Patterned Au@Ag alloy thorn-like nanoparticle film, preparation method thereof and raman detection method based on the same

Abstract

The application belongs to the technical field of nanomaterials, and relates to a patterned Au@Ag alloy thorn-shaped nanoparticle film, a preparation method thereof and a Raman detection method based on the same. The preparation method of the patterned Au@Ag alloy thorn-shaped nanoparticle film comprises the following steps: uniformly mixing a PDMS main agent and a curing agent, pouring the mixture on a patterned silicon wafer, drying, obtaining a PDMS film, and cutting the PDMS film into small squares; adding an Au@Ag alloy thorn-shaped nanoparticle solution into a beaker, injecting a mixed solution containing anhydrous ethanol, n-hexane and 1H, 1H, 2H, 2H-perfluorodecanethiol into the beaker, and standing; contacting the side with a pattern printed on the PDMS small square with the liquid surface of the beaker, standing, and obtaining the patterned Au@Ag alloy thorn-shaped nanoparticle film. Based on the bidirectional recognition sandwich nanostructure assembled by the patterned Au@Ag alloy thorn-shaped nanoparticle film, the application realizes super-sensitive detection of target molecules and ensures detection accuracy and stability.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, and relates to a patterned Au@Ag alloy spiked nanoparticle film, its preparation method, and a Raman detection method based thereon. Background Technology

[0002] Surface-enhanced Raman spectroscopy (SERS) detection, combining fingerprint characteristics with single-molecule-scale specific recognition capabilities, is widely used in bioanalytical testing. The key to improving Raman signal intensity lies in selecting a suitable substrate. Due to the strong plasmon effect and high Raman scattering performance of noble metal nanoparticles, they have become a reliable choice for preparing SERS substrates. Compared with other types of noble metal nanoparticles, Au@Ag alloy nanostructures with densely packed tips can significantly improve the detection limit and signal intensity, resulting in excellent sensitivity and specificity for detecting disease biomarkers, due to the lightning rod effect generated by the tips. Simultaneously, by utilizing the high scattering performance of silver nanoparticles and the stability of gold nanoparticles, alloy nanoparticles ensure that the Raman substrate possesses both high signal enhancement and good biocompatibility. In particular, the signal enhancement generated by the hot spots produced by the alloy nanostructures and multiple tips has a crucial impact on the detection of small molecules with low Raman scattering signals. Dickson Joseph (Joseph D, Huh YS, Han Y-Kyu, A top-down chemical approach to tuning the morphology and plasmon resonance of spiky nanostars for enriched SERS-based chemical sensing, Sensors and Actuators: B. Chemical (2019)) systematically demonstrated that the electromagnetic enhancement of SERS detection is directly related to the plasmonic properties of the substrate and its dielectric function, including the composition, morphology, size, and local dielectric environment of the nanostructure. In SERS, electromagnetic enhancement, or localized surface plasmon resonance (LSPR), amplifies excitation and emission radiation. This mechanism, with enhancement coefficients as high as 10⁶–10⁸, is generally considered the strongest source of signal amplification. The above mechanism explains the importance of the synthesis of anisotropic nanostructures for Raman detection. Due to their novel structure, branched sea urchin nanoparticles have extraordinary potential in drug delivery, in vitro bioassays, in situ cell probe tracking, Raman imaging, particle-based drug delivery, and photothermal therapy. Matthew Rycenga (Chem. Rev. 2011, 111, 3669–3712) elucidated that silver nanoparticles exhibit stronger plasmonic properties than gold nanoparticles, resulting in higher SERS enhancement. However, silver nanoparticles lack the chemical stability and biocompatibility of gold nanoparticles. These factors limit the application of silver nanoparticles in SERS detection. To achieve greater SERS enhancement while ensuring the stability and biocompatibility of gold nanoparticles, focusing on the synthesis of alloy nanoparticle materials is a feasible strategy.Jixiang Fang (Adv. Mater. 2014, 26, 2431–2439) reported a reliable method for generating a new class of SERS substrates with ultra-high sensitivity, uniformity, and reproducibility. This was achieved by using highly roughened hollow gold-silver alloy nanobranched urchin structures and dropping nanoparticles onto the substrate, thus improving the sensitivity, uniformity, and reproducibility of the SERS substrates. The increased number of nano-gap or nano-tip structures leads to greater structural complexity and an increased hotspot density of individual nanoparticles. This method demonstrates that the key to obtaining ultra-high sensitivity, good uniformity, and reproducibility is the synthesis of branched nanostructures with high hotspot density.

