A method for rapid detection of phenobarbital using dynamic SERS

Abstract

The present invention provides a method for rapidly detecting phenobarbital by using dynamic SERS, comprising the following steps: uniformly mixing a reducing agent and a silver salt solution, stirring the reaction under boiling conditions, cooling to room temperature after the reaction is completed, and obtaining a silver sol; centrifuging the silver sol, removing the supernatant, obtaining a centrifugal product, concentrating the centrifugal product, and ultrasonically dispersing it to obtain an enhanced substrate; using a solution containing phenobarbital as a sample to be tested, uniformly mixing the enhanced substrate, a coagulant and the sample to be tested, adding them to a hydrophilic flexible substrate, and then performing dynamic SERS detection; the hydrophilic flexible substrate is a nylon microporous filter membrane. The present invention replaces a hydrophobic aluminum foil substrate with a hydrophilic flexible substrate, and assembles the sample to be tested mixed with silver sol nanoparticles on a nylon microporous filter membrane by an injection method, thereby realizing rapid sensitivity optimization detection of a phenobarbital aqueous solution, and solving the problem of low sensitivity of the existing SERS in non-dynamic detection of phenobarbital on a sol substrate.

Description

Technical Field

[0001] The present invention relates to the technical field of psychotropic and anesthetic drug detection, and in particular to a method for rapidly detecting phenobarbital by utilizing dynamic SERS. Background Art

[0002] Phenobarbital is a Class II psychotropic drug with hypnotic and sedative effects. Due to its small safe dosage range, long-term abuse will produce many neurological side effects. Long-term abuse is also prone to toxic side effects. Moreover, this drug is incompatible with other drugs. Therefore, it is necessary to strictly regulate such drugs to control their use.

[0003] Currently, various detection and identification technologies for drugs and narcotics are widely used, such as chemical color testing, gas chromatography and high-performance liquid chromatography, as well as ultraviolet-visible spectrophotometry, nuclear magnetic resonance, and mass spectrometry combined with gas chromatography or high-performance liquid chromatography. However, these methods have complex and time-consuming sample preparation processes and are only suitable for laboratory testing, making it difficult to achieve large-scale screening.

[0004] Surface-Enhanced Raman Spectroscopy (SERS) is a technique that offers qualitative detection advantages over other techniques, including simplified pre-processing, simple operation, and rapid detection times. Due to its molecular specificity, SERS can potentially resolve mixtures into individual components, enabling ultra-trace analysis. Therefore, SERS is likely to become a viable method for identifying illicit drugs in complex systems, making its sensitivity crucial.

[0005] SERS detection states are mainly divided into dry state detection and wet state detection. Dry state detection is performed on the sample after it is dried. As the sample is enriched and concentrated, the sensitivity of dry state detection increases accordingly. However, conventional drying time prolongs the detection time, and the coffee ring effect generated by the substrate platform during the drying process makes the sample distribution uneven, thus affecting the stability of SERS detection. Wet state detection is performed on the sample before it is dried. Conventional wet state detection can achieve better detection uniformity and stability, but it is not conducive to rapid detection.

[0006] All of the above tests are static tests in solution or solid states. When the sample to be tested is in a sol state, the state of the sol continuously changes during the test, which is a dynamic process. Dynamic testing methods speed up the test time based on conventional wet-state testing, but this affects the test stability. Summary of the Invention

[0007] In order to solve the problem of low sensitivity of existing SERS in non-dynamic detection of phenobarbital on a sol substrate, the present invention aims to provide a method for rapid detection of phenobarbital using dynamic SERS.

[0008] To achieve the above objectives, the technical solutions of the present invention are as follows.

[0009] The present invention provides a method for rapidly detecting phenobarbital using dynamic SERS, comprising the following steps:

[0010] The reducing agent and the silver salt solution are mixed evenly, and then stirred under boiling conditions for reaction. After the reaction is completed, the solution is cooled to room temperature to obtain a silver sol; the silver sol is centrifuged, and the supernatant is removed to obtain a centrifuged product, which is concentrated and ultrasonically dispersed to obtain an enhanced substrate; a solution containing phenobarbital is used as a test sample, and the enhanced substrate, a coagulant and the test sample are mixed evenly, added to a hydrophilic flexible substrate, and then dynamic SERS detection is performed; the hydrophilic flexible substrate is a nylon microporous filter membrane.

