A method for sers detection of kanamycin sulfate

Abstract

The application discloses a SERS detection method of kanamycin sulfate, and relates to the detection technology of kanamycin sulfate. The preparation of a SERS substrate and the SERS detection of kanamycin sulfate are provided. The quantitative analysis of kanamycin sulfate is carried out by using silver nanoparticles as the SERS substrate. Under the optimized addition amount of kanamycin sulfate and the volume of added hydrochloric acid, the linear detection range of the kanamycin sulfate is 100 ppm to 10 ppb, the detection limit is 10 ppb, and the relative standard deviation of the SERS intensity is less than 10%. The silver sol as the SERS substrate has the advantages of low cost, controllability, high detection efficiency and the like, and can be popularly applied to the practical detection of antibiotics and the like in the SERS detection. The method is simple in operation, high in accuracy and sensitivity, and does not need complex operation technology, and thus meets the requirements of large-scale and rapid analysis and detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the detection technology of kanamycin sulfate, and in particular to a SERS detection method of kanamycin sulfate using silver nanoparticles as a SERS substrate. BACKGROUND

[0002] Kanamycin sulfate is an aminoglycoside antibiotic, mainly a glycoside compound formed by connecting amino cyclohexanol and amino sugar through an oxygen bridge (Ding Dalian, Richard Salvi. High concentration of Kana can cause obvious damage to the ear, kidney and other parts [J]. Chinese Journal of Otolaryngology, 2007 (02): 125-131.). Kanamycin has inhibitory effect on both gram-positive bacteria and gram-negative bacteria, and is one of the commonly used veterinary drugs in China's livestock, agriculture and aquaculture. However, its abuse can be accumulated through animal-derived food, such as pork, beef and other meat, as well as dairy products and eggs, affecting the final intake of humans. When humans consume food with excessive kanamycin, not only can it cause, for example, deafness, kidney disease and cardiovascular disease, but also the infection of animal body with drug-resistant pathogenic bacteria can cause drug resistance of normal bacteria in human body. In order to ensure food safety, many countries and regions have regulations on the maximum residue limits (MRLs) of kanamycin. The GB 31650-2019 “National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Food” stipulates that the maximum residue limit of kanamycin in milk is 150 μg / kg (Lu Yang, Chen Jin-yuan, He Zhao-yuan, Guo Ya-wen, Wang Bo, Xie Kai-zhou. Comparative study on the standards of veterinary drug residue limits in beef and mutton and their products in China and abroad [J]. Chinese Journal of Veterinary Drugs, 2020, 54 (11): 57-64.), and the European Union stipulates that the maximum residue limit of kanamycin in milk is 150 μg / kg (Official Journal of European Union. Commission Decision (EU) No 37 / 2010 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin [EB / OL]. [2009-12-22] [J]. Therefore, veterinary drug residue monitoring is an important issue to ensure food safety, and it is necessary to develop a highly sensitive and selective determination method for kanamycin drug residues.