[0003] Although many Raman substrates prepared from irregularly shaped alloy nanoparticles have been used in applications such as biosensoring and environmental monitoring, the preparation of SERS substrates with high sensitivity and stable data reproducibility remains a significant challenge. Furthermore, due to the complex environment of real-world samples, which is susceptible to influences from physicochemical conditions and impurities, the synthesis of highly sensitive substrates capable of specifically and stably recognizing target molecules is essential. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention transfers Au@Ag alloy spiky nanoparticles synthesized under dextrorotatory penicillamine to form a patterned Au@Ag alloy spiky nanoparticle film, and assembles a two-way recognition sandwich nanostructure, achieving ultrasensitive detection of dopamine and other analytes, ensuring the accuracy and stability of the detection.

[0005] One aspect of the present invention provides a method for preparing Au@Ag alloy spiked nanoparticles, comprising the following steps:

[0006] S1. Boil the first reducing agent solution, add the first silver nitrate solution to obtain the first generation silver seed solution; take a portion of the first generation silver seed solution, dilute it with water, heat it, add the second reducing agent solution, add the second silver nitrate solution after 1-3 minutes, react for 15-50 minutes to obtain the second generation silver seed solution.

[0007] S2. The second-generation silver seed solution was added to the dextrorotatory penicillamine aqueous solution (D-Pen solution) and incubated to obtain a mixed solution; chloroauric acid solution, surfactant solution, third reducing agent solution and water were mixed to obtain a growth solution; the mixed solution was added to the growth solution and grown at 28-32℃ for 1-3 hours to obtain Au@Ag alloy spiky nanoparticle solution.

[0008] In step S1, the first reducing agent solution is a solution formed by dissolving the first reducing agent in water, and the second reducing agent solution is a solution formed by dissolving the second reducing agent in water; preferably, the first reducing agent and the second reducing agent are each individually selected from one or more of sodium citrate, tannic acid, and ascorbic acid; more preferably, the first reducing agent and the second reducing agent are a mixture of sodium citrate and tannic acid.

[0009] The first silver nitrate solution is a solution formed by dissolving the first silver nitrate in water, and the second silver nitrate solution is a solution formed by dissolving the second silver nitrate in water; preferably, the concentrations of the first silver nitrate solution and the second silver nitrate solution are 10 to 50 mmol / L.

[0010] Preferably, when the first reducing agent is a mixture of sodium citrate and tannic acid, the molar ratio of sodium citrate, tannic acid and first silver nitrate is (40-80):1:(2-4).

[0011] Preferably, when the second reducing agent is a mixture of sodium citrate and tannic acid, the molar ratio of sodium citrate, tannic acid and second silver nitrate is (2-5):1:(5-8).

[0012] Preferably, a portion of the first-generation silver seed solution is 1 / 6 to 1 / 3 of the total volume of the first-generation silver seed solution.

[0013] Preferably, during the dilution process, the volume of water added is 0.5 to 1 times the volume of a portion of the first-generation silver seed solution.

[0014] Preferably, the heating temperature is 80–95°C.

[0015] Preferably, in step S2, the volume of the second-generation silver seed solution added is 10–70 μL. More preferably, the volume of the second-generation silver seed solution added is 10 μL.

[0016] Preferably, the concentration of the dextrorotatory penicillamine aqueous solution is 0.05–0.5 mmol / L.

[0017] Preferably, the volume of the dextrorotatory penicillamine aqueous solution is 40–100 μL. More preferably, the volume of the dextrorotatory penicillamine aqueous solution is 80 μL.

