SERS (Surface Enhanced Raman Scattering) detection method of sisomicin based on gold nanoparticle aggregation

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) detection method of sisomicin based on gold nanoparticle aggregation, and relates to the field of analysis and detection. The method comprises the following steps: synthesizing gold nanoparticles by adopting a hydrothermal method, inducing the gold nanoparticles to agglomerate through an electrostatic adsorption effect between amino groups on a sisomicin structure and citric acid radicals on the surfaces of the gold nanoparticles, forming an SERS enhanced region, amplifying a Raman signal of the sisomicin adsorbed on the surfaces of the gold nanoparticles, and carrying out quantitative analysis on the sisomicin. Under the optimal experiment condition, the detection linear range of sisomicin is 10 <-5 >-10 <-9 > M, and the detection limit is 4.86 * 10 <-10 > M. The gold nanoparticle sol as the SERS substrate has the advantages of low cost, controllability, high detection efficiency and the like, meets the requirements of simple operation, high accuracy and sensitivity and rapid analysis and detection without complex operation, and has good universality and practical prospects.

Description

Technical Field

[0001] The invention relates to the field of analysis and detection, and in particular to a SERS detection method of sisomicin based on gold nanoparticle agglomeration. Background Art

[0002] Aminoglycosides (AGs) antibiotics are glycosides formed by connecting two or more amino sugars to an aminocyclobutane alcohol through a glycosidic bond. They have stable chemical properties, good water solubility, and strong antibacterial ability. These antibiotics are alkaline due to the amino group. They have a static bactericidal effect and have a good inhibitory and killing effect on Gram-negative bacteria and tuberculosis. Commonly used AGs include kanamycin, neomycin, streptomycin, gentamicin, tobramycin, kanamycin, sisomicin, amikacin, netilmicin, etc. AGs have significant antibacterial effects on a variety of Gram-positive and Gram-negative bacteria, and can effectively inhibit the growth and reproduction of bacteria. They are currently one of the commonly used veterinary drugs in my country's animal husbandry, agriculture, and aquaculture. However, if antibiotics cannot be used legally, they will remain in animal food and the environment. If humans eat foods with excessive aminoglycoside antibiotics, it will not only cause deafness, kidney disease, and cardiovascular diseases, but also the drug-resistant pathogens in the animal body will infect normal human bacteria to produce drug resistance. In order to ensure food safety, my country's GB31650-2019 "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Food" stipulates that the maximum residue limit of aminoglycoside antibiotics in food animals is 100 ug / kg. Establishing rapid screening and detection of aminoglycoside drug residues in aquatic products is of great significance to maintaining human dietary health.

[0003] The detection methods of aminoglycoside antibiotics reported so far mainly include microbiological assay, gas chromatography, high performance liquid chromatography (HPLC), colorimetry, and fluorescence. Aminoglycoside antibiotics lack chromophores or fluorophores in their structure, making it difficult to detect them directly by liquid chromatography. The derivatization process of HPLC is complicated and cumbersome, not easy to operate, not stable, and easily interfered by the matrix, with a high detection limit. The colorimetric method uses unmodified gold nanoparticles as biosensors for the detection of aminoglycoside antibiotics. Aminoglycoside antibiotics can quickly induce the aggregation of gold nanoparticles, accompanied by color changes of gold nanoparticles. The fluorescence method (FL) mainly detects antibiotics by quenching or enhancing the fluorescence intensity of fluorescent nanomaterials. Although it is more widely used due to its convenient operation, simple setup, and high sensitivity, it is usually affected by photobleaching, interference, and poor stability in the environment. Therefore, it is not suitable for rapid, simple and effective screening of large quantities of samples with different characteristic peaks of the same antibiotics on the same substrate. Summary of the invention

[0004] The purpose of the present invention is to solve the above-mentioned problems in the prior art and provide a SERS detection method for sisomicin based on gold nanoparticle aggregation. The present invention induces the aggregation of gold nanoparticle sol through the electrostatic adsorption between sisomicin and gold nanoparticles. Through electrostatic interaction, sisomicin appears in the SERS enhanced hot spot area of ​​the gold nanoaggregate, and the qualitative and quantitative analysis of sisomicin is achieved under the conditions of optimal amount ratio and reaction time. The detection linear range of sisomicin is 10 -5 ~10 -9 M, the detection limit was 4.86 × 10 -10 M.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A SERS detection method for sisomicin based on gold nanoparticle agglomeration comprises the following steps:

[0007] 1) Gold nanoparticle sol was prepared by trisodium citrate hydrothermal synthesis method;

[0008] 2) mixing the sample solution containing sisomicin with the gold nanoparticle sol, inducing the aggregation of the gold nanoparticles through the electrostatic adsorption between the structure of sisomicin and the gold nanoparticles to form a SERS enhancement region;

[0009] 3) Under the optimal excitation wavelength, the mixed solution is subjected to surface enhanced Raman scattering detection, and the analysis and detection of sisomicin is achieved by measuring the Raman peak intensity of sisomicin at a specific wavelength.

[0010] The qualitative analysis of sisomicin is to mix the sample solution with the gold nanoparticle sol, perform surface enhanced Raman detection at the optimal excitation wavelength, and compare the Raman peaks of the solid powder of the sample; the SERS characteristic peaks of sisomicin and kanamycin, tobramycin, and amikacin are different, and the four are distinguished based on the different SERS characteristic peaks of sisomicin.

[0011] The quantitative analysis and detection of sisomicin is as follows: standard sample solutions of different concentrations are mixed with gold nanoparticle sol, and surface enhanced Raman detection is performed at the optimal excitation wavelength. As the concentration of the sample solution increases, the Raman peak of the sample at a specific wavenumber gradually increases. A standard curve is established using the relationship between the intensity of the SERS characteristic peak of the sample and the content of the sample, and the sample is quantitatively analyzed and detected.

[0012] The standard curve of the quantitative analysis detection is established by taking the logarithmic values ​​of the series concentrations of the standard sample solution as the abscissa and taking the peak intensity at the characteristic peak measured as the ordinate.

[0013] In the present invention, the linear range of sisomicin detection is 10 -5 ~10 -9 M, the detection limit was 4.86 × 10 -10 M.

[0014] The optimal excitation wavelength refers to the 785 nm excitation wavelength; the Raman peak of sisomicin at a specific wavelength refers to the Raman peak of sisomicin at 544 cm -1 The SERS characteristic peaks are shown at .

[0015] In the present invention, after the sample solution of sisomicin is mixed with the gold nanoparticle sol, surface enhanced Raman scattering detection is performed within 5 to 240 seconds.

[0016] In step 1), chloroauric acid solution is added to a container, heated in a water bath until boiling, and then trisodium citrate solution is added, and the heating reaction is continued to obtain a gold nanoparticle sol.

[0017] The particle size of the prepared gold nanoparticles is 45 ± 10 nm.

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

[0019] The present invention relates to a detection technology for aminoglycoside antibiotics, and utilizes the electrostatic adsorption between sisomicin and gold nanoparticle sol to induce the agglomeration of gold nanoparticle sol. Through the electrostatic interaction, sisomicin is adsorbed on the surface of gold nanoparticles, the gold nanoparticles form aggregates, a SERS enhancement region appears, and the Raman signal of sisomicin adsorbed on the surface of gold nanoparticles is enhanced and amplified, thereby achieving qualitative and quantitative analysis of sisomicin.

[0020] The invention is a method for preparing gold nanoparticles based on hydrothermal synthesis of trisodium citrate and detecting sisomicin by utilizing the agglomeration phenomenon of gold nanoparticle sol. The SERS substrate has the advantages of simple and convenient synthesis, low cost and good reproducibility, and can directly utilize the SERS characteristic peak of the sample solution to perform quantitative analysis on it. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A in the middle is the SEM image of concentrated gold nanoparticle sol; Figure 1 Middle B is a SEM image of aggregated gold nanoparticles after adding sisomicin to the concentrated gold nanoparticle sol; Figure 1 Middle C is the particle size histogram of the gold nanoparticle sol;

[0022] Figure 2 Zeta potential analysis graphs were obtained for the gold nanoparticle sol and the mixed solution of gold nanoparticle sol and sisomicin;

[0023] Figure 3 The Raman peak of sisomicin solid powder and the SERS peak of sisomicin mixed with gold nanoparticle sol;

[0024] Figure 4 A in the middle is the SERS spectra of sisomicin under different ratios of gold nanoparticle sol and sisomicin solution; Figure 4 B is the 544 cm-1 fluorescence intensity of sisomicin at different molar ratios of gold nanoparticle sol and sisomicin solution. -1 Line graph of SERS peak intensity;