[0003] The reported detection methods of kanamycin mainly include microbiological assay, chromatography, capillary electrophoresis (CE), immunoassay, fluorescence and ultraviolet spectroscopy (State Pharmacopoeia Commission of P. R. China. Chinese Pharmacopoeia [S]. Part 2. Beijing: China Medical Science Press, 2000; Zhang X., et al. Determination of Kanamycin by High Performance Liquid Chromatography [J]. Molecules, 2019, 24(10); Manyanga V., et al. Improved liquid chromatographic method with pulsed electrochemical detection for the analysis of kanamycin [J]. Journal of Chromatography A, 2010, 1217(24): 3748-3753; Long Y.H., et al. Determination of kanamycin in serum by solid-phase extraction, pre-capillary derivatization and capillary electrophoresis [J]. Journal of Chromatography B, 2003, 784(2): 255-264; Kloth K., et al. A regenerable immunochip for the rapid determination of 13 different antibiotics in raw milk [J]. Analyst, 2009, 134(7): 1433-1439; Liao Q.G., Wei B.H., Luo L.G. Aptamer based fluorometric determination of kanamycin using double-stranded DNA and carbon nanotubes [J]. Microchimica Acta, 2017, 184(2): 627-632.). Microbial assay is the traditional detection method, but it has the disadvantages of long culture time and large error; capillary electrophoresis requires expensive instruments, complex methods, time-consuming and tedious sample pretreatment, which is not suitable for routine analysis of a large number of samples. Liquid chromatography is the most commonly used method, but due to the strong polarity of kanamycin and the lack of chromophore, it needs to be derivatized; or high performance liquid chromatography combined with universal detection systems such as evaporative light scattering detection (ELSD), pulsed amperometric detector (PAD) and mass spectrometry (MS) (Megoulas N.C., Koupparis M.A. Direct determination of kanamycin in raw materials, veterinary formulation and culture media using a novel liquid chromatography-evaporative light scattering method [J]. Analytica Chimica Acta, 2005, 547(1): 64-72; Zhang Y., et al. HPLC-ELSD determination of kanamycin B in the presence of kanamycin A in fermentation broth [J]. Biomedical Chromatography, 2015, 29(3): 396-401; Hanko V.P., Rohrer J.S. Determination of tobramycin and impurities using high-performance anion exchange chromatography with integrated pulsed amperometric detection [J]. Journal of Pharmaceutical and Biomedical Analysis, 2006, 40(4): 1006-1012; Fujii Y., Kaga T., Nishimura K.Simultaneous Determination of Aminoglycoside Residues in Livestock and Fishery Products by Phenylboronic Acid Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry[J]. Analytical Sciences, 2019, 35(9): 961-966.) These detection techniques, although highly sensitive and specific, are expensive, often require liquid chromatography to use volatile ion reagents as mobile phases, and cause significant damage to instruments and chromatographic columns during analysis. Regular cleaning of ion sources and chromatographs is required to prevent contamination and sensitivity decline, making them unsuitable for large-scale sample detection.

[0004] However, noble metal nanostructure surface plasmon resonance (SPR) has attracted much attention due to its wide range of applications. It not only catalyzes certain special surface reactions, but also produces surface-enhanced Raman scattering effect (SERS), greatly enhancing the surface Raman signal of molecules (Jin X P, Li X J, Zhang C J, Yuan Y X, Yao J L. Surface-enhanced Raman spectroscopy study of decarboxylation of o-mercaptobenzoic acid induced by gold film surface plasmon resonance[J]. Spectroscopy and Spectral Analysis, 2021, 41(10): 3153-3158.).

[0005] The current reports on SERS detection of kanamycin sulfate are mainly based on some new detection methods of aptamer, for example, Zengin et al. (Zengin A., Tamer U., Caykara T. Extremely sensitive sandwich assay of kanamycin using surface-enhanced Raman scattering of 2-mercaptobenzothiazole labeled gold@silver nanoparticles [J]. Analytica Chimica Acta, 2014, 817: 33-41) disclosed a sensor for detecting kanamycin in milk by combining hybrid MNPs and 2-mercaptobenzothiazole (MBT) labeled Au@Ag NPs as SERS substrate with kanamycin aptamer, with a detection limit of 2 pg / mL; Jiang et al. (Jiang Y.F., et al. Ultrasensitive analysis of kanamycin residue in milk by SERS-based aptasensor [J]. Talanta, 2019, 197: 151-158;) disclosed double-stranded DNA binding bimetallic gold@silver nanoparticles as substrate combined with kanamycin aptamer to detect kanamycin in milk, with a detection limit of 0.90 pg / mL; Jiang et al. (Jiang Y.F., et al. A simple and sensitive aptasensor based on SERS for trace analysis of kanamycin in milk [J]. Journal of Food Measurement and Characterization, 2020, 14(6): 3184-3193) disclosed a SERS-based aptamer sensor for detecting kanamycin in milk by combining 4-MBA labeled Au@Ag NPs with kanamycin aptamer, with a detection limit of 142 pg / mL. However, most SERS detections use Au@Ag modified antibiotic aptamer molecules as SERS substrate, which has good selectivity, but the synthesis of the substrate is complex, can only detect one kind, and the structure of the nucleic acid aptamer is easily affected by the environment, and the specificity is unstable; and the specific nucleic acid aptamer obtained by the research is few in kind. SUMMARY

[0006] The application aims to provide a simple, fast, high recovery rate and reliable SERS detection method of kanamycin sulfate. The application comprises the following steps: The application comprises the following steps: 1) preparing Ag nanoparticle sol by reducing silver nitrate with sodium citrate; 2) preparing SERS substrate by mixing the Ag nanoparticle sol with kanamycin sulfate and hydrochloric acid; 3) detecting kanamycin sulfate by SERS. The application has the advantages of low cost, controllability, high detection efficiency and the like, and can be popularized and applied in the SERS detection of antibiotics and the like.