[0018] Preferably, the incubation is carried out at 28–32°C for 10–60 min; more preferably, the incubation is carried out at 28–32°C for 20 min.

[0019] Preferably, the concentration of the chloroauric acid solution is 0.008–0.012 mol / L.

[0020] Preferably, the concentration of the surfactant solution is 0.08–0.12 mol / L, and the surfactant is one or more of hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, polyvinylpyrrolidone, and sodium dodecyl sulfate.

[0021] Preferably, the concentration of the third reducing agent solution is 0.08–0.12 mol / L, and the third reducing agent is one or a mixture of sodium citrate, tannic acid, and ascorbic acid; more preferably, the third reducing agent is ascorbic acid.

[0022] Preferably, the volume ratio of chloroauric acid solution, surfactant solution, third reducing agent solution and water is 1:(6-10):(4-6):(35-45).

[0023] Preferably, the volume ratio of the mixture to the growth solution is 1:(45-65).

[0024] Another aspect of the present invention provides a patterned Au@Ag alloy spiky nanoparticle film, which is formed by transferring the above-mentioned Au@Ag alloy spiky nanoparticles onto a PDMS film.

[0025] A third aspect of this invention provides a method for preparing a patterned Au@Ag alloy spiked nanoparticle film, comprising the following steps:

[0026] Mix the PDMS base agent and curing agent evenly, pour the mixture onto a patterned silicon wafer, wait for the bubbles to disappear, dry the wafer, remove the patterned silicon wafer, and obtain the PDMS film, which is then cut into small squares.

[0027] Add the Au@Ag alloy spiked nanoparticle solution to a beaker, then inject a mixed solution containing anhydrous ethanol, n-hexane, and 1H,1H,2H,2H-perfluorodecylthiol into the beaker and let it stand for 10–24 hours.

[0028] The patterned side of the PDMS cube is placed in contact with the liquid surface in a beaker and left to stand for 3-6 seconds. Au@Ag alloy spiky nanoparticles are transferred onto the PDMS cube to obtain a patterned Au@Ag alloy spiky nanoparticle film.

[0029] The pattern on a patterned silicon wafer can be any pattern, such as one consisting of numerous raised squares with a spacing between them, ranging from 1 to 10 μm.

[0030] PDMS base agent can be listed as Dow Corning SYLGARD 184 base agent. Curing agent can be listed as SYLGARD 184 curing agent, which is used in conjunction with Dow Corning SYLGARD 184 base agent. The volume ratio of SYLGARD 184 base agent to SYLGARD 184 curing agent is (9-11):1.

[0031] Preferably, the drying temperature is 65-70℃ and the drying time is 5-12 hours.

[0032] The dimensions of the small cube can be listed as 1.0cm * 1.0cm.

[0033] Preferably, the volume ratio of the Au@Ag alloy spiked nanoparticle solution to the mixed solution is 1:(2.5~3.5).

[0034] Preferably, the volume ratio of anhydrous ethanol, n-hexane, and 1H,1H,2H,2H-perfluorodecylthiol in the mixed solution is (120-180):(300-350):1.

[0035] Preferably, the volume ratio of Au@Ag alloy spiked nanoparticle solution to anhydrous ethanol is 1:(0.9-1.2).

[0036] A fourth aspect of this invention provides a Raman detection method based on a patterned Au@Ag alloy spiked nanoparticle film, employing a bidirectional recognition sandwich structure for detection, and including the following detection steps:

[0037] The second-generation silver seed solution was mixed with 4-MPBA solution for 12-36 h to obtain Ag nanoparticle solution grafted with 4-MPBA.

[0038] Patterned Au@Ag alloy spiked nanoparticle membranes were immersed in a molecular solution containing carboxyl groups and allowed to stand for 5–20 h for grafting. The membranes were then removed, allowed to stand, and an activation solution was dropped onto the membrane surface to activate the carboxyl groups. The analyte was dissolved in a phosphate buffer solution with a pH of 7.0–8.0 and then dropped onto the membrane surface. Grafting was allowed to stand for 0.5–3 h, followed by rinsing. A solution of Ag nanoparticles grafted with 4-MPBA was dropped onto the membrane surface and incubated for 20–50 min. After rinsing, the membranes were allowed to air dry.

