Preparation of surface plasmon polariton coupled composite silver film and application of composite silver film in SERS (Surface Enhanced Raman Scattering) detection of tobramycin

Abstract

The invention discloses preparation of a surface plasmon polariton coupled composite silver film and application of the composite silver film in SERS (Surface Enhanced Raman Scattering) detection of tobramycin, and relates to the field of analysis and detection, the preparation of the composite silver film comprises preparation of two layers of silver films, one layer is a self-assembled silver nanoparticle film loaded on the front surface of PDMS (Polydimethylsiloxane), and the other layer is a silver cubic film loaded on the back surface of PDMS. The thickness of PDMS is controlled, surface plasmons of the two layers of silver films are mutually coupled, and the two layers of silver films are used as an SERS substrate for analysis and detection, so that the detection sensitivity can be greatly improved. The detection limit of tobramycin can be as low as 2.36 * 10 <-12 > M, the linear detection range is 10 <-5 >-10 <-11 > M, and the relative standard deviation of SERS intensity is less than 10%. The prepared composite silver film has the advantages of high sensitivity, good universality and good practical prospect.

Description

Technical Field

[0001] The invention relates to the field of analysis and detection, and in particular to the preparation of a surface plasmon-coupled composite silver film and its application in SERS detection of tobramycin. Background Art

[0002] Tobramycin is a glycoside antibiotic formed by aminosugar and aminocyclitol connected by oxygen bridge. Because it can inhibit protein synthesis, it is mainly suitable for the inhibition and killing of sensitive aerobic Gram-negative bacteria. However, due to its obvious ototoxicity, nephrotoxicity and neuromuscular blocking effects, tobramycin residues in animal food and the environment pose a potential threat to human health. Therefore, in order to ensure food safety, my country's GB T22969-2008 "Determination of Streptomycin, Dihydrostreptomycin and Kanamycin Residues in Milk Powder and Milk by Liquid Chromatography-Tandem Mass Spectrometry" stipulates that the maximum residue limit of aminoglycoside antibiotics in milk is 10 μg / kg. Establishing rapid screening and detection of tobramycin residues in dairy products is of great significance to maintaining human dietary health.

[0003] Currently reported detection methods for tobramycin and other similar antibiotics mainly include liquid chromatography-tandem mass spectrometry, fluorescence immunoassay and enzyme-linked immunosorbent assay. Among them, the operation process of liquid chromatography-tandem mass spectrometry is complicated and the detection time is long. Fluorescence immunoassay is costly and often suffers from photobleaching and interference, and is not suitable for rapid screening of large quantities of samples. Enzyme-linked immunosorbent assay has a high probability of false positives and low sensitivity. Summary of the invention

[0004] The object of the present invention is to solve the above problems in the prior art and to provide a preparation method of a surface plasmon coupled composite silver film and its application in surface enhanced Raman scattering (SERS) detection of tobramycin.

[0005] The present invention prepares a composite silver film coupled with surface plasmons, regulates the distance between the silver nanoparticle film and the silver cube film, improves the degree of mutual coupling of surface plasmons, and enhances the Raman spectrum of tobramycin adsorbed on the composite silver film. The film thickness of the polydimethylsiloxane (PDMS) film of the composite silver film and the pH value when detecting tobramycin are optimized. Under the conditions of the optimal PDMS film thickness and pH value, a standard curve of the intensity and concentration of the SERS characteristic peak is established to achieve quantitative analysis of tobramycin. The linear detection range of tobramycin is 10 -5 ~10 -11 M, the detection limit is 2.36×10 -12The relative standard deviation of the SERS intensity is less than 10%. The surface plasmon coupled composite silver film prepared by the present invention has high sensitivity, good universality and practical prospect.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] The method for preparing a surface plasmon-coupled composite silver film comprises the following steps:

[0008] 1) The silver nanoparticle solution is self-assembled into a silver nanoparticle interface film through liquid-liquid self-assembly, and then transferred to the PDMS membrane to complete the preparation of the silver nanoparticle film;

[0009] 2) The silver cube solution is self-assembled into a silver cube interface film through liquid-liquid self-assembly, and then transferred to the hydrophobic back side of the silver nanoparticle film to complete the preparation of the composite silver film.

