etch it at room temperature for 2 min to completely remove the natural aluminum oxide film on its surface and expose the highly reactive fresh aluminum surface. After etching, rinse repeatedly with deionized water and blow dry to ensure that the residual NaOH on the aluminum foil surface is completely removed.

[0045] Step (3): The etched aluminum foil is immersed in a mixed solution of HAuCl4 and AgNO3, wherein the concentration of HAuCl4 is 0.125 mmol / L and the concentration of AgNO3 is 0.25 mmol / L. The mixing method is to add 2 mL of 0.125 mmol / L HAuCl4 solution to 2 mL of 0.25 mmol / L AgNO3 solution. Then, the aluminum foil is quickly placed on a shaker and reacted at 550 rpm for 10 min, so that gold ions and silver ions are deposited in situ on the surface of the aluminum foil through a displacement reaction, forming a gold-silver alloy nanoflower array pattern layer with "hot spots".

[0046] Step (4): Remove the aluminum foil substrate containing the gold nanoflower array pattern layer, rinse with deionized water and dry to obtain the gold-silver alloy nanoflower SERS substrate, as shown below. Figure 1 As shown.

[0047] Specifically, Figure 2 and Figure 3 The images are SEM images of the gold-silver alloy nanoflower SERS substrate prepared using a growth solution containing 0.125 mmol / L HAuCl4 solution and 0.25 mmol / L AgNO3 solution in Example 1, at a magnification of 2.0K. Figure 3 This is a SEM image of the gold-silver alloy nanoflower SERS substrate prepared using a growth solution containing 0.125 mmol / L HAuCl4 solution and 0.25 mmol / L AgNO3 solution in Example 1, at a magnification of 5.0 K. Figure 2 and Figure 3 It can be seen that the gold-silver alloy nanoflower SERS substrate prepared under these conditions has a uniformly grown array of nanoflowers with multi-level branching structure, abundant nano-tip and obvious three-dimensional rough structure, which provides a good structural basis for the formation of high-density electromagnetic "hot spots".

[0048] Please see Figure 1 , Figure 4 , Figure 5 and Figure 6 In this Example 1, a gold-silver alloy nanoflower SERS substrate prepared using a growth solution containing 0.125 mmol / L HAuCl4 solution and 0.25 mmol / L AgNO3 solution was used to perform SERS detection on fecal occult blood. The specific detection process included:

[0049] (1) Substrate Functionalization: The gold-silver alloy nanoflower SERS substrates were cut to a size of 4mm × 4mm and divided into three groups for functionalization. Group 1: A hemoglobin antibody solution (Hb) with a concentration of 1 mg / mL was prepared using PBS buffer (phosphate buffered saline) at pH 7.4. After diluting the solution 100 times, 10 μL was added to the substrate surface and incubated at room temperature for 1 h. After incubation, the substrate was blocked in 1% BSA solution (1% bovine serum albumin solution prepared using PBS buffer) for 1 h. Group 2: The functionalization and blocking steps of Group 1 were repeated using transferrin antibody (Tf). Group 3: Hemoglobin antibody and transferrin antibody were mixed in a 1:1 ratio (Hb + Tf) and a mixed antibody solution with a concentration of 1 mg / mL was prepared using PBS buffer at pH 7.4. Subsequent operations were the same as those of Group 1.

[0050] (2) Sample detection: The antigen was dropped onto the surface of the prepared functionalized substrate and incubated at room temperature for 1 hour. After incubation, the substrate surface was thoroughly washed with PBS buffer and then immersed in the probe solution for another 1 hour. After incubation, the substrate was removed, the surface was gently rinsed with 1% BSA solution, and after drying, the SERS spectrum was collected in a portable Raman spectrometer. The obtained spectra are shown below. Figure 4 , Figure 5 and Figure 6 As shown.

[0051] Depend on Figure 4 , Figure 5 and Figure 6 It can be seen that the substrates functionalized with the three types of antibodies can specifically capture fecal occult blood targets and detect their characteristic Raman signals: among which, Figure 4 As an antibody-functionalized substrate, its detection signal corresponds to the hemoglobin (Hb) specific SERS probe MMC (mitomycin C), with a Raman intensity peak close to 6.0 × 10⁻⁶. 4 au; Figure 5 The transferrin antibody-functionalized substrate, whose detection signal corresponds to the transferrin (Tf) specific SERS probe MPY (4-mercaptopyridine), has a Raman intensity peak of approximately 5.5 × 10⁻⁶. 4 au; Figure 6 The combined detection group for Hb and Tf has a peak value of approximately 3.0 × 10⁻⁶ for the red curve.4 au, Raman intensity is significantly lower than Figure 4 , Figure 5 A single target detection group.