[0007] The application comprises the following steps:

[0008] 1) preparing Ag nanoparticle sol by reducing silver nitrate with sodium citrate;

[0009] In step 1), the Ag nanoparticle sol prepared by reducing silver nitrate with sodium citrate can be prepared by controlling the concentration of reactants, reaction temperature, reaction time and stirring speed, and the specific steps can be as follows: taking AgNO3 solution in a flask, stirring and heating to boiling reflux state, adding sodium citrate until the solution becomes opaque liquid with slight green color, continuing to heat reflux for 50-60 min, stopping heating, naturally cooling to room temperature, and sealing and storing in dark place.

[0010] The mass concentration fraction of sodium citrate is 0.8%-1.5%, and the volume ratio of sodium citrate to silver nitrate is 30-50; the concentration of silver nitrate is 1-3 mmol / L.

[0011] The reaction temperature can be 90-100℃, the reaction time is 30-80 min, and the stirring speed is 1000-1500 r / min; the Ag nanoparticle is spherical nanoparticle or elliptical nanoparticle, and the particle size of the spherical Ag nanoparticle is about 50±10 nm.

[0012] The reaction conditions for preparing Ag nanoparticle sol by reducing silver nitrate with sodium citrate are as follows: heating and refluxing to boiling under magnetic stirring, then quickly adding sodium citrate, continuing to heat and reflux for 50-60 min, and naturally cooling to room temperature after reaction.

[0013] The specific steps of preparing the Ag nanoparticle sol by reducing silver nitrate with sodium citrate can be as follows: 100 mL of AgNO3 solution is taken in a single-mouthed round-bottom flask, and the solution is stirred with a magnetic stirrer while being heated to a boiling reflux state with an electric heating mantle. Then, pre-prepared sodium citrate is rapidly added. About 1 minute after the addition, the solution changes from colorless to milky white, and then to a slightly green opaque liquid. The stirring and heating are continued for 50-60 minutes at a slight boil. Then, the heating mantle is removed, and the heating is stopped. The solution is naturally cooled to room temperature, and then the round-bottom flask is sealed with aluminum foil paper to avoid light and stored.

[0014] 2) Quantitative analysis and detection of kanamycin sulfate on the SERS substrate.

[0015] In step 2), the specific method of the quantitative analysis and detection of kanamycin sulfate on the SERS substrate is as follows: different concentrations of kanamycin sulfate solution are mixed with the SERS substrate and hydrochloric acid at an optimal combination ratio under the optimal excitation wavelength, and surface-enhanced Raman detection is performed. With the increase of the concentration of kanamycin sulfate, the Raman peak of kanamycin sulfate at a specific wavelength gradually increases. A standard curve is established by using the relationship between the intensity of the Raman characteristic peak of kanamycin sulfate and the amount of kanamycin sulfate, and the quantitative analysis and detection of kanamycin sulfate is performed.

[0016] The optimal excitation wavelength refers to a 785 nm excitation wavelength; and the Raman peak of kanamycin sulfate at a specific wavelength refers to the characteristic Raman peak of kanamycin sulfate at 456 cm -1 and 536 cm -1 ;

[0017] The quantitative analysis and detection refers to taking the logarithmic value of the concentration as the abscissa, i.e., taking the logarithm of the concentration of kanamycin sulfate as the abscissa, and taking the peak intensity of kanamycin sulfate at the strongest characteristic peaks of 456 cm -1 and 536 cm -1 as the ordinate, establishing a standard curve, quantitatively detecting kanamycin sulfate, and detecting the range of 1000 ppm-10 ppb, the linear range of 100 ppm-10 ppb, the linear correlation coefficient R 2 of 0.98785-0.99216, the detection limit of 10 ppb, and the relative standard deviation of the SERS intensity of less than 10%.