[0039] SERS detection of the analyte was performed using a Raman spectrometer.

[0040] Preferably, the concentration of the 4-MPBA solution is 0.5–5 mmol / L, and the 4-MPBA solution is prepared with sodium hydroxide solution and adjusted to a pH of 9–13.

[0041] Preferably, the volume ratio of Ag nanoparticle solution to 4-MPBA solution is (12-20):1.

[0042] Preferably, the molecule with a carboxyl group is 3-mercaptopropionic acid and / or 11-mercaptoundecanoic acid.

[0043] Preferably, the concentration of the carboxyl-containing molecular solution is 5–20 mmol / L.

[0044] Preferably, the activation solution is formed by dispersing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in a phosphate buffer solution with a pH of 7.0–8.0, wherein each 100 μL of the activation solution contains 3–7 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1–4 mg of N-hydroxysuccinimide. The activation time is 20–50 min.

[0045] Preferably, the analyte is one of dopamine, norepinephrine, 3,4-dihydroxyaniline, 4-(3-aminopropyl)benzene-1,2-diol, or 4,5-diaminocatechol.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] (1) This invention is the first to use dextrorotatory penicillamine solution to induce the generation of Au@Ag alloy spiky nanoparticles, which have a spiky structure and the particles have a large number of sharp points;

[0048] (2) The density and tip length of Au@Ag alloy spike nanoparticles can be controlled by changing the amount of second-generation silver seed solution, the amount of dextrorotatory penicillamine aqueous solution seed solution, and the incubation time.

[0049] (3) The Au@Ag alloy spiked nanoparticles of the present invention combine the advantages of multiple tips and alloy nanoparticles, thereby improving the detection sensitivity of the SERS substrate.

[0050] (4) In this invention, Au@Ag alloy spiked nanoparticles are prepared into patterned Au@Ag alloy spiked nanoparticle films. The patterning of nanoparticles can control the spacing between particles, thus ensuring the stability and reproducibility of Raman scattering signals.

[0051] (5) The present invention uses a sandwich structure based on patterned Au@Ag alloy spiked nanoparticle film for bidirectional recognition to detect the analyte. The sandwich structure with bidirectional recognition has double insurance performance to ensure binding to the target molecule and achieve specific and accurate detection of it.

[0052] (6) The present invention can achieve ultra-high sensitivity detection of dopamine, norepinephrine, 3,4-dihydroxyaniline, 4-(3-aminopropyl)benzene-1,2-diol and 4,5-diaminocatechol, and ensures the accuracy and stability of the detection. Attached Figure Description

[0053] Figure 1 Macroscopic color diagram of the second-generation silver seed solution prepared in Example 1;

[0054] Figure 2 SEM image of the silver nano-seeds in the second-generation silver seed solution prepared in Example 1;

[0055] Figure 3 The ultraviolet absorption spectrum of the nano-silver seeds in the second-generation silver seed solution prepared in Example 1;

[0056] Figure 4 SEM images of Au@Ag alloy nanoparticles with different incubation times in Examples 1-6;

[0057] Figure 5 SEM images of Au@Ag alloy nanoparticles with different amounts of D-Pen solution added in Examples 7-10;

[0058] Figure 6 SEM images of Au@Ag alloy nanoparticles with different amounts of second-generation silver seed solutions added in Examples 11-14;

[0059] Figure 7 The macroscopic morphology of the Au@Ag alloy nanoparticle monolayer film product in Example 15 is shown.

[0060] Figure 8 This is a high-magnification SEM image of the patterned Au@Ag alloy spiked nanoparticle film of Example 15;

[0061] Figure 9 This is a low-magnification SEM image of the patterned Au@Ag alloy spiked nanoparticle film of Example 15;

[0062] Figure 10 Comparison of macroscopic morphology of Au@Ag alloy nanoparticle monolayer film products obtained in Examples 15 and 16;

[0063] Figure 11 Raman spectra of different concentrations of dopamine involved in Example 17;

[0064] Figure 12 Raman spectra of different concentrations of dopamine involved in Comparative Example 1. Detailed Implementation

[0065] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the present invention. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used raw materials in the art, and the methods used in the embodiments are all conventional methods in the art.