[0052] The above results demonstrate that the gold-silver alloy sunflower SERS substrate prepared in this invention exhibits excellent recognition ability and signal response performance for both single-target and dual-target detection of fecal occult blood. For single hemoglobin (Hb) or transferrin (Tf) targets, the Raman signal intensity based on the corresponding specific probes is at a high level, verifying the substrate's high sensitivity detection capability for single targets. Although the signal intensity decreases when detecting Hb and Tf dual targets simultaneously, the characteristic peaks can still be clearly identified, indicating that the substrate can achieve parallel detection of multiple targets while maintaining good overall detection sensitivity and specificity. This fully demonstrates the feasibility and superiority of the SERS substrate in the detection of complex fecal occult blood systems and in multi-marker POCT (Point-of-Care Testing) applications.

[0053] Compared with the prior art, the present invention has the following outstanding advantages:

[0054] (1) The preparation method is simple and easy: The present invention uses aluminum foil paper substrate as carrier, and after alkali treatment, gold and silver alloy sunflower nanostructure is directly generated in situ through displacement reaction without the need for external reducing agent or complicated equipment.

[0055] (2) Low raw material cost and large-area preparation: The present invention uses ordinary aluminum foil as a substrate. The material is readily available and low in cost, making it suitable for large-scale preparation of sunflower-shaped nanostructure substrates.

[0056] (3) Synergistic enhancement of gold and silver alloy: The gold and silver alloy nanoflower structure formed by the present invention combines the good stability of gold with the high SERS signal enhancement capability of silver, thereby improving detection performance and reliability.

[0057] (4) Dense hot spots in three-dimensional sunflower nanostructure: The gold-silver alloy nanoflower structure formed by the present invention has multi-level branches, abundant tips and dense nano gaps, which can generate a large number of stable electromagnetic hot spots on the surface of aluminum foil substrate, greatly improving the SERS signal strength.

[0058] (5) Applicable to the detection of complex biological samples: This invention combines the SERS sandwich detection strategy to achieve highly sensitive detection of fecal occult blood markers in complex systems with high background such as feces.

[0059] (6) Strong clinical applicability: The gold-silver alloy nanoflower SERS substrate prepared by this invention has high detection sensitivity, is suitable for early screening of diseases such as digestive tract diseases, and meets the needs of POCT, with broad application prospects.

[0060] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0061] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a gold-silver alloy nanoflower SERS substrate, characterized in that, Includes the following steps: S10, Immerse the aluminum foil paper substrate in an etching solution for etching treatment to remove the aluminum oxide film on the surface of the aluminum foil paper substrate; S20, the etched aluminum foil substrate is immersed in a growth solution for in-situ growth reaction, forming a gold-silver alloy nanoflower array pattern layer rich in hot spots on the surface of the aluminum foil substrate; the growth solution includes at least Au. 3+ and Ag + ; S30, the aluminum foil paper substrate containing the gold and silver alloy nanoflower array pattern layer is taken out, and after cleaning and drying, the gold and silver alloy nanoflower SERS substrate is obtained. In step S20, the in-situ growth reaction time is 5–15 min, and the in-situ growth temperature is 20–30 °C; the growth solution contains Au. 3+ and Ag + The molar concentration ratio is 1:(1~2.8).

2. The method for preparing the gold-silver alloy nanoflower SERS substrate according to claim 1, characterized in that, Before step S10, the process further includes ultrasonically cleaning the aluminum foil substrate sequentially with acetone, ethanol, and deionized water.

3. The method for preparing the gold-silver alloy nanoflower SERS substrate according to claim 1, characterized in that, In step S10, the concentration of the etching solution is 0.05–0.15 mol / L, the etching solution is a NaOH solution or a KOH solution, and the etching time is 1–5 min.

4. The method for preparing the gold-silver alloy nanoflower SERS substrate according to claim 1, characterized in that, The growth solution is a mixture of 0.2–2 mmol / L HAuCl4 solution and 0.2–5.6 mmol / L AgNO3 solution.

5. The method for preparing the gold-silver alloy nanoflower SERS substrate according to claim 1, characterized in that, When Ag in the growth solution + When the molar concentration remains constant, the size and surface roughness of the gold-silver alloy nanoflower array pattern layer both increase with the increase of the molar concentration of the HAuCl4 solution.

6. The method for preparing the gold-silver alloy nanoflower SERS substrate according to claim 1, characterized in that, When Au in the growth solution 3+ When the molar concentration remains constant, the size and surface roughness of the gold-silver alloy nanoflower array pattern layer both increase with the increase of the molar concentration of AgNO3 solution.

7. A gold-silver alloy nanoflower SERS substrate, characterized in that, It is prepared by the preparation method of the gold-silver alloy nanoflower SERS substrate as described in any one of claims 1 to 6; the gold-silver alloy nanoflower SERS substrate includes the aluminum foil substrate and the gold-silver alloy nanoflower array pattern layer grown in situ on the surface of the aluminum foil substrate.

8. An application of the gold-silver alloy nanoflower SERS substrate as described in claim 7, characterized in that, The gold-silver alloy nanoflower SERS substrate is used for fecal occult blood detection based on SERS sandwich immunoassay.

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