[0018] The optimal combination ratio (silver sol: kanamycin sulfate: hydrochloric acid) is 1:0.8:0.6. The concentration of the hydrochloric acid is 1-2 mol / L, and the optimal detection time after mixing is within 0-1 minutes.

[0019] The different concentrations of kanamycin sulfate solution refer to water as the optimal solvent for preparing kanamycin sulfate.

[0020] This invention utilizes the reduction of silver nitrate with sodium citrate to prepare silver nanoparticles as a SERS substrate. The SERS substrate not only exhibits excellent SERS Raman enhancement, stability, and sensitivity, but also boasts a simple, low-cost, highly reproducible, and highly sensitive detection method that is easy to operate. Kanamycin sulfate (KANA) was selected as the target molecule, and the Raman enhancement effect of silver sol as a SERS substrate on kanamycin sulfate was tested by adding a certain volume of hydrochloric acid. Experimental results show that silver sol as a SERS substrate has excellent Raman enhancement effects. Using kanamycin sulfate at 456 cm⁻¹... -1 536cm -1 The signal intensity of the characteristic Raman peak at the concentration is directly proportional to the concentration, enabling the establishment of a simple, rapid, and low-cost analytical method that directly utilizes the SERS characteristic peak of kanamycin sulfate for quantitative analysis without complex specific modifications. The linear range of this invention is 100 ppm to 10 ppb, with a linear correlation coefficient of 0.98785 to 0.99216 and a detection limit of 10 ppb. Attached Figure Description

[0021] Figure 1 The surface plasmon resonance absorption spectra of the prepared AgNPs after the addition of kanamycin sulfate and hydrochloric acid are shown.

[0022] Figure 2 Raman spectra of AgNPs after the addition of different volumes of hydrochloric acid.

[0023] Figure 3 Raman spectra of AgNPs with different volumes of kanamycin added.

[0024] Figure 4 The Raman spectrum obtained after ten parallel measurements by KANA.

[0025] Figure 5 This is a bar chart showing the SERS peak intensities at different sites after 10 parallel measurements of 1 ppm kanamycin sulfate on a SERS substrate.

[0026] Figure 6 SERS spectra of kanamycin sulfate at different concentrations.

[0027] Figure 7 Kanamycin sulfate at the strongest Raman peak of 456 cm⁻¹ -1 and 536cm -1 A graph showing the relationship between SERS intensity and the logarithm of concentration at a given location.

[0028] Figure 8 SERS spectra of spiked extracts of kanamycin sulfate at different concentrations in milk. Detailed Implementation

[0029] The following examples further illustrate the application with reference to the accompanying drawings.

[0030] The embodiments of the present application comprise the following steps:

[0031] 1) Synthesis of Ag nanoparticles: Synthesis of Ag nanoparticles with particle size of about 55 nm: 1% by mass sodium citrate was prepared in advance, 1 mmol / L AgNO3 solution was prepared, 100 mL of prepared AgNO3 solution was taken in a single-mouth round-bottom flask, and the solution was stirred with a magnetic stirrer while heated to boiling reflux state with an electric heating mantle. 3 mL of 1% sodium citrate prepared in advance was rapidly added. About 1 min after the addition, the solution changed from colorless to milky white, and then to opaque liquid with a slight green color. The solution was continuously stirred and heated to micro-boiling for 1 h. Then the heating mantle was removed to stop heating, and the solution was naturally cooled to room temperature. The round-bottom flask was sealed with aluminum foil paper to avoid light and stored.