[0066] The commercially available patterned silicon wafer used in the following embodiments was purchased from Suzhou Jiutao Sensing Technology Co., Ltd., model number: JTWX316.

[0067] Example 1

[0068] The preparation method of Au@Ag alloy spiked nanoparticles in this embodiment includes the following steps:

[0069] S1. Prepare 100 mL of an aqueous solution containing sodium citrate (5 mM) and tannic acid (0.1 mM), and stir for 15 min under water bath heating. After boiling, add 1 mL of silver nitrate (25 mM) to the solution. The solution immediately turns bright yellow, which is the first-generation silver seed solution. Extract 19.5 mL of the first-generation silver seed solution, dilute with 16.5 mL of water, set the oil bath temperature to 90℃, and add 500 μL of sodium citrate solution (25 mM) and 1.5 mL of tannic acid solution (2.5 mM) in sequence. After 1 min, add 1.0 mL of silver nitrate (25 mM) and react for 25 min to prepare the second-generation silver seed solution with a diameter of 37 nm.

[0070] The macroscopic color of the second-generation silver seed solution is as follows: Figure 1 As shown; SEM image of the nano-silver seeds is shown below. Figure 2 As shown; the ultraviolet absorption spectrum of the silver nanoseeds is as follows. Figure 3 As shown.

[0071] S2. The second-generation silver seed solution was added to D-Pen solution (0.1mM) and incubated to obtain a mixed solution; 0.1mL of 0.010 chloroauric acid solution, 0.8mL of 0.10M hexadecyltrimethylammonium bromide solution, 0.475mL of 0.10M ascorbic acid solution and 4mL of water were mixed to obtain a growth solution; the water bath temperature was set at 30℃, the mixed solution was introduced into the growth solution, and the mixture was grown for 2h to obtain Au@Ag alloy spiky nanoparticle solution.

[0072] The incubation time, the amount of second-generation silver seed solution added, and the amount of D-Pen solution added are shown in Table 1.

[0073] Example 2-14

[0074] The preparation methods of Examples 2-14 are the same as those of Example 1, except that the incubation time, the amount of second-generation silver seed solution added, and the amount of D-Pen solution added are different. The specific values ​​are shown in Table 1.

[0075]

[0076]

[0077] Examples 1-6 analyzed the effect of different incubation times on the structure of Au@Ag alloy nanoparticles. Figure 4SEM images of Au@Ag alloy nanoparticles with different incubation times are shown in Examples 1-6. When the incubation time is 20 min, the Au@Ag alloy nanoparticles exhibit excellent structure. Examples 7-10 analyzed the effect of different D-Pen solution addition amounts on the structure of the Au@Ag alloy nanoparticles. Figure 5 SEM images of Au@Ag alloy nanoparticles with different amounts of D-Pen solution added in Examples 7-10 are shown. When the amount of D-Pen solution added is 80 μL, the Au@Ag alloy nanoparticle structure is better. Examples 11-14 analyze the effect of the amount of second-generation silver seed solution added on the structure of Au@Ag alloy nanoparticles. Figure 6 The images show SEM images of Au@Ag alloy nanoparticles with different amounts of second-generation silver seed solution added in Examples 11-14. When the amount of second-generation silver seed solution added is 10 μL, the obtained Au@Ag alloy nanoparticles have a spiky structure with a large number of sharp points, which are complex and long.

[0078] Example 15

[0079] A method for preparing patterned Au@Ag alloy spiked nanoparticle films includes the following steps:

[0080] Take 8.0 mL of PDMS base agent (Dow Corning SYLGARD 184 base agent) and 0.8 mL of curing agent (Dow Corning SYLGARD 184 curing agent) and mix them. Stir quickly with a glass rod. Pour the mixture onto a commercially available patterned silicon wafer. After the bubbles disappear, place it in an oven at 70°C to dry. Remove the patterned silicon wafer to obtain the PDMS film. Cut the PDMS film into small squares of 1.0 cm * 1.0 cm using a blade.