[0010] The surface plasmon-coupled composite silver film of the present invention is used for detecting tobramycin.

[0011] The composite silver film is immersed in a sample solution containing tobramycin, and the sample solution is subjected to surface enhanced Raman scattering detection at the optimal excitation wavelength. The analysis and detection of tobramycin is achieved by measuring the Raman peak intensity of tobramycin at a specific wavelength.

[0012] Based on the different positions of the SERS characteristic peaks of tobramycin and other aminoglycoside antibiotics, tobramycin was specifically detected.

[0013] The other aminoglycoside antibiotics include kanamycin, neomycin and streptomycin, and the SERS characteristic peaks at different positions are 606 cm -1 and 1020 cm -1 .

[0014] The composite silver film was immersed in a sample solution containing tobramycin, taken out, dried, and subjected to SERS detection at the optimal excitation wavelength. The intensity value of the SERS characteristic peak in the spectrum was recorded and a standard curve was established with the corresponding tobramycin concentration for quantitative analysis and detection.

[0015] The SERS characteristic peak in the recorded spectrum refers to the peak at 1020 cm -1 The linear detection range of tobramycin is 10 -5 ~10 -11 M, the detection limit is 2.36×10 -12 M, the relative standard deviation of SERS intensity is less than 10%.

[0016] The optimal excitation wavelength is 785 nm.

[0017] The thickness of the PDMS film is 100-500 μm; the pH value of the detection condition is 2-10.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0019] The present invention is based on the surface plasmon coupling phenomenon between the silver nanoparticle film and the silver cube film, which greatly improves the SERS intensity of tobramycin adsorbed on the composite silver film. The composite silver film prepared by the surface plasmon coupling of the present invention has the advantages of simple operation, convenience, low cost and good reproducibility in detection. The experimental results show that the linear detection range of tobramycin is 10 -5 ~10 -11 M, the detection limit is 2.36×10 -12 M, the relative standard deviation of SERS intensity is less than 10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Optical photos of PDMS films with different thicknesses;

[0021] Figure 2 A in the middle is the SEM image of the silver nanoparticle film; Figure 2 B is the SEM image of the silver cube film;

[0022] Figure 3 The SERS spectra of tobramycin detected by composite silver film under conditions of different PDMS film thicknesses;

[0023] Figure 4 The SERS spectra of tobramycin detected by composite silver film under different pH conditions;

[0024] Figure 5 A composite silver film detects tobramycin, and the SERS spectra are paralleled ten times. Figure 5 B is the composite silver film detecting tobramycin, and the SERS spectrum of 10 times is parallel to 1020 cm -1 Relative standard deviation of SERS peak intensity at ;

[0025] Figure 6 SERS spectra of different concentrations of tobramycin detected by composite silver film A. Figure 6 Medium B Tobramycin at 1020cm -1 The linear relationship between the SERS intensity at and the negative logarithm of the concentration;

[0026] Figure 7 SERS spectra of tobramycin and other aminoglycoside antibiotic solutions detected by composite silver film. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0028] The present invention comprises the following steps

[0029] 1) Preparation of PDMS membrane.

[0030] The Sylgard 184 prepolymer and the catalyst are fully mixed in a suitable mass ratio, cured in an oven, and cut into required thickness.

[0031] 2) Synthesis of silver nanoparticle solution.

[0032] Silver nanoparticles with uniform particle size were prepared by using trisodium citrate and ascorbic acid method and controlling reaction parameters.

[0033] 3) Self-assembly, transfer and preparation of silver nanoparticle films.

[0034] The silver nanoparticle solution was mixed with dichloromethane, and tetrabutylammonium nitrate solution was added to form a silver nanoparticle interface film by liquid-liquid self-assembly. The silver nanoparticle film was transferred to the front of the PDMS film using a silicon wafer to complete the preparation of the silver nanoparticle film.