[0011] The present invention primarily replaces hydrophobic aluminum foil substrates with hydrophilic flexible substrates to optimize the sensitivity of detecting the psychotropic drug phenobarbital. Through physical characterization and SERS performance testing of different hydrophilic flexible substrates, the retention mechanisms of different substrates were analyzed and studied. A comprehensive comparison of the test results revealed that nylon substrates with deep filtration and retention of silver nanoparticles exhibited the highest dynamic SERS performance enhancement factor and minimal background interference. When testing the target sample phenobarbital, compared to conventional SERS detection, the method of the present invention reduced the detection limit from 12 mg / L to 0.1 mg / L, and increased the enhancement factor by 21 times. This method addresses the low sensitivity of existing SERS methods for non-dynamic detection of phenobarbital on sol substrates.

[0012] Preferably, the temperature of the boiling condition is about 100° C. The purpose is to keep the reaction in a boiling state.

[0013] Preferably, the coagulant is an inorganic alkaline solution.

[0014] Preferably, the concentration of the inorganic alkali solution is 1 mol / L.

[0015] Preferably, the inorganic alkali solution is an aqueous sodium hydroxide solution.

[0016] Preferably, the volume ratio of the reinforcing substrate, the coagulant and the sample to be tested is 1:1:1; the concentration of phenobarbital in the sample to be tested is 0.1 mg / L to 0.3 mg / L; for example, 0.1 mg / L, 0.12 mg / L, 0.2 mg / L, 0.3 mg / L, etc.

[0017] Preferably, the reducing agent is an aqueous solution of sodium citrate; and the silver salt solution is an aqueous solution of silver nitrate.

[0018] Preferably, the concentration of the sodium citrate aqueous solution is 0.2 mg / mL; the concentration of the silver nitrate aqueous solution is 0.18 mg / mL; and the volume ratio of the sodium citrate aqueous solution to the silver nitrate aqueous solution is 1:50.

[0019] Preferably, the dynamic SERS detection conditions are a laser power of 120 mW and an integration time of 5 s. In the present invention, a hydrophilic flexible substrate is used instead of a hydrophobic aluminum foil substrate, and a sample to be tested is mixed with silver sol nanoparticles and assembled on a nylon microporous filter membrane using an injection method, thereby achieving rapid sensitivity-optimized detection of phenobarbital aqueous solution.

[0020] Preferably, the sample to be tested is an aqueous solution of sodium phenobarbital.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention uses a nylon microporous filter membrane with deep filtration and retention capabilities for silver nanoparticles as a hydrophilic flexible substrate, replacing the existing hydrophobic aluminum foil substrate. The sample to be tested and mixed silver sol nanoparticles are assembled on the nylon microporous filter membrane. A coagulant is used to change the dispersion state of the silver sol nanoparticles, thereby generating more SERS hotspots and achieving a better enhancement effect. The method of the present invention can be used for dynamic SERS detection of the psychotropic drug phenobarbital, which can improve detection sensitivity and reduce background interference.

[0023] 2. The present invention detects the target sample phenobarbital. Compared with conventional non-dynamic SERS detection, the detection limit is 0.1 mg / L and the enhancement factor is increased by 21 times; the method of the present invention is expected to be used for trace detection of such drugs.

[0024] 3. The present invention replaces the hydrophobic aluminum foil substrate with a hydrophilic flexible substrate and utilizes the injection method to assemble the sample to be tested mixed with silver sol nanoparticles onto a nylon microporous filter membrane, thereby achieving rapid sensitivity optimization detection of aqueous solutions containing phenobarbital. This is mainly because the silver aggregate detection sample has good signal stability and can serve as the basis for quantitative detection.

[0025] 4. The present invention greatly shortens the time required for conventional dynamic SERS detection to achieve effective enhancement, reducing it from nearly 30 minutes to less than 2 minutes, thereby improving time efficiency and being suitable for rapid and sensitive preliminary screening of illicit drugs of abuse. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The molecular structure of phenobarbital is shown in Figure 2. (a) is a two-dimensional molecular structure diagram, and (b) is a three-dimensional spatial structure diagram.