[0032] 2) Quantitative analysis and detection of kanamycin sulfate on the SERS substrate: Under the conditions of the optimal excitation wavelength, the optimal addition amount of kanamycin sulfate, and the optimal addition amount of hydrochloric acid, different concentrations of kanamycin sulfate solution, hydrochloric acid, and SERS substrate were mixed according to the optimal combination amount ratio for surface-enhanced Raman detection. With the increase of the concentration of kanamycin sulfate, the Raman peak of kanamycin sulfate at a specific wavelength gradually increased. The intensity of the Raman characteristic peak of kanamycin sulfate was used to establish a standard curve for the quantitative analysis and detection of kanamycin sulfate. The optimal excitation wavelength was 785 nm. Water was used as the optimal solvent for preparing kanamycin sulfate. The optimal combination amount ratio (silver sol: kanamycin sulfate: hydrochloric acid) was 1:0.8:0.6. The concentration of hydrochloric acid was 1-2 mol / L. The optimal detection time after mixing was within 0-1 min. The Raman peak of kanamycin sulfate at a specific wavelength was the characteristic Raman peak of kanamycin sulfate at 456 cm -1 , 536 cm -1 . The quantitative analysis and detection was performed by taking the logarithmic value of the concentration as the abscissa, i.e., taking the logarithm of the concentration of kanamycin sulfate as the abscissa, and taking the peak intensity of the two stronger characteristic peaks of kanamycin sulfate at 456 cm -1 , 536 cm -1 as the ordinate to establish a standard curve for the quantitative detection of kanamycin sulfate. The linear detection range was 100 ppm-10 ppb, the detection limit was 10 ppb, and the relative standard deviation of SERS intensity was less than 10%.

[0033] Example 1

[0034] 1) Synthesis of silver nanoparticles

[0035] Synthesis of Ag nanoparticles with particle size of about 55 nm: 1% sodium citrate was prepared in advance, 1 mmol / L AgN03 solution was prepared, 100 mL of prepared AgN03 solution was taken in a single round bottom flask, and the magnetic stirrer was stirred while the heating mantle was heated to boiling reflux state, 3 mL of 1% sodium citrate prepared in advance was added, and about 1 min after the addition, the solution changed from colorless to milky white, and then to opaque liquid with a little green. Continue to stir and heat to micro-boiling for 1 h, then remove the heating mantle to stop heating, and naturally cool to room temperature, then cover the round bottom flask with aluminum foil paper to seal and store in the dark.

[0036] 2) Surface plasmon resonance absorption spectrum (SPR spectrum) of silver nanoparticles

[0037] Figure 1 Surface plasmon resonance absorption spectrum (SPR) of silver nanoparticles after adding kanamycin and hydrochloric acid. As can be seen from the figure, after adding 1 ppm of kanamycin to the diluted 20 times silver nanoparticles (a), it can be seen that the surface plasmon resonance absorption intensity at 414 nm decreases significantly, and new absorption peaks are generated at wavelengths ≥ 650 nm (b). When hydrochloric acid is added, the surface plasmon resonance absorption intensity at 414 nm decreases more (c).

[0038] Example 2

[0039] 1) Synthesis of silver nanoparticles

[0040] Synthesis of Ag nanoparticles with particle size of about 55 nm: 1% sodium citrate was prepared in advance, 1 mmol / L AgN03 solution was prepared, 100 mL of prepared AgN03 solution was taken in a single round bottom flask, and the magnetic stirrer was stirred while the heating mantle was heated to boiling reflux state, 3 mL of 1% sodium citrate prepared in advance was added, and about 1 min after the addition, the solution changed from colorless to milky white, and then to opaque liquid with a little green. Continue to stir and heat to micro-boiling for 1 h, then remove the heating mantle to stop heating, and naturally cool to room temperature, then cover the round bottom flask with aluminum foil paper to seal and store in the dark.

[0041] 2) Optimization of the optimal amount of hydrochloric acid

[0042] Determine the volume of AgNPs and 100 ppb kanamycin sulfate to be added, add different volumes of hydrochloric acid, scan the surface enhanced Raman spectrum of kanamycin, and determine the optimal amount of hydrochloric acid to be added.

[0043] Figure 2The results are from Example 2, where the concentrations of AgNPs and kanamycin sulfate were kept constant (100 ppb), and their volume ratios were controlled while SERS spectra were collected. In curves a–e, the volume ratios of AgNPs to kanamycin sulfate and hydrochloric acid were 1:1:1, 1:1:0.8, 1:1:0.6, 1:1:0.4, 1:1:0.2, and 1:0:0, respectively. It can be seen that after adding different volumes of hydrochloric acid, kanamycin at 456 cm⁻¹… -1 and 536cm -1 It exhibits a strong Raman peak, with the Raman signal reaching its strongest when 60 μL of hydrochloric acid is added. Figure 2 Curve c in the figure shows that as the amount of hydrochloric acid added gradually increases to more than 60 μL ( Figure 2 In curves a and b), the Raman intensity did not increase. Therefore, in subsequent tests, the volume ratio of AgNPs to kanamycin sulfate and hydrochloric acid was kept constant at 1:1:0.6.