[0035] 4) Synthesis of silver cubes. First, prepare a gold seed solution and a growth solution, mix the two solutions and let them stand to obtain a gold core solution; then react the gold core solution with CTAC, inject silver nitrate solution and CTAC-ascorbic acid solution in sequence, and control the reaction parameters to obtain silver cubes with uniform particle size.

[0036] 5) Self-assembly and transfer of silver cube film to prepare composite silver film. First, mix silver cubes and PVP ethanol solution. Then add the silver cube PVP ethanol solution to a mixed solution of n-hexane, chloroform and ultrapure water, and liquid-liquid self-assemble to form a silver cube interface film. Transfer to the back of the PDMS film with a silicon wafer to complete the preparation of the composite silver film.

[0037] 6) SERS quantitative analysis and detection of tobramycin on composite silver film.

[0038] The composite silver film was immersed in tobramycin solutions of different concentrations, and SERS detection was performed under the optimal excitation wavelength, optimal PDMS film thickness and optimal pH conditions. The intensity value of the SERS characteristic peak in the spectrum was recorded and a standard curve was established with the corresponding tobramycin concentration for quantitative analysis. Since the positions of the SERS characteristic peaks of tobramycin and other aminoglycoside antibiotics are different, tobramycin was specifically detected.

[0039] Ultrapure water was used as solvent to prepare different concentrations of 10 -5~10 -11 M tobramycin solution; at the optimal excitation wavelength of 785 nm, the concentration of tobramycin solution was controlled to be 10 -5 M, the PDMS film thickness is 100~500 μm, and the pH is 2~10 for detection, and the optimal PDMS film thickness and the optimal pH conditions are optimized; the composite silver film is immersed in tobramycin solutions of different concentrations for SERS detection. The SERS characteristic peak 1020 cm in the recorded spectrum -1 The intensity values ​​were compared with the corresponding tobramycin concentrations to establish a standard curve for quantitative analysis. The SERS characteristic peaks (606 cm -1 , 1020 cm -1 ) and the 1020 cm -1 The intensity is much higher than that of gentamicin, amikacin, sisomicin and netilmicin, achieving specific detection of tobramycin.

[0040] Example 1

[0041] 1. Preparation of PDMS membrane.

[0042] The Sylgard 184 prepolymer and the catalyst were fully mixed at a suitable mass ratio of 10:1, cured in an oven for 0.5 to 3 hours, and cut into the required thickness of 100 to 500 μm. Specifically, in this embodiment, the curing time was 1 hour.

[0043] Figure 1 Optical photographs of PDMS films with different thicknesses. From left to right, the thicknesses are 100, 300, and 500 μm.

[0044] 2. Synthesis of silver nanoparticle solution.

[0045] Dissolve 50-100 mg of trisodium citrate and 100-500 mg of ascorbic acid in 300-500 mL of ultrapure water at the same time. Heat the mixture in a water bath at 60-80°C for 40-60 min, then add 1-5 mL of silver nitrate solution with a concentration of 0.01-0.05 mM and a volume of 0.01-0.05 mM. Heat the mixture in a water bath at 60-80°C for 20-30 min, then stop heating and cool the mixture to room temperature to obtain a uniform silver nanoparticle solution, which is then stored at 4°C away from light.

[0046] Specifically, in this embodiment, 50 mg of trisodium citrate, 100 mg of ascorbic acid, 300 mL of ultrapure water, and a silver nitrate solution with a concentration of 0.01 mM and a volume of 1 mL.

[0047] 3. Self-assembly, transfer and preparation of silver nanoparticle films.

[0048] Take 1~5 mL of silver nanoparticle solution and mix it with 1~5 mL of dichloromethane, then add 10 -3 ~10 -5 M, a volume of 100~500 μL of tetrabutylammonium nitrate solution, shake vigorously. The silver nanoparticle solution will undergo liquid-liquid self-assembly in the aqueous phase and the organic phase to form a silver nanoparticle interface film. Use a hydrophilic silicon wafer to adsorb the hydrophilic end of the silver nanoparticle interface film in the aqueous phase and transfer it to the water-air interface. Then, the front side of the hydrophobic PDMS film is contacted with the hydrophobic end of the silver nanoparticle interface film exposed to the air, peeled horizontally, rinsed with ultrapure water, and dried with nitrogen for use.