[0027] Figure 2 Comparison of the measured and calculated Raman spectra of phenobarbital. (a) is the calculated Raman spectrum curve; (b) is the measured Raman spectrum curve of phenobarbital.

[0028] Figure 3 This is a comparison curve of the local electric field intensity between silver nanospheres with different particle size combinations as the spacing changes.

[0029] Figure 4 This is a scanning electron microscope characterization image of the silver sol substrate prepared in Example 1.

[0030] Figure 5 This is a diagram showing the non-dynamic SERS enhancement effect of different coagulants on sodium phenobarbital aqueous solution.

[0031] Figure 6 This is a graph showing the relationship between the non-dynamic phenobarbital concentration of 12 mg / L to 100 mg / L and the Raman spectrum.

[0032] Figure 7 Schematic diagram of the dynamic detection injection method.

[0033] Figure 8 The following are the dynamic SERS spectra of phenobarbital at different concentrations and the fitting curve of the characteristic peak intensity changing with concentration. Among them, (a) is the dynamic SERS spectra of phenobarbital at different concentrations; (b) is the fitting curve of the characteristic peak intensity changing with concentration. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0036] Dynamic surface-enhanced Raman spectroscopy, or D-SERS, refers to the dynamic nature of the sol, which continuously changes state during the detection process. This dynamic detection method involves Raman acquisition throughout the entire transition from a wet solution to a dry solid. Currently, solution samples are typically tested on a hydrophobic substrate. As the solvent evaporates, the molecules being tested are concentrated, and during this evaporation process, the concentration of the molecules at the laser focus increases.

[0037] Achieving high sensitivity in dynamic SERS detection requires both preparing high-performance SERS substrates and selecting suitable substrates. Most SERS substrates have a sharp, densely packed shape or are densely packed in an orderly fashion, increasing the number of "hotspots" per unit volume. However, the preparation of these SERS substrates is relatively complex, and the detection results may be specific to certain molecules, making them inapplicable to all detection targets.

[0038] In response to the demand for trace drug detection, the present invention proposes a rapid dynamic SERS detection method for detecting phenobarbital, which replaces the hydrophobic aluminum foil substrate with a hydrophilic flexible substrate to optimize the detection sensitivity of the psychotropic drug phenobarbital. By performing physical characterization and SERS performance testing on different types of hydrophilic flexible precipitates, such as filter paper, mixed cellulose esters, polyethersulfone, nylon, etc., and analyzing the interception mechanism of different substrates, a comprehensive comparison of the test results found that the nylon substrate with the ability to deeply filter and intercept silver nanoparticles had the highest enhancement factor of dynamic SERS performance and less background interference. When the target sample phenobarbital was detected, compared with conventional SERS detection, the detection limit of the method of the present invention was reduced from 12mg / L to 0.1mg / L, and the enhancement factor was increased by 21 times.

[0039] The present invention conducts Raman spectrum attribution analysis research on phenobarbital and conducts electric field simulation test between silver nanosphere dimers. The specific research results are as follows:

[0040] (1) Raman spectral analysis of phenobarbital.

[0041] The Raman spectrum of phenobarbital was analyzed and the molecular structure of phenobarbital was optimized using density functional theory. Figure 1 Then, the drug molecules were quantized and the Raman spectrum of phenobarbital was obtained by simulation. The theoretical calculated spectrum was compared with the actual solid Raman spectrum. Figure 2 The main characteristic peaks were then assigned based on the solid Raman spectrum, as shown in Table 1, where the correction factor was 0.985. This laid a theoretical foundation for further qualitative and quantitative analysis of phenobarbital using SERS spectroscopy.

[0042] Table 1 Correspondence and peak position assignment of phenobarbital characteristic peaks

[0043]

[0044] Note: 6311++G(d,p) is the frequency parameter corresponding to the Raman spectral peak obtained by simulating the basis set parameter settings of the existing researchers for the basis set of similar molecular structures of substances as experimental reference; Solid is the frequency parameter corresponding to the Raman spectral peak obtained by the actual detection of solid phenobarbital samples.

[0045] The Chinese name for N-heterocycle is nitrogen heterocycle. The Chinese name for benzene ring breathing is the vibration mode of the benzene ring. The Chinese name for rocking is rocking vibration. The Chinese name for scissoring is shear vibration.