[0025] Figure 5 A in the middle is the SERS spectra of sisomicin under different reaction time conditions; Figure 5 B is the 544 cm-1 of sisomicin under different reaction time conditions -1 Line graph of SERS peak intensity;

[0026] Figure 6 A in the figure is the parallel measurement of sisomicin ten times (10 -5 M) SERS spectrum; Figure 6 B in the figure shows sisomicin measured ten times in parallel (10 -5 M) at 544 cm -1 Histogram of SERS peak intensity at ;

[0027] Figure 7 is the SERS spectra of sisomicin at different concentrations;

[0028] Figure 8 Sisomicin at 544 cm -1 The relationship between the SERS intensity at and the negative logarithm of the concentration;

[0029] Fig. 9 A in the middle is the SERS spectra of different aminoglycoside antibiotic solutions; Fig. 9 Middle B is a bar graph of sisomicin and different types of antibiotic solutions. DETAILED DESCRIPTION

[0030] 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.

[0031] The invention first synthesizes gold nanoparticle sol, optimizes the amount ratio of gold nanoparticle sol to sample solution and reaction time, and realizes analysis and detection of the sample by establishing a standard curve of intensity and concentration of SERS characteristic peak.

[0032] Example 1

[0033] This embodiment includes the following steps:

[0034] 1) Synthesis of gold nanoparticle sol

[0035] Take 100 mL of chloroauric acid solution in a single-necked round-bottom flask, stir it magnetically in a water bath, heat it to boiling, add the prepared trisodium citrate solution, continue heating for 30-40 min, stop heating, cool it naturally, and store it away from light.

[0036] The present invention can obtain gold nanoparticles with uniform particle size by controlling the concentration of reactants, reaction temperature, reaction time and stirring speed. The mass percentage concentration of the trisodium citrate solution is 0.9% to 1.2%, the mass percentage concentration of the chloroauric acid solution is 0.6% to 1.2%, and the volume ratio of the trisodium citrate solution to the chloroauric acid solution is 50 to 100: 1. The stirring speed is 400 to 600 r / min; and the reaction temperature is 90 to 100°C.

[0037] Specifically, in this embodiment, the mass percentage concentration fraction of the trisodium citrate solution is 1%, the mass percentage concentration fraction of the chloroauric acid solution is 0.8%, and the volume ratio of the trisodium citrate solution to the chloroauric acid solution is 90:1.

[0038] 2) Quantitative analysis and detection of the sample solution on the SERS substrate

[0039] The gold nanoparticle sol is concentrated first, and then the sample solution to be tested (the solvent is ultrapure water) is added, and the most suitable reaction time is waited for, and the SERS spectrum is collected by exciting with the optimal excitation light wavelength. As the concentration of the sample solution to be tested increases, the intensity of its SERS characteristic peak will increase. The standard curve is established based on the relationship between the intensity and concentration of the SERS characteristic peak of the sample solution to be tested, and the sample solution to be tested is quantitatively analyzed and tested.

[0040] Specifically, the optimal excitation wavelength refers to an excitation wavelength of 785 nm.

[0041] In this embodiment, the gold nanoparticle sol is prepared by the synthesis of step 1 to obtain a gold nanosol stock solution. The gold nanoparticles are centrifuged and concentrated at a speed of 8000 rpm for 12 min, and 10 mL of the stock solution is concentrated to 1 mL for use. The volume ratio of the sample solution to be tested to the concentrated solution of the gold nanoparticles is 1:1 (see Example 2); the appropriate reaction time refers to the detection within 120 s after the two substances are mixed (see Implementation Example 3). The Raman peak of sisomicin at a specific wavelength refers to the Raman peak of sisomicin at 544 cm -1 The SERS characteristic peak at .

[0042] Figure 1 A in the middle is the SEM image of the gold nanoparticle sol, from which it can be seen that the gold nanoparticles are evenly distributed. Figure 1Figure C is the particle size histogram of the gold nanoparticle sol, from which it can be seen that the particle size of the gold nanoparticles is approximately 45 ± 10 nm. Figure 1 Middle B is a SEM image of aggregated gold nanoparticles after adding sisomicin to the concentrated gold nanoparticle sol.