[0044] Example 3

[0045] 1) Synthesis of silver nanoparticles

[0046] Synthesizing Ag nanoparticles with a diameter of approximately 55 nm: Prepare a 1% (w / w) sodium citrate solution and a 1 mmol / L AgNO3 solution. Take 100 mL of the prepared AgNO3 solution and place it in a single-necked round-bottom flask. Stir with a magnetic stirrer while heating with a heating mantle to a boiling reflux state. Add 3 mL of the pre-prepared 1% sodium citrate solution. After about 1 minute, the solution changes from colorless to milky white, then to a slightly greenish opaque liquid. Continue stirring and heating until gently boiling for 1 hour. Remove the heating mantle and stop heating. Allow to cool naturally to room temperature. Then, cover the round-bottom flask with aluminum foil, seal it, and store it in a light-proof container.

[0047] 2) Optimization of the optimal kanamycin dosage

[0048] The optimal ratio of AgNPs and hydrochloric acid to be added was determined by scanning the surface-enhanced Raman spectrum of kanamycin by adding different volumes of kanamycin sulfate.

[0049] Figure 3 The results are from Example 2, where the concentrations of AgNPs and kanamycin sulfate were kept constant (100 ppb), their volume ratios were controlled, and SERS spectra were collected. In curves a–e, the volume ratios of AgNPs to kanamycin sulfate and hydrochloric acid were 1:1.4:0.6, 1:1:0.6, 1:0.8:0.6, 1:0.6:0.6, and 1:0.2:0.6, respectively. It can be seen that as the amount of kanamycin added gradually increases, at 456 cm⁻¹... -1 and 536cm -1 It exhibits a strong Raman peak when the amount of kanamycin added is 80 μL (Figure 3 The Raman signal peaks reached the strongest in c) of FIG. 6. However, the Raman intensity did not increase with the addition of kanamycin beyond 80 μL (d) of FIG. 6). Therefore, the volume ratio of AgNPs to kanamycin sulfate and hydrochloric acid was fixed at 1:0.8:0.6 in subsequent detection. Figure 3 The Raman signal peaks reached the strongest in c) of FIG. 6. However, the Raman intensity did not increase with the addition of kanamycin beyond 80 μL (d) of FIG. 6). Therefore, the volume ratio of AgNPs to kanamycin sulfate and hydrochloric acid was fixed at 1:0.8:0.6 in subsequent detection.

[0050] Example 4

[0051] 1) Synthesis of silver nanoparticles

[0052] Synthesis of Ag nanoparticles with a particle size of about 55 nm: 1 mmol / L of AgN03 solution was prepared in advance, 100 mL of the prepared AgN03 solution was taken in a single-mouth round-bottom flask, and the solution was stirred with a magnetic stirrer while being heated to a boiling reflux state with an electric heating mantle. 3 mL of 1% sodium citrate prepared in advance was added, and about 1 min after the addition, the solution changed from colorless to milky white, and then to opaque liquid with a slight green color. The solution was continuously stirred and heated to a slight boil for 1 h, and then the heating mantle was removed to stop heating. The solution was naturally cooled to room temperature, and then the round-bottom flask was sealed with aluminum foil paper to avoid light and stored.

[0053] 2) SERS detection of kanamycin sulfate

[0054] 100 μL of AgNPs was quickly added with 80 μL of 1 ppm kanamycin sulfate and 60 μL of 1 mol / L HCl, and then the Raman spectrum was quickly scanned. Ten parallel samples were collected, and the Raman spectrum was randomly collected at different points on the substrate with an excitation wavelength of 785 nm to obtain the surface-enhanced Raman spectrum of kanamycin corresponding to different points. The uniformity of the substrate was determined by comparing the Raman peak intensity of kanamycin collected in ten parallel samples.