[0046] From the above-mentioned Raman spectrum attribution analysis results of phenobarbital, it can be seen that phenobarbital corresponds to the strongest 640cm-1 solid Raman spectrum. -1 Position, calculated value is 615cm -1 The Raman characteristic peak near this position can be used as the basis for qualitative and quantitative analysis of the sample.

[0047] (2) Electric field simulation experiment between silver nanosphere dimers.

[0048] The FDTD method was used to simulate the electromagnetic field distribution between silver nanoparticle dimers of different sizes. Under the excitation of a light source with a wavelength of 785nm, the dielectric constant of the silver nanoparticles is 26.82. The polarization direction of the incident laser electric field is perpendicular to the long axis of the dimer. Since the shape of silver nanoparticles is quasi-spherical and the particle size is mainly distributed between 50nm and 80nm, silver nanospheres are used as the research object, and the dimer model size covers 3 diameter particle size settings: 50nm~60nm, 60nm~70nm, and 50nm~70nm. The distance between them varies from 1 to 9nm, with an interval of 1nm. The statistical results of the change of EM intensity with the distance between dimer silver nanospheres are shown as follows. Figure 3 shown.

[0049] Depend on Figure 3 The results show that as the diameter of the silver nanospheres gradually increases, the electromagnetic field distribution in the gap also gradually increases. When the silver nanospheres approach each other, the electric field between the silver nanospheres slowly increases. When the distance between the silver nanospheres is less than 3nm, the enhancement changes in a step-like manner, which also confirms the excellent sensitivity of the dynamic SERS substrate.

[0050] The electric field strength between the 50nm-60nm dimer silver nanospheres was slightly lower than that between the 50nm-70nm dimer silver nanospheres, and the electric field strength between the 50nm-70nm dimer silver nanospheres was slightly lower than that between the 60nm-70nm dimer silver nanospheres, which also proves that the larger the silver nanosphere size, the greater the influence on the electric field strength between nanoparticles. This shows that under the condition of the same distance between the dimer silver nanospheres, the larger silver nanospheres exhibit a stronger electric field strength distribution; as the distance between them continues to decrease, the electric field strength continues to increase, showing a gradual change at farther distances and a sharp change at closer distances, providing a theoretical basis for the subsequent optimization of Raman detection sensitivity.

[0051] The technical solution of the present invention is further described below through specific embodiments.

[0052] In the following embodiments of the present invention, the experimental reagents and main supplies are as follows:

[0053] Filter paper, medium speed, 12.5 cm diameter, 15–20 μm pore size, purchased from Hangzhou Specialty Paper Co., Ltd. Nylon microporous filter membrane, 0.1 μm pore size, 47 mm diameter, purchased from Filter Doctor Store and custom-made. Mixed cellulose ester, mixed fiber, 0.15 μm pore size, 13 mm diameter, purchased from Shanghai Xinya. Polyethersulfone microporous filter membrane, 0.22 μm pore size, 47 mm diameter, purchased from Beijing Inokai Technology Co., Ltd.

[0054] Potassium iodide and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (analytical grade). Hydrochloric acid was purchased from Xilong Chemical Co., Ltd. (analytical grade). A replaceable membrane syringe filter (13 mm diameter) and a 1 mL filter cartridge were purchased from Shanghai Puyi Biotechnology Co., Ltd. Rhodamine 6G (R6G) was purchased from Beijing Inokai Technology Co., Ltd. Phenobarbital sodium injection (0.1 mg / mL) was purchased from Shanghai Xinya Pharmaceutical Co., Ltd. Ultrapure water was used.

[0055] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods; the reagents and materials described are all commercially available unless otherwise specified.

[0056] The method for detecting phenobarbital using conventional non-dynamic SERS is as follows.

[0057] Example 1

[0058] A method for detecting phenobarbital using conventional non-dynamic SERS comprises the following steps:

[0059] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0060] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0061] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0062] S3. Conventional non-dynamic SERS detection: Using 1 mol / L sodium hydroxide aqueous solution as a coagulant, 100 mg / mL sodium phenobarbital aqueous solution, silver sol enhanced substrate, and 1 mol / L coagulant were mixed in a volume ratio of 1:1:1, and then 10 μL was dropped onto the aluminum foil substrate. Non-dynamic SERS detection was then performed under the following conditions: laser power of 120 mW and integration time of 5 s.