[0043] Figure 2 The results of Zeta potential analysis of gold nanoparticle sol and mixed solution of gold nanoparticle sol and sisomicin are shown. As can be seen from the figure, the gold nanoparticle sol itself has a certain Zeta potential (about -17.2 ± 1.38 mV). When sisomicin is added to the gold nanoparticle sol, the Zeta potential of the mixed solution changes to 6.93 ± 0.54 mV. This change indicates that electrostatic adsorption occurs between sisomicin and gold nanoparticles, causing the surface charge properties of gold nanoparticles to change, thereby affecting the Zeta potential of the overall system. This electrostatic adsorption is an important reason for inducing the aggregation of gold nanoparticles to form SERS enhancement areas. By changing the aggregation state of gold nanoparticles, it affects the electric field distribution on their surface, thereby enhancing the Raman signal of sisomicin adsorbed on the surface of the particles.

[0044] Figure 3 The Raman peaks of sisomicin solid powder and the SERS peaks of sisomicin after mixing with gold nanoparticle sol are presented. In the solid powder state, sisomicin has its specific Raman spectral characteristics, and these peaks reflect the chemical bond vibration information of sisomicin molecules. When sisomicin is mixed with gold nanoparticle sol, its Raman signal changes significantly, and the surface enhanced Raman scattering (SERS) effect appears. By comparison, it can be found that the SERS peak after mixing is significantly enhanced in intensity, and the peak shape also changes.

[0045] Example 2

[0046] Optimization of the optimal ratio of gold nanoparticle sol to sisomicin solution: fix the concentration of gold nanoparticle sol and the concentration of sisomicin solution, adjust the ratio of gold nanoparticle sol to sisomicin solution, scan the SERS spectrum of sisomicin, and determine the optimal ratio between gold nanoparticle sol and sisomicin solution.

[0047] Specifically, the gold nanoparticle sol was fixed to the concentration of the synthetic stock solution, and the concentration of the sisomicin solution was 10 -5 M, adjust their ratio to 0.25:1, 0.5:1, 1:1, 2:1, 3:1, collect SERS spectra, see Figure 4It can be seen that when the amount ratio of the gold nanoparticle sol to the sisomicin solution reaches 1:1, the SERS intensity of sisomicin reaches the highest, so the amount ratio of the gold nanoparticle sol to the sisomicin solution is fixed to 1:1 in the subsequent examples.

[0048] Example 3

[0049] Optimization of the optimal reaction time of gold nanoparticle sol and sisomicin solution: the molar ratio of gold nanoparticle sol to sisomicin solution was fixed at 1:1, and the reaction time of the mixed solution of gold nanoparticle sol and sisomicin solution was adjusted to 5 s, 30 s, 60 s, 120 s, 180 s, and 240 s, respectively. The SERS spectrum of sisomicin was scanned to determine the optimal reaction time.

[0050] Figure 5 A in the middle is the SERS spectra of sisomicin measured under different reaction time conditions. Figure 5 As can be seen from Figure B, when the reaction time is equal to 120 s, the SERS intensity of sisomicin is the strongest, so the optimal reaction time of the sisomicin detection system is 120 s.

[0051] Example 4

[0052] Uniformity of SERS substrate of sisomicin: Gold nanoparticle sol and 10 -5 The SERS peak intensities of sisomicin collected ten times in parallel were compared to determine the uniformity of the SERS substrate.

[0053] Figure 6 A in the figure is the experimental result of Example 4, i.e., the SERS spectrum of sisomicin was measured ten times in parallel. Figure 6 B in the figure shows ten parallel measurements of sisomicin at 544 cm -1 From the bar graph of SERS peak intensity, it can be seen that the relative standard deviation (RSD) calculated from the characteristic SERS peak intensity of 10 groups of parallel samples is 6.79%, 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 sisomicin.

[0054] Example 5

[0055] SERS detection of sisomicin: gold nanoparticle sol and sisomicin solutions of different concentrations were taken with a fixed amount ratio of 1:1, the reaction time was adjusted to 120 s, and the SERS spectra were collected under excitation with an excitation light wavelength of 785 nm to obtain the SERS spectra of sisomicin of different concentrations.

[0056] Figure 7 The experimental results of Example 5, i.e. different concentrations of 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M SERS spectrum of sisomicin. It can be seen that with the increase of concentration, the SERS intensity of sisomicin becomes stronger.