[0049] Specifically, in this embodiment, the silver nanoparticle solution is 1 mL, the dichloromethane is 1 mL, and the concentration of the tetrabutylammonium nitrate solution is 10 -3 M, volume is 100 μL.

[0050] The liquid-liquid self-assembly to form the silver nanoparticle interface film utilizes the tetrabutylammonium radical ions generated by the hydrolysis of tetrabutylammonium nitrate to spontaneously form a positive charge arrangement at the water-oil interface, and electrostatically attracts the negatively charged silver nanoparticles to cause liquid-liquid self-assembly in the aqueous phase and the organic phase.

[0051] 4. Synthesis of silver cubes.

[0052] In the first step, 0.05~0.5 mM chloroauric acid with a volume of 5~10 mL and 150~200 mM cetyltrimethylammonium bromide solution with a volume of 5~10 mL were mixed and reacted at room temperature for 30 min. Then, 10~20 mM sodium borohydride solution with a volume of 0.5~1 mL was added to complete the preparation of the gold seed solution; 150~200 mM CTAC with a volume of 15~20 mL, 60~100 mM ascorbic acid with a volume of 10~15 mL and 0.05~0.5 mM chloroauric acid with a volume of 2~20 mL were mixed as the growth solution; 0.5~2 mL of the gold seed solution was added to 27~55 mL of the growth solution and the mixture was allowed to stand for 2 h to obtain the gold core solution.

[0053] Specifically, in this embodiment, the concentration of chloroauric acid is 0.1 mM and the volume is 5 mL; the concentration of hexadecyltrimethylammonium bromide solution is 50 mM and the volume is 5 mL; the concentration of sodium borohydride solution is 10 mM and the volume is 0.5 mL; the concentration of CTAC is 150 mM and the volume is 15 mL; the concentration of ascorbic acid is 60 mM and the volume is 10 mL; the concentration of chloroauric acid is 0.1 mM and the volume is 2 mL; the gold seed solution is 0.5 mL and the growth solution is 27 mL.

[0054] In the second step, 0.1~1.5 mL of the gold core solution was reacted with CTAC with a concentration of 200~400 mM and a volume of 1.9~4.5 mL. A silver nitrate solution with a concentration of 0.5~2 mM and a volume of 1.5~5 mL and a CTAC-ascorbic acid solution composed of CTAC with a concentration of 40~80 mM and ascorbic acid with a volume ratio of 1:1 were injected in sequence. After 4~6 hours in a water bath at 60~80℃, stop heating and cool naturally to room temperature to obtain silver cubes with uniform particle size, which were stored at 4℃ away from light.

[0055] Specifically, in this embodiment, the gold core solution is 1.5 mL; the CTAC concentration is 200 mM, and the volume is 1.9 mL; the silver nitrate solution concentration is 0.5 mM, and the volume is 1.5 mL; the CTAC concentration is 40 mM; and the ascorbic acid concentration is 60 mM.

[0056] 5. Self-assembly and transfer of silver cubic films to prepare composite silver films.

[0057] The first step is to mix 10-20 mL of silver cubes and a 0.9%-1.2% PVP ethanol solution. Specifically, in this embodiment, the silver cubes are 10 mL and the PVP ethanol solution is 1% by mass.

[0058] In the second step, the obtained silver cube PVP ethanol solution is added to a 1:1:1 mixed solution of n-hexane, chloroform and ultrapure water, and slightly shaken, so that the silver cubes undergo liquid-liquid self-assembly in the aqueous phase and the organic phase to form a silver cube interface film. The hydrophilic end of the silver cube interface film in the aqueous phase is adsorbed by a hydrophilic silicon wafer and transferred to the water-air interface, and then the back of the PDMS film is contacted with the hydrophobic end of the silver cube interface film exposed to the air, and then horizontally peeled, rinsed with ultrapure water, and dried with nitrogen for use.