[0063] Example 2

[0064] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that 1 mol / L hydrochloric acid is used as a coagulant. The specific method includes the following steps:

[0065] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0066] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0067] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0068] S3. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 100 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0069] Example 3

[0070] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 1 mol / L aqueous solution of KI is used as a coagulant. The specific method includes the following steps:

[0071] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0072] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0073] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0074] S3. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 100 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0075] Example 4

[0076] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 12 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0077] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0078] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0079] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0080] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0081] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 12 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0082] Example 5

[0083] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 16 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0084] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0085] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0086] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0087] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0088] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 16 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0089] Example 6

[0090] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 20 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0091] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0092] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0093] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0094] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0095] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 20 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0096] Example 7

[0097] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 30 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0098] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0099] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0100] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0101] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0102] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 30 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0103] Example 8

[0104] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 50 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0105] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0106] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0107] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0108] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0109] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 60 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0110] Example 9

[0111] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 60 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0112] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0113] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0114] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0115] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0116] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 60 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0117] Example 10

[0118] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 70 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0119] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0120] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0121] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0122] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0123] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 70 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0124] Example 11

[0125] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that an 80 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0126] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0127] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0128] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0129] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0130] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, 80 mg / mL sodium phenobarbital aqueous solution, silver sol-enhanced substrate, and 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto the aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0131] Example 12

[0132] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 90 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0133] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0134] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0135] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0136] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0137] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 90 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0138] Example 13

[0139] A method for detecting phenobarbital using conventional non-dynamic SERS is different from Example 1 in that a 100 mg / mL aqueous solution of phenobarbital sodium is used as the test sample. The specific method includes the following steps:

[0140] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0141] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0142] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0143] S3. Prepare an aqueous solution of phenobarbital sodium as the sample to be tested: take 0.1 mg / mL phenobarbital sodium injection as the standard solution and dissolve it in ultrapure water for dilution to prepare solutions with gradient concentrations of 12 mg / L, 16 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, and 100 mg / L, respectively.

[0144] S4. Non-dynamic SERS detection: Using 1 mol / L hydrochloric acid as a coagulant, a 100 mg / mL sodium phenobarbital aqueous solution, a silver sol-enhanced substrate, and a 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then, 10 μL was dropped onto an aluminum foil substrate, and non-dynamic SERS detection was performed. The detection conditions were: laser power of 120 mW, and integration time of 5 s.

[0145] The following is a further description of the dynamic SERS method for detecting phenobarbital and a comparison with the non-dynamic SERS method for detecting phenobarbital. Figure 7 .

[0146] Example 14

[0147] A method for rapid detection of phenobarbital using dynamic SERS, using a 0.1 mg / mL aqueous solution of phenobarbital sodium as a test sample, specifically comprising the following steps:

[0148] S1. Preparation of silver sol: silver nitrate is used as the oxidant and sodium citrate is used as the reducing agent. The specific method is as follows:

[0149] Under boiling conditions, add 5 mL of 10 mg / mL sodium citrate to 250 mL of 0.18 mg / mL silver nitrate solution and keep boiling for 1 hour until the final color turns gray-green. Protect from light, cool, and refrigerate.

[0150] S2. The silver sol is centrifuged and purified to remove nanoparticles with smaller particle sizes and improve the concentration of the silver nanoparticle size distribution. The supernatant is removed, and the centrifuged product is concentrated and dispersed by ultrasonic oscillation for 3 to 5 minutes to obtain an enhanced substrate.

[0151] S3. Prepare an aqueous solution of sodium phenobarbital as the test sample: take 20 μL of 0.1 mg / mL sodium phenobarbital injection, dilute to volume with ultrapure water, prepare a standard aqueous solution with a concentration of 20 mg / L, and then dilute the standard aqueous solution to 0.1 mg / mL with ultrapure water.

[0152] S4. Dynamic SERS detection: 1 mol / L sodium hydroxide aqueous solution was used as a coagulant and a nylon microporous filter membrane was used as a hydrophilic flexible substrate. 0.1 mg / mL sodium phenobarbital aqueous solution, silver sol enhanced substrate and 1 mol / L coagulant were mixed in a volume ratio of 1:1:1. Then 1 mL was injected into the hydrophilic flexible substrate. Dynamic SERS detection was performed immediately after injection. Ten random detections were performed at different positions. The obtained 666 cm -1 The highest intensity was used as the single-piece detection result, and the average of three pieces was used as the final concentration detection result. The detection conditions were: laser power of 120mW and integration time of 5s.