[0081] A mixed solution containing 5 mL anhydrous ethanol, 10 mL n-hexane, and 0.032 mL 1H,1H,2H,2H-perfluorodecylthiol was prepared. 5 mL of the Au@Ag alloy nanoparticle solution prepared in Example 11 was added to a quartz beaker. The mixed solution was then rapidly poured into the quartz beaker and allowed to stand for 12 h until the upper n-hexane layer completely evaporated. The macroscopic morphology of the resulting Au@Ag alloy nanoparticle monolayer product is shown below. Figure 7 As shown;

[0082] Take a 1mL pipette tip and attach double-sided tape to it. Using tweezers, gently place the unpatterned side of a PDMS cube onto the tape. Invert the pipette tip so that the patterned side of the PDMS cube is in contact with the liquid surface in the beaker at a 45° angle. Then, slowly bring the PDMS cube parallel to the liquid surface in the beaker until it is in full contact. After standing for 5 seconds, the Au@Ag alloy spiked nanoparticles will be transferred onto the PDMS cube, obtaining a patterned Au@Ag alloy spiked nanoparticle film. The SEM image of the patterned Au@Ag alloy spiked nanoparticle film is shown below. Figure 8 and Figure 9 As shown, Figure 8 The distance between the nanoparticles is approximately 3 μm.

[0083] Example 16

[0084] The difference between the preparation methods of Example 16 and Example 15 is that the mixed solution contains 10 mL of anhydrous ethanol, 10 mL of n-hexane, and 0.032 mL of 1H,1H,2H,2H-perfluorodecylthiol, while the rest is the same as in Example 15. Figure 10 The images show a comparison of the macroscopic morphology of the Au@Ag alloy nanoparticle monolayer films obtained in Examples 15 and 16. When the volume of anhydrous ethanol is increased to 10 ml, excessive amounts of anhydrous ethanol can lead to lower film density and make the film more prone to disintegration.

[0085] Example 17

[0086] A Raman detection method for dopamine based on patterned Au@Ag alloy spiked nanoparticle films includes the following steps:

[0087] Take 1.5 mL of the second-generation silver seed solution prepared in Example 11 and mix it thoroughly with 100 μL of 1 mM 4-MPBA solution (pH 11). Mix for 24 h to obtain a 4-MPBA grafted Ag nanoparticle solution.

[0088] The patterned Au@Ag alloy spiked nanoparticle membrane prepared in Example 15 was immersed in 3 mL of 10 mM MPA solution and allowed to stand for 12 h. The membrane was then removed, and 12 μL of LEDC and 4 mg of NHS were dispersed in 100 μL of phosphate buffer (pH = 6.8), respectively. 30 μL of each was taken, thoroughly mixed, and dropped onto the membrane surface to activate the carboxyl groups. The activation time was 30 min, and the membrane was rinsed after activation. Dopamine of different concentrations was dissolved in phosphate buffer (pH = 7.2), and 100 μL of each was dropped onto the activated membrane surface. The membrane was allowed to stand for 1 h for grafting and then rinsed. Then, 100 μL of Ag nanoparticles grafted with 4-MPBA were dropped onto the membrane surface and incubated for 40 min. After rinsing, the membrane was allowed to air dry.

[0089] At an excitation wavelength of 785 nm, with a detection power of 50 mW and an integration time of 30,000 ms, dopamine was detected by SERS using a Raman instrument.

[0090] Figure 11 The images show Raman spectra of dopamine at different concentrations. As can be seen from the figures, a significant Raman signal can be obtained using a sandwich structure with bidirectional recognition constructed from patterned Au@Ag alloy spiked nanoparticle films.