[0059] The liquid-liquid self-assembly to form the silver cube interface film is to use PVP to partially replace the CTA on the surface of the silver cube + The groups provide hydrophobicity and reduce the surface potential at the same time. Driven by Brownian motion, the silver cubes move to the interface and self-assemble in the aqueous phase and the organic phase to form a silver cube interface film.

[0060] Figure 2 A in the middle is the SEM image of the silver nanoparticle film, and the inset is the particle size histogram of the silver nanoparticles on the film. It can be seen that the silver nanoparticles are spherical with a particle size of about 51.08±4.17 nm. Figure 2 B is the SEM image of the silver cube film, and the inset is the particle size histogram of the silver cubes on the film. It can be seen that the silver cubes have good uniformity and the particle size is about 69.96±6.56 nm.

[0061] Example 2

[0062] SERS detection of tobramycin on composite silver film under conditions of different PDMS film thicknesses.

[0063] The concentration of tobramycin solution was fixed at 10 -5 M, composite silver films were prepared with PDMS films with thicknesses of 100, 300 and 500 μm, respectively, and immersed in tobramycin solutions, taken out, dried at room temperature, and SERS spectra were collected. Figure 3 It can be seen that when the thickness of the PDMS film is 500 μm, the SERS intensity of tobramycin reaches the highest, so a composite silver film with a thickness of 500 μm is used to detect tobramycin in subsequent experiments.

[0064] Example 3

[0065] SERS spectra of tobramycin detected by composite silver film under different pH conditions.

[0066] The concentration of tobramycin solution was fixed at 10 -5 M, adjust the thickness of PDMS to 500 μm, adjust the pH of the tobramycin solution in which the composite silver film is immersed to 2, 4, 6, 8 and 10, take it out, dry it at room temperature, and collect the SERS spectrum. Figure 4 It can be seen that when pH=6, the SERS intensity of tobramycin reaches the strongest, so the optimal pH of the tobramycin detection system in subsequent experiments is 6.

[0067] Example 4

[0068] Composite silver film was used to detect the homogeneity of tobramycin.

[0069] The concentration of tobramycin solution was controlled to be 10 -5 Under the condition of M, the optimal PDMS film thickness is 500 μm, the optimal pH is 6, the surface plasmon coupled composite silver film is immersed in tobramycin solution, taken out, dried at room temperature, and SERS spectra are randomly collected ten times on the substrate with an excitation wavelength of 785 nm. The SERS peak intensities of tobramycin collected ten times in parallel are compared to determine the uniformity of the SERS substrate.

[0070] Figure 5 A is the experimental result of Example 4, i.e., the SERS spectrum of tobramycin measured by the composite silver film ten times in parallel; Figure 5 B is tobramycin at 1020 cm -1 The SERS peak intensity bar graph was obtained by measuring the SERS peak intensity ten times in parallel. It can be seen that the relative standard deviation (RSD) calculated for the characteristic SERS peak intensity of 10 groups of parallel samples is 4.35%, which is less than 10%, indicating that the uniformity of the SERS substrate is good and can be applied to the SERS quantitative analysis of tobramycin.

[0071] Example 5

[0072] Detection of tobramycin by SERS of composite silver film.

[0073] SERS quantitative analysis of tobramycin on composite silver film: ultrapure water was used as solvent to prepare different concentrations of 10 -5 ~10 -11 M tobramycin solution; Under the conditions of optimal excitation wavelength of 785 nm, optimal PDMS film thickness of 500 μm, and optimal pH = 6, the composite silver film was immersed in tobramycin solutions of different concentrations, taken out, dried at room temperature, and SERS detection was performed. The SERS characteristic peak 1020 cm in the spectrum was recorded -1 The intensity values ​​were compared with the corresponding tobramycin concentrations to establish a standard curve for quantitative analysis.