[0153] Example 15

[0154] A method for rapid detection of phenobarbital using dynamic SERS is different from Example 14 in that a 0.12 mg / mL aqueous solution of phenobarbital sodium is used as the sample to be tested.

[0155] Example 16

[0156] A method for rapid detection of phenobarbital using dynamic SERS is different from Example 14 in that a 0.2 mg / mL aqueous solution of phenobarbital sodium is used as the sample to be tested.

[0157] Example 17

[0158] A method for rapid detection of phenobarbital using dynamic SERS is different from Example 14 in that a 0.3 mg / mL aqueous solution of phenobarbital sodium is used as the sample to be tested.

[0159] Test 1: SEM analysis and UV absorption spectrum analysis of silver sol substrate.

[0160] The scanning electron microscope characterization of the silver sol substrate prepared in Example 1 is shown in FIG. Figure 4 .Depend on Figure 4 The scanning electron microscope characterization image shows the shape of silver nanoparticles, which are basically spherical and rarely mixed with substrates of other shapes.

[0161] Test 2: Analysis of the SERS enhancement effect of different coagulants on sodium phenobarbital aqueous solution.

[0162] The non-dynamic SERS enhancement effect of different coagulants on phenobarbital sodium aqueous solution is analyzed in Figure 5 The non-dynamic SERS results show that the SERS spectrum of Example 2, which was accelerated by HCl, shows almost no phenobarbital peaks, while the sample of Example 3, which was accelerated by KI, shows some characteristic peaks with weak peak intensities. In comparison, the spectrum of Example 1, which was accelerated by NaOH, shows not only three characteristic peaks, but also relatively high peak intensities. This shows that using NaOH as a coagulant can change the dispersion state of the nanoparticles in the sol, resulting in more SERS hotspots and better enhancement effects.

[0163] Test 3: Stability test.

[0164] Stability tests were performed according to the methods of Examples 6, 8, and 11. The silver sols were prepared from the same batch and used within 24 hours. The concentrations of the phenobarbital sodium aqueous solution were 20, 50, and 80 mg / L, respectively. Each sample was sampled 15 times. The data after the test were analyzed for fluctuation degree. RSD was used as the standard for measuring the data to conduct the phenobarbital SERS stability test.

[0165] According to the phenobarbital SERS stability test results, the deviation of the characteristic peak intensity in the three groups of sample data is basically within the floating range of 5%, indicating that the silver aggregate detection sample has good signal stability and can be used as the basis for quantitative detection.

[0166] Test 4: Quantitative detection and recovery analysis.

[0167] The relationship between the concentration of phenobarbital 12mg / L to 100mg / L and Raman spectrum was analyzed. Figure 6 . Figure 6 The results showed that as the concentration gradually decreased, the main characteristic peak at 666 cm -1 The peak width of the spectrum is continuously widening, and it forms a wave packet shape at 12 mg / L, which is not conducive to the confirmation of the spectrum peak. -1 The intensity of the satellite peak is approximately three times that of the background noise. Taking the above factors into consideration, 12 mg / L is used as the minimum concentration for identifying phenobarbital under the non-dynamic SERS experimental conditions.

[0168] The concentration of phenobarbital was fitted with the characteristic peak intensity. The results showed that: y = 84.896x + 1582.6, and the concentration and intensity were linearly related. In the fitting equation, y is the peak intensity at 666 cm in the surface enhanced Raman scattering spectrum of phenobarbital. -1The Raman peak intensity at is where x is the concentration of the phenobarbital aqueous solution. To ensure data reliability, each spectrum was acquired five times and averaged, with a fitting coefficient of 0.995.

[0169] The recovery rate was detected and analyzed according to the method of Examples 5, 9 and 12, as shown in Table 2.