[0055] 100 μL of AgNPs was quickly added with 80 μL of different concentrations of kanamycin sulfate solution (100 ppm, 10 ppm, 1 ppm, 100 ppb, 10 ppb) and 60 μL of 1 mol / L HCl, and then the Raman spectrum was quickly collected for quantitative detection of kanamycin sulfate.

[0056] Figure 4 Ten groups of parallel samples were collected. AgNPs and 1 ppm kanamycin sulfate and 1 mol / L HCl were mixed in a ratio of 1:0.8:0.6, and ten groups of parallel samples were continuously measured to measure the Raman peak intensity of kanamycin sulfate. The intensity of each characteristic Raman peak at different positions was plotted (FIG. 7). Figure 4 The relative standard deviation (RSD) of the Raman peak intensity of the ten groups of parallel samples was calculated from the intensity of the Raman peak, and the value was 2.21% to 3.39%, which was less than 10% (see FIG. 8). Figure 5This indicates that kanamycin sulfate exhibits good reproducibility on the SERS substrate and can be used for the quantitative analysis of kanamycin sulfate using surface-enhanced Raman spectroscopy. Using the above substrate, kanamycin sulfate at concentrations ranging from 100 ppm to 10 ppb was determined. Figure 6 ), Figure 6 Raman spectra of kanamycin sulfate aqueous solutions (a-e) at concentrations of 100 ppm, 10 ppm, 1 ppm, 100 ppb, and 10 ppb, respectively, dropped onto a SERS substrate. Each concentration was analyzed in triplicate. Higher concentrations of kanamycin sulfate resulted in higher concentrations at 456 cm⁻¹. -1 and 536cm -1 The stronger the corresponding Raman peak intensity, the more directly proportional the two are. Taking the two stronger peaks, 456 cm⁻¹... -1 and 536cm -1 Plot two standard curves with the logarithm of the peak intensity at wavenumber as the ordinate and the logarithm of the kanamycin sulfate concentration as the abscissa (see...). Figure 7 The error bars represent the standard deviation. It can be seen that there is a certain linear relationship between the logarithm of the concentration and the Raman peak intensity, with a linear range of 100 ppm to 10 ppb and a linear correlation coefficient R0. 2 The detection limit reaches 0.98785–0.99216, with a detection limit of 10 ppb. This detection limit is 1–2 orders of magnitude lower than the national standard limit (approximately 200 ppb). It is lower than the detection limit of kanamycin sulfate by liquid chromatography-tandem mass spectrometry (approximately 20 ppb). The detection range of this method is 100 ppm to 10 ppb, which fully meets the requirements of the national standard. Moreover, it is simple to operate, has low detection cost, and fast detection speed.

[0057] Example 5

[0058] 1) Synthesis of silver nanoparticles

[0059] Synthesizing Ag nanoparticles with a diameter of approximately 55 nm: Prepare a 1% (w / w) sodium citrate solution and a 1 mmol / L AgNO3 solution. Take 100 mL of the prepared AgNO3 solution and place it in a single-necked round-bottom flask. Stir with a magnetic stirrer while heating under a heating mantle until boiling and refluxed. Add 3 mL of the pre-prepared 1% sodium citrate solution. After about 1 minute, the solution changes from colorless to milky white, then to a slightly greenish opaque liquid. Continue stirring and heating until gently boiling for 1 hour. Remove the heating mantle and stop heating. Allow to cool naturally to room temperature. Then, cover the round-bottom flask with aluminum foil, seal it, and store it in a light-proof container.

[0060] 2) Spiked extraction of kanamycin from milk

[0061] In 1.5 ml centrifuge tube, 200 μL milk sample was added, 100 μL kanamycin sulfate of different concentrations (1000 ppm, 100 ppm, 10 ppm, 1 ppm, 100 ppb) was added respectively, mixed, 400 ul 1 mol / L hydrochloric acid was added, then 400 ul potassium ferrocyanide solution (10.6 g / L) was added, mixed, then 400 μL zinc acetate solution (22.0 g / L) was added, shaken for 10 s to precipitate protein, centrifuged at 8000 r / min for 2 min, the supernatant was transferred, 50 ul supernatant was added to 450 μL water to obtain the target molecule extract solution, and the Raman spectrum was collected (each concentration was measured three times).