[0074] Figure 6 A is the experimental result of Example 5, i.e. different concentrations of 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M SERS spectrum of tobramycin. It can be seen that the SERS intensity of tobramycin decreases with the decrease of concentration.

[0075] Figure 6 B is the standard curve established after detecting the standard solutions of different concentrations of tobramycin in Example 5, at 10 -5 ~10 -11 M / L range, tobramycin 1020 cm -1 The negative logarithmic relationship between the intensity of the characteristic peak and the tobramycin concentration, (y = 3053.88x + 35314.57), R 2 =0.974. In addition, the detection limit is 2.36×10 -12 M.

[0076] Example 6

[0077] Composite silver film detection of tobramycin and other aminoglycoside antibiotic solutions.

[0078] SERS specific detection of tobramycin and its homologues on composite silver film: ultrapure water was used as solvent to prepare a 10 -5M of tobramycin (TOB), kanamycin (KANA), neomycin (NEO), streptomycin (STR), gentamicin (GEN), amikacin (AMK), sisomicin (SISO) and netilmicin (MET), under the conditions of optimal excitation wavelength of 785nm, optimal PDMS film thickness of 500 μm and optimal pH = 6, the composite silver film was immersed in the above solutions respectively, taken out, dried at room temperature and carried out SERS detection.

[0079] Figure 7 The experimental results of Example 6 show that kanamycin, neomycin and streptomycin are at 606 cm -1 The aminoglycoside antibiotics, except kanamycin and streptomycin, have their own unique peaks at 1020 cm -1 The SERS intensity of tobramycin is much lower than that of tobramycin. Therefore, the composite silver film can specifically detect tobramycin in solutions of tobramycin and other aminoglycoside antibiotics.

Claims

1. A method for preparing a surface plasmon-coupled composite silver film, characterized in that: The following steps are involved: 1) The silver nanoparticle solution is self-assembled into a silver nanoparticle interface film through liquid-liquid self-assembly, and then transferred to the front side of the PDMS film to complete the preparation of the silver nanoparticle film; 2) The silver cube solution is self-assembled into a silver cube interface film through liquid-liquid self-assembly, and then transferred to the back of the PDMS membrane to complete the preparation of the composite silver film.

2. A surface plasmon-coupled composite silver film, characterized in that: The composite silver film is prepared by the method for preparing a surface plasmon-coupled composite silver film as described in claim 1.

3. Use of the surface plasmon coupled composite silver film according to claim 2 in SERS detection of tobramycin.

4. The use according to claim 3, characterized in that: The composite silver film is immersed in a sample solution containing tobramycin, taken out, dried, and subjected to surface enhanced Raman scattering detection at an optimal excitation wavelength. The analysis and detection of tobramycin is achieved by measuring the Raman peak intensity of tobramycin at a specific wavelength.

5. The use according to claim 4, characterized in that: Based on the different positions of the SERS characteristic peaks of tobramycin and other aminoglycoside antibiotics, tobramycin was specifically detected.

6. The use according to claim 5, characterized in that: The other aminoglycoside antibiotics include kanamycin, neomycin and streptomycin, and the SERS characteristic peaks at different positions are 606 cm -1 and 1020 cm -1 .

7. The use according to claim 4, characterized in that: The composite silver film was immersed in tobramycin solutions of different concentrations, and SERS detection was performed at the optimal excitation wavelength. The intensity value of the SERS characteristic peak in the spectrum was recorded and a standard curve was established with the corresponding tobramycin concentration for quantitative analysis and detection.

8. The use according to claim 7, characterized in that: The SERS characteristic peak in the recorded spectrum refers to the peak of tobramycin at 1020 cm -1 The linear detection range of tobramycin is 10 -5 ~10 -11 M, the detection limit is 2.36×10 -12 M, the relative standard deviation of SERS intensity is less than 10%.

9. The use according to claim 4, characterized in that: The optimal excitation wavelength is 785 nm.

10. The use according to claim 4, characterized in that: The thickness of the PDMS membrane was 100-500 μm; the pH of the detection conditions was 2-10.

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