[0170] Table 2 Recovery test results

[0171]

[0172]

[0173] Three concentrations within the detection range (high, medium, and low) were selected as test targets. Peak intensity results were substituted into the fitting formula as the variable y, and the concentration variable x was calculated by reverse engineering. The calculated results were then compared with the initial concentration to provide the recovery rate. Three tests were performed at each concentration. The calculated concentrations shown in Table 2 were very close to the initial concentrations, with relative standard deviations (RSDs) ranging from 3.8% to 8.7% and recoveries ranging from 91.7% to 109.2%. Under these experimental conditions, the test demonstrated good reliability and stability.

[0174] Test 5: Dynamic SERS detection and analysis of phenobarbital.

[0175] The detection spectra of dynamic SERS detection of phenobarbital at different concentrations are as follows: Figure 8 Figure (a) will detect the main characteristic peak 666cm at several concentrations. -1 The peak intensity is analyzed by linear fitting. Figure 8 Figure (b) shows that R 2 The factor is 0.568.

[0176] like Figure 8 The results of 0.15 mg / L and 0.25 mg / L were selected as the dependent variable and substituted into the fitting formula y = 94.90 + 2491.05x to determine the independent variable, concentration x. The recoveries for the corresponding concentrations were calculated. The RSDs for 0.15 mg / L and 0.25 mg / L were 15.8% and 22.2%, respectively.

[0177] Comparison of the enhancement effect of non-dynamic SERS detection and dynamic SERS detection of phenobarbital. -1 After the addition of NaOH, the Raman peak shifted to 666 cm -1 The enhancement factor of the non-dynamic SERS detection at a concentration of 12 mg / L is 2×10 3 The enhancement factor EF of dynamic SERS at a concentration of 0.12 mg / L is 4.2×10 4When the concentration is reduced by one order of magnitude, the enhancement factor of dynamic SERS detection is increased by 21 times. This promotes the optimization of the sensitivity of phenobarbital SERS detection, and only from the single main peak 666cm -1 For the analysis object, the minimum concentration of its quantitative detection may continue to drop.

[0178] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for rapid detection of phenobarbital using dynamic SERS, characterized in that: The following steps are involved: The reducing agent and the silver salt solution are mixed evenly, and then stirred under boiling conditions for reaction, and after the reaction is completed, cooled to room temperature to obtain a silver sol; Centrifuging the silver sol, removing the supernatant to obtain a centrifugal product, concentrating and ultrasonically dispersing the centrifugal product to obtain a reinforced substrate; A solution containing phenobarbital is used as a sample to be tested, and the enhanced substrate, coagulant and the sample to be tested are mixed evenly and added to a hydrophilic flexible substrate, and then dynamic SERS detection is performed; the hydrophilic flexible substrate is a nylon microporous filter membrane.

2. The method for rapid detection of phenobarbital using dynamic SERS according to claim 1, characterized in that: The coagulant is an inorganic alkaline solution.

3. The method for rapid detection of phenobarbital using dynamic SERS according to claim 2, characterized in that: The inorganic alkali solution is a sodium hydroxide aqueous solution.

4. The method for rapid detection of phenobarbital using dynamic SERS according to claim 2, characterized in that: The concentration of the inorganic alkali solution is 1 mol / L.

5. The method for rapid detection of phenobarbital using dynamic SERS according to claim 1, characterized in that: The volume ratio of the reinforcing substrate, the coagulant and the sample to be tested is 1:1:1; the concentration of phenobarbital in the sample to be tested is 0.1 mg / L to 0.3 mg / L.

6. The method for rapid detection of phenobarbital using dynamic SERS according to claim 1, characterized in that: The reducing agent is sodium citrate; the silver salt solution is a silver nitrate aqueous solution.

7. The method for rapid detection of phenobarbital using dynamic SERS according to claim 6, characterized in that: The concentration of the sodium citrate aqueous solution is 0.2 mg / mL; the concentration of the silver nitrate aqueous solution is 0.18 mg / mL; and the volume ratio of the sodium citrate aqueous solution to the silver nitrate aqueous solution is 1:

50.

8. The method for rapid detection of phenobarbital using dynamic SERS according to claim 1, characterized in that: The detection conditions of dynamic SERS detection are as follows: laser power is 120 mW and integration time is 5 s.

9. The method for rapid detection of phenobarbital using dynamic SERS according to claim 1, characterized in that: The sample to be tested is a sodium phenobarbital aqueous solution.

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