[0062] Figure 8 The SERS spectra of kanamycin sulfate of different concentrations in milk are shown from top to bottom, which are 100 ppm, 10 ppm, 1 ppm, 100 ppb, 10 ppb of kanamycin and blank extract solution. Due to the interference of other components (such as lipids) in the sample extract solution, the intensity of the measured Raman peak is weaker than that of the Raman peak of the standard solution of the same concentration, and the detection sensitivity is reduced. The Figure 7 The standard curve was used as the working curve, and the standard addition recovery results of kanamycin sulfate of different concentrations in milk were measured as shown in Table 1.

[0063] Table 1

[0064]

[0065] As shown in Table 1, the standard addition recovery rate of the sample in milk is 89.4% to 100.1%, and the relative standard deviation is 1.0% to 13%. The results show that the standard addition recovery rate of kanamycin sulfate in milk is high, the detection precision is good, the results are reliable, and AgNPs can be used as the SERS substrate for determination of kanamycin sulfate.

Claims

1. A method for SERS detection of kanamycin sulfate characterized in that The method comprises the following steps: 1) preparing Ag nanoparticle sol by reducing silver nitrate with sodium citrate, specifically, adding AgNO3 solution into a container, heating and refluxing under magnetic stirring until boiling, then adding sodium citrate and continuing heating and refluxing for 50-60 min, and naturally cooling to room temperature after reaction; by controlling the concentration of reactants, reaction temperature, reaction time and stirring speed, Ag nanoparticle sol with uniform particle size is prepared; the mass percentage concentration of sodium citrate is 0.8%-1.5%, the volume ratio of sodium citrate to silver nitrate is 30-50, and the concentration of silver nitrate is 1-3 mmol / L; 2) quantitative analysis and detection of kanamycin sulfate on the SERS substrate, specifically: under the optimal excitation wavelength, mix different concentrations of kanamycin sulfate solution with the SERS substrate and hydrochloric acid according to the optimal combination amount ratio, and perform surface-enhanced Raman detection; as the concentration of kanamycin sulfate increases, the Raman peak of kanamycin sulfate at a specific wavelength gradually increases; a standard curve is established using the relationship between the intensity of the Raman characteristic peak of kanamycin sulfate and the amount of kanamycin sulfate to quantitatively analyze and detect kanamycin sulfate; the optimal excitation wavelength refers to a 785 nm excitation wavelength; the Raman peak of kanamycin sulfate at a specific wavelength refers to the characteristic Raman peak of kanamycin sulfate at 456 cm -1 and 536 cm -1 ; The volume ratio of silver sol to kanamycin sulfate to hydrochloric acid is 1:0.8:0.6, the concentration of hydrochloric acid is 1-2 mol / L, and the optimal detection time after mixing is within 0-1 min.

2. The method of claim 1, wherein the kanamycin sulfate is detected by SERS. In step 1), the reaction temperature is 90-100℃, the reaction time is 30-80 min, and the stirring speed is 1000-1500 r / min; the Ag nanoparticle is spherical or elliptical, and the particle size of the spherical Ag nanoparticle is 50±10 nm.

3. The method of claim 1, wherein the kanamycin sulfate is detected by SERS. In step 2), the quantitative analysis detection refers to taking the logarithm of the concentration as the abscissa, i.e. taking the logarithm of the kanamycin sulfate concentration as the abscissa, and taking the peak intensity of kanamycin sulfate at the strongest characteristic peaks 456 cm -1 and 536 cm -1 as the ordinate to establish a standard curve for quantitative detection of kanamycin sulfate, with a detection range of 1000 ppm to 10 ppb, a linear range of 100 ppm to 10 ppb, a linear correlation coefficient R 2 of 0.98785 to 0.99216, a detection limit of 10 ppb, and a relative standard deviation of SERS intensity of less than 10%.

4. The method of claim 1, wherein the kanamycin sulfate is detected by SERS. In step 2), the different concentrations of kanamycin sulfate solution refer to water as the optimal solvent for preparing kanamycin sulfate.

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