[0091] Comparative Example 1

[0092] Comparative Example 1: Raman detection method for dopamine based on patterned Au@Ag alloy spiked nanoparticle film, comprising the following steps:

[0093] Take 5 mL of the second-generation silver seed solution prepared in Example 11 and mix it thoroughly with 500 μL of 10 mM MPA solution. Mix for 12 h to obtain an MPA-grafted Ag nanoparticle solution.

[0094] 1.2 μL of EDC and 0.4 mg of NHS were dispersed in 10 μL of phosphate buffer (pH = 6.8), respectively. 800 μL of MPA-grafted Ag nanoparticle solution was added, and the mixture was thoroughly mixed and allowed to stand for 30 min. Then, 50 μL of the above solution was mixed with 50 μL of dopamine solutions of different concentrations (dissolved in pH 7.2 phosphate buffer) and allowed to stand for 12 h to obtain a mixed dopamine-grafted MPA Ag nanoparticle solution.

[0095] The patterned Au@Ag alloy spiked nanoparticle membrane prepared in Example 15 was immersed in 3 mL of 1 mM 4-MPBA solution (pH 11) and allowed to stand for 24 h. The membrane was then removed, and 100 μL of a solution of Ag nanoparticles grafted with dopamine and mixed with MPA was dropped onto the membrane surface. After incubation for 40 min, the membrane was rinsed and air-dried.

[0096] At an excitation wavelength of 785 nm, with a detection power of 50 mW and an integration time of 30,000 ms, dopamine was detected by SERS using a Raman instrument.

[0097] Figure 12 The images show the Raman spectra of dopamine at different concentrations in Comparative Example 1. As can be seen from the figures, the Raman signal is very low or nonexistent after reverse assembly of the sandwich structure based on the patterned Au@Ag alloy spiky nanoparticle film for bidirectional recognition.

[0098] Finally, it should be noted that the specific embodiments described herein are merely illustrative of the spirit of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A Raman detection method based on patterned Au@Ag alloy spiked nanoparticle films, characterized in that, The detection process employs a sandwich structure with bidirectional recognition, and includes the following steps: Boil the first reducing agent solution, add the first silver nitrate solution to obtain the first generation silver seed solution; take a portion of the first generation silver seed solution, dilute with water, heat, add the second reducing agent solution, add the second silver nitrate solution after 1-3 minutes, react for 15-50 minutes to obtain the second generation silver seed solution. The second-generation silver seed solution was added to the dextrorotatory penicillamine aqueous solution and incubated to obtain a mixed solution; A growth solution is obtained by mixing chloroauric acid solution, surfactant solution, third reducing agent solution and water; the mixture is added to the growth solution and grown at 28~32℃ for 1~3h to obtain Au@Ag alloy spiky nanoparticle solution. The second-generation silver seed solution was mixed with 4-MPBA solution for 12-36 h to obtain Ag nanoparticle solution grafted with 4-MPBA. Patterned Au@Ag alloy spiked nanoparticle membranes, formed by transferring Au@Ag alloy spiked nanoparticles onto PDMS membranes, were immersed in a molecular solution containing carboxyl groups and allowed to stand for 5–20 h for grafting. The membranes were then removed, allowed to stand, and an activation solution was dropped onto the membrane surface to activate the carboxyl groups. The analyte was dissolved in a phosphate buffer solution at pH 7.0–8.0 and then dropped onto the membrane surface. Grafting was allowed to stand for 0.5–3 h, followed by rinsing. A solution of Ag nanoparticles grafted with 4-MPBA was dropped onto the membrane surface and incubated for 20–50 min. After rinsing, the membranes were allowed to air dry. SERS detection of the analyte was performed using a Raman spectrometer.

2. The Raman detection method according to claim 1, characterized in that, The first reducing agent, the second reducing agent, and the third reducing agent are each individually selected from one or more of sodium citrate, tannic acid, and ascorbic acid.

3. The Raman detection method according to claim 1, characterized in that, The concentrations of the first and second silver nitrate solutions are 10~50 mmol / L; The concentration of dextrorotatory penicillamine aqueous solution is 0.05~0.5 mmol / L; The concentration of the chloroauric acid solution is 0.008~0.012 mol / L; The concentration of the surfactant solution is 0.08~0.12 mol / L; The concentration of the third reducing agent solution is 0.08~0.12 mol / L.