[0057] Figure 8 The standard curve established after detecting different concentrations of standard solutions of sisomicin in Example 5 was -5 ~10 -9 M, sisomicin 544 cm -1 The negative logarithmic relationship between the intensity of the characteristic peak and the concentration of sisomicin, (y = 604.42 x + 6318.37), R 2 =0.9726. In addition, the detection limit (LOD) is 4.86 × 10 -10 M.

[0058] Example 6

[0059] Selectivity of gold nanoparticle sols for sisomicin, kanamycin, tobramycin, and amikacin.

[0060] The gold nanoparticle sol was mixed with a concentration of 10 -5 Under the optimal experimental conditions, surface enhanced Raman spectra of sisomicin (Siso), kanamycin (Kana), tobramycin (Tob), and amikacin (Amk) were obtained at an excitation wavelength of 785 nm.

[0061] Fig. 9 The experimental results of Example 6 are SERS spectra of sisomicin, kanamycin, tobramycin and amikacin solutions. As can be seen from the figure, the characteristic peak of sisomicin is compared with kanamycin, tobramycin and amikacin, and a unique 544 cm -1 Characteristic peaks, so sisomicin can be distinguished from sisomicin, kanamycin, tobramycin and amikacin.

Claims

1. A SERS detection method for sisomicin based on gold nanoparticle aggregation, characterized in that The following steps are involved: 1) Gold nanoparticle sol was prepared by trisodium citrate hydrothermal synthesis method; 2) mixing the sample solution containing sisomicin with the gold nanoparticle sol, inducing the aggregation of the gold nanoparticles through the electrostatic adsorption between the structure of sisomicin and the gold nanoparticles to form a SERS enhancement region; 3) Under the optimal excitation wavelength, the mixed solution is subjected to surface enhanced Raman scattering detection, and the analysis and detection of sisomicin is achieved by measuring the Raman peak intensity of sisomicin at a specific wavelength.

2. The SERS detection method of sisomicin based on gold nanoparticle aggregation according to claim 1, characterized in that: The qualitative analysis of sisomicin is to mix the sample solution with the gold nanoparticle sol, perform surface enhanced Raman detection at the optimal excitation wavelength, and compare the Raman peaks of the solid powder of the sample; the SERS characteristic peaks of sisomicin and kanamycin, tobramycin, and amikacin are different, and the four are distinguished based on the different SERS characteristic peaks of sisomicin.

3. The SERS detection method of sisomicin based on gold nanoparticle aggregation according to claim 1, characterized in that: The quantitative analysis and detection of sisomicin is as follows: sample solutions of different concentrations are mixed with gold nanoparticle sol, and surface enhanced Raman detection is performed at the optimal excitation wavelength. As the concentration of the sample solution increases, the Raman peak of the sample at a specific wavenumber gradually increases. A standard curve is established using the relationship between the intensity of the SERS characteristic peak of the sample and the content of the sample, and the sample is quantitatively analyzed and detected.

4. The SERS detection method of sisomicin based on gold nanoparticle aggregation according to claim 3, characterized in that: The standard curve of the quantitative analysis detection is established by taking the logarithmic values ​​of the series concentrations of the standard sample solution as the abscissa and taking the peak intensity at the characteristic peak measured as the ordinate.

5. The SERS detection method of sisomicin based on gold nanoparticle aggregation according to claim 1, characterized in that: The linear range of sisomicin detection is 10 -5 ~10 -9 M, the detection limit was 4.86 × 10 -10 M.

6. The SERS detection method of sisomicin based on gold nanoparticle aggregation according to claim 1, characterized in that: The optimal excitation wavelength refers to the 785 nm excitation wavelength; the Raman peak of sisomicin at a specific wavelength refers to the Raman peak of sisomicin at 544 cm -1 The SERS characteristic peaks are shown at .

7. The SERS detection method of sisomicin based on gold nanoparticle aggregation according to claim 1, characterized in that: After the sample solution of sisomicin was mixed with the gold nanoparticle sol, surface enhanced Raman scattering detection was performed within 5 to 240 s.

8. The SERS detection method of sisomicin based on gold nanoparticle aggregation according to claim 1, characterized in that: In step 1), chloroauric acid solution is added to a container, heated in a water bath until boiling, and then trisodium citrate solution is added, and the heating reaction is continued to obtain a gold nanoparticle sol.

9. The SERS detection method of sisomicin based on gold nanoparticle aggregation according to claim 1, characterized in that: The particle size of the prepared gold nanoparticles is 45 ± 10 nm.

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