4. The Raman detection method according to claim 2, characterized in that, When the first reducing agent is a mixture of sodium citrate and tannic acid, the molar ratio of sodium citrate, tannic acid and silver nitrate is (40~80):1:(2~4). When the second reducing agent is a mixture of sodium citrate and tannic acid, the molar ratio of sodium citrate, tannic acid and second silver nitrate is (2~5):1:(5~8).

5. The Raman detection method according to claim 1, characterized in that, The second-generation silver seed solution was added to a dextrorotatory penicillamine aqueous solution and incubated to obtain a mixed solution. The volume of the second-generation silver seed solution added was 10~70μL. The volume of the dextrorotatory penicillamine aqueous solution is 40~100μL.

6. The Raman detection method according to claim 5, characterized in that, The volume of the second-generation silver seed solution added was 10 μL; The volume of the dextrorotatory penicillamine aqueous solution is 80 μL.

7. The Raman detection method according to claim 1, characterized in that, The second-generation silver seed solution was added to the dextrorotatory penicillamine aqueous solution and incubated at 28-32℃ for 10-60 min to obtain a mixed solution.

8. The Raman detection method according to claim 1, characterized in that, The volume ratio of chloroauric acid solution, surfactant solution, third reducing agent solution and water is 1:(6~10):(4~6):(35~45).

9. The Raman detection method according to claim 1, characterized in that, The preparation method of patterned Au@Ag alloy spiked nanoparticle film includes the following steps: Mix the PDMS base agent and curing agent evenly, pour the mixture onto the patterned silicon wafer, wait for the bubbles to disappear, dry the wafer, remove the patterned silicon wafer, and obtain the PDMS film, which is then cut into small squares. Add the Au@Ag alloy spiked nanoparticle solution to a beaker, then inject a mixed solution containing anhydrous ethanol, n-hexane, and 1H,1H,2H,2H-perfluorodecylthiol into the beaker and let it stand for 10~24h. The patterned side of the PDMS cube is placed in contact with the liquid surface of the beaker and left to stand for 3-6 seconds. Au@Ag alloy spiky nanoparticles are transferred onto the PDMS cube to obtain a patterned Au@Ag alloy spiky nanoparticle film.

10. The Raman detection method according to claim 9, characterized in that, In the mixed solution, the volume ratio of anhydrous ethanol, n-hexane, and 1H,1H,2H,2H-perfluorodecylthiol is (120~180):(300~350):

1.

11. The Raman detection method according to claim 9, characterized in that, The volume ratio of Au@Ag alloy spiked nanoparticle solution to mixed solution is 1:(2.5~3.5). The volume ratio of Au@Ag alloy spiked nanoparticle solution to anhydrous ethanol is 1:(0.9~1.2).

12. The Raman detection method according to claim 1, characterized in that, The concentration of 4-MPBA solution is 0.5~5 mmol / L; The concentration of the solution containing carboxyl groups is 5~20 mmol / L; The activation solution is formed by dispersing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in a phosphate buffer solution with a pH of 7.0-8.

0. Each 100 μL of activation solution contains 3-7 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-4 mg of N-hydroxysuccinimide.

13. The Raman detection method according to claim 1, characterized in that, The 4-MPBA solution was prepared with sodium hydroxide solution and adjusted to a pH of 9-13.

14. The Raman detection method according to claim 1, characterized in that, The volume ratio of the second-generation silver seed solution to the 4-MPBA solution is (12~20):

1.

15. The Raman detection method according to claim 1, characterized in that, The molecules with carboxyl groups are 3-mercaptopropionic acid and / or 11-mercaptoundecanoic acid.

16. The Raman detection method according to claim 1, characterized in that, The analyte is one of dopamine, norepinephrine, 3,4-dihydroxyaniline, 4-(3-aminopropyl)benzene-1,2-diol, or 4,5-diaminocatechol.

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